先天免疫学糖生物学

Cheorl-Ho
Kim
Glycobiology
of Innate
Immunology
Glycobiology of Innate Immunology
Cheorl-Ho Kim
Glycobiology of Innate
Immunology
Cheorl-Ho Kim
Molecular and Cellular Glycobiology Lab
Sungkyunkwan University, Department of
Biological Sciences
Suwon, Korea (Republic of)
ISBN 978-981-16-9080-8 ISBN 978-981-16-9081-5 (eBook)

https://doi.org/10.1007/978-981-16-9081-5
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Contents
1 Repertoire in Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1 Historical Expansion of Defense System . . . . . . . . . . . . . . . . . 1
1.2 Columbus Era to Modern Revolution in Immunological
Defense System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3 Historical Profile of Defense Constituents and Progress
in Innate Immune Repertoire . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3.1 Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3.2 Leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3.3 Neutrophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3.4 Granulocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3.5 Monocytes and Macrophages . . . . . . . . . . . . . . . . . . 9
1.3.6 Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.3.7 Complement System . . . . . . . . . . . . . . . . . . . . . . . . 13
1.3.8 Opsonization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.3.9 Lysozyme and Salvarsan . . . . . . . . . . . . . . . . . . . . . 19
1.3.10 Progress in Innate Immune Response Since Historic
Spanish Flu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1.4 The Outline of Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . 24
1.4.1 Concept of Immune Receptors . . . . . . . . . . . . . . . . . 25
1.4.2 Host Protection from Microbial Invaders of Innate
Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
1.5 Autophagy from Microbial Invaders and Self-Associated
Molecular Patterns (SAMPs) of Innate Immune Cells . . . . . . . . 30
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2 Dendritic Cells (DCs) in Innate Immunity . . . . . . . . . . . . . . . . . . . 37
2.1 General Biology of DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
2.2 Classification and Different Function of DCs . . . . . . . . . . . . . . 40
2.2.1 pDC, Lymphoid Organ CD8α+ DC, and Tissue
CD103+ DC Interaction with Tregs . . . . . . . . . . . . . . 43
v
vi Contents
2.2.2 DCs Induce Tolerance State . . . . . . . . . . . . . . . . . . . 44

2.2.3 DC co-Stimulatory Receptors . . . . . . . . . . . . . . . . . . 45
2.2.4 Application of DCs to Human Diseases . . . . . . . . . . . 48
References . . . ........................................ . 49
3 Glycan Biosynthesis in Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.1 General Glycosylation Events . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.2 Sugar Nucleotide Transporters Deliver Donor Saccharides
to ER-Golgi Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.3 Golgi Traffic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.4 N-glycan Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.5 O-glycosylation and Multiple O-Glycan Structures . . . . . . . . . . 61
3.5.1 7 Core O-glycan Structures . . . . . . . . . . . . . . . . . . . 61
3.5.2 Modification of 7 Core O-Glycan Structures . . . . . . . 63
3.6 O-GlcNAcylation, O-Mannosylation, O-β-Glucosylation,
O-α-Fucosylation, O-β-Glucosylation, O-β-Galactosylation,
C-Glycosylation, and C-Mannosylation . . . . . . . . . . . . . . . . . . 64
3.6.1 O-GlcNAcylation . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.6.2 O-Mannosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.6.3 O-β-Glucosylation . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.6.4 O-α-Fucosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.6.5 O-β-Glucosylation . . . . . . . . . . . . . . . . . . . . . . . . . . 66
3.6.6 O-β-Galactosylation . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.6.7 C-Mannosylation and C-Glycosylation . . . . . . . . . . . 67
3.7 Function of O-Glycosylation and O-Glycans . . . . . . . . . . . . . . 68
3.8 Glycosaminoglycans (GAGs) . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.8.1 Classification and Biosynthesis of GAGs . . . . . . . . . . 70
3.8.2 Chondroitin Sulfate (CS) . . . . . . . . . . . . . . . . . . . . . 71
3.8.3 Dermatan Sulfate (DS) . . . . . . . . . . . . . . . . . . . . . . . 81
3.8.4 Keratan Sulfate (KS) . . . . . . . . . . . . . . . . . . . . . . . . 84
3.8.5 Heparin and Heparan Sulfate . . . . . . . . . . . . . . . . . . 85
3.8.6 Hyaluronic Acid (HA) or Hyaluronan . . . . . . . . . . . . 86
3.8.7 Proteoglycans (PGs) . . . . . . . . . . . . . . . . . . . . . . . . 86
3.8.8 Extracellular PGs . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
3.9 Glycosylphosphatidylinositols (GPIs) Anchor Glycosylation . . . 89
3.9.1 General Structure of GPI Anchors . . . . . . . . . . . . . . . 89
3.9.2 Function of GPI-Anchored Protein . . . . . . . . . . . . . . 91
3.9.3 Biosynthesis, Structural Assembly,
and Transportation of GPI-Anchored Protein . . . . . . . 95
3.9.4 GPIs in Parasites . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
3.9.5 GPI Interaction with TLRs in Malaria
P. falciparum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
3.9.6 GPI-Defected Disorders of Paroxysmal Nocturnal
Hemoglobinuria (PNH) and Prion Disease . . . . . . . . . 103
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Contents vii
4 Glycans in Glycoimmunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

4.1 Glycans in Cell Recognition and Evolutionary Adaptation
in Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
4.2 Changes in Glycan Structure Involved in Coregulated
Expression of Glycan-Binding Lectin Counterparts . . . . . . . . . . 116
4.3 Evolution of Lectin: Alternative Splicing Contributes
to Variation for Glycan-Binding Receptors . . . . . . . . . . . . . . . 117
4.4 E-Selectin-Binding Ligand sLex (CD15s) on Neutrophil
CD44 N-glycan and Alternatively Spliced Exon 6 Contains
Core 2 O-Glycan sLea (CD44v6) Epitope . . . . . . . . . . . . . . . . 118
4.5 Glycans Regulate T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
4.5.1 Glycans Regulate Development and Differentiation
in T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
4.5.2 Glycosylation of Notch Receptor Signaling
for Thymocyte β Selection and T Cell Function
Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
4.5.3 Alternatively Spliced Variants Produce Different
Glycan Structures of CD43 and CD45 Isoforms
in T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
4.5.4 T Cells CD43 and CD45 Interaction with Their
Counter-Receptor or Lectins to Determine
T Cell Fates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
4.5.5 TCR Glycosylation Governs Hyper-response
and Autoimmune Responses in T Cells and Tregs . . . 130
4.5.6 SAMP and N-Glycan-Dependent Modulation
of Inhibitory T Cell Receptors to Suppress T Cell
Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
4.5.7 Galectins in Suppression of T Cell Functions . . . . . . . 134
4.5.8 Glycans Regulate T Cell-Mediated Immune
Suppression and Tolerance in Tumor Progression . . . 135
4.6 Abnormal N-Glycosylation in Autoimmunity . . . . . . . . . . . . . . 136
4.7 Glycan Regulation of NK Cell Receptors . . . . . . . . . . . . . . . . . 137
4.7.1 NCRs on NK Cells . . . . . . . . . . . . . . . . . . . . . . . . . 138
4.7.2 NCR Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
4.7.3 Interaction of NCRs Ligands with Pathogens . . . . . . . 141
4.7.4 Interaction of NCRs Ligands with Self-Ligands . . . . . 142
4.7.5 NK Cells MHC-I-Independent Inhibitory Receptors
Siglec-7 and Siglec-9 . . . . . . . . . . . . . . . . . . . . . . . . 143
4.8 Carbohydrate Recognition of Target Antigens by DCs During
Infection and Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
4.8.1 Lewis Ligand Recognition by DCs . . . . . . . . . . . . . . 146
4.8.2 VIM Ceramide Dodecasaccharide . . . . . . . . . . . . . . . 149
4.9 Glycan-Specific Trafficking Receptors in DC Maturation . . . . . 152
4.10 Glycan Ligands in Trafficking of DC Migration . . . . . . . . . . . . 154
viii Contents
4.10.1 sLex-PSGL-1 Glycans in DC Trafficking . . . . . . . . . . 154

4.10.2 Ganglioside Recognition by DC Receptors
in Trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
4.11 Chemokine Receptors in DC Trafficking . . . . . . . . . . . . . . . . . 158
4.11.1 Chemokine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
4.11.2 Chemokine Receptor . . . . . . . . . . . . . . . . . . . . . . . . 159
4.11.3 Chemokine-GAG Interaction as a Type
of Protein-Glycan Interactions . . . . . . . . . . . . . . . . . 159
4.11.4 Molecular Motifs in Chemokine for GAG
Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
4.11.5 C-C Type Chemokine Receptor 4 (CCR4)
and Specific Ligand 17 (CCL17) and Specific Ligand
22 (CCL22) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
4.12 Glycan Structure-Recognizing Selectins in DC-Endothelium
Interaction During Infection and Inflammation . . . . . . . . . . . . . 164
4.12.1 3 Species of Selectins: E-, L-, and P-selectins . . . . . . 165
4.12.2 Representative Selectin Ligand PSGL-1 and Role
of PSGL-1 O-Glycan . . . . . . . . . . . . . . . . . . . . . . . . 168
4.12.3 Glycosyltransferases for Biosynthesis of PSGL-1
O-Glycan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
4.12.4 Designation of Carbohydrate Glycomimetic Drugs
and Natural Inhibitors of Selectins . . . . . . . . . . . . . . 171
4.12.5 Glycomimetic Drug Candidates . . . . . . . . . . . . . . . . 172
4.12.6 GAG-Glycomimetic Drugs . . . . . . . . . . . . . . . . . . . . 174
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
5 Pathogen-Host Infection Via Glycan Recognition
and Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
5.1 Lectin Recognition of Glycans on Cell Surface and Soluble
Glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
5.2 Innate Immune-Specific and Host Defensing Lectins of Fungal,
Protozoa, Invertebrate, and Lower Vertebrates . . . . . . . . . . . . . 204
5.3 How Do Hosts Interact with Pathogens? . . . . . . . . . . . . . . . . . 206
5.3.1 Lectin-Carbohydrate Interaction . . . . . . . . . . . . . . . . 206
5.3.2 Bacterial Glycoconjugates Interact with Host
Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
5.4 Pathogen-Producing Lectins as Receptors to Bind to the Host
Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
5.4.1 Uropathogenic E. coli (UPEC), Enterohemorrhagic
E. coli (EHEC), and Enterotoxigenic
E. coli (ETEC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
5.4.2 Lectins and Glycans of Other Pathogenic Bacteria . . . 228
5.4.3 Viral Lectins or Host Lectin-Binding Glycans . . . . . . 233
5.5 Host Lectin Defense Mechanisms in Lectin-Carbohydrate
Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
Contents ix
5.6 Pathogenic Glycans to Trigger Innate Immune Enhancement . . . 238

5.6.1 Example 1: Polysaccharides with Immune
Enhancement of Cyrtomium macrophyllum . . . . . . . . 240
5.6.2 Example 2: Activation of Macrophage by
Polysaccharide from Paecilomyces cicadae . . . . . . . . 240
5.6.3 Example 3: NK Cell-Mediated Cytotoxicity
Increased by Arabinogalactan from Anoectochilus
formosanus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
5.6.4 Example 4: Streptococcus pneumonia
Polysaccharides Activate NK Cells, NK-Like T Cells,
and Monocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
5.6.5 Example 5: C. macrophyllum Polysaccharides
(CMP) Enhance Lymphocyte Proliferation
and Macrophage Function . . . . . . . . . . . . . . . . . . . . 242
5.7 TLR4 Receptor-Activating Glycans Activate NO Production
in Macrophage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
5.8 CBPs or GBPs in Antigen Recognition . . . . . . . . . . . . . . . . . . 244
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
6 Innate Immunity Via Glycan-Binding Lectin Receptors . . . . . . . . . 261
6.1 Glycosylation Effect on Autoimmunity and Inflammation . . . . . 262
6.1.1 Glycosylation in Immunological Recognition and
Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
6.1.2 Glycosylation Effect on Autoimmunity . . . . . . . . . . . 263
6.2 Glycosylation Effect on Tumor Immunity of Immune Cells . . . . 266
6.3 Immune Tolerance and Defense Mechanisms of Innate
Immune DCs During Infection . . . . . . . . . . . . . . . . . . . . . . . . 268
6.4 How Are Pathogenic Bacteria Recognized by Receptors
of DCs of the Host Immune System? . . . . . . . . . . . . . . . . . . . . 270
6.4.1 DC Lectins for Glycan Recognition of Invasive
Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
6.4.2 Toll-Like Receptors . . . . . . . . . . . . . . . . . . . . . . . . . 273
6.4.3 Innate Immune Receptors in Malaria Infection . . . . . . 278
6.4.4 Innate Immunity Receptors in Protozoan Parasite
Toxoplasma gondii . . . . . . . . . . . . . . . . . . . . . . . . . 287
6.5 Pathogen Recognition and Adaptive Immune Responses
in Acquired Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
6.6 Galactose-Specific C-Type Lectin: Two Major ASGPR
and Macrophage Galactose Lectin (MGL) in the Human . . . . . . 295
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
7 Sialic Acid-Binding Ig-Like Lectins (Siglecs) . . . . . . . . . . . . . . . . . 311
7.1 PolySia and Host Sialic Acids Modulate Host Immune
Responses as Pathogenic Decoys . . . . . . . . . . . . . . . . . . . . . . . 313
x Contents
7.2 Sialic Acid Recognition by Siglecs for Self-

or Nonself-Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
7.3 Classification of Siglecs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
7.4 Evolution of Siglecs, Sialic Acids, and Sialic Acid
O-Acetylation as Host Ligands (Receptors) for Microbes
and Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
7.5 Microbial Sialic Acid-like Molecules Synthesis
and Recognition of Microbial Sialic Acids by DCs
and Bacteriophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
7.6 Hematopoietic System in Siglecs . . . . . . . . . . . . . . . . . . . . . . . 325
7.7 Structure of Siglecs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
7.7.1 Cytoplasmic ITIM and ITAM Domains of Siglecs . . . 327
7.7.2 Adaptor Proteins Associated with Siglecs . . . . . . . . . 329
7.7.3 SA-Recognition Tropism of Siglecs . . . . . . . . . . . . . 330
7.8 Inhibitory Signaling of DCs . . . . . . . . . . . . . . . . . . . . . . . . . . 334
7.9 Siglec-1 (CD169, Sialoadhesin/Sn) . . . . . . . . . . . . . . . . . . . . . 338
7.9.1 General SAbinding Specificity of Siglec-1 . . . . . . . . . 338
7.9.2 Siglec-1 Is a Pathogen-Binding Receptor . . . . . . . . . . 341
7.9.3 Siglec-1 Recognizes HIV and Is a Transinfection
Receptor Expressed on mDCs . . . . . . . . . . . . . . . . . 343
7.10 CD22/Siglec-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
7.10.1 General and Structural Aspects of CD22/Siglec-2 . . . 346
7.10.2 CD22 I Associated with Development of
Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . . 349
7.10.3 CD22 Function in Immune Tolerance Events . . . . . . . 351
7.10.4 Role of CD22 (Siglec-2, Mice Siglec-G) in Immune
Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
7.10.5 Model Ligands for Recognition of CD22
on B Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
7.10.6 B Cell-Targeted Immunotherapy Through
CD22-Positive Targeting of B-Cell Lymphomas . . . . 357
7.10.7 Immune Tolerance Capacity of Neu5Ac-α2,6-Gal
Ligands in DCs by ST6Gal-1 of Tumor Cells for
Immunesurveillance . . . . . . . . . . . . . . . . . . . . . . . . . 359
7.10.8 CD22 Vs. Pathogens . . . . . . . . . . . . . . . . . . . . . . . . 360
7.10.9 CD22 Application with CAR-T on Acute
Lymphoblastic Leukemia (ALL) . . . . . . . . . . . . . . . . 361
7.10.10 CD22/Siglec-2 Coreceptor, CD45 on T Cells . . . . . . . 362
7.11 Siglec-4/Myelin-Associated Glycoprotein (MAG) . . . . . . . . . . 366
7.11.1 General Aspects of MAG/Siglec-4 . . . . . . . . . . . . . . 366
7.11.2 Siglec-4/MAG in the CNS and Brain
Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
7.11.3 Siglec-4/MAG in Hippocampal Long-Term
Potentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
Contents xi
7.12 Siglec-15, Non-CD33-Related Siglecs in Humans . . . . . . . . . . 370

7.12.1 The Structure and Expression of Siglec-15, Called
Misnomer “CD33L3” in Humans . . . . . . . . . . . . . . . 370
7.12.2 DAP12-Syk Pathway in Siglec-15-Mediated
Remodeling of the Tumor Microenvironment . . . . . . 372
7.12.3 Siglec-15 Functions in Osteoclastogenesis . . . . . . . . . 372
7.13 Siglec-3 (CD33)-Related Siglecs on DCs . . . . . . . . . . . . . . . . . 373
7.13.1 Siglec-3 (CD33) . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
7.13.2 Structure, Natural Ligand, and Cellular Signaling
with SHP-1/-2 of Siglec-3/CD33 . . . . . . . . . . . . . . . 375
7.13.3 Pathogen Ligand for CD33 . . . . . . . . . . . . . . . . . . . . 377
7.13.4 Siglec-3/CD33 Is Related to SOCS3
and Internalization of CD33 . . . . . . . . . . . . . . . . . . . 378
7.13.5 Putative Functions of Siglec-3/CD33 in Alzheimer’s
Disease (AD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
7.13.6 Siglec-3-/CD33-Based Immunotherapy for AML . . . . 381
7.13.7 Siglec-5/CD170 as a CD33-Related Siglec . . . . . . . . 382
7.13.8 Siglec-6 as a CD33-Related Siglec . . . . . . . . . . . . . . 388
7.13.9 Siglec-7 (CD328) as a CD33-Related Siglec . . . . . . . 390
7.13.10 Siglec-8 as a CD33-Related Siglec and Siglec-F
as a Mouse Paralog . . . . . . . . . . . . . . . . . . . . . . . . . 399
7.13.11 Siglec-9 as a CD33-Related Siglec and Murine
Functional Counterpart, Siglec-E . . . . . . . . . . . . . . . 403
7.13.12 Siglec-10 (Mouse Ortholog Siglec-G) in Humans
as a CD33-Related Siglec . . . . . . . . . . . . . . . . . . . . . 424
7.13.13 Human Siglec-11 as a CD33-Related Siglec . . . . . . . 433
7.13.14 Siglec-14 in Humans as a CD33-Related Siglec . . . . . 438
7.13.15 Siglec-16 as a CD33-Related Siglec Is a Paired
Receptor with Siglec-11 . . . . . . . . . . . . . . . . . . . . . . 445
7.14 Mouse CD33-Related Siglecs with ITIM-Like Domains . . . . . . 447
7.14.1 mSiglec-E that Belongs to CD33-Related Siglecs . . . . 448
7.14.2 Siglec-F (Human Paralog Siglec-8) as
a CD33-Related Siglec . . . . . . . . . . . . . . . . . . . . . . . 451
7.14.3 Human Siglec-10 and Mouse Ortholog Siglec-G
as CD33-Related Siglecs . . . . . . . . . . . . . . . . . . . . . 454
7.14.4 Siglec-H as a CD33-Related Siglec . . . . . . . . . . . . . . 457
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
8 C-Type Lectin (C-Type Lectin Receptor) . . . . . . . . . . . . . . . . . . .. 497
8.1 Evolutionary Diversity of C-Type Lectins . . . . . . . . . . . . . . .. 497
8.2 Ca2+-Dependent Glycan-Binding CTLs . . . . . . . . . . . . . . . . .. 499
8.3 Myeloid CTL-Like Receptor or Myeloid-Suppressive
or Inhibitory CLR (MICL), CLEC 12A . . . . . . . . . . . . . . . . .. 502
8.4 Macrophage Inducible CTLR (Mincle, Clec4e, ClecSf9)/
Macrophage CTL (MCL, CLEC4d, ClecSf8) . . . . . . . . . . . . .. 504
xii Contents
8.4.1 Expression and Ligand-Binding Specificity

of Mincle, Clec4e, ClecSf9, and MCL . . . . . . . . . . . 504
8.4.2 Pathogenic PAMPs-Recognition of Mincle
and MCL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
8.4.3 Th1/Th17 Activation and T Cell Development
in Mincle or MCL Interaction with Host . . . . . . . . . . 507
8.5 Mannose Receptor (MR) as CLR and Macrophage Mannose
Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
8.5.1 Structural Basis and Functions of MR . . . . . . . . . . . . 510
8.5.2 MR Expression in Immune Systems and Interaction
with Helminth Flatworm Trematodes . . . . . . . . . . . . 512
8.5.3 Recognition of Pathogenic Microbes by MR . . . . . . . 514
8.6 Mannose (or Mannan)-Binding Protein (MBP) and Mannose-
Binding Lectin (MBL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515
8.6.1 Structural Basis and Glycan Ligand Binding
Specificity of MBL . . . . . . . . . . . . . . . . . . . . . . . . . 516
8.6.2 Immunoprotective Activity of MBL . . . . . . . . . . . . . 517
8.6.3 MBL Function in Diseases . . . . . . . . . . . . . . . . . . . . 519
8.7 Fucose-Binding Lectin (FBL) and Ficolin . . . . . . . . . . . . . . . . 520
8.7.1 Fucose-Binding Lectin (FBL) Diversity of F-Lectin
Repertoires . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 520
8.7.2 Specificity of Ficolins or FBL . . . . . . . . . . . . . . . . . 521
8.7.3 Ficolin Functions in the Immune Response . . . . . . . . 523
8.7.4 Ficolin Interaction with Microorganisms . . . . . . . . . . 525
8.8 Dectin 1 (CLEC-7A in Human) . . . . . . . . . . . . . . . . . . . . . . . . 526
8.8.1 Basic Function and Structure of Dectin 1 . . . . . . . . . 526
8.8.2 Dectin-1 Recognizes β1,3/β1,6-glycans in Fungi,
Plants, Bacteria, and House Dust Mite . . . . . . . . . . . 527
8.8.3 Dectin-1 Cluster Includes CTL-Like Receptor 2
(CLEC-2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 530
8.8.4 CLEC Structures and Ligand Recognition . . . . . . . . . 531
8.9 DC-Associated CTL-2 (Dectin-2) Family or CLEC4n . . . . . . . . 533
8.9.1 Structural Basis and Function of Dectin-2 . . . . . . . . . 533
8.9.2 Langerhans Cell-Specific Expression of Dectin-2
and Interaction with Fungal High-Man Glycans . . . . . 535
8.10 Dectin-3 (Clec4D, Clecsf8, MCL, Macrophage CTL) . . . . . . . . 537
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 540
9 Galectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557
9.1 General and Structural Aspects of Galectins . . . . . . . . . . . . . . . 557
9.1.1 Biological Roles of Galectins . . . . . . . . . . . . . . . . . . 558
9.1.2 Immunological Roles of Galectins . . . . . . . . . . . . . . 559
9.1.3 Classification of Galectins . . . . . . . . . . . . . . . . . . . . 561
9.1.4 Galectin Ligands in Proteins and Gangliosides . . . . . . 564
Contents xiii
9.1.5 Galectins in Lower Organisms such as Zebrafish

or Marine Oyster . . . . . . . . . . . . . . . . . . . . . . . . . . . 565
9.2 Galectin-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566
9.3 Galectin-3 and -8 Recognize GM3, But Not Galectin-4 . . . . . . . 567
9.3.1 Galectin-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567
9.3.2 Galectin-8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570
9.4 Galectin-1 and -4 Bind to GM1, But Not GM3 . . . . . . . . . . . . 570
9.4.1 Galectin-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570
9.4.2 Galectin-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
9.5 Galactine-9 and Galelctin-10 . . . . . . . . . . . . . . . . . . . . . . . . . . 576
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
10 DC-SIGNs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
10.1 DC-Specific ICAM-3-Grabbing Non-integrin, DC-SIGNB
(CD209) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
10.1.1 Molecular Characteristics of DC-SIGN . . . . . . . . . . . 585
10.1.2 General Signaling of DC-SIGN . . . . . . . . . . . . . . . . 586
10.1.3 α2,6 Sialyl IgG Fc Function by DC-SIGN
Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
10.1.4 DC-SIGN Binds to Pathogens, Antigen,
and Glycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590
10.1.5 DC-SIGN Role in DC-Mediated Viral Transmission
by HIV-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
10.1.6 DC-Mediated Immunosuppression
by Mycobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
10.1.7 DC-SIGN Recognizes Lewis Antigens Expressed
in PMN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
10.2 Other DCs-Derived Receptors . . . . . . . . . . . . . . . . . . . . . . . . . 593
10.2.1 Dendritic Cell NK Lectin Group Receptor
(DNGR-1; CLEC9A) . . . . . . . . . . . . . . . . . . . . . . . . 593
10.2.2 CTL-Like Receptor-1 (CLEC-1) . . . . . . . . . . . . . . . . 595
10.2.3 CTL-Like Receptor, CLEC12A, Known as Myeloid
Inhibitory CTL-Like Receptor (MICL), CTL-Like
Molecule-1 (CLL-1), DC-Associated CTL 2
(DCAL-2), and CD371 . . . . . . . . . . . . . . . . . . . . . . 597
10.2.4 CD161 (NKR-P1A) . . . . . . . . . . . . . . . . . . . . . . . . . 600
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
11 Toll-Like Receptors (TLRs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
11.1 TLR Molecular Structure, Subtypes, and Recognition
Ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609
11.2 Signal Initiation and Transduction of TLRs . . . . . . . . . . . . . . . 612
11.3 Glycosylation of TLRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
11.4 General TLR Functions as Pathogen and Antigen Receptors
on DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
xiv Contents
11.5 TLR-9 as a CpG DNA Receptor . . . . . . . . . . . . . . . . . . . . . . . 616

11.6 TLR-3 as a dsRNA Receptor . . . . . . . . . . . . . . . . . . . . . . . . . 617
11.7 TLR-4 as the LPS Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . 618
11.7.1 Ligands of TRL4 Recognition . . . . . . . . . . . . . . . . . 619
11.7.2 MyD88-Dependent Pathway of TLR4 . . . . . . . . . . . . 621
11.7.3 MyD88-Independent Pathway of TLR4 . . . . . . . . . . . 621
11.8 TLR11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622
11.8.1 Three Major Domains and Binding Ligand
of TLR11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623
11.8.2 TLR11 Intracellular Signal Transduction . . . . . . . . . . 624
11.9 Inhibition of TLRs by Gangliosides . . . . . . . . . . . . . . . . . . . . . 625
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626
12 CD33 and CD33-Related Siglecs in Pathogen Recognition
and Endocytosis of DC in the Innate Immune System . . . . . . . . . . 631
12.1 CD33 (Siglec-3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631
12.1.1 General Biology of CD33 . . . . . . . . . . . . . . . . . . . . 631
12.1.2 CD33 (Siglec-3)-Targeting of Acute Myeloid
Leukemia (AML) . . . . . . . . . . . . . . . . . . . . . . . . . . 635
12.1.3 CD33 (Siglec-3)-Targeting Treatment
of Alzheimer’s Disease (AD) . . . . . . . . . . . . . . . . . . 636
12.2 CD33-Related Siglecs (CD33rSiglecs) . . . . . . . . . . . . . . . . . . . 636
12.2.1 Inhibitory CD33rSiglecs in Escape from Tumor
and Bacterial Immunosurveillance . . . . . . . . . . . . . . 637
12.2.2 Activating CD33rSiglecs . . . . . . . . . . . . . . . . . . . . . 638
12.3 Pathogenic Suppression of the Pathogen-Specific Host
Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639
12.3.1 Inhibitory Receptor CD200R and CD200:CD200R1
Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640
12.3.2 Pathogenic Decoy Ligands Neutralize Host
Immunity Through Eliciting Host CD200-CD200R1
Inhibitory Signaling . . . . . . . . . . . . . . . . . . . . . . . . . 642
12.4 DCs Tumor Immunotherapy Through Sialyl Binding of DCs
to T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 648
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 650
Abbreviations
AhR Aryl hydrocarbon receptor

AICD Activation-induced cell death
ALIS Aggresome-like structure
APC Antigen-presenting cell
AR Autosomal recessive
ASGPR Asialoglycoprotein receptor (ASGPR)
ATCS Adducted thumb-clubfoot syndrome
BAG6 Bcl2-associated anthogene 6
BAT3 HLA-B-associated transcript 3
Batf3 Basic leucine zipper transcription factor ATF-like3
BGN Biglycan
C2GnT Core 2 β1,6N-GlcNAc-transferase
C4ST Chondroitin 4-O-sulfo-transferase
C6ST Chondroitin 6-O-sulfo-transferase
CAM Cell adhesion molecule
CD Clustered differentiation
CEA Carcinoembryonic antigen
CFG Consortium for functional glycomics
ChABC Chondroitinase-A, -B, and -C
ChGn Chondroitin GalNAcT
ChPF Chondroitin polymerizing factor
ChSy-1 Chondroitin synthase
CIA Collagen type II-induced arthritic disease
CLD Collagen-like domain
CLR C-type lectin receptor
CNS Central nervous system
CNTN-1 Contactin-1
CNX Calnexin
Core 1 β3Gal-T Core 1 β3-Gal-Transferase
COSMC Core 1 β3GalT-specific molecular chaperone
CR Complement receptor
xv
xvi Abbreviations
CRD Carbohydrate recognition domain

CRP Complement regulatory protein
CRP C-type receptor protein
CRT Calreticulin
CS Chondroitin sulfate
CSPG CS attached PG
CTL C-type lectin
CTL-4 Human CTL antigen-4
CTLs Cytotoxic T lymphocytes
D4ST Dermatan 4-O-sulfo-transferase
DALIS DC Aggresome-like induced structures
DAMP Damage-associated molecular pattern
DBL Duffy binding-like
DCN Decorin
DC-SIGN DC-specific intracellular adhesion molecule-3 grabbing
non-integrin
DLL Delta-like ligands
Dol-P Dolichol phosphate
DS Dermatan sulfate
DSE DS epimerase
DSE-L DSE-like gene
DTH Delay-type hypersensitivity
EDS Ehlers-Danlos syndrome
EDSKT EDS Kosho type
eIF2 Eukaryotic initiation factor 2
EMT Epithelial-mesenchymal transition
ERAD ER-associated degradation
ERGIC ER-Golgi intermediate compartment
ESAM Endothelial cell-specific adhesion molecule
EtN-P Phosphoethanolamine
Flt3L FMS-like Tyr kinase 3 ligand
FUT3 α1,3/4-Fuc-Transferase
GalNAc-4S-6ST GalNAc-4-sulfate 6-O-sulfo-transferase
GalNAcT-I N-acetylglucosaminyltransferase I
GalNAcT-II N-acetylgalactosaminyltransferase
Gal-T1 β1,4-galactosyltransferase
GBP Glycan-binding protein
GBS Group B streptococcus
GCN2 General control non-derepressible 2
GIT Gastrointestinal track
GITR Glucocorticoid-induced TNFR-related protein
GlcA Glucuronic acid
GlcAT-II Glucuronosyltransferase II
GlcNAcT-II N-acetylgalactosaminyltransferase I
Abbreviations xvii
GNE UDP-GlcNAc-2-epimerase
GPCR G-protein coupled receptor
GSK3β Glycogen synthase kinase 3β
GSL Glycophospholipid
GT Glycosyltransferase
GvHd Graft-versus-host disease
HA Hemagglutinin
HBP Hexosamine biosynthetic pathway
hIBM ITM-like hereditary inclusion-body myositis
HIV Human immunodeficiency virus
hLys Hydroxyl Lys
HNK Human natural killer
HNK-1ST HNK-1 sulfo-transferase
Hp Heparan phosphate
HPMR Hyperphosphatasia mental retardation
hPro Hydroxy-proline
HS Heparan sulfate
IBD Inflammatory bowel disease
ICOS Inducible co-stimulator
ICOS-L ICOS-ligand
ID2 DNA-binding protein 2
IDDM Insulin-dependent diabetes mellitus
IDO Indoleamine-2,3-dioxygenase
IdoA2S IdoA 2-sulfated residue
IdoUA Iduronic acid
IFN Interferon
IIM Idiopathic inflammatory myopathies
IRF8 IFN-regulatory factor 8
ITIM Immune receptor tyrosine-based inhibitory motif
KS Keratan sulfate
LacNAc Lactosamine
Lag-3 Lymphocytic activation gene-3
LAP Latency-associated protein
LLC Lewis lung carcinoma
LPS Lipopolysaccharide
MAL MYD88 adaptor-like protein
MAP MBL-associated protein
MASP MBL-associated serine protease
MBL Mannose-binding lectin
MBP Mannan-binding protein
MCEDS Musculocontractural EDS
MHC Histocompatibility complex
MK Midkine
MLL-5 Mixed-lineage leukemia-5
xviii Abbreviations
MMP Matrix metalloproteinase

MNK ManNAc kinase
moDC Monocyte-derived DC
MR Mannose receptor
MS Multiple sclerosis
NA Neuraminidase
NGS Next-generation genome sequencing
NLR NOD-like receptor
NOD Nucleotide oligomerization domain
NST Nucleotide-sugar transporter
OST Oligosaccharyltransferase
PAMP Pathogen-associated molecular pattern
PAPS 30 -Phospho-adenosine 50 -phospho-sulfate
PAPST PAPS transporter
PARP Pathogen-associated recognition
PC Phosphatidylcholine
PCNA Proliferating cell nuclear antigen
pDC Plasmacytoid DC
PD-L1 PD ligand-1
PE Phosphatidylethanolamine
PG Proteoglycan
PGAP Post-GPI-attachment to protein
PGI Protein–glycan interaction
PGRP Peptidoglycan recognition protein
PMN Polymorphonuclear neutrophils
PNH Paroxysmal nocturnal hemoglobinuria
POM-T O-mannosyl-Transferase
PrPc Proteinase-sensitive cellular prion protein
PrPres Proteinase-resistant prion protein
PRR Pattern recognition receptor
PSGL-1 P-Selectin glycoprotein ligand-1
PTN Pleiotrophin
QC Quality control
RA Retinoic acid
RAG Recombination-activating gene
RAGE Advanced glycation end products
rER Rough ER
RLR RA-inducible gene I-like receptor
SED Spondyloepiphyseal dysplasia
SIGNR1 SIGN receptor-1
SLC35D1 Solute carrier 35D1
SLE Systemic lupus erythematosus
SLRP Small leucine-rich PG
SP Surfactant protein
Abbreviations xix
ST3Gal-1 β-Gal α2,3-SA-transferase 1

T helper-3 TGF-β-expressing Th3
Teffs Effector T cells
TEP Thioester-containing protein
TF Thomsen-Friedenreich antigen
TGN Trans-Golgi network
TIGIT T cell immunoreceptor with Ig and ITIM domains (TIGIT)
Tim-3 mucin-domain-containing molecule-3
TIR Toll/IL-1R-homology domain
TLR Toll-like receptor
TM Transmembrane
TOR Target of rapamycin
TPA Tissue plasminogen activator
Tr1 T regulatory-1
TRAM TRIF-related adaptor molecule
Treg Regulatory T cell
TRIF TIR-bearing adaptor-inducing IFN-β
TRIF TIR-domain-containing adaptor protein inducing IFN
TS Thrombospondin
TSR Type 1 repeat
UA Uronic acid
UST Uronyl 2-O-sulfo-transferase
uTPA Urinary type plasminogen activator
VNTR Variable number tandem repeats
VSP Variant surface glycoprotein
Xyl-T β-xylosyltransferase
β3GnT β1,3-N-GlcNAc-transferase
Chapter 1
Repertoire in Innate Immunity
1.1 Historical Expansion of Defense System
The first historical depiction of infectious diseases was from the Epic of Gilgamesh,
an Akkadian poem, which is the oldest literature that belongs to early Sumerian
poems. The Epic of Gilgamesh is a Babylonian poem written around 2000 B.C. that
depicts the adventures of Gilgamesh, the king of Uruk. Gilgamesh is speculated to
reign sometimes between 2800 and 2500 B.C. and was deified posthumously
[1]. There are a few versions of the poem, and the oldest version of it dates back
to the seventh century B.C. In this poem, the presence of pestilences and diseases is
well recorded. In addition, the Greek mythology also illustrates infectious diseases in
the story of the Apollo and Artemis’s murder of Niobe’s sons. In the story, Apollo
and Artemis kill Niobe’s sons with the arrows of plague. Apollo, once again, sends
plague to the Greek camps during the Trojan War (from 1194 to 1184 B.C.), thereby
causing the death of many lives.
Infectious diseases have always been with human and, thus naturally, recorded
since the very beginning of the written history. Infectious diseases at the beginning
of written history were considered as curses of God or somethings mysterious.
However, it was Hippocrates, the Greek physician, who has changed the course of
medical practices entirely. Hippocrates, famed to be known as the father of modern
medicine, ingenuously observed the relationship between hygiene, immunity, and
infections. His clever observations made him possible to forecast infectious diseases,
and he even mentioned tuberculous spondylitis, malaria, and tetanus [2]. His insights
still have bearing on the understanding of innate immunity. Hippocrates’s brilliant
clinical observations are well documented in the Corpus Hippocraticum (Hippo-
cratic Collection), and the contents in it are still recognized as useful “lessons” for
current and future medical philosophy since they provide archeological portrayals of
infectious diseases. The knowledge of infectious diseases was accumulated through
the course of history and had influenced the relevant consciousness and awareness of
the modern medical practices and education. For instance, the fact that the symptoms
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 1
C.-H. Kim, Glycobiology of Innate Immunology,
https://doi.org/10.1007/978-981-16-9081-5_1
2 1 Repertoire in Innate Immunity
of diseases are associated with the disequilibrium of bodily fluids has been widely
accepted from the ancient to the modern eras. Hippocrates was the first to recognize
magical miasma was not the cause of diseases and, rather precisely, recorded
influenza pandemic [2].
Let’s take a look at how the knowledge of fever was produced to understand how
the knowledge of infectious diseases has been developed. During the time of
Hippocrates, the tertian paroxysmal and quartan fevers were recognized to be caused
by malaria [3]. The tenth-century C.E. Persian physician Akhawayni then created
fever curves for tertian and quartan fevers and recorded them in Hidayat
al-Mutaʽallemin fi al-Tibb [3]. Likewise, many ancient Greek, Roman, and medieval
savants and physicians increasingly gained knowledges about fevers and body
temperature contributing to the basic understanding of the role of fever and ther-
mometry. Even fevers as a symptom and as a disease have been distinguished during
this time. In the same ear, scholars were able to define the cause of fever and discover
methods for measuring fevers. This progression of the understanding of fever
enabled the sixteenth-century scientist Galileo to produce thermometric instruments.
For understanding fever, clinical thermometry was continuously being useful even
until the end of the eighteenth century providing useful tools to measure fever and
interpret the role of fever in human health [4].
Many more scientists kept discovering more and more, thanks to pioneers in
history. Aulus Cornelius Celsus (ca 25 B.C.–50 A.D.) defined, for the first time,
inflammation as calor (warmth), dolor (pain), tumor (swelling), and rubor (redness)
[5–8]. In 1443, Thomas of Sarzana, later known as Pope Nicholas V, found the copy
of De Medicina which describes the Greco-Roman medicine by Celsus at the library
in Milan. This book was published in 1478 and quickly gained its reputation as a
standard text of medicine. The descriptions of diseases and rational therapeutic
approaches recorded in the book had influenced practices of physicians for many
following years. For instance, in the sixteenth century, Ambroise Paré cited Celsus
for his use of vessel ligation in order to stop hemorrhage in wounds [9]. Two
centuries later, Morgagni utilized De Medicina as a standard reference to understand
diseases and develop therapeutic and surgical methods. He also attempted to corre-
late case studies done in the past with relative pathologies with the help of De
Medicina [10].
Muhammad ibn Zakariya al-Razi, who was called Rhazes (ca 850–930), was a
Persian physician who first described measles authentically in literature, and he
distinguished smallpox from measles for the first time in medical history [11–
13]. Razi wrote medical books such as Kitab al-Mansur’t (Book for al-Mansur)
and Kitab al-Hawi (Comprehensive Book on Medicine) while working at hospitals in
his hometown of Rayy, near Tehran and Baghdad. These books were known in Latin
tradition as Liber Almansoris and Continens and had influenced many European
physicians and universities [11]. The concept of diseases founded by medieval
Islamic physicians was described in the Greek philosophy and Greco-Roman med-
icine as “Arabized Galenism.” Many brilliant descriptions of diagnoses are included
in Razi’s case studies like the terms “headache caused by a yellow bile vapor” or
“illness.” Besides Razi, many other Arabic physicians like Abū al-Qāsim al-Zahrāwi
1.2 Columbus Era to Modern Revolution in Immunological Defense System 3
(known as Albucasus among Europeans), Ibn Sina (Avicenna), and Ali ibn al-'Abbas
al-Majusi (Haly Abbas) all have greatly contributed to the knowledge of infectious
diseases and inflammation.
During 1104–1110 C.E., the plague killed more than 90% of the European
population, and it was when the term Black Death was first introduced. During
1346–1353, a new plague broke out over Western Asia, the Middle East, North
Africa, and Europe which killed an estimated 75 million people. Like the Latin pestis
(curse), plagues were dreaded by the humankind throughout history. Plagues were
considered as the Apocalypse, a divine curse, which possess “the power to kill over a
fourth of the earth.” It is one of the rare epidemics that have been recurred with
highly transmissible nature resulting in brutality, high pathogenicity, strong lethality,
and great swiftness, and, moreover, there were virtually no options for preventions
and treatments until the twentieth century. Particularly, in the Western world,
epidemics have influenced the evolution of societies at both biological and cultural
levels [14]. King Philip VI in 1348 supported the study group in the medical faculty
of the University of Paris to study the relationship between the constellation of
Saturn, Jupiter, and Mars on March 20, 1345, and the dissemination of pestilence in
the air [15]. The Swiss-German physician Philippus Aureolus Theophrastus
Bombastus von Hohenheim, known as Paracelsus, introduced mercury salt to treat
syphilis. The rationale behind mercury salt that Paracelsus thought arsenic was one
of the major components of the first effective treatment for syphilis [16]. Paracelsus,
the sixteenth-century alchemist and physician, had acquired his skill from barber
surgeons, alchemists, and gypsies, and, at the same time, he was also appointed as
the professor of medicine in Basel. He was famous for his use of mercury, arsenic,
antimony, and tin salts for syphilis, intestinal worms, and endemic diseases during
medieval Europe. In assumption, he killed more patients than he cured using such
toxic metals. For hundred years later, Paul Ehrlich discovered the arsenic-containing
drug “606” (later called Salvarsan) which was the first drug for syphilis and
considered to be the “magic bullet.” The drug was utilized until the introduction of
another powerful antibiotic in the twentieth century, penicillin.
1.2 Columbus Era to Modern Revolution in Immunological

Defense System
The Columbian Exchange includes the multiple influences through exchanges of

crops, tropical diseases, religion, ideas, philosophy, and populations between the
New World and the Old World. The exchange was initiated by the voyage of
Christopher Columbus to America in 1492. Infectious diseases, such as smallpox,
measles, chicken pox-varicella zoster, Bordetella pertussis whooping, plague,
typhus, and malaria, were entered into the new lands [17]. The New World (Mundus
Novus) had gained many things described above as a byproduct of the Columbian
Exchange or interchange, but the most important and best-known gains were the
supplies of new metals. The Old World had also gained new staple crops like
cassavas, maize, potatoes, and sweet potatoes. The exchange of many stuffs like
currency was drastically increased throughout many parts of the world. The
exchange had introduced many crops of the Old World including coffee and sugar
that were well available for the soils of the New World.
Hereafter, Martin Luther (1483–1546) initiated the religious reformation after the
church sold indulgence to citizens. The first microscope was designed by Antonie
van Leeuwenhoek (1632–1723), a Dutch tradesman and scientist from Delft. Using
the microscope, he discovered the world of previously invisible creatures which he
called animalcules (small animals). van Leeuwenhoek communicated his discoveries
in 1674 to the Royal Society of London with detailed drawings of his findings.
Elie Metchnikoff, a Russian biologist, zoologist, and protozoologist, was the first
to introduce the concept of phagocytosis into broader perspectives. He is the father
of natural immunity. Together with Paul Ehrlich, Elie Metchnikoff was a recipient of
the 1908 Nobel Prize in Physiology and Medicine from their discoveries and
achievements on the conceptional antibody and the important immune aspects.
Elie Metchnikoff and Paul Ehrlich shared Nobel Prize to their pioneer works in
humoral and adaptation immunology. Metchnikoff achieved the phenomena includ-
ing leukocyte recruitment and microbe phagocytosis in the defense system of hosts
during pathogenic infections, infectious agent-induced inflammation, and systemic
immune response of hosts. His work pioneered the contemporary research on innate
immunity. During his research era, he stayed in Odessa region of Russia and the
Pasteur Institute in Paris, France. At that time, he was enriched with his complex
personality, creative ingenuity, imagination, and insight which made him as “the
father of natural immunity” although his observation that a thorn of rose caused
phagocyte recruitment in a starfish might be a myth [18, 19].
Louis Pasteur (1822–1895), a French chemist, is one of the founders of microbi-
ology. In Louis Pasteur’s scientific career, he laid the foundation for the future study
on stereochemistry. He also pointed out the importance of epidemiology and public
health. He fought against the idea of spontaneous life generation. He established the
concept of immunity and generalized the principle of vaccination. He developed the
concept of microorganism-induced infectious diseases and the concept of
vaccination [20].
Robert Koch (1843–1910), a German physician, was the first researcher to isolate
bacterial pathogen of Bacillus anthracis in 1877. He formulated the Koch postula-
tions that the microorganism is present and discoverable in every case of the disease.
He also pointed out that microorganisms need to be cultivated in a pure culture.
Inoculations from cultures must reproduce the diseases in susceptible animals. Then,
microorganisms must be re-obtained from infected animals and grown again in a
pure culture.
1.3 Historical Profile of Defense Constituents and Progress in Innate Immune. . . 5
1.3 Historical Profile of Defense Constituents and Progress

in Innate Immune Repertoire
1.3.1 Phagocytosis
Johann August Ephraim Goeze (1731–1793) is recognized to be the first researcher

ever to study phagocytic phenomenon. He first described tardigrades in 1773 and, in
1784, observed pathological outcomes of neurocysticercosis during pathogenic
larval stages [21]. He did figure many aquatic invertebrates, particularly insects
and worms. Goeze perceived the similarities of the two different sources of tape-
worm heads present in the intestinal tract of humans and Cysticercus cellulosae-
invaginated heads in pigs [22] (Fig. 1.1).
1.3.2 Leukocytes
In 1843, Gabriel Andral (1797–1878) and William Addison (1802–1881) described

for the first time the leukocytes [23], as it was almost 200 hundred years after the first
identification of the red cells by Jan Swammerdam using his designed microscope in
1658. Thereafter, Max Schultze performed functional studies on finely and coarsely
granular cells. For the leukocytes, a German anatomist Max Johann Sigismund
Schultze (1825–1874) also described for the first time four different types of
leukocytes and the existence of intracellular granules. Max Johann Sigismund
Fig. 1.1 Johann August

Ephraim Goeze
(1731–1793), a German
zoologist’s portrait
estimated to be painted by
some painter in the
eighteenth century. The
current portrait image has
been copied from the public
domain from Wikipedia in
the web address of https://it.
wikipedia.org/wiki/Johann_
August_Ephraim_Goeze#/
media/File:Johann_August_
Ephraim_Goeze1.jpg
Schultze studied on coarse granular cells to observe by a warm stage technique using
his microscope at 38 C. The finely and coarsely granular cells of Max Schultze were
shown in his article reported in 1865 [24]. He observed the amoeboid behaviors
including interacting movement and phagocytosis phenomena. These cells were a
type of granular cells like eosinophils and other blood leukocytes. Schultze joined
the medical research groups of Greifswald and Berlin. In 1854, after his appointment
of associate professorship of anatomy at Halle, he was successfully obtained his full
professor in anatomy and histology. Later, he was the director of the Anatomical
Institute, the University of Bonn. His recognition was particularly marked for his
historical study on the cellular theory. His prominent achievement is attributed to the
uniting animal sarcode conception, which was raised by Felix Dujardin, with
vegetable protoplasma, which was raised by Hugo von Mohl. His suggestion was
the terminology of protoplasm ad its identity. His achievement is his first definition
of the cells to nucleated mass of protoplasms with presence or absence of cell walls
(this is written through the description of Das Protoplasma der Rhizopoden und der
Pflanzenzellen, ein Beitrag zur Theorie der Zelle, 1863).
Max Schultze also investigated medicine field with the naturalist Fritz Müller,
who was a German biologist and doctor, to follow the debate in Europe about
evolution logics of Darwin’s theory. Max exchanged research literatures related to
the Darwin’s monograph book of On the Origin of Species, and a simple microscope
manufactured with Friedrich Wilhelm Schiek in Berlin in 1857. Using the micro-
scope, Müller insisted on his hypothesis of “all higher Crustacea probably will be
traceable to a Zoea ancestor,” which is basically originated from his own investiga-
tions. In addition, Müller insisted on his criticism of Darwin’s theory through his
book of Für Darwin, which is basically written in defense of Darwin’s theories. He
corroborated the Darwin’s theory of natural selection [23]. He established in 1865
the book series of serial articles in the Archiv für mikroskopische Anatomie, which
contained several his articles. He highly investigated into depth on his study subject
via fine refining (Fig. 1.2).
Continuously, Florence Rena Sabin (1871–1953), an American medical scientist
and one of the first women as a full professorship at Johns Hopkins School of
Medicine, elucidated the differentiation events of white blood cells in the 1920s
(Fig. 1.3). In 1924, the origins of blood, blood vessels, and blood cells have been
reported by Florence R. Sabin. Thereafter, she further examined the brain histology,
tuberculosis pathology, and immunology [26]. In 1925, after movement to the
Rockefeller Institute for Medical Research in New York City, she concentrated
and focused on the lymphatic organ system and cells. From her achievement of
tuberculosis pathology for the functional discovery of monocytes to form tubercles
known for granuloma, in 1926, she was invited to the committee of the National
Tuberculosis Association.
Fig. 1.2 Max Schultze’s granular white cells described in his article reported in 1865. The figure
has been copied from the review article of Dr. Kay AB (2015) [25], as the captions are copied from
Douglas Brewster’s translation [24]. The photo image has been copied from the public domain from
Wikipedia in the web address of https://en.wikipedia.org/wiki/Max_Schultze
Fig. 1.3 Florence Rena

Sabin. The photo image has
the web address of https://
en.wikipedia.org/wiki/
Florence_R._Sabin
1.3.3 Neutrophils
In the concept of neutrophils, James Gerald Hirsch (1922–1987), an American

physician and scientist, for the first time, reported the degranulation and phagocy-
tosis of blood cells (Fig. 1.4) [27]. Using electron microscope, he observed and
elucidated degranulation of neutrophils and eosinophils, as such events are specific
for phagocytosis. Thus, phagocytic granules are observed because they are released
into the phagocytic vacuole. Apart from the works by James Hirsch, Seymour
Klebanoff independently discovered the similar granulating events with specific
enzymes in the late 1960s. For example, some white blood cells express a
myeloperoxidase enzyme that oxidizes the cell membrane components of targets,
most well-known for killing of infected microbial pathogens [28, 29]. Therefore,
Hirsch is discriminated for his achievement of phagocyte where the white blood cells
engulf harmful microbes [26].
1.3.4 Granulocytes
For granulocytes, Niels Borregaard, a Danish physician and scientist, found several
intracellular proteins in granulocytes with the synthetic origins, intracellular granule
stores, and extracellular release (Fig. 1.5) [31]. From the electron microscopic
observation, degranulation of neutrophils and eosinophils has been observed during
phagocytosis. Granulation is a crucial process to release components from the
Fig. 1.4 James Gerald

Hirsch. The photo image has
the web address of https://
en.wikipedia.org/wiki/
James_G._Hirsch
Fig. 1.5 Niels Borregaard. The photo image has been copied from the public in the web address of
https://jlb.onlinelibrary.wiley.com/doi/full/10.1189/jlb.4LT0217-049R, as shown in Fig. 1.4, read-
ing with grandchildren. He was one of the editors of Journal of Leukocyte Biology, Society for
Leukocyte Biology [30]
neutrophils in innate immune responses at the infection sites and initiate generation
of bactericidal oxygen species for degranulation of granule subsets [32]. The gran-
ules are released into the phagocytic vacuole [33–35]. Niels graduated in 1978 from
Aarhus University with his medical doctor degree in 1981. He was especially
interested in the neutrophils among human phagocytes, as pursued. Niels isolates
neutrophil subcellular organelles toward granulopoiesis. In the 1980s, in his work on
neutrophil granules and component proteins, he isolated several neutrophil granule
proteins and antimicrobial proteins such as α-defensin [30].
1.3.5 Monocytes and Macrophages
Macrophage was initially identified as immune cells to phagocytosize infectious

agents [36] in the late nineteenth century. Since its discovery, macrophage received
largely attracted attention in its defender roles against infectious pathogens. Later it
received a spotlight for its role in tissue homeostasis in organism. The tissue
macrophages are characteristically ontogenic and diverse subpopulations with var-
ious phenotypes. In current knowledge, macrophages are present in three distinct
tissues including yolk sac, fetal liver, and hematopoiesis-lineage stem cells (SCs) in
bone marrow (BM). Most macrophage populations are present in the epidermis,
Fig. 1.6 Zanvil Alexander Cohn. The photo image has been copied from the public in the web
address of http://centennial.rucares.org/index.php?page¼Innate_Immunity, as shown in the Rocke-
feller University Hospital Centennial, Monday, January 21, 2019. Because Cohn joined the
Rockefeller University of René Dubos group in 1957 and with James G. Hirsch, he studied on
leukocyte ingestion and microbial killing
lung, liver, brain, and spleen. The macrophages are produced before host birth
through self-renewal. However, macrophages resident in the gut, dermis, and heart
are established from blood-existing monocytes [37, 38]. Macrophage phenotypes are
associated with macrophage-associated disorders. Macrophages engulf and digest
pathogens, as well as toxins and dead cells. For monocytes and macrophages, Zanvil
Alexander Cohn (1926–1993), an American cell biologist and immunologist, is the
founder of modern macrophage biology (Fig. 1.6). Dr. Siamon Gordon, born in
South Africa in Zanvil A. Cohn lab, conducted pioneer studies on the differentiation
of mature macrophages (Fig. 1.7) [39, 40]. His achievement on defense mechanism
against infectious pathogens is a spot. He claimed the endocytosis as a general
cellular function to regulate the quantal uptake of exogenous molecules via plasma
membrane-derived vesicles and vacuoles. Soluble substances are internalized by a
type of pinocytosis and particulate substances by phagocytosis to the final destina-
tion of the vacuoles. Eukaryotic cells are house-kept and essential in leukocytes,
macrophages, capillary endothelial and thyroid epithelial cells, yolk sac, and
oocytes. They are involved in host defense, immunological responses, molecular
transport, hormone transformations, and the metabolic pathways.
Fig. 1.7 Siamon Gordon.

His professorship in cellular
pathology department, the
University of Oxford is
associated with his
prominent outcomes until
retirement at the end of
2008. Currently, he is an
emeritus professor of the
University of Oxford.
Siamon Gordon has studied
on macrophage
heterogeneity and
differentiation. https://
royalsociety.org/people/
siamon-gordon-11519/
Fig. 1.8 Ralph Steinman.

The photo image has been
copied from the public in the
web address of https://en.
wikipedia.org/wiki/Ralph_
M._Steinman
1.3.6 Dendritic Cells
For DCs, Ralph Steinman (1943–2011), born in Canada, launched the concept of yet
another cell-recognizing antigens and the DCs (Fig. 1.8) [41]. He, in 1973, discov-
ered DCs during his joining as a postdoctoral fellow in Zanvil A. Cohn group
[41]. Steinman is a recipient of three co-receivers of the 2011 Nobel Prize in
Physiology and Medicine, with other two scholars as recipients. The DCs are
Fig. 1.9 Bernard Babior.

wikipedia.org/wiki/
Bernard_Babior
functionally bridging the innate to adaptive immunity because DCs are sentinels,
capable of presenting antigens through processing of captured antigens to T helper
cells. The DCs migrate to lymph nodes, lymph tissues, or antigen-reactive T-cell
clones. DCs are sensing responders due to differentiation or maturation capacity,
influencing the differentiation of Thl vs. Th2 T cells. The DCs allocate the innate
defenses to enhance cytokines and innate lymphocyte behaviors. Three innate
features of DCs influence peripheral tolerant pathway. DCs target antigens to
differentiate to matured DCs, contributing to actively stimulation of both B cells
and T cells [42]. FcR death receptors activate or inhibit DC function because most
DCs are immature and microbial stimuli mature DCs to control helper, cytotoxic,
and regulatory T cells.
Reactive oxygen species (ROS) of DCs are actual parameters to capture death
cells or debris. Manfred L. Karnovsky (1918–1998), a South African biochemist,
showed the clues how phagocytes convert oxygen to reactive species to kill bacteria
or pathogens [43]. Studies on how white blood cells covert oxygen to protect the
host by means of defenses against pathogens are his pioneering achievement.
Bernard Babior (1935–2004), an American physician and scientist, pioneered
research on the oxidase system of neutrophils (Fig. 1.9) [44]. Thus, Babior found
that free radicals are crucial for defensing mechanisms of white blood cells. High
toxic derivative, superoxide as a ROS is synthesized by a specific enzyme NAPDH
oxidase. This kills pathogenic microbes. Babior attempted to expand the NAPDH
oxidase property to genetically fatal diseases including immunodeficiency and
chronic granulomatous diseases.
Fig. 1.10 David Lambeth.

web address of http://www.
emoryhealthsciblog.com/
tag/david-lambeth/ in
Emory University
David Lambeth, MD, PhD, Emory pathologist, discovered the functional expla-
nation of reactive oxygen-producing enzymes (Fig. 1.10). In 1999, he discovered the
Duox family of ROS-generating enzymes. NADPH oxidases (Nox) are known for
plants to fight off pathogens and for fungi to induce sexual development. In flies,
NADPH oxidases also lead for egg laying, and in humans, they could sense gravity.
NADPH oxidase catalyzes the superoxide genesis from oxygen and NADPH. The
genetic disorder such as chronic granulomatous disease is an oxidase enzyme-
lacking inherited immunodeficiency. Nox (NADPH oxidase) and the related Duox
(dual oxidases) are involved in diverse responses via ROS. The Nox/Duox families
are widely identified in various organisms including fungi, green plants, fruit flies,
green plants, slime molds, nematodes, and mammals [45].
1.3.7 Complement System
Complement is a thioester-containing protein (TEPs including C3 and C4), which is

also found in the protease inhibitor a2-macroglobulin. Complement cascade consists
of collagen-tulip family molecules, α2-macroglobulin family molecules, and serine
protease family molecules. Components of complement are found widely in meta-
zoans. Compared to other defense systems in each organism, the complement
belongs to an ancient molecular machinery and innate immunity. The complement
system complements the additional functions of immunoglobulin antibodies and
phagocytic myeloid cells to make a clearance of invaded pathogenic microbes and
damaged self-cells. Eventually, the system stimulates inflammatory response. The
complement system can also be acted together with antibodies generated from the
adaptive immune cells. The complement system is composed of 40 more proteins in
Fig. 1.11 Jules Bordet. The

photo image has been
wikipedia.org/wiki/Jules_
Bordet
mammals with soluble forms in blood and some membrane bound on cells. Com-
plements act as an immune surveillance on host cells and nonself-invaders. Upon
accounting to damaged cells and microbes, they keep homeostatic status with
functionally and structurally multiple roles. The complement is crucial as PRR to
detect nonself, PAMPs, and DAMPs and as effector system in both primary innate
and adaptive immunities. It influences adaptive immunity, particularly B cells and
Ab. It affects many other biological systems as part of homeostasis [46].
Jules Bordet (1870–1961), a Belgian immunologist and microbiologist, described
for the first time during the end of the nineteenth century the bacteria-killing agents
as he found the bactericidal and bacteriolytic effects (Fig. 1.11). Such bacterial-
killing activity has been considered to be the cooperative results between the specific
serum antibody and another thermolabile substance, called alexin. Thus, Jules
Bordet is a pioneering immunologist in the era of the dawn of molecular immunol-
ogy. His works are made at l’Institut Pasteur in Paris from 1894 to 1901 and the
Pasteur Institute of Brabant in Brussels, when such works are before World War
I. His observation was that complements bind to antibody-antigen complexes,
designing the assay system of the complement fixation. For example, he identified
anaphylatoxin, conglutinin, and whooping cough-causing bacterium of Bordetella
pertussis. He identified thrombin formation, platelet clot formation, human milk
lysozyme, and bacteriophage biology. From the outcomes of his complement, Jules
Bordet was a recipient of the 1919 Nobel Prize for Physiology and Medicine
[47, 48]. Although he could not attend the award ceremony, the award was from his
contribution to the complement. However, complement seemed not to be his
research goal but for bacterial killing. Actually, he contributed to bacteriophage
life cycle and lysozyme studies. His scientific supervisor Metchnikoff also received a
Nobel Prize in 1908 in Medicine from phagocytosis discovery. Interestingly, in
1903, Almroth Wright in London rather discovered the “opsonin” reality and serum
opsonic activity [49]. Later, in 1935, Gordon and Thompson demonstrated that
complement is the same identity as “serum opsonin” [50].
1.3.7.1 Alternative Complement System
In the same era, alternative complement pathway has been appeared by Louis
Pillemer (1908–1957), who was born in South Africa. He isolated properdin from
serum. Louis Pillemer traced his ideas about the antibacterial serum component as a
complement, originated as early back as the 1790s, when John Hunter in London,
UK, observed the blood resistance against putrefaction. A century later, many
immunologists including Jozsef Fodor (Hungary), Carl Flugge (Germany), George
Nuttall (USA), and Hans Buchner (Germany) studied on antibacterial serum con-
stituents. Nuttall and Buchner reported that the blood constituents having bacteri-
cidal activity are heat-sensitive over heating at 55 C or 60 C [51, 52]. Thereafter,
Elie Metchnikoff (Ilya Ilyich Mechnikov), the Institut Pasteur, Paris, France,
co-worked on the same phenomenon with a visiting researcher Jules Bordet
[53]. In 1895, Jules Bordet discovered a heat-stable serum molecule at 56 C for
30 min from immune-injected animals with agglutinated Vibrio cholerae debris. The
isolated serum constituent is named sensibilatrices, currently named antibodies. In
contrast, a heat-labile agent from nonimmunized animals is named “alexine” by
Buchner. Alexine is currently the complement and means the Greek “to defend,”
which lyses the bacterium Vibrio cholerae. Bordet evidenced that antibody-foreign
erythrocyte complex binds to alexine which leads to lysis of the erythrocytes
[54, 55], indicating that the hemolysis indicates the bacteriolysis. Then, the comple-
ment fixation reaction enables to develop complement fixation technology to detect
the antibodies against bacterial infection including typhoid, plague, and anthrax
[56]. Louis Pillemer’s laboratory was in the Institute of Pathology, Case Western
Reserve University School of Medicine. His pioneering works largely affected to
open the alternative complement pathway, an antibody-nondependent defense sys-
tem, as he is evaluated as an early researcher of the alternative complement pathway.
He described for the first time properdin as a key component of an antibody-
independent complement activation pathway. That was his pioneering work and
allowing the discovery of the alternative complement pathway. In the 1970s, pro-
perdin was reconfirmed to be a stabilizing agent, required for the alternative pathway
convertases, which are used for the complement cascades, where properdin recog-
nizes target cells and infectious microbes to lead to phagocytic clearance. It also
recognizes ligands, phagocyte receptors, and serum-regulating proteins [57]. Later,
by his laboratory in Case Western Reserve, Irwin H. Lepow purified the C1qrs
complex and characterized its enzyme, and with the natural serum C1 esterase
inhibitor.
1.3.7.2 Lectin Pathway
The lectin pathway was first found in the 1980s, although the lectin pathway was not
solidly confirmed till the 1990s. Discovery of the mannose-binding lectin (MBL)
from the mammalian sera initiates the reality of lectin pathway in 1987 by Kawasaki
group [58]. Toshisuke Kawasaki, a Japanese glycobiologist in Kyoto, isolated and
characterized a named mannan-binding protein (MBP). The MBP was isolated as a
soluble protein from liver tissues of rabbits in 1978 (Fig. 1.12). The isolated MBP
activates complement system via the classical lectin pathway [59]. MBL activation
of complement system contributes to the diverse defense mechanism in vertebrates.
Hence, because MBL is structurally similar to Ciq in its quaternary structure, the
MBL has been considered to be one of the classical pathways. MBL binds to C1r and
C1s to activate. MBL was also isolated from human serum in 1983. The membrane
protein binds mannan. ManNAc, GlcNAc, and Man inhibit the mannan binding,
while GalNAC and M-6-P are inert. His proposed theory was that the MBP is the
Fig. 1.12 Toshisuke Kawasaki. The center is Prof. Kawasaki, and his left are Mrs. Dr. Kawasaki
and Dr. SJ Kim (SCM Biotechnology, Director, Korea) and YJ Kang. His right are Prof. Cheorl-Ho
Kim (Sungkyunkwan University, Korea), Dr. Tae-Wook Chung (GeneBioCell, Ltd., Korea, Direc-
tor), and YJ Kang in Glycoconjugate Symposium, Lubeck, Germany. The author Cheorl-ho Kim
presented his snap photo due to his personal respect
hepatic uptake protein of glycoproteins with terminally glycosylated

glycoconjugates having GlcNAc and/or Man residues.
As mentioned above, the complement also influences adaptive immune responses
operated by B cells and antibodies, affecting many other biological systems as part
of homeostasis [60]. Components of complement are found widely in metazoans and
particularly important for human resistance to bacteria [61]. Human populations
frequently exhibit specific resistant phenotypes to infection and manifest of malaria
parasites through gene polymorphisms in the antiparasitic thioester-containing pro-
tein 1 (TEP1) gene. The polymorphism is related with the variability in parasite
killing [62]. Complement is activated on the nonself cell surfaces by the classical,
lectin, or alternative pathways. Evasion of complement function is a hallmark of
invasive pathogens and hematophagous organisms. The complement system is
particularly important for human resistance to bacteria [63] and for mosquito
resistance to malaria parasites [64].
For example, the complement is also important for mosquito resistance to malaria
parasites. The transmission capacity of carrier, the mosquito Anopheles gambiae
species to protozoan parasites of Plasmodium family, is varied in each individual.
Because Plasmodium falciparum malaria is an intracellular parasite by Anopheles
mosquitoes, most deaths occur as a result of complications of anemia or cerebral
malaria named coma. The complement receptor-1 (CR1/CD35) is a regulator present
on the red cell surfaces and most leukocytes, in the malaria pathogenesis upon
P. falciparum infection. CR1 mediates the interaction between the red blood cells,
which are infected by parasites, and normal red blood cells (noninfected cells),
forming rosettes and disturbing microcapillaries. CR1 also controls complement
activation and immune complex formation during malaria infection. CR1 is a
receptor for the invasion of red cells by the parasite. Complement activation on
RBCs normally occurs via C3 cleavage and immune complexed with C3b. Com-
plement activation contributes to the loss of RBCs during P. falciparum malaria
anemia [65], which occurs via the classical pathway by antibody binding to C1q,
MBP binding to pathogenic cells, and basal cleavage of C3 on cell surfaces
[66]. RBC-surfaced complement regulatory proteins (CRPs) including CR1/CD35,
DAF/CD55, and protectin/CD59 [67] regulate the complementation. CR1 mediates
the clearance of immune complexes, and C3b-opsonized immune complexes bind to
CR1 on RBCs and endocytosed by liver or spleen macrophages [65]. The CRP
clearance reduces the RBC’s capacity to regulate complement deposition [68]. Then,
RBCs become susceptible to complement-mediated destruction by phagocytosis.
Erythrophagocytosis by macrophages is such a complement-dependent anemia
during Plasmodium infection. This process is also enhanced by CD47, a self-marker
expressed on RBC. Plasmodium parasite preferentially infects CD47-expressed
RBCs to avoid phagocytic clearance [69].
1.3.8 Opsonization
For opsonic phenomenon, Almroth Wright (1861–1947), Sir Almroth Edward

Wright, a British bacteriologist and immunologist, described that the number of
organisms ingested increased if incubated in the presence of fresh normal serum
(Fig. 1.13) [70]. He developed an antityphoid fever inoculation system in World
War I, but his name and work are not well recognized today. After born in 1861 in
Yorkshire, where his father was an Irish protestant minister and Hebrew scholar.
Almroth’s mother was a Swedish as the daughter of NW Almroth, governor of the
mint in Stockholm, Sweden [71]. His two clinical tests of the erythrocyte sedimen-
tation rate and the opsonic index are nonspecific detection parameters for patho-
physiologic serum proteins. Although infectious serum contains an increased level
of specific antibodies, in healthy subjects, Wright found that antibodies contribute
minimally to opsonic activity. That is because the opsonic activity. The activity is
present in newborn serum and increased in the acute immune phase, with less
specific. His demonstrations of complement-mediated lysis both of normal cells by
lectins and of foreign cells by animal lectins are caused by Wright’s serum opsonic
activities. His findings influenced the mechanistic understanding of complement
activation through lectin pathway by recognition of surface sugars as predictable
pathogen patterns that are quite different with the less predictable targets of the
adaptive immunity. For healthy subjects, Wright suggested that antibodies contrib-
uted to opsonic activity that is the complement-enhanced phagocytosis of
Fig. 1.13 Almroth Wright.

wikipedia.org/wiki/
Almroth_Wright
microorganisms. In 1906, he introduced the term “opsonic” (Greek “to cater for”)
[70, 72]. In the third Cold Spring Harbor Symposium on immunology in 1989, as
Charles Janeway mentioned that the immune system has evolved specifically to
interact with infectious microbes and recognizes both specific antigenic determinants
and certain characteristic patterns, this is indeed appropriate to Almroth Wright
consistence. He proposed and used the “opsonic index,” and this would be meetable
too much of Janeway’s address.
Among many mechanisms in which immune system fights pathogens, one is
opsonization. Opsonization is the immune process of recognizing and targeting
invading agents for phagocytosis [73]. Opsonization uses opsonins to tag infectious
pathogens to eliminate them by phagocytes. Opsonization of a pathogen occurs by
antibodies or the complement system, fighting off foreign pathogens like bacteria
and viruses. Opsonization supports self-tolerance and inhibits autoimmunity. Opso-
nins are used to overcome the repellent force between the negative cell walls and
promote uptake of the pathogen by the macrophage. Opsonization is an antimicro-
bial technique to kill and stop the spread of disease. For acting opsonins, C3b, C4b,
and C1q are complement molecules and serve as opsonins. In the alternative
complement pathway, the spontaneous complement activation converts C3 to an
opsonin C3b upon binding to antigen. Antibodies can also activate complement via
the classical pathway, generating C3b and C4b onto the antigen surface. C3b-bound
antigen is phagocytosed. CR1 expressed on all phagocytes recognizes complement
opsonins, including C3b and C4b which are both parts of C3-convertase. C1q as a
C1 complex binds to the Fc region of antibodies. As circulating proteins, pentraxins,
collectins, and ficolins are all opsonins and secreted PRRs. These molecules coat the
microbes as opsonins to enhance neutrophil response. Pentraxins bind to phospha-
tidylcholine (PC) and phosphatidylethanolamine (PE) on apoptotic cell membrane.
IgM also binds to PC. Currently, the known collectins are MBL, surfactant protein
(SP)-A, and SP-D. They recognize unknown ligands on apoptotic cell membranes to
interact with phagocyte receptors for phagocytosis.
1.3.9 Lysozyme and Salvarsan
For lysozyme, Alexander Fleming (1881–1955), later respected to Sir Alexander

Fleming, who is a Scottish biologist and pharmacologist, discovered in 1922 sub-
stance in nasal mucus molecules that causes bacterial cells to disintegrate. The
substance was lysozyme (from the Greek lysis), as he named the substance in
1923 (Fig. 1.14). In 1928 he isolated the antibiotic penicillin from the mold Peni-
cillium notatum. It was the world’s first antibiotic substance benzylpenicillin (pen-
icillin G) from the mold. From the discovery, he became the 1945 Nobel Prize
recipient in Physiology and Medicine, as the prize was shared with Howard Florey
and Ernst Chain [74]. Fleming was knighted for his scientific achievements in 1944
[75]. In 1999, he was nominated in Time magazine’s the 100 Most Important People
of the twentieth century. In 2002, he was chosen in the BBC television for the
Fig. 1.14 Alexander

Fleming. The photo image
has been copied from the
public in the web address of
https://en.wikipedia.org/
wiki/Alexander_Fleming
Fig. 1.15 Sahachiro Hata.

wikipedia.org/wiki/
Sahachiro_Hata
100 Greatest Britons. Again in 2009, he was selected as the third “greatest Scot” in
STV, behind the great Robert Burns and William Wallace.
For Salvarsan, Sahachiro Hata (1873–1938) (Fig. 1.15), a Japanese bacteriolo-
gist, developed Salvarsan in the lab of Paul Ehrlich in 1909. Salvarsan also known as
“Präparat 606” or the “magic bullet,” was used to treat syphilis and one of the first
modern chemotherapeutic agents [76]. He was a prominent Japanese young scientist

and bacteriologist who developed the arsphenamine in 1909 under Paul Ehrlich
(1854–1915) group. He was the 1908 Nobel Prize recipient in Physiology or
Medicine for his achievement of the antibody production theory. Historically, he
designated and discovered the antisyphilitic arsenic agent, arsphenamine, Salvarsan,
or 606, which was the most valuable chemotherapy in the twentieth century by Paul
Ehrlich group with his young members, chemist, Alfred Bertheim, and bacteriolo-
gist, Sahachiro Hata. Sahachiro Hata stayed in the Institute for Infectious Diseases
(Densenbyogenkyuzo), currently renamed Ikkaken or the Institute for Medical Sci-
ences (Ikkagakugwnkyuzo, IMS), Tokyo, Japan. The magic bullet he established was
prescribed to cure or eradicate syphilis for a half and more century until “penicillin”
discovery to WWII by Howard Florey (1898–1968). Although Ehrlich is known as
the father of chemotherapy followed by Brian Druker, Alexander Fleming
(1881–1955), and Hamao Umezawa (1914–1986), his contribution was a real trying
to application. Syphilis was used until penicillin and other antibiotic introduction in
the 1940s. However, the precise mechanism how the Salvarsan acts to eliminate the
syphilis spirochete in vivo is unclear, although other arsenic agents such as
melarsoprol and roxarsone are used to treat parasites. Interestingly, arsenic trioxide
has recently been reported to cure patients with acute promyelocytic leukemia.
Although Sahachiro Hata received a public nomination of the 1911 Nobel Prize in
Chemistry and of the 1912/1913 Nobel Prize in Physiology or Medicine [77],
unfortunately, it was not.
1.3.10 Progress in Innate Immune Response Since Historic

Spanish Flu
In influenza virus, Spanish flu (1918–1919) was pandemic, and an influenza pan-

demic killed European peoples ranged from 20 and 40 million in 1918 and 1919
[78]. Normally, virus subtypes with HA and NA combinations are rarely identified
and isolated from mammal species; virus subtypes with all 15 HA subtypes and all
9 NA subtypes were identified and isolated from avian birds. Since entrance to the
twentieth century, the emergence of different antigenic strains, which can be trans-
mitted to in human populations, though shifts in antigen genes were identified in
1918 (type of H1N1), 1957 (H2N2), 1968 (H3N2), and 1977 (H1N1) to date with a
pandemic propahatopns. The strains emerged after gene assortment of viruses of
avian and human origins. During 1996, an H7N7 influenza virus subtype, which was
an avian origin, was identified. In addition, during 1997 in Hong Kong, an H5N1
subtype of avian influenza virus was identified, and the H5N1 subtype caused death
of humans. During 1999, influenza virus H9N2 subtype was identified as forms of
two independent isolations of influenza virus from humans. The gene reassortment
could be emerged from virus to infectious cases in the human population. The
outbreak was caused by H1N1 influenza virus and the most devastating epidemic
in modern history. Hong Kong flu (1968–1969) caused by H3N2 influenza virus

caused the death of one million people. The pandemic began in 1968 in China and
spread rapidly to the Southeast Asia and Australia region. The H3N2 subtype was
again transmitted to the USA in September through military personnel returning
back from Vietnam to hometown unite states [79]. Acquired immunodeficiency
syndrome was discovered in 1981 to now. AIDS was first reported on June
5, 1981, and is caused by the human immunodeficiency virus (HIV), a lentivirus.
AIDS killed approximately 25 million people within three decades. Five million
people received HIV treatment in 2009. It has been known that 33.3 million people
were exposed to HIV in 2009 of which 2.6 million peoples were newly infected and
1.8 million peoples with AIDS-associated lethal deaths were counted [80].
During the immune receptor-ligand interaction, a provisional immunologist,
Dr. Charles Alderson Janeway Jr. (1943–2003) claimed the immunological recog-
nition through antigen binding. This largely contributed to the TLR discoveries and
prospects toward TLR-carbohydrate recognition in innate immune response. Innate
immune recognition relies on germline-encoded receptors. The immune receptors
evolve to recognize conserved molecules by pathogenic microbes. Well-defined
molecular structures or patterns are bound by the immune defense receptors, and
the structural recognition can distinguish invaded or pathogenic nonself from self.
The innate immunity molecularly discriminates the self- and nonself-antigens.
Immune system evolves basic conceptional strategies of nonself and self-
distinguishment. The innate immune discrimination of nonself and self is possible
through occasional recognition of molecular patterns appeared on the invaders and
hosts. In sides of endogenous protection of self, the innate immune recognition is
defined as discrimination of normal and abnormal self. The abnormal self frequently
define damaged self or transformed self. These patterns are deciphered by host
receptors, depending on the meaning of these signals. His description of “I believe
that immunological recognition extends beyond antigen binding by the clonally
distributed receptors ---- and there is still much to learn------I believe that the
immune system predates the development of specific immunological recognition
mediated by clonally distributed receptors ----” clearly provoked the importance in
the immune receptor-ligand interaction. This is really innovative, as implied from
“the most likely possibility is that primitive immune effector cells possess receptors
recognize certain pathogen-associated molecular pattern (PAMP) not found in the
host” and also from “What kind of ligands or patterns should such non-clonally
distributed receptors recognise? I think it is likely that such patterns in molecules are
found in many microorganisms ---. -- Complex cell wall carbohydrates or lipopoly-
saccharides are likely ligands . . .” [81–83].
On the other hand, depending on the information derived from the immune
responses, studies on the invertebrate immunity have been greatly contributed to
the mammal host immunity. For example, Clay G. Huff (1900–1982), an American
parasitologist, mentioned the Immunity in Invertebrates. Christiane Nüsslein-
Volhard, a German biologist and geneticist, published in 1985 the first article on
the Toll gene product (Fig. 1.16). Later, she received the 1995 Nobel Prize in
Physiology or Medicine for her achievements. As the light gradually shed to the
Fig. 1.16 Christiane

Nüsslein-Volhard. The
photo image has been
wikipedia.org/wiki/
Christiane_N%C3%
BCsslein-Volhard
Fig. 1.17 Jules Hoffmann.

wikipedia.org/wiki/Jules_A.
_Hoffmann
appearance of the receptors, Jules Hoffmann, born in Echternach, Luxemburg, as a

chemist and biologist together with Bruno Lemaitre, made outbreaking contributions
to the function of Toll receptors in antifungal immunity (Fig. 1.17). His finding
revolutionized the Toll functions where the dorsoventral modulating gene loci of
spaetzle-Toll-cactus modulates the antifungal immunity in the experimental eukary-
otic fly, Drosophila [84, 85]. His colleague, Ruslan Medzhitov, a biochemist from
Uzbekistan, received the Shaw Prize in Life Science and Medicine 2011 together
with Bruce Beutler and Jules Hoffmann. He depicted that the innate immunity
should be the virtues of non-clonal immune recognition with CA Jr. Janeway
[83, 86]. From the continuous progress in the immune receptors to discriminate
their ligands even including glycans, lipids, proteins, or nucleic acids, the well-
defined word of “PRR” has been come on stage. Richard Ulevitch, a biochemist
from the USA, has also focused on the signaling pathways of PRRs. He has
organized and defined the TLRs in the innate immune response [87–89]. Later,
Shizuo Akira, a Japanese scientist, conducted his research on microbial ligands of
PRRs, such as the identification of TLR9 as a CpG DNA receptor, suggesting innate
recognition of and regulation by DNA [90, 91]. Currently, the PRRs include human
TLRs 1–10, the mannose receptor (MR), Dectin 1, Dectin 2, nucleotide oligomer-
ization domain (NOD)-like receptors (NLRs), retinoic acid (RA)-inducible gene
I-like receptors (RLRs), etc.
1.4 The Outline of Innate Immunity
The concept of neutrophils, leukocytes, dendrites, monocytes, and macrophages has

been born, and their effects on innate immunity have been fairly established to these
days. The infectious pathogen-derived pressure is a powerful driving kinetic and
force, contributing to the evolution of mammals. Innate immunity is the vital
non-clonal immune response in discrimination of nonself via molecular binding.
The responses of innate immunity are rapid and nonspecific to pathogens [83]. The
immune system utilizes immune recognition to constantly select against the targets.
The target recognition is basic in innate immunity. The recognized targets then
interact not only with innate immune cell populations such as cells of innate
lymphoid, myeloid, and NK but also with nonimmune cells in specific circumstances
and the ancient humoral systems like complements. Innate immune cells capture the
invasive targets via the two known ways of encapsulation and phagocytosis. Encap-
sulation event is restricted to invertebrates in defense against foreign targets too large
for phagocytosis by their hemocytes. The encapsulation process in invertebrate
immune response requires coordinated action of cellular and humoral factors.
Another event named phagocytosis needs phagosome process. When a macrophage
ingests a pathogenic microbe, the pathogen is trapped in a phagosome and fused to a
cellular lysosome to incorporate a specific intermediate vesicle formation, named
phagolysosome. Within the phagolysosome, the incorporated pathogens are digested
by phagolysosomal enzymes and toxic peroxides.
Humoral antibodies produced by innate immune cells of vertebrates or mammals
are expressed in the four modes including neutralization, complement activation,
opsonization, and agglutination (Fig. 1.18). Neutralization indicates that antibodies
bind to viruses and toxins and then blocks the pathogens’ proliferation and replica-
tion. Complement activation indicates the classical pathway that antigen-antibody
complexes activate the complement system and dissolves the pathogens.
Opsonization suggests that phagocytic cells grab the antibody-bound surface of
1.4 The Outline of Innate Immunity 25
A) Neutralization
Virus Inactivated
Virus-infected
cell Virus
B) Complement Activation
C C
1 C C
4
Bacterial 2 3
cell
C) Opsonization
Bacterial Fc Phagocytic
cell receptor Phagocytic cell
cell
Fig. 1.18 Antibody-mediated events of innate immune cells in mammals
foreign substances for efficient phagocytosis and then eat them. Similarly, aggluti-
nation event indicates the action of an antibody when it cross-links multiple antigens,
producing clumps of antigens.
1.4.1 Concept of Immune Receptors
Innate immune responses are displayed by distinct receptors, frequently defined

pattern recognition receptors (PRRs). PPRs recognize PAMPs directly (Fig. 1.19).
Several PRRs include peptidoglycan recognition protein (PGRP), Toll-like receptor
(TLR), lipopolysaccharide (LPS) and β1,3glucan-binding protein, galectin, C-type
lectin (CTL), and thioester-containing protein (TEP). For PAMPs, examples of
Gram-negative and Gram-positive bacterial PAMPs have been illustrated in
Fig. 1.20. Each E. coli and S. aureus strain has been shown with several components
found in virus, bacteria, and fungi. Other complement receptors and Toll receptors
bind to PAMP recognition products. Among the PRRs, specific interests have been
functionally related in multiple forms of CTL, C-type lectin in myelocytes. Currently
known and representative CTLs are illustrated (Fig. 1.21). Innate immune responses
are common features, which occurred in all the metazoan organisms to protect hosts
against pathogenic invaders and opportunistic infectious pathogens because inver-
tebrates lack an adaptive immunity.
PAMPs
PRRs
Example of PRRs
Pattern recognition receptors
Cellular
PGRP Peptidoglycan Recognition Protein
signaling
TLR Toll-Like Receptor
LGBP Lipopolysaccharide, β-1,3-glucan Binding Protein
Immune
response CTL C-Type Lectin
GALE Galectin
TEP Thioester-containing Protein
Fig. 1.19 Some pattern recognition receptor (PRR) forms in myeloid lineage innate immune cells
PAMPs Example of PAMPs
Gram-negative bacteria
Lipopolysaccaride Pathogen-associated molecular patterns (PAMPs) Microbe type
E. coli
Outer membrane ssRNA Virus
peptidoglycan
Inner membrane Nucleic Acids dsRNA Virus
CpG Virus, Bacteria
Dilin
Proteins Bacteria
Flagellin
Gram-positive bacteria Glycolipid LPS Gram-negative bacteria
Cell wall lipids
S. aureus peptidoglycan
Lipoteichoic-acid Gram-positive bacteria
membrane
Mannan Fungi, Bacteria
Carbohydrates
Glucans Fungi
Fig. 1.20 PAMPs produced by Gram-negative and Gram-positive bacteria and components known
in virus, bacteria, and fungi
As a first step, metazoan organisms recognize microbial PAMP structures. CTLs

are involved in pathogen recognition, cellular adhesion, antigen uptake, and com-
plement activation. The CTLs as a superfamily of Ca2+-dependent C-type receptor
protein (CRP) recognize nonself and invader for clearance through binding to
terminal sugars on the microorganisms. For example, mollusk CTLs (CfLec-1 to
CfLec-4) identified from Chlamys farreri are involved in the immune response
against Vibrio anguillarum PAMPs via the opsonization, encapsulation, and phago-
cytosis. CfLec-1 serves as a PRR in the PAMP recognition and opsonizes it to clear
invaders. Encapsulation, different from opsonization event, is specific for inverte-
brates against large foreign agents for phagocytosis by individual hemocytes
[92]. Like phagocytosis, the cooperation of CfLec-1 and hemocytes activates
C-type lectin
Type Ⅰ
DEC-205 Type Ⅱ
S
S
DC-SIGN
MMR Langerin
S DCIR
CLEC-1
S Dectin-1
CRD
Dectin-2
DLEC
Carbohydrate
recognition domains (CRD)
Tyrosine-based motif for

targeting to coated pits and
internalization
P
I
P I
T Triad of acidic amino acids
P T
I
M A
Fibronectin type Ⅱ repeat
M
Di-leucine motif
Tandem repeat
Fig 1.21 The representative C-type lectins (CTLs) in humans
hemocyte encapsulation. CfLec-1 from C. farreri opsonizes with hemocytes against

invaders (Fig. 1.22).
Receptor engagement can transduce signals to alarm the presence of pathogens
from their innate ligands. The innate or primary immune responses are, therefore, the
first line of defense system upon infectious invasion. While innate immunity elim-
inates majority of pathogens quickly, certain infections are not cleared initially due
to the virulence factors produced by pathogens. Innate immune responses are not
specific originally and have potential to adapt. The nonspecific property of innate
immune responses is alternatively complemented and overcame by the PRR concept.
The PRR receptors are expressed on the innate immune cells to recognize specific
microbial components.
PRRs are classified into three types of humoral proteins circulating in the plasma,
endocytic receptors, and signaling receptors. Cellular PRRs, which are associated
with the innate immune responses, are expressed on antigen-presenting cells (APCs)
in adaptive immune response and on epithelial cells that are the first to encounter
pathogens during infection. TLRs are well defined in the innate immune responses
[88]. Because the innate immune-related myeloid cells respond to foreign invaders
as the first defense line during infectious diseases, the elementary mission for the
host is to bind to the pathogens. Subsequently, it exerts a rapid and fast response to
defend. TLRs or Toll-like receptor families display the primary roles in both
vertebrates and invertebrates, reflecting a functional role in host defense. Ruslan
Medzhitov, a biochemist from Uzbekistan, received the Shaw Prize in Life Science
and Medicine 2011 together with Bruce Beutler and Jules Hoffmann. Richard
Pathogen (Vibrio anguillarum)

PAMPs
C-type lectin (CfLec)
Opsonization
Encapsulation
Immune response
Fig. 1.22 Mollusk CTLs (CfLec) of Chlamys farreri are involved in the immune response against
Vibrio anguillarum PAMPs via the opsonization, encapsulation, and phagocytosis
Ulevitch is a biochemist from the USA, and he focused on the signaling pathways of
pattern recognition receptors. Shizuo Akira, a Japanese scientist, conducted a
research on microbial ligands of PRRs, such as the identification of TLR9 as a
CpG DNA receptor. Innate recognition of and regulation by DNA has been carried
out by Akira Shuzo [93].
1.4.2 Host Protection from Microbial Invaders of Innate

Immunity
During an infection, the innate immunity is the first line to be bordered, within a
short time, no longer than minutes to hours for full activation. This is because of the
host defense in the first infection phase. While innate immunity eliminates the most
pathogens quickly, certain infection is initially not cleared just due to the virulence
factors produced by pathogens. Innate immune responses are traditionally not
specific and potential to adapt. The property that innate immunity is not specific is
alternatively complemented and overcome by the PRR concept. The PRRs are
expressed on the innate immune cells to recognize specific microbial components.
Innate immune cells recognize the difference between Gram-positive and Gram-
negative bacteria but not closely related strains [94].
As a model of bacterial pathogens to evade phagocytosis/phagocytic killing, the

following mechanistic examples are raised:
1. Evasion of complement-mediated phagocytosis by expression of a polysaccha-
ride capsule common among bacterial pathogens, group B Streptococcus (GBS),
Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae:
neonatal infectious events are the most common causes of life-threatening path-
ogen infections in neonates and newborns. Meningitis, pneumonia, and septice-
mia are infections in adults as a growing clinical problem and skin infections,
urinary tract infections, and meningitis in adults with underlying illness. They are
often important virulence factors with antiphagocytic properties affecting com-
plement deposition/activation at the bacterial surface.
2. Evasion of non-opsonic phagocytosis. This is a bacterial mechanism to evade
phagocytosis/phagocytic killing and evasion of non-opsonic phagocytosis. PRRs
exhibit signaling such as TLR that triggers an intracellular signaling which
culminates in induced production of multiple mediators including chemokines,
proinflammatory cytokines, type I interferon (IFN), and subsequent activation of
phagocytes. In phagocytosis, SR-A recognizes and binds with non-opsonic
phagocytosis of pathogenic microbes. They are expressed by most macrophage
populations. Endocytosis of mLDL contributes to foam cell formation in athero-
sclerosis. They act as a PRR. Phagocytic receptor mediates direct non-opsonic
phagocytosis of several bacterial species and contributes to resistance to exper-
imental infection with Gram-positive bacteria (L. monocytogenes, S. aureus,
S. pneumoniae). Bacterial mechanisms to evade phagocytosis/phagocytic killing
include targeting of inhibitory receptors in human Siglec-5, where they are
expressed by neutrophils, monocytes, macrophages, and basophils. It consists
of the extracellular four Ig-like domains and two cytosolic immune receptor
Tyr-based inhibitory motifs (ITIMs) to act in cell-cell interactions and inhibition
of inflammatory signals. The binding protein of GBS binds to Human Siglec-5
and inhibits phagocytosis.
3. Targeting of inhibitory receptors.
4. Evasion via T3SS.
5. Increased intracellular survival.
How can we determine the evolutionary history of innate immunity? Innate
immunity displays the rapid evolution. This rapid evolution is frequently caused
by interaction between hosts and pathogens as well as pathogen-driven direct
selection [95, 96]. Many genes involved in innate immunity undergo evolution
along the mammal lineage [97]. Other characteristics of the innate immunity are
heterogeneous in immune-responding cells in the different responses against path-
ogen infection [98, 99] and diversity of PAMPs [100]. The innate immune response
against pathogens is controlled to avoid self-damage. The innate immune response is
transcriptionally and adaptationally diverse in expression of cells between species
and variability. Transcriptionally diverging genes include chemokines and cytokines
produced in myeloid cells. The gene expressions are also transcriptionally regulated
by each distinct promoter [101]. Thus, each gene expression distinctly differs among
species and conditions to evolve for adaptive and relevant response [102]. The
immune-responding genes change continuously during the evolution because of
their repeated exposure to the surrounded antigens. Diverse environmental patho-
gens influence the changes of genetic markers in each environment
[103, 104]. Prolonged adaptation to different ecological niches led to shape the
evolution of PRR such as TLRs differently even within some species [105]. The
allele frequency of sickle cell β-hemoglobin is increased in humans due to malaria-
endemic interaction. This process is well described as a commonly accepted example
of a deleterious gene polymorphism occurred via selective pressure by a certain
harmful parasite [106]. Recent next-generation genome sequencing (NGS) has
elucidated that certain immunity-associated human genes such as the viral
RNA-editing gene are continuously, but positively, being selecting [107–109] to
adapt between pathogens and their hosts. This concept is so-called Red Queen
hypothesis [94], but outlined cases between infectious pathogens and co-selection
in humans are just dawn stage.
Complement components are very ancient. The barriers include tight junctions in
epithelial cells, mucins and mucus, stomach acid, and nutrient sequestration (factors
which strongly bind iron, biotin, etc.). Cells include phagocytes and other blood cells
(macrophages, granulocytes), NK cells, αβ-NKT and γδ T cells (invariant or semi-
invariant T-cell receptors). Molecules include antimicrobial peptides (AMP, like
defensins, magainins, cecropins), complement (C, like thioester-containing proteins
including C4, C3, and C5, TEPs), lectins, ficolins and collectins (MBL), scavenger
domain-containing proteins, TLRs, NLRs, cytokines, and chemokines as well as
intracellular defenses such as PKR, interferon, lectins, TRIMs, RNAi, etc. NK cells
are also crucial for the innate immunity since NK cells as innate lymphocytes bear
many receptors with crucial roles in infectious disease, cancer, autoimmunity,
transplantation, and reproduction [110]. NK receptors have a function of ligand
recognition by lectin or immunoglobulin domains. They have a signaling either
activating or inhibitory, exhibiting highly diverse between individuals.
1.5 Autophagy from Microbial Invaders

and Self-Associated Molecular Patterns (SAMPs)
of Innate Immune Cells
PRRs such as TLR4 also induce autophagy in APCs (Fig. 1.23). Autophagy in
immune cells functions as a surveillance way of the intracellular pathogens and
SAMPs [111]. A main player of autophagy is the target of rapamycin (TOR) kinase
for growth factor receptor signaling, hypoxia, ATP levels, and insulin signaling.
TLR signaling induction elicits autophagy in APCs. In fact, autophagy is frequently
observed in macrophages treated with certain TLR agonists such as TLR4-specific
LPS [111, 112]. For the adaptive immune responses driven by T cells, the CD4+
helper T-cell subsets are normally regulated by major histocompatibility complex
References 31
(A) lassical pathway of autophagosome formaon

Nutrient depleon or stress
mTOR pathway
LC3
fusion
Phagophore MHC-II compartment
MHC-II compartment
(B) Endosome mediated autophagy in DCs
/Autolysosome
Bacteria
LPS binding to TLR receptor
Selecve autophagy
MHC-II compartment
DC aggresome-like induced structures

Early endocyc pathway
Fig. 1.23 Autophagy induction of PRRs such as TLR in APCs
(MHC) class-II (MHC-II)-presented peptidyl antigens on the APCs such as DCs.

Endocytosed antigens are delivered to MHC-II compartments, endocytic organelles
[113]. Cytosolic and nuclear proteins, including mucin-1 (MUC1) and complement
component, are also loaded on MHC-II. Cytoplasmic proteins are delivered to
lysosomes through three autophagy pathways including microautophagy,
chaperone-mediated autophagy, and macroautophagy [114]. Autophagosome is
formed with a nucleation membrane, phagophore, and fuses with an endosome-
lysosome. Importantly, autophagy in APCs is formed via TLR activation to detect
microbes, although classical autophagy is induced by rapamycin or nutrient depri-
vation. LC3, autophagosome marker, colocalizes with MHC-II compartments. LPS
treatment in DCs induces the transient protein aggregates named DC aggresome-like
induced structures (DALIS) or ubiquitin-positive aggresome-like structures (ALIS)
[114]. In LPS-induced DCs, autophagosomes formed via MHC-II compartments
bear the autophagosome markers LC3 and ATG16L [113], calling endosome-
mediated autophagy. MHC-II compartment-driven autophagosomes preferentially
engulf the LPS-induced DALIS, which become later degraded in autolysosomes.
The TLR signaling is initiated via two adaptor proteins, MyD88/Toll/IL-1R-homol-
ogy domain (TIR)-bearing adaptor-inducing IFN-β (TRIF), where they activate
protein kinases, ubiquitin ligases, and transcriptional factors.
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Chapter 2
Dendritic Cells (DCs) in Innate Immunity
2.1 General Biology of DCs
A representative innate immune cell, dendritic cells (DCs) belong to hematopoietic

cells and bridge immune responses between innate immune and adaptive immuni-
ties. DCs are the immune cells resided on the first line of interaction with antigens. In
addition, the DCs are the primary defense guards for host immune response against
pathogens and invaders. Because immune system of mammals consisted of two
fundamental immunities of (1) primary or innate and (2) acquired or adaptive ones,
DCs belonged to the primary or innate immunity groups. When foreign and invaded
antigens are encountered or exposed to the mammal immune system, initial receptors
regarding innate immunity system are broadly and systematically selected by molec-
ular and physical recognition. Then, molecular and biochemical reactions are acti-
vated. The activation reflects the downstream signaling that has extensively been
subjected for biological signal transduction. During the past 3 decades since 1985,
the huge clarification of such primary defense system has been made. Once activa-
tion of the innate immunity is performed, adaptive immune system is operated by
highly compromised and specific T-cell and B-cell receptors. Therefore, the
response style and pattern of the two immune systems are quite different. The innate
immunity is rapidly processed in short time intervals within hours to days, while
adaptive case exhibits slow reaction period such as days to weeks for longer lasting
time than innate one. Due to the specific signal transduction-based proposition,
clonal amplification of the adaptive immune-related cells is highly outstanding and
indeed explosive, but the case of innate immunity is almost constant without
amplification. At the stage of completion of the reaction, the adaptive immunity is
potentially forwarded to the memory state of the next generation of responses, but
not observed in the case of the innate immunity.
DCs fall into a heterogeneous group of myeloid lineage APC. They occupy,
throughout the entire body, 2–4% of leukocytes. DCs are defined by their surface
proteins, localization, and function. In initial defense system of the body, indeed
38 2 Dendritic Cells (DCs) in Innate Immunity
likely to the innate immunity, the first defense line in cell level is DCs in body fluids.
DCs have a diverse and big group of antigen-presenting cells (APCs), which
translate the innate immunity-derived information of invaded antigens to adaptive
immunity. The basic concept and function of DCs in transmitting the information to
the B and T cells are originally described for the initial immune response by
Steinman and Cohn in 1973 [1]. At the molecular level, DCs have a unique ability
to transmit the collected and processed information to B and T cells of the adaptive
immunity [2]. Although there are many different bone marrow-differentiated cells,
DCs are a small group of innate immune cells derived from the bone marrow through
the mammalian body system. DCs in body fluids are distinct APCs that function to
translate the foreign antigens to recognizing cells, specifically through connection of
the innate immunity to adaptive immunity. The translation process is operated by
initiating T-cell responses in a direct fashion. DCs are therefore one of the most basic
actors and the general mediators in the initial immune responses of mammals.
Besides DCs, monocyte-translated macrophages play the same roles as DCs in the
flamed tissues. In other words, DCs as an essential regulator of innate immune
response and docking to trigger the active immune responses are the major mediators
and players [2]. More specifically, antigen-captured and eventually antigen-loaded
DCs induce a stepwise antigen-specific T-cell immunity that is called adaptive
immunity. The DC’s loaded information-receiving receptor is called major histo-
compatibility complex (MHC), where MHC-II is specific for the B-cell activation,
whereas MHC-I is oriented for the tumor- or virus-dependent receptor style in
mammals. The MHC-II is expressed on DCs, macrophages, or cervical cells, while
the MHC-I is normally expressed on the somatic and nonimmune cell surfaces.
On the other hand, in organ tissues but not in body fluids, macrophages are the
major actors of the innate immune defense. Initially the macrophage’s precursors,
named monocytes, are circulating in the body fluids. Upon tissue damages by
pathogen infection and related agents, the macrophage derived from the monocytes
acts as a local mediator of the innate immunity through modulation of inflammatory
response. Through the continued innate immune responses, macrophages activate
the next step of responses, specialized for adaptive immune responses via internal-
ization of antigenic peptides, process, and present the captured foreign antigens to
the T cells. Macrophagic differentiation of monocytes or partial DCs involves in
differentially expressed phenotypic changes in cell surface antigens of clustered
differentiation (CD) markers and the production of mediators responsible for pro- or
anti-inflammatory response [3].
Apart from pathogenic invasion, DCs are applicable to the tumor immunity
because the versatile capacity of DCs can trigger antigen-specific immune responses
to adaptive immune cells. Therefore, application of DC biology has recently
received some great interests to cancer immunotherapies. The positive aspect and
merit of DCs are attributed to target cancer-associated antigens expressed on tumor
cell surfaces. This allows many researchers to the current research trend to use DCs.
DCs can be directly isolated from the body or often derived from monocytes and
in vitro cultured in order to utilize them to the cancer immunotherapy [4]. In
organisms, DCs can be found in many different types of cells, and they reasonably
2.1 General Biology of DCs 39
protect the peripheral tissues. The most general DCs can be seen on the skin, a
dermal barrier as peripheral tissues. The skin DCs are indeed non-clonal type with
diverse receptors of DCs, and they recognize specific invasive agents, antigens,
pathogen-associated recognition patterns (PARPs), or PAMPs. The peripheral DCs
ingest and digest such foreign antigens in order to present the processed antigen
fragments to adaptive immune cells such as T helper cells, as above mentioned.
When DCs present the processed antigens and carry them on MHC-II, they are ready
to migrate from the original phagocytic region, lymphatics, to the final destination in
near lymph nodes. The antigen-processed fragments are typically oligopeptides to
date, and they are subjected to load onto each MHC-II amino acid region. This
process is a classical behavior for antigenic presentation after processing to helper T
cells resident in lymphoid nodes or tissue [5]. Among the APCs in mammals,
therefore, DCs are typical and professional APCs having the co-stimulatory mole-
cules and MHC-II. DCs are consequently regarded as positive stimulators of primary
immunity. In addition, DCs are an essential regulator in initiating organism’s innate
immune responses, giving a term of “acquired immunity or secondary immunity.”
Thereafter, binding of antigen peptides to T-cell receptor (TCR), MHC-antigen
fragments, and co-stimulatory proteins on the DCs’ surface eventually activates T
cells. The T-cell activation is a multi-complexed process including phosphorylation-
based signal transduction and subsequently induction of T-cell differentiation,
giving a description of “the antigen-specific response.” On the other hand, apart
from DC activation, nonactivated and immature DCs contribute to the constitutive
presentation of self-antigen. The status indicates T-cell deletion and also differenti-
ation status of regulatory T cells (Tregs) or suppressor T cells by the interaction with
the nonactivated and immature DCs. This process eventually triggers the T-cell
behavior including the deletion of T cells and Tregs, and also differentiation of
suppressor T cells. Therefore, this status of immune suppression is called “self-
tolerance.” Thus, the mammalian immunity is established with the appropriate and
relevant response to the nonself threats from external environment. Thus, a well-
organized and target-oriented immunity is consequently ensured in the immunology,
homeostatic limiting to only foreign pathogens, invaders, or agents [6].
Apart from APC function, DCs induce effector T cells or Treg responses through
co-stimulatory signals and cytokine expression. As the most positive APC, DCs
bridge between T-cell response and immune tolerance. The consequent case is
DC-derived tolerance as a result of rare and distinct aspect of DC-regulated T
cells. DCs also influence multiple phenotypes of T cells, including development,
differentiation and function, to maintain tolerogenic state. The DC-T-cell interaction
is not regularly observed but observed by certain conditions including DC matura-
tion state or tissue microenvironments. The DC and Treg recognition is indispens-
able in induction of central and peripheral tolerance. DCs are indispensable for Treg
differentiation and homeostasis [7].
2.2 Classification and Different Function of DCs
Basically, DCs are differentiated to each specific cell type, depending on their
functions for the innate immune system. DCs develop from a common
BM-derived macrophage or progenitor DC cells, which further progress to the
differentiated cell types of monocyte and macrophage lineage or the common DCs
[8]. DCs in the BM exist as both forms of the named plasmacytoid DC (pDC) and
pre-DC progenitors [9]. Completely matured pDC in the BM moves to the blood-
stream. Prematured DCs migrate to the peripheral tissues or lymphatic organs via the
vascular vessel. The migrated prematured DCs to peripheral tissues or lymphatic
nodes are ready to differentiate into conventional DC subsets of CD8α+/CD103+
DCs or CD11b + DCs [10]. The DCs can be classified depending on their maturation
to antigen-specific cell types, presentation to T cells, or migration to lymphatic
nodes. Typically, DCs are classified into two distinct classes of (1) pDCs, which
produce type I IFN upon viral infection and (2) conventional DCs as APCs, which
activate naive T cells. Among conventional DCs, CD8α+ DCs orchestrate immune
responses upon infection of intracellular pathogens. In contrast, CD11b+ DCs fight
extracellular pathogens. Conventional DCs are classified into two basic categories of
(1) migratory DCs such as dermal DCs and Langerhans cells and (2) nonmigratory
DCs such as spleen DCs, which are resident in secondary lymphoid organs,
depending on their migratory potentials. Also, if foreign pathogens infect the body
tissues, DCs are classified into other two different categories of (i) monocyte-derived
DCs (moDCs) and (ii) pDCs as a first defense line against pathogenic invasion or
type I interferon-releasing DCs. In the meaning of diverse functional phenotypes of
DCs, DCs are defined as multifunctional-shaped and phenotyped defense cells
against pathogenic infection or agent invasion. In order to accomplish the transla-
tional behavior which transmits the information of foreign antigens to T cells as
adaptive immune cells and eventually B cells, moDCs are highly migrative to their
directed locations to account their counterpart cells such as T cells [11]. For
migrative potentials, DCs or their precursors acquire and require two different
migration steps, i.e. (i) as a first step, they leave the vascular blood to peripheral
tissues through the leukocyte homing process, and (ii) as a next second step, matured
DCs leave the peripheral tissues through the homing process to the draining lym-
phoid nodes. The process is well defined through the leukocyte homing process. DCs
and DC precursors include undifferentiated monocytic cells and extravasate to
peripheral dermis, tonsils, epidermis, and gastrointestinal tract (GIT) mucosal region
across the endothelial lines from the blood. When DCs are translocated to inflam-
matory tissues, DCs are activated by an uptake of foreign antigens or by
proinflammatory cytokines released by diverse inflammatory cells.
On the infection or injured site, leukocytes under circulation attach to endothelial
cells and platelets as well as the adhered leukocytes. When inflammatory agent or
invaded pathogens are encountered to DCs, DCs undergo their maturation process to
express their surface antigens such as CD11 series. When the immature DCs are in
stages of antigen uptake and processing, they are differentiated to active and mature
2.2 Classification and Different Function of DCs 41
Immunity
Tolerance
CD83+
Immature DC Stimuli Mature DC
CD83+
Antigen uptake and

capturing, Antigen presentation,
antigen processing to be able to
costimulate and
activate T-cell
Fig. 2.1 Maturation of dendritic cell
Pathogens
DC precursor
(CD11c+, Mac1+,
CCR5+)
Inflammatory
monocyte
(Mac1+, CCR2+)
Blood
CD83+
Lymph node
Tissue
T cells
Fig. 2.2 Maturation of DCs and migration to lymph node and T-cell activation, and chemokine-
driven migration of monocytes during pathogenic invasion
DCs. The differentiated DCs exhibit the classical APC roles including antigen
presentation, costimulation, and T-cell activation (Fig. 2.1). Thus, DCs are important
for initial stage of the immune responses, where immature DCs positively internalize
foreign antigens and differentiate to trigger T-cell responses. To accomplish such
mission, therefore, DCs serially step down into two essential migration stages.
(i) Migration into peripheral tissues is to acquire antigens on the inflamed sites,
and (ii) migration to lymph nodes is its final destination to collaborate with the
information-receiving cells like B cells and T cells (Fig. 2.2). Cell maturation,
positional migration to lymph node, and T-cell activation of DCs are all their
Complement-binding protein
like domain
wG
EGF domain
j lG Lectin domain
jG
sTzG uG
lTzG
wTzG
Fig. 2.3 Structure of selectins
pathways collaborated during pathogenic invasion or occurring of inflammatory

agents. During inflammatory status, the leukocytes such as DCs are highly activated
to express the cell adhesion molecules (CAMs) of selectin molecules, which recog-
nize and tightly bind to the sugar components on the endothelial cells.
Selectins expressed on DC surface act as rolling mediator, whereas cell surface β2
integrins act to potentiate arrest, intraluminal crawling, and slow rolling. The
adhesion cascade events are comprised of a series of various responses including
basic interaction of arrest, crawling deceleration, slow rolling, tethering, and
transendothelial migration [12]. In leukocytes, to accomplish the adhesion event,
lipid rafts in membranes are highly ordered through assembly with membrane lipid
components of cholesterol and sphingolipids, and membrane-selected proteins.
Particular membrane proteins participate in lipid rafts via hydrophobic residues in
transmembrane domains. Consequently, they are ready to interact with membrane-
located GSLs or cholesterol. N- and O-glycans on protruding proteins are also
associated with lipid rafts. The dominantly expressed leukocyte ligands are recog-
nized by their receptors of P /L-selectins. The specific P-selectin glycoprotein
ligand-1 (PSGL-1) known as the selectin ligand is recognized. Apart from the
PSGL-1, other important leukocytic ligands to recognize E-selectin are well defined
to date, and several representative candidates of CD44 and CD43 are reported
[13]. Among selectins, myeloid lineage leukocyte cells produce L-selectin form,
while activated platelets produce P-selectin form on surfaces. In addition, stimulated
endothelial cells express E- and P-selectins on their cell surfaces (Fig. 2.3). Through
such selectin-ligand interactions, the leukocytes are also ready to bind to the
s i

h w
l
h
p
h
p
p
a z
a z
Fig. 2.4 Leukocyte trafficking to inflammatory endothelium and infiltration
peripheral platelets and infiltrate into the inflammatory tissues, where the immune
cells resident in the area are also stimulated to activate by various inflammatory
mediators including cytokines of IL-1β and TNF-α as well as arachidonic prostanoid
metabolites (Fig. 2.4). Among the DC subpopulations, only matured DCs can
activate and proliferate T cells after migration to the lymph node, while immature
DCs rather induce anergy status, apoptosis, or deletion of T cells, providing the
immune tolerance status, and then allowing the immunological homeostasis.
2.2.1 pDC, Lymphoid Organ CD8α+ DC, and Tissue CD103+

DC Interaction with Tregs
The phenotype of pDCs is a characteristic of B220+ PDCA-1+ Siglec-H+ CD11clow

MHC-IIlow cells. The pDC subset development depends on E2–2 and IRF8
[14]. pDCs secrete type I IFN, and normally, pDCs are a poor type of APC, although
pathogen-stimulated pDCs can prime naive T cells. Normal mouse antigen-pulsed
splenic pDCs and human pDCs induce anergy states of antigen-specific mouse T
cells and human CD4+ T cells [15], respectively. A subset of naive T cells is
activated by CpG-induced pDCs, and activated T cells generate immunosuppressive
Treg populations [16]. Human pDCs stimulate differentiation of T cells into IL-10-
expressing Tregs, and pDCs activate CD4+CD25+ FOXP3+ Tregs.
CD8α+ DCs are lymphoid organ-specific subsets. CD103+ DCs are tissue-specific
CD103+ DCs. The cells are developed through a fms-like Tyr-kinase 3 ligand
(Flt3L), which is known as a distinct ligand of Tyr-kinase receptor [17]. In fact,
Flt3L is also known as a specific inhibitor of basic Leu zipper transcription factor
ATF-like3 (Batf3) [18], transcription factor IFN-regulatory factor 8 (IRF8), and
DNA-binding protein 2 (ID2) [2]. The CD8α+ DCs are involved in peripheral and
central tolerances through TGFβ production. This tolerance is nonresponsive to
peripheral tissue-associated SAMPs by direct recognition to self-responsive T cells
that induce apoptosis. The tolerance of CD8α+DCs is mostly strong when antigens
recognize CD205 known as DEC205. CD205 promotes clonal deletion and Treg
differentiation [19]. DEC205 + DC depletion induces thymic Treg level and main-
tenance in mucous [20].
In conventional DC subset, CD11b + DCs are resident in the spleen, lymphoid
organ, and peripheral tissues, after development with the help of GM-CSF, Flt3L,
IRF2, IRF4, LTβ, Notch2, and RelB [21]. However, CD11b+ DCs are minorly
resident in the thymus. CD11b+ DCs express endothelial cell-specific adhesion
molecule (ESAM). In spleen-resident CD11b+ DCs [12], ESAM-high-expressing
DCs are generated through differentiation from DC progenitors, whereas the ESAM-
poorly expressing DCs are derived from circulating monocytes. CD8α CD11b+
DCs exhibit cross-tolerance against intestinal antigens, while CD8α+ DCs do not.
For example, antigen-tolerogenic CD11b+ DC subpopulation is specially enriched in
the specialized organs such as Peyer’s patches when type II collagen-specific oral
tolerance responses are appeared, and therefore, they inhibit development of
collagen-induced arthritis [22].
2.2.2 DCs Induce Tolerance State
Apart from APC function, DCs can also induce immune tolerance. DCs induce
central and peripheral tolerance via T-cell interaction. Tolerance induction of DCs
indicates that the loss of DCs breaks peripheral tolerance. However, constitutive DC
depletion does not induce spontaneous autoimmunity but a myeloproliferative
disease. In normal condition, DCs maintain immunological homeostasis in organism
and also promote peripheral tolerance state of T cells when T cells are presented with
foreign non-harmful antigens or self-antigens. DCs assist T-cell development to
maintain the T-cell homeostasis in the thymus, as confirmed by two-photon imaging
and live intravital microscopic analysis via DC-T-cell binding. DC-derived T-cell
homeostasis is based on signals through MHC-TCR complex recognition [23]. DCs
are also capable of presenting self-antigens to peripherally resident T cells in the
lymphoid node drained.
Peripheral tolerance is essential due to the limited central tolerance. Peripheral
tolerance potentiates to avoid “horror autotoxicus” [24]. DC-induced T-cell toler-
ance is acquired by immune checkpoints of CTLA-4 and PD-1 specifically
expressed on CD8+ T cells, and also by Treg induction. DC self-antigens induce
CD4+ T-cell tolerance, as derived from the interaction between DC PD-L1 and T-cell
PD-1 as well as antigen-specific peripheral iTregs. Constitutive DC ablation
enhances autoimmunity upon self-antigen immunization [25]. Enhanced production
level of Flt3L in blood plasma is essential for development of the myeloid prolifer-
ative disease because the DCs are absent. The similar examples are observed in
patients defected with hereditary monocytes or DC deficiency in humans [26]. How-
ever, constitutively depleted DCs in lupus-prone MRL/lpr mice exhibit the improved
level of autoimmune response. DCs expand and differentiate T cells. DCs maintain
homeostasis of peripheral-resident T-cell populations via prevention of unwanted
activation of T cells. DCs rather induce induced type Tregs known as iTregs
[8]. Treg proliferation depends on the DCs [27]. Treg depletion accelerates DC
maturation and expansion, depending on Flt3. Lamina propria CD103+ DC subset
expands when the cells are interacted with Flt3L [27]. CD103+ DCs are the DC
subtype resident in peripheral tissues and also corresponded to CD8+ DC type
present in the lymphatic nodes or splenic tissues. Binding of Flt3 to Flt3L influences
the functions of Treg cells and CD8α+-CD103+ DCs. DCs differentiate and maintain
various types of Tregs. For example, IL-10-expressing T regulatory-1 (Tr1), T
helper-3 (TGFβ-expressing Th3), thymic-generated Tregs named nTregs and
periphery-differentiated Tregs named iTregs, and Foxp3+ T-cell subsets are affected
by DCs toward their differentiation. DCs can also induce tolerance of peripheral T
cells, and DCs generate antigen-specific peripheral iTregs by PD-L1-PD-1 binding.
2.2.3 DC co-Stimulatory Receptors
2.2.3.1 CD80/CD86
DCs express surfaced CD80/CD86. All T cells including Tregs express CD28. T-cell
CD28 binds to DCs CD80/CD86 to develop and maintain thymic and peripheral
Tregs. DC CD80/CD86 enhances Treg proliferation [28]. The DC CD80/86 signal-
ing does not affect the development of nTregs, which are derived from the thymus.
However, DC CD80/86 induces development of iTreg subsets in peripheral tissues.
2.2.3.2 CD70
DCs and mTECs express CD70, a TNF family, whereas CD70’s receptor is CD27 on
developing thymocytes. CD70-CD27 binding develops thymic-derived nTregs. In
the thymus, CD70-expressing CD8α+ DCs contribute to development of nTreg cell
population. In addition, CD70-CD27 binding positively transduces the nTreg selec-
tion and leads to prevention of apoptotic cell death [29]. Peripheral CD70 contributes
to Th1 differentiation, while it suppresses Th17 differentiation, reducing autoimmu-
nity [30]. In contrast, CD70 overexpression does not differentiate Th17 without any
effect on Treg development.
2.2.3.3 ICOS-L, PD Ligands (PD-L), IL-10, IOL-27, TGFβ, Retinoic

Acid, and β-Catenin
The cell surface protein ligands include co-stimulatory B7 family members and bind
to receptors on lymphocytes toward immune response regulation. Among them, the
inducible co-stimulator (ICOS) ligand (ICOS-L) is mainly present in APCs like B
cells, DCs, and macrophages. Moreover, the ICOS-L is also expressed from
nonimmune cells including lung epithelium, endothelium, and tumor microenviron-
ment cells. ICOS as a co-regulatory receptor of T cells provides a co-stimulatory
signal to T cells during antigen-mediated activation. ICOS is a rapidly induced
co-stimulator upon T-cell receptor cross-linking. Follicular lymphoma cells generate
Treg cells via ICOS/ICOS-L pathway, applicable to treatment by anti-ICOS/ICOS-L
therapy [31]. ICOS molecule is found in the T-cell subsets activated including CD8+
and CD4+ T cells and, also, effector T cells including CD4+ T follicular helper cells
(Tfh). ICOS-targeting therapy improves antitumor immunity. ICOS signaling acti-
vates the effector T cells of CD4+Foxp3 T cells upon tumor immune responses.
ICOS-L transfection of tumor cells enhances antitumor immunity when cells are
vaccinated with anti-CTLA-4 treatment [32]. ICOS-L also promotes antitumor
immune responses [33]. ICOS promotion of immunosuppressive Tregs may impair
tumor immunity [34].
Tregs specifically express the transcription factor Foxp3 [35]. Natural and induc-
ible Tregs express CD25, glucocorticoid-induced TNFR-related protein (GITR),
CD45RO, and CTLA-4, but lack CD127 [35]. Tregs suppress effector T cells
(Teffs) to prevent autoimmune diseases, allergies, infection-induced organ damage,
as well as transplant rejection. ICOS-L activates memory and effector T cells upon
humoral immune reaction. ICOS expression is increased in rejected allografts
[36]. In a negative viewpoint, Tregs are harmful in cancer due to its suppression of
antitumor immunity. Tregs actively accumulate in tumor microenvironments with
poor antitumor immune response and poor survival. The tumor-associated microen-
vironment (TAM) favors the phenotype conversion from CD4 + CD25-T-cell sub-
sets to inducible subsets of Tregs. ICOS protein belongs to a CD28 class, which is a
co-stimulatory protein, and maintains durable immune reactions upon binding to
ICOS-L. ICOS/ICOS-L axis promotes Treg differentiation. Normal tissues express
ICOS-L and regulate CD4+ T-cell activation and cytokine production [37]. ICOS+
Tregs dampen T-cell responses via impairing APC with IL-10. ICOS blockade
upregulates activated and pathogenic T cells. Certain cancers stimulate ICOS-L to
develop immunosuppressive CD4+ T-cell population like Tregs. Thus, tumor pro-
gression and survival require ICOS-L expression. During anticancer vaccination or
anti-CTLA-4 treatment, ICOS+ T cells exhibit the enhanced CD4+ and CD8+ subset
levels, increasing the Teffs/Tregs ratio in tumor microenvironment. Hence, ICOS/
ICOS-L binding improves cancer therapy effect.
In airway asthma, ICOS-L-expressing semi-mature DCs induce TGFβ-expressing
and antigen-specific iTregs [38]. pDC induces iTreg, an ICOS-L-dependent. In mice,
ICOS-L-deficient pDC cannot protect them against asthma. PD-L1-KO APCs
generate iTregs in vitro. PD-L1-KO APCs stimulate to differentiate the naive CD4+
T-cell subsets to iTreg cell subsets, even to a lesser extent [39]. PD-1 binding
stabilizes and strengthens DCs and T-cell recognition [40]. PD-1 and PD-L1 binding
inhibits TCR-mediated signaling [40]. DC treatment with soluble PD-1 blocks DC
maturation with IL-10 secretion [41]. PD-L1-expressing DCs induce antigen-
specific iTreg generation with dampened disease severity.
DCs and T cells express IL-10, a regulatory cytokine [42]. IL-10 regulates Treg
cells and inhibits APC function with anti-inflammatory activity. IL-10 inhibits
maturation of DCs. Moreover, IL-10 suppresses the levels of co-stimulatory pro-
teins, MHC-II, and chemokines of CXC and CC. In addition, IL-10 inhibits expres-
sion of proinflammatory cytokines in DCs [43]. IL-10 in human DCs increases the
levels of T-cell tolerance and T-cell anergy [44]. IL-10-stimulated DCs inhibit the
response level of effector T cells [45], protecting EAE symptoms and inhibiting
transplanted graft rejection in hosts [46]. IL-10 controls DCs to inhibit contact
hypersensitivity and anti-Leishmania immune response [47]. IL-10 modulation of
CD11c+ APCs maintains intestinal immune homeostasis [48]. DC IL-10 expression
is important for T-cell anergy and suppression. IL-10-expressing matured pulmonary
DCs induce tolerance event through Tr1 cells. BM-derived DCs are transmitted to
semi-matured types of DCs when GM-CSF, TNF-α, and IL-10 are present. Those
DCs trigger to differentiate suppressive T cells, which express IL-10. Dermatic DCs
known as Langerhans cells inhibit IL-10-mediated contact hypersensitivity event
and Tr1 cell differentiation [49]. DCs co-cultured with Tregs secrete TGFβ, IL-10,
and IL-27, and also generate Tr1 cells. DC IL-27 inhibits the IL-23 and IL-1β
expression but activates IL-10 expression. Hence, differentiation into more immu-
nogenic Th17 cell type and the resulting autoimmune potentials are terminated
[50]. IL-27 induces c-Maf, ICOS, and IL-21 expression in naive T cells, to collab-
oratively concert to Tr1 cells [51]. Human DC stimulation with IL-27 increases the
level of PD-L1 surface expression, without DC maturation [52]. Stimulation of DCs
with IL-27 increases CD39 and suppresses the inflammasome pathway [53]. A
regulatory and pleiotropic cytokine TGFβ is effective on T cells and on APCs.
TGFβ stimulates conversion of naive T cells, which are peripherally resident to
CD4 + CD25+ Treg cell type through Foxp3 gene expression. During treatment with
LPS, splenic DCs secrete highly TGFβ to differentiate Tr1 cells. DCs induce extra-
thymic iTreg differentiation by TGFβ assistance [8]. T-cell-specific TGFβ signaling
inhibition using a dominant-negative TGFβRII terminates differentiation of iTreg
cells [18]. The integrin-α4β8 activates TGFβ by metalloproteinase degradation of
latency-associated protein (LAP) and extracellular TGFβ release [54].
DC-produced retinoic acid (RA) induces oral tolerance by iTregs. Mucosal DCs
guide T-cell homing to the gut by DC-derived RA. DC-produced RA inhibits
TGFβ-dependent Th17 cell production and also activates Foxp3+ Treg cell differ-
entiation. RA activates iTreg differentiation by inhibition of effector memory T-cell
generation, which expresses IFN-γ or IL-21 [55]. RA-forming enzymes depended on
β-catenin known as the key canonical protein in the Wnt signaling (Wingless Int)
[56], constitutively expressed in DCs. β-Catenin regulates BM-DC maturation.
Blocking of β-catenin interaction with E-cadherin of BM-DCs increases the
expression levels of two major surfaced co-stimulatory molecules and MHC-II but
not proinflammatory cytokines, providing tolerant DCs with IL-10-expressing
Tregs. CD11c-specific deficiency of β-catenin is sensitive to colitis and Th1-/
Th17-associated EAE and prevents Foxp3+ Treg responses [57]. Wnt/β-catenin
signaling suppresses tumor-raised immunity through inhibition of CD8 T-cell-medi-
ated DC priming with IL-10 [58].
2.2.3.4 Indoleamine-2,3-Dioxygenase
A specific catabolic enzyme of a Trp catabolism, known as indoleamine-2,3-

dioxygenase (IDO), catabolizes Trp residue to its metabolite kynurenine, which
induces cell starvation caused by tryptophan loss. In addition, kynurenine activates
the general control non-derepressible 2 (GCN2) kinase. The GCN2 kinase specifi-
cally phosphorylates the eukaryotic initiation factor 2 (eIF2), and the activated
pathway generates Tregs and expands to IDO-rich condition. Kynurenine binding
to aryl hydrocarbon receptor (AhR) present in CD4+ T-cell populations inactivates
the AhR receptor. This induces immunosuppressive T cells and allows Tregs
[59]. AhR also increases IDO expression and RA-synthesizing enzymes directly
on the DCs. DC-produced IDO induces tolerance by iTreg generation. iTregs can
induce the IDO synthesis in pDC [60]. IDO expression differs between DC subsets.
CD8α+ DCs produce highly IDO, although CD8α DCs do not. CD103+ DCs
present in intestines also produce highly IDO enzyme and maintain gut homeostasis
and oral tolerance [61]. DC CD80/86 binding to CTLA-4 induces IDO expression.
2.2.4 Application of DCs to Human Diseases
DCs are candidate cells to treat human diseases including antitumor immunity and
tolerance in transplantation and autoimmunity. Tolerogenic DCs can be obtained by
deletion of co-stimulatory receptors. B7-H1 (PD-L1) deletion generates tolerogenic
DCs [62], although it is not possible in humans. Cytokine cocktails containing IL-10
or TNFα are alternative to overcome. Another overcoming strategy is generating
DCs that induce tolerance in several types of human autoimmune disorders like
graft-versus-host disease (GvHd) and collagen-type II-induced arthritic diseases
(CIA). Blocking of co-stimulatory protein expression or IDO activation or TGFβ
expression in DCs is suggested. Tolerance DCs from the patients can be acquired
using cytokine cocktails [63]. The clinical trials with DCs are limited, as boost
immunity in cancer therapy is performed currently. However, tolerogenic DCs are
not used to treat autoimmunity [64].
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Chapter 3
Glycan Biosynthesis in Eukaryotes
For three billion years, terrestrial life has been evolved to adapt to environmental
changes with energy production, reproduction, and cellular signal transduction. Each
organism has mainly used each DNA genetic code and RNA diversity, structure-
based functional proteins, lipid-based membranes, and metabolites. Using such
nucleic acid-protein-lipid axis, to some extent, they have acquired their survivals
to share with diversity, allowing terminology of evolution. However, the axis is too
limited to cover all the diversity in organisms, in addition toward the future evolution
probabilities. For organisms to protect themselves from pathogenic infections, self-
hyperreactivity, and intellectual differentiation, each organism has acquired diverse
cell surface glycans which are essential. The diversity or escape process from
pathogens is distinct from genetic code due to importance in organism survival.
Hence, any concept to explain the future unidirectional evolution is required in the
biotic and abiotic environments. Then, the appropriate field has been raised from the
dawn to create a link between water and hydrophilic environments, terming of
“glycans” as molecules and “glycobiology” as subject.
For the basic background of the steady-state investigation on the glycans, the
functional importance and structural diversity are the most well-recognized facts.
Glycans such as N-, O-glycans, GSLs, GAGs, GPI anchors, sialic acids, and
cytoplasmic and nuclear glycans are particularly characteristics of eukaryotes. In
the biosynthesis of glycans, template is not needed because such equivalent is not
utilized for the design of glycans, as this is contrast to the DNA biosynthesis. It is
reminded that DNA generates the template for the protein. The biosynthesis of
glycans consisted of three distinct steps. In the first step, sugar nucleotides are
generated in the cytoplasm and supplied. In the next second step, the sugar nucle-
otides are trafficking to organelle ER or Golgi apparatus by each specific transporter
located in the membranes. In the third step, each specific glycosyltransferase attaches
the sugars from each sugar nucleotide to an acceptor protein substrate or glycan
substrate in the ER and Golgi apparatus, toward Golgi trafficking. In eukaryotes,
subcellular organelle ER-Golgi networks create evolutionary glycan diversity and
cell surface glycans. In addition, glycan structures are diverse in different organisms.
54 3 Glycan Biosynthesis in Eukaryotes
For example, various N-glycans are structurally defined in different eukaryotes

including yeast, insect, plant, and animal (Fig. 3.1).
3.1 General Glycosylation Events
Glycosylation is a post-transcriptional modification with over 50% of total proteins

and ubiquitous process in plasma membrane glycolipids. Most cellular interactions
are mediated by glycosylation, and disease development involves changes in glycan
biosynthesis. All the N-glycans carry an identical and common saccharide linkage
generated from the same biosynthesis pathway that normally terminates in the late
Golgi step. O-glycosylation also provides diverse linkages with diverse structures in
protein glycans. N-glycosylation and O-glycosylation of glycoproteins as well as
lipid glycosylation of sphingolipid are responsive to environmental changes and are
changed in various diseases. Alterations in O-glycosylation ad N-glycosylation are
caused by changed expression levels of biosynthetic enzymes or dysfunctional
biosynthetic pathway of the ER-Golgi network. Glycoproteins only carry glycans
through three categories of N-linked (Asn), O-linked (Ser, Thr, hydroxy Lys), and
C-linked (carboxyl group of Trp). The glycosylation of O-glycans, N-glycans, and
glycosphingolipids (GSLs) is performed by the multiple machineries such as
endosomal ER and Golgi complexes. Thereafter, the synthesized glycans are local-
ized on their locations in plasma membrane or each destination (Fig. 3.2). Glyco-
sylation event occurs only to proteins in the ER-Golgi, lysosome, plasma membrane,
and extracellular protein-trafficking pathway. However, the exceptional cases are for
the cytosolic and nuclear proteins which a single O-linked GlcNAc residue is
attached. Ribosome-mediated translated proteins are sorted through the
N-glycosylation secretion way in rough ER (rER) with the N-terminal ER-specific
signal region. ER-processed proteins which are normally folded are trafficking to the
Golgi complex through transport vesicles. The defect in innate immune functions
Glucose(Glc) Galactose (Gal) Fucose (Fuc)
N-Acetyl glucosamine N-Acetyl galactosamine Mannose

(GlcNAc) (GalNAc) (Man)
Xylose Glucuronic acid Sialic (Sia) or

(Xyl) (GlcA) N-Acetyl neuraminic
acid (NeuAc) Plant Animal
Yeast Insect
A) Monosaccahrides
B) Different N-glycan structures in different organisms
Fig. 3.1 Monosaccharides and N-glycan structures in various eukaryotic organisms

3.1 General Glycosylation Events 55
A) Scheme of N- and O-glycan biosynthesis
B) Core structures of glycans

Processing enzymes
-Ser/- -Ser/- )CN )NE

Golgi /CP Thr Thr
Cer
)CN
LacCer
Flip
)NE0#E )CN0#E
GalNAc-T
-Asn- -Ser/- -Ser/-
Thr Thr
N-glycans O-glycans GSLs

ER
Ceramide Asn protein
Ser/-Thr protein Flip : flippase
C) ER/Golgi pathway of glycan synthesis
N-Glycans O-Glycans GSLs
Rough
ER
Golgi
Secretory Granule
Plasma membrane
Fig. 3.2 Mammalian glycan biosynthetic pathways. (a) Scheme of N- and O-glycan biosynthesis.
(b) Core glycan structures. (c) ER-Golgi pathway of glycan synthesis. Synthesis of three major
glycans in the ER-Golgi, and subsequent modification in the lumen. Genes or glycosylation
enzymes are well defined
does not primarily come from one of the glycantransferases or processing enzymes
such as hydrolases but is likewise originated from the sugar nucleotides formation,
transports to the ER-Golgi trafficking.
Each sugar residue has at least three or four recognition sites for glycosidic
linkage with adjacent monosaccharide residues. In addition, the resulting anomeric
α- or β-configuration is generated in each glycosidic linkage to yield each specific
branch in glycan structures. The synthesized glycans consequently exhibit structural

diversity compared to other cellular constituents. These linear chains of polymers are
only types of the simple linkages formed by peptide or nucleotide bonds. Theoret-
ically, the naturally occurring 9 monosaccharides in humans can link into more than
15 million possible tetrasaccharides. Even simple glycans can have many varieties of
forms [1]. Evolution process, therefore, exhibits inter-species and intra-species
differences, molecular similarity, structural mimicry, glycosylation adaptations of
invasive agents, each specificity of glycosyltransferase, environmental acquisition,
and natural coevolution. Differences in glycosylation pattern are found in all
eukaryota including arthropoda, deuterostomia, fungi, nematoda, and viridiplantae
[2]. Currently, the eukaryotic biosynthesis of glycans is well established, as the
representative synthetic pathways of glycans are described.
3.2 Sugar Nucleotide Transporters Deliver Donor

Saccharides to ER-Golgi Network
For supplying of sugar nucleotides required for donor substrates, monosaccharides

used for the biosynthesis are supplied from the so-called high-energy form of
nucleotide sugars. The sugar nucleotides are derived from dietary sugar sources
and salvage pathways for the formation. Monosaccharides of Fru and Glc residues
are present as the main sugar sources, and therefore, all other monosaccharides can
be converted from the two basic hexoses. Continuous enzymatic pathway including
phosphorylation, acetylation, and epimerization generates various nucleotide sugar
donors through conversion reaction. Nucleotide sugar is biosynthesized in the
cytoplasmic space. However, only CMP-SA or CMP-NeuAc is generated in the
nuclear region, indicating that synthesis of sugar nucleotides is tightly controlled
[3]. For instance, UDP-GlcNAc, an abundant form in cytosol, inhibits the enzyme
activity of Gln-Fru-6-phosphate transaminase required for the first step of synthesis
of UDP-GlcNAc in the enzyme reaction [3]. In addition, CMP-SA or CMP-NeuAc
blocks enzyme activity of the GNE/MNK known as the UDP-GlcNAc-2-epimerase
(GNE)/ManNAc kinase (MNK). The enzyme GNE/MNK is the basic two enzymes
for the CMP-SA or CMP-NeuAc biosynthesis [4]. Biosynthesis of nucleotide sugars
requires ATP, and tightly regulated synthesis of sugar nucleotides indicates that the
altered nucleotide sugar impairs cellular pathway for glycosylation.
The sugar nucleotides are generated in the cytosolic region, after monosaccha-
rides are delivered to the ER lumen side and/or Golgi apparatus. From the fact that
sugar nucleotides cannot transmigrate across the lipid bilayer of membrane, it is
suggested that Man and Glc residues transmigrate across the membrane by interac-
tion with the dolichol phosphate (Dol-P) as its specific lipid carrier. The cytoplasmic
Dol-P-Man/Dol-P-Glc synthase enzymes recognize each substrate GDP-Man or
UDP-Glc for linking the Dol-P present in the cytosolic site. The “flippase” enzyme
catalyzes the transport of the Dol-P-monosaccharides from the cytosolic side to ER
3.2 Sugar Nucleotide Transporters Deliver Donor Saccharides to ER-Golgi Network 57
luminal leaflet side. Then, the monosaccharides are ready to be utilized by various
ER-resident glycosyltransferases (GTs) [5]. The nucleotide sugar transport is spe-
cifically mediated by nucleotide sugar transporters. The nucleotide sugars can cross
the ER membranes and also Golgi membrane by means of the nucleotide sugar
transporters (NSTs) embedded on the ER and Golgi membranes. They are indeed
antiporters. Hence, nucleotide sugars enter into the ER and/or Golgi system from
cytosol, and this entry event is coupled to exit of each nucleoside monophosphate to
cytosols at the level of equimolecular level from the lumen of ER and Golgi complex
[6]. Upon the ER-Golgi lumen transportation of sugar nucleotides, each
glycosyltransferase transfers the monosaccharide residue to each glycan substrate.
Then, to be equilibrium status in the lumen, the nucleoside diphosphates are again
subjected to the molecular conversion to di-anionic nucleoside monophosphates that
are utilized by a nucleoside diphosphatase for the antiporter and inorganic phos-
phate. In the topology, several transporters including UDP-Gal transporter,
UDP-GalNAc transporter [7], UDP-glucuronic acid (GlcA) transporter,
UDP-GalNAc transporter, UDP-GlcNAc transporter [8], and UDP-Xyl transporter
[9] are multiple enzymes with two more substrate specificities. In contrast, the
CMP-NeuAc transporter [10] and GDP-Fuc transporter [3] are a mono-type enzyme
with a single specificity. Interestingly, many NST transporters such as
UDP-GlcNAc, UDP-Fuc, GDP-Xyl, and CMP-SA/NeuAc transporters are reported
to be strictly localized on Golgi membrane [6, 9, 11], while the specific transporter
for UDP-GlcA is localized in the membrane of ER [8]. UDP-Glc and UDP-Xyl are
resident in the ER region, but UDP-Glc and UDP-GlcA are detected in the Golgi
apparatus [6].
A special GSL, galactosylceramide (Gal-Cer) is synthesized by UDP-Gal-Cer
Gal-transferase present in the ER region of certain cells of myelinating cells,
spermatogenic cells, and epithelial cells. However, UDP-Gal transporter as an
NST is located in Golgi apparatus. The different localization of the two Cer-Gal-
transferase and UDP-Gal transporter is surely unmatched issue to fully agree with
the substrate and glycosyltransferase relationship. For this inconsistency, several
studies have clearly concluded to settle down the discrepancy [11, 12]. First,
ER-resident galactosyltransferase is associated with the UDP-Gal transporter
(UGT) during ER-Golgi networks, and the ER-Golgi boundary is suggested to be
flexible to fuse together [11]. Second, ER and Golgi colocalization of the UDP-Gal
transporter is also explained by RNA variants produced via its alternative spicing
with two splice forms of UGT1 and UGT2. UGT1 is suggested to be a Golgi type
and UGT2 for dual ER and Golgi type operated by a C-terminal di-lysine motif
(KVKGS) [12].
GlcNAc-phosphotransferase selectively catalyzes phosphorylation reaction of the
N-glycoproteins to move to the lysosome region [13]. Golgi-localized transferases
recognize only one monosaccharide residue, a saccharide sequence and target
peptides. For example, α2,6-sialyltransferase, ST6Gal I, binds to the terminal
LacNAc to form a linkage of the SAα2,6-LacNAc structures present in N-glycan/
O-glycan of glycoproteins and GSLs; however, the β1,4-galactosyltransferase
A) Major classes of N/O-glycans on different membrane proteins

N-Glycans O-Glycans
GSLs
High Hybrid Complex N/O Core 1 Core 2 Core 3 Core 4 GM3 GM2 GM1 GD3 GD2
mannose type
B) N/O glycans drawn on a membrane protein and GSLs 1)N[ECPU

0)N[ECPU %QTG
56
*KIJ %QTG
%QORNGZ /CPPQUGV[RG
V[RG 56
*[DTKF
V[RG
%QTG
56
0*
)CN 56 %QTG
#UP 0* )5.U
)NE 0*
#UP
/CP #UP )/ )&C )6D
)/
)CN0#E )/
)NE0#E )N[EQRTQVGKP
0GW#E
(WE
Fig. 3.3 Structures of representative O-/N-glycoproteins and glycolipids. Representative protein

N-glycosylation (complex, high-mannose, and hybrid type), mucin-type O-glycosylation, and
gangliosides on mammals are described. (a) Each specific structure drawn on each carrier. (b)
N-/O-glycans drawn on a membrane protein
(Gal-T1) catalyzes the galactosylation reaction to any terminal GlcNAc residue.

Structures of cell surface glycoproteins and glycolipids are illustrated in Fig. 3.3.
3.3 Golgi Traffic
The Golgi traffic models are not unified and differently explained by Golgi
researchers. Thus, the consensus model indicates the complexity. The Golgi com-
partments include cis, medial, and trans cisternae plus the trans-Golgi network
(TGN). For more details, the Golgi apparatus contains cisternae structures, ranged
between the nucleus and cis-Golgi system, including cis-compartment, medial-
compartment, and trans-compartment of Golgi network. All the glycosylation events
3.3 Golgi Traffic 59
are ended with the trans-Golgi system. The Golgi apparatus is kept by the cytoskel-
etal matrix with microtubules of actin-spectrin network and intermediate filaments.
They are different from their structures, Golgi-localizing enzymes, and COPI- or
clathrin-mediated vesicle-forming capacity. The filaments and Golgi membranes are
linked through membrane proteins or mechanochemical proteins including dynamin,
dynein, kinesin, and myosin [14]. The protein transport from ER to each different
Golgi compartment is started from the hundreds of releasing sites located in the ER
vesicles coated with COPII. In order to incorporate COPII vesicles, coatamer pro-
teins, termed COPs, binds to transporter molecule domain of the cytosolic tail region
in the cargo membranes. The ER export signaling proteins belong to type I mem-
brane protein family, which contains the peptide motifs containing diacidic amino
acids or di-hydrophobic amino acids. However, GTs are type II transmembrane
(TM) proteins and contain proximal amino acids of RK(X)RK sequence near in the
TM domain [15]. Soluble proteins are passively or actively exported from the ER
[16]. Golgi glycosyltransferases are polarly distributed. In species of mammals to
plants, glycosylation-beginning transferases are accumulated in cis-cisternae,
whereas late-acting glycosyltransferases are accumulated in trans-cisternae [17].
Golgi structure varies between organisms. COPI vesicles are associated with
Golgi structures in eukaryotes. COPI vesicle is an intra-Golgi carrier. Mammalian
Golgi is surrounded by thousand more COPI vesicles. COPI is conserved. COPII-
coated vesicles are fused to the specific vesicle, named ER-Golgi intermediate
compartment (ERGIC) complex. COPI vesicles are particularly important for retro-
grade traffic of the vesicles, in way of COPI vesicles in protein recycling from Golgi
complex to ER side. The ER proteins escaped or proteins misfolded are reversely
retrotransported to the ER region through vesicles coated with COPI. The cis-Golgi
system is again fused with ERGICs. Several proteins enhance smooth transports of
vesicles. ARF and Sar1 GTPases stimulate the formation of COPI and COPII
vesicles, where Sar1-GTP and ARF-GTP proteins associate with the recruited
vesicle coat proteins. Consequently, the GTPases of Rab family selectively target
vesicles. The remaining protein, SNARE, leads to fused vesicle formation, where
vesicles hold vesicle-SNARE, termed v-SNARE, for specific recognition of
tethering-SNARE form, termed t-SNARE, and the fused cargo vesicles are moved
to specific compartments [18, 19]. From the sequential glycosyltransferase catalysis,
the Golgi enzymes are particularly interested in their roles. Glycosyltransferases are
sequentially arranged in the Golgi apparatus for their complete synthetic pathway,
while early catalyzing enzymes are present in the cis-Golgi complex and intermedi-
ately catalyzing enzyme groups are found in medial Golgi complex. The
end-catalyzing enzymes are present in the trans-Golgi complex. In addition, the
glycosyltransferases present in a certain Golgi compartment are structurally
complexed to allow the steady-state location of the Golgi-resident enzymes [20].
3.4 N-glycan Synthesis
The N-glycosylation event is conserved through eukaryotes. N-glycan structures are

heterogeneously generated in the pathway and classified for their backbones to three
major types of (A) high-Man, (B) hybrid, and (C) complex. N-glycan attachment to
candidate Asn residues is carried out through the commonly observed sequence,
triamino acid sequon that is the Asn-X-Ser/Thr. “X” means any other amino acid,
barring Pro residue. The N-glycans are commonly featured with the core of Manα1,6
(Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAcβ-1-Asn-X-Ser/Thr- with two independent
antennae. The Asn-linked core structure is further processed and trimmed to three
distinct groups of N-glycans. The three groups consist of oligomannose type, which
contains only Man residues, complex type having sialyl LacNAc on each antenna,
and a Fuc residue in the Asn-linked GlcNAc. Hybrid type has a combined oligo-Man
type and complex type has Manα1,6 and Manα1,3 antennae. The precursor glycan is
serially attached to a dolichol pyrophosphate known as lipid carrier in the ER
cytoplasmic face. The first monosaccharide of GlcNAc residue is attached to
dolichol phosphate, where a phosphate group is transferred and the GlcNAc residue
is linked to yield dolichyl pyrophosphate-GlcNAc. The formed dolichyl
pyrophosphate-GlcNAc is then further used to generate dolichyl pyrophosphate-
GlcNAc2Man5. This substrate is translocated to cross the ER lumenal membrane
surface by a specific enzyme flippase. Thereafter, Man residues are serially attached,
and the Manα1,3 antenna is attached by a triglucosyl Glcα1–2Glcα1–3Glcα1–3-
cluster. In this stage, dolichol-P-Man and dolichol-P-Glc are used to be used as
donor substrates. The final dolichol-linked glycan is Glc3Man9GlcNAc2-. Using
this dolichol-linked glycan, oligosaccharyltransferase (OST) in the ER lumen
attaches it to Asn residues on target proteins, and the resulting β-N-glycosidic-linked
proteins are yielded [21]. This glycan is further subjected to trimming and processing
to mature N-glycans. All fungi, metazoans, and plants use the common biosynthetic
pathway in the ER lumen, including the trimming and processing of the sugar
structures. The ER chaperon in the pathway contributes to precise folding and
quality control of glycoproteins, processing by calnexin (CNX) and calreticulin
(CRT). During the quality control (QC) of glycoproteins, the triglucosyl residues
monitor the level by α-glucosidases I and II. Therefore, the incomplete structure of
folded proteins is α-glucosylated by a specific α-glucotransferase to transfer glucose
residue to the terminal α-1-2 Man on the α-1,3 mannosyl antenna and further cycled
via the calnexin/calreticulin interaction. If still unfolded, glycoproteins fall into the
ER-associated degradation (ERAD) machinery to eliminate them through secretory
proteasome to ubiquitination in the ER cytosols. Indeed, the N-glycan-bearing
glycoproteins disappeared during the ubiquitination, and the remained protein part
is digested in proteasomes [22, 23]. Precisely folded glycoproteins are further
glycosylated with trimming and finally sialylation. Finally, the three groups of
N-glycans are produced in Golgi apparatus via ER-Golgi network. Among them,
the Man-6-phosphate (Man-6-P)-carrying N-glycans recognize the Man-6-P
receptor that are localized on membranes of endosomes and lysosomes. Then, the
3.5 O-glycosylation and Multiple O-Glycan Structures 61
Man-6-P-glycoproteins are translocated to lysosomes. Each form of peripheral

N-glycan is different from each phylum in plants and invertebrates to mammals
[22, 23].
3.5 O-glycosylation and Multiple O-Glycan Structures
Monosaccharides of GalNAc, GlcNAc, glucose, galactose, mannose, xylose, fucose,

and arabinose are subjected to the O-glycosidic attachment to Ser/Thr of proteins.
The well-known reaction is the monosaccharide GalNAc transfer from the
UDP-GalNAc to Ser/Thr through an α-glycosidic linkage, and this is catalyzed by
GalNAc transferase. The O-glycan synthesis commences with the protein folding
process and subunit oligomerization of proteins between the ER and Golgi compart-
ments. O-glycosylation process is sequentially progressed with the pathway assem-
bled by membrane-bound glycosyl-, O-acetyl-, and sulfotransferases. The
O-glycosylation pathway is distinct for GTs, sulfotransferases, and
O-acetyltransferase, and O-glycan biosynthetic transferases are mainly localized in
the Golgi. Although they similarly catalyze, sequence homology is not high between
them. All Golgi GTs belong to the type II TM proteins and consist of a membrane-
spanning domain region, a large catalytic domain in C-terminal region and a short
cytosolic domain region in N-terminal region of the Golgi lumen. The β-Gal α2,3-
SA-transferase 1 (ST3Gal-1) catalyzes the sialylation reaction to the core
1 O-glycosylation structures. Core 1 of O-glycosylation structures is further
converted to core 2 mucin type of O-glycan structures through addition of GlcNAc
residue by a specific enzyme, core 2 β1,6 N-GlcNAc-transferase (C2GnT). The
C2GnT enzyme transfers a β1,6-GlcNAc residue using the donor substrate
UDP-GlcNAc to the acceptor substrate, core 1 structure. These core 2 O-glycans
have merits for further glycosylation and extension as the substrate, which has a
poly-lactosamine (LacNAc) sequence. In addition, core 1 β3-Gal-transferase (core
1 β3Gal-T) enzyme synthesizes core 1 mucin-type (and also core 2 type) O-glycan
structures, by means of a distinct chaperone named core 1 β3GalT-specific molecular
chaperone (COSMC). The COSMC as an ER-resident enzyme, which is also a
specific type II transmembrane protein, is required for the core 1 β3Gal-T enzyme
folding. Therefore, if the COSMC is not present in the ER, the core 1 β3Gal-T
enzyme is proteolyzed through the proteasome [24].
3.5.1 7 Core O-glycan Structures
The seven distinct O-linked glycans are described (Table 3.1), as subclassified
through the first monosaccharide residue linked to Ser/Thr residues or hydroxyl
Lys (hLys) residue of O-glycoproteins. O-glycosylation is in other word termed
“mucin-type glycosylation” through GalNAc α-linkage formation attached to the
Table 3.1 O-glycan types found in humans and different mucin-type O-glycans
Types Glycan linkage
(A) O-glycan types
Mucin-type GalNAcα1-Ser/Thr
GAG GlcAβ1,3Galβ1,3Galβ1,4Xylβ-1-Ser on proteoglycans
O-GlcNAc GlcNAcβ-1-Ser/Thr on nucleus and cytosol proteins
O-Gal Glcα1,2Galβ-1-O-Lys on collagens
O-Man NeuAcα2,3Galβ1,4GlcNAcβ1,2Manα-1-Ser/Thr on muscular
α-Dystroglycan
O-Glc Xylα1,3Xylα1,3Glcβ-1-Ser on EGF protein domains
O-Fuc NeuAcα2,6Galα1,4GlcNAcβ1,3Fucα-1-Ser/Thr on EGF domains
Glcβ1,3Fucα-1-Ser/Thr on thrombospondin (TSR) repeats
(B) Mucin-type
O-glycans
Core-1 Galβ1,3GalNAcα1-Ser/Thr
Core-2 Galβ1,3(GlcNAcβ1,6)GalNAcα1-Ser/Thr in blood cells types
Core-3 GlcNAcβ1,3GalNAcα1-Ser/Thr in colon and salivary tissue
Core-4 GlcNAcβ1,3(GlcNAcβ1,6)GalNAcα1-Ser/Thr
Core-5 GalNAcα1,3GalNAcα1-Ser/Thr
Core-6 GlcNAcβ1,6GalNAcα1-Ser/Thr in ovarian tissue
Core-7 GlcNAcα1,6GalNAcα1-Ser/Thr
Core-8 Galα1,3GalNAcα1-Ser/Thr in bronchial tissue
hydroxyl (OH)-group attached to Ser/Thr residues. The first GalNAc residue is the
first transferred sugar catalyzed by GalNAc-transferase and is thus an initiating sugar
of mucin-type O-glycans [24]. The mucins belonged to O-glycoproteins with vari-
able number of tandem repeats (VNTR), which are rich in multiple Ser-Thr-Pro-
containing domains as the O-glycan substitution sites. The O-glycosylation as a
mucin-type occurs at mucosal surfaces and protects the mucosal surfaces from
infection [2]. The mucin-type O-glycan is the most common type having a GalNAc
residue at the reducing end. Currently, eight mucin-type core structures are found
with the different saccharide at the second position and saccharide links. In humans,
the core 1 to core 6 and core 8 O-glycans are known (Table 3.1) [25, 26]. For
O-glycan consensus sites, there is the consensus amino acid region, which is the
recognition and attachment sites of the first saccharide in most O-glycoproteins, but
not known for O-GlcNAcylation and mucin-type O-glycosylation. However, spe-
cific glycosylation sites for O-Glc glycosylation and O-Fuc glycosylation have been
suggested for putative consensus sites [27, 28]. In addition, statistic analysis of the
known O-GlcNAcylated proteins and mucin-type O-glycans indicated each specific
role for each type of glycosylation. The prediction algorithms for the
O-GlcNAcylations and mucin-type O-glycans suggest a ruling mechanism. For
example, the NetOglyc 3.1 prediction program exhibits possible prediction of 76%
of the glycosyl-targeting amino acid sequences and 93% of the nonglycosylated
amino acid sequences from the known proteins [29].
3.5 O-glycosylation and Multiple O-Glycan Structures 63
Apart from mucins, some glycoproteins have only mucin-like domains, but not
the VNTR domain. The glycosylation of the mucin-like domain is absolutely the
same as the mucin-type O-glycoprotein. The mucins contain the linear core struc-
ture, named core 1 (Galβ1,3GalNAc-) and core 3 (GlcNAcβ1,3GalNAc-) structures
and also branched core 2 structure of the Galβ1,3(GlcNAcβ1,6)GalNAc- and core
4 structure of the GlcNAcβ1,3(GlcNAcβ1,6)GalNAc- sequences. Backbone units of
LacNAc types 1 and 2 (Galβ1,3GlcNAc- and Galβ1,4GlcNAc-) are further added for
length extension, respectively. The branched I and linear i antigens of
Galβ1,4GlcNAcβ1,3Galβ1,4- are also extended. Complex oligosaccharides such as
ABO and Lewis blood group structures are such extended O-glycans, and the
glycans can be further subjected to sialylation, fucosylation, and sulfation
[24, 30]. The detailed mucin-type O-glycans exhibit their diverse structures, as
described previously.
3.5.2 Modification of 7 Core O-Glycan Structures
In the seven core carbohydrates, the GalNAcα-1-Ser- or GalNAcα1-Thr-, termed

Tn-antigen, and NeuAcα2,6GalNAcα-1-Ser- or NeuAcα2,6GalNAcα-1-Thr-,
termed sialyl Tn (STn) antigenic epitopes, are known. The core carbohydrates can
be further modified by the LacNAc unit (Galβ1,4GlcNAc). The LacNAc unit is also
by a GlcNAcβ1–6 residue or repeated for the same LacNAc units, called poly-
LacNAcs. These are also linked to the AB(O)H blood group antigenic epitopes and
type 2 Lewis antigenic epitopes of LeX, sialyl LeX (SLeX), and LeY. Poly-LacNAcs
are found predominantly on core 2 structures of O-glycans. Nonreducing terminal
saccharides are GlcNAc, GalNAc, Fuc, and NeuAc (Neu-SA). Gal residue and
GlcNAc residue are also frequently sulfated at the carbon C-6 and at the C-3 or
C-6, respectively [31]. NeuAc or SA residues are often O-acetyl esterified at the
carbons of C-4, C-7, C-8, and C-9 [32]. The UDP-GalNAc to polypeptide GalNAc-
transferases, termed pp-GalNAc-transferases (EC 2.4.1.41), transfer the GalNAc
residue using the donor UDP-GalNAc through an GalNAc-α-OH-Ser/Thr residue
to produce the mucin-type O-glycosylations. Currently, the pp-GalNAc-Ts com-
prises 15 members [33, 34]. Currently, approximately 24 pp-GalNAc-Ts enzymes
are known in humans [35]. Each pp-GalNAc-Ts overlaps with different specificity
and tissue specificity [31, 34]. No consensus sequence is present in pp-GalNAc-T
enzymes, and they have their own linking sites. Ser residue and Thr residue only in
protein sequences are targeted to glycosylate, because O-glycosylation is indeed a
process after folding process. Thus, O-glycosyl attachment sites are mainly oriented
to Ser and Thr, and in certain case to Pro residues.
3.6 O-GlcNAcylation, O-Mannosylation,

O-β-Glucosylation, O-α-Fucosylation,
O-β-Glucosylation, O-β-Galactosylation,
C-Glycosylation, and C-Mannosylation
3.6.1 O-GlcNAcylation
The other five O-glycan types have one conformation in each structure. For another
type of O-glycosylation, O-β-GlcNAc as a single reside type is also linked to
hydroxyl group attached to Ser/Thr residue [35], and this type is frequently found
in cytoplasmic and nuclear proteins. Hence, it is regarded as a single GlcNAc
modification named GlcNAcylation via a β-glycosidic linkage. The frequently
occurring O-glycan synthesis is to attach a GlcNAc residue to proteins which are
resident in cytoplasm or nucleus. This posttranslational modification replaces phos-
phorylation because the process is a reversible process by O-GlcNAc transferase and
O-GlcNAcase [36]. The common Ser or Thr site for O-β-GlcNAcylation is possibly
competitive to phosphorylation event specific for the same OH-groups. The
O-phosphorylation and O-β-GlcNAc glycosylation are frequently known for nuclear
proteins. The enzymes of O-GlcNAc-transferase (OGT) and N-acetyl-D-
glucosaminidase specific for O-linkage cleavage (O-GlcNAcase) transfer and
remove the O-GlcNAc residues, which are linked to the O-GlcNAc-bound proteins,
respectively. They are conserved in all metazoans. On the other hand,
O-GlcNAcylation on the targeted amino acids linked to EGF repeats is completed
through enzymatic catalysis of an extracellularly resident O-GlcNAc transferase
enzyme [37].
3.6.2 O-Mannosylation
For minor O-glycan forms, O-mannosylation is known, and this type of glycans has
Man-αlinked Ser or Thr on proteins in the brain and muscle of metazoans
[38]. O-mannosylated glycans are minorly found in glycoproteins in the brains and
nerves as well as skeletal muscles. One representative case of the O-mannosylated
glycoproteins is α-dystroglycan in dystroproteins in extracellular matrix (ECM)
protein in the skeletal muscle [39]. Skeletal muscle glycoprotein, α-dystroglycan,
has mainly the O-mannosyl glycans. Most structures are
Neu5Acα2,3Galβ1,4GlcNAcβ1,2Manα-Ser/Thr-. This, only the
NeuAcα2,3Galβ1,4GlcNAcβ1,2Man- glycan, is reported in humans. Interestingly,
in sheep brain, the α-dystroglycan, which contains the Galβ1,4(Fucα1,3)
GlcNAcβ1,2Man-glycan, was known [40, 41]. Also, in rat brain, the
O-mannosylated glycan of HSO3-3GlcAβ1,3Galβ1,4GlcNAcβ1,2Man form was
found [41, 42]. Mammalian GlcNAc-transferase IX functions specifically to the
GlcNAcβ1,2Manα1-Ser/Thr substrates, and thus consequently the O-mannosylated
3.6 O-GlcNAcylation, O-Mannosylation, O-β-Glucosylation, O-α-Fucosylation,. . . 65
glycans with 2,6-branches are found in the brain [43], giving the diverse
O-mannosylated glycan forms in humans. However, other fucosylated chain and
GlcA-3-sulfated chain and branched chain are also related. The fly Drosophila
melanogaster O-glycan-synthetic genes contain at least two genes encoding for O-
mannosyl-transferase (POM-T1 and POM-T2). The O-mannosyl-transferase
enzymes are catalytically active when the POM-T1 and POM-T2 genes, which
encode mannosyltransferases, are co-expressed [27].
3.6.3 O-β-Glucosylation
In addition, for the rare types, two different glycan types of O-glucosylation and
O-fucosylation are present in the EGF-like homology domains (EGF domains). The
EGF-repeated domains are attached by O-Fuc, O-Glc, or O-GlcNAc. Extracellular
domain glycosylation contains up to 36 tandem EGF repeats, and they regulate
Notch signaling pathway. An EGF domain is often a motif found to involve in
interaction between protein and protein. The EGF domain repeats contains approx-
imately 30–40 amino acids in length with the conserved 6 Cys residues and 3 S-S
bonds. Glc residue is linked to the OH-group in Ser residue of the Cys1-Xaa-Ser-
Xaa-P-Cys2, which is the common consensus sequence [27]. O-Glc residue is
further linked to one or two α1,3 Xyl linkages, as found in human coagulation factor
VII, coagulation actor IX, and protein Z [28, 44]. Currently known O-fucosyl
glycoproteins contain a single Fuc-O-linkage glycan. The O-fucosyl glycoproteins
include blood coagulation factors VII and XII, tissue plasminogen activator (TPA),
and urinary-type plasminogen activator. One exceptional case, coagulation factor
IX, consists of Fuc-O linkage to Ser/Thr, and the Fucα1-Ser/Thr is further modified
to the longer tetrasaccharide structure of NeuAcα2,6Galβ1,4GlcNAcβ1,3Fucα1-Ser/
Thr-. Currently known O-fucosyl glycosylation observed in EGF domain repeats
utilizes the common motif of Cys2-Xaa3-5Ser/The-Cys3 sequence [28]. Another
specific type of O-fucosyl glycans is reported. The thrombospondin (TS) type
1 repeats (TSRs) in the human ECM contain disaccharide-bearing O-fucosylated
glycans of the Glcβ1,3Fucα1-Ser/Thr- structure [45].
3.6.4 O-α-Fucosylation
The TSR proteins are expressed in the ECM of cells. A TSR protein has about
60 amino acids in length with the conserved Arg, Cys, Ser, and Trp amino acid
residues with the putative sequence of WX5CX2/3S/TCX2G [28]. O-β-glucose and
O-α-fucose attached to Ser or Thr are also known in the EGF-repeated domains of
Notch and Cripto/FRL/Critic proteins. Notch regulator Fringe protein is a β3-
GlcNAc-transferase that O-GlcNAcylates O-Fuc residue to regulate Notch ligand
recognition. The EGF-repeated domain is O-fucosylated by a specific enzyme of
protein O-fucosyltransferase 1 (Pofut1) in mammals [46]. O-fucosylation seems to

need a consensus Cys-Xaa-Ser/Thr-Cys motif present in the domains with the
EGF-like repeats. Currently, two different protein-O-fucosyl-transferases, named
POFU-TI and POFU-TII enzymes, catalyze the transferring reaction of Fuc residue
from the GDP-Fuc as a donor substrate to acceptor substrate [47]. POFU-TI specif-
ically transfers Fuc residue to the EGF-like repeat domains, whereas the POFU-TII is
specific for the O-fucosylation reaction of TSRs. As a product, the tetrasaccharides
of Neu5Acα2,3/2,6Galβ1,4GlcNAcβ1,3Fucα-O-Ser and Neu5Acα2,3/
2,6Galβ1,4GlcNAcβ1,3Fucα-O-Thr are known in the examples of some substrates
including blood coagulation factor XII, Cripto factor IX, Delta, Notch, Serrate, and
thrombospondin type 1 repeats and urokinase, which are all known as carriers of the
EGF-like repeat domains. These proteins also contain single O-fucose residue with
the tetrasaccharide [47].
3.6.5 O-β-Glucosylation
O-β-glucosylation is also found at the hydroxyl Ser residue or Thr residue attached to
the EGF-like repeated regions with the amino acid sequence of Cys-Xaa-Ser-Xaa-
Pro-Cys as the conserved consensus sequence but different from the O-fucosylation
sites, and this contains a trisaccharide with two Xyl residues (Xylα1,3Xylα1,3Glc-
β-O-). Apart from O-Glc, the EGF repeats are also recipient to receive O-fucose or
O-GlcNAc. Several EGF-like repeated domains also have single O-β-Glc attachment
in only specialized glycoproteins including factor VII, factor IX, and Notch proteins
[48].
Protein O-glucosyltransferase (Poglut), which is a CAP10-like protein, catalyzes
the O-glucosylation reaction to the Ser residue present in the common conserved
sequences of EGF-repeated region [49]. Deficiency in the mice Poglut gene exhibits
embryonic lethal effect, which exhibits the Notch-like phenotype [50]. O-glucose
residue is attached to the terminal Xyl residue in the trisaccharyl
Xylα1,3Xylα1,3Glcβ1-O-EGF [51]. To catalyze the xylosylation reaction in
humans, two UDP-xylose to glucoside α3-xylosyltransferase genes of GXYLT1
and GXYLT2 [52], as well as an ER-localized UDP-xylose to xyloside α3-
xylosyltransferase gene of XXYLT1, are identified [53]. Unfortunately, the function
of O-glucosylation in Notch signaling is not yet elucidated, although the Notch
signaling determines cell fate during development and uncontrolled regulation of the
Notch signaling is associated with various human diseases. EGF repeats, small
domains composed of at least 40 amino acids, are present on cell surfaces and
extracellularly secreted in metazoans. It has six Cys residues with three S-S disulfide
bonds to form a characteristic three-dimensional structure. Fringe uses folded EGF
repeats as a substrate for GlcNAc attachment [54], indicating the glycosyltransferase
recognition of the three-dimensional confirmation of EGF-repeated domains. Fringe
has O-fucose specificity on some EGF repeats. Mouse Notch1 has the specific
16 amino acid sites modified with O-Glc trisaccharide [55]. The known 17th
3.6 O-GlcNAcylation, O-Mannosylation, O-β-Glucosylation, O-α-Fucosylation,. . . 67
O-glucosylation site present in the EGF9 is the consensus amino acid motif with
CASAAC sequence, indicating Ala residue replacement of Pro residue in the
consensus sequence. However, bacteria do not transfer the O-glucose glycans,
concluding their restricted role in eukaryotes [56].
3.6.6 O-β-Galactosylation
O-β-galactosylation is also known for collagen, forming the hydroxy-Lys and

hydroxy-Pro in the collagen with the disaccharide Glcα1,2Galβ-O-Hydroxyl-Lys/
Hydroxyl-Pro attachment [57]. Because collagen protein structure is a trimeric form,
which is composed of three left-handed alpha chains with the Gly-X-Y repeats.
Hydroxy-proline (hPro) and hLys residues are major amino acids at X and Y
positions. Certain hLys is attached by galactose and glucose-galactose units. Colla-
gen glycosylation occurs in the ER before triple-helix formation by β1-O-galactosyl-
and α1,2glucosyltransferase enzymes. O-Galactosylation is present only on collagen
proteins. Gal residue or Glcα1,2Gal disaccharide binds to the modified amino acid
hLys residue in collagen proteins with covalent linkages [58, 59].
3.6.7 C-Mannosylation and C-Glycosylation
Finally, a very unique type of C-mannosylation is known, and this attaches a single
Man residue to the Trp indole ring via a C-linkage in most of eukaryotes, except for
yeasts. A specific glycan linkage is formed between a carbohydrate and a protein,
and the linkage has been known as C-glycosylation that occurs at a specific Try
residue in human RNase Us type [60]. The reason why it has been termed
C-glycosylation is the saccharide linkage with the protein via a carbon-carbon
bond. Apart from regular protein glycosylation, C-mannosylation of tryptophan
residues is unique between mannose and protein. Not for the classical O-glycan
type or N-glycan type, a C-C bond is the characteristic site of a single Man
attachment. C-mannosylation event is therefore very specific among various glyco-
sylation events of proteins, which differs from types of glycosylation. It involves in a
covalent linkage for addition of an α-Man residue to the Trp indole C-2 carbon
position via a C-C link. C-mannosylation of tryptophan residues is also an
ER-localized catalysis reaction using the donor substrate, Dol-P-Man. The Dol-P
sugars are general substrates for several ER mannosyl- and glucosyltransferases for
N- and O-glycosylation and mannosyltransferases involved in GPI-anchored bio-
synthesis. The dolichol-diphosphate (Dol-P-P) oligosaccharide is also a substrate of
OSTs during N-glycan synthesis [61]. A conserved amino acid W-X-X-W motif
bears a Man residue linked to the initial Trp residue [62, 63], in the MUC5AC and
MUC5B CYS domains. The dolichol-phosphate-Man is used as the donor substrate
[62]. C-mannosylation contributes to the protein folding process. C-Man linkage is
particularly identified in thrombospondin (TSN) type 1 repeats and the WSXWS

motif present in cytokine receptors of type I. The C-mannosylase gene is not sure,
although Caenorhabditis elegans DPY-19 has been suggested to be a
C-mannosyltransferase [61]. The DPY-19-coding gene has sequential and topolog-
ical homologies with the N-glycan OST. This indicates some evolution-based
C-glycosylation creation from the house-keeping N-glycosylation. C. elegans recep-
tor proteins of MIG-21 and UNC-5 have been suggested to be acceptor substrates of
DPY-19 enzyme to secret soluble MIG-21.
3.7 Function of O-Glycosylation and O-Glycans
Regarding the roles of O-glycoproteins in cells, O-glycan-containing glycoproteins

contribute to, like N-glycan-containing glycoproteins, protein structure and protein
stability, immunity, nonspecific protein recognitions, receptor signaling, enzyme
activity, protease resistance, and cell survival [31]. The functional roles of glycans
in proteins or lipids are known to broadly involve in the bigger spectra including
development, differentiation, growth, cell function, or survival of organisms. Addi-
tionally, O-glycans of mucin-type easily interact with water molecules compared to
other types of glycans. Mucins with a heavy glycosylation in proteins, called mucin-
type O-glycan structure, are present in cell surfaces located on the genital, digestive,
and respiratory tracts. The mucins also carry clustered sialylglycans with a strong
negative charge affordable to hold water and form mucus. The gel-like sticky and
hydrophilic molecules present in nasal secretions are basically composed of secreted
MUC2 polypeptides. For the fundamental role of the mucin-type O-glycosylations,
protective function of the cells is suggested from bacterial invasions, allowing
protection of the cells from bacterial attacks [64]. Similar to O-glycans of mucin
type, another polymeric GAGs also show such negative charges from the sulfate
group. Another function of O-glycans is to recognize and interact between each
protein. Carbohydrate structures are used as substrates for nonenzymatic
carbohydrate-binding proteins, named lectins. Upon lectin binding, glycans influ-
ence the function and fate of the target proteins. Most of glycan recognition of
glycoproteins are ubiquitously taken place in the cellular system. Representatively,
selectins and galectins are well characterized to bind to carbohydrate epitopes.
Consequently, their recognition to carbohydrate ligands induces cellular events
such as apoptosis, signaling, endocytosis, proliferation, cell-to-cell recognition,
ECM-cell recognition, ECM assembly, fertilization, and differentiation [65]. In
addition, O-mannosylated glycans with sialylation play crucial roles in interaction
with ligands for complex of laminin and dystroglycan. The interaction leads to
functionality in development of the brain and muscle [41]. Furthermore, O-glycans
specifically recognize immunologic antigens in mammals. The O-linked glycosyla-
tion therefore affects the signaling molecules such as hormones and cytokines, and
certain enzymes, although the influence is not very strong but rather finely regulates
them. O-glycans of mucin type decrease the biological activity of cytokine IL-5 [66],
3.8 Glycosaminoglycans (GAGs) 69
but they activate lactase phlorizin hydrolase enzyme of humans [67]. The change in
glycan structures also specifically influences the signaling molecules.
O-fucosylation in urinary-type plasminogen activator (uTPA) activates the uTPA
receptor. Additionally, the O-fucosylations are needed for proper Notch function, as
described previously [68]. Also, the O-GlcNAc turnover modification is important
for signaling pathways of many different biological phenomena including transcrip-
tional regulation of gene expression, protein degradation via proteasome, and
insulin-receptor signaling by competing with phosphorylation. O-GlcNAc modu-
lates neutrophil motility in DCs and macrophages [69]. O-glycans are also required
for the protein expression, as found in glycophorin A with a heavy glycosylation.
The O-glycans are present on the surfaces of human erythrocytes [70]. The
O-glycans influence protein modification and proteolytic processing. Representa-
tively, insulin-like GF-II protein is degraded into the matured IGF-II as a functional
form, when amino acid Thr-75 residue is specifically O-glycosylated [71].
3.8 Glycosaminoglycans (GAGs)
Proteoglycans are highly glycosylated proteins, which contain core protein part and
GAG sequences with one or more repeats. GAGs and proteoglycans (PGs) constitute
the ECM at the cell surfaces. The attached GAG chain number is diverse. GAGs are
a different type distinct from common O-glycosylation types and structurally
diverse. GAGs are linear, unbranched, and heterogeneous sulfated glycans with
negatively charged heteropolysaccharides found in every mammalian tissue
[72]. Because sulfotransferases synthesize sulfated GAGs’ side chains present in
proteoglycans, GAGs have monosaccharide type and sulfation specificity. The GAG
backbones consist of repeated disaccharide units with alternated uronic acid
(UA) residue and hexosamine residue units. GAGs have diversity through sulfated
reaction and GlcA residue epimerization to iduronic acids. The UA-repeated units
are composed of β-D-GlcA or the epimerized form of β-D-GlcA C-5, termed α-l-
iduronic acid (IdoA). In the GAGs, the amino sugar residues are forms of
(i) Glc-derived α-D-glucosamine (GlcN)or β-D-GlcN and also (ii) Gal-derived
GalNAc residue. The permutation events of the monosaccharides, which are char-
acteristically found in the GAG-repeating units, generate distinct GAG structures.
The generated GAG structures include the glucosamine-carrying heparan sulfate
(HS) and heparan phosphate (Hp), and the GalNAc-containing dermatan sulfate
(DS) and chondroitin sulfate (CS). Meanwhile, another type of GAGs, keratan
sulfate (KS), alternates GlcNAc with Gal but does not bear UA. Hyaluronan
known as HA alternates GlcNAc with GlcA but does not bear a core protein.
GAGs consist of alternated UAs and N-acetylated hexosamine with GlcNAc or
GalNAc residue, which is combined with GlcA residue or Gal residue. The GAG
linkage tetrasaccharide is the GluAβ1,3Galβ1,3Galβ1,4Xylβ-O-serine, where the
repeated disaccharide unit of GAG is generated through the common structure
with tetrasaccharide of GlcAβ1,3Galβ1,3Galβ1,4Xylβ1-O-Ser sequence and

affected by the GTs for the linkage synthesis (Fig. 3.1).
The sqv gene products of GAG synthesis (e.g.,, HS or CS) are resident in Golgi
apparatus. SQV-1 gene encodes the UDP-GlcA decarboxylase for the UDP-xylose
genesis functioning as a first nucleotide sugar donor in GAG synthesis. SQV-6
encodes for xylosyltransferase, SQV-3 for galactosyltransferase I, SQV-2 for
galactosyltransferase II, and SQV-8 for glucuronosyltransferase I. SQV-4 gene
encodes for the enzyme UDP-Glc dehydrogenase to synthesize UDP-GlcA in the
cytoplasm. SQV-7 gene encodes for nucleotide sugar transporter protein that trans-
ports UDP-GlcA in Golgi apparatus into the lumen and also translocates UDP-Gal
residue and UDP-GalNAc residue. Glucuronosyltransferase (GlcAT-II) and
N-acetylgalactosaminyltransferase (GalNAcT-II) act as the acting enzymes for CS
chain elongation. N-acetylgalactosaminyltransferase I (GlcNAcT-II) acts in HS
synthesis. N-acetylglucosaminyltransferase I (GalNAcT-1) functions in CS chain
initiation. Mutations in CS GAG synthesis lead to human diseases such as connec-
tive tissue disorder, Ehlers-Danlos syndrome, and hereditary multiple exostoses,
which causes inappropriate chondrocyte proliferation and bone growth. Also,
defects in HS synthesis cause disorders related to intracellular signaling, Wingless,
Hedgehog, and fibroblast grow factor pathways. Defects in GAG synthesis caused
animal development abnormality and human disease. For example, mutation in
Drosophila HS GAG-synthetic enzymes causes developmental defect.
GAGs carry sulfated or non-sulfated monosaccharides. Deacetylated and
N-sulfated GlcNAc residues are also found. Most hexosamine units are acetylated
and present as GlcA-containing disaccharide repeats. Deacetyl form of hexosamine
units are regularly sulfated by sulfonylation enzymes, and they are present as
iduronate-containing disaccharide repeats. The ionic carboxylate and sulfate groups
in GAGs attract water. GAGs covalently bound to a core protein are PGs or in some
case of free chains, HA, or hyaluronan.
3.8.1 Classification and Biosynthesis of GAGs
GAGs are categorized into six classes with hyaluronan (or hyaluronic acid) (HA;
GlcA and GlcNAc), DS (iduronic acid or GlcA and GalNAc) and CS (GlcA and
GalNAc), heparin, HS (iduronic acid or GlcA and GlcNAc), and keratan sulfate (KS;
Gal and GlcNAc) [73]. From structural basis of the disaccharide repeats, GAGs are
further classified into three types of (i) DS and CS (GlcA+GalNAc), (ii) heparin-HS
(GlcA+GlcNAc), and (iii) KS (Gal+GlcNAc). The epimerization form of GlcA in
DS and heparin-HS is called “iduronate.” GAG structures are heterogeneous due to
O-sulfation [74]. Heparin is a highly sulfated GAG, whereas HS is sulfated only in a
certain region [75].
Sulfated GAG forms are generated at the region of Golgi apparatus with modi-
fication by O-sulfotransferases [76]. The biosynthesis of GAGs is quite different
from those of other O-glycans, because all the transferases are specific for GAGs,
only except for the chondroitin 6-O-sulfotransferase, KS Gal-6-O-sulfotransferase,

and GlcNAc 6-O-sulfotransferase, which transfers sulfate group to other LacNAc
extensions [77]. CS and DS as well as heparin and HS chains are generated from the
known common linker tetrasaccharide of the GlcAβ1,3Galβ1,3Galβ1,4Xyl-struc-
ture. GAG chains are assembled in ER-Golgi apparatus. GAG-protein-linked
sequence is the GlcAβ1,3Galβ1,3Galβ1,4Xylβ1-O-Ser structure as an initiating
unit. The tetrasaccharide in the linkage site is assembled by transfer of a Xyl,
2 Gal, and a GlcA residues by Xyl-T, β1,4GalT-I, β1,3Gal-T II, and
β1,3glucuronyl-T I [78], respectively.
CS and DS GAGs are generated through the GalNAc linkage, while heparin and
HS are generated if GlcNAc residue is added at the first reaction [74]. Chondroitin
GalNAc transferase-I, GalNAc transferase-II, and chondroitin synthetase initiate the
CS/DS GAG chain synthesis with the first few GalNAc residues. However, chon-
droitin synthetase functions as a co-polymerase to elongate the CS/DS forms, having
the multiple (GalNAcβ1,4GlcAβ1,3)n structure [78, 79]. HA has no core protein and
is synthesized at the extracellular surface functioning in tissue freshness and cancer
growth. KS-type GAG belongs to the sole GAG species that lacks uronic acid but
bears Gal residue in the KS disaccharide units. On the other hand, HA species
belongs to non-sulfated KS forms, which are attached to substrate proteins in a
N-glycosylation form or core 1 O-glycosylation form. GAG species have a different
chain number specificity. One chained GAG, decorin, is known as a small Leu-rich
proteoglycan (SLRP). GAG having 100 or more saccharide chains is known for
aggrecan [76].
3.8.2 Chondroitin Sulfate (CS)
All the CS-synthetic enzymes are well explained to generate diverse structural
formation of CS chains. CS-associated developmental and pathophysiological pro-
cesses include CS-recognizing molecules with CS receptors. CS is a sulfated GAG
as a linear polysaccharide with disaccharide unit repeats of uronic acid and HexNAc.
CS-attached PGs (CSPGs) have at least one side chain. CSPGs are involved in
cytokinesis, morphogenesis, and neuronal plasticity, skeletal diseases, formation of
glial scars, and pathogenic invasions such as bacteria and viruses [74, 80]. Disaccha-
ride unit is [(–4GlcAβ1–3GalNAcβ1–)n] as galactosaminoglycan (Fig. 3.4).
In C. elegans, ChSy gene is the orthologue of human ChSy. ChSy family is a
group of glycosyltransferases that conduct polymerization of chondroitin sulfate
chain through GalNAc transferase-II and GlcA transferase-II activities. Unlike
human type, ChSy of C. elegans has not only GalNAc transferase activity but also
GalNAc transferase-I activity. Normally, the GalNAc transferase-I activity can be
found in ChGn genes, which produce CS chain by addition of GalNAc to
tetrasaccharide linker of core protein. That means C. elegans ChSy is indispensable
for production of chondroitin proteoglycans. For the roles of chondroitin
proteoglycans in C. elegans, knockdown of ChSy was established by soaking in a
%J5[ %J5[
A) %J5[
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*GRCTCPUWNHCVG -GTCVCPUWNHCVG &GTOCVCPUWNHCVG *[CNWTQPCP
Fig. 3.4 GAG structures and glycosyltransferases involved in GAG synthesis. Synthesis of GAG
linkage tetrasaccharide, GluAβ1,3Galβ1,3Galβ1,4Xylβ-O-serine, and different GAGs in C. elegans
is described. HS, KS, DS, and HA GAG chains as the typical disaccharide units are described.
CS-repeated units are GalNAc residue and GlcA residue. DS is a CS stereoisomer having an IdoA
residue instead of GlcA. HS repeat units are GlcNAc residue and GlcA residue. The saccharide
residues are esterified by sulfate, while HA belongs to a linear polymer with the repeated disac-
charides of 4GlcAβ1,3GlcNAcβ1-units. Glycosyltransferases involved in GAG synthesis include
(i) GlcAT-II, which is a glucuronosyltransferase, and GalNAcT-II, which is a
N-acetylgalactosaminyltransferase for chondroitin chain elongation, (ii) GlcNAcT-II
(N-acetylgalactosaminyltransferase I) for heparan sulfate synthesis, and (iii) GalNAcT-1
(N-acetylglucosaminyltransferase II) for chondroitin chain initiation
double-stranded siRNA solution or feeding with E. coli, which produce a double-

stranded form of siRNA. As expected, these ChSy knockdown C. elegans show a
decrease of chondroitin. Interestingly, ChSy-null strains exhibit abnormal embryo-
genesis in C. elegans with incomplete mitosis and reversion of cytokinesis.
Although zygotes undergo mitosis, cytoplasms are not perfectly separated due to
mal-cytokinesis. Nonetheless, nucleus is divided to produce multinucleated cells,
leading to embryo death and abnormal reproductivity. In terms of CS or HS, similar
phenotypes are observed in blocking tetrasaccharide biosynthesis due to incomplete
chondroitin sulfate chain synthesis. But blocking of heparan sulfate chain synthesis
does not display any morphological change [81]. Therefore, chondroitin proteogly-
cans are responsible for cytokinesis of C. elegans embryos, but without detailed
mechanism of chondroitin proteoglycans in cytokinesis.
Using tetrasaccharide-O-Ser/protein, GalNAcT-I enzyme catalyzes the attach-
ment of GalNAc residue to the GlcA residue terminally present in nonreducing
end to form chondroitin stem [74]. The disaccharide GlcA-GalNAc repeats of CS are
formed by the alternative catalysis of GlcA residue and GalNAc residue through
GlcAT-II and GalNAcT-II enzymatic transfers, respectively. The tetrasaccharide
unit is also used for another sulfated GAG, HS which has disaccharide repeats of
(–4GlcAβ1,4GlcNAcα1–)n as glucosaminoglycan. Therefore, the alternate GlcNAc
forms the HS. Hence, CS and HS chain synthesis needs the first HexNAc addition
[74]. To sulfate the chondroitin backbone, sulfotransferases add sulfate group at the
Glc-A C-2/GalNAc C-4/GalNAc C-6, forming the disaccharide A, C, D, E, and O
units. In addition, the GlcA epimerization to IdoA residue is catalyzed by 2 GlcA
C-5 epimerases called DS-epi-1 and DS-epi-2 enzymes [82, 83]. The epimerases
convert CS into its stereoisomer, DS, which carries disaccharide repeats of iA, iB,
and iE.
To date, six glycosyltransferase genes for chondroitin biosynthesis of the disac-
charide repeats [(–4GlcAβ1–3GalNAcβ1–)n] were isolated for chondroitin synthase
(ChSy)-1, ChSy-2, ChSy-3, chondroitin GalNAcT (ChGn)-1, ChGn-2, and
chondroitin-polymerizing factor (ChPF). The three ChSy-1, ChSy-2, and ChSy-3
enzymes bear bifunctional glycosyltransferase enzymes known as GlcAT-II and
GalNAcT-II. Of interests, co-expression of two proteins among the four ChPF,
ChSy-1, ChSy-2, and ChSy-3 proteins upregulates the activity levels of GlcAT-II
and GalNAcT-II enzymes, increasing chain lengths. Thus, chondroitin length is
formed by the synthesizing enzyme complexes of chondroitin polymerases as well
as their combinations of four proteins including ChPF, ChSy-1, ChSy-2, and
ChSy-3. However, ChGn-1 and ChGn-2 bear both enzyme activities of GalNAcT-
I and GalNAcT-II, catalyzing initiation and elongation of chain synthesis [74].
3.8.2.1 Sulfotransferases Sulfate CS/DS Chains
Seven sulfotransferases sulfate CS and DS chains [84] utilizing the donor substrate
30 -phosphoadenosine-50 -phosphosulfate (PAPS) to supply GlcA, GalNAc, or IdoA
in CS and DS GAGs as acceptors. In sulfation of CS units, the common acceptor
substrate is an O group saccharide with non-sulfation. The monosulfated A and C
units are generated through sulfation reaction at GalNAc 4-O/6-O positions, respec-
tively. Further additional sulfation reaction of A and C saccharides yields disulfated
D and E saccharides, respectively. Hence, the sulfo-transferring reaction of chon-
droitin unit has the two different “4-O/6-O-sulfation” reactions at the initial step.
GalNAc-4-O-sylation in CS and DS sugar chains is performed by specific enzymes
of three forms, named chondroitin 4-O-sulfotransferase (C4ST)-1, C4ST-2, and
C4ST-3, for the CS, while dermatan 4-O-sulfotransferase (D4ST)-1 yields the
GalNAc 4-O-sulfation next to IdoA in DS. Therefore, it is clear that the specific
enzymes of C4STs and D4ST-1 yield the specific products, A and iA saccharide
units, respectively [74].
Chondroitin 6-O-sulfotransferase-1 (C6ST-1) sulfates to the carbon C-6 of
GalNAc residue present in C and D units in CS, but not DS. Two UST and
GalNAc-4-sulfate 6-O-sulfotransferase (GalNAc-4S-6ST) make the specific
disulfate-containing disaccharides linked to CS/DS chains. Uronyl 2-O-
sulfotransferase (UST) enzyme performs the GlcA 2-O-sulfation reaction in the
IdoA and C unit present in the iA unit, reducing the levels of iB chain and D
chain, respectively. During E/iE chain synthesis, a specific enzyme, GalNAc-4S-
6ST, catalyzes the sulfation reaction to the 4-O-sulfated 6-O position of GalNAc in
the A/iA units, which are generated by enzymes of C4-ST and D4-ST. Human
C6ST-1 (CHST3) mutation causes a genetic disorder named spondyloepiphyseal
dysplasia (SED) known as an Omani-type disease, as a chondrodysplasia disease.
CS, which is 6-O-sulfated by sulfotransferase, C6ST-1 catalysis, is important for
development and formation of skeletal bones in humans. In mice-deficient model,
mice C4ST-1 (Chst11) mutation also causes chondrodysplasia with neonatal lethal-
ity. Human D4ST-1 (CHST14) mutation causes genetic disorders called adducted
thumb-clubfoot syndrome (ATCS) and Ehlers-Danlos syndrome (EDS), which are
the EDS Kosho-type (EDSKT) and kyphoscoliosis-type EDS as
musculocontractural EDS (MCEDS) [74]. The D4ST-1 mutation yields DS defi-
ciency and a hypersynthesis of CS chains.
Chondroitin polymerization is performed by the constituent chondroitin poly-
merases. In the type 1 herpes simplex virus-resistant cells, which were originated
from mouse fibroblasts, two mutant cells named gro2C and sog9 were established.
The gro2C is HS-deficient due to lack of Ext1 for HS synthesis [84], whereas the
sog9 is CS-deficient due to C4ST-1 mutation. CS chain is important in pathogenic
infection. For example, deficiency in ER nucleotide sugar transporter gene
SLC35D1 (solute carrier 35D1) shortens CS chains with skeletal dysplasia. CS
elongation increases binding potential to atherogenic lipids and arteriosclerosis
development [85]. In fact, the abnormal CS moieties with long chains generated
by C4ST-1 and ChGn-2 are found in the atherosclerosis. TGF-β, EGF, and PDGF
induce CS chain elongation. ChGn-1 initiates CS synthesis with an increased chain
number. ChGn-1 enzyme aberrantly expressed in chondrosarcoma cells synthesizes
a CSPG named aggrecan with multiple CS chains. ChGn-1 enzyme initially synthe-
sizes the CS chains through enzymatic catalysis of the first GalNAc transfer to the
acceptor chain of tetrasaccharide. The GalNAc (4-O-sulfate)-linked pentasaccharide
carbohydrate located in the nonreducing end is preferably used as the substrate for
chondroitin polymerases. Then, the CS chain number is gradually increased. ChGn-
1 also cooperatively acts with the C4ST-2 enzyme and consequently increases the
number of CS chains. Xyl residues are frequently 2-O-phosphorylated in both HS
and CS chains. GlcAT-I enzyme catalyzes the transfer reaction of GlcA residue to
the phospho-trisaccharide-Ser of the Galβ1,3Galβ1,4Xyl-2-O-P-β1-O-Ser. In pro-
teoglycan decorin, transferring event of GlcA residue is coupled to enzymatic
dephosphorylation reaction of the 2-O-phosphorylated Xyl residue by phosphatase.
The Xyl phosphorylation reaction is important for complete linkage in the
tetrasaccharide-bearing CS and HS chains. Unlike the 2-O-phosphorylation, the
Gal sulfation of the linkage is detected only in CS and DS. The sulfate groups on
the Gal help the CS-selective assembly on the tetrasaccharide linkage region
[86]. Gal C-4 and C-6 sulfations influence GalNAcT-I enzyme activity of ChGn-1.
Gal sulfation regulates the CS chain initiation, as C6ST-1 catalyzes the Gal C-6
sulfation.
3.8.2.2 Chain Termination in CS Chains
In chain termination of CS, the nonreducing GalNAc residue is 4,6-O-disulfated,

and this disulfation reaction is a terminator in CS chain elongation. In rat CSPG
aggrecan, CS biosynthesis terminates with sulfated GalNAc in the structure of
GalNAc residue with 4,6-O-disulfation. In human aggrecan CS, the level of 4,6-O-
disulfated GalNAc is increased. Because the mammalian GalNAc4S-6ST enzyme
generates disaccharide E/iE chain units and the non-reduced 4,6-O-disulfonyl
GalNAc residue end present in the CS/DS [87], it involves in the chain termination.
In GalNAc4S-6ST-deficine mice, the nonreducing terminal sugar structures are
absent. Moreover, mast cells derived from the bone marrow, which was isolated
from the deficient mice, synthesize longer chains of CS than those produced from
mast cells derived from wild-type mice. EXTL2 acts as an inhibitor of GAG
biosynthesis where Xyl kinase (FAM20B) enhances the GAG synthesis. The
EXTL2 regulation of GAG biosynthesis is a type of “quality control” process
appeared in HSPGs and CSPGs. EXTL2-type enzyme is an enzyme produced by
the three different EXT-like genes. EXTL2 is an α1,4-HexNAc-T enzyme
possessing both α-GalNAc-T and GlcNAc-T-I activities, which transfer
α-GalNAc/α-GlcNAc residues to the acceptor of tetrasaccharide chain part, respec-
tively. A product of pentasaccharide-Ser, which is generated by α-GalNAc-T
enzyme, has a structure of GalNAcα1,4GlcAβ1,3Galβ1,3Galβ1,4Xylβ1-O-Ser, and
this pentasaccharide is not used for an acceptor substrate in CS chain synthesis,
because the binding α-GalNAc in the site of β-GalNAc inhibits the continued
elongation of CS chains. The GalNAcα capping is not found in natural GAGs.
EXTL2 transfers the GlcNAc residue to a phosphoryl tetrasaccharide chain. The
phospho-pentasaccharide produced cannot be used for an acceptor substrate in HS or
CS polymerization.
For O-sulfate-transferring reaction to nonreducing GlcA end of the chain, cell-
surfaced thrombomodulin (TM) is detected as both forms of CSPG (βTM) and
non-PG form (αTM). αTM is not substituted by CS, hence named “part-time” PG
[88]. Interestingly, two differently prepared αTM forms of the recombinant αTM
expressed by αTM gene-transfected CHO cells and the human urine-purified αTM
exhibit the tetrasaccharide unmodified linked to the GlcAβ1,3Galβ1,3Galβ1,4Xyl-
structure with GlcA 3-O-sulfation present in the nonreducing end. Human natural
killer-1 (HNK-1)-specific MAb binds to the αTM. HNK-1 sulfotransferase
(HNK-1ST) catalyzes the transfer of the sulfate group via the 3-O-sulfation reaction
to GlcA residue of the nonreducing end in the HNK-1 glycan epitope, and thus, a
unique HNK-1 epitope is generated. HNK-ST suppresses CS substitution on TM,
and the CS constituents reduce anticoagulant activity of αTM. HNK-1-posessing
TM is not used for chondroitin polymerases of ChSy-1 and ChPF. Thus, the terminal
GlcA 3-O-sulfation of the chain structure catalyzed by the specific enzyme of
HNK-first is used as a blocking mark of the CS substitution in TM.
Among several GAG species, a representative chondroitin synthesis has been in
part studied in human and C. elegans with regard to its role in developments.
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Fig. 3.5 C. elegans chondroitin synthase disruption by RNAi knockdown technology blocks the
oocyte growth and the developmental stage of 2-cell to 4-cell stage. Adopted from reference No
[89]. Chondroitin glycans are key component of embryo development
Chondroitin species are enzymatically synthesized by multiple enzyme genes

encoding for chondroitin synthase enzymes. Especially, the detailed enzymatic
characterization has been studied in C. elegans. C. elegans chondroitin synthases
have been disrupted by a RNAi technique of knockdown. The resultant strains
exhibited the defectively reduced oocyte proliferation mainly caused by blocking
the developmental stages during two cells to four cells [89]. Hence, it has been
concluded that chondroitin glycans are essential for embryo development. However,
the current status of the functional roles of the chondroitin carbohydrates is still not
well elucidated in the molecular level. Several experiments to demonstrate the
synthetic pathway of the chondroitins are partially performed in several laboratories.
In C. elegans, chondroitin-synthetic enzyme genes reported include the genes of
sqv-1 to sqv-8. These genes seem to be crucial for early embryonic development
process and postembryonic vulval morphogenesis, as adopted from the recent study
[89] (Fig. 3.5). From eight sqv genes, SQV-4 gene encodes a UDP-Glc dehydroge-
nase enzyme, which synthesizes UDP-GlcA in the cytoplasm. SQV-7 encodes a
sugar nucleotide transporter, which transports UDP-GlcA synthesized at the cyto-
plasm to the lumen of Golgi apparatus. This also transports the UDP-Gal and
UDP-GalNAc at the cytoplasm to Golgi lumen. For the roles in Golgi apparatus,
Cytosol
SQV-/-
Golgi lumen
SQV-6
Heparan Sulphate
SQV-4 SQV-1 SQV-3
GlcAT
SQV-7 GlcNAcT-II
SQV-2
…
GlcAT-I
SQV-8
Identity
37% human CS synthase
SQV-5
GalNAcT 37% Drosophila CG9220
20% human GalNAcT-I
GlcAT-II
SQV-4 : UDP-glucose dehydrogenase

SQV-7 : nucleotide-sugar transporter
SQV-1 : decarboxylase for UDP-GlcA
SQV-6 : xylosyltransferase
SQV-3 : galactosyltransferase I
SQV-2 : galactosyltransferase II Chondroitin
SQV-8 : glucuronosyltransferase I
…
Forming fluid-filled
extracellular space
Glucose Glucuronic acid Galactose Xylose
UDP GalNAc GlcNAc Protein core
Fig. 3.6 C. elegans chondroitin glycan is essential for the oocyte growth and the developmental
stage. Adopted from reference No [81, 89]. In C. elegans, chondroitin-synthetic enzyme genes are
known for the sqv-1 to sqv-8 and crucial for embryo development with postembryo vulval
morphogenesis. From 8 sqv genes, SQV-4 is a cytoplasmic UDP-Glc dehydrogenase. SQV-7
encodes a sugar nucleotide transporter protein. SQV-1 encodes a UDP-GlcA decarboxylase enzyme
to generate UDP-Xyl in Golgi apparatus. SQV-3, SQV-6, SqV-2, and SqV-8 denote for
Gal-transferase, Xyl-transferase, Gal-transferase II, and glucuronosyltransferase I enzymes
SQV-1 is a UDP-GlcA decarboxylase, and it synthesizes UDP-Xyl that is the first

nucleotide sugar donor for GAG synthesis (Fig. 3.6). Next, SQV-6 is a
Xyl-transferase and SQV-3 is a Gal-transferase. SQV-2 is Gal-transferase II and
SQV-8 glucuronosyltransferase I (Table 3.2).
3.8.2.3 Sulfated CS Function as Pathogen Receptor and Co-receptor

as Well as CS Binds to Advanced Glycation End-Product
Receptor in Metastasis
Various CS chains can directly recognize a variety of molecules. Among CS chains,

HS chains, and sulfated heparin, the sulfated CS chains are particularly important for
their binding capacity [74]. Heparin pentasaccharide binds to antithrombin [72]. Spa-
tial distribution of negative charges attached to repeated saccharide units present in
CS chains is important, as known for HS/heparin recognition to FGFR and FGFR
[72]. CS oligosaccharides interact with a pleiotrophin (PTN) known as heparin-
binding growth factor [90]. Therefore, certain pathogens including bacteria, para-
sites, and viruses utilize CS saccharide chains for attachment and infection. For
Table 3.2 CS tetrasaccharide chain linkage- and repeated disaccharide chain-synthesizing

enzymes and catabolizing enzymes in humans. Sqv genes biosynthesize GAG of HS and chondroi-
tin in Golgi apparatus and GAG linkage tetrasaccharide, GluAβ1,3Galβ1,3Galβ1,4Xylβ-O-serine.
SQV-1, UDP-GlcA decarboxylase; SQV-6, xylosyltransferase; SQV-3, galactosyltransferase I;
SQV-2, galactosyltransferase II; SQV-8, glucuronosyltransferase I; SQV-4, UDP-glucose dehydro-
genase for UDP-GlcA formation; SQV-7, nucleotide sugar transporter for UDP-GlcA transport to
Golgi apparatus lumen as well as UDP-Gal and UDP-GalNAc to Golgi. GlcAT-II
(glucuronosyltransferase) and GalNAcT-II (N-acetylgalactosaminyltransferase) are used for chon-
droitin chain elongation. GlcNAcT-II (N-acetylgalactosaminyltransferase I) is used for HS synthe-
sis. GalNAcT-1(N-acetylglucosaminyltransferase II) involves in chondroitin chain initiation
Enzymes Abbreviation Gene symbols Chromosomal loci
In tetrasaccharide linkage
Xylosyl-T XylT XYLT 16p12.3
XYLT2 17q21.33
β1,4-Galactosyl-T-I GalT-I B4GALT7 5q35.2–q35.3
β1,3-Galactosyl-T-II GalT-II B3GALT6 1p36.33
β1,3-Glucuronyl-T-I GlcAT-I B3GAT3 11q12.3
In repeated disaccharide
Chondroitin synthase ChSy-1 CHSY1 15q26.3(GalNAcT-II,
GlcAT-II)
ChSy-2 CHSY2(CSS3) 5q23.3
ChSy-3 CHSY3(CHPF2) 7q36.1 (CSGLCA-T)
Chondroitin-polymerizing ChPF CHPF(CSS2) 2q35(GalNAcT-II,
factor GlcAT-II)
Chondroitin GalNAc-T ChGn-1 CSGALNACT1 8p21.3 (GalNAcT-I,
GalNAcT-II)
ChGn-2 CSGALNACT2 10q11.21
Sulfate-transferases and epimerases
Chondroitin 4-O-sulfo-T C4ST-1 C4ST-1 (CHST11) 12q
C4ST-2 C4ST-2 (CHST12) 7p22
C4ST-3 C4ST-3(CHST13) 3q21.3
Dermatan 4-O-sulfo-T D4ST-1 D4ST1 (CHST14) 15q15.1
Chondroitin 6-O-sulfo-T C6ST-1 C6ST-1 (CHST3) 10q22.1
Uronyl 2-O-sulfo-T UST UST 6q25.1
GalNAc 4-S-6-O-sulfo-T GalNAc4S- GALNAC4S-6ST 10q26 (BRAG)
6ST (CHST15)
Glucuronyl C-5 epimerase DS-epi1 DSE (SART2)
DS-epi2 DSEL 18q22.1
Sulfate-transferases and epimerases
Glucuronyl C-5 epimerase DS-epi1 DSE (SART2) 6q22
DS-epi2 DSEL 18q22.1
Tetrasaccharide linkage-modifying enzymes
Xylose 2-O-kinase XylK FAM20B (gxk1) 1p25
Galactose 6-O-sulfo-T C6ST-1 (CHST3) 10q22.1
Exostosin-like glycosyl- EXTL2 EXTL2 1p21 (GlcNAcT-I)
T2
Uronyl 3-O-sulfo-T HNK1-ST (CHST10) 2q11.2
(continued)
Table 3.2 (continued)

Enzymes Abbreviation Gene symbols Chromosomal loci
Chondroitin sulfate hydrolases
Endo-β-N- HYAL-1 HYAL-1 3p21.3
acetylgalactosaminidase
HYAL-4 HYAL-4 (CSHY) 7q31.3
SPAM1 SPAM1 7q31.3
example, the malaria (Plasmodium falciparum)-infected erythrocytes adhere to

endothelial cells by recognition of CS saccharide units carrying a low sulfated
CS-A structure like monosulfated A unit [91]. HSV recognizes and binds to E
unit-rich CS chains as its infection receptor [92, 93]. Hence, C4ST-1 and E unit-
deficient sog9 cells cannot be infected by HSV-1. C4ST-1 gene transfection in sog9
cells produces E disaccharide and renders HSV-1 infection [93].
CS chains with D or E units bind to PTN, midkine (MK), FGF, HGF, and BDNF.
CS stimulates neurite outgrowth as well as neural stem cell and neuronal progenitor
cell growth [74], because CS saccharide chains are co-receptors or holding reservoirs
for associated molecules. CS binds to neuronal receptor, named neurite outgrowth
identified contactin-1 (CNTN-1), which is necessary for extension and regeneration
of neurons. Interestingly, CSPGs as essential CNS components inhibit axon out-
growth during CNS injury. If CS parts of CSPGs are removed in lesion sites, axon
regeneration event is induced. However, CS action in neurite outgrowth inhibition is
controversial because CS-E stimulates neurite outgrowth in the controlled experi-
ment using cultured primary neurons. Such apparent contradiction is attributed to the
structure difference in CS saccharide chains. The dominant CS forms present in
mammal tissues include the two monosulfated A and C units. The A unit-enriched
CS form (CS-A) inhibits axon guidance event and cerebellar granule neuron growth
[94]. However, C chain-enriched CS form (CS-C) does not inhibit and induce axon
regeneration [95], and their synthetic enzyme C6ST-1 expression is increased in
injury conditions. Hence, neuronal cells have distinct CS-recognizing CS
receptors [96].
In two different cell lines such as L cells of mouse fibroblast cells and mutant
sog9 cells, the CS-E is generated by C4ST-1 enzyme, and it easily recognizes
Wnt-3a. Consequently, it controls β-catenin-dependent canonical Wnt downstream
signaling [97]. However, C4ST-1-lacking sog9 cells do not recognize Wnt-3a.
C4ST-1 also involves in tumorigenesis, because C4ST-1 genes in colon adenocar-
cinoma cell and hepatocarcinoma cells of humans are not expressed, although those
cells exhibit the activated Wnt/β-catenin signaling [97]. C4ST-1 expression is also
inhibited in certain human patients with B-cell lymphoma [98]. In addition, the
C4ST-1 expression is correlated with malignant colorectal cancer [99]. A proto-
oncogene HRAS is known to diminish the C4ST-1 gene expression in the Costello
syndrome pathogenesis, which germline mutations in the HRAS generate the path-
ogenesis [100]. Therefore, C4ST-1 can be applied for therapeutic treatment of cancer
progressions associated with RAS signaling or canonical Wnt signaling in humans.
Apart from endogenous CS-E, exogenous CS-E treatment prevents accumulation of

β-catenin [97]. CS-E can be used to treat aberrant canonical Wnt-caused diseases.
A CS-E binds to its specific neuronal receptor for a CNTN-1 known as a
GPI-anchored protein of cell adhesion molecule (CAM) as the Ig superfamily
[101]. Only CS-E recognizes the CNTN-1. However, others of CS-A, CS-C, or
HS do not bind to the CNTN-1. Certain CS chains, which are resident in extracellular
region, intracellularly transduce their signaling. A transmembrane receptor protein
phosphatase known as PTPσ is also known as a CS receptor to inhibit axon
regeneration [102]. PTPσ and leukocyte-related antigen (LAR) subfamily are also
receptors for HSPGs, and they exert to potentiate synapse formation and axon
guidance at the early developmental stage [103, 104]. PTPσ regulates bimodally
extension of neurons through double actions such as inhibition of CSPG and
stimulation of HSPG. In fact, HS induces oligomerization of PTPσ ectodomains.
In contrast, CS inhibits the HS-induced oligomerization [104]. Thus, differential
action of PTPσ is mediated by their oligomerization modulation of PTPσ, which is
used for the common receptor for CSPGs and HSPGs. Currently, TM leukocyte
common antigen-related phosphatase (LAR) receptor as a subclass of LAR subfam-
ily, and two family members of Nogo receptors including NgR1 and NgR3, are also
known for the CS receptors to inhibit CSPG function [105].
CE-E unit mediates pulmonary metastasis, as confirmed by Lewis lung carcinoma
(LLC) cells. A silencing of GalNAc4S-6ST reduces the CS E units and suppresses
pulmonary metastasis in LLC cells. CS-E binds to advanced glycation end-product
(RAGE) receptor, an Ig superfamily, largely present in lung tissue [106]. RAGE
binds to sulfated GAG saccharide chains such as CS-E and HS. Anti-RAGE
antibody inhibits CS-E-involved lung metastasis of LLC cells (Fig. 3.4c), allowing
RAGE and sulfated GAG for drug targets for pulmonary metastasis.
3.8.2.4 Roles of CS in Embryogenesis and Development
Caenorhabditis elegans generates HS and chondroitin, which is not sulfated in

CS. The chondroitin and CS chains are indispensable, as confirmed in the nematode
ortholog of ChSy for sqv-5 in the vulva formation [89]. GlcAT-I (B3gat3) KO mice
are lethal at embryonic stage of development before the 8-cell stage because of
cytokinetic defaults. The two-cell embryos of wild type were treated with
chondroitin-digesting enzymes such as chondroitinase-A, chondroitinase-B, and
chondroitinase-C (ChABC) from bacterial sources also which show embryonic
lethality. Vertebrate bones are formed through the known process of endochondral
and intramembranous ossification events. Sulfation of CS/DS chains is important for
skeletal bone formation with endochondral ossification. Mouse MC3T3-E1 cells as
an osteoblastic line show intramembranous ossification. Cadherin mediates cell-cell
interaction during osteogenic differentiation because MC3T3-E1 cells produce
cadherin-11 and N-cadherin [81]. In differentiating MC3T3-E1 cells, CS-E unit is
increased, where CS-E recognizes N-cadherin or cadherin-11 and consequently
enhances osteogenic differentiation [107]. However, CS-A does not bind to the
molecules. Interaction between CS-E ligand and the cadherin-11 and N-cadherin-11
as CS receptors influences osteogenic bone formation of MC3T3-E1 cells, which is
potentially applicable for patients with osteoporosis. The enforced GalNAc4S-6ST
expression for binding ligand CS-E units may enhance adhesion to N-cadherin/
cadherin-11 in MC3T3-E1 cells.
Sulfation and CS chains synthesis are also crucial for embryonic development.
Incomplete CS synthesis at early stage of embryonic development causes cell death
due to reversed cytokinesis. Enforced reduction of CS levels induces both of
myogenic differentiation and myofiber regeneration. Therefore, a dystrophin defi-
ciency in mice, which is a typical model animal of Duchenne muscular dystrophy,
can be compensated through the intramuscular ChABC injections. This potentially
improves the dystrophy pathogenic progress of myofibers. In other words, the
abundantly synthesized CS levels are an essential factor for muscular dystrophic
protection, regeneration of skeletal muscles, disease cell differentiation, and disease
improvement.
3.8.3 Dermatan Sulfate (DS)
In the structure aspect, DS is a linear oligo- or polysaccharide, as a sulfated GAG,

with a covalent linkage to the PG core proteins [108, 109]. The DS saccharide units
have GalNAc and l-iduronic acid (IdoUA), although CS is indeed a DS stereoisomer,
consisting of D-GlcUA instead of IdoUA. Sugars are the general subjects of
esterification reaction by sulfate at multiple positions. DS chains are assembled
through cooperative action of various GTs and modifying enzymes such as epimer-
ases and sulfotransferases. For the biosynthesis of DS, first, acceptor core proteins
are generated. Second, the GAG linker chain of GlcUAβ1,3Galβ1,3Galβ1,4Xylβ1-
sequence is synthesized by specific transferases of β-xylosyltransferase (Xyl-T),
β1,4-Gal-transferase-I (Gal-T-I), β1,3-Gal-transferase-II (Gal-T-II), β1,3-GlcA-
transferase-I (GlcAT-I) using the target core proteins for linking to Ser residue.
The four different Xyl-T, GlcAT-I, GalT-I, and GalT-II enzymes commonly act to
form all GAGs including CS, DS, and HS. Upon formation of the saccharide linker
chains, chondroitin synthase enzymes make the assembly with the chondroitin units.
Then, the GlcUA epimerization as well as each sugar sulfation occur through
enzymatic catalysis of DSE, D4ST, and UST. Xyl-T, Gal-T-I, Gal-T-II, GlcAT-I,
GalNAcT-I, GlcAT-II, GalNAcT-II, DS epimerase (DSE), dermatan 4-O-
sulfotransferase (D4-ST), and UST are involved in the multiple reactions.
DS-PGs are abundant in the cartilage, skin, and aortic endothelium in humans.
DS-PGs exhibit ubiquitous expression patterns in many organs and tissues including
the brain, heart, kidney, liver, and lung. DS and DS-PG synthesis defections are
associated with development of skin and skeletal diseases. DS chain units contain
disaccharides of GalNAc and IdoUA residues, having 50–200 more repeated units
(Fig. 3.4). DS chain units are frequently targeted to sulfate at the carbon C-4 and C-2
sites both on GalNAc and IdoUA residues, respectively. Their localization in ECMs
exerts to play crucial roles in various biological pathways including growth factor-
mediated signal transduction, anticoagulation, and wound healing [110]. CS consists
of another sugar residues GlcUA and GalNAc. After the chondroitin unit formation,
the GlcUA residue is targeted to epimerization reaction to form the IdoUA residue
by a specific DSE enzyme. Then, the new type chains of CS/DS hybrid can be also
synthesized. The small Leu-rich DS-PGs include biglycan, fibromodulin, and
decorin, which carry the Leu-rich sequences with small protein core sequences
[72]. The PG-deficient KO mice are particularly featured with osteoporosis, skin
fragility, collagen fibrils, and abnormal Achilles tendon. Core protein of decorin
modulates the collagen fibrogenesis. EDS is classified to a heterogeneous diseases
and also heritable connective tissue disorders with the multiple expressions of joint
hypermobility, skin hyperextensibility, and tissue fragility.
3.8.3.1 Biosynthesis of DS
CS disaccharide contains GlcA and GalNAc, namely, 4GlcAβ1–3-GalNAcβ1-.

While in DS, β-D-GlcA is transformed to α-L-IdoA via the C5 epimerization, which
is catalyzed by an epimerase. Then, the CS/DS is frequently present as a single chain
hybrid form of the CS/DS hybrids [111]. DS is generated from CS, firstly by GlcA
epimerization to IdoA, followed by sulfo-transfer reaction in distinct carbons
[112]. The DS-epimerase (DSE) and DSEl genes encode the DS epimerase-1 and
DS epimerase-2, respectively. The DS epimerases modify CS to CS/DS hybrid units.
The produced CS/DS hybrid units are richer than CS in structure and conformation
aspects. The CS/DS hybrid units preferentially interact with ECM proteins and
related growth factors. More specifically, after chondroitin backbone synthesis,
two DSE enzymes convert GlcA residue to IdoA residue form via the C5-carboxyl
group epimerization of GlcA residue [83], forming CS/DS hybrid units with differ-
ent IdoA contents. C5-epimerized residue is O-sulfated, forming CS/DS chains. The
IdoA residue is formed by DS-epi-1 and DS-epi-2 enzymes, genetically coded by the
DSE and DSE-like genes (DSE-L), respectively [83]. Therefore, CS/DS consists of
alternated units of a hexuronic acid of GlcA or IdoA, and the amino sugar GalNAc.
The IdoA-containing units yield long blocks and often are interspersed among
unmodified GlcA saccharide units. Missense mutations of homozygous forms in
DSE gene are the causing factors of the musculocontractural type of Ehlers-Danlos
syndrome (MC-EDS), a connection tissue disease showing fragility complications
[113]. Human DSEL (C18orf4) is related to bipolar disorder [114] and depression
disorder [115]. Dse KO mice show easy skin fragility caused by the reduced IdoA
content in the CS/DS of the small Leu-rich PGs biglycan and decorin, known to
assemble collagen fibrils [116]. CS/DS IdoA-null double KO mice die upon birth
[117]. The amino sugar of the CS/DS is N-acetylgalactosamine (GalNAc), small
leucine-rich PGs that mostly carry DS chains. CS and DS GAG chains may exist as
separate entities but can also form hybrid structures along the same GAG chain. The
distribution of CS and DS domains has been shown in their tissue and regional
differences like in the brain.
The repeated disaccharide units of DS, linking to Ser residue of core proteins,
exert their functions. The common GAG-protein linker region is the tetrasaccharide
with the carbohydrate structure of GlcUA-Gal-Gal-Xyl-O-Ser- [108]. β-Xyl-T
(Xyl-T) encoded by XYLT-1 or XYLT-2 catalyzes the transferring reaction of a
Xyl residue using the donor substrate UDP-Xyl to a certain Ser residue present in the
PG core proteins, which previously is biosynthesized through ER/cis-Golgi complex
network, which commences the DS, CS, and HS chain biosynthesis [118]. Two Gal
saccharides attached to Xyl-O-Ser-core proteins are formed from the donor UDP-Gal
by Gal-T-I and Gal-T-II enzymes that are expressed from their genes of B4GALT7
and B3GALT6, respectively [119]. Thereafter, β1,3-glucuronosyl-T-I (GlcAT-I)
expressed by its gene B3GAT3 adds a GlcUA residue using the donor
UDP-GlcUA to the acceptor substrate Gal-Gal-Xyl-O-Ser. B4GALT7 (GalT-I)
deficiency in GalT-I-encoding B4GALT7 mutation causes EDS-progeroid type
1 [120]. Phenotype includes an appearance aged, craniofacial dysmorphism, delayed
development, elastic skin, short stature, osteopenia, hypermobile joints, hypotonic
muscles, and wound healing dysfunction. Furthermore, homozygous mutations
found in B4GALT7 gene exhibit the similar EDS form and reduce DS side chain
lengths of decorin. The mutated fibroblasts showed the reduced sulfation level of HS
chains with retarded wound closure [120]. Thus, EDS-progeroid type 1 is caused by
defection of HS as well as DS. Homozygous mutation in B4GALT7 generates a
certain type of disease such as Larsen syndrome found in regional Reunion Island in
France with symptoms including dwarfism, facial features, hyperlaxity, and multiple
dislocations [108]. The known Larsen syndrome clinically displays congenital joint
dislocations and craniofacial abnormal dysfunctions including dislocated hip, elbow,
foot, and knee deformities. Therefore, genetic syndromes expressed as the Larsen in
Reunion Island and EDS-progeroid type 1 show common joint dislocations, but the
reason why the B4GALT7 mutation makes the two disorders is not known. Another
GalT-I-encoding B4GALT6 synthesizes the common linker region tetrasaccharide,
GlcUA-Gal-Gal-Xyl- in CS/DS and HS saccharides (Fig. 3.5). Patient-derived
fibroblast cells, which have the heterozygous mutations in GalT-I, synthesize
low-glycosylated decorin and biglycan PG core proteins with shorter DS chains
[121]. B3GALT6 (GalT-II) deficiency shows defects in DS, HS, and CS through
mutations and influences the development of the skeleton and skin with different
symptoms.
3.8.3.2 Deficiency Syndrome of DS
The repeat of disaccharide units of the chondroitin repeat (-4GlcUAβ1–3GalNAcβ1-

)n chain is formed by enzymatic reaction of ChSy family [122]. DSE encoded by the
specific genes of DSE or DSE2 epimerizes the GlcUA residue to IdoUA residue
through the GlcUA C-5 hydroxyl group epimerization [83]. Dermatan chains are
sulfated by specific enzymes of dermatan 4-O-sulfotransferase-1 (D4ST1) and UST,
encoded by different genes of CHST14 and UST, respectively. Among them, the
enzyme D4ST1 transfers the sulfate group using the substrate PAPS to the substrate
GalNAc C-4 in DS [123], while UST transfers a sulfate group using PAPS substrate
to the substrate IdoUA C-2 of DS [124]. UST deficiency by lack of the UST gene
causes EDS-related symptoms.
DSE deficiency patients are featured of collagen bundles with collagen fibrillar
proteins, the intermittent small flowered fibrils, and granulofilamentous deposits
[125]. DSE-lacking patients show a mild symptom of the EDS musculocontractural
type, compared to the CHST14-negative patients. The DSE deficiencies influence
the DS biosynthesis. Dse/ mice mutants synthesize a less amount of IdoUA
residues in the skin tissue and consequently weaken the collagen fibril strength. In
addition, DSE enzyme is efficient in forming IdoUA blocks of DS, while DSE2 is
more effective to form a CS/DS hybrid chain compared to IdoUA units
[83]. CHST14 (D4ST1) deficiency leads to a retrograded epimerization reverse-
converting IdoUA residue to GlcUA residue, and this contributed to the
DSE-generated chondroitin production, which is 4-O-sulfated by C4ST enzyme to
GalNAc residues present in chondroitin, because CS and DS 4-O-sulfation acts as an
inhibitor of DSE [126]. Lack of CHST14 causes an autosomal recessive disorder.
Chst14/ mutant mice exhibit lower body weights, fragile skin, kinked tails, and
low fertility. Moreover, Chst14/ mutant mice are featured with the impaired
growth of neural stem cells, defected neurogenesis, and change in glial cell
populations [127]. These phenotypes are similar to the D4ST1-deficient EDS
patients.
3.8.4 Keratan Sulfate (KS)
Keratan sulfate (KS) disaccharides consist of β-D-GlcNAc and β-D-Gal units. KS is

a linear polysaccharide form of LacNAc, Galβ1-4GlcNAcβ1–3, which is Gal and
GlcNAc C-6 sulfated [128]. The KS disaccharides can have the six-positional
sulfation at most units, although sulfation at GlcNAc is much more frequent. The
KS carbohydrate backbone is elongated by two distinct GTs of β1,3-N-GlcNAc-
transferase (β3GnT) and β1,4-Gal-Transferase (β4GalT) [129]. Sulfation of the chain
is performed by two sulfotransferases like KS Gal-6-sulfotransferase (KSGal6ST) to
Gal. GlcNAc6ST-1 and GlcNAc6ST-5 (CGn6ST) sulfate GlcNAc in the brain tissue
and cornea. In humans, β3GnT has eight genes and β4GalT has seven genes. Among
these, specific β3GnT7 and β4GalT4 are known for KS chain elongation, because
β3GnT7 and β4GalT4 have higher activity for sulfated than non-sulfated substrates
[129, 130]. However, there is no direct evidence to support of this possibility. KS is
synthesized by two stages. The first stage is the GlcNAc-sulfated poly-N-
acetyllactosamine chain synthesis by β3GnT7, β4GalT4, and GlcNAc6ST-5. The
second stage is highly sulfated KS synthesis via Gal sulfation by KSG6ST. Most
cells do not express KS-specific sulfotransferases in culture [131]. KS is generated
by elongating the N-/O-glycans linked to scaffold core proteins [128]. KSPG is
mainly expressed in the ECM or extracellular surfaces in the cornea, cartilage, and
brain. The approximate MW of KS has been estimated around 20 kDa; therefore,
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approximately 45 disaccharide units are assumed as the commonest structured

MW. KS is used as an active component present in protective eye drops used for
dysfunctional vision. KS is enriched in human cornea tissue. Human central nervous
system (CNS) and peripheral nervous system also consist of the KS with low
amounts [132]. KS-PG and KS display cellular regulatory roles in epithelial cells
and mesenchymal cells as well as bone and cancers. KS contains sulfated chain with
non-sulfated poly-LacNAc, monosulfated and disulfated disaccharides. Historically,
KS was initially discovered in the corneal region for the first time by Suzuki in 1939,
as a mucoid with Gal and Glc as well as acetyl and sulfate groups. Karl Meyer
determined the mucinous mucopolysaccharides designating keratosulfate [133].
Does KS-PG protect radiation-mediated apoptosis? In human Burkitt’s lym-
phoma cells, expression of PAPS transporters (PAPSTs)-1 and -2 (PAPST1 or
PAPST2) reduced radiation-induced apoptosis. Cleavage of KS chains by keratanase
increases radiation-mediated apoptosis. The KS-mediated apoptosis depends on
6-O-sulfation of GlcNAc residues. GlcNAc-6-O-sulfotransferases (CHST2,
CHST6, and CHST7) inhibit apoptotic cell death. PAPST1 increases in the phos-
phorylation level of p38 MAPK and Akt. Therefore, GlcNAc 6-O-sulfation reaction
in KS decreases radiation-induced apoptotic cell death [134]. The sulfation in KS has
been summarized in protection from apoptosis (Fig. 3.7).
3.8.5 Heparin and Heparan Sulfate
The heparin (Hp)/heparan sulfate (HS) disaccharides consist of four-linked UA and

four-linked α-GlcN unit. HS and Hp carbohydrate structures are different from only
in their proportions of the composed monosugar and dissugar. HS bears β-D-GlcA as
a key UA form, whereas Hp bears α-l-IdoA residue. GlcA residues in HS are
replaced by GlcNAc units, but low level of GlcN-N-sulfated residue and limited
level of unsubstituted GlcN residue are also present. Hp predominantly bears IdoA
2-sulfated residue (IdoA2S) with N,6-disulfated GlcN units. The GAGs can be
utilized as potentially therapeutic drugs in the forms of the potent antithrombotic
and anticoagulant Hp [135]. In fact, mast cell-enriched tissues including swine and
cattle intestinal mucosa are the sources of such pharmacological Hp. Hp is currently
the widely used GAG substance as the most common therapeutic glycans through
the world to prevent and treat the thromboembolic prophylaxis. The Hp is simply
prepared [86], and currently 20 more mammalian HS-PG core proteins are known
[136]. Among GAGs, HS mediates intracellular signaling, Wingless, Hedgehog, and
FGF pathway. Therefore, the damaged HS synthesis causes for developmental
defect, as observed in Drosophila. During the studies, Hp has been the first GAG
having biological function. Hp is the anticoagulant. The FGF recognizes the HS
chain of the syndecan proteoglycan and consequently activates the FGFR of cells
[137]. Heparanase digestion of cell-surfaced HS and matrixed HSPGs promotes
invasion and metastasis [138].
3.8.6 Hyaluronic Acid (HA) or Hyaluronan
HA as a sole GAG form is not sulfated and consists of repeated disaccharide units of
β-D-GlcA residue and β-D-GlcNAc residue. HA has the longest chain among all the
known GAG types. The MW of HA is approximately above 100 kDa, and the
polymerization degree of HA is, therefore, in the range in about 255 disaccharide
units per chain up to several million of MW [139]. This extremely high MW
polysaccharide has high viscosity even at lower concentrations. Hyaluronan
synthase 2 overexpression promotes ErbB2 signaling and progression of breast
cancer [140], while its suppression inhibits tumorigenesis and progression
[141]. HA influences the metastasis of mouse mammary carcinoma cells [142].
3.8.7 Proteoglycans (PGs)
PGs are GAG-linked proteins. GAGs, except for keratan 6 sulfate, are linked to a Ser
in PG protein through the tetrasaccharide GlcAβ1,3Galβ1,3Galβ1,4Xyl-liker. The
GAG’s protein core simply acts as a scaffold for GAG activity. PGs are often large in
size with heavy glycosylation and membrane attachment. There are about 50 distinct
PG genes except for alternative spliced proteins [143]. Although most PGs are N-
and O-glycoproteins, however, the only PG definition is based on O-linked GAG
chains. Most PGs function mainly at the extracellular area and act for the cytokines,
chemokines, growth factors, and morphogens. They also modulate embryonic
development, pathogen-infectious inflammation, and cell-cell communication
[144]. PGs also influence growth factor action, collagen fibril formation, tumor
cell behavior, and corneal transparency for vision.
GAGs nonspecifically interact with proteins, and they are located on cell surfaces
as proteoglycan forms and adhere to soluble forms of polypeptides such as growth
factors via electrostatic interactions. For the most important cellular function, GAGs
stabilize growth factors [108]. Some GAGs act as co-receptor for the growth factors
or directly bind to cellular receptors or via growth factor sequestration. The GAGs
can be used for diagnostic markers and also for targets of potential therapy in
cancers. For the application, infrared and Raman spectroscope analysis and
bioimaging analysis have been developed to distinguish GAG class. Defect of
GAG synthesis leads to diseases such as connective tissue disorder and Ehlers-
Danlos syndrome that displays hereditary multiple exostoses. This syndrome
involves in inappropriate chondrocyte proliferation and bone growth. GAGs
increase cell adhesion and cancer cell invasion. GAGs regulate cell functions
through transforming growth factor (TGF) and FGF signaling [72]. Certain corneal
dysfunction is caused by sulfated GAGs. Mutation of CS and DS biosynthetic
enzyme genes generates connective tissue-defected diseases [145]. GAGs and pro-
teoglycans regulate cancer progression. CSPGs activate the melanoma growth
[146]. CS inhibits the migration of transendothelial monocytes and consequent
angiogenesis [108]. In stem cells, GAGs and PGs are biomarkers of progenitor
cells [147]. GAG and PGs give “stemness” of stem cells, as evidenced by CSPG
role in neural stem cells [148]. HSPG and CSPG also give stemness in hematopoietic
precursor cells [149].
3.8.7.1 Intracellular PG Type of Mast Cell Granule Serglycin
The Hp-containing PG serglycin [150] is an unusual PG type because of its

existence only in the mast cell granules and mast cell-related cells but not in the
ECM. Mast cell-secretory granules contain Hp chains attached to the small peptide
of serglycin [151]; consequently, Hp is partially fragmented during mast cell
degranulation. Extremely small Hp fragments are the LMWH products such as
enoxaparin. Mast cells contain granules packed with secretory proteins. The PG
serglycin carries GAG side chains including mainly Hp, but sometimes CS or
DS. The granules are closely packed by the help of the PG serglycin, and its GAG
side chains [152]. The intracellular GAGs are stored in mast cell granules. In rare cell
type, basophils also have granules. Serglycin is the mast cell granule PG and has a
small protein core. The human sequence is 158 amino acids long with a signal
peptide in the N-terminal region. Serglycin contains a central domain with Ser and
Gly alternate. The human sequence has 8 Ser [153]. Ser residues carry
galactosaminoglycan side chains of chondroitin-4-sulphate (CS-A) or DS (CS-B)
or GAG chains of the HS/Hp family. Rat peritoneal mast cell Hp PG, serglycin,
carries about 750 kDa with 75 kDa Hp chains attached to a protein core
[154]. Serglycin has a different GAG substitution.
3.8.7.2 PGs of Syndecans and Glypicans on Cell Surfaces
There are two distinct PGs of syndecan and glypican expressed on cell surfaces.
Several ECM PGs include small Leu-rich PGs (SLRPs) like decorin, biglycan, and
lumican as well as aggrecan. GAG chains are less dense, and the extracellular
surfaced GAGs linked to glypicans or syndecans likely act as signaling molecules,
or in tissue remodeling. The cell surface glypican PGs function as modulators or
morphogens of functional action of bone morphogenetic protein, FGF, Sonic Hedge-

hog, and Wnt. A total of six mammalian glypican genes are known for the core
proteins of glypican-1 to glypican-6. They are linked to the outer membrane region
through a C-terminal region of GPI anchor [155]. Another cell surface PG,
syndecans, includes four mammalian syndecans. Syndecans are composed of a
TM region and a cytosolic domain, which recognizes cytoskeleton proteins and
protein kinases [156]. Syndecans collaboratively with integrins and hyaluronan
signaling via CD44 increase cancer cell motility [157]. GAGs increase metastasis
and angiogenesis. Syndecan-1 increases the cancer cell adhesion to lymphatic
endothelium [158]. Decorin regulates EGFR signaling and proliferation in mela-
noma [159]. Decorin binding to VEGFR-2 indicates its antagonistic role [160],
because decorin acts as an antagonist to VEGFR-2. In fact, 12-amino acid
oligopeptide present in the decorin Leu-rich-repeated 5 domain is the binding site
for the VEGFR-2. Consequently, VEGF-VEGFR-2 binding is antagonized
[157]. Lumican glycoprotein inhibits melanoma cell migration [161]. GAGs linked
to PGs on cell surfaces help viral invasion. Enveloped proteins of yellow fever and
dengue viruses, which belonged to flavivirus family, recognize GAGs on surface
[162]. Viral carbohydrates also bind to the cell surface receptor such as
DC-SIGN [163].
3.8.8 Extracellular PGs
3.8.8.1 Aggrecan
Aggrecan is mostly abundant as PG in tissue ECMs like the cartilage, where

aggrecan associates with big HA-aggregated complexes [164]. Thus, this is a target
for regenerative medicine. For example, the CS-linked core aggrecans are abun-
dantly present.
3.8.8.2 Perlecan
Multiple domain-containing PG perlecan is present at the region of cellular base-

ment membrane or at the interspaces between pericellular region. Perlecan as a
strong HS-consisting PG function as growth factor stores like FGF and in angio-
genesis event upon binding to VEGF [164]. Perlecan loss inhibits the cancer growth
such as colon carcinoma cells and tumor angiogenic progression [165].
3.8.8.3 Small Leu-rich PGs (SLRPs) of Decorin, Lumican, and Biglycan
The SLRPs include decorin that acts to wrap tendons adjacent to D-band of
collagen fibrils [166]. SLRPs form the structure of the cornea and transparency.
3.9 Glycosylphosphatidylinositols (GPIs) Anchor Glycosylation 89
The SLRP lumican bearing three N-linked KS chains leads to ordered collagen
fibrils of the cornea [167]. Decorin (DCN) deficiency KO mice show the EDS-like
phenotype [168]. However, decorin core protein-encoding DCN gene mutations do
not cause EDS. But DCN mutation yields only the truncated decorin core protein
with limited functions of decorin. Decorin is a prototype SLRP with a DS side chain
responsible for autophagy, collagen fibrillogenesis, tumor growth, and wound repair
[169]. C-terminal domain of decorin functions for maintenance of the cornea fibrillar
reorganization. Dcn/ mutant mice are featured with impaired collagen morphol-
ogy and skin weakness and fragility [168], resembling the EDS-like symptoms.
Dcn/Bgn double deficient mutant mice also exhibit skin fragility and osteopenia
similar to the EDS-progeroid-like form [170]. The decorin KO mice exhibit abnor-
mal collagen morphology and human EDS-like pattern [168]. In addition, the double
decorin and biglycan KO mice show the human EDS-like progeroid type
[170]. Lumican has four sites for KS. The corneal lumican is a KSPG with a
molecular weight range of 70–300 kDa KS, giving corneal transparency. In contrast,
skin dermal lumican is a glycoprotein with a 57 kDa [161]. Biglycan (BGN) two
missense mutations cause the spondyloepimetaphyseal dysplasia as an X-linked
inheritance disease in tropical families including Korean, Indian, and Italian with a
short stature and joint osteoarthritis [171]. BGN-deficient mice reduce growth and
bone mass, promoting myofibroblast differentiation and proliferation through TGFβ
and SMAD2 signaling [172]. The KO mice are used as models for human
spondyloepimetaphyseal dysplasia or Meester-Loeys syndrome for therapeutic
agents for these disorders.
3.9 Glycosylphosphatidylinositols (GPIs) Anchor

Glycosylation
3.9.1 General Structure of GPI Anchors
GPIs, ubiquitous surface-anchored molecules in eukaryotes, have a basic role,

simply to anchor to cell membranes. The main question of “what is GPI membrane
anchor? is the conceptional long subject in the membrane protein diversity in
organisms. In a single word, GPI stands for glycosylphosphatidylinositol. In other
words, a phosphatidylinositol (PI) phospholipid linked via a glycosyl, or sugar
chain, component to the protein C-terminal region. What are GPI membrane
anchors? GPI stands for glycosylphosphatidylinositol. In other words, a PI phos-
pholipid linked via a glycosyl, or sugar chain, is a component of the protein in the
C-terminal region. During the transglycosylation reaction, GPI species is linked to
cell wall proteins. Cell wall β1,6-glucan is cross-linked to proteins via its GPI
glycan. How are proteins bound to membranes? Chemical and enzymatic reactions
of GPI anchors are different from humans and parasites. Differences are present in
parasite and host GPI pathways, and therefore, selective inhibitors of the T. brucei
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Membrane-linked GPI-AP with N-/O-glycans. (c) Glycan structures of basic GPIs
GPI pathway can be designed. Before understanding of the GPI structures, several
basic components are figured out. For example, phosphocholine,
phosphoethanolamine, and ethanolamine are basic components. Among them, eth-
anolamine is a functional group of phospholipids and this is abundant (Fig. 3.8).
GPI anchors are holders of surface proteins anchored in eukaryotic membranes.
GPI anchors are used for tethering candidate molecules to the exposed extracellular
leaf of the lipid bilayered plasma membrane (PM) via their carboxyl termini. GPI
anchors are frequently synthesized in most eukaryotes including fungi, invertebrates,
protozoa, plants, and mammals [173]. GPI anchors are biosynthesized via the
membrane-associated enzyme complex. Lastly, the inositol-linked palmitic acid is

cleaved off. Phospholipase C specific for phosphatidylinositol (PI-PLC) cleaves and
releases the peptide part from the cellular PM [174]. The known GPIs consist of a
common component of an ethanolamine-phosphate (EtN-P), three Man residues, a
glucosamine (GlcN) as a non-acetylated form, and inositol phospholipid (IP or PI).
The GPI anchors contain an ethanolamine-phosphate-
6Manα1,2Manα1,6Manα1,4GlcNα1,6myo-inositol-1-phosphate-lipid. Peptides are
linked to amino group in ethanolamine through the NH-CO linkage with C-terminal
carboxyl groups. GPI-tethered proteins are removed from the membrane by PI-PLC
enzyme. The substitution of palmitic acid on the myoinositol C-2 position inhibits
the PI-PLC activity. GPI-anchored proteins (GPI-Aps) are the only forms of post-
translational modification by glycolipid. Fatty acid chains, which are linked to
inositol phospholipids, are embedded into the PM outer leaflets of cells. However,
protein parts are not directly embedded into the PM of cells. More than approxi-
mately 150 proteins of human membrane proteins are reported to be such GPI-APs
[175], having their distinct functions. There are many GPI-anchored proteins known
from mammals, protozoa, plants, and lower eukaryotes including yeast, fungi, and
slime molds, as shown in Table 3.3. 40 more number enzymes are GPI-anchored and
include alkaline phosphatases; RECK protease inhibitors; transcytotic transporters;
50 -nucleotidase; dipeptidase; cellular adhesion molecules (CAMs) including CD48,
contactins, and glypicans; receptor proteins including GDNF receptor-α series,
folate receptors, and FcγR-IIIb; and complement regulatory proteins including
CD55 and CD59 as decay-accelerating proteins. GPI-Aps are often associated
with lipid raft-microdomains of cellular PM. They are transiently homodimerized
or released by enzymatic cleavage from the GPIs [167, 176, 177] and, also, sorted
apical directions in certain cells upon polarization stimulation [178]. GPI-APs are
required for fertilization, embryo formation, development, neural formation, and
immunity [84, 179–181].
3.9.2 Function of GPI-Anchored Protein
For example, CD24 is the cell’s surface antigen consisted of protein and sugar
residues. It is also localized at cell’s surface by GPI-anchored link. CD24
GPI-anchored antigen is a neural surface molecule known as a heat-stable antigen.
The CD24 has many broad roles in the cells, as many scientists work with the CD24
to identify its interaction molecules and functions. CD24 is also related to cell
proliferation, neuronal development, lymphocyte activation, and other cellular pro-
cesses. It is named a nectadrin and small cell lung cancer antigen cluster-4
[182, 183]. The roles of CD24 in neuronal development and neuronal diseases
have been explained at the level of the intracellular signaling. CD24-mediated
signaling uncovered its systemic involvement in neural migration, neurite extension,
and neurogenesis (Fig. 3.9) [183]. Overexpressed CD24 also inhibits DAMPs with
Siglec-10 cis-interaction. The multiple roles of the CD24 are caused by its glycan
Table 3.3 Examples of GPI-anchored proteins

GPI-anchored protein Function
Protozoa
T. brucei variant surface glycoprotein (VSG) Protective coat
Leishmania major promastigote surface protease (PSP) Bound complement degradation
T. cruzi GPI-anchored mucins Host cell invasion
P. falciparum merozoite surface protein 1 (MSP-1) Erythrocyte invasion
Toxoplasma gondii surface antigen 1 (SAG-1) Host cell invasion
Entamoeba histolytica GPI proteophosphoglycans Virulence factor
Yeast, fungi, and slime mold
S. cerevisiae α-agglutinin Adhesion molecule
S. cerevisiae GAS1p Cell wall biogenesis
Aspergillus fumigatus GEL 1p Cell wall biogenesis
Candida albicans HWP1 Adhesion molecule
Dictyostelium discoideum prespore antigen (PsA) Adhesion molecule
Dictyostelium discoideum contact-site A (CsA) Adhesion molecule
Plants
Pyrus arabinogalactan proteins (AGP) Cell wall biogenesis
Arabidopsis thaliana metallo- and aspartyl proteases Pollen tube development
Arabidopsis thaliana β1–3 glucanase Cell wall biogenesis
Mammals
Erythrocyte CD59 and CD55 DAF Complement regulation
Alkaline phosphatase Cell surface hydrolase
50 -Nucleotidase Cell surface hydrolase
Renal dipeptidase Cell surface hydrolase
Trehalase Cell surface hydrolase
NCAM-120 Adhesion molecule
NCAM TAG-1 Adhesion molecule
CD58 Adhesion molecule
FcgIII receptor Fc receptor
Ciliary neurotrophic factor receptor (CNTFR) a subunit Neural receptor
Glial cell-derived neurotrophic factor receptor (GDNFR) Neural receptor
CD14 LPS receptor
Prion protein, PrP Unknown
Glypican family of GPI-anchored proteoglycans ECM
CD24 (heat-stable antigen, nectadrin) SCLC antigen cluster-4
chains. The CD24 molecular weight is a 20–70 kDa protein, while its predicted
molecular weight from its DNA and amino acid sequences is 3 kDa, which is smaller
than the cellular protein forms. The difference is derived from CD24 sugar chains.
CD24 protein core is glycosylated at different sites with different sugar sequences.
Murine CD24 contains seven distinctly predicted O-glycan sites, and three N-glycan
sites. Because it has many different glycan structures, the CD24 interacts with many
different cellular receptors and shows diverse functions. The CD24 has a variety of
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Fig. 3.9 CD24 cis-recognition and interaction in neuronal development process. The illustrative
pathway has been described for CD24-involved signaling at the plasma membrane proteins and
co-stimulatory factors, signaling transducers with influenced phenotypes (deduced from Ref. [182])
roles, where one of the cells that function is neuronal cells. CD24 is known to
involve neuronal migration, neurite outgrowth, and neurogenesis. CD24 is highly
expressed in developing brain stage in the neuroepithelial migratory zone, but lowly
in the ventricular zone. CD24 expression is activated in postmitotic neurons during
the period of migration. Also, in humans CD24 is expressed highly in neural stem
cells in order to differentiate to neuroblasts and neurons. CD24 has multiple poten-
tials to activate or suppress outgrowth of neurites. CD24 expressed in cerebellar,
hippocampal, spinal, and cortical non-motor neurons promotes neurite outgrowth,
while in retinal, dorsal root ganglia, or motor neurons, it’s expression is inhibited.
Three CD24 receptors, L1, contactin, and TAG-1, are known. L1 binds to α-2,3-SA
and others bind to Lewis X antigen. TAG-1- and contactin-deficient mice do not
exhibit neurite outgrowth at cerebellar neurons. On the other hand, in dorsal root
ganglia, CD24 inhibits neurite outgrowth, and CD24 associates with clusters of L1
and contactin or L1 and TAG-1. These parts are characteristics for the inhibited
neural outgrowth and the destined nodes of Ranvier. Therefore, promotion and
inhibition of outgrowth are determined, depending on the neurons which are mye-
linated or not. CD24 also interacts with many signaling molecules such as MAPK,
NF-kB, Notch and Hedgehog, and other networks. With many signaling pathways,
CD24 potentiates cell proliferation, growth, and differentiation and sometimes
induces cancerous behaviors. Nonetheless, the relationship between neuronal behav-
ior and CD24 signaling is still not revealed. Further works will allow to know
us. Neuronal diseases such as multiple sclerosis (MS) are also related to CD24.
MS is a chronic inflammatory disease with widespread loss of myelination and
axons, as depicted to CNS and autoreactive lymphocytes. CD24 regulates T cells
and lymphocytes, as CD24 expression on CNS-resident lymphocytes increases
autoreactive T cells. In addition, in neural cancer, CD24 importantly regulates

development, invasion, and metastatic progression. In glioblastoma CD24 activates
Src kinase to upregulate the growth and invasiveness of glioma.
Another CD52 resembles to CD24. CD24 with cooperation of Siglec-10 specif-
ically suppresses damage-caused immune responses in injured tissues. CD52 is a
classical GPI-anchored glycoprotein present in hematopoietic lineage cells including
monocytes, DCs, B cells, and T cells [184–186]. In T cells, they release the soluble
form of CD52 after absolutely activation. Soluble CD52 can bind to SA-binding
immunoglobulin-like lectin (Siglec-10) with its N-linked glycan motif, and this
downstream signal attenuates the TCR signaling pathway resulting in T-cell sup-
pression. Siglec-10 has an ITIM motif and thus plays an inhibitory role due to its
ITIM motif in most of immune cells like B cells, NK cells, DCs, or monocytes. In
addition, it has a homology to a murine Siglec-G with a similar structure and
function. Siglec-10 alerts the pathogen and regulates p38 and MAPK pathway to
exert anti-inflammatory function like IL-10. Siglec-10 is a key molecule during
C. jejuni infection in humans. Moreover, Siglec-G expressed on DCs inhibits CD8
T-cell proliferation. For example, when the pathogens break into the host cells,
Siglec-G recruits the SHP-1 increasing degradation in phagosome. This pathway
inhibits T-cell proliferation. A DAMP protein, HMGB1, mediates the formation of
CD52-Siglec-10 complex. In contrast, other types of DAMPs such as HSP70 and
HSP90 cannot form the CD52-Siglec-10 complex. As the HMGB1, DAMP can
initiate the noninfectious inflammation to express proinflammatory cytokines
through MyD88 and NF-kB signaling pathway. Especially, HMGB1 interacts with
several immune-related molecules to upregulate the expression of cytokine genes.
HMGB1 has two motifs of BOX A and BOX B with each distinct function. BOX A
has a p53 transactivation-binding domain, and BOX B has a TLR4-binding cytokine
region. Interestingly, Box B is related to the Siglecc-10 functions. If T-cell immune
response is overactivated, soluble 52 in T cells binds to Siglec-10 through HMGB1
as a DAMP. In the Siglec-G-deficient mice, inflammatory cytokine expressions are
increased. Siglec-10 suppresses soluble CD52 in hyperactivated immune response.
The authors of this paper showed one of the DAMP proteins, HMGB1; no other
type of DAMPs such as HSP70 and HSP90 is required to make CD52-Siglec-10
complex. The inhibitory receptor Siglec-10 recognizes the soluble CD52 and regu-
lates activation of T cells. In fact, T cells release the soluble form of CD52 upon
activation. The N-linked glycans present in soluble form of CD52 released from T
cells are recognized by Siglec-10 and attenuate the TCR signaling for T-cell
suppression. The Box B domain, not Box A, in HMGB1 having proinflammatory
function specifically binds to CD52 and consequently elicits Siglec-10 binding to
N-glycans attached to CD52, recognizing only α-2,3 SA-Gal linkage, but not
O-linked or N-linked α-2,6 SA linkage. CD52-HMGB1-Siglec-10 trimolecular
complex recruits SHP1 and interacts with the T-cell receptor. CD52-bound Siglec-
10 is then phosphorylated in ITIM motif and associates with SHP1. The phosphor-
ylated SHP1 inhibits the TCR signaling by decreasing phosphorylation level of
TCR-associated Tyr kinases such as Lck and ZAP-70. This is the mechanism of
T-cell suppression [187]. The CD52-HMGB1-Siglec-10 interacts with the TCR and
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wiki/N-linkedglycosylation
attenuates the hyperactivated T-cell response. For the mechanism with CD52-
HMGB1-Siglec-10 complex, HMGB1 protein is essential for suppression by
CD52. Soluble CD52 can be used as a therapeutic agent. Soluble CD52 requires
HMGB1 Box B to bind to Siglec-10, and α-2,3 sialylated CD52 is crucial for
HMGB1 interaction and suppressor function. SA is required for the complex
formation. The schematic illustration of the CD52 glycan recognition to the
proinflammatory B box of HMGB1 has been expressed for engagement of the
Siglec-10 toward downregulation of human T-cell function (Fig. 3.10). This effect
is significantly decreased when HMGB1 antibody was treated. Altogether, this
mechanism underlying the T-cell suppression provides homeostasis of inflammatory
responses as a possibility as therapeutic target in inflammation-associated diseases.
3.9.3 Biosynthesis, Structural Assembly, and Transportation

of GPI-Anchored Protein
The overall GPI-AP biosynthetic pathway has been established with their structural
remodeling and transporting mechanism(s). GPI species is generated through the ER
synthetic pathway through amination by specific GPI-transamidase using en bloc
transferred target proteins. Regarding biochemical synthesis, GPI formation in the
ER is started from free phospho inositol species and transamidated en bloc to
proteins through enzymes produced by PIG genes. The initial two synthetic steps
are found in the ER cytoplasm region, and the synthesis event is continued on the ER
lumen side. For GPI attachment, precursor proteins bear their GPI-attachment
signals with three distinct regions of a ω site, a hydrophilic spacer, and a
hydrophobic region. The protein precursor candidates are independently prepared

and catalyzed by the complex of GPI-transamidase enzyme. Therefore, the precursor
proteins are posttranslationally modified for attachment to GPIs by the complex
enzyme. Later, the glycan and lipid parts are further matured through multiple genes
called post-GPI-attachment to protein (PGAP) genes. Once proteins are
posttranslationally modified with GPI, structural shift between the GPI glycan and
lipid occurs by PGAP genes. The acyl chains are generally the palmitic acid in
GPI-AP, indicating most acyl chains are palmitic acid in GPI-AP and the palmitic
acid is cleaved by a deacylase enzyme, called PGAP1. The deacylase named PGAP1
cleaves off the acyl chain from the inositol backbone. Similarly, PGAP5 cleaves a
phosphoethanolamine (EtN-P) from Man-2 residue, which is responsible for recog-
nition and delivery of p24 family protein to the ER. When GlcN-PIs are flipped
across the ER lumen side, GlcN-PIs are additionally modified with an inositol acyl
chain. PGAP5 cleaves off an EtN-P from Man-2 to afford the sorting to the
ER. During trafficking via secretory vesicles, which is initiated at the ER sites, to
the Golgi lumens and cell surfaces for fatty acid modification, COPII vesicles are
cooperated with GPI-Aps, where the COPII vesicles are associated with Sec24C and
Sec24D proteins. Therefore, the GPI-APs are fused with COPII vesicles with
cooperation with Sec24C and Sec24D isoforms, for transportation to the cell sur-
faces via the Golgi network which further remodels structures such as fatty acid
remodeling. GPI-APs delivered to the Golgi network are further remodeled through
the process of fatty acid remodeling for the GPI-AP-lipid rafts. PGAP3 removes the
unsaturated lipid chain at the sn-2, and PGAP2 adds a saturated lipid chain like
stearic acid chain. Sequential modification of the GPI lipid composition reorganizes
the membrane lipid raft-competent GPI-APs. The PI in the GPI has the sn-2 diacyl
and unsaturated lipids including 1-stearic acid and 2-arachidonic PI. For example,
1-stearic and 2-arachidonic PI are the diacyl PIs. When they are flipped into the ER
lumen side, GlcN-PIs are linked to an acylated fatty acid species, representatively
like palmitic acid, and the inositol, and thus, the lipid part is exchanged.
For synthesis of GPIs in the ER lumen, alkyl lipids such as alkyl-acyl donors
come from peroxisomes where alkyl lipids synthesized are used for the 1-alkyl-2-
acyl formation. Alkyl-acyl donors come from peroxisomes. PGAP1 cleaves an acyl
group from the GPI-AP inositol in order to recognize p24 family proteins and
GPI-AP delivery to the ERES and remodel glycans by PGAP5. GPI-APs in the
Golgi are further remodeled for fatty acid, which are associated with lipid rafts.
PGAP3 removes the unsaturated lipid linked to the sn-2 and PGAP2 adds the
saturated lipid chain. Biosynthesis of GPI is started on the ER cytoplasmic side by
GlcNAc transfer using the donor UDP-GlcNAc to the PI as the acceptor substrate.
Thus, the formed GlcNAc-PI is produced by GPI-GlcNAc transferase, a complex
monoglycosyltransferase [188]. GlcNAc-PI species is further subjected to the
deacetylation reaction to form the product glucosamine-PI by a deacetylase. Flippase
is essential for cell survival. During the GPI-AP biosynthesis, GPI is generated from
free PI resided in the ER via ten more reactions. 20 PIG genes involve in the
synthesis pathway. The initial two steps are operated on the ER cytoplasmic outlets,
and other continuing reactions occurred at the ER lumen side. Alkyl lipids are
supplied from peroxisomal organelle and converted to the 1-alkyl-2-acyl-lipid com-
ponent of GPIs anchored in the ER luminal side. Paroxysmal nocturnal hemoglo-
binuria (PNH) occurs at this step, caused by PIG-A defect. Current-reported
GPI-deficient disorder is raised by enzymatically defected PIG-M or PIG-V gene
mutations at gene levels. Among PIG genes, genetic defected deficiency in PIG-A,
PIG-M, or PIG-V gene causes for PNH and some GPI deficiency diseases. For the
GPI-synthetic gene-defected diseases, defected GPI genesis may block cell-surfaced
GPI-AP location and consequently cause early lethality in embryonic development.
However, inherited diseases also occur due to the restricted synthesis of GPI-APs.
The disease-causing factor in the patients is a point mutation of the transcriptional
factor Sp1-recognition site in the PIG-M gene 5-flanking region. The PIG-M gene
encodes the Man-transferase for the attachment of Man-1. The point mutation of
Sp1-binding cis-element on the 50 -flanking promoter region of PIG-M gene dimin-
ishes the PIG-M protein synthesis. The PIG-M promoter mutation-derived disease
phenotype expresses the seizures and venous thrombosis. Administration with a
histone deacetylase inhibitor, sodium butyrate, prevents and improves the PIG-M
phenotype with GPI-Ap distribution in cells. Another defected GPI-Ap derived from
the PIG-V gene mutation, where Man-2 is produced by the PIG-V gene for the
Man-transferase enzyme, has been known. The PIG-V gene mutation raises
hyperphosphatasia mental retardation (HPMR) disorder, termed Mabry syndrome.
The HPMR belongs to an autosomal recessive (AR) form of the inherited diseases.
The disease exhibits an abnormal retardation in mental behavior and facially abnor-
mal features. It shows extremely high alkaline phosphatase activity [189]. Just four
point mutations are known in the transmembrane domain gene and cause the reduced
PIG-V gene expression with the reduced location of surfaced GPI-Aps [190].
3.9.4 GPIs in Parasites
GPI anchors function for stable association of proteins with the plasma membrane,
but with measurable “off-rates” from the membrane and the potential to be shed by
phospholipases. The very high lateral mobility is potentially obtained on the plane of
the lipid bilayer. They insulate the protein domain from the cell interior because this
is important in protozoa. They participate in signaling through association with other
membrane-spanning components in lipid rafts. In human African sleeping sickness
of Trypanosoma brucei, Trypanosoma forms in blood smear from patient with
African trypanosomiasis. The surface coat is made of a dense monolayer of VSG.
Many protein variants are common GPI anchors. GPI anchors from T. cruzi act as
TLR2 ligands and TLR4 ligands.
With regard to innate immunity and malaria caused by Haemosporida, the life
cycle is complex. They undergo schizogony in the infected body of vertebrates and
gametogony in the intermediate hosts. Plasmodium in RBC does not interact with
cytosols but forms a called parasitophorous vacuole and proliferates in a way of
Fig. 3.11 Camillo Golgi

(1843–1926), an Italian
biologist and pathologist.
He studied on the central
nervous system at the
University of Pavia during
1860 and 1868. His
discovered a staining a black
reaction called Golgi’s
method or Golgi’s staining
in 1873. Several findings
including Golgi apparatus/
tendon organ/tendon reflex
were made. Golgi and
Santiago Ramón y Cajal was
a recipient of the 1906
Nobel Prize in Physiology
or Medicine for his
discoveries on the nervous
system structure. Adapted
from https://en.wikipedia.
org/wiki/Camillo_Golgi
schizogony. Periodically, they destroy erythrocytes or RBCs. Currently, only culti-

vable plasmodium strain is the Plasmodium falciparum, which is resistant to chlo-
roquine. In the liver, forms of sporozoites are produced, and one sporozoite produces
thousands of merozoite and they destroy the liver organ. They are released to blood
circulation and enter to RBC. This status is called malaria. Schizogony reproduces
through its multiple asexual fission. Innate immune receptors of host immune cells
rather mediate the systemic inflammation during malaria progression. Like most
infectious diseases, the pathology of malaria is also driven by inflammatory cyto-
kines. Consequently, cytokine production results in the symptoms of malaria with
fever, chills, rigors, headaches, myalgias, lethargy, and more relatedness. Malaria
pathology involves in the following three steps of the excessive production of
inflammatory cytokines with septic shock-like syndrome, severe anemia with RBC
destruction and destruction of infected red blood cells with erythropoiesis inhibition,
and adhesion of infected RBC on capillary blood vessels.
For the malaria toxin hypothesis, in 1889, Camillo Golgi (1843–1926), an Italian
biologist and pathologist, reported the malarial fever paroxysms. The paroxysms are
observed when the protozoan forms of schizonts rupture from erythrocytes of host
and release newly produced merozoites (Fig. 3.11). Innate immune receptors are
mediators of systemic inflammation and pathogenesis of malaria. Innate immune
receptors for malaria parasites define parasite targets for innate immune receptors,
identify relevant innate immune receptors, define their role on host-parasite interac-
tion and disease outcome, and elaborate prophylactic and therapeutic interventions
employing TLR agonists or antagonists. Plasmodium parasite components activate
innate immune receptors toward release of inflammatory mediators. Plasmodium
membrane GPI anchor functions as LPS-like roles and this is thus the malaria toxin.
Merozoite GPI anchor induces inflammation and TLR2 activation but to less extent
of TLR4. Malaria PAMP is the hemozoin that is the hemin known as protoporphyrin
IX’s crystalline polymer and as hemoglobin component. During the initial malaria
study, there were numerous questions on the malaria toxification. For example, how
does malaria detoxify the protoporphyrin IX (hemin)? What are the innate immune
receptors involved on hemozoin recognition?
3.9.5 GPI Interaction with TLRs in Malaria P. falciparum
What is hemozoin? It is a malaria pigment. Hemozoin is the crystalline breakdown

product of hemoglobin. It is how malaria detoxifies protoporphyrin IX, also known
as hemin. Hemozoin is present in parasitophorous vacuole. Hemozoin chemistry in
solution exhibits that hemin chloride can form β-hematin dimer by bonding between
carboxylic groups and heme synthetic hemozoin. Therefore, highly purified hemin
lacks cytokine-inducing activity. Hemozoin level in circulating phagocytes corre-
lates with disease severity. For the hypothesis, hemozoin has been regarded as a
potential candidate for a proinflammatory substance made by malaria. Toll receptors
act in the pathogenesis of malaria through the recognition. For example, the purple
sea urchin has 222 TLRs [188, 191]. Shizuo Akira discovered that TLR9 elicits
innate immune response upon binding to the malaria pigment hemozoin
[192]. When malaria parasites enter to red blood cells (RBCs), the malaria degrade
heme components of hemoglobins present in host RBCs and form a hemozoin (HZ),
which is a heme polymer with hydrophobicity. The HZ is consequently liberated to
the plasma blood and circulated through the blood. The circulating HZ species are
consequently captured by the reticuloendothelial system and concentrated in the
system. The HZ species strongly induce the host immune responses, although the
acting mechanism(s) of the HZ regulation of the innate immunity is(are) not molec-
ularly clearly studied yet. However, the precisely studies evidences indicate that HZ
species isolated from the malaria parasitic P. falciparum act as a specific ligand for
TLR9, although the HZ is not a DNA-based ligand. The HZ activity to induce the
host innate immune responses was demonstrated. The HZ induces the expression of
cytokine/chemokines responsible for inflammation response. In addition, the HZ
upregulates co-stimulating receptors. In the TLR9-deficient, TLR9/, and
MyD88/ animals, the inflammatory induction of innate immune responses is
severely impaired. However, the other receptor-deficient animals including Toll/IL-
1R domain-containing adaptor-inducing IFN-β/, TLR2/, TLR4/, or
TLR7/ mouse are not impaired for induction of the inflammatory responses.
For the reverse demonstration, the experimental results obtained from the synthetic
HZ, which is purely synthesized without any contaminant, TLR9-dependently
exhibit in vitro induction of the innate immune responses. Interestingly, the pre-
scribed anti-malaria drug, chloroquine (CQ), abrogated the HZ-induced expression
of the related cytokines. Therefore, HZ is concluded to induce the TLR9-driven and
MyD88-associated innate immune responses, which is highly sensitive to CQ drug.

In addition, HZ is a key regulator of interactions between malaria and hosts. Natural
hemozoin activates cytokine production via TLR9/MyD88. The stimulatory activity
of hemozoin was destroyed by Dnase. Bacterial DNA is non-methylated and rich in
CpG, and immunologically active CpG-rich DNA is recognized by TLR9. Shizuo
Akira discovered for the first time that hemozoin constitutes the first non-nucleotide
ligand for TLR9. Thus, the cytokine-inducing component of hemozoin is DNA. PCR
analysis of hemozoin shows that most of its DNA is malarial. Is the DNA on
hemozoin human or malarial? Can malaria DNA actually activate cells through its
specific receptor TLR9? The biological property of TLR9 is complex. Although
TLR9 resides in the ER, TLR9 translocates to the endosomal compartment to bind its
ligand known as CpG-rich DNA. Then, does malaria DNAs activate innate immune
responses? In fact, malaria DNA is stimulatory for DCs only when introduced into
endosomal compartment of cells. Malaria DNA is stimulatory for pDCs only when
introduced into cells. The malaria genome contains 269 CpG repeats. Hemozoin
functions to traffic DNA into an intracellular compartment to which TLR9 can be
recruited. However, most malaria DNA is AT-rich. The malaria genome contains the
motif ATTTTTAC over 6000 times. Microarray analysis of 14 patients with febrile
P. falciparum showed an IFN signature. Hemozoin-DNA rapidly activates IFN-β
production in PBMC, but most malaria DNA is AT-rich. In addition, AT-rich DNA
mimics malarial DNA, and transfection of AT-rich DNA drives a variety of promoter
constructs like native DNA and activates cytokines such as TNF-α, IL-1β, IL-6 and
IFN-β. When six AT-rich motifs of AT1–AT6 have been studied, AT-2 is the
GCACACATTTTTACTAAAAC. Human PBMC produces IFN-β in response to
AT-rich DNA. AT-rich DNA activates type I IFNs independently of TLRs. AT-rich
DNA activates type I IFNs independently of TLRs but dependently on IRF1.
Infection with Plasmodium leads to a proinflammatory priming and
hyperresponsiveness of TLRs. In mice, TLR9 has an important role in promoting
this proinflammatory priming, which is mediated by IL-12 release by dendritic cells
and IFN-γ release by T and NK cells. Treatment with an antagonist of nucleic acid
sensing TLRs prevents cytokinemia and lethality in a rodent model of cerebral
malaria. Can we interfere with pathogenesis of malaria by blocking MyD88 activa-
tion? Phagocytized hemozoin colocalizes with TLR9 in the lysosomes. The inflam-
matory component of hemozoin is DNA. CpG and AT-rich DNA activate different
innate immune pathways [193, 194]. In step 1, internalization of hemozoin by
phagocytes leads to activation of MyD88 via TLR9 and leads to IFN-g production,
inflammasome formation, and caspase-1 activation. In step 2, IFN-γ priming of
phagocytes enhances expression of TLRs and pro-IL-1β, TNF-α production,
caspase-1 fragmentation, and maturation for inflammation. Therapeutic modulation
of nucleic acid sensing TLRs can be the potential target to prevent cerebral and blood
malaria symptoms [195].
For malaria hypothesis, hemozoin is internalized into the phagolysosome. TLR9
can be recruited to the phagosome, thus resulting in the proinflammatory mediator
genesis such as TNF-α, which causes fever. Later, its DNA fragments are liberated
and released from the cell surfaces via the crystal forms by the enzyme activities of
Modulaon of innate immunity by African Trypanosomes

Host
• VSG: variant surface glycoprotein
Infecon progress • SR-A: Scavenger receptor A
Macrophage • ROI: Reacve oxygen intermediate
(or Dendric cell) • RNI: Reacve nitrogen intermediate
VSG VSG
Th1 cell response
Gal Man P EtN Gal Man P EtN
TLR-dependent
signaling Gal Man During infecon Gal Man
Infecon progress
Gal Gal Man Gal Gal Man
High level of IFN-γ
Glc Ino Glc Ino
Th2 cell response P P

NFκB, MAPK signaling
Homodimerizaon and
GIP-sVSG
low level of IFN-γ (glycosylinositol
phosphorylaon of STAT1
(inducon of Type 1 IFN) phosphate-sVSG)
①
Pro-inflammatory cytokines An-inflammatory cytokines GPI-mfVSG
(TNF-α, ROI, RNI) (IL-10) New VSG molecules (glycophosphadylinositol)
expressed by variants
VSG VSG VSG
① regulaon of MP and DC
secreted cytokines profiles $3&
② alteraon of DC and MP ②
angen-presenng cell funcons
Unable to present
African Trypanosome
Fig. 3.12 A proposed modulation of innate immunity by African trypanosomes. Deduced from Ref
[196]
proteases and other enzymes. Then, why do humans get fever during malaria? The
search for the malaria toxin has been started through the world. What innate immune
receptors are activated during disease and what microbial products such as malarial
toxins activate these receptors? Is the source of inflammation a component of the
parasite outer membrane? Is malaria like LPS and Gram-negative bacteria? Is the
source of inflammation a component of the merozoite outer membrane? In other
words, is the malaria toxin just like LPS? The malarial parasite is coated with a GPI,
which activates TLR2. Malaria GPI structures trigger innate immune responses in
hosts. GPI anchors from T. cruzi are also TLR2 ligand and, to a lesser extent, TLR4
ligands.
GPI-anchored proteins of P. falciparum have the TLR-inducing activity. In the
protozoan infectious diseases such as Brucei group African trypanosomes or
malaria, the host innate immunity recognizes trypanosome PAMPs expressed as a
type of GPI-anchored and shed membrane-bound variant surface glycoprotein
(VSG) molecules. GPI substituents induce proinflammatory macrophages and DC
responses [196]. Next, the polarized VSG-restricted Th cells express type 1 cyto-
kines. GPI activates the host innate immune system (Fig. 3.12). In malaria, there has
been a big question of “Is there any source of inflammation as a component of the
merozoite outer membrane? In other words, is the malaria toxin just like Gram-
negative bacterial LPS? Currently, it has been accepted that GPI anchors activate
TLR2, as the malaria is coated with a GPI anchor. For example, GPI anchors from
T. cruzi are TLR2 ligand and, to a lesser extent, TLR4 ligands. GPI anchors from
P. falciparum have the similar TLR-inducing activity. For structure of malaria GPIs,
as they trigger innate immune responses in hosts, GPIs are frequently expressed in
protozoa rather than animals with immunostimulation of the host [197]. Such known
pathogenic protozoa include Trypanosomes, Leishmania, Toxoplasma, and Plasmo-

dium species. GPIs are the malaria pathogenic factor or malaria parasite-associated
molecular patterns (malaria PAMPs). The patterns are malaria molecular signatures
on outmost membranes. The primary structural units of GPIs are composed of an
ethanolamine-phosphate-substituted sugar moiety, EtN-P-
6Manα1,2Manα1,6Manα1,4GlcN, which is attached to α1–6-glycosidic PtdIns
[198] with structural diversity.
GPI in each different organism differs in the structure and composition of the
lipids and sugars and/or ethanolamine-phosphates. GPIs vary in the lipid compo-
nents including diacylglycerol part, 1-alkyl part, 2-acylglycerol part and ceramides),
inositol acylation, and ethanolamine-phosphate groups apart from Man C-6
[199]. Malaria GPIs are anchored with several proteins such as MSP-1, MSP-2,
MSP-4, MSP-5, and MSP-10 as well as rhoptry membrane proteins, which are
required for merozoite invasion to erythrocytes. The malaria GPI structure is differ-
ent from GPI species produced by humans. Human GPIs contain alkyl moieties at
ethanolamine-phosphate substituent on GlcN and β-GalNAc-Man. The GPI acyl
groups of human sources are large size with unsaturation at the carbon sites of C22:
4, C22:5, and C22:6 [198]. The variations are related with structure diversity with
different GPI roles. During infection, the schizont burst and merozoites are released.
Some merozoites are recognized by the host innate immune cells. The merozoites
invade RBC erythrocytes to survive [200]. Schizont components induce the innate
immune responses. Malaria GPIs activate the host innate immune system [198]
during malaria infection. Malaria-infected individuals have rarely anti-GPI anti-
bodies [201], providing protective effects against malaria illness. For example,
malaria, P. falciparum-bearing GPIs are regarded as malaria-producing toxins.
Host proinflammatory responses are induced by the host upon malaria GPI recog-
nition through TLR2 interaction. GPIs also interact with NKT and B cells toward
establishment of adaptive immune responses, thereby producing GPI-binding anti-
bodies of class M or G [198]. GPIs are released during malaria schizont burst. GPI
lipid moieties are extracted with hydrophobic solvents. GPIs are cleaved to sugar
part and PtdIns lipid part [202]. The question is: how GPIs are bound by TLRs
because GPI lipid moieties are not bound by TLRs?
In the aspects of GPIs and TLR-mediated signaling, GPIs activate PTK, PKC, and
three MAPK species such as ERK, JNK, and p38 and also NF-κB/c-Rel factor
toward proinflammatory responses [202]. GPI-anchored proteins also bind to host
lectin-like receptors to activate protein tyrosine kinase (PTK). GPI phospholipase D
releases diacylglycerol and it activates PKC. Malaria GPI-induced signaling has
been known in macrophage and TLR-/MyD88-knockout mice and GPIs are bound
by TLR2 or TLR4. In human HEK cells engineered, malaria GPIs bind to the TLR2/
1 heterodimer, while the sn-2 lipid-deficient GPIs switch binding capacity to TLR2/6
from TLT2/1 [202]. The scavenger receptor CD36 is a co-receptor for diacyl
peptides, where diacyl peptides are recognized by TLR2/6 [203]. Hemozoin differ-
ent from GPIs also presents parasite DNA to TLR9. TLR9 also recognizes parasite
factors. Parasite hemozoin activates plasmacytoid DCs through TLR9 [192] in such
a way that hemozoin is inactive but presents parasitic DNA to DC TLR9 for
References 103
proinflammatory cytokine expression. Parasite hemozoin binds DNA and the com-
plex recognizes TLR9.
3.9.6 GPI-Defected Disorders of Paroxysmal Nocturnal

Hemoglobinuria (PNH) and Prion Disease
PNH as a hematopoietic stem cell disorder is originated by a defected GPI-anchored

synthesis. PIG-A is defective in PNH patients, causing that PIG-A is the causing
gene for PNH. The major PNH symptom is anemia due to hemolysis caused by
intravascular erythrocytic destruction. The pathologic cause is derived from the
failed and incomplete surface location of complement regulators or GPI-APs
CD59 and CD55 (named DAF). The incomplete or failed expression of CD55 or
CD59 raises erythrocytes destroyed due to high susception to self-complement
attack. The defected expression of the surfaced GPI-APs of CD55 and CD59 is
caused by defects of the GPI-anchored synthesis. For example, the PIG-A as the GPI
biosynthetic gene is defected in PNH patients, as found in 1993. PIG-A is a causing
factor for PNH. PIG-A gene in the X chromosome causes the PNH, among the
20 more genes thought to be associated with the GPI biosynthesis. Genetic mutation
in the PIG-A gene raises the defected GPI formation. In contrast, other related genes
encoded on autosomes exhibit two site mutations for defected GPI synthesis. For the
therapeutic agent, a humanized MAb form of termed eculizumab was generated to
bind the complement protein C5, as the anti-hemolytic agent of PNH anemia. On the
other hand, prion disease forms insoluble plaques in neuronal cells in the brain. This
is involved in neurodegenerative spongiform encephalopathy. Proteinase-sensitive
cellular prion protein (PrPc) is shifted to an insoluble and proteinase-resistant prion
protein (PrPres) as the deposited plaques. PrPres is also the plaque-causing agent.
PrPc and PrPres are GPI-Aps crucial for the pathogenesis. PrPres is generated at the
cell surface through the endocytosis. Prion endocytosis through caveolin domain/
lipid rafts requires GPI. GPI anchors propagate and spread out misfolded Sup35,
known as an amyloidogenic yeast protein. This induces prion-like protease-resistant
aggregates. GPI anchor on PrPc is crucial for the infection of cells.
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Chapter 4
Glycans in Glycoimmunology
4.1 Glycans in Cell Recognition and Evolutionary

Adaptation in Organisms
Carbohydrates (or glycans) are ubiquitous and display a broad range of biological
functions and disease expressions. Without glycan-mediated events, any biological
aspect is not possible in living organisms. For example, protein folding, cell adhe-
sion, trafficking, signaling, fertilization, embryogenesis, pathogen recognition, and
immune responses require such glycan-mediated events. The structure of glycans is
complex, which is propagated and amplified by the stereoisomers, anomeric config-
urations, branched chains, and modifications by sulfation, methylation, and phos-
phorylation. This complexity is distinguished from genomics and proteomics. The
face molecules of organisms are glucans, and the functions are dependent from
glycan-protein and glycan-lipid interactions.
Glycan carbohydrate residues are especially well fit to form a wide range of
distinct sequences, due to the specific rings and chains with axial and equatorial
presence of the hydroxyl groups. Moreover, the anomeric positioned hydroxyl group
favorably forms α- and β-glycosidic linkages. This is one of the evolutionary
selections. Then, a question how diverse glycan structures are shared with various
eukaryotes is answered by the evolution of glycan-recognizing and processing pro-
teins including glycosidases, glycosyltransferases, sugar transporters, sugar-
nucleotide transporters, and glycan-binding lectins. Such processed glycans known
in eukaryotes are N-/O-glycans, C-mannose, glycolipids, and GAGs. Synthesis of
O-/N-glycan carbohydrates is also found in the microbes. The most well-evolved
microbe with the N- and O-linked carbohydrate synthesis is the Campylobacter
species, because they generate both O- and N-linked glycans to their proteins such as
flagellin [1].
Each specific glycan exhibits a key core unit with extended core, differentiating
into strain- and phylum-depending structures. Glycans as the key molecules of
organisms face the extracellular outer world, allowing a terminology of glycan
116 4 Glycans in Glycoimmunology
diversity created in each individual environment. Therefore, diverse glycan structure

influences phenotype differences [2–7]. The glycobiological process enables possi-
bilities to acquire natural selections in exclusively allowed conditions. Evolution-
linked glycosylation creates each specific glycan to unicellular organisms with
monosaccharides. Among them, the eukaryotes utilize restricted numbers of mono-
saccharide residues without detailed modifications. Glycoforms in each eukaryotic
organism are restricted and limited due to the distinct glycosyltransferases and
glycosidases, leading to specific glycan structures in individuals through the expres-
sion pattern of the related enzymes cooperated with the spatial and temporal
expression behavior. Glycans are produced through multiple harmonists such as
glycosyltransferases and glycosidases and have various characteristics of sugar
component composition, linked sequence, and stereo-conformation depending on
cell or tissue dependency [8]. Consequently, the generated glycan structures and
patterns are the molecular bases for the non-self- and self-recognition.
4.2 Changes in Glycan Structure Involved in Coregulated

Expression of Glycan-Binding Lectin Counterparts
Glycans and lectins or glycan-binding proteins (GBP) reciprocally control innate and
adaptive immunity. The glycome is a regulator of the immunity. The development,
manifest, display, and function of the immunity rely on glycan structures and GBP as
well as their interactions. Lectins recognize glycans for diverse roles of cell sociol-
ogy. Carbohydrate recognition domain (CRD) is known in the animal or human
lectins. The examples are the C-type (referred from Ca2+-dependent glycan recog-
nition), I-type (referred from Ig-like domain specificity), and P-type (referred from
Man-6-P recognition specificity) domains. During long historic evolution, structural
variations within their genes and protein sequences have been made to the scaffold
homologous proteins, consequently generating a wide range of the C-type lectin
families [9]. In inflammatory responses, cellular glycosylation event and substitu-
tions with sulfate groups as well as selectin expression are coregulated to recognize
modified glycoprotein glycans, as known for the examples of CD34, CD43, and
CD44 (Hutch-I, Hermes antigen). In tumor metastasis, glycans of proteins in CD11/
CD18, CD24, or CD66 are coregulated to be recognized by selectins [10]. Such
example is also reported in a variant myeloperoxidase that has sialyl and fucosyl
glycans as a CD62E (E-selectin, ELAM-1, LECAM-2) counter-receptor on human
myeloid cells which are also expressed upon exposure on granulocyte colony-
stimulating factor [11]. In tumor suppression of tumor biology, the defected tumor
suppressor activity causes growth. Tumor suppressor p16INK4a is known to trigger
anoikis in pancreatic cancer cells through the galectin-1 and desialylated glycan in
α2,6-sialylation with the reduced level of the antiapoptotic galectin-3 [12–14]. In
detail, the reengineered pancreatic carcinoma cells having a tumor suppressor
p16INK4a gene expression exhibited the coregulation of glycan biosynthesis
4.3 Evolution of Lectin: Alternative Splicing Contributes to Variation for. . . 117
genes such as glycosyltransferases and sialyltransferases [15] with expression of two

lectins, thereby enabling for the tumor cells to induce lectin-dependent anoikis [12–
14]. More specifically, the reduced α2,6-sialylglycans present in the tumor cell
surface are more susceptible for the recognition of fibronectin receptor (FR; α5β1-
integrin) and galectin-1 recognition, and this interaction consequently activates
apoptotic downstream signaling [12, 14]. In addition, expression of galectin-3 as
an antagonist to galectin-1 is simultaneously decreased in order to physiologically
respond to galectin-1. The glycosylated fibronectin receptors known as α5β1-
integrin and galectin complexes activate caspase-8. Glycosylation enforced by
microenvironmental agents of NO [16] or proteins is not directly related to glyco-
sylation caused by the Rho GTPase Rac1 [17]. In those cases, surfaced α2,6-
sialylation event is regulated. Desialylation by neuraminidases regulates the
hyaluronic acid-CD44 binding [18]. For the same action, neuraminidase activity
converts GD1a ganglioside to GM1 form, which is bound by galectin-1 [19, 20]. Con-
sidering that lectin is differentially expressed between human and mouse, neither
α1,3-gal residues nor NeuGc residue sialylation of N-glycans is found in humans. In
addition, CD15 (X-hapten, LeuM1, SSEA-1, Lex), CD75 (LacNAc), and branched
N-glycans are highly present in immune cells of humans than mouse [21]. Galectin-1
can bind to GM1 ganglioside in some neuroblastoma cells and the activated effector
T cells by the microenvironmental assists of neuraminidase or sialidase that cleaves
the terminal SA residue from abundant GD1a moiety into the active counter-receptor
GM1 [22–25]. The sialic acid removal facilitates the galectin-1 binding to GM1.
Thus, dynamic reconstituting of the cell surface glycans is an effective means for the
responsiveness to distinct lectins, without any additional glycan synthesis. The
counter-receptor recognition by cellular lectins correctly translates cellular signals.
4.3 Evolution of Lectin: Alternative Splicing Contributes

to Variation for Glycan-Binding Receptors
In the side of glycans, glycan structures are timely changed, although glycan
structures created from organisms are not genetically encoded. Extracellularly,
organisms have acquired glycosylation events as a form of their adaptation to
extracellular environments and intracellularly to reflect cellular activation or differ-
entiation. In the side of glycan-binding lectin, during environment-based adaptation
and evolution, glycan affinity of lectins has been modulated by sugar structures of
monosaccharides and oligosaccharides as well as the three-dimensional recognition
of the glycan code. Glycans affect the lectin reactivities. Alternative splicing-derived
protein variations alter the acceptor specificity of the glycoprotein and explains the
glycan roles of glycoproteins [26].
4.4 E-Selectin-Binding Ligand sLex (CD15s) on Neutrophil

CD44 N-glycan and Alternatively Spliced Exon
6 Contains Core 2 O-Glycan sLea (CD44v6) Epitope
As an evolution strategy, eukaryotes have obtained alternative splicing of protein

genes. In glycan structural aspects, glycan sequence and structure can be altered by
the alternative splicing of protein genes. Thus, alternative splicing event is a
regulation type. Glycan affinity of lectins is therefore regulated by the lectin-binding
carbohydrate epitope density or number obtained by the glycan core or by branched
multiantennary epitope. For example, desialylation unmasks contact sites for a
lectin. Leukocyte transendothelial migration is a key step in their recruitment to
sites of inflammation by synergic regulation of endothelium-expressed selectins.
Selectin and immunoreceptor engagement activate leukocytic integrin affinity, while
ligand-induced integrin clustering likewise stabilizes adhesion and initiates transmi-
gration. Homing of leukocytes and their migrative extravasation are forced by
selectin-glycan bindings in the endothelial cells on vascular vessels closed to
inflammation region and infected tissues. Another case to blocks in immune
response is obtained from glycan-synthesizing enzyme-deficient cells. For example,
deficiency of GCnT-1, a core 2 β1,6-GlcNAc-transferase, exhibit decreased DCs
homing on L-, P- and E-selectin-expressing cells and blocking DCs homing to
inflammation area. The glycan-decorating epithelial barrier is a starting line of
defense from pathogenic invaders. However, GCnT-2-deficient animals, which
encodes the core 2 β1,6-GlcNAc-transferase, are susceptible to infection and
inflammation.
In addition, the distinct and dynamic contribution of P- and E-selectins mediates
β2-integrin-induced PMN transmigration [27]. E-selectin-binding leukocyte ligands
are known for CD43, CD44, and PSGL-1. For example, glycan determinants such as
CD15s (sialyl Lex) (Fig. 4.1) or CD176 [Thomsen-Friedenreich antigen (TF),
Galβ1,3GalNAcα1-O-R, mucin-type O-glycan to protein] have rather become
counter-receptors for lectins. E-selectin interacts with PSGL-1 or CD44 ligand
towards Src family kinase-integrin αLβ2-mediated leukocyte rolling. P-selectin-
PSGL-1 binding induces αMβ2-ICAM-1 interaction to potentiate adhesion [27].
CD44 is not a mucin-type protein but modified only with N- and O-glycans
[28]. CD44’s N-glycans recognize E-selectin [29]. CD44 and PSGL-1 form mem-
brane lipid rafts microdomain on leukocytic cells. Interaction between selectin and
Fig. 4.1 Sialyl-LeX

4.5 Glycans Regulate T Cells 119
CD44 or PSGL-1 expressed on myocytes or any equivalent cells contributes to SKF

phosphorylation and signaling mediators, which transduce β2-integrins into active
status with extended, intermediate affinity, slowed rolling, and contributed arrest
levels [30]. In the leukocytic rolling status, selectin recognition with CD44 or PSGL-
1 activates a signaling pathway, like the T cell receptor signaling cascade [10]. For
example, ligand clustering with non-rafted T cell receptors and with rafted
coreceptors initiates downstream signaling. In fact, the PSGL-1-/CD44-involved
signaling is mediated through the β2-integrin transduction event and rolling to
ICAM-1 [10]. P-selectin-PSGL-1 binding activates β2-integrins and initiate PMN
transmigration, whereas E-selectin engages CD44 to influence PMN transmigration,
indicating the interaction of P-/E-selectins and their ligands to promote PMN
transmigration. The glycoprotein CD44 (Hutch-I, Hermes antigen) has its globular
domain in the extracellular region and transmembrane (TM) domain, as spaced by a
stem. The conventional stem region is the site responsible for structural variation
through alternative splicing to produce variant isoforms. A single gene primary
transcript is alternatively spliced between exon No. 5 and No. 16. The CD44 isoform
is the removed form for stem extensions. CD44 N-glycans of blood polymorphonu-
clear neutrophils (PMN) contain sialyl Lex antigen known as CD15s epitope
Neu5Acα2,3Gal β1,4(Fucα1,3)GlcNAc that is a counter-receptor for CD62E
(E-selectin, ELAM-1, LECAM-2) [31], and the additional exons facilitates the
CD44 to have binding sites for O-glycans/mucin types. The exon 6 variant known
as CD44v6 is expressed in neutrophils in the pathologic lesion of ulcerative colitis,
as SLeA epitopes on core 2 O-glycans modified by α1,3/4-Fuc-transferase (FUT3).
By help of the biantennary chains, CD44 can induce mucosal inflammation during
cell detachment into the blood lumen and transepithelial migration events [32]. Thus,
alternatively spliced forms are responsible for their distinct roles.
4.5 Glycans Regulate T Cells
Glycosylation regulates and controls host immune responses through T cell regula-
tions such as thymocyte precursor development and T helper subset differentiation
[33]. N-glycan branching is associated with the immune system.
Glycosyltransferases involve in N-glycan branching and their target proteins with
various biological functions. N-glycan biosynthesis is distinctly specialized for
transfer en bloc, conserved mechanism during evolution, transmembrane GTs, and
monogenic substrate specificity. For the folding and quality control of glycoproteins,
the synthesis is also well conserved for the step-by-step transfer, interspecies vari-
ability, and type II membrane GTs with stage-specific and tissue-specific expression.
GTs biosynthesize N-glycans in rough ER and Golgi apparatus. N-glycan branching
enzymes exist in Golgi. During thymus development, the antennae N-glycans are
branched five-fold more from SP or DN thymocytes. Thereafter, during the transi-
tion stage from SP to peripheral T cell types, the N-glycan branching level is
declined two-fold more [34]. Hence, the N-glycan branching changes result in the
level of TCR clustering during thymocyte development. Autoimmune diseases are

caused by self-antigen-specific abnormal immune responses. The N-glycans are
associated in T cell-driven autoimmune responses. In fact, development and pro-
gression of inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE),
multiple sclerosis (MS), and insulin-dependent diabetes mellitus (IDDM) are related
to the N-glycans. Additionally, complex type of N-glycans essentially recognize the
MHC peptide and TCR interaction towards regular T cell development [33].
Changes in glycosylation are frequently observed during activation, develop-
ment, and differentiation into effector T cells. Ultimately, glycosylation and
glycosylated target molecules control a stage-specific pathway of T cell fate and
life. Naïve T cells are metabolically quiescent using consuming ATP produced by
oxidative phosphorylation. During early development of T cells, variously different
T cell subsets are made by distinct glycosylation patterns. Functionally, T cells are
developed, learned, educated, and matured in the thymus via final selection pathway
through TCRs to phenotypically express an antigen recognition repertoire [35]. The
stage-specifically expressed glycans influence the T cell development, and glyco-
sylation also controls thymocyte development, where T cells display alterations in
glycosylation for thymus development as well as peripheral activation and survival.
T cells alter glycosylation process during thymus development and peripheral
activation. Alteration in T cell glycosylation regulates T cell functions. For example,
glycans present in two abundant glycoproteins CD43/CD45 expressed in the T cells
are dramatically regulated during development, activation, and survival of T cells.
N-glycosylation is involved in the thymus seeding and speciation of T cell lineages.
N-glycans expressed in T cells elaborately control the T cell roles in activation,
development, differentiation, and signaling. Peripherally circulating naive T cells
and thymus-resident T cells also produce sialylated core 1 O-glycoproteins to
regulate their fates. O-glycans also influence T cell development, for example,
upon O-glycan-galectin binding. Among galectins, galectin-1 triggers apoptotic
event of immature thymocytes via galectin-1 recognition by core 2 O-glycan struc-
tures present in T cell coreceptors of CD43 and CD45. In the special condition that
CD45 bears α2,6-sialyl core 1 O-glycans and α2,6-sialyl N-glycans, CD45 on
mature thymocytes is resistant to galectin-1 binding [26]. Upon binding to the altered
glycosylate antigens, several immune events including antigen capture, antigen
presentation to Th cells, cross-presentation to CTL, and anticancer response are
progressed. GBPs recognize self by cell adhesion via DC-SIGN and ICAM-1/
ICAM-3 binding and by T cell signaling via GalNAc-bearing CD45 glycoform
and MGL binding as well as by discrimination of danger patterns via HMGB1 and
Siglec-G/CD24 binding and APC immune or tolerance regulation via galectins
binding to APC glycoproteins of Tim-3 and CD43.
4.5.1 Glycans Regulate Development and Differentiation in T

Cells
After born in BM, initiation of T cell development and thymic progenitors trafficking
to the thymus depend on the P-selectin expression in the thymus epithelial cells or
PSGL-1 present in circulating thymus progenitors [36]. α1,3Fucosyl PSGL-1 is a
binding site of P-selectin. When thymus progenitors are homing into the thymus, the
progenitors bind to α1,3Fucosyl PSGL-1, too [37]. In autoimmunity or cancer, the
question of how glycans modulate multiple steps of thymocytic development is
raised to answer. The elucidation of the glycan-encoded immune responses is
therefore a goal of this chapter. Glycosylation of surfaced membrane receptors on
T cells directs T cell functions. In addition, glycans are involved in tolerogenic and
immune suppressive responses in cancer progression and autoimmune responses.
Alteration of the glycan structures in T cell receptors and tumor cells as glycan code
of tumor can modulate the immune response to suppress immune pathways. Such
immune suppressions are frequently occurred in the tumor-associated microenviron-
ment with tumor immune escape [38, 39]. Glycans generate the lost immunological
tolerance in autoimmune response, giving tolerance in cancer progression. Glycans
may exhibit dual roles of immune inhibiting checkpoints or stimulating signals.
Elucidation of the glycan roles responsible for autoimmune response and cancer
progression will create insights into new concept of potential targets or markers for
the immunomodulatory drugs.
Immune cells are step-wisely developed from the primitive progenitor cells and
further differentiated into each functional CD-expressing cell type. For example, the
T cell subpopulation in development is mainly early thymocyte progenitors (ETP).
The ETP include various CD-specific subpopulations. Representatively, double
negative (DN)-1/DN-2/DN-3/DN-4, double positive (DP), and single positive
(SP) populations are classified. The glycosylation of SP subpopulation gives the
functional T cell function that can discriminate counterparts via its distinct
CD. However, T cell-surfaced glycans act as players in many immunological
behaviors including immune unbalance, autoimmunity incidence, and cancer pro-
gression. The causing reasons are basically based on that changes in T cell glyco-
sylation pattern often induce reprogrammed and reconstituted immune stimulation
as well as immune tolerogenic responses. The interplay of T cell glycans confers
both autoreactivity and self-tolerance of T cells. For glycoenzymological synthesis
of the T cell interplay, several glycan synthetic GTs are known to form glycans
required for T cell function.
Eukaryotic proteins are matured by Asn glycans or Ser/Thr glycans at the
ER/Golgi networks by means of posttranslational modification. N-glycans are
formed at protein N-X-S/T region and are processed by α-mannosidases and
GlcNAc-transferases (Mgat)1, Mgat2, Mgat4, and Mgat5 using donor
UDP-GlcNAc substrate via the hexosamine biosynthetic pathway (HBP). Glc,
glutamine, or GlcNAc residue is used for the UDP-GlcNAc synthesis of the HBP.
The product UDP-GlcNAc is then served for the N-glycan synthesis in the ER/Golgi
apparatus. Mgat1 transfers GlcNAc to Man5GlcNAc2-Asn to start the N-glycan

synthesis. Therefore, Mgat1 yields the first antennary. Mgat1 KO mouse embryos
lack branching of protein N-glycan with developmental lethal and retardation effects
[40]. Some Mgat1 polymorphisms upregulate the mRNA level and enzymatic
activity. Consequently, the Mgat4- and Mgat5-mediated UDP-GlcNAc usage is
reduced and decreases in N-glycan branching level [41].
Mgat2 yields β1,2 GlcNAc branch with bi-antennary N-glycans as the second
antenna. Lack of Mgat2 yields incomplete complex N-glycans with bisecting
N-glycan structure in mice [42] with gastrointestinal, hematologic, and osteogenic
dysfunctions, like human Mgat2 deficiency phenotype of CDG-II [43]. The Mgat2-
KO mice to target T cells (Mgat2f/f/Lck-Cre) show severe EAE [44]. Mgat2 lacking
remarkably reduces N-glycan branching, compared with Mgat5 deletion. In the
Mgat1 deletion [34] or Mgat2 deletion [34, 42] mice, the mice show N-glycan
branching-deficient phenotypes. In addition, the total T cell number present in the
thymus and spleen tissues is reduced. Mgat4a further branches the β1,4-GlcNAc
branches in N-glycan core Man and yields tri-antennary N-glycans. Mgat4a-formed
tri-antennary N-glycans bind to galectin-9 present in β island cells of the pancreas.
Lack of Mgat4a displays disease phenotypes such as hyperglycemia, insulin resis-
tance, and obesity in mice [45]. The β cells isolated from Mgat4a-transgenic mouse
exhibit the insulin sensitivity and ameliorates non-insulin-dependent diabetes
[46]. Hence, Mgat4a-catalized branching in Glut2 N-glycans may bind to galectin-
9 giving prolonged retention time.
Mgat5 enzyme forms tetra-antenna N-glycan structures. MGAT5 gene encodes
the β1,6-GlcNAc-transferase-V (GnT-V) enzyme, which generates a β1,6-branching
enzyme on the complex N-glycan type [47]. The GnT-V branches the antennary
structures of N-glycans and is known to control T cell functions. Mgat5-KO mice are
hypoglycemic and hypersensitive to fasting time [48, 49]; however, the insulin
responses are unchanged. The increased glucagon level indicates their lean pheno-
type in Mgat5-KO mice [49]. Mgat5-KO mice lead to inflammatory demyelination
and neurodegeneration. In addition, Mgat5-KO mice show Tim-3+ Th1 phenotyped
cells with high frequency compared to that of PL/J mouse [50]. Mgat5 expression is
increased in cancer with the enhanced cell motility, epithelial-mesenchymal transi-
tion (EMT), and invasive potentials [48]. Experimental results obtained from mice
models have revealed the T cell activation by N-glycans. T cells derived from tissues
of the Mgat5-deficient mouse exhibit the lowered activation level threshold rather
than T cells isolated from normal mice of wild type. Mgat5 deletion does not affect
the T cell number and population resident in the spleen and thymus. The N-glycans
with tetra-antennary branches contain poly-LacNAc glycan chains, which are used
as the naturally found endogenous ligands for binding to galectin-3. The mice with
Mgat5 deficiency showed increased signs of proliferative glomerulonephritis and
developed autoimmune responses. In immune synapses, Mgat5 loss reduces the
lattice generated from T cell-associated galectin-3 and increases TCR clustering
level. Therefore, EAE and delay-type hypersensitivity (DTH) are easily developed.
Mgat5-branched N-glycans further attenuate differentiation of Th1 [50] and Th17
cells [51].
In addition, in human CTL antigen (CTLA)-4, its glycans are the N-glycosylated
types at the two Asn sites. One N-glycan site-lost protein is produced from the wild
type having two Asn sites in CTLA-4 protein via the Thr17Ala mutagenic polymor-
phism which is produced in human CTLA-4. The one Asn site-restricted isoform
increases MS incidence through the suppression of its surface retention time on T
cells [41, 52, 53]. However, the branched N-glycans increased the surface retention
time of CTLA-4 [54], where CTLA-4 functions mainly in T cell arrests of hosts.
CTLA-4 exhibits a high binding affinity for the counterparts of CD80/CD86
coreceptors present in APCs and suppresses the T cell induction [55].
GnT-V enzyme acts to many T cell receptor proteins like TCR, CD25 (IL-2Rα),
and CD4. IL-2Rα known as CD25 is highly glycosylated in N-glycans and
O-glycans/mucin type [56]. IL-2Rα (CD25) consists of three distinct
N-glycosylation sites. IL-2Rα (CD25) response is activated by N-glycans. The
N-glycan number influences T cell growth and differentiation. N-glycosylation
inhibition suppresses the surface retention time of IL-2Rα (CD25) present in T
cells and IL-2 signaling. The inhibited CD25 suppresses Th-1, Th-2, and Treg cell
differentiation but activates development of Th17 cells [56, 57]. Glucosamine as the
UDP-GlcNAc substrate in HBP [58], unlike GlcNAc, interferes with
N-glycosylation [59]. Glucosamine is known to inhibit inflammatory immune
responses and autoimmune diseases [60]. For example, glucosamine administration
attenuates differentiation of T cell subsets of Th1, Th2, and Tregs but remarkably
induces Th17 cell polarization by blocking the CD25 N-glycosylation and signaling.
The attenuation effect of glucosamine is almost the same as those effects obtained
from the N-glycosylation blocker tunicamycin treatment. As expected, the restricted
glucosamine dose exacerbates the EAE level by enforcing Th17 cell differentiation
[56]. This inhibitory effect is also similar to that of non-branched N-glycan forma-
tion in EAE incidence. In addition, Glc and glutamine prevent Th17 cell differen-
tiation and also lead to switching to iTreg cells by branched N-glycan and
subsequent elevated CD25 retention on cell surface [57]. Such Glc and glutamine
supplementation activates N-glycan synthesis and hence increases the surface reten-
tion time of CD25 [57]. Tregs or activated T cells produce CD25, and the CD25
recognizes cytokine IL-2 associated with IL-2Rβ and γ chains, activating PI3K/Akt/
mTOR, MAPK, and STAT5 for growth, survival, activation-induced cell death
(AICD), and differentiation. Therefore, glycolysis and glutaminolysis events can
collaboratively regulate T cells to develop and differentiate as well as enable self-
tolerance via limited N-glycosylation. Such catalyzed GNT-V-branched products as
N-glycan branches influence to T cell phenotypes including proliferation, differen-
tiation, intracellular signaling, and inflammatory cytokine expression. In TCR sig-
naling, glycosylated surface receptor proteins are resistant to proteolysis.
4.5.2 Glycosylation of Notch Receptor Signaling

for Thymocyte β Selection and T Cell Function
Regulation
When thymus progenitors enter the thymus, thymus progenitors differentiate into
ETPs that are the CD4 CD8 DN1 subsets [26]. The Notch signaling commits
CD4 CD8 DN1 thymocytes linking to the T cell populations [61]. The Notch
receptors/ligands are glycosylated to transduce Notch signaling. The manic, radical,
and lunatic Fringe is the GlcNAc-transferases that catalyzes the GlcNAc transfer
from UDP-GlcNAc to fucosyl O-glycans in EGF-like repeated domains of the
extracellular region of Notch receptor [62, 63]. Loss of the 3 manic, radical, and
lunatic Fringe glycosyltransferases diminishes Notch binding to Delta-like ligands
(DLL) [64]. The Fringe-catalyzed Notch glycosylation develops T cells. The lunatic
Fringe gene known as Lfng is wrongly expressed by a lck promoter [65]. The
defected Notch glycosylation by missed GlcNAc residue of the EGF-like repeated
region makes a B cell subset differentiated from thymus lymphatic progenitors. Lfng
is weakly regulated in CD4 + CD8 + DP subset thymocytes. Lfng ectopic expression
increases Notch recognition with ligands present in stromal cells, inhibiting DN
development but potentiating differentiation of B cells [66]. Thus, alteration in the
Notch glycosylation affects T cell development. At DN stages, Notch binds to DLLs.
The Lfng presence on DNs increases Notch binding to DLLs, and the Lfng defect in
DPs leads to Notch-independent development of T cells. The T cell subset lineages
develop at the DN3 step, and recombination-activating genes (RAG) rearrange the
Tcrb and induce the TCR-β chain (TCR-β) expression and consequently yield a
pre-TCR complex [61, 67]. Next, in the presence of IL-7 and Notch, the pre-TCR
signaling allows β-selection through the suppressed expression of RAG complex of
quiescent DN3 (DN3a) Rag1/2. The DN3a subset differentiates into cycling DN3
thymocytes (DN3b), and this is also differentiated into DN4 cell type. Loss of
pre-TCR signaling can be rescued in lck-null cells by artificial Lfng expression.
O-GlcNAcylation also regulates the T cell development [68]. After β-selection in
DN4 thymocytes, ST6Gal-I (β-galactoside α2,6-sialyltransferase 1) expression is
significantly increased up to 10 times more, compared to the DN3 thymocytes with
α2,6-sialylglycans [69]. In ST6Gal-I KO mice, the DN subpopulations are
decreased. In the same model of ST6Gal-I KO mice, expression of CD96, a receptor
of nectin-1 in cellular migration, is decreased in the DN2 and DN3 subpopulations.
In addition, ST3Gal-I expression level is reduced in DN or DP, while ST3Gal-I
expression is increased in SP thymocytes [70]. In ST3Gal-1 KO mice, the TCR
repertoire is altered, and thymocyte selection requires sialylation [71].
The β-selected DN4 cells exhibit rapid self-renewal and differentiation into DP
CD4 + CD8+ thymocytes with the expression of TCRαß receptors [72]. TCRαβ
carries at least 7 N-glycosylation sites, and TCR-CD3 complex carries 12 N-glyco-
sylation sites responsible for TCR folding and function [73]. In addition, decreased
sialylation level in DP CD4 + CD8+ thymocytes increases binding capacity to
MHC-I, and the increased sialylation on differentiating SP CD8+ CTLs in the
thymus decreases the level of MHC-I-CD8 recognition [74]. Furthermore, lack of

the GnT-V-encoding Mgat5 gene significantly increases TCR clustering and, con-
sequently, reduces T cell activation and increases the level of autoimmune disease
development. The cytosolic regions of the CD4 and CD8 receptors recognize Lck in
the molecular level, strengthening TCR signaling to MHC antigen complex during
CD4 and CD8 coupling to the TCR [75]. N-glycan branching enhances the DN cell
maturation and TCR selection levels. Protein O-glycan and N-glycan structures
regulate T cell responses [76]. In the N-glycans on T cell receptors, N-glycans
regulate the T cell responses. MHC-I expressed in nucleated cells recognizes
TCRs present in CD8+ T cells. However, MHC-II present in APCs including B
cells, DCs, macrophages, and TECs recognizes CD4+ T cells [77]. Defection of
MHC1a N-glycan synthesis caused by Asn site mutation or conformational shift
frequently yields unfolded protein and accumulates proteins in cytosol [78]. MHC-II
consists of two α and β subunits. The α subunit carries N-glycans attached to high-
mannose and complex types. However, the β subunit contains only complex-type
N-glycans [79]. Among two different MHC-I and MHC-II, MHC-II glycosylation is
necessary in antigen recognition and microbial carbohydrate antigen presentation.
The perfect MHC-II glycosylation enables to respond downstream T cell signals.
4.5.3 Alternatively Spliced Variants Produce Different

Glycan Structures of CD43 and CD45 Isoforms in T
Cells
Glycosylation of coreceptors activates the T cells, as known in the case of

TCR-CD45 complex. TCR-CD45 complex is formed by galectin-3, because
galectin-3 recognition to poly-LacNAc sequences in branched N-glycans forms
molecular lattice. CD45 phosphatase enzyme activity prevents T cell activation
and suppresses T cell downstream activation [80]. CD45 gene is also spliced with
alternative five variant isoforms including CD45ABC, CD45AB, CD45BC, CD45B,
and CD45RO on human leukocytes [81–83]. The alternatively spliced variant
protein forms have up to 11 different site glycosylation sites. However, the 11 N-
glycosylation forms are different [84, 85] during T cell differentiation and
activation [86].
For functional regulation, alternatively spliced transcript of CD45 gene likewise
creates differential glycosylation events. CD45 is a predominant transmembrane
glycoprotein expressed on T cells, and CD45 contains the cytoplasmic Tyr phos-
phatase domains. Three exon (4,5,6)-produced protein regions differentiate the
common (RO) polypeptide formed by a Cys-rich region and three extracellular
fibronectin type III repeats. The exons produce the A, B, and C domains, and the
isoforms of RA translated from exon 4 form, RB from exon 5 form, RBC from exon
5 and exon 6 forms, and RABC from exons 4 to 6 forms are generated. The series of
CD44 variants target mucin-type O-glycans [26]. The CD45 forms have MWs
80–230 kDa, larger than the predicted MW of nascent protein not glycosylated
(123–141 kDa). Sialylation regulates the CD45 clustering on oligomerization and
TCR signaling [26, 87, 88]. During CD8+ T cell regulation, CD45 RO isoform is
expressed for higher dimerization. Because of the glycosylation, switching from
sialyl CD176 (Thomsen-Friedenreich antigen, TF) to non-sialyl CD176 decreases
electrostatic tension when CD45 is dimerized [89]. The α2,6-sialylation on
N-glycoproteins, not on core 2 O-glycoproteins, decreases the mature thymocyte
capacity of CD45-mediated apoptosis by a galectin [26, 88].
T cells regulate their CD45 glycosylation by alternatively spliced CD45 isoforms
having different glycosylation. Consequently, the T cell CD43 and CD45
glycophenotype controls interaction between T cells and endogenous lectins. Two
major glycoproteins CD43 and CD45 expressed in the T cells are differentially
expressed for the T cell lifespan. Glycans control T cell behaviors. Core 1 sialyl
O-glycan is produced on DN thymocytes which are unmatured and matured CD4 or
CD8 CTLs in the thymus, although non-sialylated core 1/core 2 O-glycosylations are
found in immatured forms of DP thymocytes [90]. Activated CD4+ T cells and
CD8+ CTLs exhibit the prevention of sialylation with increase in core
2 O-glycosylation level because of de novo hyposialylated CD43 and CD45 syn-
thesis [89, 91]. Additionally, expression of ST3Gal-1 gene is differentially modu-
lated in CD4+ T cell differentiation to Th-1 and Th-2 subsets [92]. For example, Th2
cells express ST3Gal-1 for core 1 sialyl O-glycans, whereas Th1 cells are negative
for ST3Gal-1 expression with non-sialylated core 1 O-glycans [92]. Th-1 and Th-2
cells exhibit C2GnT expression and synthesize core 2 O-glycoproteins [93].
CD43 and CD45 glycosylation in T cells are controlled in thymocyte develop-
ment and differentiation from DN thymocytic precursors to memory T cells, TCR
signaling, apoptosis, migration, and T cell activation [91, 94–96]. CD43 and CD45
glycans control migration, TCR signaling, and apoptosis, at the T cell level. T cell
life fate is thus controlled by CD43 and CD45 glycosylation. How does the
glycosylated extracellular domain of CD43 influence the CD43-mediated T cell
fates? Thymocytes and T cells express tri- or tetra-antennary types of N-glycans
due to GnT-V that adds β1,6-GlcNAc to the N-glycan Man core, and then poly-
LacNAc unit is further added and terminally sialylated in SAα2,3- or SAα2,6-
glycans. The SAα2,3- and SAα2,6-linked N-glycans are generated by ST enzymes
of ST3Gal-4 and ST6Gal-1, respectively. Sialylation reaction is influenced by
development of thymic T cells. For example, medullary and cortical thymocytes
contain α2,3-sialyl N-glycans, whereas mature medullary thymocytes possess α2,6-
sialyl N-glycans [96]. Similar to mature thymocytes, the thymus-left naïve T cells
express α2,6-sialyl N-glycans. Activated peripheral T cells increase the level of
surface complex N-glycans, decreasing α2,6-sialylation [97, 98]. ST6Gal1 increases
complex N-glycan levels on CD45. How are T cells modulated by the CD43s
80 O-glycans and 11 N-glycans as well as CD45s 8 ~ 47 O-glycans during the
life fate? How do CD43 and CD45 glycans on T cells differ from such glycans
expressed by other leukocytes of B cells or DCs?
CD43 and PSGL-1 have various O-glycan forms. Core 1-O-glycans and PSGL-1
recognize three selectin types [29, 99]. The core 1 β1,3-Gal-transferase enzyme
generates the O-glycan core 1 sugar structure of Galβ1,3GalNAcα1-Ser/Thr- and

more distal sLex are attached. Neutrophils isolated from core 1 β1,3-Gal-transferase-
deficient mice exhibit significantly defected leukocytic rolling driven on P-selectin
or E-selectin expressed on the endothelium [29]. PSGL-1 and CD43 associate to
form lipid rafts on leukocytes. E-selectin also engages in CD43 on rolling of effector
T cells [100] to activate signaling. The CD43 gene has only one exon, and CD43 has
about 80 O-glycans on T cells with core 1 O-glycan and core 2 O-glycan structures
[101]. The human CD43 extracellular region has about 80 O-glycan structures and
has one N-glycan structure. T cells express two CD43 membrane proteins, which are
sialylated in core 1 O-glycan structure (115 kDa) and core 2 O-glycan structure
(130 kDa) [76], while the predicted weight is about 44 kDa. Naive and activated
types of T cells equally produce CD43 protein with peripheral tissue core
1 O-glycosylation structure, whereas the activated type of T cells also synthesizes
CD43 protein, which has a core 2 O-glycan structure [100, 102]. The cytoplasmic
domain of CD43 as a mucin-type glycoprotein interacts with the cytoskeleton linker
proteins (ezrin–radixin–moesin family) to activate T cell activation, migration, and
survival [103, 104]. T cells and thymocytes have two different CD43 glycan
structures. The CD43 protein extends about 45 nm length from the surface of T
cells. The CD43 115 kDa glycoform is primarily expressed by mature CD4 or CD8
SP thymocytes, native T cells, and immature DN thymocytes. The CD43 130 kDa
expression is increased in the activated peripheral T cells as well as immatured DP
thymocytes.
CD45 has five isoforms on human T cell surface due to alternative splicing, and
the CD45 isoforms differ in the O-glycosylation level. How does CD45 glycosyla-
tion change in development and activation of T cells? The CD45 intracellular
domain is commonly present in all CD45 isoforms with tandem phosphatase
domains for TCR signaling [105]. Intracellularly, all CD45 isoforms have the
enzymatic active domains of phosphatase, which are named phosphatase P1 and
phosphatase P2. Among them, the phosphatase P1 has a strong enzyme activity. The
alternatively spliced form with extracellular domains of CD45 bears two different
O-glycosylation structures of core 1 O-glycan and core 2 O-glycan forms. All CD45
isoforms have commonly three regions of a membrane proximal F region, a CR for
N-glycosylation, and a terminal end region [87]. The three regions of the F region,
CR region, and terminal end region of CD45 protein have Asn sites for
N-glycosylation. The CD45 extracellular region contains three A-domain,
B-domain, and C-domain, which are formed by the alternatively spliced variants
of exon 4, exon 5, and exon 6, as O-glycosylation sites. From total five isoforms, RO
form does not contain alternatively spliced variant domains. Other forms are RB
form produced from the exon 5, and the RA form is produced from the exon 4. Also,
the RBC form is produced from the exon 5 and exon 6, while the RABC form is
produced from the exon 4, exon 5, and exon 6. T cell development and activation
regulate alternative forms of CD45. DP thymocytes express predominantly RO,
while mature SP thymocytes express mainly RB and RBC. In contrast, naive T
cells reside in peripheral region and generate mainly the RB form. The activated T
cells and memory T cells generate mainly the RO form [88]. Therefore, immature
DN T cells in the thymus generate the CD45RA/RBC/RB form, having core 1 sialyl
O-glycans. The matured CD4 SP or CD8 SP T cell subsets in the thymus generate
the CD45RB/RBC form, having core 1 sialyl O-glycans. In contrast, DP thymocytes
express the CD45RO form with both O-glycan types of core 1 O-glycan/core
2 O-glycosylation structures, which are not sialylated. Naive T cell types express
the CD45RB isoform, having a core 1 sialyl O-glycan structures in the peripheral
organs, whereas activated T cell populations like DP T cells in the thymus generate
the CD45RO form with non-sialyl forms of core 1 O-glycan and core
2 O-glycans [106].
4.5.4 T Cells CD43 and CD45 Interaction with Their

Counter-Receptor or Lectins to Determine T Cell Fates
CD43 and CD45 function with their counter-receptors or lectin proteins expressed
from neighboring cells including immune cells, endothelial cells, and cancer cells
[107]. Glycosylation of thymocytes and T cells during development and activation is
important in a fashion that T cells interact with the lectins through CD43 and CD45
glycans. Interaction between CD43 and CD45 glucans and endogenous lectins
regulates the T cell functions.
During T cell migration event, T cells normally transmigrate to inflammation
tissue regions and sites. E-selectin as an adhesion molecule is synthesized in
activated types of endothelial cells, recruitment of T cells to inflammation sites.
The CD43 form is an endogenously produced coreceptor responsible for E-selectin
recognition [108], because E-selectin binds to the T cell CD43 130 kDa glycoform
bearing sLex tetrasaccharide on core 2 O-glycans [100, 102–104]. Activated T cells
increase the synthesized carbohydrates of core 2 O-glycan linked to CD43 protein,
which have the SLeX epitopes as the E-selectin ligand. The T cells interact with
E-selectin and migrate to inflammation regions.
In TCR signaling, TCR signaling is involved in negative and positive selection of
thymocytes and peripheral T cell activation. CD45 intracellular phosphatase regu-
lates TCR signaling thresholds [105]. Intensive N- and O-glycans and sialyl residues
on the CD45 extracellular domain keeps CD45 molecule separated on the plasma
membrane, increasing TCR signaling via the CD45 intracellular phosphatase acti-
vation [87]. Reduction in SA content and/or multivalent lectin interaction with the
CD45 extracellular domain induces molecular clustering or oligomerization of
CD45 molecule, causing TCR signaling dysfunction [87, 88, 106, 107]. CD45
oligomerization also decreases the CD45 intracellular phosphatase activity
[88]. Thus, CD45 binding with lectins like placental protein 14 (PP-14) [106,
107], macrophage galactose-type lectin (MGL) [108], or galectin-1 [88] suppresses
TCR signaling because such clustered CD45 losses phosphatase activity (Table 4.1).
However, the role of T cell CD43 in TCR signaling is not certain compared to that of
CD45.
Table 4.1 Coreceptors of CD43 and CD45 expressed in thymic T cell subsets and peripheral T
cells
CD43 coreceptor Glycan Function
E-selectin SLeX on core 2 O-glycan T cell migration
Galectin-1 LacNAc T cell apoptosis
Galectin-3 LacNAc Unknown
Macrophage galactose-type lectin Terminal GalNAc Unknown
Mannose receptor Man Unknown
CD45 Coreceptor Glycan Function
Placental protein 14 LacNAc T cell apoptosis
CD22/Siglec-2 SAα2,6 T cell signaling
Macrophage galactose-type lectin Terminal GalNAc T cell apoptosis
Serum mannan-binding protein Man, GlcNAc Unknown
Mannose receptor Man Unknown
In T cell apoptosis, β-Gal recognition lectin, termed galectin-1, leads to apoptotic

cell death of immature thymocytes residing in the thymus. Apoptotic cell death of T
cells is elicited by galectin-1 action, and this is a type of negative selection
[109]. Both activated peripheral T cells and thymocytes in the thymus are subjected
to apoptosis by galectin-1 [110]. Three known counter-receptors including CD43,
CD45, and CD7 are directly associated with the regulation of the galectin-1-driven
apoptosis of T cells [111–113]. Human T cell CD7 is crucial for galectin-1-mediated
apoptosis [112]. CD43 and CD45 are originally targeted by galectin-1 for apoptosis.
However, they enhance galectin-1-induced apoptosis [114], because galectin-1
recognizes CD43 and CD45 dependently on their glycan density and type. Origi-
nally, galectin-1 preferentially binds to asialo-LacNAc unit in non-α2,6-siyalylated
complex N-glycan forms or core 2 O-glycan forms [115]. However, galectin-1 is
also capable to recognize the core 1 O-glycan structures such as mucin or CD43
[88, 114], if they are abundantly present. In contrast, core 2 O-glycan types linked to
CD45 protein induces the galectin-1-caused apoptosis of T cells [116]. CD43 and
CD45 on immature thymocytes contain galectin-1-recognizing core 2 O-glycan
forms. However, CD45 present in matured T cells in the thymus have only core
1 O-glycan types and α2,6-sialyl N-glycan type. Thus, the so-called glycophenotype
blocks galectin-1 binding, because of the absence of α2,6-sialylation and core
2 O-glycosylation. In addition, alteration of CD43 and CD45 glycosylation renders
resistant survival potentials of mature T cells in the thymus against the galectin-1-
caused apoptotic cell death of the T cells [88, 115]. In the peripheral naive T cells,
SAα2,6- and CD45 core 1 O-glycosylation are present, rendering likewise resistance
to galectin-1-driven apoptotic cell death. The activated types of CD4+ T cells and
CD8+ CTLs also express receptors for CD43 and CD45, which carry the complex
types of N-glycan and core 2 O-glycan types with decreased α2,6-SA levels,
potentiating the activated T cells to galectin-1-induced apoptotic death
[88]. Nevertheless, because CD4+ effector Th-2 cells synthesize α2,6-sialyl

N-glycan structures and sialyl core 1 O-glycan structures, galectin-1 binding capac-
ity is reduced, and galectin-1-caused apoptotic Th-2 cells death is also reduced
[117]. Therefore, even though T cells and thymocytes in the thymus can be exposed
to galectin-1 even in the peripheral tissues, certain T cell populations exhibit
resistance to galectin-1-caused cell deaths by different glycosylation in CD43
and CD45.
As a secondary surface glycoprotein of T cells, CD28 N-glycans occupy about
50% of total molecular mass. N-glycosylated human CD28 suppresses regular
CD28-drived adhesion and co-stimulation of T cells during CD28-CD80 recogni-
tion. Complete disruption or mutation of the CD28 N-glycosylating sites or treat-
ment of N-glycan synthesis inhibitors enhances the binding to CD80 on APCs in
Jurkat T cells [118]. The branched CD25 receptor N-glycans increase its cell surface
retention time and consequently confers immune tolerance by T cell differentiation.
Experimental restriction of UDP-GlcNAc and lack of branched N-glycans prevent
CD25’s cell surface retention and inhibit downstream IL-2 signaling. This conse-
quently activates a Th-17 over-induced differentiation of activated Treg cells (called
iTreg cells) [119].
4.5.5 TCR Glycosylation Governs Hyper-response

and Autoimmune Responses in T Cells and Tregs
Autoimmunity develops upon the self-tolerance absence or autoimmune responses.

Glycans determine fate of self-/non-self-antigens. Certain pathogenic glycans pri-
marily activate the innate immune system; however, the question of how glycans
discriminate self/non-self is unclear. Abnormally controlled N-glycan synthesis
induces autoimmune and exacerbated immune responses [120]. Lack of β1,6-
GlcNAc branch synthesis in Mgat5-deficient KO mouse exhibits development of
delayed-type hypersensitivity and EAE [121] and colitis [122]. The lack of β1,6
branches in N-glycans breaks normal lattice of T cell surface by abnormal TCR
clustering in Mgat5 KO mice. The TCR clustering reduces the TCR threshold and
activates T cells in the hyperimmune response in Mgat5 KO mice. Abnormal lattice
formation indicates a broken lattice between TCR-β1,6-branched N-glycans and
galectins, leading to abnormal TCR downstream signaling [44, 123]. Similarly,
lack of β3GnT2 enzyme induces T cell hypersensitivity, as shown in β3GnT2-KO
mice. β3GnT2 enzyme attaches GlcNAc residues to the N-glycan, and the GlcNAc
residues act as a galectin ligand. However, β3GnT2-KO mice, lacking the GlcNAc
residue in the N-glycans, lost galectin ligand, poly-LacNAc on the N-glycans. This
is similar to the Mgat5 KO mice [123].
Most galectins bind to cell-surfaced N-glycans to form lattices [124] and increase
glycoprotein surface retention time [54]. The galectin-glycan interaction generates
the cell surface lattice as the receptor modulator that can regulate cell proliferation
and response. Galectin-3 binds to Mgat5-synthesized N-glycans with the β1,6-

GlcNAc branch in TCR proteins of T cells. In CD8+ T cells, the GnT-V activity
catalyzes the branched N-glycan formation, and the branched N-glycans again
increased the galectin-3-mediated membrane lattice, and this downregulates the
glycoprotein recognition, ultimately increasing the T cell activation [125]. Lack of
Mgat2 reduces N-glycan branches compared to Mgat5 deficiency. Galectin-3 bind-
ing capacities to the total LacNAcs in Mgat2-deficient KO T cells and Mgat5-
deficient KO T cells are not different for each other. Lack of N-glycan branches
yields poly-LacNAc-structured linear N-glycans. Galectin-3 prefers to bind to the
poly-LacNAc carbohydrate present ion the Mgat5-driven N-glycan branches on
EGFR and TGFβRII, delaying endocytosis [126]. The GnT-V-produced β1,6-
GlcNAc branches on N-glycans activate CD4+ T cells by inhibition of self-growth
and self-signaling levels of T cells [127]. In addition to GnT-V, enzyme activities of
α-mannosidase II (α-MII), MGAT1 gene-encoded GlcNAc-transferase I (GnT-I),
and MGAT2 gene-encoded GlcNAc-transferase-II (GnT-II) compromise the T cell
functions, which initiate autoimmune responses including EAE, IBD, SLE, and
IDDM. On the other hand, the T cell self-renewal and development are regulated
by the O-GlcNAc-transferase enzyme and GnT-I enzyme, respectively. In addition,
core-fucosyl GlcNAc-Asn catalyzed by α1-6 Fuc-transferase (FUT8) affects T cell
responses in immune diseases [127, 128]. The FUT8-catalyzed core fucosylation of
TCR proteins hyper-activates CD4+ Th cells to display auto-responses of T cells,
while FUT-8 core fucosylation to the CTLA-4/PD-1 co-inhibitory receptors induces
immune tolerance status. Likely, in the α-mannosidase-II-lacking mice, glomerulo-
nephritis is increased. In addition, glomerular IgM immunocomplex deposits, com-
plement factor 3, and anti-nuclear antibodies are accumulated [129]. These
pathologic and biochemical parameters are almost like to those occurred in lupus-
like syndrome. Thus, it is considered that N-glycosylation gives a clue in manifest of
T cell immunology.
Regarding Tregs, SIGN receptor-1 (SIGNR1/CD209b), the murine homolog of
DC-SIGN, was discovered as the secretory IgA (SIgA) receptor, where SIgA inhibits
DC maturation in signr1 KO BMDCs. IgA is the most abundant antibody isotype in
mammals and maintains homeostasis at mucosal surfaces and immune protection.
IgA interacts with various receptors such as IgA Fc receptor I (FcαRI), transferrin
receptor 1 (CD71), asialoglycoprotein receptor (ASGPR), Fcα/μR, FcRL4, and
DC-SIGN/SIGNR1. Upon IgA binding to two receptors of the FcαRI and
DC-SIGN/SIGNR1, anti-inflammatory immune responses occurred. SIgA prevents
autoimmune responses through the glycan-dependent interaction with SIGNR1 on
DCs which induces an immune tolerance via Treg expansion. This sugar interaction
prevents tissue damage in multiple autoimmune and inflammatory diseases during
SIgA-DC interaction, exhibiting a tolerogenic phenotype with IL-10 expression.
These phenotype cells stimulate the IL-10-secreting Treg expansion. Therefore,
in vivo trials using the SIgA-DC administration diminish the autoimmune disease
development in EAE and IDDM [130]. SIGN1R-mediated signaling activates and
expands IL-10-secreting Treg cells. SIGNR1 also regulates colonic immune
response and peripheral immunity against systemic pathogenic infection.
Consequently, immune suppressive SIGNR1 in murine macrophage differentiation

and cancer progression is anticipated. In a recent report [131], when SIGNR1-
positive RAW264.7 macrophages were co-cultured with Lewis lung cancer cells
(LLC) with IL-4 induction, LLC-stimulated macrophages expressed IL-10. How-
ever, inhibition of SIGNR1 by SIGNR1 (CD209b)-specific antibody inhibited the
IL-10 expression. Consequently, SIGNR1 is essential for LLC-induced polarization
to M2 macrophage sub-phenotype. The polarized M2 phenotype macrophages in a
tumor-associated microenvironment induce the LLC migrative potentials, although
the migration is prevented by SIGNR1-specific antibody. In addition, LLC-activated
macrophages inhibited the activated T cell growth and IFN-γ-mediated Th1
response, while SIGNR1 inhibition rescued Th1 cell functions. Thus, murine
SIGNR1 expressed in LLC-educated macrophages induces macrophage phenotype
change to M2 polarization and helps lung cancer evasion.
4.5.6 SAMP and N-Glycan-Dependent Modulation

of Inhibitory T Cell Receptors to Suppress T Cell
Functions
SA recognition of hosts commences with the immune response during sepsis.

SA-bearing SAMPs suppress T cell-driven cytotoxic activity in cancer cells.
SA-bearing SAMPs activate potential of immune scape and growth of tumor cells.
SAMPs elicit Siglec-engaged immune evasion of cancers from T cells [132]. The
cancer immunotherapy has recently been shifted into the immune regulation strategy
using immune checkpoint inhibitors. The inhibitory immune checkpoints are CTLA-
4- and PD-1-specific antibodies with clinical benefits. Therapeutic efficiency of these
immune checkpoint inhibitors is dependent on their target immune receptors. This
circumstance restricts the tumor therapeutic outcomes obtainable for only certain
cancer patients, and unfortunately this indicates unexpected secondary resistances.
This indicates new targeting pathways for tumor-specific T cell suppression. For
compatibility to meet the desired success, T cell-specific new receptors are candi-
dates such as the CD33-related Siglecs as PRRs of immune cells, because the
sialoglycans act as SAMPs to suppress autoimmune responses. Siglecs are expressed
in both normal T cells and tumor-infiltrating T cells. For example, Siglec-9-
expressing T cells coexpress PD-1. If the sialoglycan-SAMP/Siglec pathway is
targeted, anticancer immunity is increased. Siglec-9 is a dominant inhibitory Siglec
in cancer patients, and Siglec-9 ligands have also high expression in tumor environ-
ment. In human patients of cancer, the Siglec-9 expressed in T cells reduces cancer
patient survival rate. Thus, the sialoglycan-SAMP/Siglec pathway is a target for T
cell activation in tumor immunotherapy. Siglec-9 is an inhibitory CD33rSiglec.
Interaction between SA-based SAMPs on the tumor cells and Siglec-9 suppresses
NK cell-driven killing of cancer cells and myeloid cell-mediated cancer progression.
Sig9 + CD8+ T cells express inhibitory receptors including general immune
checkpoint proteins. When stimulated by co-stimulatory receptor antibodies,

Sig9 + CD8+ TILs are highly activated compared with Sig9-TILS. Functional
PD-1-hiCD8+ TILs and Sig9 + CD8+ TIL cells are tumor-specific CD8+ TILs. In
the role of SA-SAMP/Siglec-9 in tumor environment, SA-SAMP/Siglec-9 interac-
tion decreases T cell activation in tumor microenvironments, contributing to tumor
immune evasion. Thus, targeting Sia-SAMP/Siglec-9 can be a way of cancer
immunotherapy. SA-SAMP/Siglec-9 interaction has been also demonstrated in
tumor-bearing mice models. Siglec-E, murine counterpart of Siglec-9, increased in
tumor-infiltrating T cells, and murine SigE+CD8+ TILs is similar to Sig9 + CD8+
TILs of cancer patients. Tumor growth rate is faster in HS9 CD4-Cre mice with
hSig-9 in CD4+ and CD8+ T cells, indicating inhibitory Siglecs in T cells as tumor
evasion pathway. In another mice model with Siglec-E16, T cells are more activated
and tumor growth was suppressed. Thus, Siglec-E on T cells potentiates immune
evasion through inhibition of T cells. In lung carcinoma, Siglec-9 induces lowered
survival rate, and polymorphism of Siglec-9 gene is associated with lung cancer
development. SA-SAMP/Siglec-9 binding is indeed an immune checkpoint inhibitor
in T cell activation, which is a target of cancer immunotherapy.
The co-inhibitory receptors act in a N-glycan-dependent manner. For example, T
cell inhibitory receptor of CTLA-4 consists of two N-glycan sites, and the N-glycan
confers its T cell surface retention and controls CD80-CD28 binding on APCs
[133]. The CTLA-4 N-glycans and PD-1 N-glycans regulate the inhibitory func-
tions. Other N-glycan-dependent co-inhibitory receptors include T cell
immunoreceptor with Ig and ITIM domains (TIGIT), mucin-domain containing
molecule-3 (Tim-3) and lymphocyte activation gene 3 (Lag-3) [134]. Thus,
N-glycans activate T cell functions through TCR-coreceptor signaling.
PD-1 is also a T cell inhibitory receptor for immune inhibition towards the “T cell
exhaustion” [135]. PD-1 and Tim-3 expression is increased by the core fucosylation
of FUT8 [136]. The PD-1 core fucosylation inhibition induces an anticancer immune
response through activation of T cells, indicating a new antitumor immunity,
because the PD-1 glycosylation is implicated in T cell immunosuppression. The
PD ligand-1 (PD-L1) known as a specific ligand of PD-1 stabilizes its cells. The
binding of non-glycosylated form of PD-L1 to glycogen synthase kinase 3β
(GSK3β) responsible for glycogenesis induces the PD-L1 degradation [137]. In
triple-negative breast cancer MDA cell line, the β1,3-GlcNAc-transferase
(B3GNT3), a poly-LacNAc chain-synthetic enzyme, is essential for the PD-1 bind-
ing to PD-L1 [138]. The glycosylated PD-L1 form-specific antibody blocks the PD-1
and PD-L1 binding, resulting in its digestion and internalization and consequently
inducing tumor regressive activity against triple-negative breast cancer. Thus, nor-
mal Tregs show the different N-glycosylation patterns, compared to CD4+ T cells.
The branched complex N-glycan level is correlated with the protein production
associated with Treg suppressive roles. The candidate proteins are PD-1, PD-L1,
and CTLA-4 [139]. CTLA-4 consists of multiple sites of N-/O-glycosylation, and
these sites control its T cell surface-retained time for T cell function [133]. The TCR
is activated by β1,6-GlcNAc branches on CTLA-4 N-glycans, because the β1,6-
GlcNAc-mediated N-glycosylation increases T cell-surfaced CTLA-4 retention,
consequently suppressing T cell-driven immune tolerance [140]. Thus, the Thr17Ala

polymorphism observed in CTLA-4 of human decreases the N-glycosylation sites,
limiting T cell-surfaced CTLA-4 retention [141]. Exogenously supplemented vita-
min D and GlcNAc residues enhance branched N-glycan synthesis, upregulating the
surfaced CTLA-4 retention time and the immunosuppression.
4.5.7 Galectins in Suppression of T Cell Functions
In suppression of T cell function, galectins are also another crucial checkpoint in T

cell activity. Galectin-1, galectin-3, and galectin-9 are associated with T cell immune
suppression. Galectin-1 is produced by CD4 + CD25+ T cells and tolerogenic DCs
[142, 143], inducing T cell apoptosis upon interaction with N-/O-glycans linked to
CD45/CD43 SAMP and N-glycan-dependent modulation of inhibitory T cell recep-
tors to suppress T cell functions and CD7 or upon FAS-mediated death of resting T
cells [111]. The Th-1/Th-17-activated cells are highly susceptible to apoptotic cell
death when treated with galectin-1 if the activated cells synthesize the galectin-1-
interacting carbohydrates, whereas Th-2 cells express SAα2,6-glycans to protect
from the cell death [117]. Certain tumors can produce galectin-1 to induce immu-
nosuppression via a bias towards TH2 cytokines as well as tolerogenic activation by
DCs, which express IL-27 cytokine and type 1 Treg cells, which express IL-10
cytokine [144]. Galectin-3 role is still ambiguous in T cell functions. Cytoplasmic
galectin-3 protects the cells from apoptosis through antiapoptotic pathway with
increased Bcl-2 expression [145]. However, extracellular galectin-3 rather causes
the activated T cell death by galectin-3 binding to T cell glycosyl receptors. The dual
ambiguous pathway is distinct from galectin-1 [54]. Moreover, galectin-3 can
recognize CTLA-4 N-glycans to prolong the CTLA-4 inhibitory signals [54] and
also to the CD8+ CTLs Lag-3 to suppress the CD9+ T cell functions [146]. Finally,
galectin-9/Tim-3 interaction diminishes glycan-dependently the activities of CD8+
CTLs, Th-1 cells, and Th-17 cells [147, 148]. However, galectin-9 which binds to
other receptors may regulate proinflammatory cytokine expression [149]. Galectin-1
is known as an immune-modulator in EAE. Galectin-1 (Lgals1/) KO mice
induces TH1 and TH17 responses, causing EAE [117]. Galectin-1 also controls
the CD8+ CTL activity. Galectin-1 binding to Fas ligand keeps its retention at the
CTL cell surface, and the retention hampers the cytotoxic capacity of the T cells
[150]. Thus, GBPs and glycoprotein binding are important to motivate a T cell
response.
4.5.8 Glycans Regulate T Cell-Mediated Immune

Suppression and Tolerance in Tumor Progression
The glycosylation is crucial for T cell development and functions, as glycans

develop auto-immunities and cancer survival. Consequently, the bindings suppress
the host immune responses. Glycans regulate T cell-mediated immune suppression
and tolerance in tumor progression. If abnormally glycosylated pattern of cancer
cells are targeted, introduction of antitumor immune response will be a future
promising strategy. Tumor cells express aberrant glycan structures in sialylation
and branch formation [151]. The aberrant glycosylation affects tumorigenesis and
progression in tumor cell dissociation, angiogenesis, adhesion, growth, invasion, and
metastasis; hence they escape from tumor immunoediting or immune surveillance
[152]. GBPs expressed in immune cells bind to altered carbohydrate structures on
cell surfaces of tumor to introduce the glycan information to immune cells towards
immune stimulation or immune inhibition. Tumor cells abnormally express Tn and
Lewis antigens carrying sialylglycans, and these Tn and Lewis sialylglycans are
bound to macrophage and immature DCs DC-SIGN. The binding of MGL to
terminal GalNAc residue appeared on CD45 glycoprotein downregulates TCR
signaling, consequently decreasing T cell growth and increasing T cell apoptosis
[108]. The Lewis Fuc residues induce TH2, follicular, and Treg responses
[153]. DCs recognize DC-SIGN-binding Leb/x formulated liposomes and internalize
and consequently activate CD4+ Th and CD8+ CTL [154]. Macrophage galactose
binding lectin (MGL) recognizes Tn antigen and GalNAc residue with TLR-2
binding; ultimately IL-10 and TNF-α are secreted [155]. Moreover, tumor-
infiltrating macrophages (TIL) express IL-10. If the TIL are blocked, CD8+ CTL
effectively responds. During chronic infection, IL-10 elevates the Mgat5 gene
expression to yield N-glycan branches on CD8+ CTLs. The N-glycan branches of
CD8+ CTLs suppress T cells, and hence the infected virus or pathogens can acquire
their prolonged infection status [156]. Mgat5-branched N-glycans synthesized by
IL-10 suppress CD8+ CTLs in tumor.
Fuc residues in Lex and Ley antigens are present on tumor surface proteins like
carcinoembryonic antigen (CEA) [157] and induce the IL-10 and IL-27 production,
known as anti-inflammatory cytokines, in APCs. CLRs such as MGL and DC-SIGN
alert glycosylation changes in some CEA, and MUC1, tumor-associated antigens,
occur during onco-transformation or tumorigenesis. The alterations of glycosylation
are seen in the Lewis blood group antigenic epitopes including LeX and LeY linked
with poor cancer prognosis. Sialylglycans also suppress immune responses via
DCs-expressing Siglecs due to their inhibitory domains. For example, DCs Siglec-
E binding to sialylantigens increases in antigen-specific Treg response and decreases
in antigen-specific Teff cell responses. Teff suppresses tumor proliferation and
growth [158, 159]. The sialylated tumor mucin antigens of Sialyl-Tn (STn) and
Sialyl-T (sT) are such examples. For example, MUC1 leads to tumor immune
tolerance. The Siglec-9 binding to MUC1-ST on tumor-infiltrating macrophages
initiates MEK-ERK signaling towards immune inhibition [160]. Furthermore, Siglec
binding to Mucin sTn enables DC maturation and DC-induced FOXP3+ Treg cell
activation. In addition, Siglec-Mucin sTn binding decreases the INFγ production of
T cells [161, 162]. In fact, CD8 + TIL express Siglec-9 in NSCLC patients and
protect the NSCLC from immune surveillance, consequently reducing survival rate.
Consequently, Siglec-9 polymorphisms alert the danger signal of lung and colon
cancers. Siglec-9+ CD8+ TILs also express Lag3/TIM-3/PD-1/CTLA-4 as inhibi-
tory receptors. Defected synthesis of sialyl glycans in tumor cells reduces tumor
growth through the infiltration levels of CD4+ T cells and CD8+ CTLs [163]. Hence,
from the innate immune cells, several lectins of CTLs, galectins, and Siglecs interact
with each relevant molecule as GBPs in immune responses [164, 165].
4.6 Abnormal N-Glycosylation in Autoimmunity
Abnormal N-glycosylation-associated human autoimmunity is also seen in MS

patients. PBMC from MS patients exhibits the decreased Golgi-resident β1,6-
GlcNAc-transferase (termed core 2 GlcNAc-T) activity, in contrast to normal indi-
viduals [166]. In addition, MGAT5 polymorphisms are also associated with level of
MS progression [167] with MGAT1, IL2R, and IL7R SNPs [168]. In the IBD
progression, T cells in lamina propria region prepared from patients of ulcerative
colitis (UC) lack β1,6-GlcNAc-T-catalyzed formation of branches in N-glycan
structures because of MGATt gene deficiency [169]. When the branch levels in
N-glycosylation of intestinal TCR of T cells are profiled using colon tissue biopsies
obtained from human patients of UC, UC patients have a dysregulated
N-glycosylation in TCR on lamina propria T lymphocytes. Severe UC patients
carry defected N-glycan branches in T cells due to a reduced MGAT5 transcription.
N-glycan branch-deficient TCR is a new UC pathogenic factor, which can be used as
a potential biomarker for therapeutics. If the UC patients lack the branched
N-glycans, standard therapeutic trials will be failed [170]. When T cells resident in
intestinal region isolated from human patients of UC and mice of colitis are
supplementarily enforced with GlcNAc, β1,6-branched N-glycan structures linked
to cell-surfaced proteins on T cells are increased, and consequently, TCR signaling
and TNF-α and IFN-γ synthesis, which are proinflammatory cytokines, are
suppressed. IBD and MS can be clinically modulated by N-glycans due to T cell
immune response [51, 122], towards the development of clinical agents
[119, 122]. N-glycosylation analysis can primarily be performed in autoimmune
diseases, as muscle-associated muscular dystrophy [171] and congenital disorders of
glycosylation are known [172]. The relationship between muscle cell surface gly-
cosylation alteration and inflammation is related to the interaction between muscle
glycocalyx and the extracellular molecules in autoimmune diseases including idio-
pathic inflammatory myopathies (IIM) [173]. Glycans function as determinants in
auto-responses by regulating T cells because abnormal glycoantigens unleash an
autoimmune response.
4.7 Glycan Regulation of NK Cell Receptors 137
In mouse MRL-lpr model, the mouse phenotypes are accepted as a well-

established mice SLE. The MRL-lpr mouse accumulates agalactosyl bi-antennary
glycans and high-Man and pauci-Man contents in the kidney [174]. Moreover, in
Mgat1f/fSyn1 mice, which are characteristic of the Mgat1 gene deletion at the
Synapsin I-positive cells, and Synapsin I is abundantly expressed in neuronal area
of the brain, and neuronal functions are defected with caspase-3-mediated neuronal
apoptosis [175]. The apoptotic events activate immune responses, as frequently
observed in autoimmune responses. In rare autoimmune diseases, abnormal
N-glycosylation induces the diseases. For example, IIM belong to rare types of
autoimmune diseases [176]. Glycoproteins of muscle cell surface display a muscle
homeostasis and function. The GNE enzyme as a specific ManNAc kinase is
essential in the NeuAc biosynthesis, and the GNE gene disruption yields a broken
synthesis of NeuAc and blocking of sialylation of glycoproteins. In this case,
supplementation with ManNAc as sialic acid precursor prevents ITM-like hereditary
inclusion body myositis (hIBM) [177]. Thus, N-glycosylation is perspectively
important in formation of autoantigens, since autoantigens are N-glycosylated with
multiple N-glycosylation sites.
4.7 Glycan Regulation of NK Cell Receptors
NK cells are the first defense line in tumor immunosurveillance. Changes in the
glycosylation pattern on surfaces of malignant tumor cells influence tumor immune
responses through direct interaction with each receptor, glycan-binding protein, and
lectin expressed on the immunomodulatory cells. The NK cell activation signals are
delivered from their surface molecules such as adhesion molecules known for
LFA-1. The NK cell co-stimulatory family receptors include NKG2D, DNAM-1,
and SLAM as well as activating receptors bearing the ITAMs, TCR-ζ, DAP12, and
FcεRI-γ [177, 178]. The NK cell activating receptors also include NKp30/NKp46.
Innate immune NK cells exhibit directly cytotoxic cell killing against MHC-negative
cancer cells, stressed cells, and virus-infected cells [179]. Inhibitory receptors of NK
cells include NKG2A (CD94) and KIRs as well as Tim-3, etc., providing tolerance
of immune checkpoint via the MHC-I recognition in the normal cells. There is
imbalanced expression in the activating and inhibitory NK cell receptors, providing
the NK cell dysfunction. Among human NK cell subsets, NK cell subset like
CD56dim NK cells of CD56dimCD16 + KIR+ occupies 90% more spleen, and
peripheral NK cells contain granzymes and perforin. CD56dimCD16 + KIR+ cells
are the main cytotoxic cells [180], while CD56bright NK cells of
CD56brightCD16dim/–KIR– are the main NK cells in the tonsils and lymphatic
nodes [181]. NK cell is activated by KIRs including 2B4, KIR2DS, KIR2DL4,
KIR3DS, NKG2D, NCRs, and NKp80 in humans. The predominant ITAM-bearing
activating receptors include CD94/NKG2C, FcγRIIIa/CD16, KIR receptor subfam-
ily (KIR2DS and KIR3DS), and NCRs in human NK cells.
4.7.1 NCRs on NK Cells
NCRs include three distinct types of NCR-1 (NKp46 or CD335), NCR-2 (NKp44 or
CD336), and NCR-3 (NKp30 or CD337). The three NCRs are produced by the ncr-1
gene, ncr-2 gene, and ncr-3 gene, respectively. Each NCR selectively performs
target cell lysis through perforin, granzymes, and IFN-γ [178]. The NCRs have
been isolated by Alessandro and Lorenzo Moretta during the 1990s [182–184] as
type I transmembrane glycoproteins. They are characterized to be activating recep-
tors and termed NKp30, NKp44, and NKp46 depending on each molecular weight.
The NCRs bind to carbohydrate ligands, which are the targets for recognition of NK
cells. The NKp44 and NKp30 genes (ncr2 and ncr3) are located on human chromo-
somal 6-MHC-III locus, and the NKp46 gene (ncr1) is loaded on the chromosome
19-leukocyte regulatory complex in humans [182, 185]. Interestingly, the ncr1 gene
product, NKp46, is also expressed in mice and rats [186, 187].
NKp46 is a marker of all NK cells of human and mouse and present in certain ILC
and T cell subpopulations [103, 185]. Of interests, NKp46 is absent in human and
mouse CD1d-restricting invariant NKT cells. NKp46 recognizes and kills various
tumor target cells. In mice, NKp46 develops type 1 diabetes [185]. For tumor
survival through immune suppression, tumor microenvironments display the
decreased NKp46 expression on NK cells by a tryptophan metabolite,
1-kynurenine, synthesized by IDO enzyme, which is the indoleamine
2,3-dioxygenase [188]. NKp30 is also present on most human NK cells, as NKp46
does. NKp30 mediates the NK cell and DC crosstalk through stimulating the
immature DCs to mature DCs with cytotoxic activity. NKp30 and NKp46 expres-
sion is induced by IL-2, IFN-α, and prolactin. But cortisol and methylprednisolone
suppress the expression of NKp30/NKp46 receptors [189]. The NKp30/NKp46
expression is also suppressed “memory-like” or “adaptive” NK cells, as shown in
cytomegalovirus-infected patients [190]. TGF-β suppresses the NKp30 expression
in NK cells [191]. NKp44 is quite different from other NCRs in humans because it is
present constitutively on only CD56 bright NK cells. NKp44 is distinctly present in
IL-2-induced NK cells in higher primates [184]. Cytokines IL-15/IL-1β/IL-2
enhance the NKp44 synthesis. IL-3 increases the NKp44 expression in pDCs
[192]. PGE2 and prednisolone suppress IL-2-mediated NKp44 expression on NK
cells [193].
NCRs bear Ig-like domains as the Ig superfamily. NCRs lacks ITAM and
transduce signals through adaptor proteins having ITAMs [194]. NKp30 and
NKp46 receptors are present in the activated and rested types of NK cells
[183]. Three NCRs have different structures, but functions are only similar together,
where NCR extracellular domains contain one Ig-like region for the NKp30 and
NKp44 receptors and also two Ig-like regions for NKp46, which bind to ligands
[185]. NKp30 and NKp44 are homodimerized with NKp30 to generate an I-type
Ig-like complex. Two NKp44 V-type Ig-like domains are dimerized with unique
disulfide bridging [195]. The NKp46 shows two C-2-type Ig-like domains like the
Ig-like domains of KIRs [196]. All the NCD transmembrane domains carry a basic
Lys residue for NKp44 or Arg residue for NKp30 and NKp46. They bind to Asp
residues in DAP12 transmembrane region for NKp44 or TCR-ζ transmembrane
region for NKp30 and FcεRI-γ transmembrane region for NKp46. NKp30 and
NKp46 expressions are decreased in memory-like or adapted NK cells due to the
lacked expression of FcεRI-γ [190]. Thus, adaptors are crucial for receptor transport
to the NK cell surface. Thereafter, the transmembrane adaptors also activate the
NCR signaling because the adaptor’s cytoplasmic ITAM is phosphorylated and
recruits and activates the downstream Syk and ZAP-70 protein tyrosine kinases
[185]. NKp44 expression requires three transmembrane charged residues recogni-
tion with DAP12. NKp44 extracellular domain contains a V-type Ig-like domain for
ligand binding [197, 198], a cytoplasmic ITIM, and a Lys residue-bearing trans-
membrane domain, where Lys links to a dimer of the ITAM-bearing adaptor DAP12
[199]. Therefore, NKp44 has a dual function of either inhibitory or activating
signaling in a ligand dependency. NKp44 induces cytokine release and cytotoxic
activity in human NK cells. NKp44 also bind to self-ligands of PCNA and MLL5
alternatively spliced form [200]. Variously spliced variant forms such as ncr2 and
ncr3 which are generated for NKp44 and NKp30 have been found, providing
NKp44- and NKp30-medited inhibitory signaling.
4.7.2 NCR Ligands
NCR extracellular domains bind to carbohydrates, surfaced proteins, and cytoplas-

mic proteins that are exposed on infection or transformation (Table 4.2). In viral
ligands, influenza viral hemagglutinin (HA) on the infected cells surface binds to
branched SAα-2,3-linked and SAα-2,6-SA-likned O-glycans attached to NKp46
protein. Then, human NK cells kill the influenza-infected target cells through a
NKp46 signaling [202]. Influenza virus interaction with NK cells or HA interaction
with NK cells reduces NCR cytotoxic activity through TCR-ζ expression inhibition
[203]. DCs infected by influenza virus are activated to induce IFN-γ release in the
NKp46-dependent and HA-dependent manner in NK cells. In fact, NKp46-KO mice
easily die upon influenza virus infection [204].
NKp46D2 and NKp44 recognize HA-neuraminidase (NA) protein of the Sendai
virus, Paramyxoviridae. NKp44 binds to viral hemagglutinin of the influenza virus,
Orthomyxoviridae [205], and the envelope glycoprotein (E-protein) of Flaviviridae
[206–208]. Influenza and Sendai virus HAs specifically bind to NKp44 proteins but
not NKp30 species. NKp44+ NK cells can also destroy the influenza- and Sendai
virus-infected cells [209]. NKp46D2 and NKp44 α2,6-sialylglycans bind to the
ligands on tumor cells [210] and envelope-coated membrane glycoproteins deco-
rated on dengue and West Nile viruses [208]. NKp44 binds to charged and
IdoA2SGlcNS6S heparin carbohydrates [197].
Similarly, NKp46 and NKp44 bind to avian Newcastle disease virus HA and kill
paramyxovirus-infected target cells [211]. HA species isolated from the vaccinia
virus of humans, orthopoxviruses, and ectromelia virus of mouse bind to both
Table 4.2 Lignads and roles of natural cytotoxicity receptors (NCRs)

Ligand
Ligands Location NCRs role References
Hemagglutinin (HA) Human vaccinia virus NKp30 Inhibiting [35]
Main tegument protein pp65 Human CMV NKp30 Inhibiting [37]
DBL-1α domain P. falciparum NKp30 Activating [46]
HS Membrane NKp30 Activating [201]
BAT3/BAG6 Membrane NKp30 Activating [42]
B7-H6 Tumor cell surface NKp44 [43]
HA Influenza and Sendai NKp44 Activating [31]
viruses
HA Avian Newcastle virus NKp44 Activating [34]
Domain III of WNV envelope West Nile/dengue virus NKp44 Activating [26]
protein
NKp44L Tumor membrane NKp44 Activating [38]
PCNA Plasma membrane NKp44 Inhibiting [41]
Cytoskeleton type III vimentin Infected cell surface NKp46 Activating [40]
HA Influenza virus NKp46 Activating [25]
HA Avian Newcastle NKp46 Activating [34]
disease
HA Human vaccinia virus NKp46 Activating [35]
DBL-1α domain NKp46 Activating [46]
HS GAGs NKp46 Activating [201]
HS/HSPGs Surface All Cis ligand [70]
NCRs
NKp46 and NKp30. NKp30 protein expressed in the NK cells less kill vaccinia
virus-infected cells, which are present at the late stage of life cycle, compared to
normal cells, and due to the presence of viral HA in the target cells [212]. The
vaccinia virus-infected cell HA binds to NKp30 to suppress NK cell’s activating
function or to stimulate NK cell inhibitory response, whereas NKp46 binding to
vaccinia viral HA on host cells kills the host cells. NKp44 recognizes the West Nile
and dengue viral envelope glycoproteins [213]. NKp44 protein directly recognizes
WNV envelope protein domain III but not viral HA due to NKp44 sialylation. Cells
infected with West Nile virus easily recognize the soluble NKp44 protein and
consequently activate NK cell degranulation to release cytokine IFN-γ. Dengue
viral non-structural protein expressed in the host cells prevents NK cell cytotoxic
action due to their MHC-I expression [214]. NKp30 directly binds to pp65 of human
cytomegalovirus (HCMV) [215], and consequently, the HCMV-infected host cells
are resistant against NK cell cytotoxicity. However, when the pp65-negative HCMV
infects the cells or when anti-NKp30 MAbs are treated, the targeted host cells are
readily killed. Soluble pp65 treatment with NK cells releases the TCR-ζ protein from
the associated NKp30. pp65 disrupts activation signaling through NKp30 in HCMV
infection.
Intracellular protein ligands are moved to cell surfaces in order to function as

surfaced ligands of NCRs on cells. NKp44L is a ligand for NKp44. GP41 envelop
protein of CD4+ T cells, which are infected with HIV-1, induces expression of
NKp44L [216]. The NKp44L is initially generated as a spliced variant protein of
mixed-lineage leukemia 5 (MLL5) [200]. The MLL5 protein with a full-length form
is located as a nuclear protein in the nucleus, but the spliced NKp44L variant form is
located in the tumor cellular PM and transformed cell PMs but not in normal tissues.
NKp46 mediates the NK cell-driven cytolysis of Mycobacterium tuberculosis-
infected monocytes due to binding to vimentin expressed in infected monocytes
[217], where vimentin protein belongs to a cytoskeleton type III intermediate
filament. NK cells readily kill vimentin-expressing target cells transfected. NKp44
binds to PCNA of target cells when PCNA transports to the cellular PM region and
inhibits cytotoxic cell death and IFN-γ production of NK cells [218]. NKp44 binds
to PCNA of target cells when PCNA transports to the PM of target cells and inhibits
cytotoxicity and NK cells’ IFN-γ production. The PCNA-NKp44-induced cytotox-
icity inhibition is involved in the cytoplasmic ITIM-like sequence of NKp44. The
PCNA-NKp44-induced cytotoxicity inhibition is involved in the cytoplasmic ITIM-
like sequence of NKp44. PCNA-NKp44 binding induces a conformation shift to
transduce an inhibitory signal in ILCs and pDCs [192]. PCNA-NKp44 binding
induces a conformation shift to transduce an inhibitory signal in ILCs and pDCs.
NKp30 binds to the Bcl2-associated anthanogene 6 (BAG6) and HLA-B-associ-
ated transcript 3 (BAT3) and induces NK cell cytotoxicity [219]. BAG6 and BAT3
proteins are mainly present in the nuclear region but transported to the PM of heat-
shocked cells or secreted into exosomes, which was formed by stressed and tumor
cells. BAG6/BAT3-producing exosomal vesicles induce cytokine secretion by NK
cells during their NKp30 recognitions. DCs BAT3/BAG6 activate NK cells to
interact with DCs. NKp30 binds to a cell surface B7 family, B7-H6 [220]. Interaction
between NKp30 and B7-H6 promotes NK cells’ cytotoxic activity and cytokine
expression. Although normal cells do not express B7-H6, human tumor cells
produce it or upon TLR ligand induction of monocytes and neutrophils or inflam-
matory cytokines [221]. Certain tumor cells prevent NKp30 interaction through
shedding B7-H6 with the ADAM-10 and ADAM-17 metalloproteinases [222] as
well as by reduced NKp30 production by peritoneal NK cells, because of chronic
recognition of ligand in breast cancers [223].
4.7.3 Interaction of NCRs Ligands with Pathogens
In pathogens, NCRs bind to bacterial and parasite pathogens. NKp30 and NKp46
directly recognize the Duffy binding-like (DBL)-1α region of erythrocytic cellular
membrane protein-1 produced in Plasmodium for malaria-infected erythrocyte lysis
[224]. NKp44 directly binds to Mycobacterium bovis BCG [225], and BCG
increases NKp44 production in CD56 bright NK cells [226]. Nocardia farcinica
and Pseudomonas aeruginosa bind to NKp44. NKp44 stimulation activates NK cell-

mediated autoimmunity in chronic cartilage inflammation.
4.7.4 Interaction of NCRs Ligands with Self-Ligands
Recombinant NKp46 and NKp44 extracellular domains (rNKp46, rNKp44) strongly

recognize and bind to K562 cells, where the Fuc-transferase (FUT)-3 gene synthe-
sizes the sLeX epitope of NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAc-R sugar structure
[227]. The rNKp46 and rNKp44 bind to sLeX-positive transferrin secreted by human
hepatoma cells such as HepG2 cells, but not NKp30 (rNKp30) [228, 229]. In a recent
paper [230], Globo-A also binds to rNKp44 and induces NKp44-mediated NK cell
cytotoxicity. Globo-A binds to rNKp44. rNKp44, but not rNKp30 and rNKp46,
recognizes Globo-A although the binding affinity is weak. Globo-A with the carbo-
hydrate structure of GalNAcα1,3(Fucα1,2)
Galβ1,3GalNAcβ1,3Galα1,4Galβ1,4Galβ1-Cer sugar structure is generated by the
specific enzyme of 3-α-GalNAc-transferase, which transfers GalNAc residue to
Globo-H structure of Fucα1,2Galβ1,3GalNAcβ1,3Galα1,4Galβ1,4Galβ1-Cer.
Globo-A species is found in the kidney [231] and erythrocytes [231, 232]. The
Globo-A sugar structure resembles with the blood A antigenic determinant of ABH
blood group, which has the GalNAcα1,3(Fucα1,2)Galβ1- structure. NKp44 does not
bind to Globo-H but the terminal part of NKp44 binds to Globo-A. The A antigen of
the ABH blood group is present in many human tissues and associated with invasive
progression of cancer cells. The NKp44 protein synthesis in NK cells is also
associated with the autoimmune reactions. For example, the SCLC and breast
cancers [233] exhibit the disappeared antigen of the blood group A, and this
phenotype accelerates cancer invasiveness and poor prognosis. Therefore, NKp44
to blood group A antigen interaction would be a key mechanism of tumor
progression.
In addition, NKp44 and other NCRs directly bind to HS or Hp on HSPGs. HSPG
present in NK cells binds to NKp44 present on NK cells itself in a cis type
[234, 235]. However, binding of NK cell NKp44 to target cells HSPG is a trans
type. The cis interaction NKp44 and the HSPG syndecan of NK cells affects
distribution and function of NKp44 and the NKp44 on the membrane [236]. As
NKp44, NKp46 and NKp30 are bindable to HS, all the NCRs preferably bind
sulfated HS [177, 197, 237–239]. HS on the mammalian cells is predominantly
supplied from the syndecan HSPG. KIR2DL4, NKG2D [240–242], and all three
NCRs [178, 243] also recognize HSPGs. NCRs recognize distinct HS structures
[197]. Exogenous HS interferes with endogenous binding NCR and HS present in
the NK cell surface. NKp44-HS interactions regulate the NKp44 function of NK
cells. Syndecan in B lymphocytes also result in an “activated”-like phenotype of
BCR stimulation. The membrane proximal domain 3 of CD19 binds to HS. CD19
forms BCR synapse via cytoskeleton organization in B cells [244]. HS and heparin
mimics can suppress progression and metastasis of tumors via NK cell antitumor
responses.
NK cells also self-modulate its receptor function through the cis “masking.” For
example, a “-cis” interaction of NK cell receptor with its ligand can be seen in the
Ly49 receptor binding to MHC-I ligand in the NK cells of mice. The Ly49 protein is
normally “masked” by the MHC-I of NK cells in a cis-type recognition [245],
suppressing the inhibitory potential. The –cis binding of Siglec 7 known as
CD328 and α2,8-disialyl ligand on NK cell surfaces is another type of cis interaction
[235, 245, 246]. Among GAGs, HS species can bind to KIR2DL4 protein, another
NK cell receptor. The binding regulates receptor signaling [247]. NKp44 binds to the
HSPGs known as syndecan-4 of the NK cells in a cis type on the NK cells and
modulates the receptor distribution and function. HSPG cis recognition regulates
KIR2DL4 and NCR functions via masking target cell trans-recognition of HS
species or other ligands present in cells, alerting the NCR trafficking to cytosolic
degradation site and recycling derived from internalization [248]. Thus, cis-NCR
and HSPG recognition influences the cell function.
BAT3 is the HLA-B-associated transcript 3 (BAT3), and BAG6 is the Bcl2-
associated anthanogene 6 (BAG6). They are transported from nuclear to membrane
or exosomes. DBL-1α domain is an erythrocyte membrane protein-1 of
P. falciparum. The alternatively spliced variant form, NKp44L, is the variant of
the mixed-lineage leukemia-5 protein present in the nuclear region. Proliferating cell
nuclear antigen (PCNA) is also the nuclear protein and transported to membrane.
Vimentin is the intracellular protein of cytoskeleton type III protein.
4.7.5 NK Cells MHC-I-Independent Inhibitory Receptors

Siglec-7 and Siglec-9
Siglec-7, which is the p75/AIRM1 protein, and Siglec-9 belong to MHC-I-indepen-

dent inhibitory receptors expressed in NK cells of human. In NK cells, Siglec-9
predominantly presents in CD56dim NK cells [249], and this selectivity indicates the
matured type of NK cells with chemotactic potential. In tumor patients, the level of
Siglec-9-expressing NK cell subsets is reduced [250]. Siglec-9 is an emerging
immune regulator. NK cell functions are strictly regulated by the surfaced receptors.
In virus-infected patients, Siglec-9-expressing NK cells are stimulated with activa-
tion receptors including NKG2D, NKp30, and NKp46, but with inhibition receptor
of NKG2A. Such receptors resemble known inhibitory receptors such as PD-1
[251]. Siglec-7 or Sigelc-9 ligand expression on malignant tumor cells also strongly
and directly activates primary NK cell activity in humans. Siglec-7 and Siglec-9 bind
to certain gangliosides such as GD2, GD3, and GT1b produced by human tissues
and neuroectodermal origined tumor cells, because these cells and tissues contain
Siglec-7- and Siglec-9-specific ligands [252, 253]. Using recombinant Siglec-IgG1
Fc fusion, Siglec-7 and Siglec-9 were demonstrated to bind to melanoma cells or
lesions as well as AML of CLL leukemia of patients in humans [254]. The Siglec-7-
Fc protein strongly binds to the GD3 synthase-transfected P815 cells and enhanced
NK cell cytotoxicity but not inhibit due to Siglec-7-independent efficacy
[246]. Hence, diverse ligands for Siglec-7 and Siglec-9 function in distinct tumor
forms for protection from NK cell cytotoxicity.
Interaction between Siglec-7/Siglec-9 with their ligands is applicable for the
therapeutic, diagnostic, and prognostic biomarkers of malignant cancers in humans.
Human tumor-expressed Siglec-7/Siglec-9 ligands are recognized by NK cells.
Cytotoxic NK cell-produced Siglec-9 is regarded as a pan-NK cell biomarker
[255, 256], while Siglec-9 is specifically present in the type of CD56dim NK cell
subset [257], and peripheral Siglec-9-positive NK cell subpopulation level is
decreased in cancer patients. The CD56dimSiglec-9+ NK cells exhibit the receptor
expression of inhibitory KIRs and ILT2 and consequently decreasing target cell
killing capacity. The chemotactic activity of NK cells, which express the
CD56dimSiglec-9, tropic to IL-8, is stronger than that of CD56dimSiglec-9-negative
type of NK cells. The expression level of chemokine receptors such as CXCR1
known as IL-8 receptor or CX3CR1 is much high in CD56dimSiglec-9+ NK cell
subset. Siglec agonists/antagonists are beneficial for targeting or cell-based therapies
[258, 259]. Therefore, the Siglec-7/Siglec-9-producing NK cells or their ligands
would be a potential strategy for NK cell-derived therapy to antitumor immunity
[260]. For example, Siglec-9 expressed on the tumor-associated macrophages binds
to sialyl O-glycan-bearing MUC1 (MUC1-ST) on tumor cells to acquire tumor-
associated microenvironment in invaded tissue [160]. Stem cell transplants, which
are allogeneic KIR-mismatched, in human leukemic diseases can be beneficial if
Siglec-7 and Siglec-9 expression is suppressed in donor NK cells [261]. Siglec-7/
Siglec-9 agonists as NK cell inhibitory receptors contribute to improved graft
survival in solid organ transplantation [262]. Siglec-2, Siglec-3, and Siglec-8 are
targeted with autoimmune and allergic diseases as well as non-Hodgkin lymphoma,
hair cell type leukemia, and AML [258, 259].
Siglec roles in NK cell subsets are not well understood, because Siglec-7 and
Siglec-9 ligands are present even in cancer cells or healthy cells [263, 264]. Siglec-9
binding in neutrophils makes its quiescence in the bloodstream [265], blocking its
recruitment and oxidative burst as well as cell death in inflammatory milieu or cancer
[266]. Siglec-9 is a biomarker for the lowered CD56dimSiglec-9-positive NK cells
in tumor. Siglec-9 is specifically present in CD56dim NK cells. Thus, ligand analysis
specific for Siglec-7/Siglec-9 in immune cells like CD56dimSiglec-9+ NK cells can
define distinct functionalities. For example, the ganglioside DSGb5 expressed on
renal carcinoma cells is a Siglec-7 ligand [264]. GD3 or DSGb5 expression does not
influence on NK cell function, while neuraminidase pretreatment of NK cells inhibits
NK cell cytotoxic activity due to unmasking Siglec-7.
The secreted or membrane glycosylated tumor antigens including CA125, CA19-
9, CEA, and MUC1 are recognized as tumor markers. MUC16 is a specific ligand for
Siglec-9 [257]. MUC16 on human epithelial ovarian cancer cells or shed MUC16 are
observed in serum or peritoneal fluid as the CA125 cancer marker. MHC-I-inde-
pendent Siglec-7/Siglec-9 leads to NK cell inhibition in aberrant sialoglycan ligand-
4.8 Carbohydrate Recognition of Target Antigens by DCs During Infection and. . . 145
bearing tumor cells. Siglec-7/Siglec-9 expression in human NK cells [246] resem-

bles the typical inhibitory receptors like KIRs, CD94/NKG2A, and Ig-like tran-
scripts (ILTs), because they have one or more ITIMs. NK cell receptors recognize
the MHC-I, indicating the “missing-self” theory to support the rejection concept of
the infected/stressed and tumor cells, deficient for MHC-I, and discrimination
between normal self and abnormal cells. Siglecs as ITIM-bearing and
MHC-independent inhibitory receptors negatively transduce downstream signalings
to NK cells, regardless of the condition that the cells lost the MHC class I molecule,
which is a missing-self status. Hence, virus-infected and tumor cells require Siglecs,
and therefore they evade the immune surveillances of hosts as a strategy of immune
escape. For example, hepatitis B viral patients exhibit the reduced Siglec-9 presence
in NK cells. Thus, Siglec-9 blocking activates NK cell function [267]. Inhibitory
receptors in innate NK cells and adaptive CD8+ CTL immunity strengthens anti-
cancer activity. For example, blocking KIRs, CTLA-4, or PD-1 controls inhibitory
or activating signal transduction in innate and adaptive immune cells to regress
cancer cells [260, 268, 269]. In addition, for telomeric length, CD56dim NK cells
undergo cell divisions with 5–10 times more than that of CD56bright NK cells,
causing a shortened telomeric length with the 50–100 bp lengths per each cell
division [270]. But telomeric length difference in the two cell types of
CD56dimSiglec-9-positive and CD56dimSiglec-9-negative NK cell types was not
detected, suggesting a growth-independent Siglec-9 expression [254].
4.8 Carbohydrate Recognition of Target Antigens by DCs

During Infection and Inflammation
Inflammation is a process of self-defense from invasive agents or allergens. There-

fore, this protective and alert reaction is involved by invasion of foreign agents or
pathogenic infection such as virus, viroids, prions, bacteria, yeasts, fungi, helminths,
and larger organisms like macroparasites or protists into tissues or organs of host
organisms. During the infection of foreign agents, hosts fight against those infectious
agents for defense using innate or acquired immune responses of hosts. Considering
the side of infectious agents, the host cells or organisms have to react in some ways
like altering the inflamed sites or positive defense. One of such ways during
pathogenic infectious events is the systemic induction of changes in host glycosyl-
ation level, especially in its cell surfaces.
The resulting changes in glycosylation status in cells, tissues, organs, and organ
systems of host organisms are directly linked to changes in functions and structures
of lipids, proteins, and membranes. It means that host cells are ready to allow distinct
functions against attacks. Especially, inflammation events accompany the altered
glycosylation status of lipids and proteins and are responsible for cellular functions,
giving capabilities to adapt of host immune system in mammals. In pathological
description, the status is termed “infectious diseases” or “immune dysfunction
diseases” when the host immune systems are not normally functioning. In the case of
targeting host immune system, pathogenic agents unable the host immune system.
With regard to the glycosylation-based pathology, the Glyco-Evasion hypothesis
suggests that invasive and pathogenic agents regulate host glycosylation events to
promote infection by making host immune system malfunctioning. The Glyco-
Evasion hypothesis has been suggested by Kreisman and Cobb (2012) [271],
indicating that invasive and pathogenic agents regulate host glycosylation events
to accelerate infection through host immune retardation [21]. Thus, pathogens have
been evolving to modulate host immune responses by controlling its glycosylation,
indicating that pathogens regulate the host immune response via glycosylation [272].
Surfaces of mammalian cells are covered by glycocalyx including glycolipids,
glycoproteins, glycophospholipids (GSLs), GAGs, and proteoglycans. Glycocalyx
is synthesized and matured in the ER and Golgi apparatus. Some of them are
transported to the cell PMs. As glycocalyx is biologically important for develop-
ment, growth, communication, and recognition of cells, this glycome is recognized
by surrounded or neighbored cells to interact and communicate for the multicellular
societies. This process confers the dynamic system of construction in tissues, organs,
organ systems, and individuals. If the state is in non-normal or pathological danger-
ous outcome, the process that DCs migrate to peripheral tissues requires the molec-
ular recognition of the counterpart target cells which is operated in vascular fluids
and lymphatic nodes.
4.8.1 Lewis Ligand Recognition by DCs
In the initial studies on sialic acids, sialyl ligands have been demonstrated to
modulate the leukocyte homing or trafficking. During the 1980s, factor H was the
sole intrinsic SA-binding protein. SA residue in the SAα2,3Galβ1,3/4(Fucα1,3/4)
GlcNAcβ1-R (SLeX/A) is used as intrinsic selectin ligands [273]. The sLeX/A motifs
are cooperated with the more negatively charged sulfates attached to the Gal residue
or GlcNAc residue for L-selectin ligands (Fig. 4.2a, b). Such sulfation is also found
on adjacent tyrosine residues for P-selectin ligands, allowing solely recognition sites
on mucin-type O-glycoproteins [274]. The N-terminal sulfoglycopeptide motif on
PSGL-1 is a key P-selectin ligand [275]. Although Sias are negative charge carriers
and typical ligands for selectins, the esterified sulfate at the C3 of galactose is also
used as selectin ligands [276]. Likely, although the α2,3sialyltransferases and α1,3/4
fucosyltransferases synthesize the selectin ligands, GlcNAc sulfotransferases and
tyrosine sulfotransferases are also alternates [277]. Certain 6-O-sulfated GAGs
including HSe are also alternate selectin ligands [278]. Functionally, Sias are a
negative charged pattern served in innate immunity. SAs are the SAMPs
[279]. The different amounts of sialic acid on erythrocytes of different mice strains
may reflect the control extents of the alternative complement pathway activation
[280]. Ficolin is a soluble lectin of the lectin pathway to activate complement system
u s
A) s B)
COOH
j T
E-selectin
G s

CR repeat domains
uoY
lnmG
s EGF domain
jyGG
GGGGGGGGGGGG
Carbohydrate-recognition domain
NH2 S-Lewis
Gly-CAM Gly-CAM
jvvo
hG Peripheral node HEV Endothelial Cell
G
Fig. 4.2 Leukocytes trafficking to the inflammatory endothelium by E-selectin-sialyl Lewis

interaction (a). It was also known that lymphocytes also migrate into the peripheral node HEV
endothelium (b)
(A) Sialyl LeX Fuc-α1

p
3
NeuAc-α-(2→3)Gal-β-(1→4)-GlcNAc-β-(1→3)Gal-β-1-R
(B) Sialyl Lea Fuc-α1
p
4
NeuAc-α-(2→3)Gal-β-(1→3)-GlcNAc-β-(1→3)-Gal-β-1-R
(C) VIM-2 Fuc-α1
p
3
NeuAc-α-(2→3)Gal-β-(1→4)-GlcNAc-β1→3Galβ1→4-GlcNAcβ1→3Galβ-1-R
Fig. 4.3 Some ligand sugar structures of selectins of sialyl-LeX, sialyl-Lea, and VIM-2
in circulation and recognizes sialic acids on the pathogenic bacterial surfaces with
sialylated glycans [281].
For molecular recognition of the endothelial barriers, specifically expressed
“selectin ligands” in DCs bind to its receptors, E-selectins/P-selectins, present in
vascular endothelia with attachment to the endothelial lining [282]. Selectins also
function in the DC trafficking to peripheral tissues. Immatured DCs, not matured
DCs, recognize the E-selectins/P-selectins for the migration into inflammatory skin
or tissues. Lymphocytes are also migrated into the peripheral node HEV endothe-
lium. The representative ligands of E-selectins/P-selectins are the glycan determi-
nants of SLeX or SLeA. SLeX or SLeA is peripherally present as sugar oligomers
attached on O-glycans, complex N-glycans, or glycolipids synthesized by several
glycosyltransferases such as GalT, FucT, GalNAcT, and Sia-T (Fig. 4.3). Sialyl
Lewisa (type I) and sialyl Lewisx (type II) determinants are structurally similar in
their linkages (Fig. 4.4). Some difucosyl Lex are also known, and this ligand is
A) SLeA
SiaD2 ,3GalE1,3GlcNAc E1,3Gal1-R
Ƅ Ƅ
Sialyl Lewisa Sialyl Lewisx FucD1,4
ȼ ȼ SLeX
Ƅ Ƅ SiaD2,3GalE1,4GlcNAcE1,3Gal1-R
FucD1,3
B)
NeuAcD D2 3GalE 1 3GlcNacE1 3GalE1 R
4
FucD1
ST3Gal III
(D2,3 sialyltransferase) Sialyl Lea D1,3 or 1,4 fucosyltransferase
NeuAcD2 3GalE 1 4GlcNacE1 3GalE1 R

3
FucD1
Sialyl LeX
: GlcNAc : Fucose : NeuAc : Galactose
Fig. 4.4 Structures and synthetic enzymes of sialyl Lewisa (type I) and sialyl Lewisx (type II)
determinants. (a) Schematic structure. (b) Synthetic enzymes
synthesized by α(1,3)Fuc-Ts and α(2,3)-Sialyl-T from Lex and

Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ1-R-, NeuNAcα2,3,
Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ1-R- and Galβ1,4GlcNAcβ1,3(Fuc-α1,3)-
Galβ1,4GlcNAcβ1-R-, or Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ1(Fuc-α1,3)-R-
precursor structures. The respective β1-3 and β1-4 galactosyltransferases determine
the sialyl Lewisa (type I) and sialyl Lewisx (type II) determinants, respectively
(Fig. 4.5a, b). Terminal type II disaccharides are further modified by several
enzymes including CHST2, CHST4, and CHST6; GlcNAc6ST3; FUT3–FUT7,
FUT9–FUT11, and FUT1–FUT2; ST3Gal III, ST3Gal IV, and ST3Gal VI;
Gal3ST2, Gal3ST3, and Gal3ST4; and ST6Gal I and ST6Gal II (Fig. 4.5c).
Sialyl-difucosyl LeX is synthesized from the VIM-2 glycan structure
(NeuNAcα2,3Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ1(Fuc-α1,3)-R) or sialyl-LeX
structure (NeuNAcα2,3Galβ1,4GlcNAcβ1,3(Fuc-α1,3)Galβ1,4GlcNAcβ1-R) from
the precursor glycan structure of
NeuNAcα2,3Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ1-R. Sialyl-Lea structure
(NeuNAcα2,3Galβ1,3GlcNAc(Fuc-α1,3)-R) synthesized by α(1,3/1,4)Fuc-Ts from
NeuNAcα2,3Galβ1,3GlcNAc-R as the precursor glycan, where Galβ1,3GlcNAc-R
is converted by α(2,3)Sialyl-T. Lea is directly synthesized by α(1,3/1,4)Fuc-Ts from
the precursor glycan Galβ1,3GlcNAc-R (Fig. 4.6a, b, c).
B) Sialyl Lewis A
A) FucD1
Fucosyltransferases
Ƅ Ƅ Ƅ ST
4
SiaD2 3 GalE1 3GlcNAcE1-R Type 1 chain
ȼ ȼ ȼ
E1-3 galactosyltransferase
Type Ⅰ Type Ⅱ Type Ⅲ Sialyl Lewis X

FucD1
Fucosyltransferases
n nuh nuh zh ST
3
SiaD2 3 GalE1 4GlcNAcE1-R Type 2 chain
E1-4 galactosyltransferase
C) D] ]z
]
X 1: CHST2, 4, 6, GlcNAc6ST3
Zz j 2: FUT3-7, FUT9-11
\ E 3: FUT1, 2 Gal Fuc GlcNAc NeuAc S Sulfate
Y 4: ST3Gal III, IV, VI
[ Z DZ
C Core sequence Type 2 : GalE1-4GlcNAc
5: Gal3ST2, 3, 4
DZ 6: ST6Gal I, II
DY
Fig. 4.5 (a) Type I, II, and III sialyl disaccharides. (b) β1-3 and β1-4 galactosyltransferases for
sialyl Lewisa (type I) and sialyl Lewisx (type II) determinants. (c) Glycosyltransferases forming
terminal type 2 disaccharides
4.8.2 VIM Ceramide Dodecasaccharide
VIM-3 epitope has a structure of ceramide dodecasaccharide 4c and is known as

CD65. Historically, the VIM-2 or CD65 epitope has been known from the
fucosylation mutants. There are several mammalian α(1,3)fucosyltransferases
(α1,3Fuc-Ts) [283]. In order to study the regulation of α1,3FucT expression, the
CHO mutants of α1,3FucT-VI-expressing LEC11, LEC11A, and LEC11B were
established. The two genes of Fut6A and Fut6B genes are known [284]. The genetic
mutation of the two genes produces SLeX with the structure of SAα2,3Galβ1,4
(Fucα-1,3)GlcNAc-, VIM-2 (CD65s) of the SAα2,3Galβ1,4GlcNAcβ1,3,Galβ1,4
(Fucα1,3)GlcNAc-R structure, and LeX with the Galβ1,4-(Fucα1,3)GlcNAc-struc-
tures linked to glycoproteins. Other mutants of LEC12 and LEC29 can not be
recognized by anti-SLeX antibody, because they do not fucosylate α2,3sialylated
LacNAc [285], due to non-expression of the Fut6A, Fut6B, or Fut7 genes. The
LEC12 and LEC29 exhibit different patterns of fucosylation. While LEC12 cells
produce the LeX and VIM-2 antigens, LEC29 cells are negative for the VIM-2
synthesis [285]. α1,3FucT-IX expression produces the VIM-2 antigen as oncofetal
epitope on the cell surfaces. The Fut9 gene in LEC29 mutant cells results in VIM-2
expression, as enhanced by transfection with a Fut9 cDNA. Thus, VIM-2 expression
is dependent on the α1,3FucT-IX. Using the substrate of nLc6 glycolipid,
Galβ1,4GlcNAcβ-1,3Galβ1,4GlcNAcβ1,3Galβ(1,4)Glcβ1,1-ceramide, the
α1,3FucT-IX enzyme in LEC12 cells catalyzes preferentially the fucosylation on
the terminal GlcNAc [286]. Human α1,3FucT-IX prefers LacNAc as substrate,
compared to di-LacNAc, and also prefers the terminally located GlcNAc residue
not the internal location or proximal location of GlcNAc residue of di-LacNAc
[287]. Among the tri-LacNAc [288] or tetra-LacNAc [287], human α1,3FucT-IX
zGGGGG
A)
Difucosyl LeX Difucosyl LeX
Fuc-α1 Fuc-α1
p p Fuc-α1 Fuc-α1
3 3 p p
Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→R 3 3
Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→R
α(1,3)Fuc-Ts α(1,3)Fuc-Ts
LeX
Fuc-α1 Fuc-α1
p p
3 3
Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→R Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→R
α(2,3)-Sialyl-T
α(1,3)-Fuc-Ts
NeuNAcα2,3
B) Sialyl-Difucosyl LeX
Fuc-α1 Fuc-α1
p p
3 3
NeuNAcα2→3Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→R
Sialyl-Difucosyl LeX
α(1,3)Fuc-Ts Fuc-α1 Fuc-α1
p p
3 3
VIM-2
Fuc-α1
p
3
NeuNAcα2→3Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→R α(1,3)Fuc-Ts
Sialyl-LeX
Fuc-α1
α(1,3)Fuc-Ts p
3
C) Sialyl-Lea
Fuc-α1 α(2,3)Sialyl-T
Lea
Fuc-α1
p p
4 4
NeuNAcα2→3Galβ1→3GlcNAc→R Galβ1→3GlcNAcβ1→R
α(1,3/1,4)Fuc-Ts α(1,3/1,4)Fuc-Ts
α(2,3)Sialyl-T
NeuNAcα2→3Galβ1→3GlcNAc→R Galβ1→3GlcNAc→R
Fig. 4.6 Synthesis of difucosyl LeA/X (a), sialyl-difucosyl LeA/X (b), and sialyl-LeA/X (c) as
selectin ligands during the inflammatory response towards endothelium interaction
also prefers the terminal GlcNAc residue. In contrast, for the substrate of α(2,3)
sialylated polylactosamines, human α1,3FucT-IX prefers fucosylation of internal
GlcNAc residues, which is far from the SA [287]. Thus, VIM-2 is the glycolipid
class bearing internally fucosylated sLex isomer. The VIM-2 antigenic epitope
contains an SAα-2,3- moieties with two LacN units, where the α-1,3-fucosylated
moiety is lined to the GalNAc residue of the internal LacN units. VIM-2 on
glycolipids is a binding determinant for E-selectin, and, therefore, VIM-2 carbohy-
drates potentiate E-selectin binding to the Fut-9-expressing cells [289].
The VIM-2 has the carbohydrate structure of Galβ1,4GlcNAcβ1,3Galβ1,4
(Fucα1,3)GlcNAcβ1,3Gal β1,4GlcNAc β1,3Gal β1,4GlcNAc β1,3Gal β1,4Glcβ1-
Cer. The VIM-2 epitope (CD65) also includes the carbohydrate structure of
NeuAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc. This VIM-2 structure
is frequently sulfated, and the VIM-2 epitope (CD65) is a putative ligand, but a
minor ligand of E-selectin (CD62E). CD65 is the solely independent risk factor of
AML for leukemic extravascular dissemination. The Mab VIM-2 recognizes the
human myelomonocytic lineage of blood cells [290]. VIM-2 Mab binds to the
LacNAc unit in β1,3-conjugated repeats [291]. CD65 carries single Fuc residue.
CD65s is α2,3-sialylated ceramide dodecasaccharide 4c (VIM-2) as the VIM2-
specific antigenic epitope from CML cells both with sialylation and fucosylation
[292, 293]. VIM-2 also binds mucins of gastrointestinal tumor such as adenocarci-
noma [292]. Galectins can bind to the tandem-repeat sugars in CD65. In addition, a
sialyl CD65s is a form of the 2,3-sialylated ceramide dodecasaccharide 4c or
2,3-sialyl VIM-2, having a structure of α(1,3)-fucosylated sialyllactosamines on
the Neu5Ac α2,3Galβ1,4GlcNAcβ1,3Galβ1,4-(Fuc α1,3)-GlcNAc-β
1,3Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAc β1,3Gal β1,4Glc β1-Cer. The Fucα1,3-
linked to GlcNAc is specific for the penultimate LacNAc residue of a terminal
polylactosaminyl glycan of NeuAc(SA)α2,3Galβ1,4GlcNAc-β1,3Gal-β1,4
(Fucα1,3)GlcNAc-R. Because AML is a blast cell type resided of the bone marrow
and peripheral blood. Both acute lymphoblastic leukemia (ALL) and AML are
extravascularly infiltrated. Although extravascular infiltration is predominant in
ALL, the extravascular infiltrative disease in AML is rare. As peripheral blood
leukocytes traverse the vascular endothelium, AML blast cells also undergo the
property in order to escape from the vasculature [294]. Interaction between leuko-
cytes and vascular endothelium is regulated by adhesion molecules. In the adhesive
extravascular infiltration by AML, a major E-selectin ligand CD15s is involved.
Secondly, a minor E-selectin ligand CD65 is also involved in leukemic infiltration.
In AMLs extravascular leukemic cell infiltration utilizes the CD65.
When foreign agents or pathogens are invaded, systematic glycomics are changed
because of the molecular adaptation of the cells or organisms as well as changes in
glycan-transferring, modifying, and hydrolyzing enzymes. This process allows
foreign agents or pathogenic invasion to alarm the host organism to inflammation
through changes in carbohydrate expression and immunity. During pathogenic
infection and inflammatory reaction, the glycans or carbohydrate structures and
compositions in lipids and proteins of cells are changed. The recently made word
named “glycomics or lipidomics” covers the systemic glycan structures linked to the
glycoproteins/glycolipids/glycosaminoglycans of cells and tissues or organs. For

example, pathogenic infection and consequent inflammation result in altered glyco-
sylation of hosts. This process in modulation of host glycosylation leads to evolution
and adaptation to trigger the host defense. Therefore, the evolutionary adaptation of
the protein, lipids, and function of the host is carried out by altered glycosylation. As
such phenotypes, the cells inform the invaded status to the bodies via immune cells
in immune system, as a mode of inflammation or alarms or alerts.
Many glycosyltransferase expressions are regulated during the monocytic differ-
entiation into macrophages or DCs [295, 296]. The glycosyltransferases synthesize
cell surface glycoproteins and glycolipids to regulate various cellular responses
including host defense, tissue homeostasis, immunity, inflammatory responses, or
cancer behaviors [297, 298]. Thus, the carbohydrates responsible for cellular regu-
lations such as endocytosis, intracellular trafficking of proteins, folding, cell inter-
action, adhesion, communication, signaling, apoptosis, and proliferation are recently
subjected to elucidate the functions and structures [80, 299]. Especially, the struc-
tural basis due to their diverse complexity of glycans is the next step of functional
analysis to understand cell-cell recognition and interaction [300]. Moreover, those
carbohydrates, glycans, function as receptors for specific ligands such as lower
organisms of viruses, bacteria, and helminths during infection and toxins of
GM1-specific cholera toxin [301]. Glycans are directly associated with host immune
system even in innate immunity and adaptive immunity. In addition, the glycans are
co-evolved to antigen presentation of MHC, priming of T cells, leukocyte homing to
inflammation sites, and lectin receptor function. Changes in surface gangliosides of
cells are also known with regard to function of immune cells.
4.9 Glycan-Specific Trafficking Receptors in DC

Maturation
DCs with the antigen induce only a primary immune response in resting naïve T
lymphocytes, priming a naive helper T cells. To B cells, DCs keep the B cell function
in control. Thus, antigen-specific DCs function as a tolerance initiator, and DCs are
regarded as adaptive immune response player and memory. During activation stage
of DCs in such condition of pathogenic infection, (i) DCs uptake antigens and
activate signaling pathway to induce DC maturation and receptor-mediated endocy-
tosis that maintains self-tolerance, micropinocytosis, and phagocytosis, and (ii) DCs
proceed to maturation step where immune stimuli induce changes in the phenotypes
and functions. In maturation stage, the levels of antigen uptake and lysosomal
acidification are decreased, while levels of co-stimulatory receptors of MHC-II/
CD86/CD80 and DCs-specific inflammatory cytokine production are increased.
For the final stage of migration, cDCs or inflammatory DCs with antigen are
migrated to T cell area. Chemokine-mediated cells are recruited to the lymphoid
target site with increased adhesion to the endothelium by adhesion molecules
4.9 Glycan-Specific Trafficking Receptors in DC Maturation 153
(integrin, selectin, and its ligands). When foreign agents or pathogens invade the
host organisms, immune cell trafficking to inflamed sites is a fundamental action for
detection and search of inflammatory site. To do this in inflammatory site, attractive
trafficking molecules are synthesized from the immune surveillance cells and host
target cells. By a basic mechanism of immune cells binding with trafficking mole-
cules expressed on the host cells of tissues and immune cells can enter inflammatory
site. To interact with target tissues for entering and escaping tissues, interaction of
molecule to molecule is the most fundamental recognition. Then, the question is
raised. What is the recognition molecule? The answer is that they are carbohydrate
molecules expressed in DCs and host cells together. First, ligand-specific selectin of
DCs is recognizing the glycosylation patterns. Before rolling or homing, DC cells
have resting integrin structures.
In the first process of tethering and rolling of DCs, non-specific glycoproteins or
glycolipids and PSGL-1, on basement membrane of DC cell surfaces are attached
with selectin’s ligand of sLex or sLeA. They are interacted with sLe sugar’s coun-
terparts of E-/P-selectins present in the surfaces of endothelial cells. Also, L-selectin,
47 integrin, and 41 integrin expressed on DCs surfaces interact with PNAd,
MAdCAM, and VCAM-1 expressed on endothelial cell surfaces. In the second
stage of activation of DCs, cells have active integrins and chemokine signals, and
these trigger the cell responses. More specifically, the G protein-coupled receptor
(GPCR) present in DCs or leukocytic cells is activated for downstream signaling
pathway by several low molecules including chemoattractants, chemokines, com-
plement, PAF, LTB4, formyl peptides, and other minor molecules secreted to plasma
fluids from inflammatory and injured sites. Next, leukocyte arrest to the inflamma-
tory sites is operated by help of activated GPCR signaling. For the arrest, several
known molecules were known for common trafficking molecules. They are LFA-1,
Mac-1, activated 47 integrin, and activated 41 integrin expressed on DC cell
surfaces, and they are interacted with ICAM-2/ICAM-1/MAdCAM, and VCAM-1
present on the surfaces of endothelial cells [302] (Fig. 4.7). When DCs are differ-
entiated into mature forms by antigens, PSGL-1’s sLex expression is decreased for
the easy DC migration to lymphatic nodes, processing, and antigen presentation.
This is the reason why DCs decrease sLex expression in PSGL-1. For final stage, the
arrested cells undergo polarization, diapedesis, and junctional rearrangement from
the endothelial cells of endothelium. The cells entered into the tissue sites which
induce proteolytic degradation of basement membrane by matrix metalloproteinases
(MMP) and then progressed to interstitial migration after interaction with cytokine-
stimulated parenchymal cells. Finally, the DCs make a clean through clearance of the
damaged or inflamed tissues, and the DC cells are migrated to draining lymph node
and lymph vessel. In mature DCs, CAMs of CD44 variants [303] and MMP-2 and
MMP-9 in the extracellular area are expressed [304]. If the O-glycosylated patterns
of CD44 and MMP-9 are changed, their functions will also be altered.
SLeA SLeX
SiaD2,3GalE1,4GlcNAcE1,3Gal1-R
SiaD2 ,3GalE1,3GlcNAcE1,3Gal1-R
&%
FucD1,4 FucD1,3
Tethering Rolling Activation Arrest

Activated Activated
Glycoprotein Integrin Integrin
GPCR Integrin Integrin
L-selectin DE LFA-1 Mac-1 DE DE
/ glycolipid PSGL-1 DE
%JGOQCVVTCEVCPVU
Sialyl
LeX,A

%JGOQMKPG
%QORNGOGPV
2#( .6$
HQTO[NRGRVKFG
.WOGP
E-selectin P-selectin PNAd MAdCAM VCAM-1 ICAM-2 ICAM-1 MAdCAM

VCAM-1
Hepara Extravasation
sulfate
proteoglycan Endothelium
Fig. 4.7 Common trafficking molecules during DC recruitment during general inflammation and
migration step
4.10 Glycan Ligands in Trafficking of DC Migration
The trafficking of mature DCs to the drained lymphatic nodes is essential for the
primary immune cell functions. Considering that phagocytosis-based antigen pre-
sentation is one of the actions of DCs, DCs activate adaptive immunity of T
lymphocytes. The so-called antigen-presenting immune cells (APCs) indicate the
macropinocytosis by receptor-mediated endocytosis or receptor-independent endo-
cytosis. The presentation of foreign antigens activates T cell stimulators. In order to
play that DCs function as an innate immunity actor and make a link of innate
immunity to adaptive immune response, DCs have to recognize trafficking mole-
cules expressed at the cells injured site and enter to the site and consequently uptake
antigens (Fig. 4.7) [305]. Then, finally DCs home to adjacent lymph node to translate
their antigen information to adaptive immune response and present antigens to T cell
and B cell. This stage is called “cell migration step.”
4.10.1 sLex-PSGL-1 Glycans in DC Trafficking
The most important trafficking molecules are glycans or carbohydrates called sLex
attached mainly on O-glycan, and these capture immune cells to move to inflamma-
tory site. In a minor case, N-glycan is also attached with sLex ligand. The receptor
P-selectin binds to sLex as peripheral carbohydrate in order to capture immune cells
migrated to the inflammatory site. P-selectin strongly binds to PSGL-1 expressed on
4.10 Glycan Ligands in Trafficking of DC Migration 155
Fig. 4.8 Biosynthesis of sialyl Lewis antigens from Tn antigen originated from Thr/Ser residues on
polypeptides by sLex-synthesizing glycosyltransferases such as C2GnT1 and C2GnT2. In tumor
cells, from core 1 direction, tumor-specific sialyl 6 T, sialyl 3 T, and disialyl T antigens are
produced. The name of the carbohydrate structure is indicated. An extended O-glycan contains
core 2, which carries variable lengths of stem region caused by LacNAc motif repetition (n 0) and
SLeX tetrasaccharide motif. Ser/Thr, serine or threonine
immune cells. During differentiation and maturation of DCs, sLex expression pattern
in PSGL-1 decides the cell migration levels. Immune trafficking aptitude of DCs is
thus explained by sLex pattern and sLex-synthesizing glycosyltransferase expres-
sion. Relationship between triple factors of glycosyltransferase expression pattern,
sLex expression pattern, and PSGL-1-P-selectin signal pathway is a parameter of
“DC maturation.” DCs are APCs, translating innate information to adaptive immune
response, surveilling microenvironment, carrying foreign invasive antigens to adja-
cent lymph node, and presenting to T cells. For example, sLex expression level is
decreased during differentiation and maturation of DCs (Fig. 4.7). This is regulated
by sLex-synthesizing glycosyltransferases such as C2GnT1 (1,6GlcNAc-T or core
2 synthase and 1,3GlcNAc-T (C2GnT2) (Fig. 4.8). The gene expression of the two
enzyme genes is increased during differentiation, but decreased in maturation stage,
while ST3Gal-1 expression as a key factor is increased during differentiation and
maturation of DC [306]. For molecular structure of carbohydrate ligands, DCs
express sLex on PSGL-1 for the above missions. The PSGL-1 carries sLex as a
key ligand molecule.
4.10.2 Ganglioside Recognition by DC Receptors

in Trafficking
In the cell membrane, for example, in innate immunity, GSL glycans play pivotal
roles to induce differentiation responses of primary defensing cells. Membrane-
associated GSLs confer structural integrity to the plasma membrane, GSLs agglom-
erate into cholesterol dense microdomains, or lipid rafts participate in affordable
recognition, adhesion, and signal transduction of cells. Signaling via lipid raft
microdomain-associated GSLs is an important process especially in myeloid lineage
cells for innate immune responses as well as lymphocytes and osteoclasts [307]. For
example, GM3 has well been known to regulate lipid raft- and microdomain-
associated cell signaling and cell adhesion in human lymphocytes
[308, 309]. Increased synthesis of GM3 is also a defining characteristic that marks
the differentiation and maturation of myeloid lineage precursors into monocytes and
macrophages, a process that can be promoted by addition of GM3 [310].
In addition, the membrane GSLs play crucial roles in invasion and infection of
extracellular infectious agents such as virus. For example, enveloped virus enters
into host cells through host cell attachment and fusion into membrane. Virus
adsorption occurs at the recognition of specific receptor molecules of viral attach-
ment molecules. Gangliosides are also functional components of the plasma mem-
brane, especially GM3- and 3SL-containing gangliosides. Sialic acid residues in
gangliosides expressed in the viral coats also function as capture ligands of pattern
recognition receptors of DCs [311]. Because virus captures the cell-surfaced sialyl
residues on gangliosides, the gangliosides GM3, GM1, GM2, and GD1 are
expressed on viral membrane coats or envelops, as well as known in several viruses
including HIV, SFV, VSV, and MuLV [312, 313]. Gangliosides of the
gangliotetraose series bearing the sialic acid in α2-3 linkage of GD1a, GT1b, and
GQ1b, and neolacto-series gangliosides are also known to the receptors for Sendai
virus [311]. Human parainfluenza viruses 1 and 3 recognize N-
acetyllactosaminoglycan branches with Neu5Acα2-3Gal. Sialylylated glycan resi-
dues of gangliosides have been reported to function as cell adhesion molecules as
receptors due to their hydrophilic properties. Considering that sialic acid residues on
gangliosides function as host cell receptors for pathogenic bacterial toxins such as
cholera toxin [550] and several viruses [314–316], the reverse biology is the case of
ligand function. Accordingly, the sialic acid derivatives or analogs can be designed
to inhibit the sialidase and/or receptor-sialic acid binding activity which are future
antiviral strategies.
Integrin-mediated binding of DCs with target cells or antigens is influenced by
ganglioside. In the membrane, gangliosides localize with proteins for specific amino
acid sequences. For example, GD3 is clustered with β1 integrin and affects proper-
ties controlled by integrin-mediated signaling. Chemokine receptor type 9 (CCR9)-
positive immune cells are enriched in the small intestine, and integrin α4β7-positive
cells are enriched in the small intestine and colon. Gangliosides regulate immune cell
signaling, as gangliosides are organized into microdomain (lipid rafts) and serve as
4.10 Glycan Ligands in Trafficking of DC Migration 157
Resting T-cells Activated T-cells E1,4

D2,3
NeuAcD2,3GalE1,4GlcNAc E1,6
D 2,3 E1,3 E1,6 GalE1,3GalNAcE -Thr/Ser
E1,3 D 2,6
Disialyl T Thr/Ser
(NeuAcD2,3GalE1,3 D
E1,4 D 2,3NeuAc-T
[NeuAcD2,6]GalNAcD-Thr/Ser)
GalE1,4GlcNAc E1,6
E1,3 E1,6
GalE1,3GalNAcD-Thr/Ser
Thr/Ser
Sialyl 6T D 2,6
E 1,3 E1,4Gal-T
D
Thr/Ser Core 2
E 1,3 E1,6 GlcNAc E1,6
D2,6NeuAc-T
Thr/Ser GalE1,3GalNAcD-Thr/Ser
(ST6GalNAc-I, II)
E1,6 GlcNAc-T
T antigen (Core2-synthase)
E 1,3 GalE1,3GalNAcD-Thr/Ser
Thr/Ser
Sialic acid (SA)
E 1,3Gal-T Galactose (Gal)
N-Acetylglucosamine (GlcNAc)
Tn antigen D-Thr/Ser
GalNAcD N-Acetylgalactosamine (GalNAc)
Fucose (Fuc)
Thr/Ser
DGalNAc-T
Thr/Ser
Fig. 4.9 Different glycan structures of O-glycan in resting T cells or activated T cells. Sialylation
direction of T cells determines regulation of the relevant T cell responses where they become resting
T cells or activated T cells
signaling molecules and receptor trafficking. For example, in T cells, glycoforms of

SGL-1 in resting cells and activated cells are quite different in their O-glycans
(Fig. 4.9). T lymphocyte activation pathway involves the sialylation of integrin
proteins and requires interaction between membrane gangliosides and sialylated
O-glycans (Fig. 4.9) [317, 318]. Sialylation direction of T cells determines regula-
tion of the relevant T cell responses where they become activated or resting T cells.
Core 2 glycan synthetic direction of Tn antigens on O-glycosylated proteins is
operated in the activated T cells. The O-glycosylation enzymes are 1,6 GlcNAc-T
(Core2 synthase), 1,4Gal-T, and 2,3NeuAc-T to synthesize the Gal1,3(GlcNAc1,6)
GalNAc-Thr/Ser, Gal1,3(Gal1,4GlcNAc1,6)GalNAc-Thr/Ser, and Gal1,3
(NeuAc2,3Gal1,4GlcNAc 1,6)GalNAc-Thr/Ser (Fig. 4.9). Disruption of lipid rafts
displaces cellular signaling molecules and alters immunoreceptor signal transduc-
tion. Sphingolipid depletion inhibits GPI-anchored protein trafficking in
microdomains. For example, inflamed intestinal mucosa has decreased ganglioside
content. Enrichment of intestinal mucosa with ganglioside causes a reduction in
cholesterol content. Cholesterol depletion disrupts membrane microdomain structure
and inhibits generation of proinflammatory mediators. Ganglioside inhibits signals
caused by proinflammatory stimuli TNF-α and IL-1β in rats. Ganglioside protects
the gut by attenuating proinflammatory signals.
4.11 Chemokine Receptors in DC Trafficking
4.11.1 Chemokine
Chemokine molecules are small ranged, between 8 and 10 kDa, chemoattractant

cytokines and render the chemoattraction of migratory cells [319]. They have a
similar tertiary structure. Chemokine superfamily includes chemotactic proteins as
modulators of leukocyte trafficking [320]. Chemokine signaling occurs through
chemokine receptors for immune reactions. Currently, 50 chemokines and 20 che-
mokine receptors are discovered from humans. The chemokines and chemokine
receptors are associated within cellular responses such as homeostasis and inflam-
mation [321]. Discovery of chemokines has been ascended to the initial
GAG-binding protein of platelet factor 4 (PF-4, currently termed CXCL4) which
is known for more than 40 years. The CXCL4 neutralizes Hp in coagulation [322] by
heparin affinity interaction [323]. When IFN-γ-dependent cytokine known as IP-10/
CXCL10 has been discovered in 1985 [324]. The CXCL4, CXCL10, and platelet-
derived protein β-thromboglobulin/CXCL7 commonly contain four Cys residues in
their sequences [325]. Since IL-8/CXCL8 has been defined as a neutrophil
chemoattractant, it has been classified as chemokine that denotes chemoattractant
cytokines [326]. Using the signature cysteine sequence, 50 more chemokines were
isolated as the largest cytokine sub-class.
Chemokines consist of conserved Cys residues with classification into four sub-
families of chemokines C, C-C, C-X-C, and C-X3-C, depending on the pattern of
Cys residues present in the ligands [644]. Chemokines C-X-C type (termed
α-chemokines) bear two Cys residues, which are distinctly specific with a single
variable amino acid residue and action on neutrophils, B and NK cells, and T cells.
Among them, C-C chemokines known as β-chemokines contain two adjacent Cys
residues and associate with monocytes, macrophages, T cell, NK cells, basophils,
and eosinophils in the inflammatory sites. The C chemokines known as
γ-chemokines which are further classified to lymphotactin α (XCL1) and
lymphotactin β (XCL2) have one Cys residue and act on T cells only.
The last, chemokine C-X3-C known as δ-chemokine in human CX3CL1 or
fractalkine, has N-terminal Cys residues which are distinctive of three variable
amino acid residues. CX3CL1 is found as a soluble protein and a membrane-
bound protein attached to a mucin domain. Interestingly, all the chemokine receptors
are the types of GPCR having a seven-transmembrane domain. The non-GPCR
forms are called the “decoy” receptors or atypical chemokine receptors, and these are
found in scavenger receptors [327]. In communication between chemokines and
their receptors, chemokine receptors bind to multiple Zchemokines, even in dimer-
ization with other receptor species [328]. In fact, the chemokines of CCR5 and
CXCR4 form their heterodimers with selectins, CD4, and integrins [329]. The
chemokines and receptor expressions are spatially and temporally controlled in
immune cells.
4.11 Chemokine Receptors in DC Trafficking 159
4.11.2 Chemokine Receptor
Chemokine receptor nomenclature has been made. For example, CXCL8 was
previously IL-8 and now is a CXC ligand, while CCL2 was MCP-1 and now called
a CC chemokine. Currently, 23 human chemokine receptors are known
[330]. Among them, 18 receptors are the GPCR family, whereas 5 receptors are
“atypical receptors” such as ACKR1-4 and CCRL2. The atypical receptors exhibit
chemokine scavenging and transport functions [331, 332]. PTMs including Tyr
sulfation, alternative splicing, proteolysis, ligand modification, and ligand-receptor
dimerization alters receptor ligand recognition [330]. The flexible N-terminal region
is central to receptor activation in altered leukocyte activity. Different ligands can
activate distinct signaling pathways following binding to the same receptor. In fact,
both CCL19 and CCL21 activate chemotaxis of CCR7-expressing cells, while
CCL19 induces receptor downregulation [324, 325].
4.11.3 Chemokine-GAG Interaction as a Type

of Protein-Glycan Interactions
Proteins relatively large in their sizes are frequently membrane anchored and are
glycosylated. In case of glycans, glycans are extremely heterogeneous and often hard
to characterize using the isolated glycans. Proteoglycans consist of one or more
GAG chains and core protein parts. GAGs carry various disaccharide units. GAGs
are also subclassified to several subgroups, depending on their composition of
disaccharide units. The subclasss includes chondroitin, heparin, heparan sulfate
(HS), dermatan sulfate (DS), hyaluronan, and keratan sulfate (KS). Protein-glycan
interactions (PGIs) are being understood in cellular function from molecular analy-
sis. Interaction between proteins and glycans is widespread in cellular environments,
as glycan-protein bindings are being analyzed at a molecular level and challenged.
The challenging interests are in the field of chemokine-GAG interaction as a type of
protein-glycan interactions in order to understand GAG functions in transduction of
signals through proteins that are large, membrane anchored, and often glycosylated.
Because glycans are heterogeneous and difficult to isolate and characterize, new
technologies including nuclear magnetic resonance (NMR) and mass spectropho-
tometry (MS) provide detailed structure information on moderately sized systems as
well as qualitative information with the precise structural basis. NMR specifically
contributes to qualitative receptor ligand bindings. Using MS, qualitative informa-
tion can be precisely obtained from less material but with few size limitations.
Chemokine-GAG interaction as well as N-glycosylated Ig-receptor interaction has
been analyzed using the analytic technology [333]. GAG binding of chemokines is
involved in leukocyte extravasation because GAG chains are involved in leukocyte
transmigration. GAGs mediate cell recruitment where tissue-produced chemokines
bind to cell surface GAGs. Circulating leukocytes first recognize selectins and are
rolling circle; second, they adhere to integrin ligands on the leukocytes and trans-
migrate into the tissue. Third, GAG and chemokine receptor recognitions promote
cell migration [334–337]. Chemokine receptor recognition as well as chemokine-
GAG recognition on the migrating cells determine the leukocyte type during inflam-
matory response.
The most abundant endothelial cell GAG is HS, occupying up to 90% of total
endothelial GAGs [337]. Various proteins including cytokines, adhesion molecules,
proteases, and growth factors bind to HS. Endothelial cell-associated CXCL8 binds
to GAG through its C-terminal GAG-binding domain for neutrophil migration. For
example, chemokines CCL5, CXCL8, and CXCL12γ bind to HS through
GAG-binding regions. HS-chemokine recognition has a merit to protect from pro-
teolysis [338]. Chemokine oligomerization increases chemokine activity [339]. For
example, the extracellular HSPGs such as perlecan, agrin, and type XVIII collagen
bind and sequester chemokines to allow leukocyte migration, contributing to leuko-
cyte diapedesis [340]. Hence HS-chemokine interaction is a key step of leukocyte
extravasation because neutrophil migration is depended on the CXCL8-HS
binding [341].
Chemokines require immobilization, upon oligomerization and GAG interac-
tions, on cell surface GAGs to transmigrate from leukocyte circulation. GAGs are
on cell surfaces or shed as soluble ectodomains. The chain lengths of GAGs range
between 1 and 25,000 disaccharide units with different sulfation patterns. The most
abundant form are syndecans having a TM domain on the surfaces of cells. How-
ever, the glypicans are anchored to the GPI anchors, and the other three (agrin,
collagen XVIII, and perlican) are not embedded to the cell membrane but instead
associated [342]. GAG-bearing six disaccharide units have been estimated to have at
least 12 billion more different disaccharide sequences. The GAG sequence diversity
is 100 times more than that calculated from a hexapeptide and 2,000,000 times more
than that calculate from DNA [343]. Therefore, chemokine-GAG complexes even in
a single chemokine are heterogeneous with large scale for their structure diversity,
even for a single chemokine [319]. Additionally, only hyaluronan does not exist in
C. elegans among these GAGs. Chondroitin proteoglycans regulate cell division of
C. elegans. HS PG is present on the cell surfaces and ECM of cell membranes and
regulates signaling pathways such as Hedgehog, TGF-βl, FGF, and Wnt pathways
involved in development. Heparan sulfate chains also regulate self-renewal, ES, and
pluripotency of ESCs of mice.
4.11.4 Molecular Motifs in Chemokine for GAG Recognition
The GAG-binding motif present in chemokines is the C-terminal XBBXBX in CC

chemokines [344] or C-terminal XBBBXXBX, where B indicates basic amino acids,
while X indicates any non-basic amino acid [345]. For several chemokines,
GAG-binding motifs indicate the “BBXB” motif, in which B also indicates a basic
amino acid residue and X indicates any amino acid residue. For an instance, CCL5
(RANTES) interaction with CS hexamer provides BBXB motif recognition with a

10 μM of KD. CCL5 has the 40s-loop BBXB cluster, 44RKNR47, as the crucial
GAG binding epitope [346]. Tetramer model can extend to the higher oligomers than
the tetramer. This information can rationalize interactions between GAG and recep-
tor proteins [347, 348]. Interaction between chemokine CCL5 and GAGs potentiates
adhesion on GAGs expressed cell surfaces for the initiation of the transmigration.
Oligomerization and GAG recognition are essential for transmigration of cells.
CCL5 dimerization has been experimentally evidenced [349]. However, E66S
mutation of CCL5 restricts oligomerization for such CCL5 dimer. In the CCL2,
the binding motif is rather the non-BBXB sequence with amino acid residues of R18,
K19, R24, and K49 [346]. These residues are important for receptor binding and
bind to sulfate groups attached to the GAG and Tyr sulfation-modified receptors
[350]. The GAG-binding motifs are different from the receptor binding domain.
Although positively charged chemokines favor negatively charged GAGs,
non-specific electrostatic forces are also involved. For example, acidic chemokines
of CCL3 and CCL4 also bind GAGs due to hydrogen bond and Van der Waals
forces [695]. The GAG binding chemokines are crucial for chemokine activity
[344, 351–353]. For example, CXCL8 truncated in its C-terminal HS-binding region
cannot bind to Hp and activate leukocyte and receptor binding [354] as well as
transcytosis across endothelial cells and luminal surface presentation to blood
leukocytes, resulting in blocked leukocyte transmigration [355]. The HS binding
domains are well established [345]. CCL5 is chemotactic for monocytes and CXCL8
for neutrophils, and CXCL12γ has a HS-binding C-terminus and stimulates migra-
tion of lymphocytes.
Although CCL4/MIP-1β and CCL3/MIP-1α contain the acidic amino acids on
proteins, most chemokines contain basic amino acids on proteins. This is the reason
why these exhibit the highest affinity for HS and Hp among other GAGs, which have
low sulfation levels, as in CS or DS [356]. Chemokines bind to receptors as mono-
mers and also oligomers. Many chemokines oligomerize [351, 357]. CC type of
chemokine CCL2/MCP-1 holds the canonical tertiary structure [358] with “CC
dimers.” Other CXC chemokine forms and CXCL8 associate with a “CXC dimer”
from the identical monomeric forms [350]. CXC chemokine dimer helps to select its
appropriate CXC receptor and CC chemokine dimer for CC receptors. Disulfide-
bonded CXC chemokines are also dimerized. Chemokine monomers can be acted as
receptor agonists. When dimeric form of CC chemokine and CXC dimers cannot
recognize receptors, chemokine oligomers are important for GAG recognition to
many chemokines. GAGs stabilize dimers and chemokine oligomers [359–
361]. Chemokine-GAG binding and chemokine oligomerization enforce each
other to concentrate chemokines near inflammatory sites [362]. Each chemokine
displays monomer state (CCL7/MCP-3), tetramer state (CXCL4), and polymer state
(CCL5) [363, 364]. Although CCL7/MCP-3 does not take its oligomeric forms, the
CCL7/MCP-3 binds GAGs due to its dense GAG binding epitopes, compared to
oligomerizing chemokine CCL2. Chemokine oligomer formation and GAG recog-
nition are crucial for migration capacity of cells [365–367].
Each chemokine exhibits affinity for each GAG. GAG affinities of CXCL4,
CXCL11, CCL5, and CCL21 are high, whereas the GAG affinities of CCL2 and
CXCL8 are intermediate. GAG affinities of acidic CCL3 and CCL4 chemokines are
weak [367]. The CXCL12 isoform, CXCL12α, forms dimers and polymers [368],
while another CXCL12 alternatively spliced variant, CXCL12γ, does not
oligomerize, but CXCL12γ has 30 amino acid C-terminal extension with BBXB
motifs with high affinity for GAGs [369]. Seemingly, the CCR7 receptor ligand,
CCL21, contains a basic 40-amino acid extension in C-terminus for GAG immobi-
lization and DC recruitment [370]. Thus, alternative GAG interaction controls
chemokine- and GAG-dependent migrative behavior [369]. GAG sulfation is cru-
cial, as Hp 2-O-desulfation loses chemokine-binding affinity. Although HS prefers
CCL2 dimers, Hp-bound CCL2 tetramers are rather stabilized [339, 363]. Apart
from homo-oligomers, chemokines heterodimerize [371–373] for GAG receptor
activation.
4.11.5 C-C Type Chemokine Receptor 4 (CCR4) and Specific

Ligand 17 (CCL17) and Specific Ligand 22 (CCL22)
CCL17 and CCL22 are specific ligands for C-C type chemokine receptor 4 (CCR4).
CCR4 was found from basophiles of human [374]. The murine orthologue is
predominantly present in the thymus, lymph node T cells, and peripheral T cells
[375]. CCR4 species is mainly present in T cell subpopulations of the activated types
Th-2/Treg/T cells. Th-1, Th-2, and polarized Th cells exhibit different chemokine
receptors with migration activities. Th1 cells produce CXCR3/CCR5, but Th2 cells
produce CCR4/CCR8 [376, 377]. Human and mouse Th17 cells express CCR4
[378]. Memory Th17 cells of human coexpress CCR4 and CCR6. CCR4 expression
is restricted to DCs, platelets, NK cells, monocytes, and macrophages [374, 379–
381]. CCR4 is involved in various diseases. CCR4 expression is also seen in T cell,
which are skin-homed, and in Th-2 phenotype for skin-involved allergic immune
responses [382]. The Ccr4 gene-deficient KO mice do not exhibit its phenotype
changes in a Th-2-dependent inflammation [380]. In addition, Th2 cells express
CCR4 in allergic disease, and CCR4+ T cells produce Th2 cytokines in asthmatic
patients. CCR4 is involved in activation of innate immune response and
Th2-involved immune responses [383, 384]. CCR4 is also involved in sepsis, as
Ccr4 KO mice survive even in LPS-activated endotoxic condition by decreasing the
expression of proinflammatory cytokines and function of macrophages [381].
For CCL17, in 1996, a C-C chemokine gene has been reported as activation-
regulated chemokine (TARC) and thymus-type chemokine. It has been renamed C-C
chemokine ligand 17 (CCL17). The CCL17 gene is expressed constitutively in the
thymus and activated PBMCs. CCL17 and its receptor CCR4 are expressed on DCs
and macrophages. CCL17 is expressed in murine bone marrow-derived DCs
[385]. DCs express CCL17 in homeostasis and inflammation [386]. Among classical
or conventional cDCs and pDCs, only cDC types express MHC-II antigens, which
are engaged in phagocytosis and antigen presentation. cDCs are further divided,
depending on antigen expression of its surface marker CD11b. The CD11b-
expressing cDCs stimulate CD4+ T cells. Also, CD8α-expressing cDCs act as
cross-presenting cells [387]. In other sides, DCs can also be subdivided into DC-1
and DC-2 subsets, depending on their induction abilities of Th-1 and Th-2 cell
differentiation, respectively. CCL17 is expressed predominantly by a CD11b-
positive cDC subset in lymphatic organs, not by the spleen. TLR also induces the
expression of CCL17 gene in cDC subset, which express CD11b antigen, in
lymphatic nodes, not in the spleen. α-GalCer-activated NK T cells in mice stimulate
CD8α + DCs and produce CCL17 in the cells. CCL17 is involved in various
diseases. CCL17 induces immune reactions of contact hypersensitivity, allograft
rejection event, IBD, atopic dermatitis, and atherosclerosis [386, 388]. The reduced
atherosclerotic level in Ccl17-deficient KO mice is modulated by Treg cells. The
CCL17 expressed in DCs reduces the Treg cell subset level. CCL17 reduces the cell
numbers of Treg cells; however it activates the IL-12/IL-23 cytokine releases
in DCs.
The second ligand specific for CCR4 is the C-C chemokine ligand, CCL22. The
CCL22 is also a macrophage-derived chemokine and shares 37% homology with
CCL17. Interestingly, the genes encoding CCL17 and CCL22 are proximally pre-
sent to the close CX3CL1 gene location on chromosome 16q13 of human [385]. The
two CCL17 and CCL22 are present in the myeloid cells and thymus. M2 type
macrophages involved in Th-2 responses express CCL22 [389]. Monocyte-derived
DCs also express CCL22 and thus DCs bind to activated T cells. Chemokine-
dependent T cell interaction with DCs indicates T cell priming [390]. LPS and
IL-1β, TNF-1, and CD40 ligand induce CCL22 expression in DCs. DCs which are
activated contain CCL22 and N-terminally truncated CCL22. CCL22 is indeed a
chemoattractant for antigen-presented T cells or NK cells, DCs, and monocytes
[391]. CCL22 expression is stimulated by the IL-4/IL-13, which are the Th2
cytokines, in myeloid cells and suppressed by the IFN-γ known as a Th-1 cytokine
and responding to polarized Th-2. In contrast, IL-4/IL-13 inhibit IFN-γ- and
TNF-α-induced CCL22 expression in keratinocytic cells. This indicates a cell type
expression of CCL22 gene [392]. CCL22 is also involved in various diseases.
CCL22 is involved in allergy and autoimmunity to tumor. In the lung, alveolar
macrophage and DC depletions save severe inflammatory mouse caused by IL-13
action, through inflammation protection by reduction in the CCL17 and CCL22
production [393]. CCL22 immune modulation in autoimmunity is based on regula-
tion of the Treg cells. CCL22 expression of pancreatic β cells in IDDM diabetes
eliminates Treg cells’ autoimmune attack [394]. CCL22-mediated control of Treg
cells is observed in tumor cells in humans, because cancer cells and tumor-associated
microenvironmental macrophages express the CCL22 and effectively recruit the
Treg cells, Therefore, CCL22 inhibits tumor-mediated T cell immunity. CCL22
indicates an immune escape response of tumor [395].
Expression patterns of chemokine receptors and chemokines also depend on the
cell status of maturation of each cell. Conventional cDCs are continuously supplied
by bone marrow-derived DC precursors (BMDCs) called pre-DCs. The pre-DCs

traffic through the BM-blood vessel-blood-peripheral tissue transmission. The born
pre-DCs are heterogeneous in subset population, and the currently known pre-DCs
are cDC subset-committed progenitors called pre-cDC1 and pre-cDC2. Pre-DC
subsets are continuously trafficking into their peripheral locations to afford the
host immune responses against immune SAMPs or PAMPs. However, the relation
between pre-cDC1 and pre-cDC2 in trafficking, homeostasis, and fighting against
PAMPs is not explained. A recent report discriminated the relation between the two
subsets. The pre-cDC1 but not pre-cDC2 expresses the Th1-associated chemokine
receptor CXCR3. Moreover, the CXCR3 played a cell-specific role in the trafficking
of pre-cDC1 to melanoma. Thus it is considered that each pre-cDC1 trafficking
differs from others such as pre-cDC2, indicating the different DC lineage determi-
nants [396]. For the case, CCR7 together with MHC as a chemotactic receptor is
activated and induced. These two coreceptors allow the DCs to move from the
antigen uptake region to the blood stream-based spleen or the lymph node. Such
acted CCR7 and MHC stimulate the cytokine production. Chemokines and their
receptors, for example, CCL21 and CCR7 [397], have active roles in the migration
behavior [398]. PSGL-1 is also a functional secondary receptor responsible for
CCL21 of T cells of mice, homing to lymph node [399]. Interestingly,
O-glycosylation levels of PSGL-1 modulated the level of the T cell homing event.
Intestinal DCs include two subset populations based on the presence of CD103 and
C-X3-C motif-bearing chemokine receptor 1 (CX3CR1) proteins
[400]. CD103+CX3CR1 cells are a major cell type of migratory intestinal DCs
and activate regulatory T cells. In contrast, CD103CX3CR1+ DCs are the luminal
antigen-interacting resident cells, initiating local immune responses. Similar mech-
anism is also observed in the expression of CCR7 (C-C chemokine receptor type-7)
and MMP-9 responsible for the DC migration. In case of PGE2 (prostaglandin E),
PGE2 is known to stimulate DC migration by induction of the expression of the
above genes. Glycosyltransferase inhibitor, BGN-treated DC, has been known to
decrease the DC migration effect in chemotaxis.
4.12 Glycan Structure-Recognizing Selectins

in DC-Endothelium Interaction During Infection
and Inflammation
Circulating DCs are continuously interacted along with the endothelium of vascular
vessel. This is to avoid any damages from the hemodynamic shear forces to have
anti-shear power resistance by binding between vascular walls and binding mole-
cules. The binding and interacting molecules are called selectin. The selectin is
defined as single-chain transmembrane glycoprotein, and three different selectins
such as L-, P-, and E-selectins are known. Selectins interact with ligands. Selectins
belong to C-type lectin type, which is a family of CAMs and recognizes and binds to
4.12 Glycan Structure-Recognizing Selectins in DC-Endothelium Interaction. . . 165
specific glycan determinants in a Ca2+-dependent fashion. For selectin binding,

specific carbohydrate ligand is needed [401]. Selectins are present in platelets,
endothelium, or leukocytes, and they are E-selectin, P-selectin, and L-selectin.
They recognize the SA- and Fuc-containing tetrasaccharide, where SLeX is the
main form. Ligands of selectins are present mainly in immune and endothelial
cells, which are predominantly expressed in the condition of inflammatory
responses. sLex is expressed in neutrophils, lymphocytes, and DCs as functional
selectin ligand. Using anti-sLex antibody, sLex was known to mediate the
DC-selectin binding. The selectin ligand SLeX is present in glycolipids or glycopro-
teins. The protein-linked SLeX, PSGL-1, is known in moDCs. During chemokine-
driven migration of monocytes and DC progenitors, SLeX species-coated PSGL-1
proteins are the binding ligand to P-selectin species [402].
4.12.1 3 Species of Selectins: E-, L-, and P-selectins
The storage pore of P-selectin molecules is the endothelial Weibel-Palade body and
moves to the surfaces during proinflammatory responses [403]. Cell-cell interactions
mediated by molecular recognition of P-selectin with PSGL-1 are well known. Cell-
cell interactions driven by P-selectin-PSGL-1 binding mediate leukocyte rolling
process on inflamed endothelial cells or adherent, activated platelets. Neutrophils,
or PMNs, or other leukocytes can also move through rolling by P-selectin recogni-
tion. PSGL-1-P-selectin interaction also mediates adhesion of platelets to myeloid
leukocytes to form mixed cell aggregate, and this is a responsible mechanism of
vascular rupture and atherosclerosis. In addition, the adhesion of platelets to leuko-
cytes mediated from the PSGL-1 and P-selectin interaction generates aggregation in
vascular endothelial cells, and this is a reason why tumor cells adhere to platelets and
endothelial cells. E-selectin is newly synthesized at every need without storage
pools, but L-selectin is newly generated in stimulation-dependent leukocytes only.
Selectin structures are similar in their protein architecture with the three distinct
domains structures of complement-binding protein like domain, EGF domain, and
lectin domains. They are transmembrane-anchored through the plasma membranes,
and the domain structures are topologically shredded in the extracellular regions
(Fig. 4.10).
In cancer cells, SLeX or SLeA is a carbohydrate ligand, which are interacted with
E-selectin present in blood vessel endothelial cells. Cancer cell SLeX or SLeA
epitope stimulates the cancer cell attachment to endothelial cells. The adhesion is
enhanced upon TNF-α-stimulation to endothelial cells. SLeX or SLeA present in
cancer tissues helps hematogenous metastasis with cancer prognosis (Figs. 4.11 and
4.12) [402].
Both selectins of E-/P-selectins are mainly present in the endothelial cell surfaces
upon inflammatory activation, and they bind to SLeX of the carbohydrate structure of
NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAc-R and SLeA structure of NeuAcα2,3Galβ1,3
(Fucα1,4)GlcNAc-R epitopes as well as the sulfated derivatives. The original roles
Complement-binding protein
like domain
wG
EGF domain
j lG Lectin domain
jG
sTzG uG
lTzG
wTzG
Fig. 4.10 Structure of selectins. Selectins’ structures are similar in their protein architectures with
the three distinct domain structures of complement-binding protein like domain, EGF domain, and
lectin domains
Cancer cells #FJGUKQP

Normal cells
D (-)
TNF-D
Cultured cancer cells
TNF-D (+)
Cultured endothelial cell monolayer

with TNF-D
Fig. 4.11 Tumor cell adhesion to endothelial cells stimulated with TNF-α
of the epitopes include leukocytic homing, where E-/P-selectins recognize cancer

cells harboring SLeX or SLeA antigens. The recognition initiates attachment to the
endothelial cells and transmigration to the invasive sites of target tissue.
Infiltration
Adhesion Metastasis
Intravasation Detachment Circulating
Cancer Cells
Sialyl LeX
Sialyl Lea
E-Selectin
Endothelium
Fig. 4.12 Schematic process of the complex and multistep process of hematogenous metastasis of
cancer
Hematogenous metastasis of cancer is carried out by a multistep process. Cancer

cells acquire the cell motility and invade blood vessels [404]. Through these steps,
the cancer cells migrate across the stroma and blood vessels. SLeX- or SLeA-
expressing cancer cells in the invasive sites invade a blood vessel and circulate to
organs. SLeX or SLeA selectin recognition is crucial for the construction of meta-
static foci at each organ. SLeA or SLeA expressed on cancer cells adheres to selectins
present on endothelial cells and gradually provides strong attachment and transmi-
gration. Thus, blood-circulating cancer cells with SLeX or SLeA enhance the meta-
static potential. SLeX or SLeA expressed at the invasive sites is a leading factor for
postoperative cancer recurrence of metastatic cancer cells. Hematogenous metastasis
events of cancer cells are mediated through binding of SLeX- or SLeA-positive tumor
cells to E-selectin expressed on the endothelial surfaces.
Blocking biosynthesis of SLeX or SLeA as E- or P-selectin ligand prevents the
hematogenous metastatic potential of cancer cells. SLeX or SLeA antigen is
biosynthesized on glycolipids or glycoproteins by various glycosyltransferases.
The essential reactions are completed by distinct α1,3 or α1,4-fucosyltransferases
and α2,3-STs. Currently, in humans, six fucosyltransferases of FucT-1 to FucT-7
and FucT-9 enzymes [405] and six α2,3-STs of ST3Gal-I to ST3Gal-VI [406]
catalyze the formation of sialyl Lewis antigens using type I epitope of the
Galβ1,3GlcNAcβ1-R or the Galβ1,4GlcNAcβ1-R type II substrates in the Golgi
complex compartments. The α1,2Fuc-transferase (FUT1) (E.C. 2.4.1.69) disturbs
terminal α2,3-sialylation and consequently prevents the SLeX or SLeA synthesis
[407]. Thus, FUT1 intercepts the type II precursor substrates before the type II
precursors are trafficking to the α2,3-sialyltransferase-resident compartment.
4.12.2 Representative Selectin Ligand PSGL-1 and Role

of PSGL-1 O-Glycan
SLex antigen is a tetrasaccharide sugar with SA, Gal, GlcNAc, and Fuc residues as
specific carbohydrate structures and determinants. Tetrasaccharide sLex is known to
bind three P-, E-, and L-selectins (Fig. 4.13). For selectin binding activity, addition
of the sLex tetrasaccharide to O-linked glycan is needed. SLex attached to O-glycans
present in the cell surfaces importantly function in cell-to-cell binding events, as
SLex is present in monocytes and immatured DCs but not in matured DCs. P-selectin
and PSGL-1 primarily mediate the rolling phase of the adhesion cascade. In cancer
patients, selectin ligands in cells are circulated in the plasma of blood. Many selectin
ligands exist as O-glycans of mucin-type sialylated and thus classified as sialomucin
glycoproteins, because they contain sialic acid in PSGL-1, CD34, and GlyCAM-1
carrying sLex. In protein level of PSGL-1 and P-selectin, the two amino-terminal
domains are also interacted during homing and rolling. PSGL-1 in cell membranes
has a disulfide-bridged dimerization form (s-s bond) in confirmation, and it contains
SLeX N-linked
A) B) O-linked
ST3Gal-4
E1,4GalT-1
D1,3FucT-4,
FucT-7
E3GlcNAcT
n
ST3Gal-1 n
E1,4GalT-1 n
Core 1 E1,3GalT Core 2 GlcNAcT (E1,6)

Core 1 Core 2
D GalNAcT
Thr/Ser Thr/Ser Thr/Ser
Core 4 Core 2
Asn
SO4 6-SO4-SLeX SLeX

Sialic acid (SA)
GlcNAc-6-Sulfo-T-1/2 SiaD2,3GalE1,4GlcNAcE1,3Gal1-R
Galactose (Gal)
FucD1,3
N-Acetylgalactosamine (GalNAc)
ST3Gal-1 Fucose (Fuc) SLeA
TNF-D
PGE2 D2 ,3GalE1,3GlcNAc E1,3Gal1-R
SiaD
Core 2 GlcNAcT
FucD1,4
INF-J Core 2 GlcNAcT
Fig. 4.13 The biosynthesis of carbohydrate selectin ligands, sialyl Lewis ligands, as forms of
N-glycan and O-glycan in DCs. (a) Simplified biosynthetic pathway of SLeX-decorated core 1/core
2 O-glycosylations. Simple pathway of SLeX-bearing O-type glycans. Modulation of DC’s
glycosyltransferases by maturation stimuli. PGE2, Prostaglandin E2. (b) Sialyl Lewis ligands as
forms of N-glycan and O-glycan. The name of the carbohydrate structure is indicated. An extended
O-glycan contains core 2 O-glycans. Ser/Thr, serine or threonine
the sLex component in broad ranges of carbohydrate length and residues. In mono-
cytes and immature DC, only PSGL-1 is the glycoprotein carrying sLex. sLex as
glycan acts in extravasation through selectin recognition which is expressed on
PSGL-1 in monocytes and immature DC. DCs express sLex glycan ligands present
in PSGL-1, and these ligands recognize P-selectin receptor for their functional
migration to lines of endothelial cells on vascular endothelium. When DCs are
maturated by specific antigens and inflammatory cytokines, the level of PSGL-1’s
sLex expression is decreased. By decreasing sLex expression in PSGL-1, DCs can
migrate to the lymph node for processing antigen presentation. For P-selectin
interaction with PSGL-1 ligand, O-glycan attached to PSGL-1 displays importantly
in the P-selectin adhesion. Thus, if maturation of DCs is over, sLex expression is lost,
even though these cells retained expression of PSGL-1. This suggests specific
affinity of P-selectin counter-receptor present in myeloid lineage cells. To express
function as APCs, DCs and their precursors such as progenitor DCs require trans-
location and transfer from circulating blood to the damaged peripheral tissues.
Further, when foreign antigens induce DC activation, they have to migrate using
O-glycans produced in the inflamed tissue sites to the drained lymphatic nodes. In a
similar mode, the O-glycans are also crucially associated with T cell trafficking.
Therefore, it is certain that sLex expression on PSGL-1 O-glycan is modulated
during each step of differentiation and maturation of DCs. PSGL-1 O-glycan is an
enforcing factor during the white blood cell recruitment to inflamed sites raised by
infection or inflammatory agents.
4.12.3 Glycosyltransferases for Biosynthesis of PSGL-1

O-Glycan
DC maturation is involved in the changed production levels of GTs required in

O-glycosylation process. Biosynthesis of selectin ligands is well explained in DCs.
The mechanistic perspectives of how they are modulated during diverse environ-
mental situation are still under investigation through many glycoimmunists
[402]. Biosynthesis of sLex-decorated O-glycans has been studied and synthesis of
mo-DC’s glycosyltransferases is modulated during maturation stimuli. As the most
significantly changeable glycosylation, sialylation event in DCs is increased in DCs,
and this event consequently results in the increased sialylation contents because it is
an important issue in the maturation process [408]. Sialyltransferases and sialidases
are differentially expressed during differentiation and maturation of DCs. For exam-
ple, ST3Gal-1, ST6Gal-1, Neu1, and Neu3 activities are increased during DCs
differentiation. ST6Gal-1 mediates the transfer of SA residue by an α-2,6-SA linked
to a terminal Gal residue of type 2 (Galα1,4GlcNAc) disaccharide. ST6Gal-1
enzyme is mainly present in the Golgi apparatus, extracellular region, and cell
membrane [409]. After maturation, DCs show the increased α2,3-sialyl glycans
and decreased α2,6-sialyl glycans. Especially, the ST3Gal-4 and ST3Gal-6 activities
are required for the adhesion-involved sLex antigens, and these glycans allow the DC
cell rolling and homing. The expressed sialic acids function as sialic acid-
recognizing DC receptors or the effector cells such as T cells. C2GnT1 mRNA
downregulation and enzymatic activity is correlated with ST3Gal I and ST6GalNAc
II mRNA upregulation. This change in glycosylation leads to lack in the core
2 structures and sLex structures, eventually default in the reduced P-selectin ligand
[410]. GT expression related to O-glycan synthesis is increased in differentiation and
maturation of DCs. The O-linked glycans must undergo posttranslational modifica-
tions in order to function (a counter-receptor for P-selectin) in enzymatic reaction of
α1,3-Fuc-transferase and α2,3-ST. For fucosylation of SLex antigen, α ! 1,3-
fucosyltransferases of FUT4 and FUT7 are known as key enzymes in leukocytes.
Interestingly, the glycosyltransferase expression is mediated by PGE2, which is
important for DC migration of human. O-glycosylation profile of human mDCs
and the O-glycan pattern present in mature cells are similar to the naive T cell-
expressing O-glycan types. Thus, O-glycans are suggested to exhibit a common role
in the different events of DC migration and T cell homing.
PSGL-1 carries both two types of Asn N-glycan and Thr/Ser O-glycan of
glycoproteins. Each glycan has the sLe carbohydrate ligand to bind with selectins.
In addition, some of carbohydrate selectin ligands is sulfonylated (such as
6-SO4-sLex) [403] (Fig. 4.13). Specific tyrosine residues in PSGL-1 protein are
sulfated, and the SO3-Tyr can be strongly bound with the endothelial or platelet
P-selectin when the sLe ligands are located on the surrounded region. Thus, the
interactions between tyrosine sulfate residue, sialic acid, and fucose residues in
O-glycan lead to strong binding capacity. The interactions with tyrosine sulfate
residues of PSGL-1 and the sialic acid and fucose residues of the core 2 O-glycan
are basically suggested.
From sialidase or neuraminidase treatments, the PSGL-1 activity is regulated
[411]. PSGL-1 treated with sialidase showed the abolished binding capacity to
P-selectin, while PSGL-1 treated with peptide N-glycosidase F had no effect on
recognition to P-selectin. PSGL-1 treated with the O-sialoglycoprotease, which
degrades sialylated mucins, blocks interactions with P-selectin. For example,
HL60 cells treated with benzyl-alpha-GalNAc, which inhibits extension of
O-glycans, reduce binding of cells to P-selectin. PSGL-1 treated with
endo-ß-galactosidase, which degrades type 2 polylactosamine repeats
[-3Galß1 ! 4GlcNAcß1-]n, reduces binding to P-selectin. Thus, selectin ligands
are actively interacted with its receptors for the immune cells to potentiate trafficking
and migration in inflammation; they directly control the inflammation, leukocyte
signaling, rolling, adhesion, extravasation, and inflammation-promoting factors.
They are key molecules of the cell communication in forms of glycolipids and
glycoproteins. Pathogens or proinflammatory cytokines equally lead to DCs matu-
ration and their migration to lymphoid tissue by adhesion to endothelium and
chemotaxis. This event is dependent on selectin recognition to sialofucosylated
glycans. SAs also are involved in the firm arrest events which are mediated by
chemokine receptors. SAα2,8-polysialylation formed by ST8Sia-4 of neuropilin-2
contributes to chemokine-mediated migration potentials for lymphatic nodes.
ST3Gal-4 is not related for chemokine-mediated DC homing events. ST6Gal-1

gene-deficient KO mice exhibit impaired DC migration to the drained lymphatic
nodes. Moreover, ST6Gal-1-deficient mice (together with α-2,6-cell surface sialic
acid-deficient lymphocytes) exhibited reduced B cell proliferation and decreased
antibody genesis during presentation of T cell-dependent and T cell-independent
antigens [412]. These results indicate that low or no B lymphocyte ST6Gal-1
synthesis, together with low or no α-2,6- cell surface sialic acid production, is
associated with a reduced immunoinflammatory effect.
A) B).
4.12.4 Designation of Carbohydrate Glycomimetic Drugs

and Natural Inhibitors of Selectins
Novel carbohydrate drugs are designed and synthesized to inhibit selectin ligand
binding to generate glycomimetic drugs. All cells are coated with carbohydrates.
Carbohydrates contain much structural information used in various kinds of molec-
ular recognition events. Carbohydrate structures are not directly determined by
genomics. The possible number of branched and linear isomers of a hexasaccharide
is more than 1.05 x 1012. Over 1 x1012 different structures are theoretically possible
from a hexasaccharide (Table 4.3). Considering the possible number of isomers of a
hexapeptide, only 46,656 different structures are possible. This dense structural
information is used for molecular recognition [413]. This circumstance causes the
isomer barrier, and the isomer barrier is the reason why single method is not possibly
utilized to apply for determination of whole oligosaccharide structures in low
quantity under 100 nmol amounts, which are obtained from 100 pmol amounts by
single tools such as a Edman peptide method or Sanger DNA dideoxy-sequencing
approach. Difficulty in oligosaccharide synthetic approach is therefore equally found
by the extremely multiple number. Therefore, a new class of synthetic carbohydrate
drugs are based on mainly the rational design through the structural conformation of
functional carbohydrates. Complex carbohydrates contain much structural informa-
tion in a small space. Recently, glycobiology and the functional carbohydrate
analysis invite the possible development of novel therapeutics against human dis-
eases to generate new pharmaceutical industry. The rational cases of carbohydrate
synthesis with pharmacological activities are described. P-selectin functions as a
Table 4.3 Possible number Saccharide name Hexose number Isomer number
of branched and linear isomers
Monosaccharide 1 2
of oligosaccharides
Disaccharide 2 256
Trisaccharide 3 38,016
Tetrasaccharide 4 7,602,176
Pentasaccharide 5 2,633,600,000
Hexasaccharide 6 1,053,045,031,000
A)
E, P-selectin ligand
B) sLeA (CA19-9)
sLeX (CD15s) P-selectin is stored in α-
P-selectin ligand
granule
Glycomimetics
P-selectin
leukocyte
E,P-selectin
platelet
platelet Cancer cell
P-selectin
Endothelial cell
Endothelial cell activation unactivation P-selectin store granules,
Activation Weibel-Palade bodies
Fig. 4.14 (a) Selectins E and P recognize the sLeX/A. Ligands for carbohydrate-binding proteins
during leukocyte homing to activated endothelial cells. (b) Selectin E and P ligands. sLeX/A present
in cancer cells recognizes the two selectins E and P present in endothelial cells to facilitate
metastatic invasiveness
CAM present in the activated endothelial cell surfaces and activated platelets. In
unactivated endothelial cells, P-selectin is granulated for cellular storage, termed
Weibel-Palade bodies, while in the unactivated platelets, the α-granules contains the
P-selectin for storage. Platelets bind to tumor cells via P-selectin, which increase the
potential for the tumor cells to reach a distant site. It is important to ensure the
effective arrest in capillaries and to facilitate the extravasation of the tumor cells.
Therefore, P-selectin inhibitors are candidates. For example, selectins have been
considered to be therapeutic targets for inflammatory diseases and cancers
(Fig. 4.14). PSGL-1 belongs to a native ligand for selectins and contains both
sulfated Tyr residues and SLex for pan selectin binding activity. New generation
inhibitors block both the carbohydrate- and sulfate-binding domains on P-selectin.
E- and P-selectins bind to sulfated Tyr residues and SLeX present in the native ligand
PSGL-1 protein. PSGL-1 known as a typical mucin-type O-glycan expressed in the
myeloid cell surfaces is the counter-receptor for binding of P-selectin. The PSGL-1
interaction with the P-selectin needs Ca2+ ions. Sulfated tyrosine residue and the
SLeX epitopes present on the O-glycans attached to PSGL-1 are necessary. Both P-
and L-selectins require proper sLex glycosylation and Tyr sulfation of PSGL-1 for
strong affinity binding. The sulfotyrosine-carrying PSGL-1 O-glycans with SLex
constitutes the P-selectin binding site [414]. Therefore, sulfated moiety-based
molecular designation can be used for inhibitory candidates of P-selectin. During
the sLeX modification with E-selectin-selective recognition, bioactive conformation
of parent glycomimetic 9669a has been evolved. Consequently, GMI-1014 series
show the higher stacking and is followed by 69669a during preorganization of the
bioactive conformation with selectin inhibitory activity [415]. The carbon 2-position
modification of Gal residue enhances binding affinity for E-selectin [416].
4.12.5 Glycomimetic Drug Candidates
The developed GMI-1070 is a pan selectin antagonist. GMI-1070 has been synthe-
sized through convergent steps and large scale-up chemistry development
[417]. GMI-1070 is a rationally designed agonist as a small compound, which is a

pan selectin inhibitor. Among them, the GMI-1070 antagonist molecule, which was
rationally and empirically designed, binds to all three selectins of E, P, and L types.
Scalable chemical synthesis enables to synthesize currently in kilogram scale.
Several carbohydrate companies are involved in large-scale process of production,
and they include Carbogen AC (Switzerland), Cambridge Major Laboratories
(USA), SynphaBase AG (Switzerland), Glycosyn (New Zealand), and Carbosynth
(UK). For the GMI-1070 phase I clinical trial of healthy volunteers, pharmacoki-
netics of a single dose of GMI-1070 have been obtained in healthy volunteers. In
addition, phase 1 healthy volunteers showed unremarkable safety profile for both
PH1 studies. This indicates clean safety profile in phase 1 clinical trials. Summary of
the GMI-1070-101 in a single dose described that there is no serious adverse events
(SAEs). All adverse events (AEs) were mild or moderate within grades 1 and 2. In
the multiple doses of GMI-1070-102, there was also no SAEs, and all AEs were mild
or moderate within grades 1–2, as equally described between drug group and
placebo controls. Testing effects of GM1-1070 have been demonstrated on
vaso-occlusive crisis of sickle cell patients in clinical trials. In phases I and II, sickle
cell patients improved the vaso-occlusive crisis (VOC) with no serious adverse
events (SAES) and serum half-life of 7 to 8 hrs in humans. Changes in relevant
biomarkers (PMA) have been certificated as GMI-1070 hits target in patients. This
indicates no major metabolic breakdown with 90% more excretion to extrabody.
Sickle cell VOC in animal models is specifically featured with aberrant cell adhe-
sion. This indicates that selectins are associated with the genesis of the vaso-
occlusions for the VOC expression. Homing, rolling, trafficking, and cell adhesion
of leukocytes are basic behaviors to vascular endothelial cells, which are activated
by several inflammatory agents or mediators. In addition, other cellular adhesion
molecules such as integrins are also conformationally changed to forms of high
affinity binding capacities and eventually bind to other blood cells such as RBC and
platelets to enhance the formation of occlusions [418]. Sickle cell disease (SCD)
known since 1910 is a feature of peculiar appearance of the RBC. The disease
originates from an abnormal hemoglobin. Sickle RBC stimulates endothelial cells
directly by adhesion. The activated endothelial cells abnormally recruit rolling and
adherent leukocytes with chemokines, selectins, and immunoglobulin families,
contributing to vascular occlusions. In sickle model mice, vaso-occlusion occurs
and is prevented by blocking leukocyte adhesion. Because the molecular discovery
of sickle cell disease is crucial in modern biological science using the modern tools
of molecular and cellular biological technology, this type of the carbohydrate-based
advances will innovate the field. GMI-1070 extends survival in sickle cell mice with
VOC. Treatment model for testing the effects of GMI-1070 on VOC is determined
by IVM. Age- and gender-matched SCD mice identical and genetic cohorts are
created by transplanting bone marrow cells from Berkeley SCD mice containing
human sickle hemoglobin into irradiated C57BL/6 male mice. In the VOC mice
induced by administration of TNF-a, once VOC is established, GMI-1070 is admin-
istered. The VOC mice exhibited relevant improvements with the increased survival,
improved blood flow, reduced leukocyte-endothelial interactions, and reduced
leukocyte-RBC interactions. Effects of GMI-1070 in sickle cell mice in VOC have

been confirmed even by intravital microscopy [419]. GMI-1070 has been evidenced
in pre-clinical models. In the GMI-1070-administered pre-clinical animal model,
thrombus formation has been inhibited. Other positive effects of GMI-1070 include
the increased survival in multiple myeloma and the inhibited relapse in multiple
sclerosis (EAE). In the treatment of VOC in sickle cell mice, the GMI-1070
treatment exhibited several improvements including the inhibition of infarct size in
myocardial infarct model, inhibition of delayed-type hypersensitivity reaction, inhi-
bition of epileptogenesis induced by pilocarpine, protection of mice treated with
chemotherapy, and cell cycle inhibition of hematopoietic stem cells.
Currently, the phase II trials for clinic of the sickle cell patients in crisis are being
tried in 20 hospital centers in the USA and Canada. In the industrial level, potential
glycomimetic drugs are validated from the pharmaceutical industry. For example,
Pfizer signs $340 million licensing pact with GlycoMimetics. GlycoMimetics initi-
ated a global licensing agreement with Pfizer for GMI-1070, which is mimetic drug
for an experimental treatment for vaso-occlusive crisis of erythrocytic sickle disease
of human patients. However, the contract deal seems to call for GlycoMimetics to
complete the phase II trial study for the mimetic drug. If the issue is solved, Pfizer
may further develop and commercialize it. GlycoMimetics has been known to
calculate their royalty income worth as much as $340 million [420].
4.12.6 GAG-Glycomimetic Drugs
On the other hand, the natural P-selectin inhibitor, heparin as GAG, has been known
to inhibit P-selectin. Due to high anticoagulant activity, it can potentially cause
hemorrhage. With regard to natural inhibitors, marine invertebrates are rich sources
of heparin-like molecules and sulfated polysaccharides, but less is known about the
anti P-selectin activity of sulfated fucans and sulfated galactans from sea urchins
(Fig. 4.15). Sea urchin sulfated polysaccharides such as sulfated fucan and sulfated
galactan function as a P-selectin inhibitor. Sulfated polysaccharides inhibit binding
of tumor cells to P-selectin and consequently prevent interaction of tumor cells with
platelets in vivo and in vivo through P-selectin-dependent manner in inflammatory
cell recruitment. The sulfated polysaccharides inhibit leukocyte recruitment in
P-selectin knockout mice. Fucan from S. franciscanus does not present
antimetastatic activity. The very similar sulfated polysaccharides isolated from
urchins are composed of 2-O-sulfated monosaccharide units. They also have differ-
ent anticoagulant effects. To prevent selectin-mediated metastasis, mouse models of
P-selectin-dependent tumor progression and inflammation have been used [421].
References "103, 104, 106, 107, 234,. . . 175
A)
Strongylocentrotus franciscanus
Fucan 1→ 3 linked 80kDa
Strongylocentrotus droebachiensis
Fucan 1 → 4 linked 100kDa
Echinometra lucunter
Galactan 1 → 3 linked 80kDa
B)
Hematogeneous
metastasis
Sulfated polysaccharides
Hematogeneous
metastasis
Fig. 4.15 Sulfated fucan and sulfated galactan from sea urchin
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Chapter 5
Pathogen-Host Infection Via Glycan
Recognition and Interaction
5.1 Lectin Recognition of Glycans on Cell Surface

and Soluble Glycans
The immune system selects the targets through immune recognition. Lectins in
innate immune cells recognize oligosaccharides of cell surface and soluble glycans
that encode complex information. Lectin repertoires are extremely diverse in their
nonself and self-recognitions. For example, how is diversity in “nonself” and “self”-
recognition achieved? Binding to targets is primary in innate immunity through the
innate immune cell populations of myeloid cells, NK cells, and innate lymphoid
cells. In certain circumstances, nonimmune cells and ancient type of humoral
complements can also bind to targets. How extensive are the lectin repertoires in a
certain species? Have they evolved through the functional and structural diversities?
Lectins recognize cell-surfaced and soluble glycans. Representatively, CTLs like
MBL in innate immunity directly recognize microbes with opsonic effect and
activation of complement pathways. Oligosaccharide structures of cell surface and
soluble glycans encode complex information. Like the stepwise recognition, cellular
information is being decoded by carbohydrate-binding molecules, and these carbo-
hydrates regulate the interaction between cells and cells or interaction between cells
and ECM and, eventually, cellular functions. The recognition occurs in early
development as a type of “self-recognition” and innate immunity as a type of
“nonself and self-recognition” (Table 5.1). The most specific aspects of the lectins
are in their diversity expressed as lectin repertoires in self and nonself recognition,
although it is not fully understood yet how is diversity in “self”- and “nonself”
recognition achieved? Although innate immunity eliminates most pathogens, certain
pathogens are not eliminated because the pathogens produced virulence factors.
Innate immune responses are not specific originally but adapted. How extensive or
broad are the lectin repertoires in a given species? And have they evolved during
early evolutionary phase in their functional diversity? Innate immune receptors
include a variety of lectin families as forms of soluble lectins and membrane lectin
200 5 Pathogen-Host Infection Via Glycan Recognition and Interaction
Table 5.1 Differentiation of innate and adaptive immune responses

Innate immune response Adaptive immune response
Fast (minutes to hours) Slow (4–7 days or more latently)
Diverse receptors Distinct receptors of TCR and Ig
“Hard-wired” in the germline Genetic recombination widely to count-react
Locally distributed Distinct tissue distribution
Macrophage, DCs, PMN, and NK cells B and T cells
Non-clonal cell specificity Clonal cell specificity
Non-immunologic memory Immunologic memory
receptors. Innate immune system expresses distinct receptors known as PRRs, which
directly recognize PAMPs. The direct recognizers are, for example, CD14, DEC205,
and collectins. Complement receptors and Toll receptors bind to PAMP recognition
products. Because of the nonspecificity of innate immune responses, alternative
complementation cooperates with the PRRs. The PRR receptors recognize specific
pathogenic components such as glycans because glycans face the outmost world
with diversity in the glycan structures and patterns as the nonself and self-
recognition basis.
Cell-surfaced receptors as the first defense line alarm the pathogenic presence.
Soluble lectins include ficolins, lung surfactant, pentraxins, Man-binding lectins
(MBL), etc. [1]. Currently, three known C-reactive protein (CRP), serum-amyloid
P protein (SAP), and long pentraxin 3 (PTX3) are the group of pentraxins. Lectin
membrane receptors or membrane-associated lectins include mannose receptor
(ML), DC-SIGN, Dectin, NK cell receptors, Scavenger receptors, Complement
receptors, and TLRs. Membrane-associated lectins consist of transmembrane
domain, complement-binding domain, EGF-like domain, and carbohydrate recog-
nition domain (CRD), whereas soluble or humoral lectins are comprised of Cys-rich
domain, collagen-like domain, and CRD. During the microbial infection, host innate
or acquired immune defense involves the systemic changes in host glycosylation in
cell surfaces. In sides of pathogens, they evolve to modulate host immune defenses
by modulating its glycosylation. The “self”- and “nonself” glycans are distinguished
by lectins. SAs on cell surfaces in prokaryotes are the targets for attack, but they are
eukaryotes are SAMPs. Many receptors are found to be pathogen-recognizing
receptors in innate immune cells, including TLR, C-type lectins, ML, and Siglecs.
Most TLRs are localized to the cell surfaces as immune receptors, and certain types
of TLRs are also intracellularly located as cytosolic compartments like the endo-
some. TLRs recognize PAMPs and DAMPs to afford immune responses in innate
immunity. TLRs are interacted with PAMPs or DAMPs, allowing affordable
recruiting of Toll/IL-1R (TIR) domain-carrying adaptor proteins. The well-defined
TIR-adaptor protein is MyD88, which mediates diverse signal transduction path-
ways in order to protect them from the microbial infection. However, if TLRs are not
sufficiently regulated or negatively regulated in the condition of excessive immune
responses, abnormal immune responses are displayed. The resulting diseases include
autoimmunity and inflammation.
5.1 Lectin Recognition of Glycans on Cell Surface and Soluble Glycans 201
Fig. 5.1 Lectin R-Type Lectin

classification
L-Type Lectin I-Type Lectin
Lectin
P-Type Lectin
Galectin
C-Type Lectin
Table 5.2 The animal lectin families and lectin domains with known three-dimensional structure
Family Carbohydrate specificity Fold type Ca2+
Galectins Strict Gal/Lac β-Sandwich, S-motif No
Pentraxins Often noncarbohydrate β-Sandwich, multi-domain Yes
CTL Diverse CTL C-motif Yes
I-type or Siglecs Sialic acid Ig, Ig-like domain No
P-type lectin Man-6-P M6P β-sandwich, P-motif Dependently
Calnexin Glc β-Sandwich
ERGIC Man Legume lectin β-sandwich
TNF Chitobiose TNF-β-sandwich
HGF (NK1) Heparin/heparin sulfate Basic amino acids No
Ym1 Heparin/heparin sulfate Chitinase, basic amino acids No
Cys-MR/FGF2 Sulfated glycans β-Trefoil
Tachylectin 2 GlcNAc/GalNAc β-Propeller
The “self”- and “nonself” glycans are recognized and distinguished by glycan-
binding proteins, named lectins. This indicates that lectins are variable in their
structural and functional aspects (Fig. 5.1). Lectin is a glycan-recognizing protein
that can recognize various glycoconjugates on cell surfaces and extracellular matri-
ces, ranging from the mediation of cell adhesion and promotion of cell-cell interac-
tion to the pathogenic recognition (Table 5.2). Therefore, it is ruled out that glycans
are information-carrying and third life chains, where its counterreceptors are lectins
that are tools to recognize them as their colleagues to interact. Lectins characterize
the cellular glycophenotype, phenotype-associated features, transforming faces, and
disease-associated alterations. Therefore, it is simply described that lectins are
saccharide-binding proteins. Based on structure of the CRD, animal lectins are
divided into C-type, galectins, P-type, I-type, and others including heparin-binding
proteins, pentraxin, and rhamnose-binding lectins. In addition, rhamnose-binding
lectins found in fish eggs have been regarded as one of the lectin families. Most
known animal lectins are currently one of the hot subjects of studies on immune
responses. Most known animal lectins function in the tissues or blood plasma, but
skin mucus lectins function externally to combat with the dermatic invasives. Similar
mode to MBP, skin mucus lectin may act together with other humoral factors that
exert innate immunity as immunoglobulin (Ig) and complement. The biological
functions of skin mucus lectin have not yet well elucidated. However, the
Table 5.3 Intracellular and extracellular lectins in mammalian lectins

Intracellular lectins Extracellular lectins
L-type lectins CTLs (selectin, DC-sign, ASGPR, Dectin, MBP, etc.)
P-type lectins I-type lectins (Siglecs, Ig superfamily)
Calnexin R-type lectins
Galectins
Table 5.4 Mammalian lectins as native carbohydrate-binding proteins. Currently, 100 more mam-
malian lectins or carbohydrate-binding proteins are found
Lectin groups Ca2+ Specificity Sequence specific and features
C-type Yes Variable C-type specific motif
Galectins No β-galactosides S-type specific motif
P-type
(Mannose-6-phosphate Variable Mannose-6-P P-type specific motif
receptor)
I-type No Variable Ig-like domains
Pentraxins Most PC/galactosides Multimeric recognition motif
Heparin-binding type No Heparin/heparan- Basic amino acid sequence
SO42– motifs
information of animal lectins is quite well studied to date. For example, some animal
lectins potentiate the classical complement pathway by help of MBP in innate
immunity. These lectins play crucial roles in host defense together with humoral
defense factors such as Igs, complement components, CRP, lysozyme, and hemo-
lysin [2]. To date, 100 more mammalian carbohydrate-binding proteins as lectins are
reported as forms of intracellular and extracellular lectins (Table 5.3). Currently,
100 more mammalian lectins or carbohydrate-binding proteins are found. Mamma-
lian lectins as native carbohydrate-binding proteins have specific structural features
and motifs to binding their carbohydrate ligands (Table 5.4). Vertebrate SA-specific
lectins including humans are summarized (Table 5.5).
Among them, C-type lectins are most diverse in their functions as mosaic
molecules. In innate immunity, C-type lectins directly recognize of microbes,
opsonic effect, and activation of complement pathways. For example, MBL is a
microbial surface carbohydrate-recognizing C-type lectin. From the diversity of
lectin repertoires, diversity in recognition of lectins is determined by tandemly
arrayed CRDs with different carbohydrate specificity, multiple isoforms which are
formed by conversion, homologous recombination, alternative splicing, and other
mechanisms. The lectins bear their distinct binding properties and oligomeric qua-
ternary structures with variable binding avidity and CRDs from different lectin
families and effector domains. Thus, lectins have been diverse via coevolution in
order to mediate different biological roles, immune response in inflammation and
autoimmunity, opsonization of microbial pathogens, fertilization, and cell adhesion.
5.1 Lectin Recognition of Glycans on Cell Surface and Soluble Glycans 203
Table 5.5 Vertebrate SA-specific lectins. Siglec genes are from Homo sapiens. OBBP obesity-
binding protein, AIRM adhesion inhibitory receptor molecule, MAG myelin-associated
glycoprotein.a CD33rSiglecs are distinctly located compared to the Siglec gene cluster
Lectin
(synonyms) SA specificity Expression cells
Selectins
E-selectin sLex, sLea Activated endothe-
(CD62E;ELAM- lial cells
1)
P-selectin sLex, SLea Activated endothe-
(CD62P; lial cells, platelets
GMP-140;
PADGEM)
L-selectin 60 -sulfo sLex Leucocytes
(CD62L; Mel
14 antigen)
Siglecs
Siglec-1 Neu5Acα2,3Gal > Neu5Acα2,6Gal > Neu5Acα2,8 Macrophages
(sialoadhesin/Sn;
CD169)
Siglec-2 Siaα2,6Gal B cells
(CD22)
Siglec-3 Siaα2,6Gal > Siaα2,3Gal Myeloid progenitor
(CD33) cells, monocytes,
macrophages
Siglec-4 Neu5Acα2,3Gal Oligodendrocyte,
(MAG) Schwann cells
Siglec-5 Siaα2,6Gal, Siaα2,3Gal > Neu5Acα2,8 Monocytes, neutro-
(OBBP2) phils, B cells,
macrophages
Siglec-6 Siaα2,6GalNAc (sialylTn) Trophoblasts, B
(OBBP1) cells
Siglec-7 Neu5Acα2,8 >> Siaα2,6Gal > Siaα2,3Gal Monocytes, NK
(AIRM-1) cells
Siglec- Siaα2,3Gal > Siaα2,6Gal Eosinophils, baso-
8 (mouse Siglec- phils, mast cells
F)
Siglec-9 Siaα2,3Gal, Siaα2,6Gal Monocytes, neutro-
(mouse Siglec-E) phils, NK cells, B
cells
Siglec-10 Siaα2,3Gal, Siaα2,6Gal Monocytes, NK
(mouse Siglec-G) cells, eosinophils, B
cells
Siglec-11a Neu5Acα2,8Neu5Ac Macrophages
Siglec-12 – Macrophages
Siglec-14 Siaα2,6Gal, Siα2,6Gal Unknown
Siglec-15 Siα2,6Gal Macrophages, DCs
Siglec-16 Siα2,8Gal Macrophages, DCs
(continued)

Lectin
(synonyms) SA specificity Expression cells
Others
Complement Sia Blood
factor H
Interleukin-1α Biantennary, Neu5Acα2,3Gal1,4GlcNac Blood
Interleukin-1β Neu5Acα2,3Gal1-Cer (GM4) Blood
Interleukin-2 GD1b Blood
Interleukin-4 Neu5Ac1,7lactone Blood
Interleukin-7 Siaα2,6GalNAc (sialylTn) Blood
L1 Neu5Acα2,3 Neurons, CD4+ T
cells, monocytes, B
cells
SA-binding Sia Rat sperm
proteins
Laminin Siaα2,3Gal1,4GlcNAc Extracellular matrix
Sarcolectin Neu5Ac, Neu5Gc Placenta
Calcyclin Neu5Gc Bovine heart
Calreticulin Neu5Gc, Neu5Ac Ovine placenta
cSBL Sia Frog egg
5.2 Innate Immune-Specific and Host Defensing Lectins

of Fungal, Protozoa, Invertebrate, and Lower
Vertebrates
Functions of lower fish lectins do exist in the skin mucus of certain lower vertebrates
such as fish including the windowpane flounder Lophopsetta maculata, the Arabian
Gulf catfish Arius thalassinus, the conger eel Conger myriaster, the dragonet
Repomucenus richardsonii, the loach Misgurnus anguillicaudatus, the kingklip
Genypterus capensis, and the pufferfish Fugu rubripes. Certain lower vertebrates
such as the African clawed frog (Xenopus laevis) produce galectins in the skin
mucus. Agglutinin in the moray eel (Lycodontis nudivomer) and the Arabian Gulf
catfish (A. thalassinus) and lectin in the dragonet (R. richardsonii) in the skin mucus
belonged to Galectins with Galactose specificity [1].
In the Galectin-type lectins of eel skin mucus, Conger eel congerins I and II in the
skin mucus are galactoside- and lactose-specific lectins. The lactose (Lac)-
recognizing lectins are known for AJL-1/AJL-2, which are isolated from the mucosal
skin tissues of the Japanese eel of A. japonica, are structurally studied as a Galectin
family [2]. They are expressed in the skin only, showing selective resistance to
infectious diseases with agglutinating activity against Gram-positive bacterium,
Streptococcus difficile [3, 4]. Therefore, the lectins are classified to a defensive
factor. Congerins belong to the Galectin family, but no homology with eel galectin
AJL-2. For functional aspect, kin mucus galectin, congerin, agglutinates a marine
5.2 Innate Immune-Specific and Host Defensing Lectins of Fungal, Protozoa,. . . 205
Table 5.6 Protozoa SA-specific lectins

Species Lectin SA-binding specificity
Trypanosomatidae Trypanosoma Inactive TS CD43 (leukosialin on CD4+ T cells),
cruzi (Tyr342His) (Neu5Acα2,3 > Neu5Acα2,6 > sLex)
Trichomonadidae Tritrichomonas TML Neu5Acα2,6 > Neu5Acα2,3 > Neu5Ac
mobilensis TFL Neu5Ac > Neu5Gc > Neu5Acα2,3/6
T. foetus
Plasmodiidae Plasmodium EBA-175 Neu5Acα2,3Gal (glycophorin
falciparum EBA-140, A) > Neu5Acα2,6Gal
BAEBL, Sia (glycophorin C), Sia (glycophorin
PfEBP2 B), Sia (receptor E)
RfRh1, Sia (receptor Y)
NBP1
P. knowlesi β protein Sia (rhesus erythrocytes)
Babesiidae Babesia Sia (glycophorin A and B)
divergens Neu5Acα2,3/6 and Neu5Acα2,3,
B. bovis and respectively
B. equi
pathogenic bacterium Vibrio anguillarum; however, it does not inhibit the growth of
V. anguillarum. AJL-2 also agglutinates and suppresses cell growth of E. coli K12.
Thus, skin mucus lectin participates in first line of host defense to inhibit the
bacterial growth in the mucus. Naturally occurring Galectins with different substrate
specificities are considered to effectively respond to a wide range of pathogens.
Tandem-repeated Galectin type was purified from the Oncorhynchus mykiss species
known as rainbow trout, and the coding gene was isolated from head kidney-derived
cDNA library. The tandem-repeated type Galectin exhibits homologies of 40–55%
with other known Galectin-9 of mammal sources. Its homologies have weak
19–25% ranges in the N-terminal region or 15–20% in the C-terminal region with
galectins isolated from conger and electric eels. The Galectin production is increased
when LPS was treated, and the expression is associated with the innate immune
response in eel [3].
Several types of SA-specific lectins produced by protozoa such as Trypanosoma
cruzi, Tritrichomonas mobilensis, Plasmodium falciparum, and Babesia divergens
and fungi including mushroom and pathogenic fungal species are described
(Tables 5.6 and 5.7). In addition, SA-specific lectins of invertebrate SA-specific
lectins including arthropoda, mollusca, echnodermata, and urochordata are
described (Table 5.8).
Table 5.7 Fungal SA-specific lectins. a Also binds GlcNAc. b Phytopathogenic fungus
Mushroom Hericium erinaceum and HEL Neu5Gc > Neu5Ac and
Polyporus squamosus abd Neu5Acα2,6Galβ1,4Glc/GlcNAc,
Psathyrella vetutina and PSA respectively
Agrocybe cylindracea PVL Neu5Acα2,3Galβ1,4GlcNAca and
and Neu5Acα2,3Galβ1,4Glc, respectively
ACG
Pathogenic Penicillium marneffei – Neu5Ac/laminin and fibronectin
fungi
Aspergillus fumigatus HA-A Neu5ACα2,6GalNAc/laminin, fibronec-
tin, fibrinogen, collagen
Histoplasma capsulatum – Neu5Ac/laminin
Macrophomina MPL Neu5Acα2,3Galβ1,4GlcNAc
phaseolinab
5.3 How Do Hosts Interact with Pathogens?
5.3.1 Lectin-Carbohydrate Interaction
Pathogenic microbes in intestines interact with intestinal environment. The patho-

genic surface glycoconjugates recognize their interacting molecules of host cells.
The pathogenic glycans have hugely diverse structures in a species-dependent
manner and act as host cell-binding ligands, giving their pathogenic species speci-
ficities. In the host cell infection, tight barrier of host cells protects against microbial
pathogens, but bacterial infection occurs through two major routes of trans-cellular
type and para-cellular type (Fig. 5.2).
Cell-surfaced SAs in eukaryotes and prokaryotes are the targets for attack and
SAMPs. SAs and their receptor patterns have been evolving rapidly to adapt and
survive in biological environments. Mammalian host species have also evolved self-
sialic acids as “self-patterns.” There are several surfaced sugars including Gal, Man,
Fuc, and GAGs, which function in immune responses. However, SAs are positively
adopted for natural selective roles in immunity. Pathogens initially bind to host cells
for the infection. Their binding target molecules are complex carbohydrates or
glycans surfaced on the outmost coat cells even in single cells or multiple cells in
organisms. Binding machinery molecules are lectins that recognize and interact with
specific binding target molecules or glycan patterns on host cell surfaces, mainly on
plasma membranes [5].
The lectins and lectin-recognizing glycan or carbohydrate structures represent
“receptors” and “ligands.” In eukaryotic self, ligand glycans are frequently disac-
charides on glycoconjugates including glycoproteins, glycolipids, or glycosamino-
glycans on the host cell membrane that are biosynthesized via ER-Golgi apparatus
by glycosyltransferases and glycosidases [6, 7]. The receptors have long been
recognized as lectins. Therefore, host-pathogen interactions are the most complex
events during the host tissue and pathogen interaction and infection, because cells
Table 5.8 Invertebrate SA-specific lectins. There is no available structural information on the lectins below
Arthropoda Chelicerata Limulus polyphemus Limulin Neu5Ac, Neu5Gc
Carcinoscorpius rotundicauda Carcinoscorpin Neu5Gc, Neu5Acα2,6GalNAc-ol
Heterometrus granulomanus Scorpin Neu5Ac, Neu5Gc
Crustacea Paratelphusa jacquemontii HA-A O-AC-Neu5Ac
Cancer antennarius HA-A Neu5,9Ac2, Neu4,5Ac2
Scylla serrata and Liocarcinus depurator HA-A Neu5Gc and O-Ac-Neu5Ac, respectively
Homarus americanus Lobster agglutinin I Neu5Ac
5.3 How Do Hosts Interact with Pathogens?
Litopenaeus setiferus Tracheata LsL Neu5Ac, O-Ac-Neu5Ac

Allomyrina dichotoma Allo A-II Neu5Acα2,6Gal1,4GlcNAc
Mollusca Bivalvia Modiolus modiolus and Crassostrea gigas HA-A Neu5Ac
Anadara granosa HA-A Neu5Gc
Gastropoda Cepaea hortensis and Achatina fulica AFL and agglutinin I Neu5,9Ac2
Pila globose and Limax flavus Achatinin H and PAL Neu5Gc and Neu5Ac > Neu5Gc, respectively
Echinodermata Echinoidea Hemicentrotus pulcherrimus LFA Neu5AcGlcCer,(Neu5Ac)2GlcCer
Strongylocentrotus purpuratus 350-kDa sperm-binding Neu5AcGlcCer,(Neu5Ac)2GlcCer
protein
Urochordata Halocynthia pyriformis 350-kDa sperm-binding Neu5Ac, Neu5Gc
protein
207
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Fig. 5.2 Microbial pathogens infect host cells through tight barrier interaction
are coated with carbohydrates and express carbohydrate-binding proteins (CBPs) or

glycan-binding proteins (GBPs). The immune responses are mediated by many
GBPs or lectins. Representative lectins known are the galectins, CTLs and Siglecs.
The lectin families are directly involved in host innate immunities and adaptive
immune responses. In innate immune response, APCs can discriminate self, nonself,
stressed self, and altered-self through lectin-glycan interactions. For example, the
well-studied lectins as GBPs or PRPs include Siglec-1, Dectin-1, DC-SIGN,
Galectin-9, and Galectin-3. Among them, Galectin-3 and Galectin-9 are water-
soluble forms as PPRs. The GBPs act as PRRs recognize exposed glycans of nonself
on infectious pathogens such as bacteria, yeasts, viruses, and parasites. CLRs such as
DC-SIGN particularly exhibit glycan specificity. Lewis glycans and mannosylated
glycans bind to multiple pathogens. The pathogens are diverse from viruses like
HIV, bacteria like H. pylori and M. tuberculosis, and helminths like Schistosoma
mansoni to yeasts like C. albicans.
Lectins receive and clear pathogens because virus and bacterial lectins bind host
surfaced carbohydrates. To protect hosts, host cells express soluble glycoproteins to
block the pathogen lectin to avoid the host cell interaction. Host lectins also remove
or disturb pathogen-host binding event. However, due to the structural similarity, the
host lectin-pathogen glycan binding potentiates rather invasive endocytosis of the
pathogenic agents to receive the host cells. In fact, most surfaced immune-related
receptors are glycoproteins such as PRRs. PRRs include chemokine receptors,
cytokine receptors, NOD-like receptors, TLRs, MHC-I/MHC-II proteins,
co-receptors of T cells/B cells, TCR, and BCR. For example, blocking of T-cell
N-glycan synthesis increases TCR signaling with T-cell activation [8]. Silencing of
MHC-II N-glycans prevents the presentation of bacterial polysaccharide antigens to
T cells [9], whereas experimentally silencing in glycosylation site of TLRs
dysregulates the signaling [10, 11].
5.3 How Do Hosts Interact with Pathogens? 209
Mammalian lectins are subclassified into two major types of (i) intracellular
lectins, which are resident in cytosolic endosomes and include the M-type lectins
(CRT calreticulin and CNX calnexin) in the ER-Golgi glycosylation organelle, and
(ii) membrane-type lectins, which are membrane anchors and include the C-type
lectins and Siglecs. M-type lectins play crucial roles in eukaryotic glycoprotein and
glycolipid secretions by step by step and protein maturation quality control. In
contrast, Siglecs and C-type lectins function in elimination of foreign invaders and
pathogenic agents as well as innate and acquired immune response to self-host
defenses [12].
5.3.2 Bacterial Glycoconjugates Interact with Host Lectins
Various genome data have been obtained from different research consortia including
the Korea Microbial Genome Project or EU-MetaHit and the Human Microbiome
Project. Bacterial pili and adhesive molecules also interact with the environment in
the gut [13, 14]. Glycoconjugates involve in bacteria-host interactions.
Glycoconjugates consist of lipopolysaccharides (LPS), capsular polysaccharides
(CPSs), exopolysaccharides (EPS), lipooligosaccharides, lipoglycans, glycopro-
teins, peptidoglycans, and teichoic acids with diversity [15] (Fig. 5.3). The capsule
CPS and EPS are biosynthesized by the regulation of capsule synthesis (rcs) system
(Fig. 5.4). RcsF is essential for bacterial biofilm formation and pathogenicity. RcsF
is a lipoprotein regulator of the rcs system. RcsF modulates the RcsC-D-A/B
signaling cascade as the complex pathways. RcsF lipoprotein anchored to the
outer membrane activates the rcs phosphorelay for synthesis of EPS and CPS, cell
motility, antibiotic resistance, and virulence [16]. The core of the rcs system is the
RcsB and the sensor kinase RcsC. RcsB is a DNA-binding protein and is activated
via an N-terminal phosphoreceiver domain. The histidine kinase RcsC is complexed
with RcsD. RcsC and RcsD are structurally similar with a sensor domain, a
transmembrane-spanning motif, a phosphorylation domain of C-terminal region,
and a histidine kinase domain. RcsC phosphoreceiver domain holds the phosphoryl
group and transmits to the RcsB domain in N-terminal region via the RcsD domain
in C-terminal region. Activated RcsB binds to the RcsB box [17]. rcs-dependent
promoters synthesize EPS through the RcsAB box motif bound by a RcsB and RcsA
complex [18]. These glycoconjugates are so-called surface-glycan barcodes or face
signature, giving unique specificities to the host. Thus, the host innate immune cells
interact with bacterial glycans through lectins such as DC-SIGN (Fig. 5.5), “sens-
ing” the presence of bacteria.
5.3.2.1 Glycoproteins
Bacterial glycoproteins are also a recently recognized group of glycoconjugates.

They are glycosylated virulence factors of bacterial pathogens. Bacterial GTs attach
< Microbial glycoconjugates>
Lipid
Bacterial O-Glycosylation
Ser
Peptidoglycan
SRRP Ser GtfB GtfA
Protein (Serine-rich Ser
repeat GtfA GtfB
protein)
…… Interact with
the host Ser
e.g. DC-SIGN Ser
Ser
Attached to the
GtfC
Establish species GalT1
cell surface specificity
GTs n
Ser SecY2
SecA2
Inner
membrane
n
EPS/LPS/CPS
Mn2+
: GlcNAc : Glc : Mono sugar : Amino acid
Type IV pili, Adhesin/Lectin
Flagella Glycoprotein
Fig. 5.3 Bacterial glycoconjugates of flagella, glycoproteins, pili, CPS, EPS, and LPS. Modified
from Ref. [13] Tytgat HLP, de Vos WM. 2016. Trends Microbiol. 24(11), 853–861
Fig. 5.4 Regulation of the 'PXKTQPOGPVCN5KIPCN

Rcs system for biosynthesis
of capsular polysaccharides
(CPSs) or 1WVGTOGODTCPG 4EU(
exopolysaccharide (EPS)
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glycans by O-glycosylation either to Ser, Thr, or rarely Tyr residues or by

N-glycosylation to Asn residue. Bacterial glycation is quite similar to eukaryotic
O-glycosylation and N-glycosylation. Protein glycosylation in bacteria was first
reported in the Firmicute Clostridium [19]. Many bacterial examples for the protein
Fig. 5.5 A schematic

structure of DC-SIGN
Carbohydrate recognition domain
(CRD)
Neck domain
Transmembrane domain
Tyrosine based motif
Triacidic cluster
Di-leucine-based motif
glycosylation are known in Burkholderia cenocepacia, Campylobacter jejuni,

Helicobacter pylori, Escherichia coli, Neisseria meningitidis, Francisella
tularensis, Haemophilus influenzae, Pseudomonas aeruginosa, and Porphyromonas
gingivalis [20–28]. Their representative roles in protein glycosylation include bac-
terial virulence, protein stability, and immune regulation from host immunity or
protein substrates. In addition, bacterial protein glycosylation provides ligand func-
tions to interact with the gut host. In the aspect of acquisition of glycans, some
intestinal commensal bacteria including Bacteroidetes and Firmicutes [29] acquire
and incorporate host-produced glycans into their bacterial surface glycoproteins. The
most unique sugar moiety is incorporated into the bacterial surfaces L-fucose residue
[30]. Some lactobacilli’s glycoprotein, SlpA of Lactobacillus acidophilus NCFM,
specifically recognize DC-SIGN in order to downregulate DCs and T-cell functions
[31]. Serine-rich repeat proteins (SRRPs) known as adhesin molecules are also
glycoproteins, which are frequently found in streptococci strains such as Strepto-
coccus parasanguinis, S. gordonii, S. pneumoniae, and S. agalactiae as well as
staphylococci strains such as Staphylococcus aureus. Such adhesins are
O-GlcNAcylated to attach GlcNAc, by GtfA and GtfB enzymes [32]. After
O-GlcNAcylation, GtfC and Gtf3 enzymes further attach Glc residues. The
glycosylated proteins are delivered by the SecA2-SecY2 system upon reaction by
GalT1 enzyme [33]. This type of bacterial O-glycosylation in adhesins highly
resembled to the eukaryotes (Fig. 5.3). Similarly, bacterial N-glycosylation is antic-
ipated. As L-fucose residues are abundant in the gut and mediate host-microbe
symbiosis, some commensal bacteria release fucose residues from the mucus.
5.3.2.2 Bacterial Capsule of Capsular Polysaccharide (CPS)
Some bacterial surfaces are coated with their bacterial capsules, which are the
structural architectures decorated on the cell surfaces of bacteria and fungi, for
their survivals. They prevent the microbes from the immune recognition surveillance
of the host and allow the microbes to invade the host. Most of the bacteria have
capsule polysaccharides. But the only exception is Bacillus anthracis which have a
PGA capsule. Capsules are mainly polysaccharides with huge diversity. Streptococ-
cus pyogenes are unique due to their one-capsule structure. In addition, within the
same microbial species, capsule structures are different. The diversity of capsules is
based on the different immune mechanisms. The capsule-biosynthetic genes are
classed into common gene family and the CPS type-specific gene family. The
common gene family includes the genes for capsule transportation to the cellular
membrane. The CPS type-specific gene family includes the genes necessary for each
type of capsule synthesis. Even in the same species of bacteria, different genes
generate their capsules. In addition, different bacteria bear same or similar synthe-
sizing genes of capsules. The capsule-synthetic pathway can be different among
microorganisms. The common pathway of capsule synthesis is in that all capsules
are generated through the membrane-associated acceptors. Three synthetic pathways
such as WZY-, ABC transporter-, and synthetic enzyme-dependent pathways are
involved in the genesis of capsules. Such generated bacterial capsules act as a
virulence factor and an immune escape factor for bacteria. In human application,
the capsules are used as a vaccine antigen.
Negatively charged glycoconjugates of capsular and slime polysaccharides are
distinct in microbiological world. Bacterial CPSs are produced from both types of
Gram-negative and Gram-positive bacteria. Bacterial CPS species are structurally
heterogeneous. However, some bacterial CPSs produced by Gram-positive strains of
Streptococcus and Staphylococcus families as well as Gram-negative bacteria of
Klebsiella, Neisseria, and Haemophilus families are rather homogeneous in their
structures with acidic polysaccharides [34]. Gram-positive bacterial CPSs are struc-
turally similar and immunogenic in hosts, providing the concept of CPS-based
vaccination [35].
Bacterial capsules are strictly attached, not released, as the outmost
glycoconjugates, and they are also produced by fungal cells. Other slime poly-
saccharides are detached or released from the cell surfaces. Capsule polysaccharides
(CPSs) include the mucus polysaccharide such as colanic acid or M antigen and
some pathogenic alginic acids [34, 36]. In 1928, Griffith in his “Griffith’s Experi-
ment” [37] reported avirulent “rough” (unencapsulated) and virulent “smooth”
(encapsulated) strains of S. pneumoniae. The encapsulated bacterial colonies are
called “smooth” type, while the unencapsulated colonies are called “rough” type
[37, 38]. Later, Avery et al. identified the capsules as the “genetic marker” to confirm
the genetic element [39]. CPS capsules found in S. pneumoniae stimulate host
immune responses against the pathogens, conferring the concept of CPS vaccination
to encapsulated bacteria such Group B Streptococcus (GBS) [34]. Several prosthetic
① WZY-dependent pathway
② ABC transporter-dependent pathway
③ Synthase-dependent pathway
① ②
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Fig. 5.6 Three types of CPS synthesis pathway. (1) WZY-, (2) ABC transporter-, and
(3) glycosyltransferase-dependent pathways are known. (1) In the Wzy pathway, the WbaP enzyme
starts the synthesis of sugar-Und-P attached to the lipid carrier Und-P. CPS-repeated units are
produced. The Wzx flippase and Wzy enzyme further elongate the periplasmic repeat units. The
matured CPSs are penetrated via the periplasm by Wzc and Wza and localized at the bacterial
surface by the Wzi protein [40]. (2) E. coli group 2 CPS KfiABCD and KpsCSMTED proteins
synthesize a translocation complex in the ABC transporter pathway. (3) In the glycosyltransferase
pathway, a series of KfiA, KfiB, KfiC, and KfiD enzymes are involved. Then, CPSs are delivered by
the KpsD/KpsE transporter [42, 47]
groups including O-formyl, O-acetyl, or pyruvate ketosidic groups are substituted

with the bacterial CPSs. In fact, some bacterial CPSs are frequently substituted with
O-acetyl ester linkage, O-formyl ester linkage, or pyruvate ketoside linkage
[40]. The reducing sugars present in CPSs are also diesterized with phosphatidic
acids. For example, the CPSs produced by E. coli group 2, H. influenzae type b
strain, and N. meningitidis group B species produce the diesterized CPSs, having the
phosphatidic acid [34]. The CPSs produced by E. coli K5 species are linked with
phosphatidyl-Kdo structure [41]. The CPSs produced by E. coli K30 strains, E. coli
K40 species, and Klebsiella family are LPS lipid A-bound CPS known as KLPS
forms [42]. S. agalactiae serotype III CPS contains polySialic acids with NeuNAc
and GlcNAc residues [43]. CPSs are larger than the LPS O antigen polysaccharides
in E. coli [26] with up to 100 kDa per chain.
For the CPS synthesis, three pathways of WZY, ABC transporter, and
glycosyltransferase pathway are known. First, the Wzy pathway for CPS biosynthe-
sis is also known to block polymerization in the K. aerogenes DD45 strain as the first
description [44]. The CPS-produced K. aerogenes DD45 strain contains repeated
and multiple sugar units. The CPS synthesis resembled with other LPS and pepti-
doglycans [35] (Fig. 5.6). For the initiation step of the CPS synthesis in the
K. aerogenes DD45 strain, Gal-l-P reside is transferred to the C55-lipid
undecaprenyl phosphate by the WbaP protein [44]. Sugar length elongating enzyme,
Wzy, sequentially assembles the CPS backbone through linking each repeat in the
bacterial periplasmic space [45]. Polysaccharide-specific transport protein translo-

cates the polysaccharides to the next space [46]. In Gram-negative bacteria, second-
ary transportation proteins of Wzc and Wza transport the produced CPSs to the
periplasmic space and outer membranes [47]. Wzc is a membrane periplasm acces-
sory (MPA) protein present in cytoplasmic region, while Wza is an outer membrane-
associated accessory protein (OMA). Wzi is an initial tethering point protein
[48]. For example, S. pneumoniae CPSs are polymerized by the Wzy pathway,
where each specific glycosyltransferase is involved in the CPS synthesis and the
synthesized CPSs are tethered to the bacterial cell wall with covalent bonds [49].
Secondly, most Gram-negative bacterial CPSs are synthesized by the ABC
transporter pathway [48]. In the E. coli K5 CPS as a representative case, the K5
CPS polysaccharide is specifically synthesized and polymerized by four different
proteins including KfiA, KfiB, KfiC, and KfiD [50]. The KfiA protein and KfiC
protein are GlcNAc-transferase and GlcA-transferase, respectively [51, 52]. KfiD
protein has an UDP-glucose dehydrogenase enzyme activity and forms the
UDP-GlcA substrate from the substrate UDP-glucose [53]. KfiB protein formulates
the KfiA/KfiB/KfiC complex form [51]. The final product, CPS-lipid cap, is deliv-
ered to the cytoplasm membrane by action of the ABC transporter with the KpsT and
KpsM [54, 55]. In E. coli K1 strain, the KpsT protein recognizes the poly-SA
structure, called Kl-CPS [56]. The periplasmic CPSs are delivered to the outer
membrane and then to the bacterial surface by the continued transporters of KpsE
and KpsD associated on membranes [57]. Lastly, a specific pathway, named
synthase-dependent synthesis, is known in some Gram-positive bacterial
S. pyogenes and S. pneumoniae types which add repeatedly saccharide units to the
reducing end [58]. For example, S. pyogenes HasA has two enzyme activities of a
GlcA-transferase enzyme and GlcNAc-transferase. Similarly, HasB is the UDP-Glc
dehydrogenase enzyme [59]. CPS cap structures are formed via a diester linkage to
the reducing sugars with phosphatidic acid or lipids. The E. coli K5 phosphatidyl-
Kdo is the example of CPS capping [41]. In the CPSs produced by Klebsiella strains,
E. coli K30 strain and E. coli K40 strain, lipid A core structure, is attached at their
reducing end of CPS [42]. In the Gram-positive bacteria, which lack for membranes,
CPSs are linked to the peptidoglycan or the membrane molecules. CPS produced in
the serotype III strain of S. agalactiae, the peptidoglycan, or GlcNAc is attached to
the CPS [60].
5.3.2.3 Function of Bacterial CPS
For the function of CPCs, they are virulence factors for bacterial encapsulated
pathogens. CPSs also facilitate bacterial adherence and activate dissemination.
Also, CPSs as shields protect from host recognition, complement-mediated
opsonization, and phagocytosis by innate immune cells, allowing evasion during
infections [61]. CPSs can induce IgM-type antibody through the T-cell-independent
immune response. The immunogenicity of CPSs depends on the chain length.
Certain SA-containing CPSs produced by E. coli K, E. coli K92, N. meningitidis
.25
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Fig. 5.7 CPSs in K. pneumoniae confer to antimicrobial peptide resistance and reduce the binding
of antimicrobial peptides to the bacterial surface. CPS mediates pathogenic bacterial resistance to
endogenic antibacterial peptides. Modified from [66] Llobet E, Tomás JM, Bengoechea JA. 2008.
Microbiology 154(Pt 12), 3877–86
group B, N. meningitidis group C, and S. agalactiae species disrupt the complement

B as well as complement C3b binding. Therefore, complement cascade pathway of
host defense system is avoided [62]. Some CPS structures are similar to host
carbohydrates, giving a molecular mimicry, providing an evasion of such pathogenic
bacteria from the host immunity. In addition, CPSs produced by E. coli K1 and
N. meningitidis group B strains are similar to the terminal poly-SA structures of the
neuronal cell adhesion molecule (NCAM) of the human brain [63]. The CPS of
E. coli K5 strain contains the N-acetylheparosan sugar structure [34]. The
S. pyogenes CPS contains hyaluronic acid found in human tissues [39]. The
S. agalactiae type III CPS SAs affect mice B-cell repertoires [64]. The CPS
produced by S. agalactiae type V strain lacks SA species and can induces IgM-to-
IgG switch turnover to allow immunoprotection in Rhesus macaques [65]. CPS is a
bacterial decoy for antimicrobial peptides produced by hosts [66]. Anionic bacterial
CPSs purified from the encapsulated strain K. pneumoniae increase the minimum
inhibitory concentration of polymyxin B. Anionic CPSs also block the bactericidal
activity of antimicrobial peptides by binding them in the naturally released CPSs,
which are produced by the pathogenic bacteria such as K. pneumoniae,
S. pneumoniae, and P. aeruginosa. To prevent host recognition, P. aeruginosa
adsorbs SAs released from bacterial cultures to utilize the SAs to block complement
deposition in cell surfaces. Unfortunately, the molecular mechanism(s) underlying
the P. aeruginosa strains survived in the host-defense system is not known. CPSs
can also bind to polymyxin B (Fig. 5.7).
K. pneumoniae CPS can also impede the antimicrobial peptide, β-defensin
(BD) expression in airway epithelial cells. The CPSs released bind to antimicrobial
Klebsiella pneumoniae
Lipopolysaccharide
(LPS; O antigen)
CPS Pathogenicity factor ugd atf
WcaI (kp3706) fucosyl transferase
Lipid chain - Fucose gnd Wzy (magA)
Periplasmic space - O-acetylation WcaG (kp3709) GDP-fucose synthesis wbaP ptf
GlcNAc wcaI, wzc
Other monosaccharides Atf (kp3712) fucose acetylation
Kdo wcaH wzb
wcaG wza
Outer Membrane gmd
Polysaccharide β-Defensins : antimicrobial activity

surface layer hBD1 : constitutively expression
hBD2,3 : expression by cytokine, pathogens
CPS
iE-DAP
,κBα
CYLD
Capsular polysaccharides
(CPS; K antigen) NF-κB signaling pathway hBD2
hBD3 NOD1
Glc
GlcA p38 X
Fuc MAPK signaling pathway JNK
hBD2 hBD2, hBD3
CPS anchorage protein
P44/42 hBD3
AP : antimicrobial peptide
MyD88
Blocking bactericidal activity
MKP-1
Klebsiella pneumoniae TLR : Toll-like receptor

outer membrane NOD1 : Nucleotide binding and oligomerization domain containing protein 1
NF-κB : Nuclear factor kappa-light-chain-enhancer of activated B cells
MAPK : Mitogen-activated protein kinase
iE-DAP : D-glutamyl-meso-diaminopimelic acid
Fig. 5.8 K. pneumoniae CPS facilitates pathogen survival in the hostile environment. Modified
from [67] Moranta D et al. 2010. Infect Immun.78(3), 1135–46
peptides to block the bactericidal activity (Fig. 5.8). The question of how CPSs
protect bacteria for survival in the hosts has been answered by the recent documen-
tation [67]. CPS inhibits activation of the signaling pathway involved in β-defensin
expression. The outer membrane CPS of K. pneumoniae is a toxic factor and reduces
phagocytosis of DCs or macrophages. BD is an antimicrobial peptide produced in
airway epithelial cells. Among human BDs (hBDs) of hBD1, hBD2, and hBD3,
hBD1 is constitutively produced by the respiratory tract lined with epithelial cells,
whereas the hBD2 and hBD3 expressions are induced by cytokines or pathogens.
K. pneumoniae causes pneumonia, and CPS in the outer membrane is a toxic factor
and inhibits phagocytosis of macrophages. The CPS binds to antibacterial peptide
AP to block the bactericidal action. Therefore, CPS protects bacteria through
suppression of the hBD expression in the lungs. The CPS mutant exhibits the low
survival rate and is vulnerable to β-defensin. CPS exerts a resistant activity against
hBD3. β-Defensin expression is increased only in CPS-lacking mutants as CPS
inhibits activation of the β-defensin signaling. Two signalings are known to increase
the expression of β-defensin. The first is the NF-κB signaling pathway to induce
β-defensin expression, cell survival, and immune responses to infection. The second
signaling pathway is activated by NF-κB as the MAPK signaling pathway to
increase the hBD2 and hBD3 levels. NF-κB signaling pathway which controls BD
expression is inhibited. Apart from the NF-κB signaling, the second MAPK signal-
ing pathway including P38, JNK, and p44/p42 was also inhibited, as the two
signaling pathways are commonly activated by TLR molecules. Activation of the
two signaling pathways is controlled by TLR. TLR activates through MyD88.
MyD88-TLR contributes to hBD2 activation. MyD88 expression is not associated
with the expression of hBD3. Similarly, NOD1, which activates MAPK/NF-κB axis
signaling, involves in BD3 expression in humans.
CPS is a bacterial decoy for antimicrobial peptides. Antimicrobial peptides
(APs) are cationic, and its action is initiated through interactions with the anionic
bacterial surface. Humans live with various bacterial populations associated with
mucosal surface, termed the microbiota. Microbial glycoconjugates interact with
host cells and are species-specific as ligands for host cells. Pathogenic microbes have
evolved to evade host immune surveillance and innate immune response and
phagocytosis clearance of hosts. Mostly, host and microbial cell surface are coated
with carbohydrates, resulting in microbial pattern recognition and governing normal
immune cell activities. A major strategy regarding pathogen is to define sugars
which mimics or interferes with host’s immune functions. Endogenous sialoglycans
represent the “SAMPs” which prevent myeloid immune cell activation and matura-
tion, including leukocytes. Some pathogens can mimic sialyl glycans of hosts, and
the SA-containing glycans make pathogenic bacteria to engage inhibitory Siglecs of
hosts, attenuate immune clearance, and dampen leukocyte activation. Engagement
of inhibitory Siglecs and SA mimicry have been coevolved with changes in Siglec-
binding specificity or host sialic acid repertoire. Carbohydrate can have a major role
in immune response rather than protein which is known for a major component of
immune response.
If SAs were incorporated into cell surface CPS, the events help them evade the
host immune responses. As a shield, bacterial pathogens including E. coli K1 strain,
H. influenzae strain, and S. agalactiae strain have evolved to bypass host immunity.
As bacterial virulence factor, K. pneumoniae CPSs are the infection determinants.
The high molecular weight polysaccharide CPSs contribute to the muco-phenotype.
CPS helps the bacteria evade phagocytosis and protects from bacterial clearance.
Sialic acid of CPS in K. pneumoniae strains is antiphagocytic, as Siglec-9 functions
as a receptor for the MHC-I expressed in neutrophils, which bind to SAs and
transduce the downstream signaling to dampen and suppress inflammation
[68]. Sialylated CPSs also provide a virulence factor. Sialic acids in CPSs promote
the factor H (FH)-binding activity of the alternative complements, restricting the
recognition of C3b and FB (Fig. 5.9) [69]. If carrier proteins are conjugated to CPSs,
the conjugated CPSs activate the T-cell-dependent immune response to produce IgG
class. CPSs are currently used for vaccination of several pathogenic bacteria includ-
ing H. influenzae, N. meningitidis, S. typhi, and S. pneumoniae [70]. In addition, the
CPS conjugation is also applied for some encapsulated Klebsiella and Pseudomonas
[34]. Nonetheless, the CPS vaccination is not successful in younger children under
age 2 [71]. CPS-protein conjugates thus improve the CPS immunogenicity in
younger children [72]. So-called CPS conjugate vaccines are the current licensing
replacements of encapsulated pathogenic bacteria including S. pneumoniae,
H. influenzae, and N. meningitidis. The vaccination of Hib vaccine is a representative
for the H. influenzae type b [73].
SAα2-3 Galβ1-4GlcNAcβ1
Without sialic acid, Factor B combines
with C3b to make a convertase which
Kiebsiella Penum induces a cleavage of C3
oniae = onset of opsonization and phagocytosis
(KP-M1)
High Level of
Sialic Acid in Factor H binding to C3b
CPS protects the bacteria from
opsonization and phagocy
tosis
hSigleg-9
Galactose
N-acetylglucosamine C3
Cleavage
Sialic Acid
V2 Ig-like domain
C2-set Ig-like domain &QYPTGIWNCVKQPQH6EGNNUK C3b C3a
ITIM IPCNKPI
ITIM-like +PJKDKVKQPQH0-EGNNVQZKEK Attaches to Chemotactic
cell surface protein
V[
Neutrophil
Fig. 5.9 Sialylated CPS of K. pneumoniae is involved in bacterial resistance to host neutrophil
phagocytosis. Modified from [68] Lee CH et al. 2014. Virulence 5(6), 673–9
5.4 Pathogen-Producing Lectins as Receptors to Bind

to the Host Carbohydrates
Many pathogens use host glycans as targets for adhesion. Therefore, the blockers of
carbohydrate-binding adhesins are fighters of infections. Thus, rapid glycan micro-
array approach can assess the bacterial adhesion. Pathogens are recognized by
recognition with GBPs including C-type lectins. Recent progress in glycan arrays
has accelerated the rapid deciphering of lectin-binding carbohydrate and glycans as
ligand specificity. The essential role of lectins is believed to decipher the glycocode
by specific recognition of carbohydrates. The human GIT covered by mucosal
enteroepithelial cells provides a mucosal defense barrier decorated by the
glycocalyx. Glycan-enriched glycocalyx is bound to plasma membranes of cells or
excreted into the extracellular milieu. Interaction between lectins and carbohydrate
ligands contributes to various biological responses and pathologic immune
responses. Conversely, many pathogenic viruses, bacteria, fungi, and parasites
utilize host glycans as targets for adhesion or for secreted toxins. Protein-
carbohydrate recognition covers many biological events and diseases.
Glycan-binding proteins from pathogens are well known as bacterial lectins,
adhesins, or toxins. These molecules are produced during coevolution. They specif-
ically recognize oligosaccharides expressed on host cell surfaces [74, 75]. Molecular
ligands for glycan-binding proteins can be used as therapeutic application [76]. A
special feature of protein-carbohydrate interactions is the wide prevalence of
multivalency [77]. Bridging lectin-recognizing sites by multivalent saccharides can
increase binding or inhibitory levels [78]. Bacteria and bacterial toxin’s SA-specific
5.4 Pathogen-Producing Lectins as Receptors to Bind to the Host Carbohydrates 219
Table 5.9 Bacteria and bacterial toxin’s SA-specific lectins. HA-A hemagglutinin activity
observed
Gram-negative
Escherichia SfaI, SfaII, Neu5Gcα2,3Gal; Neu5Acα2,8Neu5Ac
coli SFaS
K99 fimbriae Neu5Gcα2,3Galβ1,4Glc
Helicobacter SabA or Siaα2,3 or
pylori HP-NAP Neu5Acα2,3Galβ1,4GlcNAcβ1,3Galβ1,4
Haemophilus HiFA GM3,GM1,GM2, GDIa, GD2, GD1b
influenzae
Pasteurella Adhesin Neu5Ac
haemolytica
Bordetella SBHA and Neu5Ac and GD1a (or GT1b),
bronchiseptica HA-A respectively.
and B. avium
Neisseria OpcA (Opa; Neu5Ac and Neu5Acα2,3Galβ1,4Glc,
meningitidis and NHBA) and respectively.
N. subflava Sia-1
Pseudomonas – Sialyl-Lex or Siaα2,6
aeruginosa
Moraxella Fimbrial GM2
catarrhalis protein
Gram-positive Streptococcus GspB and Hsa Siaα2,3 Siaα2,6 and Neu5Acα2,3Gal,
gordonii respectively.
Streptococcus PAc Siaα2,6
mutans
S. mitis and SABP Neu5Acα2,3Galβ1,3GalNac and
S. suis Neu5Acα2,3Galβ1,4G1cNAcβ1-3Gal,
respectively
Mycoplasma Mycoplasma HA-A Neu5Acα2,3Galβ1,4GlcNAcb1,3
gallisepticum
Bacterial toxins Vibrio Cholerae toxin GM1
cholerae
Vibrio mimicus Haemolysin GD1a, GT1b
Clostridium Neurotoxin 1b series gangliosides
botulinum A-F
Clostridium Tetanus toxin GT1b, GQ1b
tetani
C. perfringens Delta toxin GM2 and GM1, respectively.
and E. coli and heat-labile
enterotoxin
Bordetella Pertussis toxin GD1a; Neu5Acα2,6Galβ1,4GlcNAc
pertussis
lectins are described (Table 5.9). Gram-negative bacterial strains including E. coli,
H. pylori, H. influenzae, Pasteurella haemolytica, Bordetella bronchiseptica,
B. avium, N. meningitidis, N. subflava, P. aeruginosa, and Moraxella catarrhalis
produce their specific lectins. Gram-positive bacteria include S. gordonii, S. mutans,

S. mitis, and S. suis. Mycoplasma gallisepticum also bear its specific lectin-like
protein. Bacterial toxins produced by Vibrio cholerae, V. mimicus, C. botulinum,
C. tetani, C. perfringens, E. coli, and Bordetella pertussis have also lectin activities.
Glycomic analysis studies using glycan microarrays have resolved the
glycobiological aspects in lectin-binding sites. Multivalent carbohydrate-lectin inter-
actions between host and pathogen establish infections. Therefore, pathogen
adhesion-preventive competitive agents can be potential antimicrobial drugs. Such
non-covalent carbohydrate-protein binding is very weak with disassociating con-
stants ranging from 103 to 104 [79]. However, nature compensates this restricted
limitation through the multivalent glycan interaction in ligand-receptor recognition.
The representative is lectin with “glycan multivalent” effect in enhanced binding
capacity. The multivalent mimics of natural glycans are inhibitors of carbohydrate-
protein interaction as therapeutic drugs in usages of antimicrobial, anti-
inflammatory, and antitumor potential [80]. Such developed multivalent scaffolds
with carbohydrate epitopes are glycodendrimers, glycopeptides, glycopolymers,
glycodynamers, fullerenes, calixarenes, etc. [79]. The known human ABH blood
group glycoconjugates are also used as ligands of bacterial lectins. ABH blood group
antigens are fucosylated glycans in endothelial and erythrocytic cells. In addition,
Lewis antigenic epitopes are such categories as tissue histo-blood types. The ABO
and Lewis type sugars were correlated in human population-dependent susceptibility
to certain pathogenic bacterial diseases in humans [81]. For example, the blood
O-phenotype individuals are susceptible to cholera toxins [82] and Norwalk viral
gastroenteritis [83].
5.4.1 Uropathogenic E. coli (UPEC), Enterohemorrhagic

E. coli (EHEC), and Enterotoxigenic E. coli (ETEC)
E. coli strains are classified, in general, to facultative anaerobic bacteria, which are
originally discovered and isolated from the gastrointestinal tract (GIT) of humans.
From the virulence consideration, E. coli strains are further subclassified to the two
distinct parameters of pathogenic or virulent E. coli group and nonpathogenic or
avirulent E. coli group. Pathogenic virulent E. coli group is zoonotic for wide
infectious diseases including diarrhea, sepsis, and meningitis. Currently, the patho-
genic E. coli family is further subclassified into five subfamilies of enterotoxigenic
E. coli (ETEC), enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli
(EAggEC), enteroinvasive E. coli (EIEC), and enteropathogenic E. coli (EPEC)
[84]. From the five groups, the EHEC group is a major causing group for epidem-
ically and sporadic E. coli infections.
5.4.1.1 Uropathogenic E. coli (UPEC)
Bacterial lectins are basically hairlike proteins, more specifically known to pili or
fimbriae. They interact with the cell outmost coats. The Gram-negative E. coli
species express type 1 pili or fimbriae as host receptor recognition and attachment
proteins [85]. Pathogenic E. coli FimH variations influence bacterial adhesion. The
Man-specific adhesin of E. coli or V. cholerae toxin binds to ganglioside GM1 as
relevant examples. Such E. coli cells migrate to the kidneys and shed the type
1 fimbriae and are shifted to the alternative expression of PapG lectins on pin-like
P pili. For example, mannose-specific adhesin FimH is located on fimbriae. Direct
glycan array to mannan or related glycans printed onto glycan microarray wells is
applicable for searching the adhesions if we know the target glycan. The fimbrial
lectin FimH from UPEC and ETEC binds to nanomolar affinity to Manα1,3Man
dimannosides at their nonreducing end, but only with micromolar affinities to
Manα1,2Man.
FimH develops infection by adherent-invasive E. coli, Crohn’s disease (CD),
urinary tract infections (UTI), enteritis, diarrhea, sepsis, and meningitis [86]. In
uropathogenic E. coli (UPEC), adhesins are FimH displayed at the type 1 fimbriae
tip in a way of a single FimH molecule per fimbria. It binds to terminal mannosides
of the mannosylated glycoprotein uroplakin on bladder urothelial cells. The
Man-dependent hemagglutination by the strains is indeed an indicator of the pres-
ence of type 1 fimbrial adhesins [87]. Uropathogenic E. coli expresses the adhesin
FimH lectins as two-domain adhesin on the terminal portion of hairlike “type 1”
fimbriae. The FimH lectin is connected to a pilin anchored with FimG and FimF. It
specifically binds to Man residue of bladder epithelial cell membranes to potentiate
the invasion to the human urinary bladder [88–91]. The fimbrial adhesin or lectin
FimH of uro- or enteropathogenic E. coli binds to high-mannosylated glycoprotein
(MGP) receptor molecules exposed on the epithelial cell surfaces resided in oropha-
ryngeal, urinary, or GITs [92]. FimH consists of (1) two Ig-like domains of the lectin
or carbohydrate recognition domain and (2) the pilin domain [93]. The lectin or
carbohydrate recognition domain (amino acids 1–157) acts using a short flexible
linker made by Thr158, Gly159, and Gly160. The pilin domain (aa. 161–276) links
FimH to the other pilins to form the fimbrial rod. FimH adheres to MGP
carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), which is
excessively overexpressed by GIT epithelial cells of the CD patients as well as
EHEC and ETEC [94]. FimH also adheres to the MGP uroplakin Ia (UPIa) on the
urinary tract epithelial umbrella cells. UPIa contain high-mannosyl N-glycans
[95]. Human CEACAM6 contains two high-mannosylated N-glycosylation
sites [86].
From analysis of the FimH lectin structure obtained from crystals, ligand recog-
nition of the recognition pocket of FimH lectin evolved. It binds to the terminal
Manα1,2-, Manα1,3 and Manα1,6-linked N-glycans, to oligomannose glycans of
Manα1,3Man di-Mannosides, and slightly to Manα1,2Man dimannosides. The two
dimannoses are important for infection by E. coli. Manα1,2Man shielding the
Manα1,3Man glycan is the best link for bacterial adhesion and invasion. Among the
di-Man (Manα1,2Man and Manα1,3Man), FimH lectin prefers to the dimannose
Manα1,3Man, not for Manα1,2Man. There are ligand-binding shifts in the associ-
ation equilibrium between the FimH lectin and the FimH lectin pilin domain. A
single N-glycan binds to three FimH lectin molecules, although cell surface
N-glycans favor monovalent FimH lectin binding [96]. A single N-glycan binds
up to three FimH lectin molecules, so that excess of N-glycans over FimH on the cell
surface strongly binds to FimH. Similarly, F9 pili specifically bind to Gal, GalNAc,
or Thomsen-Friedenreich (TF) antigen (Galβ1,3GalNAcα)- epitopes on the kidney
and inflamed bladder. FmlH binds to TF within naive or infected kidneys and also to
Thomsen nouvelle (Tn) antigen (GalNAc) within the inflamed bladder epithelium.
Experimental silencing of FmlH in the urosepsis strains blocks the ability. Further-
more, challenging vaccination with the LD of FmlH (FmlHLD) inhibits the
urosepsis [97].
5.4.1.2 Enterotoxigenic E. coli (ETEC)
ETEC is a type of E. coli, and the diarrhea-causing bacterium frequently occurs in

the developing countries. ETEC pass through the mucus layer of human intestinal
epithelial cells. ETEC infection ranges from symptomless to serious cholera-like
diseases. The main hallmarks are enterotoxin and fimbriae, which is an attachment
pilus with adhesin and used for attachment to host intestinal cells. ETEC pass
through the mucus layer of mucin domain in intestinal epithelial cells. ETEC
secretes EtpA proteins to intestinal epithelial cells through fimbriae. EtpAs specif-
ically opsonize RBCs by binding to terminal sugar, GalNAc, of blood type A
glycans. Blood agglutination and ETEC enterotoxin cause diarrhea. It has been
found that ETEC H10407 strains separated from cholera-like diseases caused type
A blood group specificity. ETEC strains secrete EtpA-bearing molecules specific for
red blood cells. ETEC pass through the mucus layer of human intestinal epithelial
cells and secretes EtpAs to intestinal epithelial cells through fimbriae which specif-
ically opsonize RBCs via terminal GalNAc of blood type A glycan [98]. The specific
binding of ETEC EtpA with GalNac mediates bacteria to host interaction in the
intestine. The main hallmarks include enterotoxins, fimbriae as the attachment pilus,
and adhesin used for attachment to host intestinal cells. EtpA is a lectin,
carbohydrate-binding protein, which recognizes the host glycans on the cellular
and molecular levels. It mediates attachment and binding of bacteria and viruses to
their intended targets. EtpA is a representatively secreted adhesin molecule. Entero-
toxins include heat-labile (LT)/heat-stable (ST) forms.
During enteropathogen’s attachment and colonization to the epithelium in intes-
tines, enteric pathogens like ETEC use a long proteinaceous fiber termed type IVb
pilus (T4bP) [99]. ETEC E. coli-blood group A interactions intensify diarrheal
severity. ETEC strain H10407 induces in blood group A human severe diarrhea
more than other blood groups [98]. ETEC infection displays serious cholera-like
diseases. ETEC H10407 strains cause type A blood group-specific diarrhea. ETEC
H10407 secretes EtpA proteins to intestinal epithelial cells through fimbriae which
specifically opsonize condense and RBCs through binding to terminal GalNAc
residue of blood type A carbohydrate. This binding consequently causes diarrhea
in humans. Specific interaction of ETEC-secreted EtpA protein with GalNac induces
severe ETEC-mediated diarrhea in the intestine. ETEC strain H10407 secretes the
EtpA adhesin molecule. In glycan arrays, EtpA is an ETEC blood group A-specific
lectin or hemagglutinin. EtpA binds to the glycans on intestinal epithelial cells from
blood group A individuals for adhesion and delivery of both the ETEC LT and ST
toxins. Therefore, ETEC is defined by the plasmid-encoded LT and/or ST entero-
toxins [100]. The toxins are easily transported to cognate receptor molecules
expressed on the intestinal epithelial cell surfaces and allowing net salt and water
efflux into the lumen of the small intestine. This is diarrhea. ETEC is identified
person suffering from severe cholera-like diarrhea. In ETEC and intestinal epithelia
interaction, EtpA is conserved among ETEC strains. FUT2 α1,2 fucosyltransferase
synthesizes ABO blood group antigens on intestinal epithelia [101]. Blood group-
dependent microbial-host interactions indicate specificity of gastrointestinal patho-
gens. In H. pylori, the bacterial BabA adhesin molecule attaches to the gastric
mucosal ABO and Leb. Similarly, V. cholerae infections are linked to the O blood
group [102]. Both CT and LT toxins of ETEC share with different binding of blood
group antigen. They favor blood group O enterocytes [103]. EtpA enhanced viru-
lence in blood group A hosts. The secreted EtpA lectin acts with two additional
glycan-binding tip adhesins of ETEC fimbriae. Genomic FimH as a type 1 pili binds
to mannosyl proteins on epithelial cells, and the plasmid CfaE adhesin lectin
combined with CFA/I pili binds to sialylglycoproteins. They are not bound to
blood groups. EtpA is the only blood group A-specific lectin identified in ETEC
[100]. EtpA-producing ETEC pass through the mucus layer of mucin domain in
intestinal epithelial cells. Then, the ETEC secretes EtpA proteins to intestinal
epithelial cells through fimbriae. EtpA specifically opsonizes RBCs by binding to
terminal sugar, GalNAc, of blood type A glycans. Eventually, blood agglutination
and ETEC enterotoxin cause diarrhea. However, blood groups N and O do not cause
agglutination of RBCs. Or α-N-acetylgalactosidase treatment to RBC abolishes the
EtpA’s agglutination capacity, as illustrated in Fig. 5.10.
The colonization factor antigen/III (CFA/III) is called a T4bP of ETEC. It bears a
minor pilin, CofB, containing an H-type lectin domain. CofB is needed for pilus
assembly with a trimeric complex. But bacterial attachment mechanism is not
defined. For bacterial attachment, T4bP needs a secreted CofJ encoded on the
same CFA/III operon. CofJ binds to CofB by N-terminal extension linked into the
glycan-binding site of the CofB H-type lectin domain. The CofJ-CFA/III pilus
complex is a target against ETEC infection. Bacterial pathogens have evolved
surface organelles to synthesize the filamentous protein polymers, simply to say
pili or fimbriae. Enterotoxigenic E. coli (ETEC) causes diarrhea by at least 22 types
of pilus-related colonization factors (CFs) of ETEC. They are called CF antigens
(CFAs) or coli surface antigens (CSs). Among 22 CFs, 17 are complex polymerized
via chaperone-usher (CU) pathway of major and minor pilus subunits named pilins.
ETEC pili adhesins have two Ig-like domains and N-terminal receptor-binding lectin
RBC α-N-Acetylgalatosidase
A
Agglutination Abolished
NO AGGLUTINATION
Æ Blood Group B, O
Bool group glycans of RBCs
# $ O
Glycan Terminal
GlycanTerminal Glycan Terminal
sugar
sugar sugar
: GalNAc
: Gal :-
Fig. 5.10 Enterotoxigenic Escherichia coli (ETEC) EtpA proteins recognize intestinal epithelial
cells through fimbriae. EtpAs specifically opsonize RBCs by binding to terminal sugar, GalNAc, of
blood type A glycans
domain that recognizes glycoconjugates or glycosphingolipids. Therefore, host

glycan and ETEC binding is key step for ETEC infection [104, 105]. CofB
C-terminal domain adopts an H-type lectin [106]. As H-type lectins typically bind
to N-GalNAc molecules, CofB is a lectin targeting the small intestinal mucosal
glycome. In CFA/III-mediated attachment, it requires a secreted protein, CofJ, at the
GalNAc-binding sites of the CofB trimer. This is important for ETEC infection.
5.4.1.3 Enterohemorrhagic E. coli (EHEC)
EHEC is a major causing agent of gastrointestinal diseases. During enteric bacterial

infection, pathogens such as EHEC recognize gut microbiota known to be relatively
resistant to bacterial pathogens. After interaction with the host microbiota, the EHEC
attach to the gut intestine via the lectin-glycan interaction to the host cells. For the
known EHEC, E. coli as a subpopulation group of Shiga toxin (STx)-forming E. coli
(STEC) is a causing agent of systemic hemolytic uremic syndrome (HUS), hemor-
rhagic colitis (HC), and diarrhea in humans. Two main STx species of EHEC are
designated STx1 and STx2, as the virulence factors. Low doses of EHEC infection
cause disease development. There is no biochemical difference known to distinguish

general EHEC from nonpathogenic E. coli. Thus, specific detection of EHEC is
difficult. The O:H serotype E. coli strains are associated with STEC infection in
humans. Among O:H serotypes, HC and HUS diseases are mainly developed by
certain serotypes including O26:H11, O103:H2, O145:H28, O157:H7, and O111:
H8 strains. Among EHEC, the E. coli O157:H7 strain defines a major foodborne
pathogen as a virulent serotype [107]. Due to its pathogenic property, O157:H7
strain of EHEC E. coli is regarded to be a dangerous bioweapon. Pathogenically, the
strain is survived and expresses bacterial virulence gene in the human GIT
[108]. EHEC strains cover 400 more serotypes. Among them, O157:H7 is a repre-
sentative [109]. EHEC colonizes intestinal epithelium by microvilli loss and adhe-
sion to the cell surfaces of hosts.
Recently, to investigate the information on the lectin-glycan interaction of EHEC,
a genome-wide analysis of putative adhesins or lectin-like proteins has been
searched using lectin-glycan interaction network. Several lectin candidates were
isolated through comparative transcriptome and proteome analysis in order to
allow mucin recognition in EHEC [110]. For example, these lectin-glycan interac-
tions are mediated by glycosylated mucins such as MUC2, MUC5 (A/B), and MUC6
in the human GIT. Bacterial pathogens express lectin-like virulent factors, and the
virulent proteins are potentially used for vaccination and drug targets. Lectin-like
proteins include pathogenic adhesins with tropism for host cell recognition via
distinct architectures, including capsules, enzymes, fimbriae, pili, and vesicles.
The lectin-like proteins interact with receptor proteins on host cell surfaces to
allow cross-membrane invasiveness. Some adhesions activate host immune
responses by binding to Man residue. As lectin-like proteins, E. coli surface antigen
20 (CS20); fimbriae (FimH, Yad) and SfaS; N. meningitidis autotransporter adhesin;
S. enterica serovar Enteritidis ShdA, MisL, Sad, and BapA; and S. epidermidis
polysaccharide intercellular adhesin (PIA) are known [110]. Structural domain and
3D structure can be integrated into multiple omics with transcriptome microarray
information, proteome interaction, and genome data. Because membrane proteins
recognize lectin-related proteins, homology analysis and B-cell epitope analysis
combined with the integrated multiple omics information will predict successful
vaccination or medicine development against pathogens such as EHEC to validate
the predicted adhesins.
Infection of the O157:H7 strain of EHEC E. coli involves in the colonized mucus
layer, evasion of the immune defense, growth, and tissue damage of host [111]. Sev-
eral types of virulence factors such as intimin, Shiga toxin, intimin, and pathoge-
nicity islands (PAIs) induce the EHEC virulence [112–124]. As the major two
different lectins or adhesin proteins, (1) intimin and (2) Shiga toxin are known.
More totally, Curli fibers, EPEC intimin, EHEC intimin, EPEC intimin, EHEC
O157:H7 CsgA, Lpp proteins, and type III secretion system are known to influence
EHEC E. coli O157:H7 or EPEC E. coli internalization into host cells [115–
117]. First, the eae gene encodes the intimin protein of MW 94-kDa, and intimin
mediates the adherence and invasion of EHEC E. coli O157:H7 and EPEC E. coli
strains to hosts [118]. The adhesin intimin is a primary lectin with a minor long polar
fimbria (Lpf) [119]. Intimin lectin is the bacterial outer membrane protein. It initiates
infection of EHEC E. coli strain during translocation of the intimin receptor (Tir) and
the intimin interaction with the surface proteins of host cells, which is encoded on the
bacteria genome. Intimin is an antibacterial drug target. Virulence of EHEC is
related to factors encoded to the locus of enterocyte effacement (LEE), which is a
specific pathogenic island for the intestinal mucosal adhesion and the attaching and
effacing (A/E) lesion formation. The LEE gene locus encodes the eae gene for the
outer membrane intimin and adhesin and also a type III secretion system (T3SS).
The LEE products lead to interaction between EHEC (or EPEC) and intestinal
epithelial cells [120]. Two Tir and intimin genes are located on 43 kb length
sequence of the PAI, as the LEE with the A/E lesion formation [113]. The bacterium
and host interaction are very interesting [121]. Intimin expressed by EHEC and
EPEC mediates bacterial adhesion to the cell surfaces of hosts and A/E lesion
generation [122]. Tir enters into the cells through the T3SS and functions as an
intimin receptor expressed on the host cells [122, 123]. The intimin binding to Tir
accelerates the pathogen adhesion to host cell [124].
At least 18 intimin subtypes [125] are found. As the adhesin protein, intimin is
subdivided into five distinct forms of intimin-α, intimin-β, intimin-γ, intimin-δ, and
intimin-ε by their C-terminus domains [126, 127]. Among these, the eae-γ1 subtype
is common in O157:H7 and O145:H28 strains of EHEC. However, the eae subtypes
of β1-, ε-, and θ- types are common in O26:H11, O103:H2, and O111:H8 strains of
EHEC, respectively. The γ-type intimin-γ is the EHEC O157:H7 intimin, whereas
the intimin-α is an EPEC type. Tir protein produced from EHEC O157:H7 is distinct
from other EPEC-produced forms, in their phosphorylation patterns when they
infiltrate into host cells [122]. EPEC intimin binding domain has been analyzed
[128, 129]. The Tir-recognition domain in the intimin protein (Int188) produced
from O157:H7 strain of EHEC E. coli is elucidated for its structural basis [107]. The
four major structural variations have been known between intimins produced by
EHEC E. coli and EPEC E. coli strains. From their structures, domain I has been
known as an Ig-like domain and domain II is a C-type lectin-like domain.
Mucin-digesting zinc metalloprotease named StcE helps infiltrative penetration of
the adhesion protein and mucus layer [130]. After colonization, STx lectin is the
main virulence factors of EHEC, because it causes the hemolytic symptom in
kidneys and brain [131]. STx gene is located on the lambdoid bacteriophage
genome. STx consists of five B subunits, which interact with
globotriaosylceramide-3 (Gb3) as receptor present in the surfaces of endothelial
cells. One catalytic subunit A targets eukaryotic ribosomes to inhibit protein bio-
synthesis [132]. The STx family consists of the two major types of STx1 and STx2
forms. STx2 is only produced during lytic cycle of phage, whereas STx1 type is
produced during phage cycle and an iron-regulated promoter [133]. Therefore, the
regulator protein of ferric uptake event, named Fur, suppresses the STx1 gene
expression when iron levels are excessive. Thus, EHEC virulence is seen by multiple
factors, not by single gene or gene product.
EHEC STx lectin or intimin lectin binds to mucin sugars of gut mucus layer. The
intestinal epithelium is decorated by secreted oligomeric mucins with heavy
O-glycan proteins [134]. The main monosaccharides on human intestinal mucins are
Gal, GalNAc, Fuc, and Neu5Ac [135], while EHEC consumes Fuc, Gal, and
GlcNAc for gut colonization [136]. EHEC strain but not commensal E. coli releases
mucin sugars by mucin-degrading enzymes such as metalloprotease StcE [137, 138]
and esterase. Esterase is expressed by the prophage-carrying nanS genes known as
nanS-p, which is located on the E. coli O157:H7 EDL933 strain genome and E. coli
O104:H4 LB226692 strain genome. The esterase deacetylates 5N-acetyl-9-O-acetyl
SA (Neu5,9Ac2) present in mucus, and thus the released Neu5Ac form is metabo-
lized by the pathogen. EHEC E. coli has two the nanS-p gene and stcE gene what
they are co-expressed with the stx2 gene and LEE gene, respectively. For digestion
of sulfated intestinal mucins, sulfatase gene co-regulates the LEE genes in EHEC.
Thus, EHEC has evolved acquisition to utilize mucin sugars and to colonize the GIT.
For adhesion to the intestine epithelial cells, EHEC enters the mucus layer of
epithelium. Adhesion level of EHEC to mucin-coated epithelial cells is higher than
the mucin-negative cells, as also observed in other intestinal enteropathogenic E. coli
(EPEC) and S. enterica. An interesting aspect is that EHEC binding to mucus-
positive cells does not require any specific adhesion. However, flagella must be in
the absence for this interaction. The reason is because a ΔfliC mutant adheres
effectively rather than the wild types [138], and flagellin-synthesizing genes are
not expressed during mucus recognition [139]. Metalloprotease StcE reduces mucin
levels since a ΔstcE mutant cannot disrupt the epithelial cell mucus layers [139]. A
cytotoxin named subtilase of certain non-O157 EHEC E. coli strains depletes mucin
or mucus layers [140]. EHEC-altered mucus layer allows easy interaction with
EHEC to bind to enterocytes. EHEC also synthesizes virulence factors for bacterial
adhesion. NagC protein represses GlcNAc residue and Gal residue catabolism in
E. coli. NagC regulates the LEE gene expression by binding to the LEE1
transcription-regulation region. If GlcNAc or Neu5Ac residues are present in the
medium, EHEC adhesion to epithelial cells is decreased.
5.4.1.4 EHEC O157:H7 or EPEC Recognition of Core 2 O-Glycans

of Mucin Type on Cell Membrane of Host
The relationship between host glycosylation and infection of the O157:H7 strains of
EHEC E. coli or EPEC E. coli strains is still unknown. O-glycans are related to
attachment and infection of the O157:H7 of EHEC E. coli and EPEC E. coli to host
cells [141]. The O157:H7 of EHEC E. coli or EPEC E. coli infection and invasive-
ness to HT-29 cells are dependent on the host O-glycosylation status. O-glycans of
mucin type have eight major groups (cores 1–8) by linkage of carbohydrate residues.
Core 2 O-glycans of mucin type are synthesized by a specific GT enzyme of the core
2 β1,6-GlcNAc-transferase 2 (C2GnT2) [142, 143]. O-glycans are associated with
the commensal microbial flora in the distal colon. O-glycans of Galβ1,4GlcNAcβ1,6
(Galβ1,3)GalNAc structure (called core 2) are converted to the Galβ1,3GalNAc
structure (called core 1) in the MUC1 synthesis [144]. O-glycans help the intracel-
lular delivery of the glycoproteins in intestinal epithelial cells, which are polarized
[145]. O-glycans are a binding site for many bacteria [146]. HT-29 cells, which are
deficient for the core 2 O-glycan structures, showed the reduced level of EHEC
O157:H7 or EPEC adherence. In addition, the upregulated MUC3 in HT-29-Gal
cells diminishes EHEC E. coli O157:H7 or EPEC E. coli attachment
[147, 148]. Core 2 O-glycans of mucin type produced in epithelial HT-29 cells are
the sites for the EHEC O157:H7 or EPEC invasion and infection.
Mucin-type O-glycan is synthesized by serial enzyme reaction from GalNAc on
Ser/Thr residues on proteins. For example, core 1 synthase (C1GnT) transfers the
Gal residue to the acceptors and generates the core 1 sugar of the Galβ1,3GalNAcα1-
Ser/Thr structure. Similarly, core 3 synthase (C3GnT) transfers the GlcNAc residue
to its acceptors and forms the core 3 sugar of the GlcNAcβ1,3GalNAcα1-Ser/Thr
structure (termed core 3 structure). The core 1 sugar chains are modified to the core
2 sugar chain through multiple enzymatic conversion of GTs including C2GnT-1,
C2GnT-2, and C2GnT-3 enzymes. Among them, the core 3 chain can also be
converted to the core 4 chain by the specific GT enzyme of C2GnT-2. If Man,
Fuc, Glc, or GlcNAc residues are attached to Ser/Thr protein, the core 5 to core
8 chains are yielded.
5.4.2 Lectins and Glycans of Other Pathogenic Bacteria

5.4.2.1 Legionella pneumophila, K. pneumoniae, S. pneumoniae,
and B. cepacia Complex
The PapG lectins specifically recognize the “Gal-based disaccharides” on the termi-
nal or distal ends of glycolipid oligosaccharides of renal cells [89, 91, 149, 150]. If
lectins are produced from Legionella pneumophila, K. pneumoniae, S. pneumoniae,
P. aeruginosa, and B. pertussis, the lectins specifically recognize the “GalNAc (beta
1-4) Gal” on the human respiratory tract [151–154]. Bacterial lectins can recognize
fucosyl human histo-blood group carbohydrates. Human fucosyl oligosaccharide-
recognizing soluble lectins are reported in P. aeruginosa and B. cepacia complex of
B. ambifaria strain and B. cenocepacia strain [154–156]. P. aeruginosa LecB,
named PA-IIL, is a tetrameric lectin structure with an affinity for L-Fuc and two
Ca2+ ions [157]. B. ambifaria BambL lectins consist of a trimeric structure through
β-propeller configuration with two Fuc-recognition sites in each monomer [82]. Both
lectins also have higher affinity toward the LeA antigenic epitope, and BambL has a
strong affinity for the epitope of H-type 2 antigen of AB(O)H blood group of
humans. From the above lectin specificity, the host-pathogen lectin-carbohydrate
interactions give some insights into the preventive infection if inhibitors are avail-
able. For example, an antiadhesive agents or adhesion-inhibitory agents can disturb
or block the GalNAc (beta 1,4) Gal interaction, giving potential anti-pathogen-
infection drugs.
5.4.2.2 Non-typeable H. influenzae and Acinetobacter baumannii
Non-typeable H. influenzae (NTHi) is a host-adapted pathogen present in the human

respiratory tract. The incidence of invasive NTHi infections is rather increasing due
to the vaccination against H. influenzae b type and S. pneumoniae. NTHi vaccine is
absent. NTHi expresses surface factors to adhere to host surfaces. For example,
major outer membrane proteins (OMPs) P1 bind to carcinoembryonic antigen
(CEA)-related CAM-1 (CEACAM-1) [83]. OMP P4 recognizes the ECM compo-
nents including fibronectin, laminin, and vitronectin [158]. H. influenzae surface-
adhesin protein E is also known to inhibit the binding activity. Acinetobacter
baumannii PilA glycoprotein binds to selectins and CEACAMs of surfaces of host
cells. Type IV pili bind to intercellular adhesion molecule 1 (ICAM-1) [159]. Fim-
briae bind to human mucins [160, 161]. Adhesins of HMW1 and HMW2 also bind to
carbohydrates. HMW1 binds to α2–3 sialyl-lactosamine, although the host receptor
for HMW2 is not known. However, simply, various host glycans can act as HMW2
receptors. The results obtained from glycan-binding HMW2 microarray and surface
plasmon resonance (SPR) techniques indicate that HMW2 recognizes the
NeuAcα2,6 linkage, not the non-human SA form, NeuGc [162].
5.4.2.3 H. pylori
In H. pylori infection, H. pylori has been suggested to evolve for the 30,000 years
with the humans as host in a way of coevolution. H. pylori has acquired from
the learning to adapt its antigenic structures such as human Lewis antigen to evade
the immune surveillance of hosts. In the human host, humans may also adapt to the
commensal symbiosis with the pathogen for its contribution to provide antibiotic and
probiotic-like components which are produced for regulatory control of H. pylori
[163]. H. pylori is classified to the human carcinogenic class 1. H. pylori recognizes
human blood group antigens (HBGAs) produced in O-glycan structures of mucous
surfaces of human epithelial cells, as a pathogen binding and infection site. Human
stomach tissues also express blood group antigen-binding adhesin (BabA). Recently,
another LabA lectin has been discovered, and the LabA binds to LacdiNAc structure
of GalNAcβ1, 4GlcNAc-carbohydrates. This LabA lectin recognizes a glycan
sequence, which is different from those of BabA and SabA [164]. LabA adhesins
also recognize LeB and LacdiNAc. This specificity leads to H. pylori colocalization
in the mucin MUC5AC in gastric epithelial surfaces, but not in MUC6. These
adhesins recognize HBGA-related glycans present in gastric mucosal epithelial
cells. HBGA-recognizing adhesin known as BabA and the SA-recognizing adhesin
known as SabA are also lectins produced by Helicobacter outer membrane protein
group attached to host cells. BabA binds to fucosyl-type 1 glycan structure of the
human AB(O)H blood group antigens and Lewis antigens expressed on glycolipids,
mucins, and glycoproteins expressed in the GITs [165]. BabA recognizes the
Fucα1–2 linked type 1 epitope of Gal1-3GlcNAc core in the O blood group,
which is the same structure as the H1 antigen in LeB antigen. If Fucα1–4 is linked to
the GlcNAc residue present in the type I sugar chain, the sugar antigenic type is
called the H1 antigen of ABO blood group. BabA binds to A/B type blood group
antigens with the terminated sugar of GalNAcα1,3 and Galα1,3 residues present in
the H1 epitope, respectively. They are Lewis b antigens [166]. Contrary to BabA, the
SabA binds to sialyl-dimeric LeX (sdiLeX) on glycolipids and GSLs as well as to the
monomer SLeX and the derivative SLeA forms [167]. Sialylglycans are not enriched
in normal and healthy stomach of humans [168, 169], but gastric inflammatory event
and malignantly transformed cells develop sialylated Lewis antigens [170–172] and
lost neutral blood group ABH antigens. Simultaneously, pathogens adapt SabA
adhesin [167]. Helicobacter exploits the receptors such as selectin mimicry through
the sialyl-(di)-LeX/A GSL recognition, because inflammatory intestinal and stomach
express sialyl Le glycans expressed in the leukocytic cells, which recognize
selectins. The other lectins of OipA, HopZ, and AlpA/B are known [163].
5.4.2.4 P. aeruginosa
In P. aeruginosa, the bacteria adhere to epithelial cells through cell-surfaced

adhesins such as LecA known as PA-IL and LecB known as PA-IIL as well as
type IV pilus known as T4P. They all recognize carbohydrate ligands present in
epithelial cell surfaces [173–175]. Among them, LecA and LecB also activate cell
functions of host [175, 176]. Soluble homotetrameric fucophilic lectin LecB forms
biofilm, bacterium/host, and bacterium/bacterium interaction potentiating its cyto-
toxicity and inhibition of ciliated removal [177, 178]. Four subunits of 11.7 kDa,
which is composed of 114 amino acids, are bridged with 2 calcium atoms resided in
the recognition sites. The two Ca2+ atoms chemically bind to the Fuc residue for a
monomeric ligand [179]. LecB binds to LewisA (LeA) 1 [180], 3-fucosyllactose
[180], SLeA [181], antigen H [182], LNFP-II, LNnFP-V, Lex [183], SLex [181], and
mannan [184]. LecB can bind to both monosaccharides of L-Fuc and D-Man. LecB
prefers to recognize Fucα1,4->Fucα1,3-> Fucα1,2-carbohydrates. From the Fuc
linkages, the nonreducing end-linked Fucα1,2 carbohydrate prevents binding of
LecB to Fucα1,3/4. Carbohydrate structure of Galβ1,3(Fucα1,4)GlcNAcβ1,3Gal-R
more potently recognizes the LecB, when compared to the carbohydrate structure of
the Fucα1,2Galβ1,3(Fucα1,4)GlcNAcβ1,3Gal-R. The LecB preferentially recog-
nizes the LeA antigens and 3-fucosyllactose (3FL) oligosaccharides [181]. The
co-crystal structure of complex LecB and LeA with the structure of Galβ1,3
(Fucα1,4)GlcNAc- suggests that component monosugars in Lea bind to the LecB
surface. 3FL oligosaccharide differs from Lex antigen due to loss of the N-acetyl
modification on the Glc residue. This difference induces an increased affinity
for LecB.
Lectin LecA is also a virulence factor in the adhesion, biofilm synthesis, and lung
injury [185, 186]. Gram-negative pathogenic P. aeruginosa in dermatitis, keratitis,
pancreatitis, urinary infections, and respiratory tract infections causes immunocom-
promised death. P. aeruginosa lectin LecA binds to glycosylated targets on the cell
surface [187]. Globotriaosylceramide Gb3 globoseries is indeed a major ligand of

LecA lectin and frequently found in epithelial cells of murine lungs and human
intestines [188, 189]. Gb3 can activate LecA attachment in a similar fashion of
cholera toxin subunit B (CTB), as a hetero-multivalent binding is also observed in
CTB [190]. Hetero-multivalent binding of LecA to Gb3 induces a LecA signaling to
trigger CrkII phosphorylation [191]. LecA lectin has a homotetrameric structure, and
each monomer carries each single carbohydrate-recognition site. LecA lectin carries
two pairs of the adjacent recognition site [192]. LecA preferentially binds to
α-Gal-terminated glycolipids. Typical one is globotriaosylceramide Gb3 globoseries
with the structure of Galα1,4Galβ1,4Glc-Cer as the LecA ligand [193–195]. LecA
lectin can also bind to other glycolipids of Galβ- and GalNAc-terminated glyco-
lipids, but with lower affinities than Gb3 [193, 196]. Therefore, inhibition of LecA
prevents adhesion of the bacteria. This concept [197] was expanded to the therapeu-
tic Gal solution usage against P. aeruginosa pneumonia in murine models and
human patients due to inhibition of the LecA binding to its glycosylated targets
[198]. Anti-infectious drugs include antiadhesive molecules in P. aeruginosa
through multivalent ligands of LecA and LecB. A series of 27 LecA-targeting
glycoclusters were synthesized. A low-nanomolar (Kd ¼ 19 nm) ligand with a
Tyr-based linker bridge was found in a study of the structure-activity relationship.
Molecular binding between the glycoclusters and the lectin tetramer was
elucidated [199].
5.4.2.5 N. gonorrhoeae, N. meningitidis, S. aureus, Chlamydia

pneumoniae and Vibrio parahaemolyticus
However, tissues frequently secrete glycan fluids to inhibit the lectins of bacterial
and viral pathogens and prevent pathogenic adhesion and recognition to cell mem-
brane glycans [200, 201]. Some representative secreted glycan is the adhesive
glycoprotein, human kidney uromodulin known as Tamm-Horsfall protein. The
uromodulin carries branched oligosaccharide glycans on lectin glycosyl moiety
expressed by some pathogens such as N. gonorrhoeae and S. aureus [202, 203].
Immunomodulatory molecules are associated with cardiovascular diseases,
including apolipoprotein-E (Apo-E), CRP, and Man-binding lectin (MBL)
[204, 205]. MBL as a circulatory and soluble C-type lectin functions in nonself-
versus self-recognition. The lectin MBL distinguishes and identifies dead cells,
dying cells, or cancer cells to capture and make clearance by phagocytic cells of
host [206]. MBL recognizes PAMPs to destroy pathogen [207]. MBL2 expression is
dysregulated in abnormal conditions such as coronary heart disease, and abnormal
function of MBL2 increases the host susceptibility to pathogenic infection
[208, 209]. In cardiovascular inflammatory pathogen Chlamydia pneumoniae
(CP), an obligate intracellular bacterium, it invades human endothelial cells
[210]. C. pneumoniae with circulating phagocytes induces plaque forming vascular
coronary heart disease. The serum MBL2 in human blood functions as a binding
receptor for Man and NAcGlc on the C. pneumoniae membrane. Detected MBL
contributes coronary disease severity in C. pneumoniae patients [211]. Alternative

splicing variants or different promoter-transcribed variants in the MBL2 gene are
expressed as altered coding missense SNPs, defecting blood MBL level-based
diseases [212]. An associate between C. pneumoniae and MBL2 is indicated within
the coronary disease cohort. C. pneumoniae-reactive antibody production in humans
is controlled by the transcription promoter variant XY as well as the allele number of
MBL2-A and MBL2-B. Therefore, human diseases are developed by the interaction
between the host immune cells and pathogen C. pneumoniae. The frequency of the
coding missense SNP alleles depends on population origin. MBL2 genotypes are
associated with coronary artery diseases [213], derived by the relationship between
MBL2 genotypes and the immune response to C. pneumoniae [214].
Meningococcal adhesins are known for pili (type IV fimbriae) and opacity
protein (Opc). Meningococcal pili as high molecular heteropolymeric glycoproteins
protrude the capsule and have a capacity to recognize host endothelial and epithelial
cells [215]. This crucial adhesin, pili, binds to the platelet activating factor receptor
of host cells via the pilin-linked glycans [216]. Nonencapsulated pili mutant cannot
recognize asialo-GM1 and GalNAcβ1–4 Gal. Therefore, meningococcal pili are
essential to bind to the host. Asialo-GM1 is abundant in regenerating epithelia and
a host receptor for meningococcal adhesion. In fact, certain pathogens present in the
respiratory tract bind to the GalNAcβ1–4 Gal epitope in asialo-GM1 or asialo-GM2
[215]. In P. aeruginosa, pili35 binds to the asialo-GM1. However, Vibrio
parahaemolyticus mannose-sensitive hemagglutinin (MSHA)-lacking pilus does
not bind to asialo-GM1 [217]. Additionally, the surface components of meningo-
coccal bacteria include capsule, LOS, Opc, and pili. Pathogenic meningococcal
bacteria bind to the GAGs for adherence and invasion. The nasopharyngeal epithe-
lial cells are enriched with mucus and mucins [214]. The CNS is also enriched with
gangliosides and chondroitin sulfate. Neisseria meningitidis strains consist of 13 cap-
sular serogroups. Six (A, B, C, W, X, Y) capsular serogroups cause diseases
[218]. N. meningitidis cause bacterial meningitis and sepsis with about 10%
mortality.
Meningococcal virulence factors such as Opc 13, 16, Opc 13, Opc 16, and NHBA
specifically bind to host glycans. The opacity protein, Opc, is integral and outer
membrane protein [219]. Opc 16 binds to sialylated monosaccharides. Opc recog-
nizes human ECM GAGs of the heparan sulfate and the fibronectin and vitronectin
of endothelial cells [220]. Neisseria heparin binding antigen (NHBA) binds to
heparin residues [221]. Meningococcal Opc attaches to the CNS cells during men-
ingitis. The main chondroitin-6-sulfate GAG expressed in the CNS is recognized by
Opc. In fact, Opc binds to chondroitin-6-sulfate structures and lacto-N-neotetraose
structures. Lacto-N-neotetraose is also known as a component of lacto-neo series
GSLs such as paragloboside. Lacto-N-neotetraose structure is used as the precursor
of the ABO and P1 blood group antigens. These GSLs and blood group antigens are
also expressed in neuronal and erythrocytes, respectively. The lacto-N-neotetraose
structure on nasopharyngeal epithelial cells is the binding site for Streptococcus
pneumoniae. Free lacto-N-neotetraose inhibits pneumococcal binding to human and
animal nasopharyngeal cells. Capsule-based vaccines can be produced by capsular
serogroups A/C/W/Y. Capsular B serogroup is used for outer membrane protein-

based vaccine [222]. N. meningitidis glycosyltransferase genes of lst, lgtB, lgtA, and
lgtE generate LOS species. The capsule and LOS modulate pathogen-host interac-
tions and virulence. Meningococcal LOS consists of two oligosaccharide chains
linked to heptose residues [223] with different types of L1–L12 [224]. LOS L3
immunotype binds to Thomsen-Friedenreich (TF) antigen. The N. gonorrhoeae LOS
binds to the ASGPR (asialoglycoprotein receptor) on epithelial cells.
5.4.3 Viral Lectins or Host Lectin-Binding Glycans
For viral infection receptors, many viruses produce lectins for their infection.
Currently, well-known virus SA-specific lectins are summarized (Table 5.10). In
the case of viruses, most pathogenic virus carries hemagglutinin (HA) lectins, and
the term of HA agglutinates red blood cells, as mainly studied on human cases.
Viruses have lectin genes to express them on their surfaces for cell targeting.
Influenza, noroviruses, and rotaviruses are studied for their lectins. Also, in dengue
viruses, they use a DC-SIGN, one of C-type lectin family, expressed on DCs for
entrance to the cells and replication [225].
In structural aspects, norovirus-encoded lectins bind to the carbohydrate receptors
of HBGA. The surfaced capsid proteins of norovirus function as a lectin-like
receptor. Norovirus is a non-enveloped ssRNA virus of the Caliciviridae family.
Noroviruses transmit person-to-person spread. The norovirus virion is composed of
the assembled structure with 180 capsid protein (VP1) copies. The capsid VPI is
dimerized to an icosahedron core [226]. The monomeric protein, capsid VP1,
consists of two different structures of domains named (i) shell domain (S) and
(ii) protruding (P) domain. P domain involves in lectin-like receptor binding with
diversity in strains. The norovirus capsid VP1 is a lectin which binds to sialyl
glycans, while human norovirus VP1 P domain binds to polymorphic HBGAs
[227–231]. Human noroviruses bind to their HBGA partners, giving their infections
and spreading. The noroviral interaction with HBGA is evolutionarily linked with
genetic traits. Fuc residue in Lewis antigens and histo-group antigens of humans is a
key recognition site for the lectin of the VP1 of GII norovirus strains. For the
fucosylation reaction of Fuc residues in the Lewis antigens and ABO blood group
antigens, the two known fucosyltransferases of FUT-2 and FUT-3 enzymes catalyze
the Fuc residue transfer. For therapeutic application, besides vaccine-based combat-
ing the virus, a strategy to prevent norovirus infection is to use human milk
oligosaccharides (HMOs). HMOs contain competing agents against virus-producing
carbohydrate receptors, which mimic the O-glycans of mucin type, which are
reactive to blood group antigens of humans. In the human HMOs, several trisaccha-
rides such as 3SL, 6SL, and 20 -fucosyllactose (2FL) are included in the glycolipid
forms or free oligosaccharide forms. Among them, 2FL prevents norovirus binding
and has gained market approval [163, 232, 233]. Oligofucoses in hepta- to
decasaccharides promote competitive effects on norovirus binding. L-fucose
Table 5.10 Virus SA-specific lectins. HA-A HA activity observed, HE HA esterase, HN HA neuraminidase
234

Orthomyxoviridae Influenza virus A Human HA Neu5Acα2,6Gal
Avian and Neu5Acα2,3Gal
equine
Porcine Neu5Acα2,3Gal, Neu5Acα2,6Gal
Influenza virus B and C HA and HE Neu5Acα2,6Gal and Neu5,9Ac2, respectively
Paramyxoviridae Newcastle disease virus HN GM3, GM2, GM1, GD1a, GD1b, GT1b, N-glycans
Sendai virus HN NeuAcα2,3Galβ1,3GalNac/4GlcNAc
Human parainfluenza virus type 1 and HN NeuAcα2,3Galβ1,4GlcNAc and NeuAc/Neu5Gcα2,3/
type 3 6Galβ1,4GlcNAc
Porcine rubula virus LPM HN Neu5Acα2,3Gal
Polyomaviridae Murine polyoma virus (large and small- VP1 Neu5Acα2,3Galβ1,3GalNA; Neu5Acα2,3Galβ1,3
plaque) [Neu5Acα2,6]GalNAc
Simian virus 40 GM1
Human polyoma virus JC and BK VP1 Siaα2,6 and Siaα2,3, respectively.
Coronaviridae Bovine coronavirus S protein, Neu5,9Ac2α2,3Gal Neu5,9Ac2α2,6Gal
HE
Human coronavirus OC43 S protein Neu5,9Ac2α2,6Gal Neu5,9Ac2α2,3Gal
Porcine haemagglutinating encephalo- HA-A Neu5,9Ac2
myelitis virus
Porcine transmissible gastroenteritis S protein Neu5Gcα2,3 Neu5Acα2,3
corona virus
Avian infectious bronchitits coronavirus HA-A Neu5Acα2,3
Murine hepatitis virus HE Neu4,5Ac2
Reoviridae Reovirus type 1 σ1 Siaα2,3
Porcine rotavirus group A OSU VP4 Neu5Gc-GM3 Neu5Ac-GM3
Human rotavirus KUN, MO VP4 GM1
5 Pathogen-Host Infection Via Glycan Recognition and Interaction
Rhesus rotavirus VP4 Neu5Ac > Neu5Gc
Bovine rotavirus NCDV VP4 Neu5Gc-GM3
Bluetongue virus VP4 Neu5Ac, Neu5Gc
Adenoviridae Adenovirus type 37 Fiber knob Siaα2,3
Picornaviridae Theiler’s murine encephalomyelitis virus VP2 Siaα2,3
Human enterovirus type 70 Siaα2,3
Parvoviridae Bovine parvovirus HA-A Neu5Acα2,3Gal
Adeno-associated virus serotype 4 and 5 HA-A Neu5Acα2,3Gal and Neu5Acα2,6Gal, respectively
Herpesviridae Murine and human cytomegalovirus Neu5Ac and Neu5Ac > Neu5Gc, respectively
Hepdnaviridae Hepatitis B virus Small S Neu5Ac
protein
5.4 Pathogen-Producing Lectins as Receptors to Bind to the Host Carbohydrates
235
dendrimers of α-L-fucose residues are natural polysaccharides of polyfucoses or

fucans. Fucoidans from brown algae exert beneficial effects [234]. In a blood group-
independent manner, anti-norovirus activities of αL-Fuc residue in fucoidan in algae
and desulfated form of Fuc residues present recognition of viral capsid proteins with
mucins present in human GIT [163].
SA-containing glycans of influenza virus HA. Hemagglutination allows easy
detection of influenza virus strains, as they agglutinate erythrocytes of special
avian birds and mammals [235]. For example, distinguishment between the H5N1
avian influenza virus and infectious H1N1 swine influenza virus strains is based on
the hemagglutinin experiment. H5N1 lectins specifically bind to the α2,3 sialyl-Gal
disaccharide on chicken erythrocyte membranes and airway epithelial cells of the
lower human respiratory tract. Lectins of the H1N1 swine influenza virus typically
bind to the α2,6 sialyl-Gal on pig erythrocyte membranes and airway epithelial cells,
which are lined in the upper human respiratory tracts [236].
5.5 Host Lectin Defense Mechanisms

in Lectin-Carbohydrate Interactions
Host defenses against pathogen infections depend on a systemic collaboration of

innate (nonspecific) and adaptive (specific) components. The organisms are facing
potentially life-threatening pathogenic infections by microbial pathogens. The sur-
vival game of the host depends on how they well recognize the pathogenic microbial
infections and how to lead to defense responses [237]. The immune response of
innate immune cells senses foreign invading agents to follow immune responses.
Innate defense is the major host defense line to restrict the pathogenic propagation in
host tissues prior to the transduction of adaptive immunity. Serum and tissues
contain a series of lectins to recognize and bind the pathogens. The host defense
response is initiated by innate immune sensors of danger signals, via pattern
recognition receptors (PRRs) since proposal in 2002. Microbial sensors including
CTL receptor (CTLR), complement system, TLR, the RIG-I-like helicase, and the
nucleotide-binding oligomerization domain-like receptor are all defensing receptors.
Ligand-activated PRR triggers to intracellular signaling events with the
co-stimulatory molecule expression and the innate immune responses, and eventu-
ally induces adaptive immunity of host. Proinflammatory or anti-inflammatory
reactions are well associated with the innate immune responses. Although four key
life molecules are nucleic acids, proteins, carbohydrates, and lipids, the different
subunits of sugars, amino acids, nucleotides, and fatty acids are linked to different
component, structure, and distribution. Diverse pathogens consist of diverse
PAMPs. The major PAMPS of pathogens include cell wall lipoproteins, lipoteichoic
acid, LPS, and peptidoglycans. β-Glucan and mannan are the fungal PAMPs. RNA
and DNA frequently mimicked by poly I:C or CpG are targeted by the immune
system to recognize virus [237].
5.5 Host Lectin Defense Mechanisms in Lectin-Carbohydrate Interactions 237
Carbohydrates (glycans) are utilized for antigen formation and innate immune
recognition [238]. The glycans are either secreted or located on the cell surfaces.
Therefore, they are the components of pathogen-host interaction and pathogen
infection as well as host innate responses [74]. Because recognition event is the
most basic step for the different hosts-pathogen interaction, glycans show diverse
nonself recognition molecules [239]. Carbohydrate recognition during host-microbe
interaction is much more elucidated by basis of genome information in hosts and
glycan diversity in pathogens. Carbohydrate-lectin interaction bonds are very weak,
compared to covalent chemical bonds. Thus, it seems that relatively high numbers of
lectins and carbohydrates are interacted together to give the desired physiological
cellular interactions. Bacterial cell coats thus present their lectins along with the
shafts of fimbriae, as surrounded like fur, to give clustered adhesive affinity and
force by multiple lectin-carbohydrate interactions on cell surfaces [240, 241]. Such
clustered with glycans are well described in the result that the FimH lectin-
expressing E. coli cells readily and strongly bind to mannose on glycoprotein film-
coated micelles [242]. Among the carbohydrate-binding molecules, a representative
member is a CLR of DC-SIGN present on the DC surfaces. The DC-SIGN binds to
various Man- and Fuc-carrying ligands in the envelope of HIV [243]. Other cases
include Siglecs, CD22, and BCR complex, which uptakes antigen and activates B
cells [244]. Additionally, the galectins associate with host glycans, to be organized
into receptor lattices [245, 246]. In microbes, their immunogens are mainly glycans,
where bacterial and virus coats are decorated with a sugar coat known as glycocalyx.
For example, gp120 protein of HIV coat protein is a representative glycoprotein.
Some bacteria are encapsulated with polysaccharides, glycolipids, or endotoxins.
Peptidoglycans of Gram-positive bacteria are also the representatively exposed
coated molecule. The PAMPs are such endotoxin LPS, bacterial capsules, muramyl
dipeptide, and viral coats. Therefore, pathogenic microbes have evolved to produce
host-similar glycans on their surfaces of cells to mimic the cell surfaces of hosts. In
addition, such glycans evade immune surveillance [247], indicating the carbohydrate
immunogen’ roles in the immune system.
As described above, host lectins are often used as receptors of pathogenic
infection agents. However, reversely, some host lectins are well designed to avoid
and prevent pathogenic infections. Surfaces of most microbes express mannose
residues, and some mammals often express mannose-binding lectins to perform
the innate immunity [248]. Reversely, however, some pathogens utilize such lectins
engaged in the innate immunity rather to invade host cells to cause severe fatal
infections. Such a well-known case is the glycans on the Ebola virus envelope, and
this lectin readily recognizes the CTL-like domain family 4, member g (Clec4g)
known as LSECtin present in endothelial cells of human lymph nodes and liver. The
glycan-LSECtin binding induces endocytosis-based infection and death of the host
cells [249, 250]. Also, a Man-binding lectin (MBL) binds to the Ebola virus
envelope glycoprotein glycan to block the interaction between the glycans and
other mannose-binding lectins expressed on host cell membrane [251]. Thus, high
doses of MBL can protect mice infected with the Ebola virus [252]. Such inhibitory
agents of host-pathogen lectin-carbohydrate interactions can be served as anti-

infectious agents in humans.
5.6 Pathogenic Glycans to Trigger Innate Immune

Enhancement
In infection, microbial pathogens of bacteria or fungi cause pathogen-borne diseases

by glycan recognition and intracellular signaling events as well as pathogenic
surface expression of mammalian-associated glycans to escape from host’s immune
system. Host defense system against pathogens can be improved by immune-
enhancing polysaccharides through regulating the levels of cytokines, macrophage,
and lymphocyte. Macrophages play important roles especially in innate immune
system such as microbial infection, producing inflammatory mediators, cytokines,
and phagocytic activities. The mediators of NO, ROS, IL-1β, and TNF-α are
increased upon stimulation of macrophages with treatment of LPS. Various host
defense functions of activated macrophages are mediated by NO that leads to
signaling molecule, and inflammatory mediator mainly regulates immune responses.
To trigger cellular signaling response, many external stimuli bind to PRRs which
recognizes surface of microorganisms and induce secretion of cytokines or TLRs on
surface of DCs or macrophage and then trigger various signaling pathways.
With T lymphocytes, NK cells in innate immunity are also important for tumor
cells and infectious pathogens by binding to MHC molecule not binding to a specific
antigen. Cytotoxic NK cells release IFN-γ cytokine and are the major IFN-γ pro-
ducer in humans. Deficiency in IFN-γ production of NK cells can cause malignancy
and infection. IFN-γ exhibits anti-infection activity through tumor surveillance and
induces tumor apoptosis. Moreover, NK cell-generated IFN-γ promotes macro-
phage, DC, and T-cell activation during inflammation and malignant diseases. NK
cells exhibit spontaneous cytotoxicity against transformed cells. NK cells are also
upregulated by IFN species or upregulated in an indirect manner by IFN-inducing
agents of bacterial immunogens, mitogens, and viral components. IFN-γ-induced
NK cell activation and resulting killing activity are crucial for cancer immunother-
apy of NK cells. IFN-γ expression is regulated by MAPK and NF-κB pathway,
inducing phosphorylation of p38, ERK, and c-Jun NH2-teminal kinase (JNK).
NF-κB activates IFN-γ transcription.
Glycans or polysaccharides as natural macromolecules are glycosidic bonded
carbohydrates or covalently bonded glycoproteins or glycolipids existed in
microbes, plants, and animals. Polysaccharides can regulate antimicrobial, antioxi-
dant, antiviral, or antitumor activity. Glycans of plants and bacteria have also
immune-enhancing activity among diverse immune responses, as activate innate
immune cells specifically. The roles of glycans are recently revisited for the func-
tional diversity and specificity of glycan-recognizing components of the innate
immune system. In mammals, glycans have diverse functions including apoptotic
5.6 Pathogenic Glycans to Trigger Innate Immune Enhancement 239
induction for mutant cells, clearance, self/nonself-discrimination, cell-cell com-

munication, and cell signaling. Glycosylation in humans is frequently linked to
inflammation, cellular stress, aging, or cancer, as the importance of glycans is in
recognition and interaction.
The immunological role of glycans observed in various bacteria and fungi has
been defined. Considered function of polysaccharides in immune system, they seem
mainly to function in activation of innate immune cell and increasing levels of
cytokine, but minor effect of humoral immunity. Because innate and humoral
immunities are cooperative, the effect of polysaccharide in innate immunity is
regarded as the major point. Among many innate immune cells, polysaccharides
activate commonly macrophages or DCs. The first defense line of hosts in patho-
genic infections and cancer is macrophage or DC, as they release inflammatory
mediators and cytokines upon stimulation with LPS and various polysaccharides.
They are IL-6, TNF-α, and IL-1α, which directly elevate the tumoricidal activity of
B- and T-cell proliferation as well as macrophage growth.
Additionally, from the studies on T-cell-independent antigen, many pathogenic
polysaccharides regulate TLRs expressed in macrophage or DC surface and recog-
nize pathogen surface patterns, following signaling pathway-activated MAPK and
transcription factor NF-κB. For example, the pneumococcal C-polysaccharide and
capsular polysaccharides activate the immune cells and cytokine release upon
stimulation of whole blood. Streptococcus pneumonia is mainly causative of bacte-
rial pneumonia, meningitis, and sepsis, causing a pneumococcal invasion disease in
humans including adults and children. Bacterial Gram-positive S. pneumoniae strain
expresses capsular polysaccharides on the bacterial surface with each specific
serotype. In all serotypes, the cell surface wall polysaccharides are structurally
similar as a common marker for the species. Because of current lack of effective
pneumococcal vaccines and antibiotic resistance, treatment of the pneumococcal
disease is dependent on the vaccination. The polysaccharides of S. pneumoniae
stimulate class switch promoting T cells and non-T cells like NK cells and mono-
cytes. To date, two different vaccines are designed using the polysaccharides.
Immunoglobulin class switching from IgM to IgG takes place in response to the
unconjugated polysaccharide vaccine due to T-cell-independent (type 2 thymus-
independent) antigen. Some immune-related cells such as CD4, CD4+, NK-like T
cells, NK-like T cells, and monocytes are activated by CPSs. These cells protect
against pneumococcal infection via B-cell function of antibody production and
induction of immunoglobulin class switching, which is a phenomenon in the
T-cell-independent antigens such as capsular polysaccharides. One vaccine type
contains only the polysaccharides, while the other type contains proteins plus poly-
saccharides. As the polysaccharides are T-cell-independent antigens, the polysac-
charides directly stimulate B-cell responses to produce antibodies. However, as
proteins are T-cell-dependent antigen, the protein-conjugated capsular polysaccha-
rides stimulate T-cell response via MHC presentation of protein antigen with
memory B/T cells and antibodies. Although such vaccination to pneumonia is
available, there is still concern that the capsular polysaccharide and other poly-
saccharides may associate with infection of host cells and recognize distinct
immune-related cells. The C-polysaccharide and capsular polysaccharide modulate

individual immune cell functions and cytokine expression.
On the other hand, some polysaccharides give immune-enhancing activity. For
example, the polysaccharides from Cyrtomium macrophyllum (family of plant) or
from Paecilomyces cicadae (family of Eumycetes) enhance immune responses
through TLR4 activation of macrophage. Anoectochilus formosanus (family of
plant like orchid) type II AGAF is a potent immunomodulator, having the signaling
actions of immunomodulatory activity, upregulating NK cell-mediated cytotoxicity
via expression of IFN-γ to stimulate the innate immune system. NK cell-mediated
cytotoxic activity is enhanced by type II arabinogalactan glycans produced by
A. formosanus. Thus, different glycans exhibit common activation of innate immune
cells including macrophage, NK cell, NK-like cell, or CD+4 cells. From the current
glycan-immune cell interaction, the basic question is raised: how glycans activate
innate immune cells, if they have distinct mechanism including signaling pathways
associated with glycan activation?
5.6.1 Example 1: Polysaccharides with Immune

Enhancement of Cyrtomium macrophyllum
Cyrtomium macrophyllum is a family of plant, and the rhizomes of Cyrtomium

contain pharmacological anti-parasite, antibacterial, antiviral, and anticancer capac-
ities, giving an immune-enhancing activity. The glycans activate TLR4 of innate
immune cells such as macrophage.
5.6.2 Example 2: Activation of Macrophage by

Polysaccharide from Paecilomyces cicadae
Polysaccharides isolated from plant Paecilomyces cicadae (PCP) have an immuno-

modulatory property on the macrophage [253]. PCO increases in interferon-γ pro-
duction by Peyer’s patch cells, phagocytosis by macrophage, spleen cell
proliferation, and dendritic cell maturation with immune-stimulatory activity on
macrophage and dendritic cell maturation. Another example, Cordyceps cicadae is
a parasitic fungus and functions for curing of malaria, enhancing blood aggregation,
and antitumor activity. PCP induced macrophage activation through TLR4 pathway.
Polysaccharide cannot penetrate cells, due to their large molecular mass, so the first
step in the modulation of cellular events is binding to receptors. The role of TLR4 as
the PCP receptor was confirmed in macrophages in C3H/HeJ mice and NO produc-
tion and cytokine gene expression. Because the activity of PCP is similar to LPS,
differences between LPS and PCP were also observed. Aside from activation
macrophage and TLR4 receptor, signaling pathways including MAPKs and
5.6 Pathogenic Glycans to Trigger Innate Immune Enhancement 241
NF-κB were activated by binding of TLR4 with PCP. Activation of MAPKs is

significant in the induction of NO as it controls the NF-κB activation. Here, it has
been confirmed that NF-κB and p38 are important for NO production by
macrophages. PCP firstly bound to membrane receptors including TLR4, and
activated MAPK and NF-κB signaling, finally increased the production of NO and
various cytokines IL-6, IL-1β, and TNF-α.
5.6.3 Example 3: NK Cell-Mediated Cytotoxicity Increased by

Arabinogalactan from Anoectochilus formosanus
Anoectochilus formosanus is an Orchidaceae plant having anti-asthma,

hepatoprotective, antitumor, and immunomodulatory effects [254]. A. formosanus
polysaccharide is characterized by a type II arabinogalactan (AGAF) and has a MW
of 29 kDa. The AGAF has an immunomodulatory effect against colon cancer in
mice. AGAF promotes splenocyte cytotoxicity and elevates the relative cell number
of CD4+ T cell and CD8+ CTLs, giving antitumor potentials because NK cells and
CTLs are critical in anticancer activity. The type II arabinogalactan AGAF has the
increasing activity of NK cell-mediated cytotoxicity. NK cells also activate the
innate immune responses, although the cells can directly kill pathogenic invaders
and tumor cells. Tumor cells and virus-infected cells are easily controlled by NK
cells through the proinflammatory cytokines of IFN-γ and TNF-α and also the
released granules with cytotoxic activity. For signaling pathway associated with
IFN-γ, the glycans act as TLR ligands of the innate immune cells and NK cells.
These molecules activate NF-κB. The NK cell-mediated cytotoxic activity is regu-
lated by cytokines of IFN-γ/TNF-α and perforin. However, only IFN-γ regulates the
NK cell cytotoxic activity enhanced by AGAF. Also, the intracellular signaling
through MAPK activates phosphorylation of transcription factors during AGAF-
elicited IFN-γ gene expression. For example, AGAF induces the NK-92MI cells to
express the IFN-γ via the NF-κB. AGAF-elicited IFN-γ expression is correlated with
the increased cytotoxic activity, which is associated with JAK2/STAT3 signaling of
the NK cells. Therefore, for the mechanistic explanation of the AGAF-activated NK
cell function, multiple pathway for the IFN-γ expression and autocrine cytotoxic
activity is suggested because the cytokine IFN-γ is an acting factor for the NK cell
cytotoxic activity. The polysaccharides have also high affinity for TLR2 and TLR4.
Polysaccharides activate macrophages via the TLR2 and TLR4 signalings. Silencing
of the TLR genes using siRNA knockdown technology demonstrates that both
AGAG upregulate the TLR2 and TLR4 signalings to enhance IFN-γ release and
NK cell cytotoxic activity. When the PRRs present in the cell surfaces are stimu-
lated, the MAPK subfamilies such as the ERK, p38, and JNK are activated by innate
immune cells. AGAF activates the phosphorylation of IKK and Iκα/β with NF-κB
translocation into the nucleus. Moreover, IFN-γ triggers AGAF-elicited NK cell
cytotoxic activity via the JAK2/STAT3 pathway. In the downstream pathway of
IFN-γ during the NK cell cytotoxic activity, another pathway of JAK2/STAT3 is

closely involved in the signaling cascade.
5.6.4 Example 4: Streptococcus pneumonia Polysaccharides

Activate NK Cells, NK-Like T Cells, and Monocytes
Streptococcus pneumonia glycans activate leukocytes, T lymphocytes, and NK cells,

as CWPS (cell wall polysaccharide) and three capsular polysaccharides (types 3, 9,
23) [255]. After activation with CWPS treatments, the CD69 values were observed
for monocytes, NK cells, CD4- (negative), CD4+, and CD56+ T cells. CWPS is a
stronger stimulator than the capsule. Among the capsules, type 23 is the strongest
stimulator followed by type 9 and type 3. With immune cell activation, cytokine
secretion is also increased by activated innate immune cell. CWPS stimulation was
observed for IL-8 production and followed by TNF-α, IFN-γ, and IL-10. CWPS is
also known as teichoic acid, structurally similar (especially in S. pneumoniae) to
lipoteichoic acid and a well-known ligand for TLR2. Also, TLRs as innate immune
components bind to the conserved molecular patterns or PAMPS on various path-
ogens. So, TLR was speculated as the main component of downstream mechanism.
Capsular polysaccharides are T-cell-independent B-cell antigens, and their capacity
to induce protective antibody responses is the main reason they are used for
vaccination. It is less clear how the capsular polysaccharides may stimulate the
immune cells investigated in this study, since they were not ligands for TLRs. It is
known that even highly purified pneumococcal capsular polysaccharides contain
some amount of CWPS. Thus, it seems that CWPS is an active component to
activate innate immune cells. Activation of cells could only be stimulated by direct
TLR contact. NK cells, NK-like T cells, and monocytes express TLRs. However,
ordinary (CD56-) CD4- (negative) or CD4+ T cells do not produce TLRs. An
indirect activation such as cytokines from directly activated cells may explain the
stimulation of T cells which is not expressing TLRs. During vaccination or a live
S. pneumoniae infection, TLR-expressing immune cells like macrophages and DCs
are present in the peripheral tissues but not in peripheral blood, and they are likely to
be activated. In many situations, a combined activation with direct and indirect (via
cell-cell contact or secreted mediators) mediators of immune cells occurs.
5.6.5 Example 5: C. macrophyllum Polysaccharides (CMP)

Enhance Lymphocyte Proliferation and Macrophage
Function
In Cyrtomium macrophyllum, T lymphocyte differentiation is stimulated by poly-

saccharide. In details, the percentages of CD4+ T/CD8+ CTLs and the CD4+/CD8+
5.7 TLR4 Receptor-Activating Glycans Activate NO Production in Macrophage 243
CTL ratio were remarkably increased in the groups treated with CMP [256]. With
the increasing of phagocytic activity in macrophage, the production of cytokine was
also increased. In detail, the production of IL-2 and IL-6 was significantly higher
than control group. Similarly, the concentration of IL-2 was also significantly higher
than control group. Surprisingly, not only cytokines but also immunoglobulin levels
are also increased in CMP-treated groups in experiment. In detail, mice treated with
CMP-L, CMP-M, and CMP-H groups were observed with significant enhancements
in IgM and IgG levels. Moreover, the NO production in RAW 264.7 macrophages
was increased by CMP. Nitrite secretion was significantly increased by CMP
treatment. In addition, CMP-treated-RAW 264.7 cells significantly secreted more
TNF-α. Thus, CMP enhances the NO production and cytokine secretion as well as
the iNOS protein expression from RAW 264.7 cells with NF-κB (in nuclear) protein
expression. iNOS and NF-κB proteins produced by RAW 264.7 cell were signifi-
cantly upregulated by LPS. In other words, CMP and LPS are thought to stimulate
production of cytokines and immunoglobulin via NO which is a critical component
of signaling pathway. In immune-enhancing activity of CMP in mice as well as
RAW264.7 cells, CY can act as a chemotherapeutic drug for cancers, but long-term
administration can cause immune suppression. The effects of immune suppression
on the development of immune organs such as the spleen and thymus could be
resisted by CMP. Another result, recovery of splenocyte-proliferative responses to
both T and B lymphocytes was promoted with CMP in mice. Aside from enhancing
the proliferation of immune cells, the IL-2 and IL-6 secretion by Th1/Th2 cells was
also stimulated by CMP. IL-2 mediates cellular immunity by promoting proliferation
and differentiation of T cell. IL-6 mediates humoral immunity by B-cell growth and
Ig production. Especially, IgG subtype and IgM subtype are the major Igs, which
activates the complement system, antigen opsonization, and toxin neutralization of
toxins as major responses in innate immune system. The levels of IgG and IgM in
CY-treated mice were increased by CMP. Humoral immunity as well as innate
immunity could be enhanced by CMP. Moreover, production of NO which contrib-
utes to killing of infected cells, tumor cells, and some pathogens and production of
cytokines were induced by activated macrophage.
5.7 TLR4 Receptor-Activating Glycans Activate NO

Production in Macrophage
In Paecilomyces cicadae, PCP (Paecilomyces cicadae polysaccharide) can activate

macrophage. Upon exposure to PCP, the NO production was increased by macro-
phages. Gene expression of iNOS was increased by PCP. The gene expression of
IL-16, TNF-α, and IL-1β, which are inflammatory cytokines, was also increased by
PCP in a similar pattern with iNOS. So how PCP can be recognized by macrophages
despite large molecule size? Because PCP cannot penetrate cells due to its large
molecular size, PCP-activated macrophage is followed by PCP binding to specific
membrane receptors. Among the specific membrane receptors, TLR4 activates

macrophages. For the TLR4 downstream signaling pathway, TLR4 signaling is
associated with the MyD88- and TRIF, but they commonly activate MAPKs and
NF-κB signaling. Both MAPKs and NF-κB signaling play major roles in macro-
phage activation in a TLR4-dependent mechanism. The phosphorylation of ERK,
JNK, and p38 which are associated with MAPK signaling was induced by PCP. PCP
can help the nuclear translocation of NF-κBp50/p65 by degradation IκB α/β block
protein, as it is demonstrated by the increased levels of nuclear p50/p65 protein. PCP
binds to TLR4, followed by the activation of p38 and NF-κB.
5.8 CBPs or GBPs in Antigen Recognition
GBPs expressed on the immune cells regulate immune responses of both innate and
adaptive immunities. The families of CLRs, galectins, TLRs, and Siglecs are well
known for the GBPs. GBPs contain one or more CRD. CLRs as a group of PRRs
influence TLR signaling. CLRs are Ca2+ dependent for their carbohydrate bindings,
capturing the carbohydrate ligand and a Ca2+ ion. Macrophages, DCs, and other
APCs of innate immunity express the CLTs on their surfaces. The CLRs play a role
to deliver immune tolerogenic signals upon antigen recognition, but the mechanisms
are not largely known yet. As Ca2+-dependent glycan-binding proteins, they contain
two distinct motifs for glycan recognition and Ca2+ ion engagement. The human
DC-SIGN (CD209) is present in DCs and macrophages [257, 258]. CLRs contain a
Glu-Pro-Asn (EPN) sequence as carbohydrate recognition domains (CRDs), for
Man- or Fuc-carrying glycans such as Lea,b,x,y carbohydrate antigens [259]. The
CLRs also include the DC-SIGN, mannose receptor (MR), and langerin. Among
CLRs, there is another type of Gal-specific CLRs, and these include macrophage Gal
lectin (MGL). L-SIGN/DCSIGNR is included with terminal Man- and/or Fuc
glycan-binding specificity [260]. However, Gal-binding C-type lectins include
MGL and DCASGPR [261]. They have the three amino acid sequence of Gln-Pro-
Asp (QPD) present in the CRD region, with terminal Gal residue or GalNAc residue-
containing glycan-binding specificity. Certain CLRs such as Dectin-1 do not require
Ca2+ for glycan recognition. Dectin-1 binds to β-glucan sugars present in yeasts. For
the type II subfamily of CLRs, 17 human subfamily is present in APCs of macro-
phages and DCs and also other endothelial or NK cells [262]. Most C-type lectin
family contains cytoplasmic endocytosis motifs for internalization in the tail region
and uptake glycosylated antigens [263]. CLRs also function in antigen presentation
to the MHC-II for CD4+ T-cell activation specific for antigens [264]. Certain DC
C-type lectins intracellularly shuttle foreign antigens to the MHC-I receptor to
trigger antigen-specific responses of CD8+ T cells. CLRs consist of both inhibition
and activation motifs in the cytoplasmic tail [260, 265]. CLRs regulate processing
and presentation of antigens for various immune reactions by T cells.
Apart from humans, mice also express C-type lectins of DC-SIGN homologues
such as mDC-SIGN, SIGNR1, and SIGNR3 [266]. The mouse SIGN-R1 (CD209b)
References 245
is the homologue of human DC-SIGN known as CD209 [267, 268]. Among several
different homologues, three mouse type DC-SIGN species of the SIGN-R1
(CD209b), SIGN-R3 (CD209d), and DC-SIGN (CD209a) are present in DCs and
macrophages of mouse [269, 270]. However, the three mouse DC-SIGN species lack
glycan recognition sites. Mouse SIGN-R1 species resembles DC-SIGN species of
humans, and mouse SIGN-R1 is present in marginal macrophages of spleens and
macrophages of lymphatic node medulla. Mouse SIGN-R1 species captures various
microbial organisms, which have polysaccharides like CPSs or dextran of
S. pneumoniae [271, 272]. SIGN-R1 involves in clearance of infected pathogens
in host. Defected SIGN-R1 lacks macrophage phagocytosis ability against patho-
gens such as S. pneumoniae. In addition, the SIGN-R1 directly helps CPS
opsonization by C3. SIGN-R1 defection blocks complement-mediated CPS capture
on splenic follicles, resulting in prevalent pneumococcal infection to mice
[273, 274]. Moreover, mouse SIGN-R1 recognition with CPS activates the classical
complement pathway which is Ig-independent for the C3 opsonization on CPS.
SIGN-R1 also removes the apoptotic cells by C1q-SIGN-R1 interaction via the C3
opsonization on the apoptotic target cells, resulting in reduction of autoimmunity
[275]. SIGN-R1 has a specificity to recognize α2,6 sialyl-antibodies, but not α2,3
sialyl-antibodies. SIGN-R1 binds to terminal α2,6 SAs through the carboxylate
moiety. α2,6 SA-bearing IgG is thus recognized by SIGN-R1. Thus, SIGN-R1 is
anti-inflammatory against intravenous Ig (IVIG) engagement. SIGN-R1 expressed
in macrophages resident in splenic marginal zones binds to the sialyl-Fc. Defected
for of SIGN-R1 species loss its anti-inflammatory response of intravenous sialyl-Fc
and IVIG [276]. The SIGN-R1 binding to sialylated Fcs elicits the anti-inflammatory
response by a Th2 cytokine pathway [277], because the IVIG has anti-inflammatory
activity through the IgG Fc-α2,6-SA [278]. The SIGN-R1 CRD binds to dextran
sulfate (DexS) and NeuAc [279]. The sialylated Fc-SIGN-R1 binding is based on the
recognition of α2,6-SA, eliciting anti-inflammatory response of IVIG. The sialylated
C1q and Ig bind to SIGN-R1 in a Ca2+-dependent CRD manner. The SIGN-R1
captures C1r-bound C1q and C1s-bound C1q by a SIGN-R1 CRD and the α2,6 sialyl
C1q, while SIGN-R1 recognizes the pathogen patterns through another
carbohydrate-binding site, as the SIGN-R1 in a Ca2+-independent way
[279]. Mouse SIGN-R3 binds only distinct ligands [280].
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Chapter 6
Innate Immunity Via Glycan-Binding
Lectin Receptors
Recognition of glycans and transfer of information contained in the glycan structures

are performed by carbohydrate-recognizing proteins of lectins or GAG-recognizing
proteins. Lectins bind to N-glycans, O-glycans, and GSLs, while GAG-binding
proteins easily bind sulfated GAGs. Lectin receptors are innate immune receptors
and include Siglec, C-type lectin, galectin, DC-SIGN, and TLRs in DCs during
pathogenic infection and immune tolerogenic homeostasis. The CTL roles are to
directly recognize invaders of microbes and also contribute to opsonic effect via
activation of complement pathways. Innate immune cells survey their habitat to
recognize pathogens by means of PRRs, where PRRs selectively bind PAMPs.
PAMPs are heterogeneous and homogeneous mannose oligomers and polymers,
β-glucans, and chitins in the fungi surface as well as carbohydrate moieties including
GlcNAc derivatives in bacteria. Innate immunity system represents our first host
defense line where the innate pattern recognition receptors/molecules (PRRs/PRMs)
encounter, recognize, and bind conserved motifs of microbial invaders or PAMPs.
Therefore, the PRRs/PRMs function as the initiator of innate immunity to microbial
invaders. Upon interaction with their invading pattern molecules as ligands, PRRs/
PRMs enter into signal transduction pathways and activate diverse downstream
kinases and transcription factors to lead to inflammatory response and immune
responses, depending on defense circumstances. For example, LPS, CpG DNA,
dsRNA, ssRNA, rRNA, or pathogenic surface glycans bind TLR4, TLR9, TLR3,
TLR7/TLR8, or TLR13 to elicit expression of cytokines of type I IFN/TNF-α/IL-6,
which are inflammatory.
Lectin is defined as proteins that recognize and bind glycan carbohydrates, and
the lectin-carbohydrate ligand binding potentiates various biological processes in
internalization and intercellular signaling. Lectins as carbohydrate-binding proteins
are specific for their binding ligands of sugar moieties and thus specifically recognize
and bind with cellular and molecular signaling properties. In intracellular roles, they
act as ER-Golgi key regulators of protein glycosylation and maturation. Protein
glycosylation-based interaction of calreticulin and calectin are ER-specific. Among
many lectin families, Siglec, C-type lectin DC-SIGN, galectin, and TLR are
262 6 Innate Immunity Via Glycan-Binding Lectin Receptors
representatively studied in intercellular signaling of phagocytosis and inflammatory

process of innate immune cells of myelocytic cells. To date, lectin receptors
expressed on DCs include Siglecs, dectin-1/dectin-2, MICL, Mincle/MCL,
mannose-binding protein (MBP), macrophage mannose receptor, and collectin.
6.1 Glycosylation Effect on Autoimmunity

and Inflammation
6.1.1 Glycosylation in Immunological Recognition

and Inflammation
Glycans are involved in multiple biological events and regulate the immune
responses. The diversity of glycans and lectins is also regulated by the immune
cells, as well described for DC functions. The changes of DCs’ glycan phenotype are
observed during differentiation and maturation. The cell surface glycosylation and
lectin-glycan interaction regulate the immune tolerance and homeostasis. Lectin-
glycan interactions also contribute to immune regulation in mammalian system and
activate tolerogenic system towards autoimmunity or antitumor immunity depending
on the downstream signaling. In the mammal system, immune cells are kept for their
authenticities through the whole organism. Hence, self-reactive T cells or auto-
reactive immune T cells are strictly controlled to be eliminated via apoptotic events
in the thymus. In autoimmune diseases, such thymus works are incomplete. To
compensate the undesired autoreactiveness, peripheral regulatory system is opera-
tive, and the system dampens the undesired and harmful reactions. Globally, such
system is suggested to be homeostasis. Overreaction or undesired responses in
immunity should be regulated by immunosuppressive cytokines or inhibitory recep-
tors, totally operative in tolerance system. The tolerance system includes T cell
anergy, deletion, and Treg cell expansion. These behaviors eventually prevent tissue
damages. In cellular level, regulatory system includes trafficking, clustering, and
signaling receptor internalization to control the immune cells [1]. Eukaryotic cells,
protein receptors, and immune mediators including MHC-I and MHC-II, TCR and
BCR, chemokine and cytokine receptors, and antibodies are normally glycosylated
by covalent linkages. This is because glycans are involved in major biological events
in almost every biological process. They are directly linked with a number of
inflammatory conditions [2], autoimmune diseases, and hematological cancers
[3, 4], as glycans seem to be associated with almost every human disease [5].
The discrimination of “self” from “non-self” is the essential driving force and the
most important aspect to defend the hosts in organisms. From the glycome diversity
and the glycan-biosynthetic immunological, glycosylation is directly associated with
immune cell networks. Glycan-related genes are phylogenetically conserved to
consist of the glycosylation system. However, variations at the intra- and interspe-
cies levels are observed among synthesized glycans from each organism. This
6.1 Glycosylation Effect on Autoimmunity and Inflammation 263
reflects that the glycans can act as danger-associated molecular patterns (DAMPs)
and PAMPs, as a key parameter of non-self- and self-discrimination in immunity.
The lectin-glycan recognition events contribute to pathogen-based immune cell
trafficking [6]. The lectin recognition of its ligand is indeed a multidirectional action
in detection of the structure, number, and glycan density in multivalent carbohy-
drates expressed in the cell surfaces. Resultant power is expressed as the type of
lectin-glycan interactions [7]. The molecular interactions form multi-dimensional
adjustments of glycans and lectins, called “lattices.” Lectins recognize glycans with
specific affinity in the very low molar levels. In immune system, lectin-interacting
surface glycoproteins include the TCR, BCR, and specific cytokine receptors. GBPs
or lectins can enhance or disrupt immune tolerance in DCs, T cells, or B cells,
because the roles of glycosylation in pathogen recognition and lectin-glycan inter-
actions are reality in regulation of immune tolerance, autoimmunity, and inflamma-
tion. In addition, glycosylation regulates inflammation and autoimmunity by
immune homeostasis. Cell surface glycans are changed from normal to inflammation
and transformation status [8]. In the inflammation status, the environmental inflam-
matory changes influence migration and trafficking of innate immune cells to disease
sites. The well-defined example is selectins as the recognition lectin receptors for the
immune cell migration, because they bind to sialyl- and fucosyl-glycans of sLex and
sulphated Le antigens on leukocytes and endothelial cells [9]. Thus, to bloc inflam-
matory response in anti-inflammatory therapy, interaction of selectins and Lewis
glycans on leukocytes and endothelia can be inhibited.
6.1.2 Glycosylation Effect on Autoimmunity
On the other hand, in autoimmunity status, T cells express altered surfaced glycans
such as terminally GalNAcylated or Galβ1,4GlcNAcylated structures, exhibiting
characteristic of desialylated residues that lack terminal sialic acid residues. For
example, terminally desialylated glycans are frequently observed in systemic lupus
erythematosus (SLE) and RA [10]. Such altered glycosylation seems to influence the
T cell immunological action in terms of TCR synapse behavior, and T cells regulate
the adaptive immunity and autoimmune responses. The desialylated glycans are
easily targeted by several lectins such as MGL and galectin. The MGL and galectins
specifically recognize such glycan structures of terminal GalNAc and
Galβ1,4GlcNAc structures without sialic acids, and consequently, TCR downstream
signaling is suppressed, and CD45 phosphatase activity is changed [11–13]. Specif-
ically, the GnT-5, mannose β1,6GlcNAcTransferase-5 (or Mgat5), yields the
β1,6GlcNAc branches structure on glycoproteins such as TCR. GnT-5 (or Mgat5)
adds the β1,6-GlcNAc residue to mannose residue of N-glycan core structures in
the Golgi apparatus. β1,6-GlcNAcylation in N-glycan by GnT-5 or Mgat5 yields the
galectin-binding ligands on surface glycoproteins. Galectins use the
N-acetyllactosamine (LacNAc) repeats as ligand substrates. The galectin-glycan
lattice alters surface glycan concentration and consequently affects cell proliferation
and differentiation. For example, GnT-5-KO T cells exhibit the reduced T cell
activation level, and GnT-5-KO mice show the increased delayed-type hypersensi-
tivity and susceptible autoimmune responses. Mgat5-deficient experimental model
animals such as mice exhibit severely type 4, delayed-type hypersensitivity and
autoimmunity such as autoimmune encephalomyelitis (EAE). The mice exhibit
EAE, glomerulonephritis, and immune complex deposition [12]. GlcNAc treatment
increased in GnT-5-mediated N-glycan β1,6GlcNAc branches and blocked activa-
tion of TCR in autoimmune EAE and T2DM models [14]. For more evidenced
results, genetically controlled, EAE-susceptible mice exhibit reduced N-glycan
branching in T cells when compared with EAE-resistant mice such as BALB/c
mouse [15].
Glycosylation also influences both the adaptive immunity and the autoimmune
diseases via innate immune to autoimmune and inflammatory development. Silenc-
ing ER-resident α-mannosidase-II (αM-II) impairs N-glycan branching and induces
an autoimmune disease in αM-II KO mice like human SLE [16], independent of the
adaptive immunity, but dependent of innate immune activation. Endogenous lectins
such as mannose receptor (MR) may recognize the mannose-deficient N-glycans and
induce innate immune responses even in the condition without infection, developing
lupus-like autoimmune disease [16].
Aside from N-glycans in the onset of autoimmune response, O-glycan changes
also lead to such similar autoimmune responses. For example, IgA nephropathy is
well known to related with the exposed GlcNAc residue linked to O-glycans in IgA
because IgA complexes are often deposited in the inflamed kidney glomerular
nephritis [17]. Human monomeric IgA1 bears two distinct sites of N-glycosylation
in the CH2 domain and C-terminal tail piece of the α-heavy chain. IgA1 contains an
extended hinge region with the nine different sites of O-glycosylation in the constant
domains. IgA1 produced by the healthy individuals has six sites of the nine O-glycan
sites which have mono- or di-sialylated core 1 O-glycan structures. Specific types of
Gal-deficient O-glycans are also detected in disease status [18]. The galactose-
deficient O-glycans are caused by dysfunctional C1GalT1 or its chaperon Cosmc
and aberrantly expressed N-acetylgalactosaminide α2,6-sialyltransferase I/II
(ST6GalNAc-I/II) enzyme specific for sialyl Tn synthesis [19]. Considering IgA
and IgG glycans, the presence of α2,6 sialic acid in the IgG Fc is a key anti-
inflammatory decision factor in autoimmune diseases [20]. Totally, changes in N-
and O-glycan-branched glycans can result in abnormal innate or adaptive immune
responses.
In mice, loss of T antigen causes thrombocytopenia, and the human Tn syndrome
is displayed in the thrombocytopenia and leukocytopenia, caused by the COSMC
gene mutation for β1,3GalT (T-synthase) or core 1 β1,3-GalT (C1β3GalT), for
O-glycan biosynthesis [21]. The C1β3GalT enzyme is an evolutionarily conserved
enzyme that transfers Gal to a mucin-type O-glycan GalNAcα1-O-Ser/Thr glycan
(Tn antigen), to form a Galβ1,3GalNAcα1-O-Ser/Thr (T antigen). The functions of T
antigen were known because deficiency of C1β3GalT1 displays many defects in
developmental diseases. Representatively in mice, lacking T antigen displays throm-
bocytopenia [22] and vascular vessel dysfunction [23], while in humans, Tn
6.1 Glycosylation Effect on Autoimmunity and Inflammation 265
syndrome is caused for thrombocytopenia and hemolytic anemia [24], with dys-
functional Cosmc, a chaperone protein for human C1β3GalT. Tn syndrome also
exhibits malfunction in the hematopoietic stem cells, although the roles of T antigen
are not known for hematopoiesis. However, T antigen has been suggested to be
conserved in blood cell lineages among species.
The surface glycan repertoires of immune cell types are also related to immune
diseases. T cell glycosylation regulates T cell functions like activation and differen-
tiation through cis- or trans-masking or demasking of the ligands for endogenous
lectins. For example, α2,6 SA content is increased on the Th-2 cell surface. However
the α2,6 SA levels are not increased in Th-1 or Th-17 cells. The difference in the
α2,6 SA contents indicates the T cells’ susceptibility to the GBP galectin-1 that
endogenously recognizes the altered glycosylation of cell surface glycoproteins and
induces cell death of activated lymphocytes [25]. Therefore, galectin-1 silencing KO
mice (Lgals1/) express autoimmune status of autoimmune EAE, caused by
expansion of antigen-specific Th-1/Th-17 cells and DCs immune response
[23, 25]. Thus, galectin-1 therapeutic treatment can restore immune tolerance and
consequently inhibit chronic inflammation in autoimmune diseases of diabetes,
EAE, hepatitis, IBD, RA, and uveitis. Also, prevention of fetal loss and
graft vs. host disease (GvHd) can be obtained through Th1 and Th17 suppression
by Treg cell expansion and tolerogenic DC supplementation. Similar to autoimmune
diseases, in HIV-infected T cells, T cell surface glycosylation is also altered and
increases susceptibility to apoptotic death of T cells, which is induced by galectin-1.
Hence, galectin-1 influences the pathogenic features of AIDS. O-glycan modifica-
tions of peripheral lymphocytes from AIDS patients are such altered glycosylation
types. HIV-1-infected T cells and AIDS patients-derived peripheral CD4 T cells and
CD8 T cells exhibited exposed lactosamine residues and the lactosamine residues
triggered to enhanced susceptibility of the cytotoxic death of T cells by galectin-1
[24]. Altered surface glycosylation level of T cells caused by infection of HIV-1
contributes to the increased T cell death via galectin-1-mediated signaling. In the
galectin-1-interacting proteins, galectin-1 has been known to cause the immature
thymocyte death and activated peripheral T cell death by directing recognition with
CD7, CD45, and CD43 glycans on T cells. The CD7 and CD45 functional roles are
not known when galectin-1 promotes apoptotic T cell death. During galectin-1-
mediated cell death of T cells, galectin-1 interacts with CD43 glycans, and thus
CD43 is suggested to be the subject for the galectin-mediated T cell death. Heavily
O-glycosylated CD43 functions as the galectin-1 recognition sites of T cells. Core
1 O-glycosylation or core 2 O-glycosylation structures in CD43 glycoprotein regu-
late galectin-1-mediated T cell susceptibility, indicating that T cell glycans are the
galectin-1-binding targets [26].
The tandem-repeat galectin-9 can also confer such suppression of progressed
autoimmune responses via galectin-9 binding to the Tim-3 glycoreceptor
[27]. Galectin-9 is a soluble lectin and forms lattices between galectin-9 and
glycoprotein of the surfaces. Interaction between galectin-9 and its ligands causes
death of CD4-positive Th1 cells, but not of CD4-positive Th-2 cells, simply due to
different glycan structures [28]. Galectin-9 is specifically expressed in T cells,
eosinophils, DCs, endothelial cells, and macrophages. It causes the cell deaths of T
cells and thymocytes with specificity to CD4-positive Th-1 cells but spares
CD4-positive Th-2. The resistance of CD4-positive Th-2 to galectin-9 is based on
the α2,6-SA linkages present in the surfaces of Th2 cells. The cell surface α2,6-sialic
acids block galectin-9 recognition to glycan ligands crucial for cell death. C-type
lectins can also modulate inflammation and autoimmune diseases. Blocking of
mannosyl encephalitogenic peptide inhibits the EAE development, because the
immature DCs MR loss its Man ligand. Thus, mannose-supplemented administra-
tion can induce oral tolerance and generate IL-10-producing Treg cells through the
CLR SIGNR1 on DCs [29]. Also, Siglec-2 (CD22) inhibits B cell signals, as
confirmed by the results that Siglec-G/CD22-deficient double KO mouse exhibits
the B cell-dependent autoimmune disease spontaneously developed. The double KO
mouse generates anti-DNA and anti-nuclear autoantibodies [30].
6.2 Glycosylation Effect on Tumor Immunity

of Immune Cells
The tumor-associated microenvironment (TAM) is a symphonic orchestrate of

cellular networks of stromal cells, tumor cells, and infiltrating immune cells. In the
host, the immune system generally modulates development of cancer cells to a
certain extent in host system, as settled down for the immunosurveillance theory
[30]. Thus, the host immune system detects and eradicates transformed tumors.
However, the immunosurveillance in the current tumor biology does not sufficiently
eliminate such transformed tumor cells. Then to explain the tumor survivals from the
immune system, tumor immunoediting theory has been newly conceptional [31],
where tumor cells acquire the evasion potentials from immune responses through
lacking immune recognition as well as increasing immune regression and immuno-
suppressive microenvironment. In tumor patients, tumor-specific immune status is
believed to be a parameter for prediction of prognosis and therapeutic monitoring.
The theoretical immunoscore is one of such predictable unit that is calculated by the
infiltration of immune cells towards immunotherapeutic consideration. For solid
tumors, the immunoscore levels of tumor-infiltrating lymphocytes are associated
with the tumor survival rate. High proportional rates of memory T cells or antitumor
CD8+ CTLs are indicative of the reduced invasion of solid tumor cells [32]. In
parallel, the immunoscore of peripheral-blood lymphocytes is also indicative of the
reduced tumor developments [33]. Although CTLs/NK cells induce the cytotoxic
killing of tumor cells, in the hypoxic tumor microenvironment, NK cell receptors are
not expressed, indicating no cytotoxic effect of NK cells [34]. Therefore, DCs in
collaboration with naive CD4+ T cells and CD8+ CTLs predominantly contribute to
the adaptive immunity.
Certain patterns of glycosylation present in the cell surfaces of transformed cells
influence host immune modulation and survival potentials of malignant tumors.
6.2 Glycosylation Effect on Tumor Immunity of Immune Cells 267
Tumor cells proliferate through the immune evasion, which is accompanied by

immune checkpoint inhibitors like PD-1 of hosts. Transformed cells exhibit the
surface glycosylation changes in the plasma membrane-associated glycoproteins.
Currently, representatively certain tumor-associated antigens (TAAs) such as CEA
and MUC1 are mostly well studied for the altered glycosylation to date [35],
although they are present in normal colonic mucosa and epithelial cells. However,
the precise roles of tumor glycosylation in immune evasion has not well been
defined. However, the aspect describing that aberrant tumor glycosylation potenti-
ates the immune escape from immune surveillance under the host immune system
provides valuable insights into consideration. It is noted that consequent immuno-
suppressive events are obtained via carbohydrate-recognizing receptors. Some car-
bohydrate patterns expressed on tumor cells is also another type of immune
checkpoint, because glycosylation of tumor cells is used as T cell targets to tumor-
specific recognition. Then, a recent study describes how the tumor-yielded “glycan
code” alters the host immune system, and if it goes through, such targeting glycans
are the future avenue for the therapeutic clues [36].
For tumor detection in microenvironment, APCs express diverse glycan-binding
receptors like CLRs and Siglecs. CLRs can internalize the bound ligands to APCs
and associate to process and present the digested antigens to T cells. The CLRs also
modify DC functions and macrophage functions. The macrophage Gal-binding
lectin known as MGL/CD301 of human CLR binds to terminal GalNAc residues
linked to Tn and STn antigens of the aberrantly expressed O-glycans in tumor cells
[37]. MGL is specifically present in tolerogenic or immature DCs or macrophages of
myeloid innate immune cells. The MGL expressed in human is a valuable biomarker
for TAM presence [38]. An immunomodulatory activity of MGL is also clinically
dedicated to the high MGL association in cancer patients with a poor tumor-free
survival [39]. For example, Lewis blood group of Lex and Ley expressions indicate
poor prognosis of the tumor cells. CLRs of DC-SIGN and MGL on DCs recognize
the Lewis glycan structures attached to CEA or MUC1, but not recognize normal
type of CEA species or MUC1 produced by colon tissues [39]. CLR-glycan binding
can trigger antitumor responses and trigger antigen-targeting autoimmune responses.
Another CLR, CLEC9A predominantly expressed on human and mice DCs
phagocytically digest dead cells for MHC-I-based presentation of the antigen epi-
topes and activation of CD8+ CTLs. However, currently, the CLEC9A-binding
ligands are not clear yet. The CLEC9A upon antibody-antigen conjugate treatment
induces antigen endocytosis to activate CD4+ T cell and CD8+ CTL proliferation.
Furthermore, CLEC9A in cancer cells raises T cell-driven rejection of tumor cells
[40]. For another case, high Man-modified antigen GP100 of melanoma cells
stimulates both T cell types of GP100-specific CD4+ T cells and CD8+ CTLs,
since high Man-type structures recognize DC-SIGN, potentiating endosomal-
dependent antigen presentation [41]. Two other Man receptor-specific ligands,
such as sulfated LeA or GlcNAc, enhance the MR activity upon sulfate LeA or
GlcNAc ligation with protein antigens, increasing antigen presentation to T cells
[42]. Tumor cell exploits lectin-carbohydrate recognition to evade the host immune
responses [43]. Tumor-produced galectin-1 can inhibit host immune responses, as
evidenced by many tumor cells including lung carcinoma cell, Hodgkin’s lymphoma
cells, melanoma cells, neuroblastoma cells, and pancreatic carcinoma cells.
Galectin-1 modulates T cell and DCs [44–46] through the Th2-dominant cytokines
and tolerogenic activation by IL-10-expressing type 1 Treg cells and IL-27-
expressing DCs [47]. Galectin-9 also increases the number of myeloid suppressor
cells with CD11b + Ly-6G+ granulocytic phenotype in order to inhibit antitumor
activity [48], while the galectin-3 controls the anergic T cells status [49]. Thus,
selectively expressed galectin family is responsible for blocking immunosuppression
at tumor growth sites through targeting immunoevasion. Glycosylation-dependent
tumor immune escape has also been observed in bladder tumor cells. The bladder
tumor cells aberrantly express the core 2 β1,6GlcNAc-transferase (GCNT1) which is
encoded by C2GnT gene. The GCNT1 catalyzes a GlcNAc branching on GalNAc in
core 2 O-glycans. The tumor cells have highly metastatic potential form because
they evade the host NK cell immunity. Galectin-3 binding to poly-LacNAc units
attached to mucin-type core 2 O-glycosylations of cancer-involved MHC-I-related
chain A (MICA) decreases the NK receptor NKG2D-activating MICA affinity and
consequently impairs the NK cell function of anti-cancer activity [50]. Moreover, the
tumor cell-produced disialyl GD3 also controls NK cell cytotoxic activity through
Siglec-7 signaling [51]. Mucins produced from tumor patients modulate the DC
immunogenicity via Siglec-9 in human [52]. The lectin repertoires and the cellular
glycosylation patterns in the TAM influence immune cell fate and tumor survivals.
The importance of carbohydrates opens new vista on the mechanism of how
autoimmunity, development, cancer, lymphocyte differentiation, and host-microbe
interaction undergo. The glycan-focused immunology, glycoimmunology, will be
interested in the field that carbohydrates influence immune responses because
carbohydrates are integral to immune pathways.
6.3 Immune Tolerance and Defense Mechanisms of Innate

Immune DCs During Infection
DCs bridge the innate immune response to the adaptive immunity, and this indicates
its key regulators of the host immune system. In addition, they determine whether
direct immunity or tolerance is generated in the body [53], since its discovery in
1868 by Paul Langerhans [54] at Berlin. Historically, in 1973, DCs were found in the
spleens of mice [55]. After another 12 years, Langerhans cells (LCs) were defined as
one group of the DCs, as in 1985, Gerold Schuler and Ralph Steinman reported the
seminar title of “Epidermal Langerhans cells in murine mature into potent
immunostimulatory DCs in vitro” [56]. Thus, a series of discoveries has been
recognized as a landmark in milstone [57]. In 1985, DCs meets a new era to shape
the LC and other Langerin + DC [58], establishing the “LC paradigm” theory [59].
Immune homeostasis is operated and kept by the coordination of innate and
adaptive immune cells and epithelial cells. As the major innate immune cell and
6.3 Immune Tolerance and Defense Mechanisms of Innate Immune DCs During Infection 269
APC, DCs uptake, digest, process, and present foreign antigenic molecules to naive
T cells to start specific immune responses against pathogens. In the side of virus,
however, DCs are also used as target cells for certain viral infections. This indicates
that infected virus-derived immune escape behavior hampers the T cell-activating
capacity of DCs. Most of DC subsets have tolerogenic roles. Tolerogenicity of DCs
is a fundamental phenotype and induces T cell anergy and Treg and T cell deletion
[59]. Tolerogenic DCs have been described ex vivo for the first time, from the
finding that UV-irradiated Langerhans cells induce T cell anergy [60]. UV-mediated
apoptosis indicates DC induction of tolerance. Immature DCs in peripheral tissues
function as immune sensors for pathogens. Pathogens, danger or inflammatory
signals, induce maturation and activation DCs, and the matured DCs are now
ready to migrate into the draining lymph nodes. DC presentation of pathogen-
processed antigens stimulates T cells by means of costimulation and
proinflammatory cytokine production [61]. The most important role of tolerogenic
DCs is to control dysregulated T cell responses against harmless or self-antigens.
The tolerance capacity in the inflammation status will add new prospects for treating
patients with autoimmunity and hypersensitivity. Tolerogenic DCs exhibit an anti-
inflammatory phenotype through lowered synthesis of co-stimulatory proteins and
Treg induction. Most infectious microbes are co-evolved to interact with their hosts,
inducing a balance between a pathogen-triggered protective response and immune
tolerance to prevent microbial elimination. Non-adapted microbial infections cause
the host cell death or are eliminated by the host’s immune reaction. If adapted, the
microbes can chronically infect without symptomatic infection by means of the
host’s immune tolerance during pathogen-host coevolution.
In the inflammation status, effector T cells are functionally dampened by sialyl
antigen-accounted DCs. DCs are tolerogenic when they are accounted with soluble
sialyl antigens. Sialylated antigen-specific immune tolerance is also induced in the
inflammatory status. Sialyl antigen-accounted DCs elicit polarization of naive CD4+
T cells for Tregs. Although DC uptake of sia antigens in vitro is associated with
surface marker synthesis, referring to “classic” tolerogenic DCs, DCs are function-
ally tolerogenic when sialyl antigens are endocytosed. The phenotype of moDCs of
human is tolerogenic when the cells are incubated with the highly sialyl pathogens.
Sialyl glycan-induced DCs elicit Treg functions and block effector T cell population
through binding of receptor to ligand, but not by anti-inflammatory cytokines.
Interestingly, the same glycan-pulsed DCs do not affect CD4+ T cells. The immune
tolerance adaptation is obtained by innate immune DCs and macrophages because
they stimulate immune tolerance and sense antigens. The immune surveillance
system of the innate immunity involves in suppressive tolerogenic and
proinflammatory responses by extracellular mediators to regulate the suppressive
and promotive balances of immune signals. Most food-borne antigens are evolu-
tionarily immune tolerant to commensal microbial antigens. Wnt/β-catenin signaling
mediates DC morphogenesis and development [62]. The Wnt signaling in DC
function regulates the stromal cells and mucosal cells to activate DCs. For immune
tolerance mechanism of DCs, the DCs migrate to lymph nodes to induce the
generation of Tregs and cytokines. Then, the Treg cells and the cytokines induce
the immunologic tolerance of self-antigens or commensal symbionts. In contrast,

when inflammatory agents or infectious pathogens are encountered, the immuno-
logic defense system is operated, where DCs and macrophages initiate inflammatory
responses to facilitate the activation of adaptive immunity [63].
During invasion and infection, pathogens produce several agonists to bind DC
receptors such as TLRs. The agonists induce classical DC maturation, and the
matured DCs produce effector T cells and potentiate inflammatory immune
responses [64]. For example, DCs, intestinal epithelial cells, and lamina propria-
resident leukocytes bind to TLRs as pattern recognition receptors, subsequently
allowing innate immune responses. Although the DCs migrated to lymph nodes
activate regulatory T cells and cytokine production for immune tolerance of self-
antigens or commensal symbionts, if this process is failed, unexpected immune
disorder, inflammatory bowel disease (IBD) is the case, as seen in the gut intestine
system. This is an exceptional case that an organism’s self-immune cells attack the
self-antigens in the intestine. Therefore, classical IBD is classified into the chronic
inflammatory disorders with organ-replacing properties, and UC and Crohn’s dis-
ease (CD) belong to the disease. Actually, the diseases are severely inflammatory
without any drug effectiveness for immunosuppressive agents or steroid-based
preventive and therapeutic agents [65]. Thus, ulcerative colitis is clinically cared
and treated through surgical removal of the bowel disease-developing organ or
region. The diseases are characterized with onset of the familial history as a key
risk factor for IBD progression.
6.4 How Are Pathogenic Bacteria Recognized by Receptors

of DCs of the Host Immune System?
6.4.1 DC Lectins for Glycan Recognition of Invasive Agents
Lectins as glycan recognition proteins display many different biological roles

including cell-to-cell recognition and binding. Lectins can be further categorized
into many different types depending on their various characteristics. Lectin can
recognize various glycoconjugates on cell surfaces and extracellular matrices, rang-
ing from the mediation of adhesion and promotion of cellular recognition pathogens.
The different families of animal lectins and lectin domains are known with their
three-dimensional structures. Lectins are found from mammals and plants and some
lower invertebrates such as nematodes. From genome analysis, each organism-
specific lectin profile has been made. Lower organisms have abundantly C-type
lectins, and plant-specific lectin domains are not found in animals but found in some
lower species such as nematodes. Common lectins between plant and animal origins
are legume-like, ricin-B, and class V chitinase-like lectins, and they are also
observed in nematodes. Plant lectin-specific sequence is not found in animals.
However, they are found in some nematode species. The immunoglobulin
6.4 How Are Pathogenic Bacteria Recognized by Receptors of DCs of the Host. . . 271
(Ig) superfamily is named by Alan F. Williams in 1988 for recognition molecules,

which he found then from the immune cells. The Ig region is used to bind molecular
structures because of their highly well-rounded fold. Lectin as a carbohydrate-
binding protein mediates protein-carbohydrate interaction including cellular adhe-
sion or recognition events. Lectin is divided into three classes of C-, P-, and I-types.
The Ig-type or I-type lectins belong to the Ig superfamily in recognition with distinct
structural diversity of glycan binding. One of them, the Siglecs, are the most well-
known I-type lectins.
As the first defense line during pathogen invasion, the innate immune cells
effectively respond to inflammation and tissue damage. DCs, macrophages, and
non-expert cells including fibroblast cells, endothelial cells, and epithelial cells are
involved in pathogen recognition [20]. Innate immune DCs have evolved to sense
pathogenic agents via PRRs, which interact with PAMPs to transduce microbial
pattern signs into immune cells to trigger their downstream activation. Apart from
importance of lectin-glycan recognition, secreted glycoproteins such as antibodies in
the immune system exert their functional activities through O- or N-glycans attached
to the IgA hinge regions or IgG Fc regions with modulating antibody activity
[20, 66], where terminal SAs give an inhibitory signal to immune cells, whereas
the defected terminal SAs give activating signals. The glycosylated ligands includ-
ing N- and O-glycan branching, LacNAc levels, and the balance of α2,3-SA and
α2,6-SA regulate lectin recognition with their counter-receptors, indicating crucial
roles of the specific glycans in lectin-binding partners in patterns of proper orienta-
tion and glycan clustering on multiple side chains. When lectins are released from
the cells, they are accumulated on the cell surface matrix to increase in local
concentration. Thus, lectins build multiple forms and cross-link glycoconjugates
on the cell surfaces to modulate downstream intracellular signaling towards a variety
of cellular events. Cellular responses including receptor expression, protein synthe-
sis, Golgi-specific enzyme behavior, glycan biosynthesis, and glycan density regu-
late lectin-glycan interaction.
During biological adaptation and evolution, innate immune cells mostly
co-evolve to sense pathogenic microbes through host PRRs, which recognize the
conserved PAMPs that independently evolved, and consequently in order to trans-
duce pathogen information into host responses. PRRs also recognize endogenous
DAMPs such as alarmins produced from invasion-derived inflammatory responses,
autoimmune responses, inflammation, or tumor growth. Representatively, TLRs
mediate the recognition events in way of lectin-glycan interactions. However, it
became apparent that the glycan epitopes expressed on pathogenic agents are also
expressed on the host surfaces and involved in cellular functions. Pathogenic agents
basically mimic the host cell surfaced glycans to avoid the host immunity. Then a
question is raised. Therefore, the molecular mechanisms responsible for pathogenic
glycan binding by host lectins are not clearly discriminated, remaining questions to
answer. How? PAMPs are molecular signature of pathogens and PRRs recognize the
pathogen patterns. In spite of the TLRs in antigen recognition, lectin-glycan bind-
ings promote pathogen sensing and related responses of immunity. The glycans and
lectins are acting elements of pathogen binding and starting the immune responses of
innate immune cells. Pathogens and host cells produce such acting elements. Endog-
enous carbohydrate-recognizing proteins or lectins decode the information. The
number, density, composition, and distribution of carbohydrate antigens of multi-
valent carbohydrate moieties in glycoproteins, glycolipids, and GAG decide the fate
of lectin-binding affinity. Most patterns are composed of glycans during evolutional
adaptation. Currently, several PRRs are identified for TLRs, CLRs, RLRs, NLRs,
and the AIM2-like receptor (ALR). The major decoding elements include C-type
lectins, galectin, and Siglecs. For the general recognition receptors of DCs and their
ligands, C-type lectins such as selectins recognize the glycans of bacteria, while
galectins recognize the glycans of bacteria. Siglecs also recognize the glycans of
bacteria. Pattern recognition of lectins in innate immune responses indicates a glycan
recognition property through a density-dependent manner. In innate immune cells,
the surfaced Siglecs and C-type lectins specifically bind to glycans of microbes,
while galectins preferentially recognize soluble targets.
Apart from the soluble target-recognizing lectins, there are also soluble glycan-
recognizing PRRs that include ficolins in which three types of ficolin-1, ficolin-2,
and ficolin-3 are known and MBL. The lectin pathway associated with the comple-
ment system depends on PPRs to help the clearance of microbial invaders. For
example, ficolins belong to a PPR family and lectin pathway component. The
ficolins are the secreted components of complement in epithelial cells, endothelial
cells, and immune cells of human [67]. Ficolins recognize GlcNAc, GalNAc, and
acetyl glycans of target cells [68]. In the side of pathogenic bacteria, they evolved to
have complement evasion strategies to mimic and recruit complement factors or
degrade complement factors [69]. Both Gram-positive and Gram-negative bacteria
can evade complementation by recruitment of complement regulators. The repre-
sentative regulators are the factor H- and C4b-binding protein (C4BP) [70]. The
neonatal meningitis-causing E. coli K1 utilizes the Omp known as outer membrane
protein A to protect the bacteria itself from the host killing activity through comple-
ments. For example, the C4BP binds to bacterial OmpA protein [71]. Immune
evasion is also mediated in pathogenic EAEC strain by proteolytic degradation of
host complement proteins using a serine protease known as Pic [72]. Pic protease
blocks complement activation by enzymatic inactivation of complement compo-
nents of C2, C3, C3b, and C4 [73]. Also, biofilm production is a way of evading
complement of hosts [74]. In ficolin escape, some bacterial strains show the changed
surface composition to avoid binding to ficolin-2, eventually escaping the host
complement [75].
On the other hand, two types of collectin-10, termed as CL-10 and CL-L1/
collectin-11, termed as CL-11/CL-K1, also exert the similar type of complement
activation [76, 77]. They recognize pathogen-associated PRRs on the microbial
pathogenic surfaces and stimulate the lectin pathway via lectin pathway-involved
serine proteases or MBL/ficolin-associated ser-proteases (MASPs) [78]. They are
indeed soluble pattern recognition molecules, which depend on glycan recognition,
the target foreign microorganisms or altered host cells induce the complement
cascade reaction of host via the alternative lectin pathway [79]. This is a distinct
type of innate immune response of mammals. Once soluble forms of PRRs are
bound to a ligand, the downstream cascade is started by the MASPs, which cleave
and activate complement factors C2 and C4, contributing to the C3 convertase
formation known as C4b2a. The active C3 convertase enzyme degrades C3 form
to C3a form, known as anaphylatoxin, and the opsonin factor C3b activates com-
plement factor C3 [80]. Therefore, in the lectin pathway of the MBP and ficolins,
complement activation, therefore, differs from the classical C1q activation pathway
[81]. The alternative lectin pathway is thus initiated by C3 degradation and the
opsonin factor C3b binding. The C3 can be further degraded by factor D, yielding
the C3 convertase, known as C3bBb. The alternative lectin pathway includes C3b
genesis, and the increased C3b factor consequently forms the C5 convertase enzyme
for the C4b2aC3b/C3bBb3b. This process yields the C5b-9 membrane attack com-
plement (MAC) complex, which has the activity of terminal lysis [75]. The com-
plement activation of classical pathway commences with unique binding of antibody
to antigen, and this consequently interacts with the PRR C1q and cleaves C2 and C4
by specific C1r/C1s proteases and deposits C3b opsonin factor.
6.4.2 Toll-Like Receptors
TLRs are historically introduced from the long investigation on innate immunity.
The most remarkable milestone in innate immunity study indicates the 2011 Nobel
laureates of Drs. Bruce A. Beutler, Ralph M. Steinman, and Jules A. Hoffmann in
physiology or medicine for their DC findings and discovery with roles in adaptive
immunity as well as for their discoveries concerning the innate immunity activation.
TLRs have been termed from their similar protein to the Toll gene in Drosophila
[82]. The Toll found in Drosophila regulates development of embryo- and fungi-
specific immune responses [82, 83]. TLRs have Leu-rich repeated ectodomains and
Toll-IL-1R (TIR) domain in the intracellular region. The known TLRs are the TLR1
to TLR10 and the TLR11 to TLR13. The TLR1–TLR10 members are isolated from
human and the TLR12, TLR1 to TLR9, and TLR11 to TLR13 are isolated from
murine [84]. TLRs recognize PAMPs. TLR2/TLR1 and TLR2/TLR6 recognize
lipoproteins as well as diacyl lipopeptides and triacyl lipopeptides. TLR2 binds to
fungal zymosan, lipoteichoic acid, and peptidoglycans. TLR3 binds to dsRNA
species, TLR5 binds to flagellin, and TLR9 binds to unmethylated CpG DNA.
TLR8 also recognizes various synthetic molecules like guanosine analogues and
imidazoquinolines. The TLR alone or TLR with co-receptors binds to pattern
molecules [84].
For the TLRs of DCs and their ligands, the interactions are outlined and summa-
rized below:
– TLR6, TLR2, and TLR1 recognize peptidoglycan (Gram + positive bacteria),
lipoprotein, diacyl lipopeptides, triacyl lipopeptides, lipoteichoic acid,
lipoarabinomannan (mycobacteria), LPS (Leptospira), LPS (porphyromonas),
GPI (Trypanosoma cruzi), and fungal zymosan (yeast).
– TLR4 and CD14 recognize Gram-negative bacterial LPS, Gram-positive bacterial

lipoteichoic acid, and RSV F protein.
– TLR3 recognizes dsDNA, dsRNA, a viral product.
– TLR5 recognizes flagellin.
– TLR7 recognizes small synthetic immune modifiers, imiquimod, R-848,
loxoribine, and bropirimine.
– TLR9 recognizes unmethylated CpG DNA.
– TLR8 recognizes imidazoquinolines and guanosine analogues.
6.4.2.1 DC-Receptor-Specific Pathogenic Ligands
GBPs recognize and bind to pathogens as PRRs through the pathogen-exposed

glycans. As PRRs, TLR and Nod-like receptors of DCs recognize bacterial patterns.
The well-known bacterial patterns are peptidoglycan as TLR2 ligand, LPS as TLR4
ligand, flagellin as TLR5 ligand, unmethylated CpG DNA sequences as TLR9
ligand, and muramyl di- and tripeptides (Nod1 and Nod2) to date [85]. Other glycans
produced by pathogenic agents can also be recognized by other kinds of DC
receptors. Those DC receptors are C-type lectins, galectins, and Siglecs. These
three kinds of receptors are also expressed by intestinal epithelial cells and APCs
[86]. For example, it is reported that DC-SIGN directly binds to α-glucan and
mannose-containing glycans of mycobacterial cellular capsules, and these glycans
induce immune suppression of host immune cells upon encounter with the
mycobacteria. The binding proteins such as DC-SIGN bind Lewis and mannose-
containing carbohydrates exposed on pathogens [87–89]. HIV-1, bacteria
M. tuberculosis and H. pylori, helminth Schistosoma mansoni, and yeast Candida
albicans are such examples (Fig. 6.1). More specifically, DC-SIGN recognizes their
high Man, LeX or LeY, or LDNF. Pathogen glycan-bound CLRs are internalization
receptors, due to the pathogen uptake by APCs [90]. Because CLRs thus are PRRs
and similar to TLRs. The functional difference between CLRs and TLRs is in that
TLRs do not internalize antigens to present by MHC-I or MHC-II. The different
signaling function is in that CLRs interfere with Fc receptor (FcR) or TLR activity.
CLRs transduce signals via the Raf-1 or Syl signaling pathways during binding to
glycans [87, 91, 92]. CLRs form normally complex machines with cytoplasmic
signaling motifs and adaptor proteins. Exceptionally, the DC-SIGN elicits expres-
sion of target genes upon binding to PRRs. For fungal β-glucans, Dectin-1, a specific
CLR, transduces downstream signaling via the Syl tyrosine kinase and ERK-JNK-
NF-κB axis pathway [87, 93]. This signaling is cooperated with the adaptor CARD9,
independently to TLR signaling. Again, DC-SIGN also recognizes Mycobacterium
tuberculosis strains through the ManLAM carbohydrates produced by Mycobacte-
rium strains and activates IL-6 and IL-12 releases, which are proinflammatory
cytokines, through a Raf-1 downstream signaling. However, if DC-SIGN recognizes
Fuc residue, Raf-1-independent signaling induces inflammatory IL-10 expression
but inhibits IL-6 and IL-12 gene expressions, which are proinflammatory cytokines
[92]. Therefore, each carbohydrate signature of pathogenic agents triggers each
Fig. 6.1 DC-SIGN

signaling in HIV-1 or
M. tuberculosis infection HIV-1 M. tuberculosis
DC-SIGN
6UF 3DN
5DV
Tyr340/341 Ser338
F\WRVRO
5DI
6HU
QXFOHXV
1)ƈ%
distinct signaling response, even via the same CLRs. The GBPs also interact with
self-glycans with mannose/fucose or GalNAc glycan structures [94]. Tumor antigen
MUC-1 binds to MGL and CEA recognizes DC-SIGN [35]. GBPs bind to single
carbohydrate structures. In fact, DC-SIGN does not bind to sialylated carbohydrates
but binds to sialylated IgGFc, contributing to the anti-inflammatory response of
IVIGs and to the inhibition response of FcR FcyRIIB signaling during the IL-33
expression in Th-2 and IL-4 expression in basophils [20].
Myelin oligodendrocyte glycoprotein (MOG) acts as an autoantigenic factor of
the neuro-inflammatory MS event. Hman myelin-located MOG is a fucosyl type of
N-glycans, which is interacted with the DC-SIGN present in microglia cells and
DCs. The binding of MOG to DC-SIGN during TLR4 activation induces secretion of
IL-10 and inhibits T cell growth. Inflammatory oligodendrocytes suppress the
fucosyltransferase expression. Fucose residue loss on myelin decreases DC-SIGN-
driven homeostasis with activation of inflammasome, growth of T cells, and differ-
entiation of Th17. DC-SIGN ligands include the CEA and CEA-CAM1. The ligands
activate the DC response to the TLR4 ligand LPS like M. tuberculosis ManLAM to
increase the LPS-induced IL-10 production. Thus, tumor and pathogens can escape
immune response via the host receptor tolerogenesis (Fig. 6.2). DC-SIGN is a
homeostatic receptor by pathogens and tumors via their glycan structure
change [96].
In other example of bacterial pathogen, C. jejuni-produced α2,3-SA-containing
glycans can be bound by Siglec-7, which is present in DC cell surfaces. Siglec-7 is
compatible for its recognition capability with both α2,3- and α2,8-SA-containing
glycans. Therefore, the α2,3-SA-bound Siglec-7 expressed in the DC cell surfaces
stimulates T helper-2 responses, whereas α2,8-sialic acid glycan-bound Siglec-7 on
the DC stimulates T helper-1 responses [97].
Fig. 6.2 DC-SIGN action as a homeostatic receptor in T cell responses. MOG, myelin oligoden-
drocyte glycoprotein; CEA, carcinoembryonic antigen; CEACAM1, CEA-related CAM-1.
Adopted from ref. [95] García-Vallejo JJ et al. J Exp Med. 211(7):1465–83
Pathogens express their own specific glycans. Helminth glycans are carbohydrate
structures such as GBPs-binding LDNF with the structure of GalNAcβ1,4(Fucα1,3)
GlcNAc-R and LDN with the structure of GalNAcβ1,4GlcNAc-R- [89]. HIV-1
expresses high Man structures not pathogen-specific but recognized by certain
GBPs of the CLRs like DCIR, DC-SIGN, Langerin, and MR [98–100]. DC-SIGN,
MR, or MGL easily bind endogenous glycans, and thus they are adhesion molecules,
antigen uptake mediators, or signaling receptors [101]. DC-SIGN recognizes the
ICAM-2 and ICAM-3 during cell-cell interaction and DC homing via LeY epitope
expressed in vascular endothelial cells. DC-SIGN also recognizes the LeX and LeY
glycans expressed in Mac-1 and CEA-CAM-1 receptors expressed in neutrophils
and thus positively regulates adhesion of neutrophils and DCs [102]. GalNAc
residue linked to CD45 present in the effector memory cells of CD4+ T cells and
CD8+ CTLs recognizes the MGL, a CTL dominantly present in tolerogenic APCs.
The recognition events induce apoptotic cell death proliferation of these cells, giving
a homeostasis-keeping function of CD45 carbohydrates [13]. Furthermore, similar
CTLs such as DC-SIGN, MR, and Langerin also recognize tissue antigens including
collagen type I, Fc-IgG, plasma hydrolases, and tissue-type plasminogen activator
TPA. DC-SIGN, Langerin, and MR can internalize, serving to homeostatic surveil-
lance by APCs of DCs and macrophages [103].
SAs are frequently expressed on infectious pathogens, as known in many differ-
ent pathogenic bacteria including C. jejuni, H. influenzae, N. gonorrhoeae,
N. meningitidis, and Pasteurella multocida [97, 104–106]. In fact, the incorporation
of host-derived SAα2,3-linkage into H. influenzae is reported to be a major virulence
factor for experimental otitis [104]. Because the sialyl moieties of the pathogenic
bacteria are structural mimics of the human sialyl glycans, these are evolutionized to
use to avoid or escape the host immune system. Although α2,3-sialyllactose or α2,6-
sialiclactose motif interacts with TLR4 on DCs, the Gram-negative bacterial nega-
tive LPS or sialylated bacterial glycans targets the TLR4 of DCs for their protection
from the hosts. For example, α2,3-sialylated LPS on C. jejuni binds to the TLR4 of
DCs to escape the host defense immunity [107].
6.4.2.2 Signaling upon Receptor-Ligand Interaction
TLR signaling initiated by TLR dimerization induced by the ectodomain consists of

two main pathways of the TRIF/MyD88 pathways [108]. From the knowledge on
the interaction of PRRs such as TLRs with PAMPs, the adaptor protein MyD88 is
involved in the downstream signaling towards NF-κB activation. NF-κB is conse-
quently activated to express the inflammatory genes of chemokines,
immunoreceptors, and cytokines [109]. MyD88 is conserved in TLRs, not for
TLR3 [110]. TLR3/TLR4 uses the TRIF pathway, initiated by dsRNA and LPS,
respectively [111, 112]. Myd88 factor recruits several adaptor proteins such as
IL-1R-associated kinase protein (IRAK)-1/IRAK-4/TNFR-associated factor
6 (TRAF6) [113]. IRAK-1 protein ubiquitinates TRAF6 and stimulates
TGF-β-activated kinase 1 (TAK1) [114]. TAK1 kinase elicits NF-κB and MAPK
axis with IKK kinase complex with subunits of the IKKα/IKKβ catalysis subunits as
well as regulatory IKKγ subunit. The IKK enzyme degrades NF-κB inhibitor IκBα
or IκB families like p105. Then, canonical NF-κB family is released and
translocated. For example, the complex dimer formed between NF-κB1 p50-c-
REL and NF-κB1 p50-RELA induces expression of proinflammatory gene. TAK1
also activates MAPK family including ERK1/ERK2, JNK, and p38 to elicit the
transcription factor AP-1. TAK1/p38/NF-κB axis signaling is required for stimula-
tion of microneme proteins.
TLR extracellular ectodomains expose leucine-rich repeats (LRR) binding to
other functional amino acids to confer the domain capacity. In addition,
heterodimerization and co-receptor recruitment increase the recognition capability
of various ligands. TLR2 and TLR4 extracellular ectodomains carry four and nine
N-glycans, respectively. N-glycosylated TLRs represent lectin-binding abilities as
protein-carbohydrate interactions (PCI) [115]. The TLR heterodimers include TLR4/
TLR6, TLR2/TLR1, and TLR2/TLR6 [116–118]. Homodimeric TLRs are TLR5/
TLR5, TLR4/TLR4, TLR3/TLR3, and TLR2/TLR2 [119–121]. TLR2 complexes
incorporate their co-receptors such as CD14 [122] and CD36 [123], forming three
distinct heterocomplexes including CD14-TLR2-TLR1 complex for triacyl lipopro-
teins as ligand and bacterial Salmonella curli fibers [124], TLR1/TLR2/CD14/CD36
complex for mycobacterial lipoarabinomannan and lipomannan as ligands [125], as
well as CD14/CD36/TLR6/TLR2 complex for LTA and diacyl lipoprotein
ligands [123].
Currently known PAMPs are the DNA unmethylated with CpG motifs, bacterial
LPS, and bacterial peptidoglycans. TLRs binding to PAMPs elicit antimicrobial
responses via adaptive Th1 immunity. TLR agonists are subject to study
prophylactic therapy and therapy to prevent pathogenic infection or neoplastic

transformation [126]. The extracellular domain of TLR architecture has the
Leu-rich repeats (LRR), and LRR can recognize broad ranges of PAMPs. The
recognition event deals with the PAMP structure, the TLR type, the
heterodimerization, the recruitment of co-receptors, and carbohydrate-binding lectin
of parasites. TLR N-glycans are targeted by the lectin CRD. Lectin binding elicits
cell signaling for proinflammatory cytokines, and this lectin response resembles with
PAMP recognition response. Thus, the lectins are functional TLRs agonists. PAMP
ligands of TLR2 and TLR4 elicit dimer formation of the ectodomains, leading to
conformational shifts for homodimer formation of the cytoplasmic TIR domains and
assembly with MyD88.
The TIR domain-containing adapter molecule (TICAM)-1, which is also known
as TRIF, recruits its specific TLRs. TLR2, CD282, recognizes foreign substances of
LTA. TLR2 recognizes and develops immune responses against Gram-positive
bacteria via peptidoglycan, lipoprotein, and LTA, upregulating expression of the
chemokines of CCL2 and CXCL8 to recruit immature subset of DCs via upregulated
NOD2 expression [127]. TLR2 is involved in the functions of TLR1 and TLR6 to
form heterodimers for bacterial lipoproteins and lipopeptide recognition
[128]. TLR2 and TLR6 recognize the diacyl lipoprotein forms produced in Myco-
plasma fermentans [129]. TLR4 specifically recognizes the Gram-negative bacterial
LPS [130]. Upon activation, TLR4 induces the proinflammatory cytokine expres-
sion. TLR2 is expressed on macrophages and DCs. The TLR5 ligand is the bacterial
flagellum required for bacterial motility [131]. Of interests, several pathogens
including Bartonella bacilliformis, H. pylori, and C. jejuni strains express flagellins
but not recognized by TLR5 because of their mutations of amino acid sequences
[132]. The TLR family of TLR3, TLR7, TLR8, and TLR9 are not expressed on
surfaces, but expressed on intracellular, cytoplasmic endosomes. They recognize
DNAs and RNAs. For example, TLR3/TLR7/TLR8 recognize the ds-/ssRNA spe-
cies. TLR9 recognizes unmethylated CpG DNA [133]. The dsRNA is recognized by
TLR3 and this is a viral receptor. TLR7/TLR8 are structurally similar, and they
recognize the same ligands such as the nucleic acid species of viruses. TLR7 in
murine DCs triggers the regular responses of IFN-α to influenza virus, while TLR8
in human recognizes the oligo-NTPs. Mouse TLR9 binds to the ssRNA virus and
vesicular stomatitis virus to induce IFN-α secretion, and they distinguish self-RNA
species from non-self-RNA species. TLR7 also recognizes synthesized guanine
analogues like imiquimod, loxoribine, and resiquimod, which are applicable for
antiviral agents.
6.4.3 Innate Immune Receptors in Malaria Infection
Malaria is caused by Plasmodium parasites that are transmitted to people through the
female Anopheles mosquitoes. The five parasite species that generate human malaria
include Plasmodium falciparum, P. knowlesi, P. malariae, P. vivax, and P. ovale.
Plasmodium parasites bind to RBC surface receptors sialylated. Plasmodium

knowlesi is a zoonotic parasite that is transmitted from long-tailed and pig-tailed
macaques causing malaria in humans in Southeast Asia. There seems to be a
congenital resistance in malaria infection, indicating that the malaria infection
requires its infection receptors to enter the red blood cells (RBC). Erythrocyte
surface receptor, for example, Duffy negative genotypes are resistant to malaria
infection. The genotype of Duffy (FyFy) population frequently found in Africa is
resistant to the infection. In contrast, the genotypes of FyaFy, FyFyb, or FyaFyb
are susceptible for infection. In the strains, P. vivax mainly infects reticulocytes,
P. malariae prefers to infect the mature RBCs, and P. falciparum infects all RBCs in
individuals. Intraerythrocytic factors which are resistant to malaria infection are
known. For example, individuals carrying abnormal Hb types including HbS,
HbC, HbE, thalassemia, sickler, glucose-6-phosphate dehydrogenase (G-6-PD) defi-
ciency, PABA deficiency, ATP deficiency, etc. are rather resistant to the infection.
Therefore, individuals having genetic disorders in RBC function exhibit the resis-
tance against malaria infection, and normal individuals are infected to die, terming
“adverse selection.” In addition, immature RBCs are preferentially targeted by
malaria infection. The immature RBCs are not easy to be cultured, and the reticu-
locytes are easy host for the infection once isolated. In addition, because the malaria
infection is not successful to infection in animals, the malaria research is not easy
compared to other infectious diseases.
On the other hand, acquired immunity in malaria is not recognized, because the
individuals who were infected once before can be also reinfected by malaria,
although antibody responses and cellular responses occur. However, the “premuni-
tion” is acceptable for malaria infection, as premunition indicates that the current
malaria patients can defense reinfection of the same strain to a less extent. This
indicates the species-specific or strain-specific concomitant immunity. In contrast,
mixed infection is also observed. Currently, there is no anti-malaria vaccination, but
only antigenic proteins and vaccine candidates are known for circumsporozoite
surface protein (CSP) that is a sporozoite surface protein antigen, Duffy binding
protein (DBP), merozoite surface proteins (MSP-1, MSP-3α, MSP-3β, and
MSP-3γ), and apical membrane antigen-1 (AMA-1). One question is raised: why
is it difficult to design such vaccination? The answers are reasonably based on the
immune evading mechanisms of malaria during infection: (1) intracellular location
(within RBC), (2) antigenic variation (from sporozoites to merozoites and gameto-
cytes), (3) immunosuppression (splenomegaly and dysfunction), and (4) interference
with immune effecter mechanisms, as the number of macrophages are decreased and
antigen presentation level is also decreased, called the “smoke screen theory.” If
merozoites are exited from RBCs, the merozoites have high motility and at fast enter
to the other RBCs.
6.4.3.1 Pathogenic Process in Malaria Plasmodium falciparum Infection
For pathogenesis and pathology, the primary events are involved in destruction of
RBC to cause severe anemia and blockage of capillary due to knobbing of infected
RBCs which tend to attach to the capillary wall (e.g., brain capillary in
P. falciparum). Knobbing events are involved in the severe headache, and the
infected RBCs are knobbing to block the blood circulation through adhesion to the
capillary blood vessels. The strain of P. falciparum is the most causative agent of
such disease phenotypes, but not P. vivax. Secondary events include anoxemic
impairment, leading to tissue dysfunction, cellular reactions, and immunopathology
(e.g., brain cell damage by TNF-α). During cellular reaction, many chemical com-
pounds such as TNF-α are released, and they damage the neuronal cells for immu-
nopathology. Therefore, knobbing causes brain damage and immunopathology of
cerebral malaria.
Receptors in innate immunity mediate systemic inflammation. Like most infec-
tious diseases, the pathology of malaria is driven by cytokines. Cytokine production
results in the symptoms of malaria that include fever, chills, rigors, headaches,
myalgias, lethargy, and more. Several strains of Plasmodium falciparum, Plasmo-
dium vivax, Plasmodium malariae, and Plasmodium ovale cause the malaria symp-
toms. To prevent and treat malaria, the first drug of choice is chloroquine, but the
recent chloroquine resistance issue is raised. The second drug is Fansidar which is
pyrimethamine or sulfadoxine. The mitochondria-acting agents are Halfan or
halofantrine as a 9-phenanthrene with an expensive price.
The first discovered drug to prevent and treat malaria was quinine, as the action
mechanisms are absolutely the same as that of chloroquine. Thereafter, artemisinin
(a sesquiterpene lactone peroxide), artemether, and artesunate were isolated from the
Artemisia plants. They are commonly linked with endoperoxides, and this endoper-
oxide is essential for the anti-malaria efficacy, because malaria protozoan heme
molecule interacts with artemisinin, artemether, or aresunate to yield the endoper-
oxide species, and endoperoxide alkylates the malaria proteins and peroxidates the
malaria lipids. At the present time, anti-malaria drugs are representatively prescribed
for atovaquone, chloroquine, doxycycline, mefloquine, primaquine, proguanil, and
hydroxychloroquine. In vertebrates, malaria are reproduced by a schizogony type,
while in intermediate hosts, they reproduce by a gametogony. With regard to the
innate immune receptors for malaria parasites, several important aspects should be
considered: (1) Define parasite targets for innate immune receptors, (2) identify
relevant innate immune receptors, (3) define their role on host/parasite interaction
and disease outcome, and (4) elaborate prophylactic/therapeutic interventions
employing TLR agonists or antagonists.
The malaria pathology has been associated with the three major steps of an
excessive production of inflammatory cytokines and septic shock-like syndrome,
progress in severe anemia, and adhesive accumulation of the infected RBCs to
capillary vessel walls, contributing to the RBC destroy and damaged erythropoiesis.
Malaria toxin hypothesis has been suggested in 1889 by Golgi. Fever-causing factor
Protein
Gal Man P EtN
Gal Man • PLC: Phosphadyl lipase C

• EtN: Ethanolamine
Gal Gal Man
GPI-anchored protein
(glycophosphadylinositol)
GlcNAc Ino
Fig. 6.3 The Plasmodium parasite of malaria protozoa is coated with a GPI anchor
has been mentioned to be pyrogenic toxin by a mechanism(s) that Plasmodium

parasite constituents activate innate immune receptors to generate the inflammatory
mediators. Why do humans get fever during malaria? The search for the “malaria
toxin” has been done. What innate immune receptors are activated during disease
and what microbial products (“malarial toxins”) activate these receptors? Is the
source of inflammation a molecule of the parasite membrane? Do bear malaria like
Gram-negative bacterial LPS? Is the source of inflammation a membrane molecule
of the merozoite? In other words, is the malaria toxin just like LPS? Plasmodium
strains are decorated with specific GPI-Aps, as known for variable surface glyco-
proteins of other protozoan strains in order to escape from host immunity (Fig. 6.3).
GPI-APs activate TLR2 signaling in hosts. For example, the GPI-APs of T. cruzi
activate mainly TLR2 signaling and partly TLR4 signaling. P. falciparum GPI-APs
also exhibit the TLR stimulating activity in the human myeloid cells.
6.4.3.2 P. falciparum GPI Anchor Glycosylation
General functions of GPI anchors are depicted in the following four main subjects of
(1) stable association of proteins with the plasma membrane, but with measurable
“off-rates” from the membrane and the potential to be shed by phospholipases,
(2) the potential for very high lateral mobility on the plane of the lipid bilayer,
(3) the potential to insulate the protein domain from the cell interior (may be
important in protozoa), and (4) the potential to participate in signaling through
association with other membrane-spanning components in lipid rafts.
GPI-anchored glycoproteins are mainly located as GPI membrane anchors. In other

words, GPI stands for a phosphatidylinositol (PI) phospholipid linked via a glycosyl,
or sugar chain, component to the C-terminal protein region. GPI-anchored proteins
are processed by GPI transamidase. Proteins are bound to membranes, and many
GPI-anchored proteins are functional on the plasma membranes [134]. The overall
GPI-anchored protein biosynthetic pathway has been reported with the structural
remodeling and transport [135]. GPI is biosynthesized using free PI in the ER region
via 10 more steps. The en bloc transferring of proteins requires multiples genes of
20 more genes, which are termed to PIG genes. In the cytosolic region of ER, the two
steps are involved in initiation of protein transferring reaction. Thereafter, the next
steps take place in the luminal side of ER. Alkylated lipid species initially generated
in the peroxisomal organelle are supplied to the ER lumen. The alkylated lipids are
then modified to the 1-alkyl-2-acyl group linked to GPIs. Genetic disorders such as
PNH- and GPI-deficient diseases are attributed to defects of the PIG-A gene and
PIG-M or PIG-V gene, respectively. For linkage of the GPIs and proteins, naïve
protein forms are then subjected to modification by GPI through their specific sites
for GPI linking. The GPI linking sites consist of a ω site, a space with hydrophilic
region, and a hydrophobic region. The native protein forms are biosynthesized
independently from the GPI synthesis. The transamidation reaction is catalyzed by
the complex enzyme of GPI transamidase. The native protein modification with GPI
species is one of the post-translational modifications (PTMs).
The GPI remodeling in their structures occurs in the combination of the GPI lipid
portions with glycans, by means of catalytic activity of the post-GPI attachment to
proteins (PGAPs). A fatty acyl group, which is a palmitic acid in most GPI-Aps of
eukaryotes, is cleaved from the acylated inositol by a deacylase termed PGAP-1
enzyme. Thereafter, another PGAP-5 enzyme cleaves to an EtN-P from Man-2
structure. The Man-2 binds to p24 family proteins and efficiently sorts the p24
family proteins to the ER region. During gradual budding out in the ER region, the
produced GPI-APs are incorporated to small delivery vesicle of COPII by means of
specific transportation proteins such as Sec24C and Sec24D delivery proteins.
Sequentially, the GPI-Aps formed are then delivered to the surfaces or membranes
via the Golgi complex. In the Golgi complex, the GPI-APs are further modified for
their specific structures, mainly in their remodelings of fatty acids [135]. The
remodeling of the GPI-APs includes changes in the composition of the GPI lipid
parts, to fit lipid raft-competent GPI-Aps of membranes. The free PI is used as a PI
source, as the PI consists of diacyl fatty chain and unsaturated lipids at the sn-2
position. The representative diacyl unsaturated lipid is 1-stearoyl, 2-arachidonoyl
PI. They are translocated to the ER luminal side by flippase activity, GlcN-PI species
are remodeled by fatty acyl groups such as palmitic acid to the inositol, the lipid part
is exchanged, and peroxisome-borne alkyl-acyl donors are used. PGAP1 enzyme
eliminates a fatty acyl group, which is linked to the inositol of GPI-APs, and the
GPI-APs are sorted to the ERES. GPI-APs are delivered to the Golgi apparatus to
receive further structural remodeling, termed “remodeling of fatty acids,” for for-
mation of GPI-AP-lipid raft complex [136]. However, the unsaturated lipid group
attached to the position of sn-2 is cleaved off by PGAP3, and the saturated lipid
chain like stearoyl chain is added by PGAP2, although the enzyme activities of
PGAP2 and PGAP3 are not yet defined. Transglycosylation reaction results in that a
GPI cell wall protein becomes cross-linked to cell wall β1,6-glucan via its GPI
glycan [137].
Defect of GPI biosynthesis in tissues abolishes GPI-APs surfaced expression and
causes lethality in the early embryonic development. The partially displaying
patients have been reported to be caused by a point mutation within the Sp1
transcriptional factor binding site in the PIG-M gene promoter for the
mannosyltransferase for Man-1. This point mutation severely reduces the expression
levels of the PIG-M protein, displaying the onset of venous thrombosis and seizures.
Sodium butyrate, a histone deacetylase inhibitor, improved the PIG-M expression
and the surface expression of GPI-APs. Another PIG-V gene encodes the
mannosyltransferase for Man-2, and the gene is often mutated in hyperphosphatasia
mental retardation (HPMR) syndrome (or Mabry syndrome), an autosomal recessive
form of mental retardation with elevated serum alkaline phosphatase. The reduced
expression of PIG-V and surface GPI-Aps are associated with the onset of the
disease. Human African sleeping sickness is caused by Trypanosoma brucei.
Trypanosoma forms in blood smear from patient with African trypanosomiasis by
many protein variants in common GPI anchors, where the surface coat is made of a
dense monolayer of variant surface glycoprotein (VSG) [138].
In 2004, R. Gazzinelli and his colleagues began purifying P. falciparum parasites
to study [139]. Unfortunately, purified P. falciparum, the parasites without the RBC
membranes or other debris, failed to induce cytokines from PBMC or mouse
macrophages. He found that the malarial parasite is coated with a GPI-Aps.
6.4.3.3 GPI Anchor on the Merozoite Surface in Inflammatory

and TLR2, TLR4, and TLR9
The malarial parasites are coated with a variety of GPI anchors. Therefore, the
question of “Is the inflammation source, malaria toxin, a component of the outer
membrane just like LPS?” was raised for a while? If GPI-APs of P. falciparum bear
TLR activation activity, it is now assumed that the GPI anchors present on Plasmo-
dium’ cellular membrane function as LPS that is a “malaria toxin” [140]. GPI anchor
on the merozoite surface induces inflammatory response and activates TLR2, but
weakly activates TLR4, as GPI anchors from T. cruzi are also TLR2 ligands. Malaria
PAMP is a malaria pigment, hemozoin, that is a hemin’s crystalline polymer as a
degraded hemoglobin. The produced level of hemozoin indicates the severity of the
malaria disease. Then, there was basic question on “What are the innate immune
receptors in innate immune recognition system involved on hemozoin recognition?”
The question has been raised because the innate immune responses are well con-
served even to human through Drosophila. The next question was “How does
malaria detoxify protoporphyrin IX (hemin)?” The inflammatory component of
hemozoin has been known to be DNA. For the hypothesis, hemozoin is internalized
into the phagolysosome, and TLR9 can be recruited to the phagosome thus causing
for the expression of proinflammatory cytokine of TNF-α (which causes fever).

Later, its DNA is liberated from the crystal surface by the proteases and other
enzymes. What is hemozoin? Hemozoin is the crystalline breakdown product of
hemoglobin. The levels of hemozoin in circulating phagocytes correlates with
disease severity. It is how malaria detoxifies protoporphyrin IX, also known as
hemin. Hemozoin chemistry made clear in solution that hemin chloride can form
β-hematin dimer by bonding between carboxylic groups and heme. However,
synthetic hemozoin, if manufactured from highly purified hemin, lacked cytokine-
inducing activity. There was a big hypothesis as Christiane Nüsslein-Volhard, Nobel
laureate (Medicine, 1995), suggested that hemozoin is a potential candidate for a
proinflammatory substance made by malaria. Toll receptors are likely to crucially act
in the pathogenesis of malaria through the recognition of hypothesis of hemozoin.
Her question was “What is a Toll receptor?” Nüsslein-Volhard is associated with the
discovery of Toll, which led to the identification of TLRs. In the dictionary, “Toll”
means amazing, bodacious, cool, corky, crazy, frantic, furious, great, like blazes,
mad, madcap, screaming, super, wild, or wow. Later, Toll was cloned by Schneider
and Anderson and recognized to be a type I transmembrane (TM) receptor with a
high homology to the cytosolic IL-1R (TIR domain) region [141]. The observation
that the Toll had a domain in common with the IL-1R led to the hypothesis that IL-1
is associated with innate immunity. The innate immune responses are actually well
conserved between Drosophila and humans. For example, the purple sea urchin has
222 TLRs [142].
Another answer has been quoted from the results that phagocytized hemozoin
colocalizes with TLR9 in the lysosomes, where CpG and AT-rich DNA activate
different innate immune pathways. TLR9 activates host innate immune response by
binding to hemozoin of the malaria pigments [143]. Plasmodium parasites in the red
blood cells degrade hemoglobins to a polymeric form known as hemozoin. The
hemozoins are exposed in the plasma blood and the reticuloendothelium captures the
hemozoins. Hemozoin is immunologically active and modulates the innate immune
system. The purified hemozoin is a TLR9 ligand and activates innate immune
responses. The hemozoin responses are blocked in TLR9-deficient null mice and
MyD88-deficient null mice. However, the hemozoin-induced responses are not
found in the several null mice. For example, the Toll-IL-1R domain-containing
adaptor-inducing IFN-β, TLR2, TLR4, or TLR7-deficient null mice are not
responding to the hemozoin. Chemically pure hemozoin synthesized also activates
innate immune responses in vivo by a TLR9. Bacterial DNA is non-methylated, rich
in CpG, and immunologically active, but CpG-rich DNA is recognized by TLR9.
Natural hemozoin activates cytokine production via TLR9/MyD88. But the stimu-
latory activity of hemozoin was destroyed by DNase. Thus, the cytokine-inducing
component of hemozoin has been suggested to be DNA. Is the DNA on hemozoin
originated from human or malarial? PCR analysis of hemozoin showed that most of
its DNA is malarial-based. Does malaria DNA activate innate immune responses?
Can malaria DNA actually stimulate cells, presumably via TLR9? The cell biology
of TLR9 is complex. TLR9 resides in the ER organelle. TLR9 translocates to the
endosomal compartment to bind its ligand (CpG-rich DNA). Malaria DNA is
stimulatory for DCs only when introduced into cells directly with the transfection
reagent (endosomal compartment). The malaria genome contains 269 CpG repeats.
Hemozoin functions to delivery DNAs to a TLR9-recruited vesicles of cytosolic
compartments. However, most malaria DNA is AT-rich. The malaria genome
contains the motif ATTTTTAC over 6000 times, and AT-rich DNA mimics malarial
DNA. Transfection of AT-rich DNA drives a variety of promoter constructs like
native DNA and activates IL-6/IL-1β/IFN-β/TNF-α cytokines. Six AT-rich motifs
have been studied (AT1-AT6), where AT-2 sequence is
GCACACATTTTTACTAAAAC. Microarray analysis of 14 patients with febrile
P. falciparum showed an “IFN signature.” Hemozoin and DNA complex rapidly
activates IFN-β production from PBMC. From the discovery of Dr. Akira Shizuo, it
has been clear that hemozoin constitutes the first non-nucleotide ligand for TLR9.
Therefore, the malaria infection is involved in step 1; internalization of hemozoin by
phagocytes leads to activation of MyD88 via TLR9, and this event results in IFN-γ
production, inflammasome formation, and caspase-1 activation. Therefore, many
researchers have assumed that can we interfere with pathogenesis of malaria by
blocking MyD88 activation [144]. Step 2: IFN-γ priming of phagocytes induces the
enhanced TLR expression, TNF-α production, and maximal production of pro-IL-
1β. When caspase-1 is cleaved and matured, the inflammatory reaction is progressed.
6.4.3.4 Inflammasome and Sialic Acid Tropism in Malaria Recognition
The next question was that “Is the inflammasome involved in malaria recognition?
What is a inflammasome?” And the answer was that the inflammasome is a complex
form of multiproteins, which is associated with IL-1β production. Therefore, in
activation of IL-1β via caspase-1, malarial AT-rich DNA appears to induce the
inflammasome. For the role of fever-causing IL-1β, AT-rich motifs have been
suggested to be a major way in which malaria causes fever. Then, the generation
of type I interferons has been assumed to involve the inflammasome, because
activation of type I interferons requires the inflammasome component Nalp3, but
not ASC. Continuously, a basic question is raised. How do the hemozoin-surfaced
DNAs migrate from the phagosomes to the cytosolic area? Different to IFN-β, IL-1β
activation requires caspase-1. Phagocytosis of inert particles causes leakage of
phagolysosomes, releasing several molecules such as asbestos, SAs, uric acid, or
amyloid-β peptides (Aβ) known as a plaque rupture factor of Alzheimer’s
disease (AD).
The Neu5Gc is a factor for P. knowlesi invasion of human RBCs. While
macaques synthesize the Neu5Gc, humans are mutated in the CMAH enzyme to
synthesize Neu5Gc. P. knowlesi is restricted in its invasion of human RBCs due to
the absence of Neu5Gc. Plasmodium infection pathway is involved in attachment,
reorientation, and invasion. When the chimpanzee CMAH gene (PtCMAH) has been
transfected in human CD34+ hematopoietic stem cells, the transfected human RBCs
express different SA variants. P. knowlesi invades Neu5Gc-positive rhesus macaque
RBCs but not Neu5Ac-producing human RBCs. P. knowlesi invades Neu5Gc-
Fig. 6.4 Human-adapted

P. knowlesi invasion is invasion
Plasmodium Chimpanzees
Neu5Gc-independent by the Knowlesi, reichenowi (Neu5Gc+)
mechanism that has adapted (Laverania)
to use NeuAc-binding
ligands and SA-independent
DBP gene duplication
2. Evolution 1. CMAH Loss
Plasmodium
invasion Human
falciparum
(Neu5Gc-)
producing PtCMAH cRBCs, but little Neu5Ac-positive cRBCs. Duffy antigen/

chemokine receptor (DARC) is the human receptor for P. knowlesi invasion.
Duffy binding proteins (DBP) act as ligands. Glycophorin A (GPA) and GPC are
used as glycosylated receptors. For the determinants of SA-dependent invasion in
P. knowlesi, the binding domains of the P. knowlesi DBP ligands, PkDBPa,
PkDBPb, and PkDBPg, were analyzed. PkDBPb and PkDBPg did not bind to
neuraminidase-treated macaque RBC ghosts. PkDBPb and PkDBPg bind to
PtCMAH-transfected cells. PkDBPb and PkDBPg are Neu5Gc-binding ligands for
invasion into Neu5Gc-expressing RBCs. Thus, P. knowlesi invades human RBCs
via surface Neu5Gc, when P. knowlesi strain H(Pk H) adaptation to human RBCs is
generated. In the invasion of P. knowlesi strain Pk H into PtCMAH cRBCs, how
does Neu5Gc engage in the human-adapted P. knowlesi strain? P. knowlesi strain Pk
H invades human and macaque RBCs equally. The question is “How does
P. knowlesi evolve to infect human RBCs?” Duplication of the SA-independent
PkDBPa gene as well as a complete deletion of the SA-dependent PkDBPg gene was
detected in P. knowlesi Pk YH1. However, it evolves to invade human RBCs in a
SA-independent manner. Neu5Gc is a key host determinant of the tropism by
P. knowlesi. Human-adapted P. knowlesi invasion is Neu5Gc-independent. This is
the mechanism that P. knowlesi Pk YH1 has adapted to use NeuAc-binding ligands
for human infection, preventing the NeuGc dependence for invasion (Fig. 6.4)
[145]. Evolution seems to be progressed to the direction of infected mosquito !
monkey ! human ! human/monkey.
In summary, for the malaria infection, (1) infection with Plasmodium leads to a
proinflammatory priming and hyper-responsiveness of TLRs; (2) in mice, TLR9 has
an important role in promoting this proinflammatory priming, which is mediated by
DC-released IL-12 and NK cell/T cell-produced IFN-γ; and (3) treatment with an
antagonist of nucleic acid sensing TLRs prevents cytokinemia and lethality in a
rodent model of cerebral malaria.
6.4.4 Innate Immunity Receptors in Protozoan Parasite

Toxoplasma gondii
Toxoplasma gondii, a protozoa parasite, is a group of the phylum Apicomplexa as a

coccidian parasite and causes toxoplasmosis. The protozoan parasite hosts include
vertebrates such as humans with one third of the worldwide population infection.
During T. gondii life cycle, parasites change in cellular stages with characteristic
phenotypes including morphology and physiology. The cycled stages are the
tachyzoites, merozoites, tissue-type bradyzoites, and sporozoites. Tachyzoite stage
is specific in phenotypes such as motile and multiple population in the host.
Tachyzoites spread through the whole body and convert to stage of tissue-locating
bradyzoites. Merozoites fast multiplies the parasite population within the cat intes-
tine. Bradyzoites convert into merozoites within intestinal epithelial cells.
Bradyzoites are the slowly dividing stage of parasites to form tissue cysts. Sporo-
zoite stage indicates the stage of the parasite present in oocysts. Host innate immune
cells produce IL-12 upon infection of T. gondii, and the produced IL-12 elicits
activation of NK cells. Tryptophan is a key amino acid for T. gondii. IFN-γ elicits the
indole-amine-2,3-dioxygenase (IDO) and Trp-2,3-DO (TDO). Both enzymes of IDO
and TDO degrade Tryptophan. The strains exhibit their specific endemicity and
transmissibility in epidemiological aspects [146], occupying about one third carriers
of the T. gondii-infected human population [147]. The major symptoms include
immune compromise [148], after the protozoan invasion to host cells through the
tachyzoite’s motility via two apically located organelle of rhoptries and
micronemes [149].
6.4.4.1 Host Carbohydrate-Binding Domain of Toxoplasma

gondii-Secreted Proteins
For the host infection, first, membrane proteins are involved. T. gondii infection of
host cells occurs by secreted microneme proteins (MICs) as well as ROP and RON
rhoptry proteins. Lac-recognition fraction (Lac+) of T. gondii extracts contains
MIC1 and MIC4, and they activate splenic immune cells to express IL-12. Immu-
nization of mice with Lac+, MIC1, or MIC4 protects the hosts from T. gondii
infection. The well-known PRRs of TLRs transduce their signals through MyD88
adaptor protein and downstream signaling for NF-κB activation. Thus, the
MyD88–67 KO mice are highly susceptible to infection of T. gondii because
TLRs act as receptor for the parasitic glycans or antigens, functioning as a modulator
of the innate immunity.
T. gondii adheres and invades host cell through carbohydrate recognition by MIC
as CRD. MICs are expressed as parasite membrane complexes from distinct organ-
elles with MIC1, MIC4, and MIC6. MIC1, MIC4, and MIC6 are preset as complexes
with other T. gondii proteins for the host adhesion and invasion as well as the
virulence factors. The three MIC molecules of MIC1, MIC4, and MIC6 are
assembled with adhesin complex resided on cell surfaces. The MIC6 is a parasite
surface-embedded form as transmembrane complex. MIC1, MIC4, and MIC6 of
T. gondii tachyzoites are trafficking to membrane-containing microneme organelles
once complexed in ER. MIC1 and MIC4 are extracellularly exposed and directly
bind to surface receptors of host cells. MIC1 and MIC4 play an integral role for the
first step of host cell-tachyzoite attachment and adhesion. MIC1 and MIC4 bind to
terminal SA residues and Gal residues, respectively, due to their lectin domains.
MIC1 [150–152] and MIC4 [150, 153] are soluble adhesins and membrane-
spanning soluble adhesin proteins. The MIC1–MIC4 and MIC6 complex adheres
to host cells. The MIC proteins from oligomers with different molecules increase the
parasite repertoire to invade host cells. Interaction between MIC1 and MIC4 with the
host cells triggers to respond immune cells. MIC1 and MIC4 were later evidenced as
TLR2-binding ligand and TLR4-binding ligand. The two MIC molecules of MIC1
and MIC4 elicit IL-12 gene expression in myeloid immune cells. T. gondii MIC1 and
MIC4 proteins enhance the expression of IL-12 from adherent cells of splenocytes.
Substantially, immunization with the lactose-binding recombinant MIC1 or recom-
binant MIC4 prevents T. gondii infection in mice. MIC1 and MIC4 activate DCs and
macrophages through TLR2 and TLR4 in a way of sugar recognition. They contain
CRDs. The MIC1 and MIC4 recognize the N-glycans linked to TLR2 and TLR4.
MIC1 is versatile in changing oh host cells for T. gondii infection competency.
MIC1 and MIC4 immunomodulate the hosts for Th1 protective behavior. The Th1
cells express IL-12, and the produced IL-12 activates NK/T cells and consequently
expresses IFN-γ in Th1 cells and contributes to host protection against intracellularly
infected pathogens. This MIC1 and MIC4 activate TLR axis signaling pathway to
elicit proinflammatory cytokine release in macrophages and DCs. MIC1 and MIC4
immunization protects against T. gondii infection, due to effects of the microneme
MIC proteins such as MIC3, MIC6, MIC8, MIC11, and MIC13 [154].
In molecular level, MIC1 and MIC4 proteins have distinct carbohydrate recog-
nition domains (CRD) [150, 155, 156]. For the IL-12 expression in response to
several infections, the innate immune receptors of TLR2 and TLR4 are selected.
Especially, TLR2 Leu-rich repeat domain in the extracellular region consists of four
N-glycans, and TLR4 contains nine N-glycans [157]. MIC1 and MIC4 can recognize
the TLR2 and TLR4 N-glycans on innate immune cells such as APCs and APCs
which produce IL-12. Thus, the first step of the parasite infection is that MIC1 and
MIC4 bind to TLR2/TLR4. MIC1/MIC4 target TLR2/TLR4 through the lectin-
carbohydrate interactions or PCI. Among them, the MIC1 lectin is more competent
for T. gondii infection and virulence. MIC1 binds to terminal α2,3-SA residue
attached to β-Gal [141, 158], while MIC4 binds to terminal β1,4- or β1,3-Gal
[150, 151, 159]. Binding of MIC1 or MIC4 activates immune cells. T. gondii
molecules activate TLR signaling [154, 160]. In binding specificity of MICs to
TLR2, MIC1 binds to the second, third, and fourth numbers of its TLR2 N-glycans,
whereas MIC4 specifically recognizes the third number of TLR2 N-glycans. During
TLR2 and lipopeptide agonist interaction, heterodimers between TLR2 and TLR1 or
TLR6 are formed. Consequent heterodimerization complex increases in the number
of accessible TLR2 agonists and potentiates the multiple association with
co-receptor CD14, GPI-anchored protein, and B scavenger receptor CD36. CD14 is

the known molecule in LPS binding of TLR4 ligand. The CD14 as a co-receptor
associates with TLR4 and consequently complexes with LPS, and CD14 recognizes
Mycobacterium tuberculosis lipoproteins by TLR2. This event enhances the TLR2-
mediated signaling. The MIC1 and MIC4 lectin domains bind to α2,3-sialyl-LacNac
and β1,3- or β1,4-GalNAc of N-glycans in host surface molecules [150, 151],
respectively. MIC1 and MIC4 lectin domains bind to N-glycans linked to the
ectodomain parts of TLR2 and TLR4 in host macrophages and DC for IL-12 release.
Pathogen lectin binding to TLR2 N-glycans elicits signaling which is well reported
in the typical lipopeptide agonists. TLR bindings to PAMPs elicit receptor dimer-
ization and consequently associate with TIR-1 domains towards NF-κB-involved
synthesis of chemokines, proinflammatory cytokines, and type I IFNs. RAW264.7
macrophages treated with MIC1 or MIC4 exhibit NF-κB, and the response resem-
bles with the PAMP action. Binding of MIC1 or MIC4 to homodimeric or
heterodimeric TLR2 elicits Myd-associated endosome complex. TAK1 activates
the canonical MAPK and NF-κB signalings for the IL-12 expression by MIC1 and
MIC4. BMDMs incubated with the TAK1 inhibitor and induced by microneme MIC
proteins block the IL-12 expression; thus, canonical NF-κB activation, but not
non-canonical, is relevant for the MIC1- or MIC4-activated response in cells. For
the mechanism to address the TLR role in T. gondii infection, MIC1 or MIC4 elicits
formation of TLR2/TLR1 as a heterodimer and heterodimer TLR2/TLR6. TLR2-
involved heterodimerization stimulates MIC response in TLR2-expressing HEK
cells. This is different from the results obtained from Mycobacterium leprae binding
to TLR2 homodimer or TLR2/TLR1 heterodimer. Ligand binding to TLR
ectodomain induces receptor dimerization.
6.4.4.2 T. gondii GPI-Anchored Protein Recognition of TLR2, TLR4,

and Galectins of Host APCs
GPI-anchored proteins are mainly present on the T. gondii tachyzoite surface.

Second, GPI-anchored proteins bind to TLR2 and TLR4 as well as galectins on
the host APCs [159]. GPI-anchored proteins are mainly present on the T. gondii
tachyzoite surface. T. gondii GPI glycans and lipids elicit the TNF-α releases in
macrophages through TLR2 and TLR4 in the NF-κB signaling when exposed to
T. gondii GPIs. Although CD14 is also involved in TLR2-mediated macrophage
behavior against Trypanosoma cruzi GPIs [158], T. gondii GPIs do not affect the
CD14 responses during the TNF-α expression in macrophages
[161]. Diacylglycerols derived from the T. gondii GPIs cleavage can activate
macrophages to produce TNF-α through TLR2 and TLR4. But only the TLR2 is
activated by GPIa or the whole GPIs. In contrast, CD14 is not involved in TNF-α
expression during T. gondii GPI treatment [161]. T. gondii GPIs act as ligands of
galectin-3, where galectin-3 has long glycan-binding sites and binds to a broad range
of carbohydrate structures. T. gondii parasite expresses six GPIs on surface. GPIs III
and VI contain a GalNAcβ1,4 linked to Man residue of the core glycan. GPI-I,
GPI-II, GPI-IV, and GPI-V contain an additional Glcα1,4 linked to the GalNAc
residue. Binding of GalNAcβ1,4 carbohydrates is unusual to galectins, as galectin-3
only binds to GalNAcβ1–4GlcNAc carbohydrates produced by S. mansoni strain
[162]. Galectin-3 associates with its binding to the N-acetyl group in GalNAc O-2
position, but other galectins do not. Lactose prevents galectin-3 recognition with
GalNAcβ1,4GlcNAc structure. Galectin-3 binds to the polygalactose (Galβ1,3)n
structure seen on Leishmania major lipophosphoglycans, but galectin-1 does not
recognize the L. major. Thus, polygalactose structure recognizes the galectin-3
binding site. The polygalactose species are not present in Leishmania donovani
strains. Therefore, galectin-3 differentially recognizes the two parasites. However,
GPI species present on virulent T. gondii and nonvirulent T. gondii strains are
different in their structures, but galectin-3 binds to all the GPIs. The diacylglycerol
species linked to the T. gondii GPIs can recognize galectin-1. Galectin-1 recognizes
Davanat, having β1,4-D-Man residues and D-Gal residues α1,6 linkage [163]. The
β-Gal-binding domain still holds the galectin-1-Davanat complex. Galectin-1 is the
tissue plasminogen activator (TPA) receptor. Binding of TPA to galectin-1 is
stronger than to galectin-3 [164]. This indicates that galectins are not always binding
to β-Gal.
GPI-AP species produced by T. gondii strains act as ligands for galectin-3 and
induce TNF-α expression in macrophages by galectin-3. Galectin-3 is a TLR-related
molecule responding to the T. gondii GPIs. T. gondii GPI-elicited TNF-α expression
is mediated by TLR2 and TLR4 in macrophages [161]. S. mansoni- or T. gondii-
infected mice, which are galectin-3 deficient, show high Th-1 cell-mediated immune
responses and reduce inflammatory response [165]. The galectin-3 deficiency pro-
duced high levels of Th1 cytokines, because galectin-3 controls Th1 cytokine
production. Galectin-3-deficient macrophages express lowly TNF-α upon Candida
albicans treatment [166]. Galectin-3-deficient macrophages can not produce TNF-α
during treatment with T. gondii GPI species. Most immune cells, except for macro-
phages, highly produce inflammatory Th-1 cell cytokines in galectin-3-negative KO
animals. The galectin-3 binds to N-glycans in TLRs [167]. The surfaced expression
of TLR2 is increased in the galectin-3 null macrophages, which are negative for the
galectin-3 expression [168]. Galectin-1 recognizes protozoan parasites. Expression
of the endogenous galectin-1 is enhanced by T. cruzi and also in cardiologic organ
shown by chronic Chagas disease [169]. The T. cruzi adhesion to smooth muscle
cells or muscle cells of the heart is regulated by GPI, and heart galectin-1 enables
parasite invasion [170]. In the human cervical cells, galectin-1 expressed on the
epithelial cells acts also as the protozoa Trichomonas vaginalis receptor through
binding to parasite lipophosphoglycans. Thus, host galectin-1 attaches to T. gondii.
During Candida albicans infection, galectin-3, which binds to β-Gal residues, is
associated with TLR2 on PMA-induced human THP-1 macrophage differentiation,
and galectin-3 is co-expressed with TLR4 on macrophages derived from BM
[171]. The galectin-3-recruited effector cells protect against parasite infection.
Indeed, galectin-3 is expressed on neighbored egg cells or worms in S. mansoni
infection [162]. Galectin-3 is specifically co-localized with carbohydrate structure of
GalNAcβ1,4GlcNAc seen on the egg surfaces to bind to GalNAcβ1,4GlcNAc and
subsequent macrophagic phagocytosis. Thus, GalNAcβ1,4GlcNAc is a molecular

pattern of parasites in immune recognition by galectin-3. For the galectin-3-deficient
mouse infected with S. mansoni strain, their egg sizes are reduced [172]. Galectin-3
deficiency also influences the cell numbers of the spleen-resident B cells and T cells
in S. mansoni-infected mouse but not influence the DC cell number. In mice tissues,
T. gondii infection elicits galectin-3 expression. T. gondii-infected mouse with
galectin-3 deficiency exhibits diminished inflammation responses in tissues.
Galectin-3-deficient mice showed high mortality with deficient macrophages and
neutrophils in the peritoneal region, while galectin-3-expressing mice, each
T. gondii-infected, showed high survival. Galectin-3 modulates through interference
with the prolonged life span and neutrophil activations in the T. gondii
infections [173].
TLR followed MyD88-deficient mice are easily infected with T. gondii [154]
with eliciting innate immune response. From T. gondii, several TLRs agonist
candidates are exemplified to date. Profilin has been suggested to be the mouse
TLR11-specific ligand and TLR12-specific ligand [174] and also as human TLR5-
specific ligand [175]. GPI-anchored proteins have also been regarded as TLR2- and
TLR4-specific ligands [161]. In addition, certain parasite nucleic acids are known as
TLR7 and TLR9 ligands [174]. Until now, the known agonists recognized by
T. gondii TLRs include murine TLR11 and TLR12 ligands as well as human
TLR-5 ligand and profilin. In addition, human GPI-APs are the ligands for TLR2
and TLR4 activation, and DNAs are for TLR7/TLR9 recognition. MIC1 and MIC4
are ligands of TLR2 and TLR4. In MIC-TLR2 binding, MIC1 binds to the first,
second, and fourth N-glycans linked to the receptor, while MIC4 recognizes the third
N-glycans linked to TLR2. TLR2 binds to lipopeptide. TLR1–TLR2 or TLT1–TLR6
heterodimerization is formed. TLR co-receptors such as GPI-AP CD14 and B
scavenger receptor CD36 also enhance TLR2 signaling.
Pathogen and plant lectin binding to glycans elicit their signalings. Like
non-canonical ligands including cationic lipids and nickel ions, lectins help the
homodimerization to the receptor ectodomains [176]. The TLR immune cells
response has been shown in the ArtinM, a plant lectin [177], which is a Man
recognition lectin. The lectin binds to the TLR2 N-glycans and its CD14
co-receptor [178]. The first N-glycan-site is found on the LRR solenoid. The second
and third N-glycan sites are located on the LRR concave surface. The fourth is
located on the LRR16 of the inner surface [157]. TLR2 heterodimerizes with TLR1
or TLR6. ArtinM does not prevent the heterodimerization responses [178]. ArtinM
lectin specific for the tri-Man core in N-glycan-induced macrophages expresses
IL-12 through ArtinM interaction with N-glycans on TLR2 and CD14. The
ArtinM-TLR2 N-glycan or ArtinM-CD14 N-glycan membrane complex undergo
cell signaling [179]. CD14 and CD36 are associated with MIC1- or MIC4-involved
TLR2/TLR1 or TLR2/TLR6 signaling. Without CD14 or CD36 co-receptors, the
TLR2/TLR1- or TLR2/TLR6-expressing HEK293T cells are activated by the MIC
protein, through IL-8 and NF-κB signaling [180].
Apart from the above parasite molecules to interact with host cells, some
T. gondii proteins TLR-dependently stimulate innate immune cells, but without
sugar interaction. For example, profilin (TgPRF) is essentially involved in the
T. gondii parasite’s gliding motility via actin polymerization, and T. gondii profilin
binds to TLR11 [181] and TLR12 [182, 183]. Also, T. gondii parasite RNA binds to
TLR7 of the host cells, and DNA binds to TLR9 as ligands [183]. T. gondii-derived
GPIs activate TLR2 and TLR4 [184]. All of the TLRs induced by the T. gondii
ligands eventually culminate in MyD88 pathway stimulation towards IL-12 release.
Some other T. gondii proteins elicit the proinflammatory IL-12 release, without
cooperation of TLRs. In fact, GRA-7 molecule, known as the dense granule
protein-7, elicits the MyD88-NF-kB axis signaling towards IL-6/IL-12/TNF-α
release [185]. MIC3 elicits TNF-α release and M1 macrophage polarization [186],
but T. gondii type II strain GRA15 elicits NF-kB towards IL-12 release. The
T. gondii GRA24 elicits the p38 MAP kinase autophosphorylation and downstream
signaling towards proinflammatory cytokine and chemokine release. In contrary,
T. gondii TgIST prevents IFN-γ release through inhibition of STAT1-involved
proinflammatory gene expression. Thus, T. gondii has both induction and suppres-
sion activities against the host immunity to control T. gondii pathogenesis in hosts
[187]. TLR-exogenous ligand recognition opens new ways for the therapeutic drug
design. Lectins are used as TLR agonists to treat infections or tumors in immuno-
compromised patients. Lectins are also used as adjuvants to boost Th1 and Th17
responses. Paracoccin (PCN) lectin of Paracoccidioides brasiliensis also elicits
immune responses through TLR4 and TLR2 N-glycan recognition. The TLR2
N-glycans elicit the PCN response [188]. PCN targets the fourth N-glycan on
TLR2. The TLR2 N-glycans in numbers 1 to 4 are attached to Asn116, Asn199,
Asn416, and Asn442.
6.5 Pathogen Recognition and Adaptive Immune Responses

in Acquired Immunity
Although vertebrates such as mammals are often exposed to infectious pathogens,

relevant resistant mechanism(s) are normally explained through the innate immunity
and continued adaptive immunity. The two innate and acquired immunities differ
functionally in their actions. The innate immune-specific responses are classified to
an indeed inborn immunity or the first defense line immunity, which is also regarded
as a dispensable defense line. From the recent studies, it has also been argued to have
the adaptive immunity-like activity, as it is also distinguishable self-antigens from
non-self-antigens as well as activates adaptive immunity. The immune system
recognizes conserved molecular pattern structures on the pathogen surfaces such
PAMPs, where PAMPS include extracellular components of microbes such as cell
wall molecules and DNAs. Molecular recognition is mediated by transmembrane or
soluble receptors, in which germline subsets encode the PRR proteins. The best-
defined receptors are the TLRs and the CLRs. Upon engagement of PRRs with
PAMPs, immune cells produce humoral mediators of cytokines, chemokines, or
6.5 Pathogen Recognition and Adaptive Immune Responses in Acquired Immunity 293
complement elements to eliminate the pathogen and to initiate the adaptive

responses. PRRs recognize microbial elements as well as structure-mimic shapes
of damaged cells of hosts. Endogenous ligands for PRR recognition include
DAMPs, which are served for the contextual cell death and damage
[189]. DAMPS and PAMPs induce proinflammatory and anti-pathogenic signaling
for innate myeloid cells, consequently inducing the adaptive immune response
[190]. The innate myeloid cells detect microbial products and respond to adaptive
CD8 CTLs. The CD8 CTLs strictly control pathogen infections like virus. Naive
CD8 CTLs initially respond to molecules alarmed by danger signals such as
molecular patterns of dsRNA or LPS. CD8+ T cells contact infected host cells for
pathogen peptides presented by MHC-I and target cell proteins, and memory T cells
remain when infection is cleared.
Maturation of DCs triggers to initiate adaptive response mediated by T cell
immunity [191]. PRRs mediate cytokine induction and co-stimulatory molecule
expression, as well known for TLRs [189]. The fundamental role of DCs is to load
microbial peptides presented to MHC-I or MHC-II receptors, as a type of T cell
antigenic presentation [192, 193]. For another role of DCs, they additionally express
surfaced co-stimulatory receptors of CD80, which is a B7–1. In addition, another
CD86 known as a B7–2, which activate T cell function, is expressed [194]. The third
role of DCs is to express cytokines like IL-12, which upregulates function of
adaptive immune cells [195]. Collaboratively, the multiple roles of DCs enable
naive T cells to differentiate to the effector cell or memory cell types of T cells
[191]. For example, upon microbial pattern interaction, TLRs form the myddosome
known as cytoplasm supramolecular organizing center (SMOC), and the SMOC
activates proinflammatory NF-κB/AP-1 factors [196]. The NF-κB/AP-1 activates
adaptive immunity such as T cell activation. Therefore, the innate immunity stimu-
lates the adaptive immune response basically by action of the APCs. Professional
DCs as an APC process and present immune-stimulatory peptides on MHC-I
molecule or MHC-II molecule. By MHC contexts, regular T cells and Tregs
propagate or regulate immune responses.
Proteasome can process antigens and present the fragments on MHC-I for CD8
CTLs [197]. T cells are present in a naive functional cell type state prior to antigen
interaction [198]. Antigen-exposed CD8 CTLs directly recognize and kill
transformed cells or infected cells via MHC-I action [199]. CD8 T cells also express
cytokine IFN-γ. Another subpopulation, named NK T cells, utilizes αβTCR chains
to target cells and produce cytokine [200]. NK T cell forms contain semi-invariant
TCRs to recognize lipid-related antigens rather than protein antigens. MHC-2-
loaded peptides are presented to CD4 T cells [197, 198], where naive CD4+ T
cells are differentiated to effector cell types such as Th-1/Th-2 and Th-17 cells. Two
IL-12/IFN-γ cytokines stimulate naive CD4 T cells to initiate the differentiation to
Th-1 cells via costimulation with TCR [198, 201], indicating the essential roles of
IL-12 and IFN-γ for genesis of the effector Th-1 cells. IL-4 through costimulation
with TCR induces differentiation of the naive CD4+ T cells to Th-2 cells as the
effector cell types. The differentiated Th-1 and Th-2 cells are important for the
parasites including helminths and allergy. Then, Th-2 cells express its type 2-related
responses through release of the type 2 IL-4/IL-5/IL-13 cytokines [200]. On the
other hand, cytokines IL-6/TGF-β with TCR costimulation activate the naive T cells
to differentiate into Th-17 cells [202]. The Th-17 type differentiation from the naïve
T cells requires IL-1 when fungal-infected conditions are associated with autoim-
mune responses. Eventually, the Th-17 cells express IL-17 [203]. After differentia-
tion, various effector T cells are phenotypically shifted to the memory T cell types
that are ready to reactivate in an antigen-dependent manner upon pathogenic rein-
fection [204]. The γδ-T cells use the TCR γ and δ chains, which are opposite to the
TCR αβ chains constituted for all regular T cells. Such T cells express IL-17 and
IFN-γ [205]. Apart from T cell subpopulations, B cell subpopulations can occupy the
acquired immunity, recognizing three-dimensional and conformational epitopes of
antigens [206]. Specific subpopulations of B cells are generated through
hypermutation of the somatic chromosome with class switch recombination adjusted
for antigen-B cell receptor interaction towards antibody effector function. Such
adapted B cell receptors are secreted as antibodies upon differentiation to plasma
cells. The antibodies indicate humoral immunity [207].
Adaptive immune responses protect against many pathogens, but not all. CD+ T
cells lead to the adaptive responses to both intracellular and extracellular pathogens
because the naive CD4+ T cells are differentiated into the effector-type T cells such
as Th1/Th2. The CD4+ T cells provide vast help to other lymphocytes by cytokines
and co-stimulatory molecules and generate antibody responses to pathogens with
memory B and plasma cells with long life span. The CD4+ T cells do not induce the
CTLs against intracellular pathogenic viruses. However, the CD4+ T cells induce to
differentiate the memory CD8 T cells towards expansion during secondary exposure
to the pathogen. CD4+ T cells are needed to eliminate chronic viral infections. Using
secreted cytokines, effector CD4+ T cells can invite monocytes and peripheral
neutrophils to the infection sites for inflamed reaction [208]. CD4+ T cells regulate
dampening immune responses through the thymus-derived Tregs or anti-
inflammatory IL-10 cytokine [209].
1. CD8 T cells---Cytotoxic CD8 CTLs---–Kills virus infected cells or viruses.
2. CD4 T cells are classified into three subsets:
---T helper 1 cell (Th1)----IFN-r—Intracellular pathogens/mycobacteria
---T helper 2 cell (Th2)----IL-4—Parasites
---T helper 17 cell (Th17)---IL-17—Extracellular bacteria/fungi
NK T cells---Lipid antigens.
6.6 Galactose-Specific C-Type Lectin: Two Major ASGPR and Macrophage Galactose. . . 295
6.6 Galactose-Specific C-Type Lectin: Two Major ASGPR

and Macrophage Galactose Lectin (MGL)
in the Human
Carbohydrate recognition involves cellular homeostasis in the body by

carbohydrate-lectin interaction. SAs act as biological masks. SA is a survival
indicator of circulating blood cells and glycoconjugates. STs catalyze SA transfer
using the donor substrate of CMP-SA to acceptor substrate in order to terminate the
stable negative charged sialic acids and consequently protect them from the apopto-
sis and clearance. Human C-type lectins are representatively mediating the mission,
following cellular clearance events such as endocytosis [210]. This leads to cell-cell
interactions because SAs are served as specific lectin ligands of selectins and
Siglecs. Circulating blood senescent cells are specifically eliminated by the scav-
enging machinery of the host cells, which make clear and induce apoptosis [211]. To
perform the roles in the cell level, they are expressed on the DCs and macrophage
surfaces. Disappearance of surfaced sialic acids accelerates the cell clearance, as in
elimination of erythrocytes and plasma glycoproteins by β-Gal recognition. It has
been suggested the SAs have dual roles as biological masks and binding sites
[212]. For example, the asialo-glycoprotein receptor (ASGPR) known as “Ashwell
receptor” is present in hepatocytes [213]. The exposed β-Gal residue which SAs are
removed is the ASGPR target. The ASGPR is different from the Gal-binding
receptors such as soluble galectins that bind to Gal residues seen on surfaces of
cells [214, 215]. Galectins prevent endocytotic internalization of the surfaced pro-
teins of cells in the immune system [216]. Most galectins prefer LacNAc disaccha-
ride ligands as the terminal β-Gal residues without SA residues. However, galectin-
8 and galectin-9 prefer to bind SAα2,3-LacNAcs [217].
Two major Gal-recognizing CTLs are lectin ASGPR and MGL known in the
human [218]. The recognition allows the phagocytosis by the lectin ASGPR
[219]. ASGPR-1 is generally expressed by liver Kupffer macrophage cells, liver
hepatocytes, and liver sinusoidal endothelial cells and recognizes galactose on
human platelets when Neu5Ac species are cleaved off by human sialidases
(NEU1–4). SA species removal from the cells leads to exposure of subjected glycans
that are then recognized, bound, and interacted with galactose-binding lectin recep-
tors of macrophages such as ASGPR-1. Such status induces phagocytosis of the used
human platelets as a life span. C-type lectins of DC-SIGN and Man receptor (MR or
CD206) consist of the specific “Glu-Pro-Asn” motif (EPN motif) triamino acids in
the CRD region. They preferentially recognize Man residue, L-Fuc residue, and
GlcNAc residue [220]. MGL present on the macrophage and DC surfaces stimulate
T cell signaling. The mechanistic signaling is further described in the other
section [221].
Masking of sugar chains by SA species blocks interaction with the ASGPR,
which is an abundant lectin receptor present on hepatocytes and macrophages.
ASGPR as a liver-specific C-type lectin is also present in sinusoidal surfaces of
hepatocytic cells. The major role of the ASGPR is clearance of apoptotic cells and
wG
hGG G
wG
wG G
G
wG

G

t
hT G

hGG

n

m w TuT G
uT G G

G
t
zG t
Fig. 6.5 Recognition mechanism of macrophage against apoptotic cells in blood and hepatic
regions
vascular homeostasis [222], through binding to terminal sugars of Gal/GalNAc

residues, which are predominantly present in senescent cells and desialylated car-
bohydrates of blood glycoproteins and platelets in circulatory track [223]. Hepatic
macrophages or hepatocytes recognize apoptotic cells in blood and hepatic regions
using poly-N-acetyllactosamine-type glycan receptor or ASGPR, as illustrated in
Fig. 6.5. The examples are elimination of erythrocytes and platelets [224, 225], as
nonsialylated glycan chains are involved in clearance of senescent blood cells
[226, 227]. As β-Gal residue serves as the internal ASGPR ligands, the appearance
of the Gal residues upon bacterial infection results in septic condition [228]. The
ASGPR recognizes and removes the exposed carbohydrates, which have the β-Gal
and GalNAc residues. In addition, they also do not recognize the SAα2,6GalNAc
and SAα2,6Gal [229, 230]. Large carbohydrate ligands are endocytosed through the
ASGPR present on macrophages [231]. As the reverse demonstration of the role of
ASGPR in mice, the ASGPR-depleted macrophages in experimental mice exhibit
the level of prolonged circulation of ST3Gal-IV-deficient platelets. Hepatocytes
mediate the platelet clearance, which are deficient in sialylation of platelets. Hepatic
ASGPRs are directly involved in clearance of cells and desialylated glycoproteins in
sera. In addition, hepatic macrophages also endocytose senescent cells for clearance
from plasma and desialylated platelets by trans-sialidases or sialidases of bacterial
pathogens [232]. The St3gal4 ST gene-deficient mice (ST3Gal-IV/ mouse)
References 297
expose the terminal Gal residues on cells, resulting in cell clearance by ASGP
receptor of macrophages, as the ST3Gal-IV-deficient platelets are eliminated by
hepatic macrophages [211]. Depletion of macrophages rather increase the survival
rate of the ST3Gal-IV/ platelets in wild-type mice [228]. ASGPRs expressed in
both hepatocytes and macrophages bind to desialylated platelets and clear the
defected platelets in sialylation. Compared to another soluble lectin of MGL, the
ASGPR has been isolated much earlier than the MGL [233]. Normal ASGPR protein
is a heterogenous oligomer, which contains two distinct H1 and H2 subunits
[234]. ASGPR ligands are known to be the Gal residue linked to the tri-antennary
and tetra-antennary structures of N-glycans [235]. B cells produce a BCR (sIgM)
that binds to glycans that enter the tolerization status and are normally eliminated
before leaving from the bone marrow. Host SAs can be regarded as “immunosup-
pressive” for the hosts.
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Chapter 7
Sialic Acid-Binding Ig-Like Lectins (Siglecs)
During the mid-1980s, in vitro rosette formation between macrophages and sheep
erythrocytes was reported; however, sialidase treatment on erythrocytes abolished
the rosettes [1]. Rosette formation is based on sialic acid-binding receptors expressed
by macrophages. Later, these receptors were identified to be sialoadhesin that binds
to sialic acids as ligands [2]. The SA-recognizing property of the innate immune
system of vertebrates led to the discovery of SA-bearing glycans as the ligands for
lectins. The binding of SA ligands to Siglecs with immune inhibitory properties
leads to suppressed immune functions. The SA-to-Siglec recognition implicates
immune activation to limit self-recognition and to destroy the defense mechanism
in hosts. Siglecs attenuate ‘self’-inflammatory triggers called DAMPs. During the
early 1990s, the first sialyl carbohydrate-binding receptor protein called Siglecs was
discovered, for example, Siglec-2 (or CD22) present on B cells. Siglec-1, known as
sialoadhesin present on macrophage surfaces, was found. The Ig-like domains of
such lectins are different from those of other known C-type lectins or Ca2+-depen-
dent lectins. Among them, I-type lectins are named I-type because they belong to the
varied immunoglobulin superfamily of proteins. As I-type lectins share similar
characteristics with the immunoglobulin superfamily of proteins, they contain Ig
folds, which consist of antiparallel β-sheets. I-type lectins can be broken down into
many different regions: V-set, C1 and C2 sets, and ITIM and ITIM-like domains.
The V-set domain is the primary site of ligand binding and recognition. The C1 and
C2 sets act as spacers and are believed to control the entire length of lectins. ITIM
and ITIM-like domains (in some cases, ITAM domain) are essentially tyrosine-based
signaling motifs that inhibit (or, in the case of the ITAM domain, activate) down-
stream signaling and thereby modulate cell activities. More than 16 I-type lectins,
such as Siglecs, recognize diverse sialoglycans by immune cells. Even among I-type
lectins, there are many different subtypes and the best characterized subtype is called
Siglecs. Siglecs are SA-binding lectins and they are the most well-studied I-type
lectins. Like any other lectins, Siglecs are composed of many different domains,
namely, the V-set, C1 and C2 sets, and mostly the ITIM domains.
312 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
In B cells, a sialyl ligand-binding receptor such as CD22 was found as a

SA-binding lectin. The CD22 ligand binds α2,6-SAs and not α2,3-SAs [3]. SA is
widely distributed in animal tissues. α2,3/2,6 sialic acid linkages are general bonds
in connection with Gal or GalNAc. α2,3-SA-linked glycans are recognized by
Siglec-4, α2,6-linked sialosides by Siglec-2, and α2,8-sialosides by Siglec-7 [4]. In
evolution, Siglecs are divided into two subgroups, namely, Sn/CD22/MAG and
CD33rSiglecs. The Sn/CD22/MAG group has a single ortholog in many species,
whereas the CD33rSiglecs group is not clear in differentiating between primates and
rodents in their clear-cut orthologs. The specificity of ligand binding of the
Sn/CD22/MAG group is highly conserved with conserved domain structures and
minor variations, whereas that in the CD33rSiglecs group is poorly conserved
toward rapid evolution. Interestingly, the Sn/CD22/MAG group exhibits a cell
type-specific pattern of expression. In fact, CD22 expression is observed in B
cells, MAG expression in glial cells, and Sn expression in macrophages.
Siglecs are further subdivided into two different subgroups based on the genome
similarity of each Siglec. The first of such subgroups is composed of Siglec-1
(termed “Sn”), Siglec-2 (termed “CD22”), Siglec-4 (termed “MAG”), and
Siglec-15. Another subgroup is the CD33-related Siglec group (CD33rSiglecs) and
is composed of CD33, Siglec-5, -6, -7, -8, -9, -10, -11, -14, and -16. Siglec-1 is
associated with the phagocytic clearance of SA-covered pathogens. The CD33-
related Siglec group suppresses inflammatory reaction through recognition of
ligands present on the cell surfaces of the same cells, which is termed “cis-interac-
tions,” or on cell surfaces of other cells, which is termed “trans-interactions.” Most
of them consist of ITIM-bearing Siglec-3 and (CD33)-related Siglec groups. Many
of such Siglecs are coupled so that one Siglec has inhibitory effects, whereas the
other has activating effects on the same cell. These coupled Siglecs work in tandem
to modulate downstream signaling.
Most Siglecs comprise one or multiple numbers of ITIMs in the cytosolic region.
ITIMs often mediate to counteract activation signals that are triggered by
immunoreceptor tyrosine-based activation motif (ITAM)-carrying receptors. In the
aspect of signal transducing motifs, the Sn/CD22/MAG group is highly specific for
the ITIM expression. hCD33rSiglecs are engaged in innate cells via interaction with
sialylated antigens to downregulate the roles of proinflammatory APCs. The role of
hCD33rSiglecs in T-cell responses is not yet clearly understood. For example, only
CD22 contains ITIMs. However, Sn and MAG both do not contain ITIMs. Only
MAG contains a Tyr-based amino-acid sequence, which is quite similar to the
known CD33rSiglec sequence. In contrast, the typical CD33rSiglecs consist of the
conserved common ITIM sequences and ITIM-like sequences. With regard to the
structural aspect, the Siglec groups belong to the type I TM proteins, which contain
an extracellular region. The extracellular region comprises an N-terminal
SA-recognizing V-set Ig-like domain and multiple 1–16 C2-set Ig-like domains.
The cytosolic tail regions of all Siglec forms, but not that of Siglec-1 (Sn), have one
or more Tyr residues as signaling motifs. Arginine (Arg97) forms a salt bridge
complex with the COOH group of SAs. The Siglec V-set region and the adjacent
C2-set region both have cysteines to form disulfide bonds in the V-set region and an
7.1 PolySia and Host Sialic Acids Modulate Host Immune Responses as Pathogenic. . . 313
Table 7.1 Mammalian orthologs of Siglecs in baboons, chimpanzees, humans, mice, and rats
Human Chimpanzee Baboon Mouse and rat
Sn/Siglec-1 Sn/Siglec-1 ? Sn/Siglec-1
CD22/Siglec-2 CD22/Siglec-2 ? CD22/Siglec-2
MAG/Siglec-4 MAG/Siglec-4 ? MAG/Siglec-4
CD33/Siglec-3 CD33/Siglec-3 CD33/Siglec-3 CD33/Siglec-3
Siglec-5 (OBBP-2) Siglec-Va Siglec-5 Siglec-F
Siglec-6 (OBBP-1) Siglec-6 Siglec-VIa
Siglec-7 (AIRM-1) Siglec-7 NF NF
5iglec-8 Siglec-8 Siglec-8 NF
Siglec-9 Siglec-9 Siglec-9 Siglec-E
Siglec-10 Siglec-10 Siglec-10 Siglec-G
Siglec-llb Siglec-11b ? NF
Siglec-XII (Siglec-L1, SV2)a Siglec-12 NF NF
NF Siglec-13 Siglec-13 NF
NF ? ? Siglec-H (rata)b
AIRM adhesion inhibitory receptor molecule, OBBP, obesity-binding protein, NF V-set domains,
not found
a
Siglec-like proteins lacking the “Arg residue” for binding
b
CD33rSiglecs located outside the Siglec gene cluster (modified from Glycobiology (2006)
16, 1R-27R) [5]
S–S bond in each domain. The structural characteristics of SAs affect Siglec
recognition. Many orthologs of Siglecs have been found in mammalian species
such as baboons, chimpanzees, rats, mice, and humans. The structural orthologs
corresponding to their mammalian Siglecs such as baboons, chimpanzees, humans,
rats, and mice are summarized (Table 7.1) [5].
7.1 PolySia and Host Sialic Acids Modulate Host Immune

Responses as Pathogenic Decoys
SAs are terminally present as monosaccharides found in glycans. Aside from this,
polysialic acid (PolySia, PSA) is a linear homopolymer of α2,8-linked sialic acid
residues that are repeatedly linked on the cell surfaces of proteins. Polysialic acid
consisting of α2,8-Neu5Ac units is an essential nutrient in brain development,
morphogenesis, and neural systems [6, 7]. Some immune cells also produce
polysialic acids in a subpopulation of DCs [8, 9] and T cells [10, 11], where the
polysialic acid is attached to neuropilins. The polySia expression on a NCAM has
long been studied in the initial stages of neural progenitor cells, and successfully
matured neuronal cells express excess amounts of α2,8-linked poly SAs on the
NCAM protein (Fig. 7.1). The role of PSA-NCAM in neuronal genesis has been
attributed to its antiadhesion property (Fig. 7.2). In neuronal progression, polySia
functions as a contact-dependent differentiation inducer. Therefore, a malformed
vo vo
ov v
_ jvvT
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ov _
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jvvT
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ov _
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jvvT
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Fig. 7.1 Schematic structure of PSA (a) and NCAM (b). Polysialic acid (PolySia, PSA) is an α2,8-
SA linear homopolymer present on protein cell surfaces
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h
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u

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Fig. 7.2 Function of polysialic acid in a NCAM for an antiadhesion effect

7.1 PolySia and Host Sialic Acids Modulate Host Immune Responses as Pathogenic. . . 315
default expression of polySia indicates the prematuration and dysmaturation of

neuronal cells simply due to the absence of polySia. PolySia also increases cell
proliferation. In the immune system, it increases the migration level of hematopoietic
progenitor cells from the bone marrow to the lymphatic node and the thymus.
ST8Sia-IV synthesizes N-linked polySia. PolySia also stimulates DC migration to
the lymphoid organ. PolySia is relatively large in size with repeated negative charges
due to carboxylic acid, and it acts as a cell–cell interaction regulator and inhibitor of
adhesion molecule interaction. For a specific condition, such as growth factor,
polySia interaction induces signal transduction. For example, polySia stimulates
CCL21-dependent migration of DCs to the secondary lymphoid organ. As NK cells
express NCAM (CD56), the cells can migrate to the lymphoid organ. In cancer cells
and embryonic development, polySia potentiates the metastatic progression of
cancer cells including lung carcinomas, myelomas, neuroblastomas, gliomas, and
Wilms’ tumor, as well as trophoblastic movement of fused blastocysts to placental
locations. In blastosis, polySia is expressed in the early implantation stage.
Ig-like lectins (Siglecs) are also abundantly expressed in plants, fungi, and
bacteria. Siglecs also trans-recognize SA ligands, on adjacent, neighboring, and
encountering cells, or pathogen-expressing ligands. This event leads to Siglec
signaling and endocytosis. A series of SA-dependent adhesion receptors has recog-
nized these SAs present on glycoproteins and glycolipids. Siglecs contain variable
C2-set domains in all V-set Ig-like domains of the N-terminal region and subsequent
extracellular regions where 17 human Siglecs have been confirmed. The moiety that
recognizes sialic acid is present in the V-set Ig-like domain, which has two aromatic
residues and one arginine motif conserved in all signals. Sialic acid is abundantly
present in various cell membranes and can affect the functional properties of cells
(cis and trans) by binding to ligands.
Host SAs are usually the binding targets or receptors for viruses, toxins, and some
bacteria, indicating PolySia as a decoy. Thus, viruses or sialic acid-binding parasites
approach the host mucosal surface to encounter mucin-type glycoproteins. Mucins
are highly sialylated glycoproteins. As decoys, mucins protect the organism from
pathogens [12]. Thus, loss of O-linked mucins increases colitis diseases
[13]. SA-binding pathogens encounter the heavily sialylated glycoproteins before
reaching their target. Erythrocytes can be used as another type of decoy for the host
due to their non-nuclear cells. Erythrocytes occupy about 50% of the total blood
volume. A SA-binding virus, influenza virus, enters into the bloodstream and
encounters this extensive cell surface. Hemagglutination defines the binding speci-
ficity of viruses. Malarial parasites use SAs for invading erythrocytes [14].
7.2 Sialic Acid Recognition by Siglecs

for Self- or Nonself-Antigens
Cell surface sialylation and sialoglycan-recognizing receptors function in immune

responses of DCs. Sialoglycosylation is thus considered to be important in DC
function and in immunological defense of mammals. Mammalian SAs are
substituted at the position of R. Like CLRs, Siglecs such as cell surface GBPs
have both positive and negative immune responses. Siglecs are members of an
immunomodulatory and transmembrane lectin family expressed prominently on
leukocytes. SAs are made up of 9-C saccharides that exist on the cell surfaces of
glycolipids or glycopeptides. SA induces a regulatory mechanism with antigens
through linkages to SAα2,3, SAα2,6, or SAα2,8 in mammalian cells. Desialylation
activates signaling pathways related to self-eating, and this activation enhances the
ability of autophagy to lessen apoptotic cells [15]. Therefore, sialic acid is an
important marker of the self-immune response system. In the recognition of patho-
gens by receptors, many distinct molecular events mediate adhesion, migration,
interaction, costimulation, and inhibition of cell signaling.
Siglecs are cell surface proteins that bind SAs and are mostly found on immune
cells. Each Siglec has a unique specificity for sialylated ligands, as each protein
mediates a distinct, partially overlapping function. Siglecs recognize glycoproteins
and glycolipids from pathogens as cell surface molecules. They were initially studied
from a macrophage lectin-like adhesion protein, sialoadhesin, later termed “Sn” [1],
and CD22, which is B cell-specific [16]. Later, two other members of the Ig
superfamily, CD33 and MAG proteins, were discovered [17, 18]. Currently, the
above four Siglecs represent sialic acid-recognizing lectins; however, another type,
CD83, is found to be expressed on mature DCs, and CD83 is a specific Siglec type
with the capacity of sialidase-sensitive interaction [19]. Immune receptors generally
exhibit high maturational structural similarity, having the properties of antagonistic
signaling known in coupled receptors. Immune receptors are considered to balance
immune homeostasis in tolerance and inflammation. Siglecs, a group of the immu-
noglobulin superfamily, can bind several types of sialylated glycans. The most well-
evolved immune receptors of human Siglecs are reported to have 14 types of Siglecs.
Mammalian immunity has evolved to distinguish the “self” from “nonself,” and this
contributes to protection from pathogenic and foreign infectious agents. When the
transformed tumors are accounted for, the immune system eliminates them because
they are recognized as “foreign agents” during immunosurveillance [20]. Until now,
various receptors for eliminating these foreign agents have been identified. Foreign
agents are treated as nonself-molecules or self-mimicking molecules [21, 22]. Thus,
most immune-related cells synthesize a series of Ig family receptors, which inhibit
the functions of immune cells in the host. Among molecules that regulate the level of
inflammation, the Siglec families are mainly present on the surfaces of leukocytes
that bind to sialylated glycans and translate glycan binding into changes in immune
cell function. Structurally, the representative inhibitory receptors are the
SA-recognizing Ig-like lectin (Siglecs) group (Fig. 7.3). Siglecs, as the
7.2 Sialic Acid Recognition by Siglecs for Self- or Nonself-Antigens 317
5KINGE
1+
9VHWLPPXQRJOREXOLQGRPDLQ
6LDOLFDFLGELQGLQJVLWH
6.4
&VHWLPPXQRJOREXOLQGRPDLQ
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2
+6+/ 2
5*2
5*2
,7,0PRWLI7\URVLQHNLQDVHWDUJHWVLWH
,7,0OLNH 5KCNKE CEKF

8UGVKOOWPQINQDWNKPFQOCKP
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+6+/
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Fig. 7.3 Basic structure of Siglecs. Siglecs on cell surfaces are morphologically extended by
C2-type Ig domains, which have no glycan-recognition capacity. Human and mouse type I Siglecs
contain a V-set Ig domain in the N-terminal region for SA recognition and multiple C2-set Ig
domains. CD33 consists of cytoplasmic Tyr signaling motifs. The C2-type domain number is
different. The inhibitory receptor function suppresses activation signals linked to the ITAM–Tyr–
phosphatase axis. The activated SHP motif represses the ITAM activation and the TLR signaling
pathway. As a result, the NF-κb pathway and immune responses such as inflammation are
suppressed
SA-recognizing lectin family, have been recently defined and predominantly found
on immune-related cell surfaces and mediate downstream immunomodulatory sig-
naling through ligand bindings.
The C2-type Ig domains of the known Siglecs are apically extended from the
surfaces of immune cells because they do not have the glycan-recognition capacity.
Different numbers of C2-type domains are found in each Siglec. Siglecs as type
1 membrane proteins carry the N-terminal V-set Ig domain and this domain is the SA
recognition site. The N-terminal V-set Ig domain consists of multiple C2-set Ig
domains. CD33-related Siglecs have diverse binding properties to their ligands,
depending on the host species with sequence similarities in the ExT region and a
conserved Tyr-based signaling motif in the cytoplasmic region. Different mamma-
lian CD33-related Siglec species include CD22, MAG, sialoadhesin (Sn), and
Siglec-15. They differ in sequence homologies from those of CD33rSiglecs.
CD33rSiglec receptors recruit Tyr phosphatases by inhibiting the activation signals,
which are mediated by ITAM signaling.
Siglecs have α-helix structures. They are type 1 membrane polypeptides with
glycoprotein and glycolipid sialic acid-binding capacities. Siglecs contain multiple
extracellular Ig-like domains that mediate glycan binding and have short cytoplas-
mic tails. They are characteristically composed of a V-set Ig-like domain in the
N-terminal region, which binds SA residues that are abundantly present in mammals
but are relatively low in prokaryotes, and a C2-set Ig-like domain [23]. The first
V-set Ig-like domain binds to carbohydrate ligands, and the second Ig-like domain
may also contribute to the binding. The N-terminal region is linked to multiple Ig
C2-set domains, a TM domain, and a tail in the cytoplasmic region. The number of
C2-set immunoglobulin domains is diverse depending on each Siglec. Siglecs are

extended by C2-type Ig domains, which have no SA-recognition capacity. Siglecs
have different numbers of C2-type domains. Cell surface Siglecs that are normally
masked by the self-SA species via cis-recognition regulate the function of cis-
ligands. Thus, endogenous cis-ligand identification is important for Siglecs to
elucidate the immune function.
7.3 Classification of Siglecs
The Siglecs are a family of SA-binding Ig-like lectins that recognizes SA-terminated
glycans of glycoproteins or glycolipids. Siglec families have been well established in
both humans and mice. Siglecs belong to the type 1 membrane proteins. As
expected, they bear a V-set immunoglobulin domain in the N-terminal region, and
this domain recognizes SA residues on the sialoconjugates. Siglecs are convention-
ally grouped into two main groups, depending on sequence and evolution conser-
vation [23]. For example, Siglec-1, -2, -4, and -15 have homologues across species
and thus belong to the same group. Siglecs are further classified into two groups:
(1) inhibitory Siglecs (Siglec-5, -7, -9, and -10), with the transducing capacity of
inhibitory signals in cellular functions by intracellular ITIM domains, and (2) acti-
vating Siglecs (Siglec-14), with the activating capacity of cells in Siglec-dependent
responses by intracellular adaptor proteins such as DAP12 recruitment. To date,
14 mammalian Siglecs have been reported. Most of them are present in specific cell
types on immune-related cells. Immune cells express them on their surfaces.
Depending on their finding history, name of Siglecs has been made. Although
most Siglecs are negative signaling regulators due to ITIMs, others activate the
immune cell function. Structurally, Siglecs belong to the Ig superfamily and are
classified into two distinct groups, in which the first is the low-homologous group
with 25–35% homology. They are Siglec-1 (Sn), -2 (CD22), and -4 (MAG).
The common Siglec forms in mammals are Siglec-1, -2, -4, -15, and the Siglec-3
(CD33)-related family. Among them, CD33 (also named Siglec-3) and its related
Siglecs, i.e., Siglec-5, -6, -7, -8, -9, -10, -11, -14, -15, and -16, are involved in the
innate immune system [24] (Fig. 7.4). CD22 is a B cell-specific glycoprotein and is a
membrane-bound glycan-recognition protein like the BCR complex. Thus, CD22 is
a SA-recognizing lectin. In B cells, if α2,6-SAs are ablated, the basic immune
functions of B cells are effected. For example, BCR signaling suppression, IgM
reduction, and antigen responses in T cell-dependent and T cell-independent mech-
anisms are completely effected [25]. The second distinct group is the fast-evolving
high-homologous group with 50–90% homology and is known as CD33-related
Siglecs (CD33rSiglecs). There are mammalian orthologs of Siglec-1, CD22, Siglec-
4, and Siglec-15 with low homology and similarity. Interestingly, the Siglec family
is well conserved between the two mammalian Siglecs, Siglec-1 and -2, and also
between the vertebrate Siglecs, Siglec-4 and -15. However, a family of a large
cluster, which is located on human chromosome 19q, varies in each different species
7.3 Classification of Siglecs 319
Siglecs common to mammals Sialoadhesion (Siglec-1/CD169)

(Conserved Siglecs) CD22 (Siglec-2)
CD33-related Siglecs MAG (Siglec-4)
E-selectin
Name Sialoadhesion CD22 MAG Siglec-15 Siglec-3 Siglec-5 Siglec-6 Siglec-7 Siglec-8 Siglec-9 Siglec-10 Siglec-11 Siglec-14 Siglec-16
(Siglec-1/CD169 Siglec-2 Siglec-4 - CD33 CD170 CD327 CD328 - CD329 - - - - )
Expression Macrophage B Oligodendrite - Myeloid Mo B, Tropho Mo(NK) Eo(Ba) Mo, DC B Mac

SC Mo
Mouse CD33-related Siglecs V-set: Ig domain (Sialic acid binding)

C2-set: Ig domain
ITIM-SHP-1/SHP-2 binding motif
ITIM-Like
Grb2 binding motif
Fyn kinase phosphorylation site
CD33
Basic amino acid residue in transmembrane domain
(Siglec-3) Siglec-E Siglec-F Siglec-G Siglec-H
Fig. 7.4 Siglec family proteins and expression in humans
[26]. This concept led to a new terminology, “CD33-related Siglecs”

(or CD33rSiglecs).
CD33rSiglecs include human Siglec-3, -5, -7, -8, -9, -10, and -11, and mouse
Siglec-14 and -E, -F, -G, and -H. The second group of Siglecs is highly specific for
SA-containing glycans toward α2,3-SA, α2,6-SA, and α2,8-SA linkages. They
branch from two subsets. The first one is based on their sequence similarities and
the second one is based on their evolutionary conservation. (1) The first group
includes Siglec-1, -2, -4, and -15. They are highly homologous across species and
are conserved from humans to vertebrates as all mammalian species orthologs. They
are related in only 25–30% sequence identities of Siglecs and have definite mam-
malian orthologs. Siglec-1 is also known as sialoadhesin (or CD169) and is a
macrophage-specific adhesion lectin. Siglec-2, also called CD22, is expressed on
mature B cell surfaces with sialic acid-binding capacity. (2) The second group
includes human CD33 (Siglec-3) and CD33rSiglecs as well as Siglec-5, -6, and -7
(another name: adhesion inhibitory receptor molecule 1, AIRM1), Siglec-8, -9, and
-10 (in mouse, Siglec-G), -11, -14, -E, -F, Siglec-H, Siglec-16, and Siglec-17. This
group is characterized by rapid evolutionary stages and high species variations
[23, 24]. Different from Siglec-1, -2, -4, and -15, CD33rSiglecs have 50–99%
similarity and are under rapid evolution through gene conversion, duplication,
exon acquisition, and exon loss or exon shuffling. CD33Siglecs have evolved
from SIGLEC genes.
Many mammals express CD33-related Siglecs than mice and rats, showing a loss
of Siglec genes in rodents. Moreover, SD33-related Siglecs are difficult-to-classify
orthologs. For example, several mouse CD33-related Siglecs have been discovered.
As Siglecs exhibit high structural homology, they are recognized as the “Siglec
family.” Siglec-7/-9 and -E show a high sequence similarity with mice Siglec-E,
where Siglec-7 binds to α2,8 sialic acid and Siglec-9 recognizes α2,3-linked sialic
acid, whereas Siglec-E recognizes both α2,3-SA and α2,8-SA. Siglec-8 and Siglec-F
&RQVHUYHG 6LJOHFV +XPDQ &'UHODWHG 6LJOHFV 0RXVH &'UHODWHG 6LJOHFV

6LDORDGKHQVLQ 6Q
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&' (
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1HXW 1HXW (RVL '&V
(RVL 1HXW 0DFUR 0DFUR %FHOO
6FKZ 0DFUR %FHOO 1HXW 0RQR 0RQR %FHOO %FHOO
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α2-6 α2-8
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2 6HU7KU *OF1$F
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Fig. 7.5 Human and rodent Siglecs. Conserved Siglecs are found in mammals. CD33-related
Siglecs exhibit evolutionary phenotypes. Eosi, eosinophil; Macro, macrophage; Mono, monocyte;
Neur, neutrophil; Oligo, oligodendrocyte; Osteo, osteoclast; DCs, dendritic cells; Placen, placental
syncytiotrophoblast; Schw, Schwann cell. Siglec-7/-9 and -E show a high sequence similarity with
mice Siglec-E, where Siglec-7 recognizes α2,8-SA and Siglec-9 recognizes α2,3-linked SA,
whereas Siglec-E binds to both α2,3-SA and α2,8-SA. Siglec-8 and -F share a similar degree of
sequences, where Siglec-8 and -F recognize 60 -sulfo-sLeX during eosinophil apoptosis, inhibiting
mast-cell-mediator release in eosinophilic asthma. Similarly, Siglec-10 and -G recognize Neu5Gc in
α2,3 and α2,6 linkages (modified from Ref. [27] Brown GD, Crocker PR. 2016. Microbiol Spectr.
4(5)
share a similar degree of sequences, whereas Siglec-8 and -F recognize 60 -sulfo-sLeX

during eosinophil apoptosis, inhibiting mast-cell-mediator release in eosinophilic
asthma. Similarly, Siglec-10 and -G recognize Neu5Gc in α2,3 and α2,6 linkages, as
shown in Fig. 7.5 [27]. Among Siglecs, mouse-specific mSiglec-E was isolated by a
yeast two-hybrid search utilizing the SHP-1 gene for the fifth mouse Siglec.
CD33rSiglecs contain one or more cytoplasmic ITIMs, allowing them to act as
inhibitory receptors to suppress receptor ITAM activation signals through the
recruitment of Tyr and inositol phosphatase. CD33rSiglecs are divided into two
groups: (i) ITIM-carrying cytosolic domains with a tyrosine phosphorylation site,
and as a result recruiting tyrosine phosphatases like SHP-1 and SHP-2 [28–31]. This
dephosphorylates phosphor-Tyr on receptor kinases. Thus, this type is called “inhib-
itory CD33rSiglecs” and negatively regulates hyperreactive innate immune
responses on SAMPs, thereby inhibiting unwanted responses such as inflammation
in self-tissue damage [31]. Some CD33rSiglecs also have other cytosolic ITIM-like
domains. In addition, some inhibitory Siglecs function independently of the
tyrosine-based motifs [32, 33]. (ii) Contrary to the inhibitory Siglecs, the other
type of CD33rSiglecs is conceptional, i.e., the so-called “activating CD33rSiglecs,”
which contain amino acids with positive charges in the transmembrane domain. The
7.4 Evolution of Siglecs, Sialic Acids, and Sialic Acid O-Acetylation as Host. . . 321
activating CD33rSiglecs recruit ITAM-containing adapters such as DAP12. DAP12

recruits the Tyr kinase, Syk, to phosphorylate Tyr residues [34, 35]. Therefore, the
N-terminal domains serve to regulate the inhibition or activation properties of
CD33rSiglecs. The activating Siglecs seem to progressively evolve over bacteria
with inhibitory Siglecs [34, 36].
7.4 Evolution of Siglecs, Sialic Acids, and Sialic Acid

O-Acetylation as Host Ligands (Receptors)
for Microbes and Innate Immunity
Innate immune responses reflect the resistance against pathogenic infections by

sensing PAMPs via signaling for proinflammatory cytokine secretion to provide
the first line of defense against foreign agents. Siglecs primarily bind “cis-ligands”
sialylated on cell surfaces [37]. If sialyl ligands are surrounded on the surfaces of
other cells or other soluble molecules, they can competitively bind to the cis-ligands
[38, 39]. The V-set Ig-like domain in the N-terminal region consists of a SA binding
site with an Arg residue conserved as the binding site to carboxylate the sialylated
ligands [40, 41]. Interestingly, the Arg residue is easily subjected to mutation,
possibly creating a new type of Siglecs specific to certain species [34, 41] in a
random manner because the Arg residue has been suggested to be mutable more than
the other codons. For example, the Arg residue once mutated mutates once again for
Siglec-5 and -14 in humans and great apes, thus inching toward an evolutionary
recognition between sialylated microbes and Siglecs [34].
Viruses and bacteria as well as bacterial toxins recognize sialylated targets for
recognition [42]. A Sia-binding virus uses hemagglutinin and sialidase (neuramin-
idase) to cleave the same receptor [43]. Viruses are basically defined by the hemag-
glutinin (H), neuraminidase (N), antigenicity, serology, or RNA genotypes. Some
viruses bind to O-acetyl-SAs and cleave off the O-acetyl group [44–46]. Some
protozoa also employ SA binding (the malarial merozoite) [47–49]. A bacterial
SubAb toxin specifically binds Neu5Gc SAs [50]. In fact, H. influenzae utilizes
SAs as a C-sourced energy in the breakdown of Neu5Gc to form pyruvic acid and
ManNAc residues. ManNAc is further converted to GlcNAc [51]. Free SA can be
used as a signaling factor in some infectious pathogens. Pneumococcus [52] forms
biofilms to colonize using SAs. For SAs as a Siglec-binding SAMP [22], bacterial
desialylation perturbs self-recognition events and, consequently, increases inflam-
matory responses by desialyl DAMPS [53, 54]. SAs on certain bacterial polysac-
charides are O-acetylated, thus dually affecting the host–pathogen interaction. The
O-acetyl Sias reduce recognition by CD33rSiglecs and also enhance immunogenic-
ity [55]. Therefore, O-acetylation helps the bacteria survive under certain conditions,
protecting them from microbial neuraminidases or bacteriophage-recognizing pro-
teins. One exceptional case is sialoadhesin where O-acetylation blocks sialoadhesin
interaction [56]. This helps the bacterium to avoid phagocytosis [55, 57]. Unlike
selectins, Siglecs recognize Sia C7–C9. O-acetylation of Sia C7–C9 blocks Siglec
recognition [56, 58]. Thus, sialic acid O-acetylation modulates Siglec function.
O-acetylation in the ganglioside GD3 is known for a melanoma-specific antigen
[59]. Although T cells also express O-acetyl GD3 and GD3 has apoptosis-inducing
potential, O-acetyl GD3 has antiapoptotic effects [60].
7.5 Microbial Sialic Acid-like Molecules Synthesis

and Recognition of Microbial Sialic Acids by DCs
and Bacteriophages
Some bacterial pathogens express surface Sias [61]. Bacterial sialic acid biosynthesis
has adopted a bidirectional parallel evolution of recruitment and modification of
bacterial nonulosonic acid synthesis [62–64] to acquire Sias. Evolved
sialyltransferases were independently combined to recreate sialylated glycans.
Based on the roles of Sias and SAMPs, microbes synthesize host-like sialyl glycans.
Organisms synthesize the ancient family of 9-C saccharides, namely, nonulosonic
acids (NulO), including legionaminic acid and pseudaminic acid [64]. Bacteria
persisted in ancient nonulosonic acid biosynthesis for the production of vertebrate-
like Sias [62]. Thus, bacterial Sias include nonulosonic acid (NulO). If bacteria
express vertebrate cell Sias, then one must question how the immune system
distinguishes sialylated pathogens (or nonulosonic acids), even after maintaining
tolerance to self-sialyl ligands. CD33rSiglecs easily bind sialylated pathogens and
potentiate endocytosis to capture and internalize the cells [65–68]. However, Siglec-
1 known as sialoadhesin has no signaling capacity, but due to its size, length, and
structure, it phagocytoses them. Siglec-1 has a well-conserved and common speci-
ficity for Neu5Ac-α2,3 or Neu5Ac-α2,8, but not for the Neu5Gc species, binding;
this property explains this concept. Bacteria escape from bacteriophages by binding
to bacterial glycans. In addition, bacteriophages evolve much faster than prokaryotes
such as bacteria, as certain bacteriophages evolve to bind to SA-containing bacterial
capsules [69, 70]. Gram-negative bacterial membranes are characteristic of O- and
K-antigens linked by lipids or free surface polysaccharides. These K- and O-antigens
consist of a thick layer to cover the cell surface and are called a capsule. In
meningitis, pneumonia, and septicemia-causing bacteria, the K- and O-antigen
complex capsules determine the pathogenicity [71]. CPSs shield bacteria from the
host immune system. Surface CPSs protect bacteria from bacteriophage infection.
On the other hand, CPSs increase the pathogenic capacity by suppressing the
function of the host innate immune system. Certain pathogenic CPSs are identical
to glycans produced by mammalian host cells, resulting in protection by molecular
mimicry in the neuroinvasive Neisseria meningitidis serogroup B and E. coli K1.
The capsules of both organisms contain α2,8-poly Sia that is abundant on human cell
surfaces [72]. PolySia is an α2,8-NeuAc chain polymer that is present on the cell
surfaces of eukaryotes and also bacteria. CPSs also serve as receptors for specialized
7.5 Microbial Sialic Acid-like Molecules Synthesis and Recognition of. . . 323
bacteriophages [73]. Bacterial CPSs often interact with bacteriophages. Capsule

phages contain spikes and function as an adsorption receptor and as a capsule-
degrading enzyme [74]. The degrading enzymes also bind to the capsular polysac-
charide. The CPS of enteropathogenic E. coli K92 is composed of poly SAs with
SAα2,8 and SAα2,9 linkages. For the E. coli Kl polysialic acid capsule, certain Kl
phages recognize the capsular polysialic acid as a cell surface receptor by their
capsule-degrading endosialidase. For example, group B Streptococcus (GBS) has
evolved to form distinct polysaccharide-producing types, terminating with the
sialylated trisaccharide [75]. The GM1-binding cholera toxin B is encoded by
nonlysogenic phage in V. cholerae [76].
As one of the viridans streptococci species, the specific strain S. mitis causes
infective endocarditis. The bacteria bind to human platelets through the bacterial
proteins Pbl-A and Pbl-B, which cause pathogenic disorders [77]. The bacteria
recognize the sialyl-oligosaccharides present in host cells and colonize the oral
cavity by bacterial adherence to salivary glycoproteins [78]. The SA species linked
to the host glycolipids and glycoproteins are used as bacteria-binding receptors for
bacterial adhesion. The bacteria attach the SAs of the GP1b glycoprotein present in
platelets [79]. As gangliosides serve as receptors for many bacteria or toxins
including the cholera toxins, H. influenzae, H. pylori, and N. meningitidis [78],
two gangliosides (GM3 and GD3) are predominantly expressed in the membranes of
human platelets [80, 81]. The GM3 ganglioside is enzymatically converted to the
disialyl form of GD3 in the early step of activation and differentiation of platelets
before calcium homeostasis and granule release. The GD3 ganglioside directly
participates in FcλRIIa receptor signaling in human platelets [82]. The interaction
of platelets with the SF100 protein is predominantly induced by the bacterial pro-
teins Pbl-A and Pbl-B, which target the SAα2,8-linked membrane GD3 present in
platelets [40]. The basic innate immunity protects against pathogenic microorgan-
isms such as S. pneumoniae [82] via the classical complement pathway as an
activation mechanism. As the key pathway, the C3 convertase forms C3 fragments
that bind to microbes for leukocytes such as DCs and macrophages. Therefore, the
deposited C3 convertase on the bacterial surface triggers the elimination of
microbes. However, if microbes are encapsulated, as frequently found in
S. pneumoniae, the complement pathway is restricted by a reduction of C3 deposi-
tion [83]. Hosts have, therefore, evolved to acquire an alternative strategy to detect
pathogens by PRRs, which distinguish the PAMPs expressed on pathogens and,
most alternatively, allow differential binding of pathogens and the released products
in the immune response [84].
The obligate human pathogens of N. gonorrhoeae and N. meningitidis have
evolved to evade cytotoxicity by the hosts. Therapeutic antibiotic-resistant
N. gonorrhoeae strains are increasingly problematic and are classified as a “life-
threatening bacterium” by the Centers for Disease Control (CDC) and CDC Preven-
tion (CDCP). An innovative therapeutic strategy against the strains is necessary for
combating antibiotic-resistant N. gonorrhoeae strains. As a complement is the line of
defense against N. meningitidis and N. gonorrhoeae, such classical and alternative
pathways amplify complement component 3 (C3) deposition. Inhibition of such
pathways increases serum resistance of the strains. The strains have evolved to
acquire resistance to evade the complement in an alternative pathway of the host.
LOS sialylation of gonococci strains induces serum-sensitive types to acquire serum-
resistant types, reduces the capacity of target-recognizing antibodies, and evades
DCs, neutrophils, and antimicrobial peptides. Sialylation of LOS is pivotal to
N. gonorrhoeae survival from the host immune responses. N. gonorrhoeae sialyl
LOS protects host complement-mediated cytotoxicity through recognition enhance-
ment of the complement inhibitor, which is known as factor H (FH). Sialylation of
LOS contributes to acquisition of N. meningitidis resistance. Diminished LOS
sialylation reduces FH binding [85]. N. meningitidis and N. gonorrhoeae LOSs are
frequently terminal-modified in lacto-N-neotetraose (LNT) residues with sialic acid.
LOS α-2,3 sialyltransferase (Lst) adds sialic acids. Lst enzymes scavenge the host
sialic acids to incorporate into their LOS. Gonococci scavenge CMP-NeuAc
(SA) from the human host and sialylate the terminal Gal residue of the LOS LNT.
The Sia-bound LOSs decrease antibody recognition [86] and increase the host
alternative pathway inhibitor, factor H (FH) [87]. N. meningitidis inhibits the
alternative pathway using CPSs [88], FH-recognizing proteins, including Neisseria
surface protein A (NspA)/PorB2/FH-binding protein (FHbp) [87–91], and Neisseria
sialyl LOSs [92]. The sialylation of LOS components protects the N. gonorrhoeae
strains from complement-involved cytotoxicity, and thus resistant N. gonorrhoeae
strains have evolved to acquire higher Lst activity to combat host immune protection
than sensitive strains (Fig. 7.6).
Complement pathway
Neu5Ac
Sialylation Classical Lectin
Alternative
pathway (AP) pathway pathway
CMP-NANA ( human host)

+ C3
terminal galactose Factor H (FH) FH
FH C3 convertase
(LNT in N. gonorrhoeae) FH
① Acting as cofactor of C3b to iC3b ¢ iC3b
C3b
② Dissociation AP C3 convertase (inactive form)
Human host ③ Competing with factor B for C3B binding C3a
¤
CMP-NANA
C3b
FB
CMP-NANA : cytidine monophospho-N-acetylneuraminic
t l i i acid
i
C3b FH
FB
LNT: lacto-N-neotetraose £ FB FB
FB
LOS: lipooligosaccharide Sialylation
Lst : LOS α-2,3-sialyltransferase FD
Lst Bb
LNT C3b
C3 convertase
C5
Neisseria Neisseria
meningitidis gonorrhoeae
P P
C5 convertase C5b
GlcN
Kdo
Hep
Gal MAC
Glc
Sialic acid (NeuAc)
Fig. 7.6 Neisseria gonorrhoeae LOS α2,3 sialylation potentiates complement resistance and
evasion from killing of the host defense (illustrated from Ref. No. [85] Lewis LA et al. 2015.
MBio 6(1),pii: e02465–14)
7.6 Hematopoietic System in Siglecs 325
7.6 Hematopoietic System in Siglecs
In mammals, surface glycans contain SA residues present in the terminal

nonreducing end of glycans, which are located in the distal region. Sialylated
glycans on cell surfaces have modulated cellular events for more than 500 million
years since the appearance of the first vertebrates in the Paleozoic Era, which is
estimated to be approximately 500 million years ago [93, 94]. In early mammals,
CD33-related Siglecs likely manifested for more 200 million years [95]. The phy-
logenetic pedigree of mammalian Siglecs indicates the coevolutionary relationship
between Siglec-1, -2, -4, and -15. However, CD33rSiglecs are independently
adapted to evolve [96]. The high sequence similarity between CD33rSiglecs is likely
caused by pathogenic attraction to SA mimic selection during invasion and infection
(called the “Red Queen effect”), as argued by Varki’s group [96, 97]. SA
glycoconjugates belong to SAMPs, which are recognized by inhibitory receptors
to dampen immune responses via the interaction of Siglecs with distinct sialylated
glycans. Eukaryotes and certain prokaryotes can decorate them with distinct
sialylglycans, and immune-involved cells recognize sialylglycans as endogenous
ligands for self- or nonself-pathogens. Siglecs as immune-regulatory receptors are
mainly present on the hematopoietic cells of vertebrates. Detailed prospects and
outlines have been made by Varki’s group on specific sialylated glycans expressed
on self-endogenous cells and nonself-exogenous cells such as pathogens. The
sialylated glycan-recognizing Siglec families have coevolved toward both inhibitory
and activating roles, as the Siglec expression is largely elucidated in mice and
humans. In addition, Siglec expression is still under study in many vertebrates
including amphibians, birds, fishes, and reptiles with a coevolutionary progress
[96]. This is why certain Siglec groups, i.e., Siglec-1, -2, -4, and -15, are structurally
conserved in the above fish. Bornhöfft et al. (2018) analyzed BLAST data of human
and mice sequences and found the presence of Siglecs both in vertebrates and in
invertebrates [96], where they found the two Siglec-2 and -15 types from vertebrates
as proinflammatory and anti-inflammatory receptors, respectively. They are all
found in the five vertebrate species of amphibians, birds, fishes, mammals, and
reptiles. Siglec-1 and Siglec-4 have been suggested to be ancient receptors, with
the Siglec-2 and -15 forms having the same vertebrate ancestors. Siglec-2 specifi-
cally exhibits an autoimmunity-prevention activity [98], whereas Siglec-4 that is
expressed on the inner side of the myelin wrap helps in myelin-axon recognition
[99]. Siglec-15 regulates bone resorption [100]. Thus, in the vertebrate evolution,
Siglec-2, -4, and -15 are suggested to be crosslinked to BCR differentiation,
myelination, and bone formation, respectively [96]. The evolution of Siglecs is
also related to lactation, which induces the mammalian CD33-related Siglec
appearance.
All human Siglecs are found on hematopoietic cells, except for MAG (Siglec-2).
Siglec-1 promotes intercellular interaction through binding to α2,3-linked SA and is
expressed on CD169+ macrophages. Siglec-5 and -14 are myeloid progenitor
markers. Siglec-5 is inhibitory, whereas Siglec-14 is an activating receptor. They
bind to the sialyl-Tn structure. In Siglec-7/-9 and -E, Siglec-7 binds to α2,8-linked
SA, whereas Siglec-9 binds to α2,3-linked SA. Siglec-E binds to both of them and
suppresses proinflammatory cytokines. Siglec-8 and -F bind to 60 -sulfo-sLeX and
induce eosinophil apoptosis but inhibit mast-cell-mediator release. Eosinophilic
asthma is thus regulated by them. Siglec-10 and -G bind to Neu5Gc in α2,3 and
α2,6 linkages. Siglec-10 induces NK cells, whereas Siglec-G induces B cells for
antibody production. In Siglec-11 and -16, Siglec-11 inhibits the microglia via α2,8-
linked SA but Siglec-16 activates it. Siglec-15 binds the sialyl-Tn structure on tumor
cells and macrophages and suppresses osteoclast differentiation.
Some Siglecs are widely expressed on hematopoietic cells. The V-set Ig-like
domains of Siglecs recognize different sialylated glycans and activate or inhibit the
immune responses. Siglecs are mainly expressed on hematopoietic cell surfaces, but
the extracellular region of the immune cells also produces Siglecs. In fact, Siglec-4,
known as MAG, is mainly present on Schwann cells and oligodendrocytes [101]. Sn
is preferentially produced in macrophages, whereas CD22 is produced in B cells.
However, Siglec-8 is expressed on eosinophils [23]. The two homologues of Siglec-
9 and -E are differentially present on myeloid-derived DCs (mDCs) of human and
mouse mDCs, respectively. However, Siglec-5 is present on human pDCs and
Siglec-H is detected on mouse DCs [23]. Siglec coreceptors are FccRIIB1, CD22,
paired Ig-like receptor B, and killer cell inhibitory receptor. Except for MAG, all
Siglecs are found in the hematopoietic cell lineage. Some Siglec expressions are cell
type-dependent. In fact, sialoadhesin is specifically expressed on macrophages and is
a type of adhesion molecule. CD22 is an inhibitory receptor of B cells with cellular
activation and survival signals. However, CD33rSiglecs are present as complex
forms on innate immune cells. CD33-related Siglec–ligand interaction leads to
phosphorylation of the Tyr residue of ITIMs, which is catalyzed by Tyr kinases
such as Src, and recruits tyrosine phosphatases, namely, SHP-1 and SHP-2. There-
fore, Siglec receptor function is modulated by inhibition of Tyr phosphorylation
[102]. Human Siglec-10 and mouse homologue of Siglec-G bind to CD24 and
distinguish the PAMPs from the DAMPS [31]. Similar to other CLRs, Siglec-H
functions as an endocytosis receptor of pDCs, when the cells capture pathogenic
viruses and bacteria for internalization to intracellular TLRs and antiviral immune
responses [103]. Siglec-H is deficient in Tyr-involved sequences and associates with
the adaptor DAP12 [103]. Thus, each specific Siglec differentially functions in
immune cell signaling. From the results obtained on Siglecs of various species
from evolutionary distinct clades, it has been determined that the conserved forms
of Siglec-1, -2, -4, and -15 exist in the same ancestral vertebrates before the division
of ancestors into tetrapod species-like mammals such as humans and teleost fishes
such as salmon about 400 million years ago [104–106]. This indicates the existence
of evolutionary pressure on the four receptors; however, the CD33rSiglec genes are
expanded with the coevolution of mammals [107]. On the other hand, the absence of
the activating Siglec-14 in some human individuals provokes the group B Strepto-
coccus suppression of neutrophil function [108]. Loss or lack of Siglec-14 increases
the risk of prematurity, since the placental amniotic epithelium produces Siglec-14
as the target for the invasive group B Streptococcus [109].
7.7 Structure of Siglecs 327
7.7 Structure of Siglecs
As cell surface receptors, Siglecs are Ig-like lectins of the Ig superfamily of verte-
brates that bind sialylated glycans and are involved in many physiological events
including glycoprotein turnover, intracellular trafficking, and pathogen interaction.
Their extracellular regions comprise multiple numbers of ‘C2-set’ Ig-like domains,
which exhibit a conserved IgFc-like sequence, an Ig-like domain in the N-terminal
region, and a V-set domain, which is highly similar to the IgV region [110]. This
domain has the SA-binding domain [111]. The multiple numbers of C2-set Ig-like
domains imply the SA-recognizing tendency of Siglecs expressed on the surfaces of
the same cells in a cis-type interaction or on adjacent neighboring cells in a trans-
type interaction. The known Siglec-1 form consists of 15 Ig domains that bind
carbohydrates in a trans-type. The Siglec-3, -8, and -15 forms, having Ig domains,
recognize sialyl glycans in a cis-interaction manner [112]. As all the Siglecs carry an
Ig-like domain in the N-terminal region with nine β-strands, they are similar to the
IgV-set domain. The Arg residue in this IgV-set domain is essential because it forms
a salt bridge with the COOH group that is linked to SAs. One salt bridge interacts
with SAs. In human Siglec-1 (hSiglec-1), the SA-recognizing domain is thus present
in the N-terminal region. The R116 guanidine group mediates a salt-based binding to
the NeuAc COOH group. The acetyl group attached to Neu5Ac binds by van der
Waals forces to the W21-attached indole ring. In addition, the SA C9-attached
glycerol group binds to the W125 aromatic group [96]. The recognition of
hSiglec-5 and SAs is mediated by a salt bridge formed with the Arg (R124)–
COOH group of Neu5Ac [113], similar to Siglec-1. K132 and S134 also recognize
the NeuAc C8 secondary amine and the hydroxyl groups, respectively, by hydrogen
bonds. Van der Waals actions occur between the Y133 aromatic group and C9 of
NeuAc [113].
7.7.1 Cytoplasmic ITIM and ITAM Domains of Siglecs
In view of the protein structures, Siglecs have generally cytoplasmic ITIMs and they
are well conserved in the Siglec forms expressed on the hematopoietic cell system
(Fig. 7.7). ITIMs are specialized domains for inhibitory signaling on stimulation
with self-antigens, allowing immune tolerance. As representative cases,
CD33rSiglecs and CD22 consist of one or multiple cytoplasmic ITIMs to effectively
perform the inhibition of tolerance. Generally, ITIMs function as suppressive medi-
ators of activation signals that initiate from immunoreceptor tyrosine-based activa-
tion motifs (ITAMs), which recruit tyrosine. Most Siglecs consist of an intracellular
domain ITIM and ITIM-like domains with the (I/V/L/S)-X-Y-X-X-(L/V) sequence
to counteract immune activation via ITAM-involved signaling inhibition. Among
the Siglec forms, Siglec-14/-15/-16 specifically recognize the DNAX activation
protein (DAP)10/12 to induce immune responses, where DAP10/12 consists of
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Fig. 7.7 Immunoreceptor ITIMs as domains for inhibitory signals and ITAM as the domain for
activating signals of Siglecs
ITAM domains [35, 114, 115], with the amino-acid sequence Y-X-X-L/I-X6-8Y-X-
X-X-L/I (where X indicates any undefined amino acid) [116]. DAP10/12 recognition
is mediated by the transmembrane region’s amino acids with positive charges in
Siglecs [115, 117]. Exceptionally, CD33 and Siglec-H of mouse and human Siglec-
14 and Siglec-15 are deficient in ITIMs for unknown reasons but seem to occur
throughout the evolutionary stage. As Siglecs have ITIMs, they can send negative
signals through the recruitment of the negatively working region, the SH2 domain,
which contains both SHP-1 and SHP-2. The activated ITIM inhibits receptor
signaling operated within the ITAM. Therefore, the receptors are called inhibitory
receptors due to suppression of activation signals caused by ITAM-carrying recep-
tors through Tyr phosphatase recruitment. Siglec-14, -15, and -16 are closely related
to the ITAM activity and the DAP12 adaptor protein by a transmembrane region
composed of positive amino acids in Siglecs. The activating receptor signals recruit
some spleen tyrosine kinase (SYK). In humans, two different pairs of Siglecs are
expressed. Siglecs transmit intracellular signals upon interaction with multivalent
“trans-“ligands derived from nonself or self.
7.7.2 Adaptor Proteins Associated with Siglecs
Among the three Siglecs, Siglec-15 is well characterized as DAP12- and DAP10-
associated Siglecs. The Asp residue in the DAP12 transmembrane domain is known
to interact with the Lys residue in the Siglec-15 transmembrane domain [35]. Upon
Siglec-15 interaction with sialylated glycans, the Src kinases phosphorylate the Tyr
residues found in the ITAM, which then serves as a docking site for the ZAP70 SH2
domains, and, as a result, Syks exert immune activation [35, 116]. With regard to the
functional and immunological roles of DAP-associated Siglecs, for example, the
Siglec-15 form regulates the fate of osteoclastic differentiation [118] in bone resorp-
tion [119]. Siglec-15 binding to DAP12 after SA recognition induces differentiation
of osteoclasts into multinucleated cell types that are functional for bone resorption
[118]. Anti-Siglec-15 antibodies block the differentiation of osteoclasts through
dimerization, internalization, and degradation of Siglec-15 dimers [120]. On the
other hand, apart from the DAP-involved Siglecs, ITIM-having Siglecs or ITIM-
containing Siglecs can silence ITAM-triggered immune reactions. The interaction of
sialyl glycans phosphorylates the intracellular Tyr residues of Siglecs by Src kinases.
Phospho-Siglecs can recruit SHP-1 and SHP-2, capable of receptor interaction, and
can inhibit kinase-dependent pathways [23]. For example, antibody production is
inhibited in a Siglec-mediated manner, when the BCR binds to a counterpart antigen.
B cells differentiate into Ab-expressing plasma cells to produce antibodies. How-
ever, if the target antigen is copresented with sialyl glycans on endogenous cells, the
B-cell Siglec-2 binds to sialyl glycans [121, 122]. Then, the BCR and Siglec-2
clusters recruit SHP-1 and SHP-2 and, consequently, the kinase-dependent signaling
pathway is inhibited, thus resulting in blocked antibody production against the
autoantigen. On the other hand, Siglec-2 can also recruit several activator proteins
such as GFR-bound protein 2 (GRB2), PLC-γ2, PI3K, and the SH2-domain-
containing transforming protein C (SHC), indicating the dual capacity of Siglec-2
to act on the respective B cells [122].
For the ITIM-having Siglecs, Siglec-9-suppressed immunity was reported by
Varki’s group [123]. Siglec-9 suppresses neutrophil function upon recognition of
sialyl glycoproteins on erythrocytes, indicating that sialyl glycans function as a
SAMP on erythrocytes. This can be applied to tumor cells for Siglec-involved
roles. AML cells and CLL cells largely express sialylated glycans as Siglec-7/-9
ligands, residing on the surfaces of NK cells. Therefore, Siglec-7/-9-specific SAs
ligands present on tumor cells suppress the activation of NK cells, allowing cancer
growth [100, 124, 125]. In addition, it is important to keep a balance in the Siglec-
related action to maintain a constant healthy status, as any imbalance may provoke
any disease status like neurodegenerative diseases [126–129]. Representatively,
Siglec-3 is a possible risk factor for Alzheimer’s because microglial cells known
as tissue macrophages of the neuronal system increasingly express Siglec-3,
resulting in an insufficient capture of amyloid-β plaques [130, 131]. Moreover, the
polymorphic Siglec-8 gene pattern is linked to the onset of allergic asthma [132], as
Siglec-8 is known to be mainly expressed on human eosinophils. Siglec-8–ligand
interaction on eosinophils in tissues promotes eosinophil apoptotic cell death. In an

airway inflammation animal model induced by ovalbumin, mucins and epithelial
cells contain an excess level of Siglec-F ligands, where Siglec-F is a mice homo-
logue of human Siglec-8, allowing a reduced level of the invading eosinophils
[100, 133].
ITIM-bearing Siglecs control the immune response and may have therapeutic
potential. However, pathogens can also utilize Siglecs. As mentioned earlier, path-
ogenic microorganisms including N. meningitides, H. influenza, C. jejuni,
P. aeruginosa, and group B Streptococcus express sialylglycans to interact with
Siglecs [134]. More specifically, group B Streptococcus produces a capsular
SAα2,3-Galβ1,4-GlcNAc ligand for Siglec-9. The binding of Sia α2,3-Galβ1,4-
GlcNAc to Siglec-9 dramatically reduces the function of the neutrophil–myeloid–
oxidative axis to increase the survival rate of pathogens [68]. Mutation results from
CD33-related Siglecs show that the ITIM is predominantly dominant over the other
ITIM-like domains and that it recruits the adaptor phosphatases SHP-1 and SHP-2.
The recognition capacity of SHP-1 and SHP-2 needs the phosphor-Tyr residue to be
attached to the ITIM and ITIM-like domains. However, Siglec-5 weakly recognizes
SHP-1, but it is not phosphorylated due to Ala replacement from Tyr [135].
7.7.3 SA-Recognition Tropism of Siglecs
SAs on immune cells can be defined as biological masking agents and cell pattern-
recognizing agents. SA-containing carbohydrates present on DCs are recognized by
Siglec receptor-holding effector cells. Although there are many SA species in
vertebrates, and they have good interactions with Siglecs [136] (Table 7.2), two
common SA glycans recognized as ligands by Siglecs are shown (Fig. 7.8a, b). For
example, high α2,6-SA contents of tolerogenic DCs and immature DCs are recog-
nized by inhibiting Siglecs produced by effector T cells and this is indeed a host
tolerance-induced mechanism. The enhanced binding capacities of Siglec-1, Siglec-
2, and Siglec-7 are correlated with the high SA levels of mature DCs. DCs also
express themselves as Siglecs on their surfaces. For biological masking functions,
SAs shield the host cells from pathogenic binding and thus consequently prevent
autoimmune responses. Therefore, the concentration of SAs expressed on human
cell surfaces is relatively increased. High SA contents are caused as a result of acute-
phase responses. SAs also upregulate immune cell activities and discriminate the
“self-” antigens from the “nonself” ones. The majority of DC Siglecs bind in the cis-
type. However, sialidases can inhibit Siglec–SA binding through the extrinsic or
intrinsic action of sialidases. More specifically, sialidases can improve phagocytosis
operated by human monocyte-derived DCs (mo-DCs) and mature mo-DCs.
Sialidase treatment increases the immune response of MDDCs [137]. E. coli phago-
cytosis is also improved by SA moieties. Sialidase treatment improves the capacity
of mo-DCs to phagocytose pathogenic E. coli isolates, as removal of SAs from the
surfaces of mo-DCs highly sialylates induced mo-DC maturation and decreased
Table 7.2 Human Siglec family and binding of sialic acids to SA-recognition molecules. Words in red indicate CD33-related Siglecs. , Sialic acid (SA);
, galactose (Gal); , N-acetylglucosamine (GlcNAc); , N-acetylgalactosamine (GalNAc); , fucose (Fuc)
Siglec Cognate
(CD number), glycan Tyrosine
No. of Ig domains structure Distributed cells Sialyl linkage motifs Pathogen binding Lectin Related disease
Siglec-1, Macrophages Sia α2,3- – Avian influenza A Maackia Autoimmunity
sialoadhesin (activated Gal>α2,6 amurensis HIV-1, PRRSV
7.7 Structure of Siglecs
(CD169), 16 Ig D monocytes) leukoagglutinin infection

(MAL) C. jejuni, group B
streptococcus
infection
Siglec-2 (CD22), B cells Sia α2,6-Gal ITIM Human influenza Sambucus nigra Lymphoma, leuke-
6 Ig D A agglutinin mia, SLE, rheuma-
toid arthritis
Siglec-4 (MAG), Oligodendrocytes Sia α2,3-Gal FYN- Plasmodium Maackia Neurodegeneration
4 Ig D Schwann cells kinase falciparum mero- amurensis hem-
site zoite EBA-175 agglutinin
(MAH)
Siglec-3 (CD33), Myeloid progenitors Sia α2,6-Gal ITIM- Acute myeloid leu-
1 Ig D Macrophages like kemia (AML),
Monocytes Alzheimer’s disease
(microglia,
Granulocytes)
Siglec-5 Neutrophils, mono- Sia α2,8Sia ITIM Group B Streptococ-
(CD170), 3 Ig D cytes (B cells) Sia α2,6-GalNac cus infection
Siglec-6 Trophoblasts Sia α2,6-GalNac ITIM Preeclampsia

(CD327), 2 Ig D (B cells)
Siglec-7 NK cells (mono- Sia α2,8Sia2,3- ITIM Cancer
(CD328), 2 Ig D cytes, mast cells) Gal C. jejuni infection
331
(continued)
332
Siglec Cognate
(CD number), glycan Tyrosine
No. of Ig domains structure Distributed cells Sialyl linkage motifs Pathogen binding Lectin Related disease
Siglec-8, 2 Ig D Eosinophils, (mast Sia α2,3-Gal ITIM Eosinophilia, asthma
cells, basophils)
Siglec-9 (CD329) Neutrophils, mono- Sia α2,3- ITIM Chronic lung
2 Ig D cytes, dendritic cells GalGalNac Sia inflammation
(NK cells) α2,6
Siglec-10, 4 Ig D B cells (monocytes, Sia α2,6-Gal ITIM Lymphoma, leuke-
Eosinophils) mia, eosinophilia,
allergy
Siglec-11, 4 Ig D Macrophages Sia α2,8Sia α2,3- ITIM Neuroinflammation
(microglia) Gal
Siglec-14, 2 Ig D Neutrophils Sia α2,8Sia α2,3- – Group B Streptococ-

Monocytes Gal cus infection COPD
Sia α2,6-Gal
Siglec-15 Osteoclasts Sia α2,6-Gal – Osteopetrosis
Macrophages, den-
dritic cells
Siglec-16 Macrophages Sia α2,8Sia2,3- – Viral infection
Microglia Gal Neuroinflammation
E-selectin Sia α2,3- Pseudomonas –

Gal1,4GlcNAc aeruginosa
(Fucα1,3)β1,3- mucoid strain
Gal1-R 8830
Anaplasma
phagocytophilum
7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
R’ OH CO2H
A) Sialic acid R R’
OH Neu5Ac CH3 OH
HO O Neu5Gc CH2OH OH
Neu5,9Ac CH3 OAc
HN HO Neu5Gc9Ac CH2OH OAc
R C
O
OH OH CO2H
B)
O
HO O α 2,6Gal-glycans
Neu5Acα
HN HO HO
O O01IN[ECPU
R C
O HO
HO Neu5Acα 2,3Gal-glycans
HO
OH OH CO2H O O01IN[ECPU
HO O HO
O
HN HO
R C
O
Fig. 7.8 Sialic acid recognition of Siglecs. (a) SA is substituted in mammals at the positions of R
and R0 . (b) SA–Gal linkages: Recognition of two SA ligands by many Siglecs. The sialic acid
species are diverse in vertebrates. The sialic acid structure is substituted at the carbon positions of R
and R0 by amino group halogenation and acylation. Various sialic acids are found in vertebrates
(adopted from Crocker PR et al. 2007. Nat Rev Immunol. 7(4), 255–66 [23] with a slight
modification)
micropinocytosis [138]. ST6Gal-1-null BMDCs have a higher phagocytic activity

compared to that of normal cell types [23]. Desialylated MDDCs increase the
phagocytic capacity of E. coli. Treatment of MDDCs with sialidase increases the
level of NF-κB activation and cytokine gene expression, consequently increasing the
phagocytic capacity of the cells, indicating that desialylated mo-DCs enhance
immunological functions including cytokine gene expression and NF-κB activation,
as well as IFN-γ gene production by T cells. E. coli phagocytic capacity is influenced
by the SA species. BMDCs isolated from ST6Gal-1 KO mice increase the phago-
cytic activity against E. coli. Neuraminidase influences the reorganization of the
cytoskeleton and activates Rho-GTPases. Phagocytosis remodels the actin cytoskel-
eton and activates Rho small-type GTPases [137].
7.8 Inhibitory Signaling of DCs
SA-containing glycans cover the DC surface to be implicated in DC biology.

Sialoglycans regulate DC function. Siglecs are variable in their ligand-binding
specificity for SA ligands. Their immune cell expression is of relevance both in
health and in disease. In the historical aspect, the Siglec-type CD22 was first reported
for the CD22-encoding cDNA clone, which is primarily expressed on B cells,
indicating that it bears multiple Ig-like domains [16] and that CD22 binds to two
sialoglycoproteins, as reported in 1991 [139]. In the same year, the sheep erythrocyte
receptor on macrophages was reported to be renamed sialoadhesin due to its
sialoglycoconjugate-binding capacity [2]. Sialoadhesin and MAG were also reported
as SA-binding lectins and a type I membrane protein integrated into the PM. They
have Ig-like extracellular domains [140, 141]. The lectin CD22 belongs to the
sialoadhesin lectin family [141], Ig-like lectin family of the I-type lectin family
[142], which is different from C-type lectins or Ca2+-depending lectins. On the basis
of the work by Crocker and Varki in 1998, the family of SA-binding Ig domain-
carrying lectins is termed “Siglecs” [143]. Certain Siglecs, however, do not bind to
sialyl ligands. Detailed information on the specificity of Siglecs is currently in the
editing stage in a step-by-step process due to their rapid evolution [104].
Some Siglecs are classified as inhibitory receptors of innate immune cells due to
their regulation property of inflammation caused by DAMPs and PAMPs. They are
involved in adhesion and phagocytosis through association with the ITAM-carrying
DAP12 adaptor. As activating receptors, Siglecs inhibit immune cells by recogniz-
ing cis-ligands, which are expressed in identical or endogenous cells, as well as by
interacting with pathogenic sialoglycans. They also maintain tolerance in B cells,
regulate conventional and plasmacytoid DCs, and regulate T-cell functions
[117]. Desialylation with neuraminidases induces the response of DCs to endotoxin
LPSs and T-cell activation [144–148]. Thus, SAs limit DC response to
TLR-mediated signaling. For example, a synthetic and fluorine derivative SA
mimetic, Ac53FaxNeu5Ac, is reported to block SA synthesis in human mo-DCs
[149]. Blocking the SA expression increases the response of mo-DCs to TLR
signaling and T-cell activation. Mo-DCs express sialic acids and also the immune
inhibitory receptors of Siglecs-3, -5, -7, -9, and -10 that bind to sialic acids.
Some Siglecs have inhibitory signaling sequences as amino-acid motifs in their
cytosolic region. Such Siglecs are crucial molecules in inhibitory signaling, which
suppress immune responses and dampen inflammatory responses in specialized
conditions like lymphocyte exhaustion or tissue cell apoptosis. Inhibitory signaling
motif-lacking Siglecs are involved in other roles in sialic acid recognition. This
discriminates how Siglecs control immune responses. For example, human cells
mostly express two pairs of Siglecs. CD33rSiglecs and CD22 consist of one or
multiple cytoplasmic immune receptors known as ITIMs. In mice, Siglec-E is an
inhibitory CD33rSiglec receptor. The presence of Siglec-E is mainly observed in
macrophage and neutrophil subsets. Generally, ITIMs act as inhibitors of activation
signaling induced by immunoreceptor tyrosine-based activation motifs (ITAMs).
7.8 Inhibitory Signaling of DCs 335
ITAMs recruit Tyr-based molecules. The inhibitory receptors of Siglecs regulate

TLR signaling, as, recently, CD33rSiglecs have been considered to have evolved
and are mainly present on clusters of innate immune cells. They consist of an ITIM
in the cytoplasmic region and an ITIM-like domain. SA recognition by inhibitory
Siglecs is mediated in a cis-interaction manner with SAs in leukocytes. Siglecs can
also recognize SA ligands in a trans-manner on adjacent cells or ligand-carrying
pathogens to elicit Siglec-dependent endocytotic signaling [149]. Exceptionally,
murine Siglec-H and CD33 as well as human Siglec-14 and -15 are deficient in
ITIMs. Due to ITIMs, Siglecs can send a negative signal through recruitment of the
SHP-1- and SHP-2-containing SH2 domain. Activator receptors are based on spleen
tyrosine kinase (SYK) recruitment.
A model mouse Siglec-E is predominantly present on tissue macrophages, neu-
trophils, and splenic DCs [150, 151], as inhibitory CD33-related Siglec, Siglec-E,
and its human homologue Siglec-9 regulate TLR-4-involved cytokine production in
DCs and macrophages. In murine BMDMs, binding of Siglec-E to antibodies
decreases the production of IL-6, TNF-α, and RANTES upon LPS stimulation
[152]. An enhanced expression of Siglec-9 of human origin in RAW264 and
THP-1 macrophages decreases proinflammatory cytokine production upon LPS
treatment [153]. Siglec-E also suppresses proinflammatory cytokine expression in
macrophages during treatment of sialyl capsules of group B Streptococcus
[154]. TLRs can directly interact with Siglec-E [155]. The cis-type Siglec-E binding
to TLR-4 is a prerequisite for TLR-4-mediated endocytotic internalization during
E. coli capture and downregulates TLR-4-mediated inflammatory responses
[155, 156]. Siglec-E induces the differentiation of macrophages upon LPS treatment,
thus weakly modulating TLR-4-involved signaling in macrophages or DCs [157].
Siglecs expressed on DC cell surfaces present ITIMs in their cytosolic portion.
Since DCs modulate both B and T cells, Siglecs regulate host tolerances (Fig. 7.7).
Thus, such inhibitory activated signals can induce immunomodulatory roles in the
cells. For example, ST6Gal-I is one such representative. Some pathogens mimic
masking to evade the immune system. In case of recognizable cell patterns of
Trypanosoma spp., they are recognized by several cell surface receptors like CLRs
and Siglecs. Two big Siglecs are members of the CD22 family and the CD33-related
family. These recognize and bind ligands in a trans- and cis-manner. In addition,
Siglecs present ITIMs or ITAMs, regulating and discriminating between SAMPs and
PAMPs. In a knockout mice study, ST6Gal-1 KO mice showed decreased humoral
immune responses. As CD22 recognizes ST6Gal-1-mediated glycans, sialic acid 2,6
is known to regulate several B-cell functions. In addition, the soluble form of
ST6Gal-1 regulates myelopoiesis upon acute inflammation. In the case of ST3Gal-
1 KO mice, α2,3-sialyl-O-glycans maintain CD8+ T-cell homeostasis. Human and
mouse Siglecs in immune cells have been well studied (Table 7.3). Siglec families
have been well established with SA-binding specificities, C2 Ig domain numbers,
and ITIM structures (Table 7.4). Human Siglecs and their mouse orthologs are
known. Sequences of Siglec-1, -2, -4, and -15 exhibit conserved common homolo-
gies in both mice and humans. Other Siglecs such as members of the CD33 (Siglec-
336
Table 7.3 Patterns of Siglec expressions on different immune cells in humans and mice
B cells T cells NK cells Monocytes Macrophages DCs Neutrophiles Eosinophiles Basophiles
Human Siglec-2 (CD22) Siglec-7 Siglec-7 CD33 Sialoadhesion CD33 Siglec-5 Siglec-8 Siglec-5
Siglec-5 (CD170) Siglec-9 Siglec-9 Siglec-5 CD33 Siglec-7 Siglec-9 Siglec-10 Siglec-8
Siglec-6 (CD327) Siglec-10 Siglec-7 Siglec-5 Siglec-9 Siglec-14
Siglec-9 (CD329) Siglec-9 Siglec-11 Siglec-10
Siglec-10 Siglec-10, Siglec-14 Siglec-15, Siglec-16 Siglec-15
Mouse CD22/Siglec-2 Siglec-E Siglec-E Sialoadhesion Siglec-E CD33 Siglec-F
Siglec-G Siglec-F Siglec-H Siglec-E
7.8 Inhibitory Signaling of DCs 337
Table 7.4 SA-binding specificities, C2 Ig domain numbers, and ITIMs or SA-binding residues of
different Siglecs
C2-Ig ITIM or
domain SA-binding
Name Distributed cells SA specificity number residue
Sn/Siglec-1 Macrophage α2,3 > α2,6 16 None
(CD169)
CD22/Siglec-2 B cells α2,6 6S 6 ITIM
MAG/Siglec-4 Myelin α2,3 > α2,6 4 None
Siglec-15 Macrophage, DCs α2,6 1 Lys
CD33/Siglec-3 Myeloid progeni- α2,6 > α2,3 1 ITIM
tors, monocytes
Siglec-5 Neutrophiles, α2,3 3 ITIM
monocytes
Siglec-6 Trophoblast α2,6 2 ITIM
Siglec-7 NK cells α2,8 > α2,6 > α2,3 2 ITIM
Siglec-8 (mouse Eosinophils α2,3 > α2,6 2 ITIM
Siglec F)
Siglec-9 (mouse Monocytes, neu- α2,6 ¼ α2,3 2 ITIM
Siglec E) trophils, DCs (prefers sulfated
residues)
Siglec-10 B cells α2,6 ¼ α2,3 4 ITIM
(mouse Siglec
G)
Siglec-11 B cells α2,8 4 ITIM
Siglec-12 Macrophages No recognition 2 ITIM
Siglec-14 Unknown α2,6, α2,8 2 Arg
Siglec-16 Macrophage, DCs α2,8
3) family and their mice orthologs are named as Siglec-E. CD33 and its related
Siglecs are broadly expressed in the innate immune system (Fig. 7.8).
Sialoglycan structures are present in epithelial cells, most immune cells, and
tumor cells. Some microbial agents take up SAs from the host cells to escape from
the Siglec-based immune recognition of hosts. The well-defined examples include
GBS, C. jejuni, and N. meningitides. They recognize CD33rSiglecs [66]. Siglecs are
involved in cis- and trans-binding to sialylated glycans. The cis-bindings of Siglecs
are often masked by sialyl ligands with low affinities toward the adjacent receptors
and they cannot block trans-binding to other cells [23].
7.9 Siglec-1 (CD169, Sialoadhesin/Sn)
7.9.1 General SAbinding Specificity of Siglec-1
Siglec-1/sialoadhesin (Sn/CD169) belongs to a cell surface CAM expressed on

macrophages among immune cells. In the beginning of the 1980s, historically,
Kraal and Janse discovered Siglec-1/CD169+ macrophages using mAb MOMA-1
against mouse lymph node cells [158]. At that time, Siglec-1/CD169, known as Sn,
was initially recognized as an unopsonized sheep erythrocyte-binding receptor
present in stromal macrophages in tissues [1, 159]. The Siglec-1 (CD169) gene
was isolated as the sole Siglec form activated during LPS-treated DC maturation.
Currently, Siglec-1 is renamed as a specific receptor for some exosomes [160, 161],
certain retroviruses [162–164], and sialylated bacterial pathogens [165, 166]. Like
other Siglecs, Sn, Siglec-1/CD169 binds to SAs and prefers SA conjugates such as
SAα2,3-Gal-linked forms. CD169/Siglec-1 binds to α2,3-Neu5Ac-sialylated mole-
cules found on pathogens and the host’s glycosphingolipid GM3 [112]. Siglec-1 is
an I-type lectin because it has 17 Ig domains, i.e., 1 variable domain and 16 constant
domains, as the Ig superfamily. For binding of Siglec-1/CD169 to sialyl ligands,
proteins form a salt bridge between a conserved Arg residue from the V-set domain
to the α2,3-sialyl linkage and the carboxylate group in SAs. Since Siglec-1/CD169
binds to SAs with its N-terminal IgV domain, it is named the Siglec family. Siglec-1/
CD169 predominantly recognizes neutrophils and also recognizes monocytes, B
cells, NKs, and CTL subpopulations through interaction between SA ligands on cell
surfaces. Siglec-1/CD169+ macrophages trans-signal to undergo cell-to-cell com-
munication and to maintain hematopoietic stem cell retention at the niche.
Siglec-1-positive macrophages are located on the interspaced area between cir-
culating fluids and the local tissue, allowing easy interaction with target molecules in
the tissue and consequent transduction of information to the adjacent immune cells
[167]. At the interspace of the circulating fluids and tissue, Siglec-1/CD169+
macrophages capture and internalize blood- and lymphoid-produced molecules in
the lymphatic organs. Antigen information transduced by Siglec-1/CD169+ macro-
phages to the adjacent immune cells elevates antimicrobial immune responses upon
recognition of the invasive pathogen. In tumor antigen recognition, Siglec-1/
SC169+ macrophages transduce information to immune cells to exert both antitumor
immunity and tolerance. Macrophages are indeed distinguished from conventional
macrophage subsets to ontogeny-based macrophage subpopulations. Therefore, a
precise classification of the macrophages is performed using certain parameters of
functional analysis including function, localization, and surface phenotype
[168]. From the characteristic properties of Siglec-1/CD169+ macrophages differ-
entiated during antigen stimulation, Siglec-1/CD169+ cells utilize distinct endoge-
nous transcription factors and exogenous local signals generated from the tissues for
their existence. Therefore, the specific expression of the Siglec-1/CD169+ cell
phenotype is dependent on local tissues. Then, a question arises: how are tissue-
specific phenotypes displayed or developed? To answer this question, specific
7.9 Siglec-1 (CD169, Sialoadhesin/Sn) 339
transcription factors develop Siglec-1/CD169+ macrophages, as reported from the

fact that liver X-receptor α-/β-lacking mouse do not develop Siglec-1/CD169+
marginal zone macrophages [169]. Lymphotoxin (LT) α1β2, a B cell-derived cyto-
kine, functions during the phenotype change in lymphatic node-resident macro-
phages but not in medullar sinus-resident macrophages and Siglec-1/CD169-
positive gut macrophages [170].
In inflammatory responses, Siglec-1/CD169+ macrophages modulate immune
responses and maintain tolerance in the spleen. Siglec-1/CD169+ macrophages
capture apoptotic cells to induce cellular antigen-specific tolerance. They also
exhibit the proinflammatory phenotype, although proinflammatory cytokines IL-6/
IL-23/IL-1β are expressed in low levels in Siglec-1/CD169+ macrophages than in
Siglec-1/CD169 cells. Inflammatory cells are rarely infiltrated in the absence of
Siglec-1/CD169+ macrophages. CCL2 is the monocyte chemoattractant and is not
expressed differently in both Siglec-1/CD169-positive and Siglec-1/CD169-nega-
tive macrophages. In the colon tissue, CCL8 expression, which is a monocyte
chemoattractant protein 2, is enhanced in Siglec-1/CD169+ macrophages and for
CCL8 mRNA expression. In the intestinal tract, Siglec-1/CD169+ macrophages
exacerbate inflammation by CCL8 production to recruit monocytes. Therefore,
Siglec-1/CD169+ macrophages can be used in tissue-targeting therapies for treating
both immune and nonimmune diseases [167]. Siglec-1/CD169 mediates innate-like
lymphocytic adhesion in the sinus lymphatic nodes and blocks the loss of cellular
adhesion capacity during flow through the lymphoid line [171]. However, Siglec-1/
CD169-deficient mice show a normal phenotype and are fertile [172]. Siglec-1/
CD169+ macrophages express the chemokine CCL8 as a chemoattractant to recruit
inflammatory monocytes. For example, CCL8 expression in Siglec-1/CD169+ mac-
rophages, but not CCL2, recruits monocytes during inflammation. CCL8 regulates
Langerhans cell, small intestinal macrophage, or Th2 cell trafficking [173, 174],
regardless of Siglec-1/CD169 and Siglec-1/CD169+ populations. Therefore,
CCL8 is regarded as a therapeutic target candidate for inflammatory disorders
such as colitis. Siglec-1/CD169+ macrophage-produced CCL8 aggravates colitis,
although pathogenic bacteria and endogenous adjuvants lead to CCL8 expression.
Bone marrow cells cultured with BMDMs produce Siglec-1/CD169. LPSs and poly
(I:C) also induce CCL8 expression in BMDMs. A DAMP, namely, HMGB1 of
necrotic cells, also elicits CCL8 expression in BMDMs. Siglec-1/CD169+ macro-
phages can express CCL8 upon treatments with both PAMPs and DAMPs. To block
the CCL8 role in vivo, CCL8-specific antibodies block the transmigration of mono-
cytic cells toward CCL8. An intraperitoneal CCL8-specific antibody injection sup-
presses colitis development with decreased tissue injury in mice. Therefore, it is
concluded that the Siglec-1/CD169-positive macrophage–CCL8 axis is a future
candidate for injured inflammatory responses in the mucosal tissues.
In cancer immune responses, CD169+ macrophages potentiate antitumor immu-
nity. Siglec-1/CD169+ macrophages also induce antigen-targeting immune
responses at the cellular level [175]. Therefore, CD169+ macrophages are
prognostically important if they reside in the tumor-draining lymphatic nodes.
CD169 expression level is a prognostic factor in tumor-specific survival. CD169+
macrophages in the lymphatic nodes enhance anticancer immunity by expanding

CD8+ T cells [176]. Subcutaneous injections of tumor cells are subjected to capture
by sinus-lining Siglec-1/CD169+ macrophages. They internalize dead tumor cell
antigens to present their information to the adjacent CD8+ CTLs to suppress tumor
growth. Siglec-1/CD169+ macrophages recruit monocytes by CCL8 expression;
therefore, the depleted Siglec-1/CD169+ macrophages decrease in monocyte level.
Siglec-1/CD169-positve macrophages that are present in the tumor-draining lym-
phatic nodes decrease the number of cancer survivals [177–181]. Siglec-1/CD169
expression does not indicate prognosis in human breast cancer patients. However,
the expression indicates the level of tumor-infiltrating CD8 T cells [182]. It is known
that CD169-expressing lymph node sinus macrophages present in the regional
lymphatic nodes elicit an antitumor immune response in the host. The lymph node
macrophagic CD169 expression is linked to longer cancer cell survival. Thus,
Siglec-1/CD169+ macrophages indeed indicate the level of antitumor immunity
[183], as described by a result that a subcutaneous injection of cancer antigen
leads to internalization by medullary sinus macrophages and inhibition of tumor
growth [184, 185]. The level of tumor-infiltrating lymphocytes in a cancerous tissue
and the macrophagic Siglec-1/CD169 expression are linked in cancer patients. The
lymph node macrophagic expression of Siglec-1/CD169 and indolamine
2,3-dioxygenase is increased by IFNs [185]. Therefore, Siglec-1/CD169 expression
in macrophages contributes to immune responses against human tumors. The Siglec-
1/CD169 expression level can be evaluated to check anticancer immunity. Siglec-1/
CD169+ macrophages in the lymphatic node sinus activate anticancer immunity.
The structural basis of Siglec-1/CD169 is that the cytoplasmic tail is not con-
served with other known Siglecs and lacks tyrosine-based signaling motifs such as
ITIMs. Sn has its own specific structure bearing 17 distinct Ig-like domains in its
extracellular region. The extracellular region has been suggested to extend its length
of approximately 40 nm from the cell surface areas. This basis suggests that Siglec-1/
CD169 is involved more specifically in ligand recognition and cell–cell communi-
cation than in intracellular signaling to elicit immune responses. As Siglec-1
(CD169) is indeed a family of macrophage-restricted Siglecs, inflammatory and
autoimmune diseases are regulated by Siglec-1/CD169. The CD169/Siglec-1
expression level is also increased upon type I IFN treatment on innate immune
cells of macrophages and DCs [186]. For example, Siglec-1/CD169 levels are
frequently increased in macrophages activated in the inflamed local organs and
tissues of known inflammatory diseases [187]. The inflammatory examples are
RA, experimental autoimmune encephalomyelitis (EAE), systemic lupus
erythematosus (SLE), Sjögren’s syndrome, and experimental autoimmune
uveoretinitis (EAU) [112, 188]. Rheumatoid arthritis (RA) patients highly express
Siglec-1/CD169 on macrophages of the local tissues. Siglec-1/CD169-deficient mice
exhibit reduced disease levels of inflammatory diseases [188]. These syndromes
develop autoantibodies by IFN signaling and type I IFN-affected Siglec-1/CD169
gene expression. How is Siglec-1/CD169 expressed by IFNs and how do the
phenotype changes of macrophages occur to the express the Siglec-1/CD169 gene?
TLR ligation of macrophages with certain antigens induces an IFN-affected gene
expression such as Siglec-1/CD169. CD45 + CD11c + and CD45 + CD11c- human

leukocytes obtained from patients with autoimmune congenital heart blocks express
high levels of Siglec-1/CD169 by type I IFN stimulation [189].
In certain pathogenic bacteria, sialic acids are decorated on their surfaces. Such
sialic acids on pathogens aid in pathogenic virulence through complement inactiva-
tion or Siglec engagement on host leukocytes. Siglec-1/CD169 is involved in the
engagement of Siglecs on macrophages with pathogenic sialyl ligands to provoke
inflammation. Sia α(2,3)Gal glycans are frequently found on pathogenic bacterial
surfaces. Siglec-1/CD169+ macrophages internalize sialylated N. meningitidis via a
Siglec-1/CD169–sialic acid interaction. In GBS-causing C. jejuni strains, Sia α(2,3)
Gal glycans attached on the LOSs are associated with the pathogenicity of C. jejuni,
causing autoimmune GBS and Miller–Fisher syndrome (MFS) [65]. Siglec-1/
CD169 captures the killed sialylated C. jejuni, thus activating proinflammatory
cytokine production and responses to type I IFNs [190]. However, Siglec-1 and
invasive sialylated pathogen binding is not demonstrated during infection. In
sialylated GBS pathogens, Siglec-1/CD169 exerts bactericidal activity via Siglec-
1/CD169–sialyl ligand recognition. Sialoadhesin on the spleen marginal zone
metallophillic macrophages captures circulating GBS and prevents the GBS from
manifesting to the adjacent tissues or organs. This effect prevents GBS mortality
from tissue to tissue [191]. In the marginal sinuses, marginal zone macrophages
highly express Siglec-1/CD169 to recognize sialylated GBS. Currently, Siglec-1/
CD169 recognizes sialylated ligands expressed on several sialylated pathogens,
including protozoa [192], bacteria [65, 187], and enveloped viruses [193–195].
In a virus, HIV-1 recognizes Siglec-1/CD169 via gp120-attached sialic acids.
This interaction stimulates infection and transinfections in IFN-α-induced CD14+
monocytes. The gene expression of Siglec-1/CD169 is increased in CD14+ mono-
cytes obtained from HIV-1-positive patients [193]. When mo-DCs are treated with
LPSs, the inflammatory IL-1β/TNF-α cytokines, TGF-β1 and Siglec-1, are produced
with an increased capturing ability of HIV particles. CD169 is the major receptor for
HIV-1 infection and is produced by DC-associated mucosal environments. DCs
capture the HIV virus and transmit it to the host cells. TGF-β1 induces the expression
of CD169, but not DC-SIGN or other Siglecs on DCs, in the HIV-1 virion capture by
the APCs [196].
7.9.2 Siglec-1 Is a Pathogen-Binding Receptor
Siglec-1 is expressed on the myeloid lineage in humans, mice, rats, and pigs. Its
presence is restricted to macrophage subpopulations, and, thus, sialoadhesin is a
macrophage-specific receptor that binds to sialyl glycan ligands on the host cells and
pathogens. However, the functional roles of pathogen engagement by Siglec-1 are
unclear. Siglec-1 is largely detected on marginal zone macrophage subpopulations in
the spleen tissue and also on subcapsular sinus macrophages of the lymphatic nodes
in mice [197]. Siglec-1 is physiologically involved in many types of signaling, but its
role is still under study. It was regarded as a hematopoiesis regulator; however, KO

mice results showed only changes in T- and B-cell populations [172]. Siglec-1 may
modulate inflammatory and autoimmune responses. It is also involved in different
processes such as viral infection, viral transinfection, or viral clearance with
sialylated pathogenic HIV [198], as Siglec-1 is a porcine viral infection receptor in
alveolar macrophages. However, a question arose in the study of Siglec-1 KO pigs
[199]. Both hSiglec-1 and mSiglec-1 capture the murine leukemia virus (MLV) in a
ganglioside-requiring manner [200]. Siglec-1 as a receptor also recognizes and takes
up bacteria such as N. meningitides, C. jejuni, and T. cruzi, as well as, protozoa,
which are sialylated on their cell surfaces [201].
For defining the roles of Sn in the interaction and capture of bacterial C. jejuni,
Siglec-1-expressing mice are used [201]. The Gram-negative spiral bacterium,
C. jejuni, shares interesting glycosylation properties with CPSs,
lipooligosaccharides (LOSs), and sialylated glycoconjugates, which are capable of
modulation in host immune responses. The bacterium has been specially outlined in
serious neurological complications, namely, the Miller–Fisher and Guillain–Barré
syndromes. Two protein glycosylation events are known in the pathogenic bacteria,
C. jejuni human strains. First, an O-glycosylation event is found in Ser/Thr residues
attached to flagellin proteins. Second, a N-glycosylation event is observed in Asn
residues linked to various C. jejuni proteins. A lack of N-glycosylation activates
inflammatory responses in hosts. Among them, the CPS species belong to the
serotype-determining polysaccharides. In fact, C. jejuni strains deficient for CPS
biosynthesis are negative for serotype determination, which is detectable by the
Penner Scheme [202], and inhibit inflammation reaction, as a CPS-deficient C. jejuni
strain produces cytokines [203]. Surface glycolipid LOSs consist of a lipid A
attached to oligosaccharides. C. jejuni strains produce LPS-like high-molecular-
weight components, where lipid A is an inflammatory endotoxin. Synthesis of
sialylated LOSs in bacteria enhances bacterial capture and synthesis of inflammatory
cytokines in the host immune cells [204]. Interestingly, the sialyloligosaccharides
produced by C. jejuni are similar in structural basis to GM1 gangliosides produced in
humans. The sialosaccharides cause the axonal Guillain–Barré syndrome (GBS) due
to ganglioside-specific antibodies. GBS-causing C. jejuni strains interact with
sialoadhesin (Siglec-1) [205]. In addition, disialylated LOS-expressing C. jejuni
strains can recognize Siglec-7 [66]. Siglec-7 is also reported to specifically recognize
C. jejuni associated with GBS or MFS oculomotor weakness [206].
Sn rapidly takes up the C. jejuni GB11 strain, which synthesizes GD1a- and
GM1-like carbohydrates. Sialylated bacteria rapidly migrate to the spleen and liver
tissues and produce high cytokine levels of IFN-β/TNF-α. Sn recognizes pathogens
as a pathogen-recognition molecule for sialylated bacteria with type I IFN responses
that are crucial in host defense. C. jejuni sialylation is essential for macrophage
capture and Sn-dependent expression of cytokines. The immune responses were well
explained in C. jejuni sialylation in a previous mouse model [201]. Sn-dependent
cytokine production can also be observed in other bacterial sialylations including
GBS and N. meningitidis, as the Sn receptor cooperates with TLRs. Sn-dependent
cytokine responses may develop postinfectious GBS.
In the V-set, Siglec-1, Arg97, and Trp-2 or Trp-106 amino-acid residues interact
with SAs. Siglec-1 binds Neu5Ac in an α2,3 linkage to the D-Gal residue but not
Neu5Gc-Gal or Neu5Ac9Ac-Gal linkages [56]. The ganglioside sialyl glycan head
groups contain the HIV-1 binding sites as confirmed by human Siglec-1-arranged
liposomes [207]. Two different gangliosides, GM3 and GM2, which contain
sialyllactose (SL) consisting of a SA-Gal-Glc link to ceramides, are recognized by
HIV/Siglec-1 [208, 209]. Viral membranes carry sialylated gangliosides synthesized
from the host ER–Golgi system. There are many examples known in retroviruses
such as HIV-1, murine leukemic virus (MLV), Semliki forest virus, and stomatitis
virus. For example, during MLV infection, MLV is captured by Siglec-1 in mDCs.
In the capture process of viral infection, Siglec-1 functions as a receptor for
molecular binding of several enveloped viruses via mDC viral captures. Moreover,
Siglec-1 is involved in the antivirus responses of immune cells to antigen-captured
immune cells. Moreover, Siglec-1-positive myeloid cells efficiently take up the
vesicular stomatitis virus with antivirus immune responses of B cells and prevention
of virus neuroinvasion through type I IFNs. Elimination of sialyllactose-bearing
GM3 or larger gangliosides, rather than that of monosialosyl motives, from viral
budding coats or sialidase treatment with desialylating activity of viral membrane
gangliosides, inhibits capacities of the mDC capture and immune recognition. In
another case of sialic acids, certain influenza viruses are often resistant to ganglioside
GM3 due to the viral neuraminidase enzyme activity in HA types. Therefore, it is
interesting to see whether the SA-defective events are related to escape from immune
recognition via Siglecs, such as Siglec-1, expressed on innate immune cells of the
host. This is explained by the fact that sialylglycans are ligands for Siglecs produced
on mDC surfaces, allowing pathogen capture and clearance. mDC capacity to
capture sialic acid-containing gangliosides in viral coat membranes via Siglec-1 is
believed to be an evolved result of sialylated pathogens.
7.9.3 Siglec-1 Recognizes HIV and Is a Transinfection

Receptor Expressed on mDCs
Siglec-1/CD169 is also an HIV-1-capturing receptor expressed on mDCs

[210]. Siglecs interact with gp120 on host cells and on monocyte-derived macro-
phages (mo-Ms). Siglec–gp120 binding to HIV-1 leads to host infections [195]. Mac-
rophage cell surfaces produce many CTLs like DEC-205 or MR, which capture
HIV-1 through the oligoman glycan link to the envelope protein, leading to virus
endocytosis or infection. Siglecs bind to surface mucins, which heavily glycosylate,
and mucin-like sequences. For glycans in infection, the gp120 mutation conferring
the defected N-glycan sites on HIV results in noninfectious viruses due to higher
sensitivity to neutralization. Thus, viral glycans are crucial for shielding against host
immune detection. In addition, multiple glycan mutations increase lower infection
activity or syncytia-generating capacity rather than the WT virus types, implying
glycans’ role in viral entry. Siglecs bind gp120 of HIV-1. The surface envelop
protein gp120 on the CCR5- (R4) and CXCR4-tropic (X-4) strains of the HIV-1
virus consists of more than 20 N-glycosylation sites with terminal SAs. SAs linked
to gp120 glycoproteins potentiate virus adhesion, attachment, and virus entry via
interaction with Siglecs. Results obtained from a direct interaction between the
gp120 protein and monocytic Siglec-1, -3, -5, -7, and -9 of humans show that
Siglec-1- and Siglec-9-Fc-fused receptors are reported to bind to the gp120 envelope
protein, with affinities between 0.01 and 1 μM [211]. HIV-1 uses its SA-containing
glycans linked to the envelope protein for virus interaction with Siglec-1 on macro-
phages and for improving the recognition level with CD4 for constant entry. In
addition, HIV-1 glycans shield HIV from host immune surveillance and also serve as
an infection tool for host entry. gp120 glycans are the sites for complex, high
mannose, and hybrid glycan types. Glycans linked to envelope glycoproteins of
gp41 and gp120 trimers of HIV-1 are distributed in CD4 complex forms. The four
domains in the CD4 protein are located on the complex associated with the two
domains in the N-terminal region.
The number of blood CD169-positive cells is increased in HIV-infected patients.
HIV-1-infected humans exhibit CD169 expression on monocytes in peripheral
blood. In addition, the CD169 level is increased in the early infectious stage
[212]. Furthermore, a recent report [195, 210] has updated a key function of
CD169-positive cells in retroviral infection spread [212]. Therefore, CD169 can be
a marker of the infectious pathogen lenti virus, leading to HIV-1 pathogenic
progression. Thus, CD169 is a receptor of lentiviral infections and accelerates
HIV-1 pathogenesis in vivo. Unmasking Siglecs on the surfaces of cells increases
their recognition with virus SAs. Between Siglec-1 and Siglec-9, Siglec-1 exists as
an unmasked type because of its relatively large (17) Ig domains. Moreover, Siglec-9
expresses better adhesiveness only after neuraminidase digestion despite its affinity
toward SAs, indicating that surface receptors expressed on cells are masked by SAs.
In addition, type I IFN-induced CD169 diminishes the antivirus capacity of type I
IFNs via HIV infection enhancement in myeloid cells [213]. When monocytic
THP-1, as a model cell, is incubated with IFN-α and the induced expression level
of CD169 is examined, then normal-type HIV-1 amplification is increased even in
the condition of IFN-α treatment. CD169 increases viral fusion and entry to host
cells. The IFN-α-activated antivirus condition helps to evade mo-Ms and CD169
involved in HIV-1 fusion in MDMs. Moreover, CD169 overexpression on inflam-
matory DCs increases at the time of virus entry by a DC-involved mechanism, such
as a transinfection event. In addition, the CD169 expression potentiates viral ampli-
fication in the host CD4+ T cells, regardless of the presence of type I IFNs. This
indicates the important roles of type I IFN-induced CD169 and the suppressive roles
of antivirus type I IFN in myeloid cells, which inhibit the viral amplification of
HIV-1.
CD169 takes up CD169-bound HIV-1 and HIV-1 associated with membranes of
THP-1 cells and DCs as intermediate hosts. HIV-1 and CD169 clustering events
increase in complex formation between viruses and host cells to enhance the
efficiency of HIV-1 viral endocytosis to hosts. Apart from cis-type infection,
HIV-1 can enter the host CD4+ T cells through DC-involved transinfection events.
HIV-1 transinfection or transmission between T cells and DCs takes place at the
tightly joined “cell-to-cell junction,” also known as the viral synapse, which con-
centrates viral particles of HIV-1 to enhance the fusion between HIV-1 and the host
T cells [214]. CD169 macrophages are constitutively present in lymphatic node
tissues, including the subcapsular sinus, and perifollicular and medullary macro-
phages. CD169+ cells like tissue-resident macrophages are involved in viral dis-
semination even in the condition of IFN-α. Lymphatic node-resident macrophages
take up most pathogenic agents, and CD169 is important for the GM3-dependent
uptake of HIV-1 [186, 209, 215, 216].
The SL portion of viral GSLs is bound during mDC HIV-1 uptake. The adhesion
and uptake receptor is a SA-binding molecule. Siglecs bind to sialyl ligands, driving
cell recognition and immune responses. mDCs, not iDCs, in lymphatic tissues,
efficiently transmit the HIV-1 virus to the host T cells. The virus–host interaction
is the key transmission factor in the highly dense lymphatic tissues. The recognition
of mDCs with CD4 + T cells creates an infectious synapse environment. Although
the DC-SIGN level is decreased during DC maturation, the HIV-1 capture-based
infection is increased. Mannan or DC-SIGN-specific antibodies such as DC-SIGN
blockers exhibit only low activities on HIV capture-based infection by mDCs.
However, DC-SIGN-transfected mDCs show completely blocked HIV infection.
Moreover, although DC-SIGN is not present in myeloid DCs of blood and
Langerhans cells, these cells take up and transinfect HIV. Thus, the DC-SIGN
receptor is not required for HIV virus uptake in mDCs, indicating that the HIV
capture by mDCs is performed by other molecules because viral envelope glyco-
proteins are not important for mDC HIV-1 capture and DC-SIGN recognizes only
the HIV-1 gp120 glycoprotein [217].
Viral envelope proteins are not linked for mDC uptake. Instead, other compo-
nents on viral surfaces will be more important for mDC capture. When HIV-1-
positive or exosome-positive cells are treated with GSL-synthesizing enzyme inhib-
itors, the mDC capture capacity is decreased. Thus, sialic acid-containing
glycosphingolipids are important for mDC capture and infection of HIV-1, indicat-
ing the capture capacity of ganglioside-dependent mDCs and sialylated carbohydrate
as the recognition region [207]. Sialic acids on cellular membranes are used as a
binding and adhesion receptor by several pathogenic agents and toxins. Removing
sialic acid from viruses by sialidase treatment or asialo-ganglioside-reconstituted
exosomes abolishes mDC uptake because SA is important for recognition of mDCs.
mDC capture of a virus is observed when the PMs contain GM1, GM2, or GM3
gangliosides. Interestingly, GM4, a SA-bound Gal moiety, has no capacity to
capture a virus by mDCs. The fact that GM3, GM1, and GM2, SA-bound to the
lactose head group, have mDC-recognition capacity indicates that the sialyllactose
head group is responsible for mDC recognition. In fact, sialyllactose prevents HIV-1
uptake by mDCs, although an efficient capture needs membrane gangliosides,
sialyllactose-bound ceramides. Between GM1 and GM3, GM3 is strongly recog-
nized by mDCs. When HIV-1 is exposed to human genital mucosal epithelial cells,
the cells mediate DC maturation by thymic stromal lymphopoietin (TSLP). Then,
DCd-involved HIV-1 infection is accelerated in the host cells, CD4+ T cells, where
Siglec-1 drives the infection of HIV-1 to vaginal Langerhans cells or DCs. In
contrast, intact viruses are endocytosed in a CLR-independent manner. When
mucosal inflammation is raised by other bacteria, fungi, or viruses, maturation of
the resident DCs or newly migrated DCs can be stimulated by binding to the
pathogens or by chemokines and inflammatory cytokines [218]. These events
accelerate the transinfection events of HIV-1. Chronic inflammation or systemic
inflammation is also the key inducer of increased infection of HIV-1, and various
proinflammatory agents activate Siglec-1 production, thus increasing HIV-1
transinfection. LPSs induce HIV-1 infection, stimulating the systemic maturation
of DCs and enhancing viral spread. IFN-α is an antivirus cytokine generated by
pDCs upon HIV-1 infection due to IFN-α-stimulated Siglec-1 generation in mDCs
or monocytes. Therefore, although IFN-α is an antiviral agent, it induces HIV-1
transinfection even in antiviral conditions. A higher HIV-1 viral titer is linked to
increased Siglec-1 expression in monocytes, as enhanced by plasmacytoid DCs,
which express IFN-α upon HIV-1 infection and elicit DC maturation [186].
7.10 CD22/Siglec-2
7.10.1 General and Structural Aspects of CD22/Siglec-2
Among immune cells, only B cells predominantly express Siglec-2/CD22, and

Siglec-2 regulates the septic balance of immune responses by B-cell modulation. It
also functions for adjusting chemokine production. B cells undergo humoral
immune responses through BCR signaling. Therefore, Siglec-2/CD22 is a specific
modulator of B cells among Siglecs. Functionally, Siglec-2/CD22 belongs to a
B-cell inhibitory receptor, which modulates various types of B-cell behaviors in
both survival and activation thresholds. Positive activating BCR signaling frequently
participates in lectin–glycan interactions, which are managed by sialoglycans and
sialic acid-binding lectins, namely, Siglecs. In contrast, negative inhibitory BCR
signaling is also found. For example, two different Siglec forms such as human
Siglec-2 (CD22) and human Siglec-10 (mouse Siglec-G) are BCR inhibitory
coreceptors because they control B-cell activation and peripheral tolerance. There-
fore, such BCR inhibitory receptor-negative individuals are susceptible to autoim-
munity. CD22 is indeed a BCR-signaling suppressor in conventional B cells, B2
cells. For example, mice lacking CD22 or Siglec-G or those lacking both develop
symptoms of autoimmune diseases [219]. Genetically deficient mice with congenital
mutations in the biosynthesis of CD22 sialic acid ligands are well known for
conferring an autoimmune status on humans [220]. CD22 is a membrane glycopro-
tein receptor of the immunoglobulin (Ig) superfamily (IgSF) and is predominantly
expressed on mature B lymphocytes and not on premature cells. CD22 is a glyco-
protein of a 140 kDa and has a single-spanning region that spans the plasma
membrane. From the elucidated human CD22 crystal structure, it is known that the
7.10 CD22/Siglec-2 347
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5*2$KPFKPI.QECVKQP
Fig. 7.9 Structure of CD22
binding specificity for α2,6-SA ligands originates from a β-hairpin structure for
recognition [221]. The D1 domain has been shown to adopt a V-type fold as well as
C1 and C2 strands to yield a β-hairpin structure. The extracellular domain of CD22
has hairpin-like Ig regions and 12 N-glycosylation sites. The outermost domains of
CD22 are named D1 and D2. Both D1 and D2 that are the most N-terminally located
Ig domain regions are binding sites for α2,6-linked SAs [104, 222]. Structurally, the
SA-binding domain has nine β-strands and Ig V-set domains. The V-set domain
specifically recognizes the α2,6-sialyl linkages on glycan structures. The β-hairpin
structure is involved in the binding of sialic acids (Fig. 7.9) [223]. The surface-
exposed arginine residues in the glycan-binding receptor family are key factors for
binding NH2 to form a salt bridge with the COOH group linked to SAs. Thus, the
Arg residue in the receptor and the sialic acid residue in the ligand are counterparts.
CD22 is distinct in terms of its structure and is not homologous to other known
Siglecs, although many Siglecs in all mammals have been reported to date. In fact,
the four Siglecs, Sd, CD22, MAG, and Siglec-15, which are expressed in all
mammals, are not homologous. Regarding the aspect of sequence homology, Siglecs
are classified into two major groups and are subject to investigation to determine
their evolutionary adaptation [224]. For example, CD33-related Siglecs in many
organisms are diverse in their structural homologies due to constant evolution,
keeping the conserved region in tyrosine-based signaling motifs at the extracellular
domain regions. CD22 has long been regarded as an adhesion molecule of SAs
[225], with a specificity to various α2,6-linked SAs as ligands [226]. The preferred
ligand for human CD22 is the N-acetylneuraminic acid (Neu5Ac) form of SAs on
N-/O-glycans and glycolipids. Specifically, in humans and mice, 9-O-acetylated
sialic acid is known as the common form of sialic acids and is linked to autoimmune
response and substitution [227]. The specific binding of CD22 to 9-O-acetylated
SAs is a possible explanation for why SA acetylation on self-antigens can prevent
CD22 recognition and consequently increase the level of B cell-mediated autoim-

mune responses. Human CD22 is known to preferentially bind to Neu5Ac, whereas
murine CD22 specifically binds to N-glycolyl neuraminic acid (Neu5Gc), also
known as the nonhuman type of sialic acid. This indicates that recognition of
CD22 SA ligands has evolved in each species in a CD22-dependent evolutionary
manner [228].
CD22 functions depend on its ligand-binding ability. CD22 (Siglec-2) regulates
B cell-dependent immune responses including prevention of autoimmunity. In
B-cell circulation, CD22 strictly controls the B-cell homing event to the bone
marrow in recirculating B-cell subsets, as the CD22 (Siglec-2) expression is
observed only in B cells. In addition to its ligand-binding affinity to sialic acids,
CD22 plays many roles in B cells. For example, CD22 modulates BCR signal
transduction as the most important function in B cells [225]. BCR signaling is
initiated by interactions with specific antigens. When antigens bind to BCRs,
BCRs initiate the signaling pathway to activate related transcription factors includ-
ing c-Myc and ATF-2 to upregulate gene expressions toward activation of B cells
[225]. CD22, upon binding to the soluble sialic acid ligand, activates various
signaling protein molecules to transduce inhibitory signals downstream of the
BCR signaling pathway. The known recruiting adaptor molecules such as SHIP
and SHP-17 have been well studied in inhibitory BCR signaling. The final inhibition
of BCR signaling is directed by the so-called ITIMs. ITIMs are suppressed by a
Src-family kinase, Lyn, upon CD22 activation of BCRs, consequently activating and
recruiting SHP-1 [228]. CD22 is known to have at least two cytoplasmic, three
inhibitory domains, ITIMs. The cytoplasmic tail of ITIMs inhibits BCR signaling.
Using ITIMs, CD22 can activate and differentiate B cells into any specific cell type
[229]. In B cells, SHP-1 initiates and maintains the BCR-induced Ca2+-dependent
pathway [229]. CD22 interaction with glycoprotein ligands expressed in T cells also
downregulates downstream signaling of T cells.
Using ITIMs, CD22 recruits various dephosphorylases such as phosphatases that
remove phosphorus from signaling protein molecules, which are subjected to kinase
action during the BCR signal transduction of B cells. Thus, BCR signaling is
deactivated and terminated [230], indicating that CD22 is an apical regulator of
homeostasis in adaptive immunity. It is summarized that CD22 is a keeper of B-cell
suppression and a holder of adaptive immunity.
Lectin CD22 is regulated by the membrane-bound glycan ligands on the same
cells, and these types of ligands are called cis-ligands. Biologically, it is an inhibitory
coreceptor of BCRs because it downregulates diverse B-cell functions. As a B
lymphocyte-restricted membrane receptor, it recognizes α2,6-linked SAs as endog-
enous self-ligands. CD22 has been a model receptor of cis-ligands, as an inhibitory
coreceptor of a BCR specifically recognizes α2,6 sialic acids with a lectin domain
capable of binding α2,6 sialic acids in the extracellular region. The cytoplasmic
region contains ITIMs to negatively regulate BCR signaling [231]. The functional
CD22 domain seems likely to be occupied by cis-ligands. If the sialyltransferase
ST6Gal-I gene that is responsible for α2,6 sialylation is deficient in B cells or if B
cells are deficient in CD22 lectin activity that is responsible for sialic acid
7.10 CD22/Siglec-2 349
recognition due to genetic mutation of the CD22 gene, then BCR signaling will be
terminated [232]. Such an assumption can be made by the fact that cis-ligands
regulate CD22 functions.
As described above, the extracellular Ig domain-1 of CD22 binds to Sia α2,6-
Galβ1,4 residues. CD22 is also self-sialylated and can form cis-homo-oligomers on
the surfaces of B cells. CD22 functions are operated through the intracellular
recruitment of phosphatases for stimulatory coreceptor dephosphorylation. Apart
from the self-sialylation on CD22, CD45 is a known CD22 ligand in the cis-form
[121]. At a cellular level, the affinity for Sia α2,6-Galβ1,4 is known to be low
[233]. Furthermore, because of the high contents of SAs on B- cell surfaces, the Sia
α2,6-Galβ1,4 binding sites of CD22 are masked in a cis-manner by SAα2,6-Galβ1,4
residues expressed on B-cell surfaces [234].
7.10.2 CD22 I Associated with Development of Autoimmune

Diseases
CD22 is indeed a surface glycoprotein, which is restrictively expressed on B cells,

and is a component of the BCR complex. CD22 has now been recognized as a
SA-binding lectin. CD22 is present on B cells, and, when subjected to certain ligands
such as sialic acids, it can inhibit the B-cell receptor signaling pathway by activating
certain phosphatases. This mechanism is shown to be crucial in many defense
mechanisms of our bodies. First and foremost, CD22 plays a crucial role in
preventing autoimmunity by stopping B cells from producing antibodies against
one’s own body. B cells are important for protecting our bodies from harmful
pathogens by producing highly specific antibodies against various pathogens
(Fig. 7.10). However, B cells sometimes recognize “self” as a harmful agent and
thus produce autoantibodies, which cause autoimmune diseases [235]. CD22 that
plays an inhibitory role in B cells also affects autoimmunity. The loss of CD22 in a
certain breed of mice can increase susceptibility to autoimmune diseases [236]. For
example, an autoimmune SLE is categorized as a member of the autoimmune disease
family, and antinuclear antibodies produced by B cells in patients with SLEs are the
hallmarks of the disease [237]. As self-recognizing antibodies cause damage to
patients with SLE, many researchers are focusing on manipulating B cells to cure
SLE. Epratuzumab is a CD22 IgG antibody that binds to CD22 and enables the
B-cell receptor to be internalized, thus resulting in the reduction of B-cell activities
[238]. Since epratuzumab reduces the activities of B cells by modulating CD22, it
can be used to treat many different autoimmune diseases such as SLE but not
restricted to only SLE. Although the efficacy and safety of epratuzumab are still
questionable, many clinical trials are being undertaken to examine the efficacy of
CD22 antibodies against various autoimmune diseases including bowel diseases and
autoimmune arthritis [239–241]. As a result, CD22 is a specialized receptor for
regulating B-cell functions and immune responses mediated by B cells. In highly
Ƅ Ƅ
#PVKIGP
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5GNHH

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5GNH

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2CVJQIGPKE
2CVJQIGP
GPKE
+PVGTCEV
< < < <

5*2
5*2
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Fig. 7.10 CD22 inhibition of B cell-mediated autoimmunity and pathogen recognition
evolved organisms such as humans, CD22 is effectively designed to prevent unex-

pected and undesired autoimmune responses in mammals. In addition, such evolu-
tionary and acquired logics can highlight the important role of CD22 in regulating
the homing phenomenon of circulating B cells. Consequently, CD22 potentiates B
cells to recirculate to the peripheral tissues and the bone marrow. If CD22 binds to
sialic acid residues of glycoprotein or glycolipid ligands on T cells, T-cell signaling
is also markedly modulated. CD22 itself cannot work, indicating that, fundamen-
tally, CD22 functions depending only on its binding of sialic acid ligands. Then, the
action property of CD22 provides a functional name to the negative regulator in
BCR signaling upon recognition between the BCR and CD22. In the immunological
aspect, the concept of inhibitory coreceptors of the BCR comes from the implicated
downregulation of immune responses. Keeping or maintaining immune tolerance
against self-antigens or autoantigens is mostly desired in evolved mammals. Indeed,
CD22 is a highly inhibitory coreceptor of the BCR. Therefore, CD22 is frequently
present on normal B cells and specified B-cell subsets. CD22 endocytosis in B cells
occurs easily upon binding to its ligands. Most likely, due to its role in B cell-
restricted antigens, CD22 is therapeutically considered to be a specific target against
dysfunctional B cells. For example, B cell-derived autoimmune diseases and blood
tumors are representative targets. To target dysfunctional B cells, CD22-specific
antibodies or CD22 antibody-based immunotoxins have been designed and devel-
oped to regress and destroy B-cell tumor cells such as B lymphomas and B cell-
derived autoimmune diseases. CD22-deficient animals, for example, CD22-deficient
KO mice, exhibited a diminished onset of autoimmune diseases and hyperreactive B
cells [242].
The CD22 ectodomain binds to trans-SA ligands to prevent autoimmune
responses in the host. From the current information on different autoimmune
7.10 CD22/Siglec-2 351
diseases, it is evident that autoimmunity-causing factors and molecular mechanisms

have not yet been well clarified. However, some common causative factors and traits
are known to share diverse autoimmune responses when B cells are persistently
activated. As described above, CD22 aids in the suppression of BCR signaling.
Therefore, it is a good candidate for a possible drug design or a target against diverse
autoimmune diseases such as SLE or rheumatoid arthritis (RA). There are several
therapeutic monoclonal antibodies targeting CD22. A monoclonal antibody,
epratuzumab, is one such therapeutic antibody. Alternatively, antibody–drug conju-
gates against CD22 are also known. One example is inotuzumab ozogamicin (INO),
which is composed of the IgG4 antibody against CD22 and a cytotoxic agent called
calicheamicin. INO has been designed to bind to CD22, and, thus, it specifically
binds to CD22 and internalizes to target B cells, and releases the drug compound,
calicheamicin, which damages DNA and induces apoptosis of the targeted B cells
[243]. This particular designed drug is considered to be a potential cure for acute
lymphoblastic leukemia and is under thorough investigation in clinical trials
[98, 244].
7.10.3 CD22 Function in Immune Tolerance Events
B-cell tolerance is known as the process by which autoreactive B cells are no longer
silenced on stimulation. Distinguishing self-antigens from nonself-antigens is essen-
tial for keeping and maintaining tolerance of B cells. Normally, individuals of
mammals display immune tolerance against himself, where himselves are expressed
by auto- or self-antigens. Immune tolerance events recognize and distinguish self-
from nonself-antigens of the body. Apart from the self-antigens, foreign antigens or
nonself antigens can also induce immune tolerance, from time to time, depending on
the injection pathway of antigens. Thus, if immune tolerance events occur inappro-
priately, then autoimmunity will be a problem in the organism. The immune
reactions of specific lymphocytes are generally suppressed by each different uptake
pathway, consequently inducing immune tolerance. Thus, immune tolerance can be
induced for usage in therapeutic strategies against unnecessary or undesired immune
responses in organisms. Such immune tolerance induction includes transplantation
rejection, autoimmunity diseases, gene therapy, and supplementation of defected
proteins. T/B lymphocytes are tolerant to immune reactions because these lympho-
cytes have evolved to induce a tolerance mechanism; consequently, tolerance acqui-
sition is beneficial to overcome detrimental autoimmune diseases by distinguishing
between self- and nonself-antigens by hosts. In B cells, keeping and maintaining
B-cell tolerance without distinguishing between nonself- and self-antigens in the
peripheral tissues is an important issue.
The inhibitory behavior and restriction of B cells indicate that CD22 can be used
as a therapeutic target for B-cell subset depletion in autoreactive immunity and
B-cell lymphomas. CD22 sialic acid cis-binding regulates BCR signaling to sialic
acids. The cis-ligand binding of CD22 forms CD22 homo-oligomers. CD22
Left: Right
Adjacent cells
5KINGE%&JQOQQNKIQOGT
5KCNKECEKFU
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Siglec-2
(CD22)
2JQURJQT[NCVKQPD[.[P
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Fig. 7.11 Left: The cis-ligand recognition of CD22 forms CD22 homo-oligomers, and CD22–SA
cis-binding regulates BCR signaling. During antigen recognition of the BCR, CD22 homo-
oligomers are associated with the BCR and SHP-1 is assembled with the phospho-ITIMs to inhibit
Ca2+ signaling. Right: Tolerance-keeping CD22 to trans-ligands on self-antigens. An autoreactive
BCR recognizes α2,3-SA or α2,6-SA ligands on adjacent cell autoantigens, and recruiting CD22 to
the BCR suppresses autoantigen-mediated BCR signaling by SHP-1 (adopted from Nitschke (2014)
[231] and Liu et al. (2017)) [245]
regulates B-cell tolerance, and its sialic acid ligand-bound inhibitory signaling is
associated with autoimmune diseases in individuals. CD22 expression is specific for
B cell-related leukemia, B-cell lymphomas, and B cell-mediated autoimmunity.
CD22 homo-oligomers are formed when α2,6-linked SA bind on CD22 of adjacent
cells. Homo-oligomeric CD22 is located independently from the location of the
BCR. During BCR antigen recognition, the BCR intracellularly recruits homo-
oligomeric CD22 and C-terminal tail ITIMs are phosphorylated by specific kinases.
As a phosphatase, SHP-1 is assembled with phospho-ITIMs to inhibit Ca2+ signaling
in a downstream pathway. CD22 maintains immune tolerance to trans-bound sialic
acid ligands on self-antigens. An autoreactive BCR binds α2,3-SA or α2,6-SA
ligands on autoantigens of the adjacent neighboring cells and recruits CD22 to the
BCR to inhibit autoantigen-induced BCR signaling through the phosphatase
enzyme, SHP-1 (Figs. 7.11 and 7.12). CD22 interacts with α2,6 sialyl glycans
between B cells and membrane autoantigens of the surrounding cells. In the synapse,
CD22 with the BCR inhibits BCR signaling (Fig. 7.13).
The suppression of CD22-mediated B-cell functions can induce immune toler-
ance or immune anergy [247] or removal of unnecessary B cells. CD22 clathrin-
mediated endocytosis [248] is used in immunotoxin internalization to cure B cell-
mediated autoactive autoimmunity diseases and blood tumors. The CD22 inhibitory
function of BCR signaling has been investigated in many rodent mice models
7.10 CD22/Siglec-2 353
Fig. 7.12 Response of CD22 to nonself-cells and comparison with Siglec-10
0HPEUDQHDQWLJHQ
*O\FRSURWHLQ DQWLJHQ
&' &'
BCR
3 3 3 3 3 3
3 3 3 3
3 3 3 3 3 3
3 3 3 3 3 3
6+3
Fig. 7.13 CD22 interaction with α2,6 sialyl glycans between B cells and membrane autoantigens
of the surrounding cells. CD22 in the synapse with the BCR inhibits BCR signaling. α2,6 sialic acid
residue. P indicates phosphate (modified from the article by Enterina JR, Jung J, Macauley
MS. 2019) [246]
[231]. Using the extracellular domain of human CD22 or the ligand α2,6
sialyllactose (SL) complex form, the CD22 target site has been elucidated for
efficacy of the epratuzumab treatment. The N-glycosylation site is important for
therapeutic antibody engagement and CD22 recognition on dysfunctional B cells.
Similar to CD22 in humans, Siglec-G in mouse is a known independent inhibitory
coreceptor of B cells. The two receptors of CD22 and Sigel-G function as a tolerance
keeper and as a maintainer of self-antigens. When B cells encounter antigens, they
are stimulated and differentiate into Ab-secreting plasma B cells. During this stage,
B cells prevent autoimmune responses by distinguishing the nonself-antigens from
the self-antigens. This process is managed and operated by BCR-regulatory
coreceptors such as CD22, which suppress nonself-antigen-induced and self-
antigen-induced immune reactions. As CD22 has an ITIM on the cytoplasmic tail,
it inhibits BCR downstream signaling. For phosphorylation, the Src kinase phos-
phorylates the ITIMs and the phosphorylated residues recruit the phosphatase,
SHP-1, to inhibit BCR downstream signaling. In CD22 KO mice, autoantibodies
are easily accumulated. Similar to human CD22, mouse Siglec-G is known, but the
downstream signaling of Siglec-G has not yet been well studied, compared to human
CD22. However, the known mouse Siglec-G ortholog is human Siglec-10. Human
Siglec-1 is well known as an inhibitor of BCR signaling, similar to the CD22 case.
Here, the reason why Siglec-G has not been well studied is interesting from a
mechanistic perspective. The simple reason is the deficiency of specific Siglec-G
ligands to date. B-cell induction by the BCR engagement of its cognate antigen
triggers a complex signal transduction cascade downstream. Thereafter,
BCR-engaged B cells differentiate into Ab-secreting plasma cells. As a BCR
inhibitory coreceptor, CD22 determines how to bind its SA ligands in a cis- and
trans-manner. However, the mechanism is currently unknown.
7.10.4 Role of CD22 (Siglec-2, Mice Siglec-G) in Immune

Responses
CD22 is specifically expressed on B cells in the differentiation stage from the

precursor type to the mature B-cell type. CD22 expression is downregulated on
plasma cells. In autoimmunity development, T/B lymphocytes are essentially
involved in detrimental autoimmune responses. Expression of B-cell tolerance is
directed by Siglec-2 (CD22). For the body to escape from detrimental autoimmune
responses, distinguishing between self- and nonself-antigens is important, and this
discriminating mechanism is known as tolerance. A Siglec family CD22 (Siglec-G
in mouse) is a glycoprotein, and CD22 knockout mice exhibit autoantibody forma-
tion and subsequent accumulation. Therefore, this indicates that CD22 induces
tolerance of B cells [249]. CD22 suppresses proliferation and activation of B cells
and also alters Siglec endocytosis in B cells. From an immunity perspective, sialic
acid functions as a biological masking agent and recognizes cellular molecular
patterns. For biological masking functions, SAs shield the host cells from pathogen
binding and this process prevents autoimmune responses. The increased expression
level of sialic acid content indicates the acute-phase response of inflammation and
helps the immune system to discriminate “self-” antigens from “nonself-” antigens.
In fact, the ST6Gal-I enzyme is a representative ST enzyme of α2,6 sialylate
7.10 CD22/Siglec-2 355
substrates. In knockout mice studies, ST6Gal-1 KO mice showed a decreased level

of humoral immune responses because CD22 recognizes ST6Gal-1-mediated
sialylglycans and regulates B-cell functions. For example, CD11 function was
downregulated. It is known that the soluble ST6Gal-1 enzyme regulates
myelopoiesis in an acute-phase inflammatory state. In ST3Gal-1 KO mice, it was
demonstrated that α2,3-SA-O-glycan sugars are essential for the maintenance and
homeostasis of CD8+ CTL cell types.
Some bacterial pathogens mimic the molecular pattern of host sialic acids by
masking to evade the immune system of the hosts. For example, Trypanosoma spp.
recognizes host cell patterns. They recognize the host patterns by several cell surface
receptors, which are similar to CLRs and Siglecs. This recognizes and binds sugar
ligands in a trans- and cis-manner. Moreover, Siglecs present ITIMs or ITAMs,
regulating and discriminating between SAMPs and PAMPs. In a knockout mice
study, ST6Gal-1 KO mice showed a decreased level of humoral immune responses.
As CD22 recognizes ST6Gal-1-mediated glycans, sialic acid α2,6 is known to
regulate several B-cell functions, as soluble ST6Gal-1 mediates myelopoiesis during
acute inflammation.
For the structural basis for binding to CD22, CD22 has a unique preference for
Neu5Gc-α2,6Gal and Neu5Ac-α2,6-Gal glycan structures (Fig. 7.14a). They struc-
turally mimic CD22-specific inhibitors and are therefore beneficial tools for eluci-
dating B-cell function and for controlling B cell-dependent immune responses. Such
inhibitors can specifically deliver therapeutic drugs or toxins to B cells. Especially,
cancerous B cells can be used in B-cell targeting [141, 245]. CD22 is expressed
throughout B-cell differentiation. The juxtaposition of CD22 and BCR contributes to
the inhibited Ca2+ signaling of B1 and B2 cells [251]. B1 cells largely produce IgM
rather than IgG, and B1 cell receptors are polyspecific, indicating that they have low
affinities for antigens. However, conventional B2 cells are indeed unqualified “B
cells.” B-cell activation is broadly inhibited in a SHP-1-dependent manner
(Fig. 7.14b). CD22-specific ligands decrease the levels of in vitro growth and
survival of B cells. Cotreatment of CD22 ligands and antigens induces tolerance
in vivo. CD22, an inhibitory coreceptor of the BCR, acts to maintain tolerance to
self-antigens, as CD22 expression is seen during the developmental stage of
precursor-type B cells, which express CD22. Development of a selective CD22-
binding ligand is important. CD22/Siglec-2-engaging, tolerance-inducing antigenic
agents are the causes of the CD22 ligand. CD22 has at least one or more ITIMs in the
cytoplasmic tail, which inhibits BCR-mediated signaling. To date, CD22 and CD22-
specific antigens have been able to induce tolerance. CD22 can distinguish danger-
ous structures from PAMPs. Especially, CD22 functions together with TLR-4 and
CD24. CD22 does not sensitize PAMPs, which are not bound to CD24 yet. Inter-
estingly, in a sepsis model, SA-induced recognition of CD24 with Siglec-G is
inhibited by treatment with bacterial neuraminidases, and this consequently accel-
erates severe inflammation. Thus, CD22 is a regulator of inhibitory BCR signaling.
Recent data obtained from a series of experiments including NMR analysis,
computational docking model simulation, X-ray crystallographic analysis of a com-
plex of α2,3 sialyllactose, and sialoadhesin (Sn)/Siglec-1 have revealed that Sn
A) Siaα 2-6 Gal

B Cell Receptor CD22 (Neu5AC)
(Siglec-2)
CD45
B) B Cell Receptor
CD22
(Siglec-2)
Siaα 2-6 Gal
(Neu5AC)
Y Y
Y Y Y Y Lyn
Phosphatase
Y Y
Y SHP- Y
1 SHP-
1
Cellular activation and signaling inhibition
Fig. 7.14 B-cell receptor regulation by CD22 α2,6 sialic acid–Gal interaction in natural conditions
(a) and B-cell regulation by CD22 in a SHP-1-dependent manner (b). Tyr phosphatase. Src
homology 2(SH2) domain (adopted and modified from Kelm S. et al. (2002). J Exp Med.
195, 1207–1213) [250]
mainly recognizes Neu5Ac and a small part of the Gal moiety attached to ligand
glycans. The interaction of Siglecs with sialoglycans modified at the fifth position of
the sialic acid moiety varies among various Siglecs. While mouse CD22 strongly
prefers the nonhuman form of Neu5Gc, human CD22 equally binds to Neu5Gc and
Neu5Ac. CD22 binding to ligands, like all other Siglecs, requires the recognition of
both the negative charge in SAs and the side chains of the carbon C-7/C-8/C-9
positions. The SA-OH group attached to 9-C is strictly required for its recognition.
Therefore, halogen substitution of the SA C-9 OH group or the C-9 OH group
esterification diminishes binding, indicating the C-9 hydrogen donor as a key point
(Fig. 7.8). Furthermore, amino group substitution of this hydroxyl group enhances
7.10 CD22/Siglec-2 357
binding. In addition, amino group acylation of this group by hydrophobic moieties

increases binding to CD22 as well as other Siglecs such as MAG and sialoadhesin.
A chemically synthesized analogue, 9-biphenyl-4-carboxamido, a derivative of
Neu5Ac (BPC-Neu5Ac), binds to human CD22 with a high affinity and selectivity.
Thus, the analogue compound augments BCR signaling due to its blocking activity
of CD22-driven signals and in turn enhances B-cell response. In contrast, another
analogue, a biphenylacetamido derivative (BPA-Neu5Ac), is a more potent and
selective inhibitor of mouse CD22 than BPC-Neu5Ac. When BPC-Neu5Ac and
BPA-Neu5Gc are chemoenzymatically assembled into the long-chained compounds
of 9-BPC-NeuAc-α2,6-Gal1,4GlcNAc ethylamine (abbreviated as BPC-Neu5Ac-
LNAcR) and 9-BPA-NeuGc-α2,6-Gal1,4GlcNAc ethylamine (BPA-Neu5Gc-
LNAcR), it is strongly bound to CD22.
7.10.5 Model Ligands for Recognition of CD22 on B Cells
It is an interesting finding that an alternative antibody-mediated therapy for targeting

CD22 can be used as the B-cell lymphoma therapy through specific toxin conjuga-
tion with these sialoside probes. For example, sialo–ligand conjugation of probes to
saporin enhances toxin uptake and toxin-mediated killing of B-cell lymphomas. A
therapeutic agent or a radioisotope can target the B cells by linking to CD22 ligands
with high affinities [250, 252]. Thus, synthetic CD22 ligands could be useful in
developing a novel strategy for regulating immune responses and can also be used as
delivery agents (Table 7.5).
7.10.6 B Cell-Targeted Immunotherapy Through

CD22-Positive Targeting of B-Cell Lymphomas
The cis-bound Sia α2,6-Galβ1,4 residues with CD22 can be degraded by sialidase
action. As described above, CD22 lectin is present in both precursor B cells and
mature B cells; however, CD22 is not observed in antibody-secreting plasma cells.
This fact makes CD22 an applicable target for treating B-cell lymphomas and
systemic autoimmune diseases. For example, anti-CD22 antibodies such as
epratuzumab are under investigation in phase I/II clinical trials for non-Hodgkin’s
lymphoma (NHL) treatment and SLE (Fig. 7.15) [253]. Other trials have been
conducted in conjugates of single-chain anti-CD22 antibodies with RNAses [254]
and also with Pseudomonas exotoxin-A [248]. Sialoside ligand-bound Pseudomo-
nas exotoxin-A is therefore called a CD22 ligand-used immunotoxin. The created
sialoside ligand–exotoxin-A conjugates can induce the cell death of CD22-
expressing B-cell lymphomas because CD22-expressing lymphoma B cells are
recognized rather than endogenously present in cis-type ligands on the surfaces of
358
Table 7.5 Human Siglec-2/CD22 and selective recognition of Sia α2,6-Gal

Human Siglec Expressed Recognition of Sialyl Pathogen-binding original Tyrosine
(CD number) cell linkage receptor (Extrinsic) Lectin motifs Related disease
Siglec-2 (CD22) B cell V-set Ig domain for Human influenza A Sambucus nigra ITIM Lymphoma, leukemia, SLE,
SA recognition agglutinin (SNA) ITIM- rheumatoid arthritis
like
7.10 CD22/Siglec-2 359
Fig. 7.15 Mechanism of 'RTCVW\WOCD

the CD22 antibody
(epratuzumab)
<<
5*2
5*2
*2

%C D

B cells. The sialylated ligand–exotoxin-A conjugates are effectively endocytosed

into B-cell lymphomas. This strategy can be extended to therapeutic candidate
compounds to specifically target CD22 expressed on B cells if humans, which are
transformed types of autoimmune diseases induced by B cells or B-cell lymphomas.
Another CD22 property is its endocytic internalization capacity upon binding to
soluble sialyl ligands [23], as observed in CD33. During B-cell tolerance, CD22-
binding agents can be developed. Similar to CD22, human Siglec-10 is also related
to inhibitory BCR signaling. Conjugation between the sialyl ligands and the toxin
saporin enhances the capture capacity of the toxin and toxin-mediated cytotoxic
killing of B-cell lymphomas, thus offering an alternative antibody-mediated therapy
to target CD22. Thus, the therapeutic treatment of B-cell lymphomas is the current
issue [252].
7.10.7 Immune Tolerance Capacity of Neu5Ac-α2,6-Gal

Ligands in DCs by ST6Gal-1 of Tumor Cells
for Immunesurveillance
Expressions of α2,6-SA glycans in immature and tolerogenic DCs are correlated

with the tumor cell presence of α2,6-SA glycans. ST6Gal-1 activates cell migration
and invasion. The enhanced migratory potentials seem to be due to ST6Gal-1-
mediated sialylation of the β1-integrin receptor. Sialyltransferase, β-galactoside
α-2,6-sialyltransferase 1 (ST6Gal-1), expression is reported to be increased in
human colon tumors. ST6Gal-1 is an enzyme that is encoded by the ST6Gal-1
gene (Fig. 7.16). The ST6Gal-1 gene-encoded enzyme, as a type II membrane
protein, transfers the SA residue from CMP-SA of donor substrates to
Gal-attached glycans of acceptor substrates. The protein is normally present in the
Golgi apparatus. In some cases, the protein is proteolytically modified and processed
to be released in a soluble form. The soluble form generates cell surface glycan
determinants and differentiation antigens, HB-6, CDw75, and CD76. If the transfer
of SA from CMP-SA is inhibited, metastasis to other organs, for example, the liver,
N-linked O-linked
α3 α3
ST6Gal-1
α6 α3
α6 α3
β4 β 4 β 4 β4
α6
β2 β2
α3 α6
Thr/Ser Thr/Ser
α6
Core 4 Core 2
Asn
Fig. 7.16 β-Galactoside α2,6-sialyltransferase-1 (ST6Gal-1) reaction in N-/O-glycans of tumor

cells
is decreased. Therefore, it is concluded that ST6Gal-1 promotes tumorigenesis and

serves as a regulator of the stem cell phenotype in both normal and cancer cell
populations. The mechanism(s) of why or how ST6Gal-1 expression is increased in
cancer cells is explained by the ras oncogene’s downstream signaling, although
many other types of adopted signaling may be involved.
α2,6 sialylation of the Fas receptor is reported to be increased in patients with
human colon carcinoma [255]. ST6Gal-1 is overexpressed in many human cancer
types such as colon, breast, and ovarian tumors. The increased expression correlates
with increased metastatic potential with poor prognosis. ST6Gal-1 upregulation in
human malignant tissues of colon tumors targets the high level of the α2,6-sialylated
Fas receptor [255]. ST6Gal-1 is also known to be highly expressed on human-
induced pluripotent stem (iPS) cells and various epithelial tumors but not on
human foreskin fibroblast cells and nonepithelial tumors. As ST6Gal-1 expression
is observed to be localized to the stem or progenitor cell compartment in the
epithelia, it is considered that the ST6Gal-1 expression enriches stem cell numbers
in colon carcinoma cells such as SW948, SW948, and resistant cells of the
camptothecin analogue, irinotecan, known to be a drug for treating colon carcinoma
cells. Metastatic murine cells seem to be more largely sialylated than nonmetastatic
parental cells [256].
7.10.8 CD22 Vs. Pathogens
Sialic acids are quite exclusively expressed in most mammalian species. Therefore,
they can be used as markers for recognizing ‘self’ in mammalian species.
7.10 CD22/Siglec-2 361
Exogenous receptor
E. coli K1
Pathogenes Meningococcus
Campylobacter
Trypanosoma
Group B Streptococcus
……
Self Self
Endogenous receptor
Sialylated glycan
Fig. 7.17 Sialic acids in self-recognition by endogenous receptors and pathogenic mimics and
exogenous receptors
Microorganisms including bacteria tend not to express SAs on their surfaces, and,
therefore, such SAs can be considered as foreign objects [257]. Our body effectively
fights against many different pathogens using sialic acids as molecular markers for
distinguishing self from nonself, but some pathogens have evolved and have learnt
to overcome this problem by expressing sialic acids on their own (Fig. 7.17). In some
pathogenic bacteria-infected sepsis models, microbial sialidases liberate sialic acids
on the patterns and consequently block the sialic acid–CD22 interaction. This event
accelerates inflammation. There are many evidences to suggest that some pathogens
that adopt and produce sialic acids can dampen the activity of B cells, thereby
avoiding massive attacks by immune cells [258].
7.10.9 CD22 Application with CAR-T on Acute

Lymphoblastic Leukemia (ALL)
CD22 also plays an important role in recognizing foreign and potentially harmful
pathogens due to the fact that pathogens usually do not have surfaces enriched with
sialic acids. By recognizing and reacting to sialic acids, CD22 acts as a checkpoint to
determine whether what it encounters is friendly. As CD22 is exclusively present on
B cells, it has become a target for drug designations and has indeed shown to be a
useful target for CAR therapy and immunotherapy. Acute lymphoblastic leukemia
(ALL) is specific for a disease phenotype as a cancer of immature lymphocytes and
intervenes with the immune system. The major ALL types consist of the B-cell
lineage type with the fact that the predominant immature lymphocytes are immature
B cells [259]. Annually, about 6000 ALL patients are newly reported and many of
them are under the age of 20 years. ALL symptoms are severe in most children and
adolescents. ALL is the most diagnosed cancer type among them [260]. Hence,
many attempts have been made to develop novel and therapeutic drugs to treat ALL.
CAR-T, first developed in 1989, indicates chimeric antigen receptor therapy. A
chimeric antigen receptor is a recombinant receptor designed and programmed to
be expressed on T-cell surfaces drawn from patient blood samples. T cells designed
and programmed for chimeric antigen receptors get injected into the patient’s blood
and cause killing of target cells [261]. CD22, expressed only on B cells, is a good
target of CAR-T because it specifically targets B cells [262]. Even though anti-CD19
CAR-T is successful, some ALL patients cannot be treated with this therapy. For
those who cannot be cured with anti-CD19 CAR, anti-CD22 CAR therapy is
alternatively used both successfully and safely [263]. In summary, CD22 is
expressed on B cells and, and when subjected to certain ligands (e.g., sialic acids),
it can inhibit the B-cell receptor signaling pathway by activating certain phospha-
tases. This mechanism is shown to be crucial in many defense mechanisms of our
body. First and foremost, CD22 plays a crucial role in preventing autoimmunity by
stopping B cells from producing antibodies against one’s own body. In addition,
CD22 also plays an important role in recognizing foreign and potentially harmful
pathogens due to the fact that pathogens usually do not have surfaces enriched with
sialic acids. CD22 by recognizing and reacting to sialic acids acts as a checkpoint to
determine whether what it encounters is friendly. As CD22 is exclusively present on
B cells, it has become a target for extensive drug researches and has indeed shown to
be a useful target for CAR therapy and immunotherapy.
7.10.10 CD22/Siglec-2 Coreceptor, CD45 on T Cells
Glycosylation highly controls the immune system. Despite the importance of gly-
cans in immunity, glycosylation regulation has been highlighted both in adaptive
and in innate immune responses in autoimmune diseases and cancers. CD22 is
classified as an inhibitory coreceptor of the BCR, and CD34 is a coreceptor of
CD22. CD34 is a pan-immune cell marker protein, acting in a manner such that
glycosylation changes in CD45 regulate T-cell behaviors, such as migration, TCR
signaling, and apoptosis. The most well-studied field of cellular glycosylation is
immune cells. For example, lymphocyte cell surface proteins affect diverse cellular
behaviors including pathogen recognition, leukocyte migration, tumor immunolog-
ical escape, and evasion. Glycans regulate development of T cells and thymocyte
selection via differentiation of T cells. T cell-produced glycans function as determi-
nants of either self-tolerance or T-cell hyperresponsiveness, which ultimately induce
tolerance in cancers or loss of immunological tolerance in autoimmune diseases.
Glycoproteins expressed in T cells contextually undergo glycosylation during thy-
mus development and peripheral lymph node activation, as well as blood circulation.
Therefore, glycosylation affects T-cell function and fate. T-cell glycosylation is
associated with a process displayed for immune responses mediated by T cells
during antigen-based cooperation.
7.10 CD22/Siglec-2 363
One of the T-cell glycoproteins, CD45, is glycosylated during T-cell activation

and thymocyte development. Among the proteins expressed on T-cell surfaces,
CD45 is a highly glycosylated surface proteins with both O-glycans and
N-glycans. Since CD45 glycosylation is functionally crucial for the adjacent neigh-
boring proteins, herein, the CD45 protein, T cells are stressed to regulate CD45
glycosylation by alternative splicing of CD45 isoforms, consequently through the
different glycan attachment sites on the protein. The differentially glycosylated
CD45 forms on T cells determine the T cells’ fate and function of how to interact
with extracellular environmental proteins and endogenous lectins, which recognize
the CD45 glycans.
7.10.10.1 Existence of CD45 Isoforms on T Cells
Five isoforms of CD45 are found on human T cells. The intracellular region of CD45
has in common the same sequence as all other CD45 isoforms found. The cytoplas-
mic region has tandem phosphatase domains crucial for TCR signaling [264]. In the
extracellular region, five isoforms of CD45 have in common three fibronectin type
III-repeated domains in the proximal membrane region and a Cys-rich domain. The
domains contain Asn residues for N-linked glycosylation sites [265]. The extracel-
lular region of CD45 has three distinct domains, i.e., domains A, B, and C, specific
for the Thr/Ser O-glycosylation sites, as generated by alternatively spliced exon-4,
-5, and -6, respectively [266]. Alternative splicing of the expressed CD45 isoforms
are strictly regulated during activation and development of T cells. Attached O-/N-
glycans contribute to molecular mass variation of the CD45 isoforms; hence, the
MWs of the CD45 glycoprotein range between 120 and 140 kDa [267]. The
experimentally observed molecular weight of CD45 was reported to range between
180 and 230 kDa, with glycans occupying 32–36% of the CD45 molecular mass. All
isoforms of CD45 have a total of 11 N-glycosylations linked to Asn residues of the
membrane proximal part of the extracellular domain. In addition, the alternative
variant forms, which are spliced in the CD45 extracellular domain, contain core-1
O-glycosylations and core-2 O-glycosylations [268]. However, each CD45 isoform
is not consistent and differs in the O-glycosylation level. ST6Gal-1 further modifies
complex N-glycans in CD45 as an acceptor substrate with sialylation
[269, 270]. Therefore, both mature thymocytes and naive T cells in the peripheral
tissues express α2,6 sialyl N-glycosylated CD45. In contrast, there is a big difference
in CD45 glyosylation between nonactivated and activated T cells. Indeed, N-glycan
structures of CD45 expressed in activated T cells do not contain α2,6 sialyl
structures.
7.10.10.2 CD45 Function Is Displayed in a Glycan-Dependent Manner

on T Cells
T-cell CD45 glycans recognize endogenous lectins to modulate T-cell functions.

Thus, expression of CD45 glycans is controlled in the development and activation of
T cells. TCR signaling is well-known to be active in positive and negative
thymocytic selections and T-cell activation in the peripheral tissues. Here, CD22 is
a coreceptor of CD45. CD45 is indeed the prototype of TM receptor-like protein Tyr
phosphatases (RPTPs), functioning in immune responses. The cytoplasmic region of
CD45 consists of two homologous protein tyrosine phosphatase domains. Among
them, one domain is active and the other is nonactive. CD45 is an evolutionarily
conserved receptor protein tyrosine phosphatase expressed on the hematopoietic
system. CD45 is expressed by several isoforms in a cell type-specific manner during
cell development. CD45 is a key player in the initiation of TCR signaling by the
Lck/Fyn protein Tyr kinases, which are a family of Src kinases. CD45 defect is
associated with T- and B-lymphocyte dysfunction. Autoimmune diseases and can-
cers as well as infectious diseases are also linked to CD45 dysfunction.
Glycans regulate phosphatase activity in the CD45 intracellular domain. The
CD45 intracellular phosphatase domain regulates TCR through interaction with
CD22, a B-cell adhesion molecule mentioned in the previous chapter
[139, 271]. Hyperglycosylated N- and O-glycans and Sas with negative charges
are characteristic of the extracellular domain of CD45. For TCR signaling activation,
glycan heterogeneity expressed on the extracellular domain of CD45 keeps CD45 on
the PM, thus increasing the level of TCR signaling by activation of the intracellular
phosphatase domains [272]. Conversely, for the downregulation of TCR signaling,
reduced sialic acid content and multivalent lectin interaction with the CD45 extra-
cellular domain induce CD45 oligomerization and this diminishes TCR signaling
[272, 273]. Thus, CD45 glycans are bound by several lectins including macrophage
galactose-type lectin (MGL) [274] or galectin-1 [273]. The lectin-bound CD45 is
consequently inactive in its phosphatase domains and thus negatively regulates TCR
signaling (Table 7.6). In T-cell apoptosis, certain thymocytes and activated T cells in
the peripheral tissues undergo galectin-1-involved apoptotic cell death during
β-galactoside binding. The glycoprotein counter-receptor CD45 and other counter-
receptors CD43/CD7 also regulate galectin-1-driven apoptotic cell death of T cells
[275]. Galectin-1 lectin recognizes lactosamine carbohydrates that lack α2,6-linked
SAs [269] on core-2 mucin-type O-glycan structures and N-glycosylations. CD45
core-2 O-glycan structures play an essential role in galectin-1-mediated apoptosis of
T cells [276]. CD45 on mature thymocytes contains α2,6-linked sialyl N-glycans
Table 7.6 Lectin coreceptors of CD45 on T cells and thymocytes

CD45 coreceptor Glycan Role References
Galectin-1 and -3 Lactosamine T-cell apoptosis [13]
CD22/Siglec-2 α2,6 sialic acid T-cell signaling [8, 9]
Macrophage gal-lectin GalNAc T-cell apoptosis [12]
7.10 CD22/Siglec-2 365
and core-1 O-glycans. Thus, this “specialized glycosyl phenotype” inhibits the
interaction between galectin-1 and CD45 present on T cells, due to downregulated
core-2 O-glycans and upregulated α2,6 sialylation. In the peripheral tissues and in
circulation, CD45 with core-1 O-glycan types and SAα2,6 linkages are present on
naive T cells. The carbohydrate structure is resistant to apoptotic cell death, which is
induced by galectin-1. During activation of both CD4+ and CD8+ CTLs, they
equally express CD45 isoforms, which contain reduced α2,6-linked SA levels in
complex N-glycan types and core-2 O-glycan types. This type of activated T cells is
susceptible to apoptotic death induced by galectin-1 [273]. In summary, through the
T-cell surface glycoproteins, CD45 glycans, T cells modulate binding of the extra-
cellular environment to endogenous lectins of T cells. Thus, migratory events, TCR
signaling, and apoptotic responses of T cells are controlled by CD45 glycans at the
T-cell level.
7.10.10.3 Two Glycoproteins of CD43 and CD45 in Glycan-Dependent

Functions on T Cells
T cells bind to endogenous lectins as extracellular environmental factors. T-cell

endogenous lectins recognize the abundance and type of carbohydrates linked to the
CD43 and CD45 glycoproteins present on cell surfaces, indicating that T-cell
regulation is controlled by glycan changes in CD45 and CD43. Therefore, T-cell
behaviors including migration, TCR signaling, and apoptosis are influenced by
CD43- and CD45-bearing glycans of the T cells. Then, in T cells, a fundamental
question is raised: how do glycosylation changes in the CD43 and CD45 extracel-
lular domains influence the functions of CD43 and CD45? Unlike CD43, the glycans
linked to the CD45 extracellular region regulate CD45 intracellular phosphatase
activity. With regard to the role of CD43 glycans, a similar prospect has been
considered. How are the glycosylations of each individual O-glycan linked to
CD43, as well as those of the O-glycan and N-glycan, linked to CD45, regulated
in the lifespan of T cells? What is the role of the microheterogeneity of glycans
linked to CD43 and CD45? Regarding the comparison of the roles between CD43
and CD45 in T cells and other leukocytes, different glycosylations of CD43 and
CD45 affect the functions of T cells and thymocytes. A comparison of the role of
glycans in CD43 and CD45 present on other leukocytes such as B cells and DCs
exhibits their different functions. If such questions are answered, we can easily
understand how the fundamental property of CD43 and CD45 glycosylation influ-
ences T-cell development and function.
Like CD45, CD43 is also a T-cell surface glycoprotein and is expressed during
development of T cells from thymocytes with a double-negative (DN) phenotype to
memory T cells [277]. Receptors or lectins expressed by host endothelial cells,
diverse immune cells, and cancer cells can recognize glycans linked to both CD43
and CD45 glycoproteins [278]. Thus, glycosylation of the two glycoproteins, CD43
and CD45, affects recognition and interaction between T cells. CD43 is a mucin-type
O-glycoprotein because the extracellular region of T-cell surface CD43 is
O-glycosylated. CD45 expression is restricted to hematopoietic cells and it has five

isoforms on human lymphocytes [279]. The cytoplasmic region of CD45 contains
TCR signaling [264]. An alternative formation of CD45 isoforms is regulated during
activation and development of T cells. CD45 functions in a galectin-mediated and
glycosylation-dependent manner. All isoforms of CD45 lack terminal α2,6-SA
linkage in CD45. CD45 promotes apoptotic Th1 and Th17 cell deaths, whereas
Th2 cells are protected from apoptotic death due to the sialylated surface glycans.
Therefore, the CD45 glycosylation type in T cells is a galectin-dependent regulator
during T-cell development and apoptosis. Th2 cells (but not Th1 cells) carry CD45
with α2,6-linked SAs. The α2,6 sialylation of CD45 affects the binding capacity of
galectin-1 to CD45. A lack in terminal α2,6-linked SAs on CD45 induces the
apoptosis of Th1 and Th17 cells. In contrast, Th2 cells are not α2,6 sialylated on
the surface glycans of CD43 and CD45, consequently without any apoptotic death of
Th2 cells. These indicate the glycan-requiring functions of CD43 and CD45.
7.11 Siglec-4/Myelin-Associated Glycoprotein (MAG)
7.11.1 General Aspects of MAG/Siglec-4
Known Siglecs are reported to be mainly expressed on the cell surfaces of hemato-
poietic lineages. Currently reported Siglecs are present on immune cells, mainly
innate immune cells. However, some specific Siglecs are also present on tissue cells
as nonimmune cells, which are not related to immune responses outside the immu-
nological system. A representative example is Siglec-4, which is characteristically
known as MAG that is mainly expressed on glial cells, such as Schwann cells, and
on neuronal oligodendrocytes [101]. Myelin is a dielectric material that promotes
impulse propagation. Myelination is a process in which fatty-layered myelins accu-
mulate around neuronal cells. Myelin enables neuronal cells to transmit signal
information to the adjacent neuronal cells with high speed and brain networks.
Thus, this process is essential for normal CNS function. MAG belongs to the Siglec
family and is a binding ligand specific for the NOGO-66 receptor (NgR). Arg118
forms specific hydrogen bonds between the SA and COOH groups. A 100 kDa
protein is located on Schwann cells in the periaxonal area and in the oligodendroglial
membrane region in myelin sheaths. Its function is associated with interactions
between the glia and axons in the CNS and the PNS. MAG–axon binding inhibits
neurite outgrowth from CNS neurons. MAG is involved in myelination during nerve
regeneration (Fig.7.18). For a better understanding of neural gangliosides and MAG
interaction, as described in Fig. 7.18, gangliosides have diverse biological functions.
Siglec-4 plays a main role in myelin sheath stabilization in vertebrates
[96]. Among vertebrates, jawless fishes are solely known to lack myelin and are
thus called nonmyelin fish. These fishes lack Siglec-4 [280, 281]. Thus, the axons are
surrounded only by glial cells in invertebrates like jawless fishes. However, oligo-
dendrocytes and Schwann cells form the myelin and its sheath through myelination
7.11 Siglec-4/Myelin-Associated Glycoprotein (MAG) 367
nȼXSZnuhȼXS[nȼXS[nȼXSX˅j simple complex myelin associated

Z gangliosides gangliosides glycoprotein (MAG)
u\hȻY GM1
nȼXSZnuhȼXS[nȼXS[nȼXSX˅j Complex gangliosides MAG
ZGGGGGGGGGGGGGGGGGGGGGGGGGZ
u\hȻYGGGGGGGGGGGGu\hȻY GD1a
nȼXSZnuhȼXS[nȼXS[nȼXSXNj GD1a
ZGGGGGGGGGGGGGGGGGGGGGGGGGZG Gangliosides
u\hȻYGGGGGGGGGGGGGGGGGGGGGGGGGGGG MAG (GD1a/GT1b)
u\hȻY_u\hȻY GT1b
GT1b
nȼXSZnuhȼXS[nȼXS[nȼXSXNj MAG
ZGGGGGGGGGGGGGGGGGGGGGGGGGZG
u\hȻYS_u\hȻYGGGGGGGGGGGGGGGGGGGGGGGGGGGG
u\hȻY_u\hȻY GQ1b
Fig. 7.18 Sialylglycans such as gangliosides on neurons interact with MAG and inhibit
MAG-mediated neurite growth. MAG–ganglioside interaction is shown. GD1a and GT1b are the
major GSLs in the brain and recognize MAG
in higher animals. Therefore, the two distinct cells, oligodendrocytes and Schwann
cells, are called myelin-forming cells or myelination cells [280, 281]. Sharks belong
to nonbony fishes also known as cartilaginous fishes. These fishes are the “oldest”
fish after jawless fishes. It has been suggested that Siglec-4 initiates myelination and
the development of myelination coevolves with Siglec-4 emergence in vertebrates
[96]. In addition, Siglec-4 seems to control the physiological systems in fishes than
those in terrestrial vertebrates [106]. The SA ligand-recognition domains are strictly
conserved in many other Siglec-4 orthologs. For example, the motif RAIW domain
in rock bream share corresponding sequences in Siglec-4 in fishes such as medaka,
zebrafish, and coelacanth, as well as in higher vertebrates [96]. The GRT motif
sequence of Siglec-4 is also conserved in coelacanth, medaka, rock beam, zebrafish,
and other vertebrates [96]. The coelacanth species is an ancient fish that exist even
today.
7.11.2 Siglec-4/MAG in the CNS and Brain Development
MAG as a member of Siglecs is a plasma membrane glycoprotein. As a cell surface

protein, it binds to sialic acids to cause myelination in neurons, with fatty-layered
myelin accumulating around neuronal cells. The molecular mass of MAG/Siglec-4 is
100 kDa. MAG/Siglec-4 potentiates neuronal cells to transmit any electrically
pretransmitted signals for complex processes of brain functions. Therefore, the
transmission event is actively essential for a normal CNS to function in brain
development; the information transmission is carried out through myelinated mac-
romolecules because myelination enables nerve cells to transmit any given informa-
tion. The myelin components are composed of a dielectric material and promote
impulse propagation [282]. Glia cells actively participate in myelination. They are
non-neuronal cells and are involved in homeostasis, myelin formation, and signal
transmission. They are known to induce a series of neuron-related events including
filopodia formation and differentiation of axon- and dendrite-like neurites. MAG

belongs to the first myelin-derived growth-inhibitory molecule [283]. MAG–axon
binding and interaction inhibit neurite outgrowth from CNS neurons. Studies on
myelination and MAG led to the discovery of the Nogo protein, and a structural
comparison between MAG and Nogo exhibited similar growth-inhibitory potentials
between the two molecules [284]. Now, it is concluded that both MAG and Nogo are
similar growth-inhibitory proteins. Embryonic and neonatal neurons are reported to
differently exhibit their responses to MAG [285]. The growth and functions of adult
neurons are inhibited by MAG. Peripheral nerve myelins are absent due to macro-
phagic phagocytic clearance before axonal regeneration. Genetic mutation in
myelination genes increases the negative clearance of myelins and nonregenerative
axons simply due to myelin presence. Regarding the relationship between MAG and
NOGO receptor, it is known that MAG functions as the NgR ligand and is present in
Schwann cells of the periaxonal region and plasma membranes of oligodendroglial
cells are present in myelin sheaths. It participates in glia binding to the axon region in
the CNS and the PNS. It is also involved in myelination during nerve
regeneration [286].
MAG binds to sialylglycans and sialoglycoproteins [287]. Sialylglycans on
neurons interact with MAG and exhibit MAG inhibition of neurite outgrowth.
Therefore, recognition and interaction of MAG with sialic acid ligands are the
fundamental basis on which the biological activity in the myelination of axons can
be demonstrated. For sialic acids in the brain tissue, the major types of brain
gangliosides, GD1a and GT1b, are reported to interact with MAG. Neurite out-
growth is suppressed in neurons treated with GD1a and GT1b. In addition, treatment
with anti-GD1a antibodies abolishes their endogenous binding of GD1a to MAG
and consequently inhibits the growth by MAG [288]. Therefore, GT1b functions as a
MAG receptor. The question is, how do neuronal gangliosides, produced on the
surface, function as MAG receptors for the interaction pathway? If they truly
function as MAG receptors, the neuronal cell surface gangliosides recognize the
MAG receptor. Then, these gangliosides contribute to growth inhibition when
activated by intracellular signaling. The Rho protein acts as the key signaling
intermediate in intracellular signaling. Rho, a small GTPase protein, mediates
growth inhibition triggered by MAG. This event mediates the growth-inhibitory
process. Essential evidences explaining the interaction of gangliosides GD1a and
GT1b with MAG have been provided [289]. From these results, it is surely accepted
that MAG interacts with GT1b to inhibit neurite outgrowth. At a molecular level, the
Arg118 residue of MAG forms hydrogen bonds with the sialic acid carboxylate
group. Gangliosides are clustered in membrane lipid rafts, forming a glycosynapse,
with several signaling proteins such as Rho in the cytoplasmic domain [290]. In
signaling, Rho specifically activates a serine/threonine kinase called the Rho kinase.
Furthermore, cAMP is known to promote protein kinase A (PKA) and the promoted
PKA further catalyzes Rho phosphorylation. Thereafter, the phosphorylated Rho is
dissociated from the membrane ganglioside-enriched lipid raft [291]. Therefore, it is
suggested that cAMP-related effects are associated with the blocking of MAG
inhibition, where increased cAMP indirectly attenuates the Rho signaling pathway.
7.11 Siglec-4/Myelin-Associated Glycoprotein (MAG) 369
Stimulations of neurogenesis, synaptogenesis, and synaptic transmission are

suggested in the central nervous system (CNS) [292]. In the CNS of mammals,
expression levels and carbohydrate structures of gangliosides are highly conserved.
For example, the CNS predominantly contains four different gangliosides such as
GT1b, GD1b, GD1a, and GM1. A functional understanding of gangliosides was
carried out using engineered mice deficient in GTs required for the ganglioside
biosynthesis pathway [293]. One unique human inherited disease was clearly under-
stood from an evidenced ganglioside-generating defect of GM3 synthase deficiency
found in the Old Order Amish population. The clinical feature is a severe neurolog-
ical syndrome with intractable seizures. However, mouse can compensate for the
symptom through O-series GSL biosynthesis. GM1 binds to galectin-1
[294]. SAα2,3 linkages are bound by Siglec-4, SAα2,6 linkages are bound by
Siglec-2, and SAα2,8 linkages are bound by Siglec-7 [4]. GM3 binds to GA2
(gangliotriaosylceramide, GalNAcβ1,4-Galβ1,4Glcβ1,1Cer) [295] and mediates
adhesion. Within the CNS, GSLs function as a “trans”-type binder to the MAG.
MAG binds to NeuAc-α2,3-Galβ1,3GalNAc linkages present in axon GSLs and the
bindings stabilize the axon and myelin interaction with healthy axonal
regeneration [293].
7.11.3 Siglec-4/MAG in Hippocampal Long-Term

Potentiation
In CA1 neurons of the hippocampus, a long-term potentiation (LTP) event is

generated by a neuron-dependent action. LTP is caused by enhanced GM2 produc-
tion or enforced expression of GM1-synthesizing enzymes [296]. Incubation of
hippocampal slices with exogenous GM1 enhances the LTP level [297]. LTP induc-
tion is also enhanced by GM1 or GQ1b treatment. Although NMDA receptors are
known as blocking agents against LTP induction, an NMDA receptor antagonist
exhibits the expected blocking effect on LTP induction. However, this blocking
effect is reversed by incubation with exogenous GQ1b treatment. In a recent report
on ganglioside-treated hippocampal CA1 neurons, it has been suggested that gan-
gliosides participate in activity-dependent LTP induction and that LTP induction has
been shown via the regulation of NMDA receptors and Ca2+ channels
[292]. Myelination products such as fatty-layered myelin accumulate around neuro-
nal cells/neurons, thus allowing neuronal cells to rapidly transduce their information
to the CNS, which are essential for brain functions such as learning, education, and
memory process. Such brain and neuronal functions are developed via three distinct
steps: (1) acquisition of a new experience (first step), (2) temporary neuronal
communication or short-term memory (STM) of the acquired new experience
(second step), and (3) stable and long-term memory (LTM) (third step) by permanent
setting of the neuronal structure and expression of functional molecules. During the
three steps of neurotransmission and memory, the hippocampal region is the most
nȼXSZnuhȼXS[nȼXS[nȼXSXNj
Z
GM1 £
u\hȻY
GQ1b nȼXSZnuhȼXS[nȼXS[nȼXSXNj
ZGGGGGGGGGGGGGGGGGGGGGGGGGGZG
u\hȻYS_u\hȻYGGGGGGGGGGGGGGGGGGGGGGGGGGGG
u\hȻY_u\hȻY
yG
Ghw\
Learning & memory
Learning wGG
GrY\Y
utkhG
short term memory (STM)
jYR
long term memory (LTM) n

ntXGMGnxX
wktw
Fig. 7.19 Learning and memory process progress in three distinct steps: (1) acquiring a new
experience, (2) short-term memory (STM) as the unstable form for the acquired experience with
temporary neuronal communication, and (3) long-term memory (LTM) as the stable form with
protein expression and permanent change in the neuronal structure. Gangliosides GM1 and GG1b
enhance LTP induction in hippocampal CA1 neurons of the CNSs. In hippocampal CA1 neurons,
GM1 and GM1 enhance the activity-dependent LTP. Antagonists of NMDA receptors block LTP
activation, and the blocking effects are reversed by GQ1b incubation. Thus, ganglio-specific GSLs
are involved in activity-dependent LTP events by regulation of the NMDA receptor functions/Ca2+
ion-channel polarization
important part of the cerebral cortex because this region is directly involved in the
potentials of learning and memory (Fig. 7.19). Actively formed hippocampal syn-
apses function to perform actions based on repeated stimuli such as electric
impulses. The persistently performed synaptic enhancement is LTP. Therefore,
GSLs of GM1 and GQ1b induce an ATP-driven LTP in CA1 neurons of the
hippocampal region of the brain (Fig. 7.18).
7.12 Siglec-15, Non-CD33-Related Siglecs in Humans

7.12.1 The Structure and Expression of Siglec-15, Called
Misnomer “CD33L3” in Humans
Siglec-15 is quite a lonely Siglec because it is not phylogenetically related to the

other known CD33-related Siglecs. The human Siglec-15 gene in human chromo-
somes is also independently located, differing from the other CD33-related Siglec
7.12 Siglec-15, Non-CD33-Related Siglecs in Humans 371
genes to date. Therefore, Siglec-15 belongs to the CD33rSiglec subfamily, and it is

also called “CD33L3” as a misnomer. Although the precise function of Siglec-15 has
not yet been clarified and is thus uncertain till now, an essential biological function
of Sigec-15 has been suggested in vertebrates because of the highly conserved
sequence of the Siglec-15 gene during vertebrate evolution. The structure of the
Siglec-15 gene is unique, when the gene is compared to the known Siglec genes. In
fact, the Siglec-15 gene has been known to have two exon structures compared to
other Siglec genes, which have 0 or 1 exon structure. The two exons precede the
exon region, which encodes the first Ig-like region, whereas another second Ig-like
region-coding exon includes the encoded transmembrane region. From the currently
known Siglec genes, the transmembrane region-coding exons and the Ig-like region-
coding exons are separately located on the Siglec genes but with a unique GC-rich
specificity.
Siglec-15 expression is also found mainly on immune cells of a myeloid lineage
such as DCs and macrophages. Siglec-15 is expressed on DCs and/or macrophages
of the spleen, testis, and small intestine in humans. Among these, the level of Siglec-
15 expression is uniquely high in the human spleen. Interestingly, Siglec-15 expres-
sion is correlated with the expression pattern of the DC-SIGN as a CLR group. In the
human spleen and lymph nodes, Siglec-15-expressing cells also express DC-SIGN,
indicating their correlation of expression. Conversely, it is suggested that approxi-
mately half of the DC-SIGN-expressing cells are also Siglec-15-expressing cells in
specific regions like the lymphatic nodes and the splenic tissues [117]. However, one
point to be noted is that Siglec-15-expressing cells among DC-SIGN-expressing
cells are quite different from many other cell types. The TM regional Lys residue of
Siglec-15 is conserved and the Lys residue seems to be functional in its interaction
with ligands. To date, it is known that proteins with amino acids of Lys or Arg with a
positive charge in the TM domain are associated with several adaptor molecules
such as 12 kDa DNAX activation proteins, such as DAP12, DAP10, and FcR.
Adaptor proteins such as DAP10, DAP12, and FcR associate with Siglec-15 on
cell surfaces because these three proteins are coimmunoprecipitated. Siglec-15 is
associated with DAP12, although the level of DAP12 expression is low than that of
DAP10 or FcR.
Reiterating that immune cells express most of the known Siglecs in cytoplasmic
ITIM domains, it is considered that such Siglecs can suppress immune cell functions.
However, Siglec-15 interacts with DAP12 and this binding to DAP12 activates
immune cell functions through ITAM-mediated signaling. Siglec-15 can also medi-
ate molecular interactions between monocytes/macrophages and cancer cells.
Immune-activating receptors complement inhibitory receptors with a high similarity
of sequences. In fact, an inhibitory receptor Siglec-5 in humans and an activating
receptor Siglec-14 in humans are both complement receptors generated through
coevolution [35]. Like in the case of Siglec-5 and Siglec-14, the activating group
of the Ly49 family has a motif similar to that of the inhibitory group and, hence,
counters inhibitory group-exploring viral pathogens. However, mammalian Siglec-
15 has no inhibitory region because the inhibitory motif of Siglec-15 has evolution-
arily been lost in mammals.
7.12.2 DAP12-Syk Pathway in Siglec-15-Mediated

Remodeling of the Tumor Microenvironment
Siglec-15 mediates binding of monocytes/macrophages to tumor cells. Human

Siglec-15 is present on macrophages. Among macrophages, tumor-associated mac-
rophages (TAMs) present in most tumor tissues produce Siglec-15 in humans.
Therefore, the expression level of Siglec-15 is specifically increased in M-CSF-
induced M2-type macrophages. M2-type macrophage cells express TGF-β in
sTn-positive tumor cells. Coculture with human THP-1 cells, known as monocytic
leukemic cells with a macrophage phenotype, and with human H157 cells, known as
lung carcinoma cells, exhibits a typical mimicking phenomenon of the recognition of
monocytes/macrophages in tumor cells. This is because THP-1 cells, which express
Siglec-15 on their cell surface, recapitulate the enhanced TGF-β production and
recognize sTn-positive H157 cancer cells. The elevated production of TGF-β needs a
binding of the two cells via SA recognition. Siglec-15 associates with the adaptor
protein DAP12 via the Lys274 residue as the binding-site determinant in the
transmembrane region. Then, this association contributes to signal transduction to
Syk. For experimental inhibition of TGF-β secretion, the increased TGF-β secretion
can be inhibited by Syk inhibitor addition in THP-1 cells. If the Siglec-15 Lys274
residue is substituted with Ala, the Ala residue disrupts the binding of Siglec-15 to
DAP12 and, consequently, inhibits TGF-β secretion [298]. This indicates that
Siglec-15 binds to sTn-present tumor cells and then transduces its own signal to
increase TGF-β secretion in TAMs. Therefore, it can be further suggested that
Siglec-15 expressed on macrophages potentiate cancer invasion and progression
through remodeling of the TGF-β-driven tumor microenvironments.
7.12.3 Siglec-15 Functions in Osteoclastogenesis
The immunoreceptor Siglec-15 has also been reported to function as an

osteoclastogenic factor [299]. Siglec-15 may induce differentiation of osteoclasts.
Osteoclasts, also known as bone-resorbing cells in bone remodeling, are originally
differentiated from precursors of hematopoietic cells such as monocytes or macro-
phages. There are two known factors that can induce osteoclastic differentiation of
precursor cells. The factors are cytokines, termed “M-CSF,” and receptor activator
of NF-κB ligand (RANKL), a member of the TNF family. The two cytokines
stimulate osteoclasts in vitro to induce osteoclastogenesis, but osteoclastogenesis
requires ITAM signaling. DAP12 modulates RANKL signaling to promote osteo-
clast development. DAP12-deficient macrophages undergo osteoclastogenesis, but
DAP12 promotes bone resorption [300]. In mouse BMMs, Siglec-15 expression is
reported to increase by RANKL stimulation. Before RANKL stimulation, a low
level of Siglec-15 expression is reported on the BMM surface. The Siglec-15/
7.13 Siglec-3 (CD33)-Related Siglecs on DCs 373
DAP12 complex is responsible for both cytoskeletal organization and differentiation

of osteoclasts.
7.13 Siglec-3 (CD33)-Related Siglecs on DCs
Membrane proteins of most immune cells have cytosolic ITIMs with negative
signaling through activation receptors. One example is Siglecs having inhibitory
receptors involved in TLR signaling. CD33rSiglecs, which is a recently evolved
Siglec subset of innate immune cells, contain a cytosolic ITIM and an ITIM-like
sequence, following Tyr phosphorylation by Src-family kinases. CD33rSiglecs
confer inhibitory signaling on cells, recruiting and activating Tyr phosphatases
SHP-1/-2 to the complex signaling machinery. The SA recognition sites of leuko-
cytic inhibitory Siglecs interact with SAs that serve as self-antigens in a cis-binding
manner.
Host SAs are indeed ubiquitously expressed as SAMPs [22]. Inhibitory receptors,
CD33rSiglecs, interact with the SAMPs to maintain and keep the normal state of
immune responses, not to be activated [24, 117]. CD33rSiglecs are thus regulators of
immune responses to maintain homeostasis in the host [117], where sialic acids as
ubiquitous SAMPs are recognized by CD33rSiglecs. This is a mechanistic homeo-
stasis in immunity: not responding to the self but maintaining the basal and quiescent
states of innate immunity. The Siglec–self-antigen SA interaction induces controlled
inflammatory responses, suppressing unwanted inflammatory events of hosts, which
can be provoked by DAMPs [301]. DAMPs include high-mobility group box-1
(HMGB1) [302] or PAMPs of LPSs, peptidoglycans, and flagellins [303]. Although
the original CD33 is expressed on myeloid cells, CD33-related Siglecs are present on
myeloid lineage immune cells. Moreover, they have CRD and ITIM. After the CRD
recognizes specific glycan structures, the inhibition signals work to deliver into the
cells via the cytoplasmic ITIM. If inhibitory CD33rSiglecs do not function, then
inflammation or similar pathogenic disease-like symptoms will be displayed, as
found in experiments using mSiglec-F KO mice [304], mSiglec-G-deficient mice
[305], and in human Siglec-8 polymorphism-derived asthmatic diseases [305] or
human Siglec-9 gene polymorphism-derived exaggerated T-cell responses
[306]. Microglial cells isolated from mSiglec-deficient mice exhibited elevated
inflammation and neurotoxicity in neuronal cocultured conditions [307]. Human
Siglec-10 is also a specific regulator to respond against the DAMPs and HMGB1
expressed by necrosed cells [307]. For example, the expression level of inhibitory
CD33rSiglecs present in peripheral CD4+ T cells is increased upon HIV infection.
This indicates that inhibitory Siglec receptors regulate T cell-mediated immunity and
can be combined with the reduced level of CD33rSiglecs expression in T cells
residing in the periphery of normal human and mouse tissues [308].
7.13.1 Siglec-3 (CD33)
CD33, a TM receptor, is expressed on myeloid immune cells [23]. CD33 or Siglec-3

with a 67-kDa glycoprotein is a member of the Siglec family, in turn belonging to the
Ig superfamily. CD33 is a receptor for commitment of hematopoietic stem cells to
the myeloid-monocytic lineage. These CD33s are present on myeloid lineage
immune cells [309]. Therefore, high levels of CD33 expression can be observed in
the bone marrow and circulating mononuclear cells. CD33 expression is
downregulated from peripheral granulocytes and resident macrophages to low levels
but is constitutive in DCs. These expressions suggest that CD33 participates in the
differentiation of myeloid immune cells such as mononuclear cells and DCs. Mye-
loid progenitors, monocytes, and myeloid DCs are prominent cells that express
CD33, whereas macrophages and granulocytes are also capable of expression at
low levels [309]. Thus, the CD33 expression is restricted to the myeloid lineage.
However, some lymphoid cells including the earlier precursors of human fetal
thymocytes and human CD34(+) postnatal thymocytes weakly express CD33
[310]. Moreover, activated NK cells and activated T cells can express CD33 at a
lesser level [311]. CD33 is expressed on leukemic AML blasts and normal myeloid
lineage progenitors. The level of CD33 correlates with the disease prognosis
[312, 313]. Apart from CD33, CD15 (3α-fucosyl-N-acetyl lactosamine), also called
Lewis X or SSEA-1, is also a carbohydrate antigen expressed on most AML cells
and in some populations of NK cells, T cells, monocytes, neutrophils, and eosino-
phils. CD15 expression on AML blasts is associated with a favorable prognosis such
as continuous complete remission and longer survival. As AML is common in acute
leukemia in adults, the AML cell surface marker CD33 is therefore a therapeutic
target, and many anti-CD33 immunotherapies are new trial agents in AML.
Loan eye members excluding Siglec-1 (Sd), Siglec 14, and Siglec 15, etc. are
suppression receptors presumed to regulate immunological functions. The functions
and role of CD33 are associated with immune responses in inflammation based on
the modulation of Tyr-kinase downstream signaling. Numerous Siglecs contain
multiple ITIMs. CD33 (Siglec-3) belongs to the smallest family and bears the two
extracellular Ig-type domains with the two cytoplasmic ITIM-type domains
[314]. These ITIMs recruit adaptor molecules of Tyr phosphatase SHP-1/-2 for
dephosphorylation after phosphorylation with SA stimulation. Anti-CD33 monoclo-
nal antibody (mAb) induces cell death and prevents the formation of DCs from
mononuclear cells and myeloid CD34 precursor cells. In addition, the role of protein
kinase C (PKC) in bone marrow cells is to phosphorylate the CD33 Ser residues and
activate its CD33 lectin activity. It was demonstrated that CD33 functions are
dependent on ligands and SOCS3 signaling. Conversely, the levels of IL-1β/TNF-
α/IL-8 production, which are proinflammatory cytokines, are decreased when the
cell surface expression of CD33 is reduced through RNA interference or
SA-recognition interruption. If CD33 expression is reduced, p38 MAPK activity is
increased and cytokine secretion is induced with cytokine-induced cellular
proliferation [315].
7.13.2 Structure, Natural Ligand, and Cellular Signaling

with SHP-1/-2 of Siglec-3/CD33
The Siglec-3/CD33 extracellular domains comprise one N-terminal SA-binding

V-type domain and one C2-type Ig-like domain linked by a helical transmembrane
sequence to a cytosolic tail containing two ITIMs. The Src family kinases phos-
phorylate the Tyr residues and the phosphorylated residues recruit and activate both
SHP-1 and SHP-2. Although CD33 signaling is not well elucidated, SHP-1/-2 may
control the signaling. CD33 has a subset of the Ig superfamily molecules [316]. The
cytoplasmic domain of CD33 has two inhibitory signaling motifs to be phosphory-
lated by Src kinases. Consequently, later, this region recruits and activates dephos-
phorylation of SHP-1 and SHP-2. To regulate ITIM signaling, a different adaptor,
termed “SOCS3,” competes with phosphatases SHP-1/SHP-2 at the CD33 recogni-
tion site, which is phosphorylated, and this event leads to ubiquitination of CD33 via
proteasomal degradation. Clearly, the CD33-SHP-1/SHP-2 complex form is gener-
ated and the complex induces inhibitory downstream signaling. Although down-
stream CD33 signaling has been poorly understood to date, SHP-1 and SHP-2 may
relate to the mechanism. Moreover, SOCS3 competes with Tyr phosphatases SHP-1
and SHP-2 at the binding site of phospho-CD33, which ultimately causes
ubiquitination and then accelerated proteasomal degradation of CD33.
There are currently two different known Tyr-based motifs and they are CD33
membranous ITIM and ITIM-like sequences, which have the conserved common
Glu/Asp-Tyr-X-Glu-Val/Ile-Arg/Lys sequence and are present in the distal mem-
brane [317] (Fig. 7.20). The distal sequence is expressed similarly as the signal motif
found in the receptors of the signaling lymphocytic activation molecule (SLAM)
Fig. 7.20 Human and NH2

rodent Siglec family V-set immunoglobulin domain (Sialic acid
proteins. Siglecs as type binding site)
1 TM proteins consist of a
V-set Ig domain in the
N-terminal region that is
involved in SA recognition
and different C2-set Ig C2-set immunoglobulin domain
domain numbers. CD33
consists of conserved
Tyr-based signaling
sequences in the cellular
region
ITIM motif : Tyrosine kinase target
site
ITIM like
COOH
+6#/ UKIPCNKPI +6+/ UKIPCNKPI
NKICPF NKICPF
#EVKXCVQTTGEGRVQT +PJKDKVQT TGEGRVQT
#FCRVQT RTQVGKP
/GODTCPG
U[M
3 3 5-( 5*2 3
3 3 3
#EVKXCVKQP +PJKDKVKQP
UKIPCN UKIPCN
Fig. 7.21 Downstream signaling of Sigec-3/CD33. An active SHP motif represses ITAM activa-
tion and the TLR signaling pathway. The NF-κB pathway is suppressed and an immune response
such as inflammation is also suppressed
group, which is associated with the SLAM-associated protein (SAP) and its homol-
ogous protein, Ewing’s sarcoma-associated transcript 2 (EAT 2). Whilst, the signal
of CD33 does not interact with SAP and EAT2 adapter, the function of recruitment
and suppression signal of ITIM’s SHP1, SHP2 appears mainly. The ITIM of CD33 is
important for other functions such as sialylated ligand and Siglec-dependent adhe-
sion inhibition of endocytosis. It strongly binds to SHP-1 and SHP-2 via the
phosphorylated Tyr residues on ITIM and ITIM-like domains. This combined SHP
is activated to suppress the ITAM downstream, and TLR mediates NF-κb signaling
[317]. As a result, inflammation is suppressed, and the immune response decreases
(Fig. 7.21).
On a structural basis, CD33, a 67 kDa transmembrane glycoprotein, is an Ig
superfamily molecule. It consists of two different domains including a V-set Ig-like
domain with a SA recognition region in the N-terminal region and an extracellular
C2- set Ig-like domain (Fig. 7.22). Alternatively spliced CD33 forms generate
various isoforms present on cell surfaces. Alternative splicing of CD33 can be
used in the development of CD33-related drugs. Tyrosine-phosphorylated CD33
in myeloid cells can be activated by pervanadate and also inhibited by the specific
inhibitor PP2 of Src family tyrosine kinases. Phospho-CD33 can associate with the
Tyr phosphatase SHP-1/-2. The first CD33 phosphor-Tyr motif is dominant in the
interactions between CD33 and SHP-1/SHP-2. Tyr-340 mutation in the C-terminal
region of CD33 significantly decreases the binding capacity to SHP-1 and SHP-2,
whereas Tyr-358 mutation has no effect on the binding capacity. Therefore, CD33
Ig-like-V-type (19-135)
1 mplllllpll wagalamdpn fwlqvqesvt vqeglcvlvp ctffhpipyy dknspvhgyw
9VHWLPPXQRJOREXOLQGRPDLQ Sialic acid binding site
6LDOLFDFLGELQGLQJVLWH 61 fregaiisrd spvatnkldq evqeetqgrf rllgdpsrnn cslsivdarr rdngsyffrm
Ig-like-C2-type (145-228)
121 ergstkysyk spqlsvhvtd lthrpkilip gtlepghskn ltcsvswace qgtppifswl
&VHWLPPXQRJOREXOLQGRPDLQ
181 saaptslgpr tthssvliit prpqdhgtnl tcqvkfagag vttertiqln vtyvpqnptt
Transmembrane region (260-282)
241 gifpgdgsgk qetragvvhg aiggagvtal lalclcliff ivkthrrkaa rtavgrndth
ITIM mof (338-343) ITIM like mof (356-361)
,7,0PRWLI7\URVLQHNLQDVHWDUJHWVLWH
301 pttgsaspkh qkksklhgpt etsscsgaap tvemdeelhy aslnfhgmnp skdtsteyse
,7,0OLNH
361 vrtq
1 mplllllpll wagalamdpn fwlqvqesvt vqeglcvlvp ctffhpipyy dknspvhgyw Sialic acid binding site
61 fregaiisrd spvatnkldq evqeetqgrf rllgdpsrnn cslsivdarr rdngsyffrm Ig-like-V-type
121 ergstkysyk spqlsvhvtd lthrpkilip gtlepghskn ltcsvswace qgtppifswl Ig-like-C2-type
181 saaptslgpr tthssvliit prpqdhgtnl tcqvkfagag vttertiqln vtyvpqnptt Transmembrane region
241 gifpgdgsgk qetragvvhg aiggagvtal lalclcliff ivkthrrkaa rtavgrndth ITIM motif
301 pttgsaspkh qkksklhgpt etsscsgaap tvemdeelhy aslnfhgmnp skdtsteyse ITIM-like motif
361 vrtq
Fig. 7.22 Structure of Siglec-3
signaling recruits SHP-1/SHP-2 and regulates its ligand recognition activity. Alter-
native splicing of CD33 leads to an isoform present on the cell surface. Splicing of
CD33 may be used in the development of CD33-related drugs, although the process
mechanism is unknown. Specifically, a majority of CD33 antibodies recognize the
dominant epitope that is located in the V-set Ig-like domain.
In general, Siglecs have a low affinity for the galactose residue. SAs of Neu5Ac-α
2,3 and α2,6 linkages (Neu5Ac2-α2-3-Gal and Neu5Ac2-Gal) are preferred for
Siglec binding. These SAs are ubiquitously attached to the glycan terminals of
glycolipids and glycoproteins in mammalian cells, and Siglecs have specificity for
SA-bearing glycans. For the binding ligands for Siglec-3/CD33, Siglec-3/CD33
prefers sialosides with Neu5Ac-α2-6Gal and Neu5Ac-α2,3-Gal structures [318].
7.13.3 Pathogen Ligand for CD33
Many pathogenic microorganisms including N. gonorrhoeae, N. meningitidis, GBS,

H. influenzae, C. jejuni, and several E. coli strains have evolved for the synthesis or
acquisition of SAs from the host and integrated them into their party conjugates. The
sialylglycans of these pathogenic glycoconjugates seem to mimic the surfaces of
host cells and are involved in the survival of the host by avoiding immunity attacks
[65, 319–322]. It is also known that the negative charge of SAs decreases recogni-
tion of the pathogen by the host cells, due to the negative charge-derived electrostatic
repulsion, and consequently suppresses the complement activation pathway. CD33-
related Siglecs can bind to SAs of N. meningitidis, C. jejuni, GBS, and T. cruzi.
T. cruzi associates with sialoadhesin to protect mice macrophages.
For the acquisition of sialic acids by pathogenic bacteria, lipooligosaccharide
(LOS) sialylation has been reported in a recent publication [323]. In a human
pathogen, Neisseria gonorrhoeae, LOS is the most abundant gonococcal surface
molecule as a pathogenicity-causing factor. The molecular mimicry of host-like
glycans by LOS is found in N. gonorrhoeae. Neisserial LOS extending from the
heptose I residue (HepI) is a mimic of host glycans. The first studied structure of
Neisserial LOS is lacto-N-neotetraose (LNnT; Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1),
which is the same structure as that of the human terminal tetrasaccharide of
paragloboside pGb. This structure is also a precursor of the human blood group
antigens [324]. Another structure is globotriose Gb3 (Galα1,4Galβ1-4Glcβ1), which
is the same structure as that of the terminal globotriose trisaccharide Gb3 of the
PK-like blood group antigen [325]. The two structures of the LNnT
(or paragloboside pGb) and PK-like antigen globotriose (Gb3) are further sialylated
by the Neisserial strains [326]. Such sialyl LOSs inhibit complement activation
[87]. Other pathogenic bacteria also display sialylation for their benefits to escape
from host immunity by mimicking SAMPs. Among the Neisseria genus, the gono-
coccus species have unique Lac residues on HepII using LOS glycosyltransferase
lgtG, as certified in clinical N. gonorrhoeae. Interestingly, such Neisserial species
can bind to Siglec-3 upon growth with a CMP-Neu5Ac-containing medium
[327]. The sialylated gonococcal LOS lactose termini enable complement evasion
and virulence. Sialylated LNnT catalyzed by gonococcal LOS sialyltransferases
(Lst) induces pathogenesis. HepII lactose Neu5Ac is resistant to α2,3 neuraminidase,
thus keeping a α2,6 linkage.
7.13.4 Siglec-3/CD33 Is Related to SOCS3

and Internalization of CD33
Most Siglecs, as endocytic receptors, function by circulating between cell surfaces

and intracellular small vesicle-like endosomes. Certain Siglecs undergo endocytosis
upon binding of their ligands, which are known for antibodies and natural sialyl
glycans. On a structural basis, cytoplasmic tyrosine-based motifs bear the endocy-
tosis capacity of Siglecs. For example, binding CD33 to its specific antibodies
induces CD33 internalization in an endocytotic manner. This event indicates a
possible strategy for a therapeutic application using Siglec-3/CD33. However, the
mechanisms of Siglec endocytosis have not been clearly elucidated [328]. Cytokines,
LPSs, and some PAMPs can stimulate suppressor of cytokine signaling 3 (SOCS3).
SOCS3 inhibits downstream signaling upon binding to its target. SOCS3 competes
with the SHP-1/-2 binding site that contains ITIMs of cytokine receptors. SOCS3
has a specific binding affinity for the phosphor-ITIM in Siglec-3/CD33. Tyrosine
residues in Siglec-3/CD33 are related to SOCS3 for proteolytic degradation. SOCS3
inhibits Siglec-3/CD33 blocking of cytokine-induced proliferation. Therefore,
Siglec-3/CD33 is considered as the first receptor to be degraded by SOCS3. During
inflammation, the inhibitory receptor Siglec-3/CD33 does not function and the
SOCS3 expression is both prerequisite and crucial for Siglec-3/CD33-based clinical
therapy. The intracellular cytoplasmic region of Siglec-3/CD33 is the subject of
tyrosine phosphorylation and ubiquitination. The endocytotic capacity of Siglec-3/
CD33 is proved by certain binding antibodies that lead to the modulation of Siglec-3/
Fig. 7.23 Putative functions of CD33 (modified from [329])
CD33 function through a decrease in Siglec-3/CD33 cell surface levels. Putative

functions and roles of Siglec-3/CD33 have been suggested in inflammatory and
immune responses, which are basically streamed from tyrosine kinase signaling. For
example, some articles demonstrated that Siglec-3/CD33 function is dependent on
its ligand and SOCS3. Conversely, IL-1β/TNF-α/IL-8 cytokines, which are
proinflammatory cytokines, are suppressed if the expression of cell surface Siglec-
3/CD33 is reduced, as observed from the results of RNA interference and SA
interaction blocking. Reduction of Siglec-3/CD33 expression enhances the p38
MAPK activity with induced secretion of cytokines and proliferation (Fig. 7.23).
The expression site of Siglec-3/CD33 is restricted to certain cells or tissues, and this
feature may render Siglec-3/CD33-directed therapeutics more efficient.
7.13.5 Putative Functions of Siglec-3/CD33 in Alzheimer’s

Disease (AD)
Siglec-3/CD33 is an immunomodulatory receptor. It is also an influencing key factor

in Alzheimer’s disease (AD), characterized by a late stage of onset. Late-onset AD
(LOAD) is officially classified as a multiple symptom-causing neurodegenerative
disorder, and this type is the most common form of AD [330]. In the CNS of
mammals, microglial cells are resident macrophages, which are differentiated into
or are derived from bone marrow monocytes. AD resembles the inflammation
process, although microglial cells act as macrophages in the brain. Thus, microglial
cell function contributes to scavenger amyloid plaques. Although microglial cells
protect neuronal cells by removal of plaques such as amyloid-β (Aβ) deposits and
damaged neuronal deposits, in inflammatory environments, they promote inflam-
matory responses and consequently induce neurotoxicity. Human microglial cells
express several Siglec forms, including Siglec-3/CD33, Siglec-11, and Siglec-16,
which are considered to be pathologically related in neurodegenerative diseases
[331]. In the CNS, microglial cells acting as neuronal-resident macrophages remove

plaques and damaged neurons. The microglia also express several Siglec forms,
including human Siglec-3/CD33, -11, and -16, as well as mouse Siglec-3, -E, -F, and
-H. They are associated with neurodegenerative diseases. Siglec-3 inhibits phago-
cytosis. Microglial cells of AD patients show increased CD33 expression and this
CD33 acts as a risk factor of AD. Thus, CD33 inhibition is a novel microglia-specific
therapy for AD.
Through genome-wide analyses, certain genetic loci involved in AD-related
inflammation have been discovered. Distinct genetic loci including several variants,
such as rs3865444 CD33, rs3764650 ABCA7, rs6656401 CR1, and rs610932
MS4A6A, have been found in LOAD-associated microglial genes [330]. These
variants cleared the Aβ peptides in a massive population-based analysis. Among
them, rs3865444 CD33 was selected as a protection factor of LOAD. Moreover,
production of two alternatively spliced variants is known for Siglec-3/CD33. The
gene product translated from the alternatively spliced variants is an AD risk factor.
The CD33 gene is spliced with the two alternatively spliced variants of a full-length
CD33m transcript, which is known as the product of AD risk alleles. The shorter
CD33m isoform lacks the SA-recognition domain, whereas the AD-protective allele
encodes the domain. The two human-specific variants are rs3865444A and
rs12459419T [332]. Therefore, it is suggested that CD33 is a negative regulator of
the phagocytic ability of the microglia against Aβ peptides as a toxic agent. Further-
more, a specific gene allele expresses a V-set region-defective Siglec-3/CD33
protein. The defective allele–ligand binding leads to suppression of Aβ-based plaque
uptake.
A CD33m-favoring SNP is associated with higher microglial activity than the
CD33M transcript [331]. Therefore, the CD33m-expressing variant is more benefi-
cial in decreasing the accumulation of Aβ peptides, and, thus, this variant is called a
protective isoform with ignorable levels of AD risk. For CD33m isoform-based
protection of AD development, the truncated CD33m variant is localized in perox-
isomes. The CD33m variant does not directly recognize Aβ peptide plaques. Instead,
peroxisomal CD33m abolishes CD33M expression with an increased clearance level
of Aβ peptides. Conclusively, the protective allele CD33m improves the CD33M
and CD33m ratio and contributes to the increase in the CD33m level [26]. As
CD33m lacks the V-set domain in the N-terminal region, the higher level of
CD33m compared to that of CD33M improves the protection level of AD develop-
ment. This differential ratio of CD33m and CD33M is suggested to resemble the
case of chimpanzees. Therefore, it is considered that this type of occurrence of a new
allele in CD33 may coevolve to compensate for other acquisitions or loss in the
human lineage [332]. In general, such new allele-forming mutations have much
improved native genetic traits that provide certain health benefits and protect from
diseases. The CD33m isoform may engage in certain interactions with Aβ peptides.
Using a CD33m-specific mAb, CD33 is demonstrated to be the only peroxisome-
localizing Siglec through a peroxisome-recognition sequence. This sequence has
convergently evolved in toothed whales [129].
7.13.6 Siglec-3-/CD33-Based Immunotherapy for AML
As described above, the Siglec family of SA-recognizing proteins belongs to a group

of endocytic immune cell receptors. As Siglec-3/CD33 is a specific antigen that
governs myeloid lineage differentiation and can function as an endocytic receptor,
CD33 expression in leukemic stem cells or AML cells is extremely limited. The
endocytic property indicates that Siglecs can be bound as therapeutic targets in cell-
driven treatments. For CD33-directed therapeutics of AML, the following two
approaches have been developed: i) An unconjugated antibody was the first trial
in vitro to reduce cell proliferation and induce apoptosis in AML. No clinical
efficacy was observed. In 2010, it was dropped. ii) As antibody drug conjugate,
GO, was developed in 2010. CD33-targeting drugs, including gemtuzumab
ozogamicin (GO), were developed and their activity relies on Siglecs’ endocytosis
or internalization function of the toxin drug to the targeting cells.
Siglec-3/CD33 is regarded as a prototype of AML therapy. In addition, Siglec-3/
CD33 is also a valid candidate for targeting AML due to its restricted expression on
myeloid cells. Apart from Siglec-3/CD33, another CD22 is known to be a prototyp-
ical member and valid candidate for targeting non-Hodgkin’s B-cell lymphoma
because B-cell lymphomas express CD22. Therefore, Siglec-3/CD33 is the best
target for therapeutic antibodies for AML. For this, an anti-Siglec-3/CD33 mono-
clonal antibody, gemtuzumab, was developed. Furthermore, gemtuzumab
ozogamicin (GO), the conjugate of an antibody drug, was designed to increase the
survival rate of AML patients [333]. GO, an anti-Siglec-3/CD33 mAb conjugated to
the immunotoxin calicheamicin, has been the only approved drug for AML treat-
ment in the last two decades. However, the conjugated drug was once withdrawn in
2010 due to unclear postmarketing techniques. Currently, GO is partly successful
and several Siglec-3/CD33-targeted drugs are in early-phase trials. Nevertheless,
Siglec-3/CD33 seems to be a potential therapeutic target in AML. GO is recorded as
the first ADC-approved drug in the USA for AML. GO is an ADC. GO consists of a
CD33-specific mAb, which is chemically linked to a calicheamicin derivative that is
a cell killer. In 2017, newly developed and clinically efficient as well as safe GO was
reapproved for AML. Fractionated dosing and clinical development for GO need to
be explored further because the approval of GO in 2017 highlights the need for rapid
AML characterization and combined incorporation into artificially intelligent
algorithms.
In 2019, CD33-targeted drugs, such as Fc-engineered unconjugated antibodies
(named BI 836858, using mAb33.1), antibody–drug conjugates (SGN-CD33A,
named vadastuximab talirine, IMGN779), radioimmunoconjugates (named 225Ac-
lintuzumab), and bi- and tri-specific antibodies (named AMG 330, AMG
673, AMV564, and 161,533 TriKE-fused protein), and chimeric antigen receptor
(CAR)-adapted T cells, were clinically developed. Clinical data on SGN-CD33A are
available and SGN-CD33A exhibits a single-agent activity when it is used in
combination with azanucleoside or other chemotherapeutic drugs [334]. On the
other hand, after immune effector T cells bearing CAR (CAR-T cells) technology
has been developed, double trials using Siglec-3/CD33 and CAR-T can be further
explored in AML treatment. CARs are artificially synthesized membrane proteins of
an extracellular domain, which are formed by fusing a single-chain variable frag-
ment (scFv), which was constructed from the established mAb, a hinge region, a TM
domain, and an intracellular signaling region with a costimulatory domain
[335, 336]. Reconstructed CAR-based GO scFv (referred to as CART33 in the
original study) was used as a therapeutic candidate for immune cells. CART33
cells can semi-clinically eradicate human AML and myelodysplastic blasts, with
toxicity against myeloid lineage cells in implanted mouse xenografts. CAR-T cells
are versatile in terms of their efficacy because similar to mAb, they too function as
effector T cells. This property renders CAR-T superior to the conventional therapies
of AML that have many limitations [337], thus presenting CAR-T cell therapy as an
emerging therapy for treating many malignant human cancers. Although several
clinical trials with unconjugated or conjugated mAbs with Siglec-3/CD33 have
failed, during CD33-targeted AML treatment, cytotoxic effects on normal myeloid
cells are observed. From the above status, a collaborative application of Siglec-3/
CD33 and CAR-T would be used. CD33-targeted therapeutics are anticipated in the
next few years [338].
7.13.7 Siglec-5/CD170 as a CD33-Related Siglec

7.13.7.1 Paired Siglec-5/Siglec-14 Receptors
Two Siglec forms, Siglec-5 and -14, are classified as coupled receptors in various
functions. They are mainly expressed on monocytes and neutrophils. They are
functionally antagonistic. As paired receptors, the Siglec-5 and -14 forms are nearly
identical in their ligand-binding domains. Siglec-5 bears an ITIM, which inhibits
immune activation. However, Siglec-14 acts in an associated form with activating
adaptor proteins such as DAP12, which bears an activating ITAM, rather than an
inhibitory ITIM, in the cytoplasmic region. In evolution, some human Siglec-14
forms are not functional. The human Siglec-14 and Siglec-5 forms are identical to
Siglec-5 in the coding region but their gene expressions are regulated under a Siglec-
14 promoter. In other words, the two Siglec-5 and Siglec-14 receptors are polymor-
phic. They regulate neutrophil and amnion signaling functions upon pathogenic
interactions with the hosts. Siglec-5 (CD170) is one of the human CD33-related
Siglecs with two ITIMs in their cytoplasmic tails. Structural and binding analysis of
Siglec-5/CD170 has revealed that Siglec-5/CD170 recognizes α2,3-SA and α2,6-SA
linkages through the conserved SA-binding sequence containing the arginine resi-
due. The Arg residue interacts with the SA-COOH group in the conformational
G-strand spatial region, which makes hydrogen bonds with glycerol side chains
attach to SAs [339]. It has an inhibitory signaling through SHP-1/-2 recruitments,
and its clustering induces phagocytosis. It binds to RBCs. Upon SA ligand binding,
ITIMs of Siglec-5 are phosphorylated, which subsequently recruits SHP-1/-2
proteins for its inhibitory signaling, and consequently, inhibits the activation of other
downstream signaling molecules.
Although the function of Siglec-5/CD170 in DCs is not fully understood, the
immunosuppressive activity of Siglec-5/CD170 to date reflects on the control
capacity of immune activation against ‘self.’ Especially, Siglec-5/CD170-mediated
phagocytic functions of DCs upon sialic-acid interaction if DCs do not other ITIMs-
containing Siglecs is related to immune tolerance development against self. Conse-
quently, Siglec-5/CD170 in DCs may be crucial for immune regulation against self/
nonself.
Paired Siglec-5/Siglec-14 receptors each contain highly similar extracellular
domains but with different and opposing immune responses through intracellular
signaling. Therefore, Siglec-14 and -5 are simultaneously controlled for their
coexpression in the same tissues. The paired receptors Siglec-5 and Siglec-14 are
coupled together [108]. Siglec-14 was first identified in humans in 2006 by Takashi
Angata. The Siglec-5 and Siglec-14 genes exhibit polymorphism. Siglec-14 is
mainly present on monocytes and granulocytes, whereas Siglec-5 is mainly present
on granulocytes and expressed only on B cells. Siglec-5 has an ITIM-bearing
domain. The ligand-recognition domains of Siglec-14/-5 are identical. However,
Siglec-14 forms a complex associated with the adaptor protein DAP12 with the
ITAM domain instead of the inhibitory ITIM domain in the cytoplasmic region.
Interestingly, the first two Ig-like domains present in Siglec-14 and -5 of humans are
almost completely identical in their sequences. Therefore, Siglec-14 and -5 are
regarded as the first coupled receptors for carbohydrate recognition in humans.
Counteraction between Siglec-5 and -14 balances cell activation and dampening.
Siglec-14 and -5 are not observed in murine homologues. Certain human individuals
are not expressed for functional Siglec-14 because the two genes encoding for
Siglec-5/-14 are fused, generating a converged Siglec-14/-5 gene. The conversion
of Siglec-5/-14 is identical to that of Siglec-5 in the coding region, but the gene
expression is regulated by the Siglec-14 transcriptional promoter. This indicates a
polymorphism in the human population. Individuals who bear both Siglec-5 and
Singlec-14 lack one or both Siglec-14 alleles since the Siglec-5/-14 gene has been
replaced. A Siglec-14-null polymorphism causes Siglec-14 absence in some
humans, which makes the host neutrophils more susceptible to immune subversion
of the GBS. The expression of Siglec-5 or Siglec-14 in humans is normally observed
in the amniotic epithelium; however, opportunistic Siglec-5/-14 expressions lead to
GBS invasion of the fetus. Therefore, the expression of polymorphic genes of
Siglec-14 or Siglec-5 influences the risks of prematurity in human fetuses of mothers
colonized and infected with certain bacteria such as GBS.
Siglec-5 extracellular domain-binding mAb also binds to the domain of Siglec-14
expressed on the human macrophage cell line, THP-1 cells, which are deficient in the
Siglec-14 form but express the Siglec-5 form [108]. The expression levels of Siglec-
5 and -14 are increased, responding to cigarette smoke (CS) in THP cells [340],
anticipating different immune responses derived from a reverse signaling pathway of
different Siglecs and suppression of phagocytosis. In humans, the genes SIGLEC-5
and SIGLEC-14 are fused and this event causes functional disruption of Siglec-14
through gene deletion [341]. From the obtained results, it can be observed that the
Siglec-5-positive Asian population is suggested to have a relatively higher suscep-
tibility toward certain pathogens that interact with Siglec-5, consequently decreasing
pathogenic and phagocytic levels of the host cells in humans. Pathogenic evasion of
the host in the innate immune system has been explained for GBS, N. meningitidis,
P. aeruginosa, C. jejuni, PRRSV, and HIV [134]. On the contrary, the European
population has been reported to predominantly express Siglec-14, which recognizes
H. influenza strains. Airway pathway infection by bacteria is known to be caused by
H. influenza strains. Hypersialylated NTHi stimulates Siglec-14 expression on
macrophages to express proinflammatory cytokine genes. In contrast, Japanese
patients with airway pathway inflammatory COPD showed a complete loss of
Siglec-14 expression with reduced COPD levels [342]. Therefore, phagocytosis
and inflammation can be modulated by immune-related cells through their bindings
to the “eat-me” signature and the “don’t-eat-me” molecular patterns via different
Siglec species and SA ligands. Similar results are also shown in Sigle-11 and
mSiglec-E, where the polysialyl glycocalyx on neurons suppressed phagocytosis
through inhibitory receptors Siglec-11 and mSiglec-E in human and mouse
microglia, respectively [343]. In contrast, desialylated glycans activate the “eat-
me” signals.
Siglec-5 expressed on DCs potentiates phagocytic capacity. Siglec-5 that is
mainly present on immune cells of neutrophils and monocytes is also found on
two DC subsets of in vitro generated monocyte-derived DCs (mDCs) and
plasmacytoid DCs (pDCs) [344]. In the expression of the SIGLEC-5 gene in
mDCs with IL-4 and GM-CSF treatments, mDCs exhibit high immunogenic prop-
erties upon TLR stimulation such as TLR-4 agonist LPS and TLR-9 agonist CPG,
through inflammatory cytokine secretion, and express TLR-1 to TLR-8 but not
TLR-9 and TLR-10. SIGLEC-5 strongly binds to various TLR proteins and hence
negatively regulates their functions. Therefore, TLR-rich DCs have protective
immunomodulatory functions against ‘self’ through SIGLEC-5. mDCs express
SIGLEC-5 with unchanged patterns after LPS stimulation, whereas SIGLEC-7 and
-9 expressions are significantly decreased. Therefore, SIGLEC-5 acts as a final break
to protect ‘self’ by suppressing uncontrolled inflammation. mo-DCs express Siglec-
5 and other Siglecs with ITIMs. The expression of Siglec-5/CD170 on mo-DCs is
persistent even in the presence or absence of LPSs, whereas those of Siglec-7 and -9
are decreased. Thus, Siglec-5 seems to modulate a proinflammatory reaction of
mo-DCs, protecting the ‘self.’ A persistent and prolonged chronic inflammatory
reaction is often related to autoimmune diseases. As a coupled receptor of Siglec-5,
Siglec-14 has atypical ITAMs and enhances TLR-controlled NF-κB phosphoryla-
tion and transcriptional activation, while Siglec-5 itself inhibits NF-κB signaling
[108, 345]. Therefore, Siglec-5 equilibrates the functional status of mo-DCs during
binding to self-sialic acids and this indicates Siglec-5’s protective capacity of the
host organism. Apart from immune cells, Siglec-5 is reported to bind to sialic acids
on red blood cells, the most common cells in the human body [346]. On the other
hand, plasmacytoid DCs (pDCs) mainly express type I interferons upon virus
infection. pDCs express TLR-7, -9, and -10, whereas mDCs express only
SIGLEC-5 but not others with ITIMs. SIGLEC-5 prevents excessive interferon
signaling. pDC-expressed SIGLEC-5 contributes to phagocytosis of self-derived
antigens and central tolerance, where pDCs deliver self-derived antigens in the
periphery to the thymus. SIGLEC-5 in pDCs contributes to the reduction of self-
reactive T cells through a clonal deletion process and also induces natural Tregs in
the thymus. Then, pDCs are regulators of central tolerance.
7.13.7.2 Role of Siglec-5/CD170 in DCs and Modulation of Immune

Responses
Among DC subsets, mo-DCs and pDCs express Siglec-5. pDCs express Siglec-5/
CD170, and Siglec-5 is rapidly internalized in adherent monocytes following
antibody-mediated clustering. During steady states, pDCs take up and deliver
peripheral self-antigens to the thymus, thus contributing to clonal deletion of T
cells, which are reactive to self-antigens [347]. pDCs as unusual DCs is a major
producer of type I IFNs during an early stage of infection of viruses. pDCs express
only Siglec-5/CD170, but not other Siglecs with an ITIM, and it is, therefore,
considered that Siglec-5/CD170 possibly prevents excessive interferon signaling
and regulates activation in pDCs. Another function of pDCs is the control of ‘central
tolerance.’ pDCs also induce natural regulatory T cells in the thymus, which induces
immune tolerance in an antigen-specific manner [348]. Siglec-5/CD170 on pDCs not
only acts as inhibitory molecules but also facilitates endocytosis of antigens through
ligand binding-mediated clustering of Siglec-5/CD170 [344]. As pDCs induce
immune tolerance and Siglec-5/CD170 has a role in endocytosis, Siglec-5/CD170
facilitates central tolerance through endocytosis of sialic acid-carrying self-antigens.
Although SAs are the ligands for Siglec-5/CD170, exogenous Hsp70 secreted
from necrotic cell deaths also bind to Siglec-5/CD170 and Siglec-14 [345]. In this
process of Hsp70 recognition by Siglec-14, Siglec-14 recognizes Hsp70 as a DAMP,
inducing inflammation. Siglecs of Siglec-1 (Sd) and DAP12-associated Siglec-14
can bind to pathogenic SAs and induce host defense responses. Siglec-5 and Siglec-
14 are designed to counteract each other to balance the dampening and cellular
stimulation. Therefore, Hsp70 is a SA-independent Siglec-5 ligand. Extracellular
Hsp70 is a potential DAMP when it binds human monocytes and activates extracel-
lular release of inflammatory TNF-α. Elevation of the Hsp70 level in
bronchoalveolar lavages is associated with lung inflammation. In extracellular fluids,
Hsp70 functions as a DAMP component when it interacts with monocytes and
releases inflammatory TNF-α cytokines in humans. However, the functional role
of Hsp70 is not well understood in immune responses, inflammatory process, and
cytokine production. Hsp70 level is elevated in bronchoalveolar lavages with lung
inflammation. Hsp70 colocalizes with Siglec-5 and Siglec-14 as ligands and directly
interacts with Siglec-5/-14. At the molecular level, Hsp70 binds to the V-set domain
regions in Siglec-5/-14. Then, the bound Hsp70 complex suppresses inflammatory
responses via Siglec-5, as confirmed in macrophage THP-1 cells. Hsp70 modulates
human monocytes, depending on SIGLEC-14. The Hsp70 and Siglec-5/-14 ligands
Hsp70
5+).'%
Sialic acid binding site Siglec-14 SA-Independent Siglec-5
1 mlpllllpll wggslqekpv yelqvqksvt vqeglcvlvp csfsypwrsw ysspplyvyw
61 frdgeipyya evvatnnpdr rvkpetqgrf rllgdvqkkn cslsigdarm edtgsyffrv
121 ergrdvkysy qqnklnlevt aliekpdihf leplesgrpt rlscslpgsc eagppltfsw
181 tgnalspldp ettrsseltl tprpedhgtn ltcqmkrqga qvttertvql nvsyapqtit DAP12 TLR extracellular
241 ifrngialei lqntsylpvl egqalrllcd apsnppahls wfqgspalna tpisntgile
301 lrrvrsaeeg gftcraqhpl gflqiflnls vyslpqllgp scsweaeglh crcsfrarpa
361 pslcwrleek plegnssqgs fkvnsssagp wansslilhg glssdlkvsc kawniygsqs
cytosol
421 gsvlllqgrs nlgtgvvpaa lggagvmall ciclcliffl ivkarrkqaa grpekmdded
481 pimgtitsgs rkkpwpdspg dqasppgdap pleeqkelhy aslsfsemks repkdqeaps
ITAM ITIM
541 tteyseikts k
Inhibion
Acvaon
Monocyte
INFLAMMATION
Fig. 7.24 Siglec-5 sequence as well as Hsp70 and Siglec-5/Siglec-14 interaction. Hsp70 binds to
the V-set domain regions of Siglec-5/-14 and inhibits inflammation through Siglec-5 in immune
cells
are colocalized in lung bronchioles [345]. Hsp70 and Siglec-5/Siglec-14 interact

directly. Hsp70 recognizes the Siglc-5/-15 V-set domain regions, inhibiting inflam-
matory responses via Siglec-5 present on THP-1 cells. However, Siglec-5/CD170
considers Hsp70 as a SAMP, inhibiting inflammation. Therefore, Siglec-5/CD170
on DCs contributes to self-antigen recognition and subsequent immune inhibition.
Hsp70 as an endogenously produced ligand responsible for Siglec-5/-14 differen-
tially controls the responses of innate immune cells. This answers how and why
human Hsp70 expressed in the extracellular region recognizes the immune receptors
of Siglec-5/-14 in humans (Fig. 7.24).
The Arg residue mutation in Siglec-5 and Siglec-14 results in an extremely weak
binding capacity between Hsp70 and Siglecs. Double mutations of the Arg residue
(119) and the Lys residue (134) in the V-set domains both in Siglec-5 and -14
abrogate the interaction with Hsp70. Hsp70 directly recognizes the V-set domains in
Siglec-5/-14. When the Hsp70 protein binds to Siglec-5/-14, the Siglec-5–Hsp70
interaction inhibits immune responses. However, Hsp70 activates immune responses
when it binds to Siglec-14. In human monocytes, there is a polymorphism in the
Siglec-14 allele. The TNF-α secretion level is increased by Hsp70 treatment but not
DnaK, known as an E. coli Hsp70 homologue, in Siglec-14 +/+ primary monocytes.
TNF-α secretion is increased gradually according to the number of Siglec-14-
positive alleles by Hsp70 treatment. Thus, Hsp70 binds to Siglec-5/-14. When
Hsp70 binds to Siglec-5, immune responses are inhibited. However, Hsp70 activates
the immune responses through Siglec-14.
Pathogens such as bacteria express little or no sialic acids on their surfaces;
therefore, human sialic acid-recognizing lectin SIGLECs on immune cells are
responsible for distinguishing self from nonself. Many bacteria living in association
with animals as hosts use sialic acid in their lifestyle biology. Some bacteria
incorporate sialic acids into cell surfaces in the form of LPSs and capsules, which
help them evade the innate immunity of the host, thus protecting them [349]. Sialic
acids can be used to evade the complement by hiding mannose antigens on the host
cell surfaces or bacteria from MBL. Therefore, a tight barrier protects against
microbial pathogens in bacterial infection. Two general routes of infection are
considered by the transcellular type in intestinal bacteria including Salmonella,
Shigella, Listeria, and Yersinia, which invade nonphagocytic epithelial cells by
subverting host cytoskeleton dynamics. Another paracellular type is observed in
certain bacteria including V. cholerae, C. jejuni, and P. aeruginosa, which perturb
epithelial integrity to facilitate translocation by disrupting the tight junctions of cells.
Human pathogens such as GBS, H. influenzae, N. gonorrhoeae, and E. coli K1
incorporate SAs into the ends of glycan chains by scavenging or synthesis. One of
the examples is a GBS β-protein. When the GBS β-protein associates with Siglec-5,
immune responses are inhibited. However, it activates immune responses through
Siglec-14. Ligands can engage the V-set domain of Siglecs through two methods.
One method is by recognizing SAs (SA-dependent), and the other is by protein–
protein interaction (SA-independent). The SA-independent ligand is Siglec-5. The
SA-independent ligand is associated with Siglec-5 in multiple tissues, where Hsp70
acts as a Siglec-5 ligand. As Hsp70 is a potential DAMP, it directly colocalizes with
Siglec-5/-14. The GBS-produced β-protein recognizes Siglec-5 and -14 expressed
on neutrophils, making their involvement counteract pathogenic invader-causing
immune suppression by p38 and AKT signaling in the hosts.
Pathogens have evolved by applying immune escape mechanisms utilizing
Siglec-5/CD170. The Siglec-5/-14 forms, which are polymorphic and paired recep-
tors with coupling, regulate functions of neutrophils during GBS colonization
[108]. If the GBS-produced β-protein recognizes Siglec-5, the rate of bacterial
survival and evasion is increased. However, if the GBS-produced β-protein recog-
nizes both Siglec-5 and -14, the pathogen death level is increased. Siglec-14 affects
the host phagocytic activity against GBS, which produces the Siglec-5-recognizing
β-protein on GBS surfaces. Gene loci of Siglec-14 receptors in blood macrophages
of humans are known. Siglec-14 expressed on THP-1 monocytes increases immune
responses against LPS and GBS. The Siglec-5 and -14 genotypes affect the mono-
cytic cell responses of humans to endotoxin LPS and pathogen GBS. Then, the
mechanism underlying the polymorphism of human Siglec-14 and -5 genes affects
the monocytic immune responses to pathogenic bacteria in humans and is explained
by the fact that Siglec-14 induces inflammatory immune responses to the pathogen
GBS and bacterial endotoxins such as LPS through the AKT/p38 MAPK axis
pathway. Therefore, the Siglec-5-borne opposing power is considered, as the ITIM
region recruits the Tyr phosphatases, SHP-1 and -2. The Siglec-5/Siglec-14 geno-
types also affect responses of neutrophils to the GBS infection in humans. The TLR
priming process also increases the suppressed neutrophil response by GBS in the
Siglec-14/ genotype but not in the Siglec-14+/+ genotype. The β-protein-produc-
ing GBS recognizes Siglec-5 and -14 present on immune cells such as neutrophils.
p38 MAPK and AKT signaling in late immune suppression inhibits GBS-induced
host immune responses. In individuals with only Siglec-5- or Siglec-14-null neutro-
phils, where Siglec-14 does not function, GBS immune subversion is more suscep-
tible. Siglec-5/-14 expressions in human amniotic epithelial cells are considered to
be the sites of initial contact for invading the fetus with GBS; this is also known as
Siglec-5/-14 polymorphism and influences the prematurity risk of human fetuses of

GBS-carrying mothers. For instance, Neisseria meningitidis expresses distinct
sialylated LPSs to bind to SIGLEC-5, eventually escaping from immune surveil-
lance through ITIM-mediated immune suppression against the bacteria. Moreover,
surface β-proteins of GBS strains bind to Siglec-5/CD170 in a Sia-independent mode
to inhibit phagocytosis [108, 350]. α2,3- and/or α2,6-linked SAs in other pathogens
also serve as defense tools in Siglec-5/CD170-mediated immune escape. These
pathogenic bacterial sialyl residues easily disrupt the host’s self and nonself
distinction.
Siglec-9 has also been targeted for the role of sialic acid in evading the human
immune system during hypervirulent Klebsiella pneumoniae (hvKP) infection. The
K. pneumoniae capsule impedes adhesion and invasion to epithelial cells
[351]. Treatment of neuraminidase enzymatically cleaves the α2,3/α2,6 sialic acid
chain on the outmost capsular polysaccharide (CPS) and reduces the mucoviscosity
of hvKP and increases the activity of human Siglec-9. It is obvious that
hypermucoviscosity is dependent on sialic acid glycan decoration and CPSs with
the immune evasion of hvKP. Further insights into the glycobiology of antibiotic
resistance are needed, where sialic acid is the important component of immune
escape of hypervirulent K. pneumoniae. The K. pneumoniae hypervirulent mucoid
phenotype is based on sialic acid [352]. Future studies are required to investigate the
structure of CPSs, especially their chemical bonding of sialic acid.
Some Siglecs can directly interact with TLRs. Similar to Siglec-9, Siglec-5 also
binds to various TLR family proteins, which negatively regulate their functions
[353]. DCs also express many TLRs. Mo-DCs express all TLR families, except for
TLR-9 and TLR-10, whereas pDCs express TLR-7, -9, and -10. Thus, Siglec-5 is
suggested to have a protective immunomodulatory function against ‘self.’
7.13.8 Siglec-6 as a CD33-Related Siglec
Siglec-6 belongs to the CD33-related Siglec family as a TM receptor. It binds leptin

and SA glycans. Siglec-6 binds to the sialyl-Tn antigen and leptin even in conditions
where there is no leptin modification with sTn [354]. Siglec-6 expression is present
on immune cells in humans and great apes. However, Siglec-6 expression is seen
only on trophoblast cells of the human placenta, and not on the ape placentae, thus
indicating only human specificity. This may be caused by the recognition site present
in the Siglec-6 transcription region for human-specific transcription factors. Natural
Siglec-6 ligands are expressed in the human placenta, human uterine endometrium,
and trophoblastic or endometrial cells. Placental Siglec-6 interacts with sialyl ligands
for negative signaling. Siglec-6 is lowly expressed on the placentae, and negative
signaling of Siglec-6 indicates the slow temporal process in human births. Siglec-6
binding to leptin also reflects this hypothesis. However, little is known about Siglec-
6 binding to leptin. Interestingly, human hydatidiform mole patients exhibit high
serum levels of leptin when compared to normal pregnancies. Leptin has thus been
suggested to localize on trophoblasts in placentas. Siglec-6 is not homologous with

the leptin receptor LepR. Leptin as an obesity hormone is largely expressed in
trophoblastic diseases of gestation. Placental formation is the complex between the
maternal decidua and the fetal trophoblasts. In the placentation process,
cytotrophoblasts are differentiated into villous cytotrophoblastic cells and
extravillous cytotrophoblastic cells. The fetal trophoblasts invade the decidualized
endometrial region, allowing placentation with the formation of chorionic villi and
anchoring villi. Among gestational diseases, gestational trophoblastic neoplasias
exhibit aggressive invasion. Siglec-6 has been suggested to accelerate the invasive-
ness in a manner such that Siglec-6 and leptin stimulate growth and progression.
Only human placentas are known to express Siglec-6 among eutherian mammals,
but its function is currently unknown. Siglec-6 binds sialyl-Tn glycans and leptin
[354]. Leptin is a placenta- and adipocyte-derived peptide hormone. The gene is
expressed only in primates with a tissue-specific expression pattern in B cells. In
humans, the human placenta expresses the Siglec-6 gene due to its promoter
sequence [355] and soluble Siglec-6 is found in maternal blood [354, 356]. One
Siglec-6 mRNA variant produced by alternative splicing directs the soluble variant.
Siglec-6 protein in the placenta has been detected in trophoblasts through patholog-
ical processes with the Siglec-6 ligand, leptin [357]. Siglec-6 protein expression is
seen in the placentas of viable human pregnancies during gestation, disease state,
and biological chorionic villi. Siglec-6 is related to the defects of trophoblast
differentiation, and overexpression is caused by the placental environment of the
pathological process [358].
In a healthy placenta, the expression level of Siglec-6 is decreased during the
early gestation period for 8 weeks. In gestational trophoblast disease, Siglec-6
expression is limited. Leptin treatment induces invasion of Siglec-6-deficient cells,
due to binding to the leptin receptor. Siglec-6 induces proliferation in a leptin-
dependent manner, preventing apoptotic cell death, but it stimulates a leptin-
independent invasion of cells. Leptin and Siglec-6 are involved in gestational
trophoblast disease [359]. Siglec-6 is overexpressed in two disorders, namely,
placentation of choriocarcinoma [360] and preeclampsia [361]. The overexpression
of Siglec-6 mRNA in choriocarcinoma is not yet confirmed, although choriocarci-
noma and preeclampsia are the representative human placental diseases limited to
pregnancy. The SIGLEC-6 gene near 19q13.4 is close to the gene loci of familial
hydatidiform moles [359]. Leptin and Siglec-6 levels indicate the progression levels
of trophoblast diseases, as Siglec-6 is predicted to be expressed and its expression in
the normal placenta and hydropic abortus is ignored. Using BeWO choriocarcinoma
cells, Siglec-6 expression has been confirmed to be increased. The leptin and Siglec-
6 expressions enhance the growth and invasion levels of BeWO cells [359].
A secretory glycodelin is a uterine glycoprotein that functions for feto-maternal
defense [362, 363]. It is secreted in the endometrial glands and the decidua but not in
the trophoblasts. Glycodelin is a paracrine regulator of trophoblast invasion,
suppressing the invasion of extravillous cytotrophoblast cells via suppression of
MMP-2/-9 expression and uPA activation [364]. Glycodelin-mediated MMP sup-
pression indicates that glycodelin suppresses trophoblast invasion by the suppressive
expression of MMP2 and uPA [364] via inhibition of ERK phosphorylation in

trophoblast cells. Glycodelin contains sialylglycans [365]. Glycodelin sialic acid
residues contribute to suppression of trophoblast invasion. Glycodelin sialic acids
suppress the invasion of trophoblast cells. Glycodelin, but not the nonsialylated
glycodelin-S, possesses immunosuppressive activities on lymphocytes. The activity
is abolished by desialylation [366]. Other sugar residues in glycodelin also have
other biological activities. For example, glycodelin-F, but not glycodelin, has the
suppression activity of progesterone-induced acrosome reaction in spermatozoa
[367, 368]. Sialic acid-binding selectins and Siglecs are expressed on trophoblasts
[369]. Siglec-6 serves as a glycodelin-binding protein on human trophoblasts. The
Siglec-6 ligand, leptin, significantly reduces the binding of GdA to cells. Siglec
function is not known in trophoblasts. Most Siglecs are found in the immune and
hematopoietic systems. Siglec-6 is the only Siglec family member present on
trophoblasts. The ligands for Siglec-6 are glycoproteins but not glycolipids. The
preferred ligand for Siglec-6 is terminal Neu5Ac-α2,6-GalNAc [354], present in the
glycan terminals of GdA [366]. Leptin reduces the binding of GdA to trophoblasts.
Leptin binds more strongly to its own receptor than Siglec-6. Trophoblastic Siglec-6
increases the binding of GdA to trophoblast cells. GdA–Siglec-6 interaction could
cause a characteristic shallow invasion in preeclamptic patients [370]. Siglec-6
synthesis is observed only in humans, not in nonhuman primates. Preeclampsia
disease is found only in humans [371].
7.13.9 Siglec-7 (CD328) as a CD33-Related Siglec
7.13.9.1 Basic Properties of Siglec-7 in DCs
As a Siglec-3 (CD33)-related protein, Siglec-7 (CD328) is one of the CTLRs. There

are many myeloid cells of DCs, monocytes, macrophages, mast cells, NK cells, etc.
It is reported that Siglec-7 expression is found on NK cells, monocytes, mast cells,
basophils, platelets, and DCs [372]. However, Siglec-7 present on NK cells is mainly
studied because most researches of Siglec-7 are conducted on NK cells, although
Siglec-7 exists in a minor population of monocytes, DCs, and CD8 T cells
[373]. Recently, Siglec-7 expression has also been detected on mucosal macro-
phages [374]. In case of DCs, Siglec-7 expression is found on DC marker CD11c
+-positive cells of the lamina propria, tonsils, and blood [375]. In addition, when a
Siglec-7-targeting liposome is treated with CD1d+-positive and monocyte-derived
DCs (mo-DCs), the cells bind to the liposome [376]. It suppresses NK cell activa-
tion, controlling apoptosis, and also mast-cell and basophil activation. It also acti-
vates monocyte-mediated inflammation and increases production of cytokines and
chemokines.
Carbohydrate recognition of Siglec-7 in innate immune cells is known. Usually, it
was reported that many Siglec molecules recognize α2,3-SA or α2,6-SA in various
sialylglycan structures of antigens. Siglec-7 has a unique glycan structural
Fig. 7.25 Siglec 7 (CD328) and membrane topology. Siglec-7 as a CD33-related CTLR has the
carbohydrate recognition domain and inhibitor motif. Siglec-7 as a type I TM protein has a V-set
Ig-like domain in the N-terminal region, multiple numbers 1–16 C2-set Ig-like domains, a TM
domain, and a tail in the cytoplasmic region. It recognizes glycans in NK cells and monocytes.
Arg124 residue is a SA binding site. Siglec-7 recognizes disialic α2,3-SA and α2,8-SA linkages
rather than α2,3-SA or α2,6-SA linkage. Siglec-7 is an inhibitory receptor with an ITIM and
SHP-1/-2. Siglec-7 blocking activates NK cells and increases proinflammatory cytokine expression
in monocytes. SA analogues target Siglec-7 on DCs. DC Siglec-7 activation activates IFN-γ and T
cells
recognition such as α2,8 sialic acid. Transfer of α2,8-SA can be achieved by various
types of α2,8 sialyltransferases and different substrates. Although Siglec-7 also
recognizes α2,3-SA and α2,6-SA, α2,8 disialic acid is well recognized and bound
by Siglec-7 [372]. To perform inhibitory signaling, Siglec-7 comprises four distinct
regions, including the V-set and the C2-set Ig domains as well as an ITIM as an
inhibitory motif and an ITIM-like putative signaling motif (Fig. 7.25). The
β-sandwich feature of the Siglec-7 structure is formed by the disulfide bond of
Cys46 and Cys106. The Arg124 residue of Siglec-7 is a primary binding site to
sialic acid [377]. In fact, mutation of Arg124 to Lys124 shows impairment of
binding to the sialylated ligand [378]. Therefore, it is considered that these structures
might form a binding site of sialylated glycan structures, but it is not known why
α2,8-SA is preferred over α2,3-SA and α2,6-SA.
In C. jejuni infection, where C. jejuni is a representative and common cause of
food-borne poisoning and gastroenteritis, sialylated lipooligosaccharides (LOSs) can
be expressed on C. jejuni. The C. jejuni strains produce monosialyl and disialyl
LOSs with SAα2,3 and SAα2,3/SAα2,8 residues, respectively. Although
nonsialylated LOSs cannot bind to any Siglec molecule, sialylated LOSs show a
specific binding affinity to Siglec-7. Especially, α2,8-sialylated LOS has a higher
affinity to Siglec-7 than α2,3-sialylated LOS. In addition, a mixture of α2,8- and
α2,3-sialylated LOSs shows synergetic binding [66]. In DCs, a sialic acid analogue-
containing liposome is recognized by Siglec-7 [376]. As the analogue seems to be
similar to α2,8-SA linkage, Siglec-7 can recognize and is bound to the analogue.
Siglec-7 specifically binds to C. jejuni [206] (Fig. 7.26). Siglec-7 binds to C. jejuni,
which generates autoimmune diseases known as oculomotor weakness syndrome in
GBS and MFS. In autoimmune diseases, bacterial infection with C. jejuni causes
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Fig. 7.26 Siglec-7 and α2,3/α2,6 sialyl mimics in MFS and GBS. Disialylated C. jejuni GQ1b
mimic sugar (GD1c/GD3)-reactive antibodies are observed in MFS patients and antibodies are
directed against GQ1b. Moreover, monosialylated C. jejuni and stool and serum samples of patients
with GBS raises antibodies against monosialylated GM1a and GM1b and disialyl GD1a and
GalNAc-GD1a. MFS is a restricted GBS variant with paralysis in the eye muscles like oculomotor
weakness
GBS, an immune disorder that emerges postinfectiously and causes defects in the
peripheral nerve roots and the CNS. GBS is expressed through antibody-driven
autoimmune responses, eventually causing disorders mainly in the peripheral nerves.
However, the initiation of the disease mainly arises through gastrointestinal tract
infection (GIT). The characteristic progression of GBS is in its rapid propagation of
acute ascending paralysis and sequentially systemic paralysis symptoms, requiring
artificial respiration. GBS is as an autoimmune disease, which shows inflammation
in motor neurons; the inflammation starts from the limbs and progresses to the
central body. The cause of GBS is not understood; however, it is known that some
people show symptoms of GBS with antibodies of their own neural gangliosides.
C. jejuni LOSs mimic human neural gangliosides. Presumably, the autoimmune
effects between antibodies produced from bacterial infection and host neural gan-
gliosides can be the cause of GBS symptoms. In a recent paper [322], it has been
found that C. jejuni LOSs bind to Siglecs expressed on the membranes of DCs and
that the sialylation pattern is important for the binding. The GQ1b-like sequences
expressed on the C. jejuni surface are recognized by Siglec-7, allowing the speci-
ficity of the epitope by Siglec-7 and connecting to GBS and MFS. Due to molecular
similarity, C. jejuni LOSs induce neuronal ganglioside-reactive antibodies. This
contributes to GBS. Monosialyl glycan-bearing C. jejuni strains can be easily
found in GBS patient stool samples. GBS is an antibody-caused peripheral nerve
disease that is an autoimmune disease, eventually leading to paralysis. A variant of
GBS, MFS, is also similarly featured with eye muscle paralysis, oculomotor weak-
ness, and loss of tendon reflexes [379]. Binding of disialylated ganglioside mimics to
Siglec-7 connects with the symptoms in GBS and MFS cases.
7.13.9.2 Siglec-7 Recognition of Pathogenic C. jejuni Sialyl LOSs

and Ganglioside Mimics
LOSs mimic gangliosides, which affects the immunogenicity and the type of
neurological defects in GBS patients. In the case of MFS as a variant of GBS,
MFS gets affected by antibodies to the ganglioside GQ1b. C. jejuni LOSs are
sialylated to form GD1a/GM1a. GD1a/GM1a mimics have α2,3-SAs and GD1c
mimics have α2,8-SAs. Antibodies raised against monosialylated gangliosides such
as GalNAc-GD1a, GM1a, GM1b, and GD1a are often observed in the sera of GBS
patients [380, 381]. However, pathogenic C. jejuni strains decorated with disialyl
LOSs, which resemble the GQ1b carbohydrate structure, such as disialyl GD3 and
GD1c, also exhibit symptoms similar to those of MFS patients. MFS patients
frequently possess GQ1b-reactive antibodies in their sera [382, 383]. Oculomotor
nerves are normally innervated to the eye muscles in humans, and, therefore, GQ1b-
specific antibodies influence MFS progression, probably due to the high contents of
GQ1b in the muscles. This fact may indicate possible damage caused by anti-GQ1b
antibodies [382]. Ganglioside GQ1b-binding antibodies are directly linked to ocu-
lomotor weakness onset in GBS patients and its similar disease, MFS. GQ1b-like
epitopes produced by C. jejuni are recognized by Siglec-7. The C. jejuni-binding
specificity to Siglec-7 indicates that Siglec-7 exclusively recognizes the C. jejuni
ganglioside mimics, which are terminally sialylated. Siglec-7 binding is linked to
GQ1b-reactive antibodies in the patient sera. Siglec-7 as a Siglec family receptor is
present on DCs; its binding to C. jejuni needs SAs, as ST-deficient strains cannot
recognize Siglec-7, whereas wild-type strains can recognize Siglec-7. C. jejuni sialyl
LOS is an essential factor for GBS and MFS progression [384]. Therefore, Siglecs
present on immune cells may have an essential role in recognition. Siglecs, therefore,
are involved in SA-dependent cell–cell recognition and cell–ligand recognition
[23]. Furthermore, Siglecs act as endocytosis receptors during bacterial and viral
recognition [65, 66]. Siglec-7 prefers to bind disialyl SA conjugates like GQ1b
[385]. In fact, GD1c- or GD3-like disialyl gangliosides are produced in C. jejuni.
Like GQ1b, GD1c and GD3 are disialyl-Gal-containing glycans. GD1c- or
GD3-producing C. jejuni infection elicits the production of GQ1b-specific anti-
bodies in MFS or GBS patients [383, 386, 387]. Siglec-7 binds to certain strains
of C. jejuni infection in MFS or GBS patients. Thus, Siglec-7 interaction with
C. jejuni is a confirmative signature for MFS or GBS patients with eye oculomotor
defects such as weakness.
LOS-specific binding to Siglec-7 may be the commencement of immune recog-
nition for production of GQ1b-specific antibodies and for oculomotor defects in
MFS and GBS patients. Siglec-7 recognizes terminal disialyl residues in ganglio-
sides of GD3, GT1b, and GQ1b [387, 388]. Siglec-7 binding to disialyl-LOS surface
on C. jejuni strains was previously evidenced [65]. The consequential outcome of the
interaction between C. jejuni and Siglec-7 is poorly understood, regardless of the
fact that Siglec-7 belongs to CD33-related Siglecs with cytoplasmic tail ITIMs.
However, the pathogenic existence of the ITIM indicates that Siglec-7 may elicit
an inhibitory immune response by ITIM signaling and Siglec internalization via
ITIM phosphorylation [389]. If the reported fact of Siglec-related pathogen uptake is
considered [65], it is speculated that Siglec-7 cis-binding to self-ligands contributes
to a suppressed cellular response, while pathogenic recognition to Siglec-7 reverses
the inhibitory signaling through coreceptor activation and cytokine
production [390].
C. jejuni LOSs are routinely sialylated to form GD1a/GM1a. GD1a/GM1a
mimics have α2,3-linked SAs and GD1c mimics have α2,8-linked SAs. Cst-II is
responsible for C. jejuni LOS synthesis. Deficiency of the SA species linked to LOS
by mutation of the ST enzyme cst-II displays lowered DC activation levels and
B-cell responses induced by DCs. Therefore, the SA linkage could be an essential
cause of induction of GBS. Although it has been demonstrated that α2,3-linked SAs
bind to Siglec 1 and α2,8-linked SAs bind to Siglec 7, how the human body responds
to each sialic acid has not yet been uncovered. C. jejuni LOSs, which have an α2,3
linkage, strongly bind to Siglec-1(CD169), and C. jejuni LOSs, which have an α2,8
linkage, strongly bind to Siglec-7 (CD328). The specific binding causes different
effects on activation of DCs. DCs treated with α2,3 linkage LOS produce OX40L,
which is a ligand for naive T-cell receptors and is the marker of T helper 2 cell
differentiation. However, DCs treated with α2,8 linkage LOS produce IL-12, which
is also a ligand for naive TCRs and it is the marker of T helper 1 cell differentiation.
Furthermore, coculture of DCs and naive T cells treated with each α2,3 and α2,8
linkage LOS shows specific differentiation of T helper cells. α2,3 linkage LOS
treatment results in T cells that produce IL-4, which is a marker of T helper 2 cell
activation, and α2,8 linkage LOS treatment results in T cells that produce IFN-γ, a
biomarker of T helper 1 cell activation. As a Siglec-7-elicited immune response,
Siglec-7 bindings increase the phenotypic shift of DCs to Th1 polarization, via
LOS-involved OX40 ligand induction [322].
During host immune responses to C. jejuni LOSs, with different linkages, it has
been found that T-cell differentiation is followed according to different sialic acid
linkages. Cst-II responsible for C. jejuni LOS SAs is a sialyltransferase. α2,3-SAs
bind to Siglec 1, whereas α2,8-SAs bind to Sn and Siglec 7. The immune response to
C. jejuni LOSs with different linkages includes T-cell differentiation. When GB11
strains with α2,3-SAs are treated with DCs, GB11 cells bind to Siglec 1 and
unknown receptors. When GB11 strains with α2,3-SAs are treated with DCs,
GB11 cells bind to Siglec 1. GB11 induces DCs to express OX40L, leading to
Th2 differentiation. When GB16 with α2,8-SAs are treated with DCs, they bind to
Siglec 7. GB16 induces DCs to express IL-12, and naive T cells differentiate into the
Th1 type. Therefore, the events of specific Th differentiation depend on SA linkages
of C. jejuni LOSs through distinct DCs Siglecs. Th1 responses were viewed when
Siglec-7 and α2,8-sialylated LOS were targeted, whereas Th2 responses were
viewed when Siglec-7 and α2,3-linked sialic acid were targeted. Different Th
responses were initiated according to the sialylation of C. jejuni LOS components by

Siglecs expressed on DCs [322]. Therefore, C. jejuni LOSs that are deficient in a SA
linkage modulate DC-induced polarization of T cells.
Given that in GBS, the similarity between LOS glycan structures produced in
C. jejuni strains and human neural gangliosides causes pathogenesis, GBS patients
express specific immunogenic and neurological defects. However, the connection
between LOS and Siglecs is still poorly understood, although α2,3-SA-GM1a/GD1a
mimics and α2,8-SA-GD1c mimics of the LOS glycan structures are considered to
bind to Sn and Siglec-7, respectively. Targeting of Siglec-7 and α2,8-sialyl LOS
induces Th1 responses, whereas targeting of Siglec-7 and α2,3-sialyl glycans
induces Th2 responses [377], thus producing a different Th response by the sialyl
residues of C. jejuni LOSs and Siglecs on DCs (Fig. 7.27). However, immune
responses of ganglioside-reactive antibodies in GBS or MFS with oculomotor
weakness remain unresolved. Still, no direct evidences have been obtained in
C. jejuni-caused infection and GBS progression. However, C. jejuni LOSs, which
mimic human neural gangliosides, can modulate DC-mediated T-cell differentiation
and are dependent on sialic acid linkage. This finding suggests a new insight into
immunology because it shows that carbohydrates can have major roles in immune
responses rather than protein, which is known as a major component of immune
responses.
Siglec-7 has a unique preference for α2,8-SA-containing glycans. α2,8-SA addi-
tion is catalyzed by α2,8 sialyltransferases, which are referred to as ST8Sia and are
divided into six types [372]. α2,3-SA or α2,6-SA transfer is catalyzed by α2,3/α2,6
sialyltransferases for recognition of other Siglecs, and ST8Sia is a different
sialyltransferase. ST8Sia-I and -V use glycolipid substrates and have a role in
GD3/GT3 and GT1a/GQ1b/GT3 syntheses and ST8Sia-II, -III, and -IV attach the
SA chain to glycoproteins [391]. ST8Sia-VI is related to O-linked glycans and is
involved in the transfer a single α2,8 sialic acid to Neu5Ac-α2,3-GalβNAc, which is
a trisaccharide [206]. In another study, it was found that ST8Sia-IV, -V, and -VI
expressed on NK cells are associated with catalysis of α2,8 di-SA transfer on the
specific sites of glycoproteins and glycolipids [372]. The transferred α2,8-SA
residue is bound by Siglec-7.
DCs, compared to other immune cells, show opposing responses to Siglec-7
recognition. Siglec-7 on DCs actually increases DC-mediated T-cell responses. In
contrast to the existence of an ITIM, it is not known whether Siglec-7 recognition
promotes DC functions, but not all Siglecs contain inhibitory signaling motifs.
Siglec-7 on various cells, except NK cells, is currently unclear. Thus, more com-
prehensive research studies on various immune cells are required. Then, the exact
information will provide answers regarding the characteristics and opposing func-
tions of Siglec-7 in different immune cells. In monocytes, treatment of Siglec-7 with
a neutralizing anti-Siglec-7 antibody increases the expression levels of IL-6/IL-8/IL-
1β/TNF-α, which are proinflammatory cytokines; phosphorylation of ERK1/2 for
cell activation is also increased [392]. Therefore, Siglec-7 is an inhibitory receptor
due to changes in the ITIM and SHP-1 axis after sialic acid chain recognition. The
role of Siglec-7 expression in DCs is to activate Siglec-7. For example, when
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Fig. 7.27 Schematic illustration of the bacterial and mutant glycan structures in GBS patients. (a)
Campylobacter jejuni LOS modulation of DC-driven T-cell polarization in a SA linkage-specific
manner. (b) Carbohydrate mimicry. (c) Summary of GB11 and GB2 effects on T-cell differentiation
via DC regulation. GB11 and GB2 carry SAα2,3 linkages and GD1a mimics. Other strains of GB16
and GB19 carry SAα2,8 linkages and GD1c mimics (adopted and modified from Ref. [322])
mo-DCs are treated with Siglec-7-targeted liposomes, the liposomes are delivered
into the cytoplasm and DC-mediated CD1b-restricted T cells are induced via
increased expression of IFN-γ. In addition, anti-Siglec-7 treatment decreases
IFN-γ expression in DC-mediated T-cell activation [376]. Thus, Siglec-7 recognition
in DCs activates DCs. However, the opposing function of Siglec-7 between DCs and
other immune cells (NK cells, monocyte, etc.) is still unclear in terms of its causative
factor.
7.13.9.3 Function of Inhibitory Siglec-7 on NK Cells
As innate immune lymphocytes, NK cells undergo pyroptosis upon bacterial infec-

tion. NK cells recognize and destroy abnormal or damaged host cells of DAMPs,
including virus-infected cells or tumor cells. To explain the mechanisms of recog-
nition and destruction, it is considered that NK cells express activation receptors to
recognize DAMPs or stressed molecules on cell surfaces. In addition, a molecular
balance is generated through inhibitory receptors to recognize self-associated molec-
ular patterns or SAMPs. However, the balance regulation produced by both activat-
ing and inhibitory receptors expressed in NK cells, as well as the manner by which to
recognize microbial patterns, is not yet known. This indicates the current inadequate
information obtained from interactions between NK cells and extracellular bacterial
patterns. However, in the human pathogenic pneumonia bacteria and Siglec-7
interaction, GBS directly engages NK cells in humans. Through interactions with
the streptococcal β-protein B6N and the V-set domain in the N-terminal region of the
inhibitory receptor Siglec-7, GBS evades NK cell surveillance [4]. GBS seems to
evolve β-protein involvement of inhibitory receptor Siglec-7 in humans. This inter-
action inhibits the pyroptotic event of NK cells to suppress innate immune
responses. This event is referred to using a special expression, i.e., “silencing the
sentinel.” Therefore, Siglec-7 on NK cells interacts with the GBS β-protein, and this
process has been evolutionarily adapted in the long human history of biology
because GBS infection leads to expression of survival signals in the hosts. The
β-protein interaction with Siglec-7 blocks the harmful responses of the host defense
barrier of NK cells. Such “sentinel silencing” should be recognized as one of
mechanistic explanations of paradoxical “protective” means obtained from the
binding on NK cells. Siglec-7 engagement is indeed a blocker of inflammasome
activation in NK cells by such harmful pathogens. Therefore, it is paradoxical to
describe that the GBS bacterial cell wall-embedded β-protein hijacks Siglec-7 on NK
cells. Certainly, Siglec-7 is the inhibitory receptor in inhibiting GBS-involved
pyroptosis of NK cells. The signaling pathway after NK cell recognition is inhibitory
in several immune cells such as NK cells, DCs, monocytes, and CD8 + -positive T
cells.
In NK cells, α2,8 disialic acid on NK cell surface molecules is recognized and
bound by Siglec-7 and the NK cells are masked. It is known that the α2,8 disialyl
motif of glycoproteins and glycolipids is naturally expressed [372]. When sialidase
is treated, the binding affinity is dramatically decreased in NK cells and C. jejuni
LOSs [66, 372]. The inhibitory signaling by Siglec-7 is caused via an ITIM and the
interaction of SHP-1/-2 on it (Fig. 7.28). SHP-1/-2 molecules are adaptor proteins
with tyrosine-specific phosphatases. When cells are activated, related molecules
such as Siglecs are phosphorylated. Then, SHP-1/-2 dissociates phosphate from
the phosphorylated molecules, and the receptor molecules are inactivated. Thus,
NK cells, monocytes, DCs, and CD8 T cells are functionally inhibited by binding to
Siglec-7 and the ligand via these mechanisms. Many Siglec types have an ITIM. The
ITIM can interact with SHP-1/-2. The interacted SHP-1/-2 is then phosphorylated
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Fig. 7.28 Activating and inhibitory receptors of Siglec-7 in NK cells and the signaling pathway
after recognition. The inhibitory signal is formed by the ITIM and the interaction of SHP-1/-2 on
the ITIM
and activated. Activated SHP-1/-2 can dissociate phosphate from phosphorylated

proteins and consequently block cell proliferation and functions. Siglec-7 also has an
ITIM on the amino acid 435–440 site and the sequence is IQYAPL [393]. In
addition, Siglec-7 and SHP-1 interact. This interaction inhibits cellular activation
events in immune-related DCs, monocytes, NK cells, and CD8+ CTLs.
For example, sialylated glycan recognition of Siglec-7 and -9 leads to inhibited
NK cell functions in NK cells. This is demonstrated in the experiment of sialidase
treatment. Sialidase treatment increases NK cell functional cytokines, IFN-γ and
TNF-α [394]. If glycans are engineered to target hyposialylated NK cells, then NK
cell activity is decreased [395], thus helping the inhibition of self-activity of NK cells
due to the missing self. On the other hand, cancer cells that are strongly
hypersialylated bind to Siglec-7 than normal cell binding. Thus, through the
hypersialylated glycans on cancer cell surfaces, cancer cells can easily evade NK
cell activity. Siglec-7 expression is also restricted to DCs, monocytes, NK cells, and
certain CD8 CTL subsets. Although Siglec-7-expressing CD8 T cells are minor,
Siglec-7 is considered to inhibit cytotoxic T-cell functions via the ITIM [372]. In
conclusion, C. jejuni LOSs mimic human neural gangliosides. Therefore, the auto-
immune effects between antibodies produced from bacterial infection and host
neural gangliosides can be the cause of GBS symptoms.
7.13.10 Siglec-8 as a CD33-Related Siglec and Siglec-F

as a Mouse Paralog
7.13.10.1 Siglec-8-Binding Glycans on Basophils, Eosinophils, and Mast

Cells of Humans
The human Siglec-8 form is mainly present on basophils, mast cells, and eosinophils
involved in allergic inflammation, where Siglec-8 ligand binding elicits apoptotic
cell death of eosinophils and inhibits release of mast-cell mediators. As a functional
paralog of human Siglec-8, Siglec-F is predominantly present on mouse but not on
mast cells. Siglec-F and Siglec-8 have a similar preference for recognizing α2,3-
linked SAs. Eosinophil apoptosis inhibits mast-cell-mediator release and hence
eosinophilic asthma. In an allergic inflammation model, the Siglec-8 paralog Siglec-
F-lacking mice show exacerbated eosinophilic infiltration. Therefore, the Siglec-
8 form is regarded as a target therapeutic candidate for reducing the levels of allergic
immune disorders because Siglec-8 is aberrantly present on the surfaces of baso-
phils, eosinophils, and mast cells in humans. Nonself-signals like allergens and
pathogens induce activation of the innate immune systems.
Several Siglecs are homologous to Siglec-3 or CD33 in their structures and,
therefore, these Siglecs are referred to by the common name “CD33-related Siglecs”
[396]. Siglec-8 is a CD33-related Siglec. In 2000, from studies by Kristine K. Kikly’s
group on cell surface proteins of basophils, eosinophils, and mast cells, a CD33
homology protein was identified from eosinophils isolated from an idiopathic
hypereosinophilic syndrome patient and named Siglec-8, which had 49% homology
to CD33 and 68% to Siglec-7 [397]. Siglec-8 consisting of 431 amino acids has
2 alternatively spliced variants. Unlike most Siglecs, the original Siglec-8 had no
signaling motif [398], whereas the spliced variant, Siglec-8 L, consisted of a
cytoplasmic tail with two Tyr-based motifs, namely, an ITIM and a signaling
lymphocyte activation molecule-like motif (Fig. 7.29) [399]. Binding between
Siglec-8 and ligands induces the intracellular signaling pathway. In eosinophils,
activated Siglec-8 triggers phosphorylation of ITIM/ITIM-like domains and conse-
quently allows ROS production, mitochondria membrane dissipation, and caspase
Fig. 7.29 Siglec-8 of Siglec-8 (Human)

humans and its paralog V-set lg domain
Siglec-F of mouse. The
Siglec-F (Mouse)
inhibitory signal is formed C2-set lg domain
by the ITIM and ITIM-like
domains. Mouse eosinophil ITIM domain
Siglec-8 can be used as the
Siglec-8 target for ITIM like domain
eosinophilic inflammation.
Mouse Siglec-8 induces
eosinophil apoptosis and
Membrane
blocks allergic mediators
from mast cells
Cre-loxP recombination system
+
SIGLEC8LSL eoCre SIGLEC8Eo (Express human Siglec-8)
To confirm that this mice expresses siglec-8
FACS (Tissue sampling)

Only eosinophil expresses siglec-8.
Engagement of receptor (Using antibody)
Siglec-F antibody cannot affect to the
SIGLEC8Eo Disease (Asthma) SIGLEC8Eo.
Cell apoptosis (Viability)
Fig. 7.30 Development of the human Siglec-8-expressing mice using the Cre-loxP recombination
system applied for SIGLEC8LSL mouse and eoCre mouse. The resulting mice, SIGLEC8Eo, express
human Siglec-8 and are target eosinophilic disease models
activation, inducing eosinophil apoptosis. In mast cells, Siglec-8 activation inhibits

FcεRl-dependent release of PGD2 and histamine, and this consequently inactivates
mast cells.
It is unclear how the Siglec-8 surface expression is related to inflammatory
responses. Siglec-8 seems to be in part masked by the cis-type sialylation of ligands
on the cell surfaces of human eosinophils [400], as the inflammation-protective role.
Mice eosinophils mask Siglec-8 on the surface, similar to human eosinophils. Siglec-
8 is subjected to endocytosis upon ligand engagement in tissues [401]. Ligand-
induced endocytosis is applicable for specific molecules to target Siglec-8-
expressing cells. To establish human Siglec-8-expressing mice, a Cre-loxP recom-
bination system was applied using SIGLEC8LSL mouse and eoCre mouse. The
resulting mice, SIGLEC8Eo, express human Siglec-8 [402]. The established mice are
applicable for functional analysis of Siglec-8 and for targeting in various eosino-
philic disease models because the results show that (1) only eosinophils expresses
Siglec-8 and (2) the Siglec-F antibody cannot affect SIGLEC8Eo (Fig. 7.30).
Siglec-8 engagement does not induce mast-cell death but inhibits mediator
releases. Endocytosis of Siglec-8 with a toxin can induce mast-cell death
[403]. Siglec-8 is present in the lung, PBMCs, spleen, and kidney. Using a mono-
clonal antibody, 2C4, it was demonstrated that eosinophils and mast cells strongly
express Siglec-8 but basophils weakly express it, whereas the following cells of
neutrophils, monocytes, B cells, and T cells do not express Siglec-8 [404]. Most
Siglecs are known to have a specific glycan-binding region in the extracellular
N-terminal lectin domain. Currently, endogenous sialoglycan ligands responsible
for Siglec-8 recognition are not clearly clarified. However, as a candidate ligand for
Siglec-8 recognition, NeuAc-α2,3(6-O-sulfo)Galβ1,4(Fucα1,3)GlcNAc-R, which is
60 -sulfo-SLeX, has been suggested [405]. This indicates that Siglec-8 ligands include
60 -sulfo-sialyl Lex [406, 407]. Siglec-8 lectin favors a SAα2,3-Gal- or its sulfate ester
on its C6-OH group with the SAα2,3(6S)Galβ1,4GlcNAc-R structure
Structure Name Siglec-8 recognition Siglec-F recognition
6S
α3 β4 6’-S-Siayl-LacNAc Yes Yes
6S
1HX$F
α3 β4
6’-S-Sialyl-Lex Yes Yes *OF1$F
α3
*DO
)XF
0DQ
Tri-antennary No Yes
Tetra-antennary
No Yes
bisected
Fig. 7.31 Ligand-binding Siglec-8 specificity of human and mice paralog Siglec-F. The glycan
structure of NeuAc-α2,3(6-O-sulfo)Galβ1,4(Fucα1,3)GlcNAc-R, which is known as 60 -sulfo-SLeX
is bound by Siglec 8
[405, 408]. Siglec-8 recognizes 60 -sulfo-SLeX with the structure of SAα2,3(6S)

Galβ1,4(Fucα1,3)GlcNAc-R, where the Gal residue-linked sulfate group binds to
an Arg residue (R56) [409]. This indicates that sialyl 60 -sulfated carbohydrates
present in target cells are the functional ligands for Siglec-8 (Fig. 7.31). Activated
eosinophils or mast cells promote proinflammatory cytokines, and these cytokines
increase the expression of endogenous Siglec-8 glycan ligands such as 6’S sLex
glycan epitopes in airway tissues [410].
The real endogenously produced sialoglycan ligands that link to Siglec-8 have
not been identified. Siglec-8 is expressed in great apes (humans, orangutans, and
chimpanzees) but not in dogs, rodents, or monkeys, [411]. Interestingly, rodent mice
consist of Siglec-F, which is functionally a paralog of human Siglec-8, on their
eosinophils, while Siglec-8-recognizing Sialyl ligands are not found in mice airways
[408] and Siglec-F-involved apoptotic cell death of mouse eosinophils is relatively
lowly operated than human eosinophils [412], indicating that human airways are
therefore enriched for Siglec-8 ligands. Recently, the sialylated keratan sulfate
(KS) structure of SAα2,3(6S)Galβ1,4(6S)GlcNAc-R in the terminal sequence has
been suggested to be an endogenous ligand for Siglec-8, expressed in the airway
track pathways in humans [413]. From an array analysis using glycans, which is
available from the CFG (Consortium for Functional Glycomics, USA) (www.
functionalglycomics.org) [413], it has been found that Siglec-8 recognizes the
glycan structure of the 60 -sulfo sialyl LacNAc, with the SAα2,3(6S)Galβ1,4GlcNAc
and 60 -sulfo SLeX structure of SAα2,3(6S)Galβ1,4(Fucα1,3)GlcNAc-R. If the sul-
fate group of the (6S)Galβ1,4GlcNAc-R structure or the SA residue of the SAα2,3-
Galβ1,4GlcNAc (SAα2,3-Galβ1,4[Fucα1,3]GlcNAc-R) structure is absent, then
ligand binding is impaired. In addition, the structure that sulfate is attached on the
6-OH attached to GlcNAc residue instead of the GalSAα2,3-Galβ1,4(6S)GlcNAc-R
structure, SAα2,3-Galβ1,4(Fucα1,3)(6S)GlcNAc-R epitope is not bound to Siglec-
8 [413].
7.13.10.2 Siglec-8 Is an Eosinophil-Specific CD33-Related Siglec

and Functions in Mast Cells
Siglec-8 is predominantly expressed on human eosinophils, with low expression on

blood basophils. Involvement of blood eosinophil Siglec-8 is internalized and
induces apoptosis. Siglec-8 is therefore regarded as a candidate for therapeutic
application to prevent eosinophilic asthma. Siglec-8 L has an ITIM and signaling
lymphocyte activation molecule-like sequences with signal transduction activity.
ITIM-carrying Siglecs (Siglec-3 and Siglec-7) exhibit inhibitory capacities of both
growth and survival of myeloid leukemia when specific antibodies are treated.
Therefore, it is hypothesized that binding of Siglec-8 with its glycan ligand inhibits
eosinophil survival. Ligands specific for Siglec-8 recognition are predominantly
produced in bronchial and tracheal submucosal glands or cartilages [408]. Indeed,
crosslinking of 2E2, a Siglec-8 monoclonal antibody, induces eosinophil death and
apoptosis. Interestingly, IL-5, IL-33, and GM-CSF as antiapoptotic cytokines for
eosinophils increase this apoptotic effect [414], as Siglec-8-dependent death is based
on activation of eosinophils by such cytokines [414–416], or allergic inflammation.
The inhibitory motif in the Siglec-8 cytoplasmic region leads to negative signaling
[415]. Siglec-8 primed by IL-5 initiates a signaling event leading toward eosinophil
apoptosis through signaling protein phosphorylation and β2-integrin-dependent
adhesion [415, 417, 418]. In 2005, it was demonstrated that Siglec-8-induced
apoptosis in eosinophils is suppressed by caspase inhibitors [419]. Next, Esra
Nutku’s group [420] reported that IL-5 increases apoptosis of eosinophils. When
IL-5 is treated as a priming stimulator, eosinophil cell death is accelerated by
Siglec-8. After IL-5 priming, caspase inhibitors prevent apoptosis [420]. For
Siglec-8-driven apoptosis in IL-5-stimulated eosinophils, costimulation of the IL-5
receptor and Siglec-8 leads to cell death resembling necrotosis, which is promoted
by MEK1/ERK signaling. ROS enhances IL-5-induced ERK phosphorylation and
this is an acting mechanism of eosinophil cell death, indicating that IL-5-activated
eosinophils are programmed to die [421]. Siglec-8 crosslinking on mast cells or on
eosinophils induces apoptotic cell death. Surprisingly, Siglec-8 activation does not
cause apoptosis in mast cells. However, activation of Siglec-8 inhibits an FcεRl-
dependent release of prostaglandin D2 and histamine. Siglec-8 activation suppresses
FcεRl-dependent contraction of smooth muscle cells (SMCs) in the human airway
track. A mutation in the residues from Tyr to Phe in the ITIM domain of Siglec-
8 exhibits that the signaling pathway of Siglec-8-mediated FcεRl-dependent Ca2+
flux reduction is linked to the ITIM of Siglec-8 [422], indicating that Siglec-8, which
is expressed on eosinophils and mast cells, is different in its functions.
Even though Siglec-8 is selectively expressed on only mast cells, basophils, and
eosinophils of humans, the main studies are focused on eosinophils. Asthma is
mainly caused by eosinophil-airway inflammatory responses [422]. Airway eosino-
philia indicates asthma exacerbations and induces airway remodeling
[423, 424]. Activated eosinophils move to the bronchial lumen and tissues to
increase the survival of asthma-related cells [425, 426]. Cell surface glycans mod-
ulate eosinophils with recruitment and survival, providing a possibility of therapeu-
tic clues to allergic asthma responses and eosinophil-related diseases [415]. Siglec-
8 on the surfaces of human mast cells is lowly expressed on basophils in blood.
Although Siglec-8 expression levels are not defined in airway cells, Siglec-8 is
regarded as a potentially therapeutic candidate. Siglec-8 engagement in eosinophils
of blood induces endocytosis to the target cells and causes cell death [414, 415,
427]. Siglec-8-directed therapeutics are thus possible for application. For example, a
phase 1 trial conducted on a volunteer group comprising healthy adult humans, who
received a humanized type of Siglec-8-specific afucosylated IgG1 mAb (named
mAb AK002), exhibited a depletion of blood eosinophils [428]. With Siglec-8-
expression on mast cells and eosinophils, the mAb AK002 is under investigation
in clinical trials for patients with eosinophil- or mast-cell-driven inflammatory
disorders, such as allergic conjunctivitis (clinical trial no. NCT03379311), eosino-
philic gastritis (clinical trial no. NCT03496571), indolent systemic mastocytosis
(clinical trial no. NCT02808793), and refractory chronic urticaria (under the national
clinical trial no. NCT03436797) [429, 430].
7.13.11 Siglec-9 as a CD33-Related Siglec and Murine

Functional Counterpart, Siglec-E
7.13.11.1 Siglec-9 Function in Immune Surveillance and Survival
By binding to the terminal SAs of carrier sialyl glycans, Siglecs control the immune
duration and intensity during immune responses. Siglecs potentiate the evasion of
the host immune system for survival of tumor cells or virus-infected host cells.
Siglec-9 is a lectin, which is located on neutrophil surfaces. As Siglecs are mainly
present on innate immune cell surfaces, Siglec-9 expression is also found on mono-
cytes, DCs, NK cells, and neutrophils. Siglec-9 recognizes SA-specific ligands like
SAα2,3 and SAα2,6 glycans (Neu5Ac-α2,3/α2,6-Galβ1,4-Glc). The Siglec-9 struc-
ture is composed of a SA-recognition domain, an Ig domain, and ITIMs. Siglec-9
recognizes SAs via the V-set domain in the N-terminal region, and it has Tyr-based
inhibitory ITIMs in its cytosolic domain. The inhibitory signal is formed by the ITIM
domain (Fig. 7.32). For the mechanistic action of Siglec-9 on macrophage cells, it
has been proposed that Siglec-9 signaling induces IL-10 production, while reducing
the synthesis and release of TNF-α. In addition, Siglec-9 can also bind to several
TLR agonists, which mimic sialic acids.
404
/*8TGSWKTGHWPEVKQPCN%&%&4UKIPCNKPI
Neuron, Astrocyte +PHNWGP\C#%&-1OKEGKPETGCUGFOQTVCNKV[
Epithelial cell, Endothelial cell, *58
Fibroblast Virus
Lymphoid cell C/EBPβ
Viral Orthologs
vOX2 (HHV8)
NAG65 CD200 R15 (RRV) 'PJCPEGT %&
NAG73 M141 (Myxoma virus) NF-kB binding site
NAG151 IFNγ-activation site(GAS)
NAG160
IFN-stimulatory response element-2
NAG69 CD200R1
NAG80 NAG168
NAG127 N135
NAG44
Myeloid cell
(NAG20) T-cell subsets
N77
N198
Upregulation N183
CD200 <
< Dok2
Upregulation
< Ras
Bacterial & CD200R1
RasGAP
Parasitic
Pathogens DAP12 Myeloid cell function
5TE MKPCUG (cytokine production, Ca2+ release,
Proliferation, etc.)
CD200R2
(CD200RLa) NAG : N-Acetylglucosamine
HHV8 : Human herpesvirus 8
RRV : Rhesus Rhadinovirus
Dok2 : Downstream of tyrosine kinase 2
6IQPFKK %&4
/KETQINKC%&4
$NQQFXGUUGNGPFQVJGNKCNEGNNUȡ RasGAP : Ras-GTPase activating protein
.COC\QPGPUKU %&
$QPGOCTTQYOCETQRJCIGȡ MHV : Mouse hepatitis corona virus
HSV-1 : Herpes simplex virus-1
0OGPKPIKVKFKU %&
$QPGOCTTQYOCETQRJCIGȡ C/EBPβ : CCAAT/enhancer binding protein β
Fig. 7.32 Presumptive mechanism of CD200–CD200R1 interaction between immune cells and related cells. CD200–CD200R1 recognition and the CD200R
activation are displayed. The Tyr residues of CD200R recruit Dok2 and RasGAP to inhibit Ras activation and anti-inflammatory signals (modified from Ref. No)
[431]
When LPSs are recognized by TLR-4, Siglec-9 is induced. Induced Siglec-9

negatively regulates TLR-4 responses. Moreover, Siglec-9 helps to internalize
TLR-4, when cells are infected by E. coli. If TLR-4 is not internalized,
proinflammatory cytokines are increased. When neutrophils are stimulated by
LPSs, Siglec-9 inhibits neutrophil recruitment to inflammation sites such as the
lung. It also specifically binds to VAP-1 and aids in granulocyte migration to the
inflammation sites. Siglec-9 mediates cell death responses in neutrophils during a
septic shock. The pathogen-synthesized molecular mimicry of host sialylglycans
potentiates pathogenic invaders to engage Siglec-9 expressed on neutrophils. Con-
sequently, Siglec-9 engagement dampens the responses of innate immunity of hosts.
Siglec-9 is a lectin, which is located on neutrophil surfaces. T cells in tumors can be
activated by an interaction between SA-SAMP and Siglec-9. Siglec-9 recognizes
self by recognizing and binding to SAs. If it recognizes self, it inhibits neutrophils’
killing activity. Bacterial pathogens use this feature to avoid the host’s immune
mechanism. Therefore, they have sialic acids similar to hosts.
Siglec-9 is homologous to Siglec-7 with 80% identity in the protein sequence,
and the two genes have been developed quite recently through gene duplication or
conversion. A positive and negative balancing of inflammatory signaling is crucial
for pathogenic clearance without autoimmune and hyperinflammatory responses
[432]. The inflammation-regulatory Siglec family, as cell surface molecules on
leukocytes, tranduces glycan binding to immune cell functions. A relevant balancing
of inflammatory signals leads to pathogenic clearance and a relevant inflammation
status; therefore, Siglecs as cell surface molecules on leukocytes should bind to
sialylated glycans. For example, in allergic inflammation, Siglec-8 (Siglec-F in
mouse) KO mice display exacerbated eosinophilic infiltration [433]. Siglec-9 present
on neutrophils, monocytes, DCs, and NK cells binds to its ligands on neutrophils and
induces neutrophil cell death and apoptosis [434]. The human Siglec-9 homologue
(Siglec-E in mice) KO mice exhibit exaggerated recruitment of neutrophils to the
lung sites in acute inflammatory responses treated with LPSs [435]. Regarding the
functions of Siglec-8 and -9, sialylated ligands are predominantly present in the
inflamed airways compared with normal airways in humans. The sialylated ligands
responsible for Siglec-8 and -9 interactions are expressed in increasing levels in the
upper respiratory airway track of human patients with chronic allergic response. This
indicates that sialoglycans are inflammatory molecules.
7.13.11.2 Immune Suppression of Tumor Cells by Siglec-9–SA

Interaction
In tumor cells, the SA structure-based ligands for Siglec-9 are frequently found on
carcinoma cells and they suppress the innate immune responses. Previously
transformed tumor cells are eliminated by immune lymphatic, myelomonocytic,
and NK cells through cancer immunosurveillance. Siglec-binding tumor ligands
positively affect immune responses to cancer. In malignant progression, tumor
cells escape from immune responses and surveillance. Importantly, in the escaped
tumors, immune cells can help in tumor progression through chronic inflammatory
responses. Myeloid-monocytic lineage cells such as neutrophils and monocytes/
macrophages are used as antitumor or protumor collaborators in the tumor progres-
sion stage and tumor microenvironment. Neutrophils also have either cancer-killing
or cancer-promoting roles. Hypersialylation is a classically aberrant transformation.
Highly sialylated tumor cells can transduce signals in tumor-associated macrophage
(TAM) conditions via SA-interacting lectins including selectins present in endothe-
lial cells, leukocytes, and platelets. In tumors, immune-deficient mice express low
sialylation of the target substrates. Effectively enhancing antitumor activity is the
most important aim in clinical cancer biology. The tumor microenvironment (TME)
is associated with effective chemotherapy since the TME consists of immune cells
with diverse phenotypes to regulate chemotherapeutic potentials [436]. The thera-
peutic efficacy of tumors can be achieved by cancer cell-immune cell binding in the
TME. In fact, antitumor therapy is effective in only a limited number of cancer
patients, with about 50% efficacy rate because cancer cells acquire drug resistance
during chemotherapy [437]. Cancer cells interact with immune cells of the TME,
exclusively with TAMs [438]. TAMs are heterogeneous and phenotypically plastic.
They act as innate immune regulators in solid tumors, where they regulate the solid
tumor progression and the efficacy of cancer chemotherapeutic drugs through mono-
cytes/macrophages, leading to phenotypic resistance against antitumor drugs. Two
phenotypic macrophages with different polarizations are known, such as i) the
classical M1-type macrophage, which is the activated macrophage type under
proinflammatory conditions, with antitumor activities, and ii) the alternative
M2-type macrophage, which is the activated macrophage type under anti-
inflammatory conditions, with the protection activities of tumors. M1 macrophages
accelerate clearance of tumor cells, whereas M2 phenotype cells induce tumor
progression.
In solid tumors, TAMs consist of M2 phenotypes responsible for tumor progres-
sion. The TAM effect shown in invasion, metastasis, and progression of tumors
depends on each tumor type, TAM form, and TAM position [439]. Thus, phenotype
conversion of the M2-type macrophage to the proinflammatory M1-type macro-
phage is of strategic interest in cancer therapy. TAMs are primarily derived from two
independent cell types: i) macrophages, which are regular residents of tissues
differentiated from the embryonic yolk sac and ii) monocyte-differentiated macro-
phages derived from the bone marrow, which migrated to tumorous tissues by
attractive molecules such as M-CSF, CCL2, and CCL5 [440]. The TME influences
the reprogramming of both types of resident and migrated macrophages. TAMs of
the resident macrophages are reprogrammed by neighboring tumors to protumoral
phenotypes. TAMs of the resident macrophages participate in DNA damage, trans-
formation, tumor survival, and inflammation. To recruit Tregs, which suppress
CD4+ and CD8+ T cells, to tumor sites, TAMs also express various kinds of
chemokines including CCL3, CCL4, CCL5, and CCL22, as well as cytokine
IL-10 [438]. Monocytes/macrophages that migrate to the tumorous sites further
induce tissue remodeling, progression, invasion, and immune suppression as well
as growth, survival, angiogenesis, and lymphangiogenesis of tumors [441]. To
perform the above-mentioned functions, TAMs express various growth factors

including EGF, FGF, and TGF-β1 with VEGF-C, VEGF-D, and CXCL8 for
tumor tissue remodeling. Proteolytic enzymes of plasmin, uPA, MMPs (MMP-2
and MMP-9), and cathepsin B are also expressed. The TAM function is also
associated with VEGF-A, TNF-α, FGF, PDGF, thymidine phosphorylase, uPA,
adrenomedullin, and semaphorin 4D for angiogenic events [442]. TAMs also sup-
port cancer stem cells [441]. In cancerous tissues of non-small-cell lung carcinomas
(NSCLCs), TAMs induce cancer stemness via Sox2 and NF-κB as inflammatory
molecules [443]. In brain tissues with glioma cells, the M2 phenotype of TAMs
induces cancer stemness and migrative potentials, which is potentiated by TGF-β1
[444]. The tumor-protecting TAMs protect malignant cancer cells including breast,
cervical, esophageal lung adenocarcinoma, ovarian, melanoma, renal carcinoma,
and prostate from immune surveillance of the host.
Macrophage CD68 expression indicates poor prognosis of tumor cells with a
decreased survival rate in many malignant cancer cells including breast, melanoma,
ovarian, NSCLC, and stomach. In malignant NSCLC cells, the high cell density of
the CD68-positive macrophages with tumor-infiltrating activity reduces the survival
rate of cancer patients, increasing cancer angiogenic potentials via tumor-produced
IL-8 activity, leading to metastasis with poor prognosis and strong EMT features
such as E-cadherin loss and vimentin expression in cancer. CD68-expressing mac-
rophages also indicate a poor prognosis in the melanoma tissue and mortality as well
as breast cancers (BCs). The CD68 expression level is lower in lymph node
(LN) metastases [445]. An antitumor effect of macrophages is also detected in
colorectal cancer, when TAMs have proinflammatory responses via cytotoxic
Th1-elicting IFN-γ/IL-1/IL-6 cytokines to exert antitumor immunity. High CD68+
TAM levels in the tumor/stroma region are associated with high survival in colon
cancer patients [446]. M2-type macrophages of TAMs also indicate decreased
survival rates in many malignant human cancer cells including BCs, gastric, hepa-
tocellular carcinoma, multiple myeloma, ovarian, renal carcinoma, and osteosar-
coma cells in carcinoma patients [447–450]. CD206 and CD163 are biomarkers of
the M2-type TAMs in tumor tissues [446]. Reduction in the CD206+ TAM number
in gastric cancer indicates a prognostic factor [448]. Infiltration of CD206+ -positive
macrophages of TAMs also indicates decreased survival rates in several human
carcinoma patients with hepatoma carcinoma cells, renal carcinoma cells, and
ovarian cancer cells [449]. Increase in the CD163+ TAM number indicates the
progression-free survival of several cancer patients with different carcinoma cells
such as BCs, gastric, multiple myeloma, osteosarcoma, and ovarian [450, 451].
Neutrophils are also one of the most abundant immune cells that are not only a
vital component of innate immunity but also contribute to adaptive immunity
regulation by interacting with various adaptive immune cells [452, 453]. The mul-
tifaceted roles of neutrophils in contributing to cancer mainly result from the
secretion of different effector factors under different statuses. Two neutrophil phe-
notypes, N1 and N2, have been used to define neutrophil subtypes with tumor-
suppressing or tumor-promoting properties due to their immunostimulating and
immunosuppressive activities, respectively, in the TME [454, 455]. The number of
N2 neutrophils in the TME implies poor prognosis for patients with cancer. Neutro-
phils are mainly located in tumor margins in early stages, but they can easily
infiltrate the center sites at later stages. In addition, N1 neutrophils present in an
early-stage tumor then transform into a tumor-promoting N2 phenotype during
tumor progression. Such phenotypic conversion is induced by factors derived from
cancer or stroma cells in the TME. N2 neutrophil polarization also expresses high
levels of arginase, CCL2, CXCR4, and MMP-9.
7.13.11.3 Siglec-9 and its Ligand Mucin in Tumor Biology
Cancer cells evade immune responses and create a permissive microenvironment in

tissues. To construct a stable and affordable microenvironment in their wanted
tissues, cancer cells recruit and educate monocytes and repolarize resident macro-
phages to the M2-macrophage type. These tumor-associated M1-type macrophages
promote the behaviors of cancer cells. Aberrant glycosylation is characteristic of all
types of cancer cells. Increased sialyl or hypersialyl glycans present in tumor cells
suppress complement cascade activation via recruitment of factor H [456]. Interac-
tions of CD33-related Siglecs with tumor-associated Siglec sialyl ligands are
reported to be related to NK cell-mediated killing. Siglec-9 and its sialyl ligand
interaction in cancer activate tumor growth. The sialyl ligand is mucin 1. Colon
cancer cells express specific binding ligands for Siglec-7 and -9 and they are
associated with generation of TAMs. In addition, inhibitory Siglec-7/-9 Siglecs are
present on NK cells [125]. Thus, introduction of sialoglycoconjugates onto the
surface of cells results in increased sialylation and, consequently, leads to suppressed
NK cell activation. Binding of Siglec-7 and Siglec-9 receptors to ligands expressed
on naive tumor cells modulate NK cell-dependent cancer immunosurveillance. Thus,
NK cell–Siglec interactions might be working for it [457]. In addition, Siglec-9 is
also present in a subset of CD8 CTLs.
NK cell Siglec-9 expression is decreased in patients with hepatitis B, which is rich
in HBV viremia. Defected Siglec-9 function exhibits suppressed NK cell function
and HBV activation [458]. ITIM-like or SLAM-like sequence-carrying Siglec-9 is
strongly present on B cells, granulocytes, monocytes, and CD56+ NK cells, whereas
it is weakly synthesized on CD4+ T cells and CD8+ CTLs. Siglec-9 binds sialyl
α2,3-Gal residues and recruits SHP-1 and -2 phosphatases. Siglec-9 also suppresses
the cellular roles of many innate immune-related cells [458]. Crosslinked Siglec-9
binding by neutrophils helps in maintaining the bloodstream flow [459] and also
inhibits apoptotic cell death in cancer cells or induces inflammation milieu
[460]. The tumor MUC1 protein with sialyl O-glycans (MUC1-ST) enables the
acquirement of the TAMs’ tumor-like phenotype via Siglec-9 recognition
[461]. Siglec-9 executes NK cell functions and is specifically present on CD56dim
NK cells [462]. Siglec-9 expression indicates the presence of a certain NK cell
population, while Siglec-9+ NK cell subpopulation level is decreased in tumor
patients because Siglec-9 is an immunomodulator in physio-pathological
conditions [463].
The expression of MUC1 has short-sialyl core 1 glycan structures of NeuAc-α

2,3-Galβ1,3-GalNAc (MUC1-S) and enhances tumor growth [463, 464]. When the
O-glycan-specific MUC1 mucin is present in tumor cells, MUC1 is further coated
with sialyl O-glycans of MUC1-S. The cancer-specific MUC1-S engages Siglec-9
on immune cells such as myeloid cells to release the tumor microenvironment-
associated factors. Moreover, MUC1-bound Siglec-9 induces β-catenin recruitment
to the Siglec-9 extracellular motif to inhibit nucleus localization. MUC1 is aberrantly
expressed on tumor cells, thus providing a poor prognostic diagnosis. MUC1 is
degraded in the ER to the two parts of the N-terminal and C-terminal regions. The
two regions form a heterodimeric complex. The N-terminal ectodomain (MUC1-
ND) is a heavily glycosylated form by O-glycans. The C-terminal domain of MUC1,
termed “MUC1-CD,” is linked to the MUC1-ND, which is present on the surface of
cells. β-Catenin is key factor for cellular growth and nucleus localization. MUC1
binding to immune cells is caused by Siglec-9 engagement. After MUC1 recogni-
tion, immune cells such as neutrophils and DCs cannot grow because of recruitment
of β-catenin to the Siglec-9 motif [465]. In addition, MUC1-S induces macrophages
to display a TAM phenotype. MUC1-S interaction with Siglec-9 does not recruit the
phosphatases SHP-1 and SHP-2. In addition, when MUC1-S recognizes Siglec-9
present on primary macrophages, the macrophages have been changed to the TAM
phenotype and CD8+ T-cell growth is inhibited. Moreover, IDO, CD163, CD206,
and checkpoint ligand PD-L1 are upregulated [466]. Thus, pathogens and tumor
microenvironments expressing MUC1 can inhibit the growth of immune cells.
Aberrant sialoglycosylation of MUC1 activates the tumor progression pathway,
following engagement of Siglec-9 [467]. From the recent issues of increased ther-
apeutic efficiency, it has been observed that emerging cancer immunotherapy may
fuse MUC1-related Siglecs with the immune checkpoints of TIM-3, CTLA-4, PD-1,
and LAG-3 proteins. Since successful cancer immunotherapy is obtained from usage
of immune checkpoint inhibitors or blockade resistances, clarification of new path-
ways to suppress tumor-specific T cells will open new avenues in the therapeutic
efficiency of immune checkpoint inhibitor-targeting strategies.
7.13.11.4 Siglec-9 Linkage to the CD200–CD200R Axis

for Inflammation Inhibition in M1- and M2-Type
Macrophages
Currently, many immune inhibitory proteins including SA, HMGB1, CD47, CD200,
and CD95L as well as complement-regulatory proteins (C3a, fH, CD55, and CD46)
are known. Their roles are to protect normal cells such as neurons from apoptosis,
infection, or inflammatory damage by phagocytes or infiltrated immune cells
[468]. Normal cells or neurons express CD47 and CD200. These receptors are the
“don’t-eat-me” signals, which can protect them from macrophagic or microglial
inflammatory responses [469]. Phagocytes including macrophages and neuronal
microglial cells can distinguish between “self-” and “nonself-” signals and molecular
patterns. Macrophage cells phagocytose pathogens and infected or damaged cells
through the discriminated “nonself-” and “altered-self” molecular patterns. Others

such as microglial cells, astrocytes, oligodendrocytes, and neurons can also recog-
nize such altered molecular patterns [470, 471]. For example, neurons or normal
cells express membrane-bound or soluble “don’t-eat-me” patterns or signals, such as
SAMPs, which interact with specific inhibitory PRRs [471, 472]. The “normal-self”
molecular pattern mostly appears as sialic acids that bind to inhibitory receptors,
Siglecs [473]. For example, microglial Siglecs recognize the neuronal glycocalyx. In
contrast, the “altered-self” patterns promote phagocytosis.
Medzhitov and Janeway [470] called PAMPs as the “eat-me” signals on patho-
gens and damaged cells. In prokaryotes, the most well-characterized PAMP is the
Gram-negative bacterial endotoxin, LPS. In eukaryotes, apoptotic cell-associated
molecular patterns (ACAMPs) and DAMPs are well-known PAMPs [474]. The
known ACAMPs are altered membrane electrical charges, oxidized forms of
low-density proteins, nucleic acids, and phosphatidylserine [471, 475]. DAMPs
include adenosine, ATP, galectins, heat-shock proteins (HSPs), HMGB1, and
thioredoxin [476]. Phagocytic PRRs bind to PAMPs, ACAMPs, or DAMPs
[474]. Various PPRs are known for CD14, CD36, complement receptors, TLRs,
scavengers, mannose receptors (MPs), milk fat globule-epidermal growth factor-
8 (MFG-EGF8), and phosphatidylserine receptor (PSR) [469].
Macrophages induce inflammation in immune responses. In vitro, bone marrow-
borne macrophages are classified into two polarizing activation states. The two
categories are governed by genes and cytokines that are expressed by each macro-
phage [477, 478]. Typically, macrophages have two opposing phenotypes of (1) clas-
sic M1 cells and (2) alternative M2 cells [479]. Basically, macrophages induced with
treatment of LPSs with or without IFN-γ are the classically activated type or M1
macrophages. M1 macrophages predominantly express proinflammatory cytokines
and exhibit bacteria killing and proinflammatory phenotypes. In contrast, anti-
inflammatory IL-13/IL-10/IL-4 cytokines promote alternatively activated types or
M2 macrophages with anti-inflammatory phenotypes. Over the last two decades,
tissue macrophage phenotypes have been explained in vivo by the M1–M2 dichot-
omy. However, tissue macrophages do not always show an apparent M1 or M2
phenotype in vivo because TAMs frequently exhibit both phenotypes of M1 and M2
[480]. Tissue macrophages often transit from the activation state to the quiescent
state to adapt to microenvironments. Therefore, the M1–M2 phenotype difference
cannot always explain the state of macrophage activation in vivo. The tissue
macrophage subtypes that exhibit either the M1 or the M2 phenotype express the
CD169 antigen.
Human monocytes and macrophages express CD33-related Siglecs, depending
on M1- or M2-specific ligand binding in different macrophage phenotypes. M1
macrophages are the classical type to induce inflammation, whereas M2 macro-
phages are the alternative or activated type to display anti-inflammatory responses or
wound healing [481]. CD200 inhibits the classical macrophage activation and
CD200R is present on M2 cells in humans and mice [482]. Therefore, CD200R
immunosuppresses the immune inhibitory M2 macrophages. Among mouse macro-
phages, BM-derived macrophages lowly produce Siglecs. However, mouse Siglecs
are largely expressed upon binding to certain TLR ligands or viral infections
[483]. In contrast, human monocytes in blood constitutively produce certain Siglecs
[344]. There might be links between Siglec expression and M1/M2 polarization. M1
is activated by IFN-γ with TLR ligand LPS, whereas M2 macrophage types are
activated by specific IL-4 and IL-13 type 2 cytokines. Currently, the question of how
M1 or M2 polarization is molecularly linked is unanswered, although distinct signal
transduction pathways regulate each phenotype. The answer comes from, for exam-
ple, the fact that Siglec-9 expression inhibits inflammation through expression of
CD200R, a surface protein of myeloid lineages, on human macrophages [484]. The
question of how Siglec-9 downregulates the inflammation is interesting. The mech-
anistic answer is that Siglec-9 stimulates CD200R production upon IL-4 treatment in
human macrophages [484]. The binding between CD200R and its ligand CD200
prevents inflammatory autoimmune diseases such as CIA and EAE [485]. Therefore,
the hidden clue for Siglec-9-mediated anti-inflammation is in its downstream sig-
naling of the CD200R–CD200 interaction. Siglec-9 suppresses inflammation via
LPS-raised CCR7 inhibition and IL-4-elicited CD200R production in macrophages.
As CD33rSiglecs belong to rapidly evolving genes and they have 5 mouse members
and 11 human members, they have 2 distinct motifs: i) ITIMs that suppress innate
and adapted immune responses and ii) instead of lacking ITIMs, ITAMs contain
specific amino-acid motifs in the TM region with positive charges of amino acids.
These positive amino-acid motifs are associated with DNAX-activating protein
(12 kDa) and activate ITAMs. Therefore, both ITIMs and ITAMs competitively
and antagonistically control each other. The two types of Siglecs expressions are
often overlapped in immune cells.
Siglec-9 blocks LPS-stimulated CCR7 and IL-4-stimulated CD200R expression
on macrophages [484]. In detail, mouse RAW264 macrophage cells transfected with
human Siglec-9 exhibit reduced IL-6/TNF-α levels as inflammatory cytokines upon
treatment with TLR ligands like LPSs and peptidoglycans [153]. In addition, the
murine functional counterpart Siglec-E crosslinked also inhibits proinflammatory
IL-6/TNF-α cytokine synthesis in LPS-treated RAW264 macrophages [483]. As
TLRs are receptors for bacteria, fungi, protozoa, and viruses, they are the first line of
defense against innate immune responses and inflammatory responses. Thus, the
host Siglecs modulate TLR signaling, as confirmed by the fact that two Siglecs,
Siglec-7 and Siglec-9, are increasingly expressed upon cotreatment with M-CSF and
CM-CSF, respectively, on human macrophages. Human macrophages, which are
induced to differentiate by M-CSF/CM-CSF, IL-4 treatment, strongly increase
Siglec-10 expression. Upon Siglec-10 binding to the C. jejuni flagellin in a
SA-independent manner, Siglec-10 upregulates IL-10 production [222]. Therefore,
IL-4 increases IL-10 expression through Siglec-10 under specific conditions. Siglec-
7 expression is decreased upon treatment with LPS plus IFN-γ on macrophages.
However, Siglec-9 expression is kept the same during M1- and M2-specific stimu-
lations [486]. As M1- and M2-specific genes are found [481], Siglec-9 expression
downregulates LPS-stimulated TNF-α expression as human and mouse M1 markers
in RAW264 cells. Upregulated IL-4 elicits Arg 1 expression, known as a M2
biomarker in mice [487]. However, Siglec-9 silencing does not affect the
LPS-induced mRNA expression level of TNF-α, regardless of treatment with IFN-γ

in human macrophages. The Mrc1 mRNA expression level as another human and
mice M2 biomarker is different between mice and humans, although Mrc1 is
induced by IL-4. Siglec-9 silencing induces the LPS-induced CCR7 mRNA expres-
sion, a chemokine receptor enabled to present DCs, neutrophils, and naive T cells to
the lymphatic nodes [488]. Siglec-9 stimulates the expression level of IL-4-stimu-
lated CD200R mRNA on human THP-1 macrophages [484]. The mRNA and pro-
teins levels of CCR7 and CD200 R are correlated [482]. Siglec-9 silencing also alters
CCR7 and CD200R protein levels, indicating that Siglec-9 function is linked to the
expression of CCR7 and CD200R. Thus, Siglec-9 blocks inflammation via
LPS-stimulated CCR7 inhibition and via IL-4-stimulated CD200R expression on
macrophages.
7.13.11.5 Inhibitory Receptor Engagement in Autoimmune Diseases

Linked to the Siglec-9–CD200R–CD200 Axis
IBD is a gastrointestinal tract autoimmune disease and has two types, CD and UC
[489], without etiology. Genetic susceptibility to intestinal bacterial factors contrib-
utes to an imbalanced mucosal immunity to overexpress inflammatory cytokines and
induce infiltration of immune cells of myeloid and lymphoid lineages [490]. The
acute inflammatory cells that induce IBD are macrophages and neutrophils [491]. A
damaged mucosal barrier and dysregulated responses of immune cells to bacterial
antigens may cause IBD. For example, dextran sodium sulfate (DSS) destroys the
mucosal epithelial barrier of colon epithelial cells, potentiating the mucosal entry of
luminal bacteria and inflammation [492]. DSS is an inducing agent of experimental
colitis, which is a similar phenotype to human IBD. TLR KO mice are susceptible to
DSS-induced colitis [493]. Proinflammatory cytokines induce IBD in colonic epi-
thelia [494]. More specifically, IL-4/IL-17/IL-22/IFN-γ, which are generated in Th
cell populations such as Th1, Th2, and Th17 cells, are involved in IBD development.
Certain IL-6/IL-12/TNF-α cytokines are also related to IBD [494]. Hematopoietic
cells, such as innate lymphoid cells (ILCs), also contribute to the development IBD.
An RA receptor (RAR)-involved orphan receptor-γt-dependent subset expresses
Th17 cells like cytokines and induces immune responses in the intestines
[495]. Type 1 TM proteins of T-cell Ig and Tim-3 regulate autoimmune IBD
[496]. As an immune inhibitory protein or “don’t-eat-me” signal, the CD200–
CD200R pairing protects from inflammation. The CD200–CD200R interaction
rescues a proinflammatory environment. CD200R signaling also suppresses demy-
elination and axonal damage, activating M2 macrophages with reduced inflamma-
tory cytokines. T-cell CD200R expression is restricted to Th2 cells [497].
CD200R1 as an inhibitory receptor belongs to the Ig superfamily with two Ig
superfamily (IgSF) domains. CD200 has a MW of 41–47 kDa as a type I membrane
glycoprotein [498, 499]. It immuneregulates after interaction with its receptor
CD200R [498]. CD200 is regarded as an immune inhibitory molecule. It is
expressed in neurons, the endothelium [500, 501], peripheral cells, DCs,
thymocytes, T and B cells [499, 502], brain astrocytes, and oligodendrocytes [503],
whereas CD200R has two IgSF regions and a cytosolic tail with three Tyr residues
including the NPXY motif and an ITIM domain. CD200R is present on microglial
cells, myeloid cells [501], thymocytes [504], T cells, and B cells [499, 505]. CD200
in the CNS indicates immune suppression, as the blocker of interaction between
CD200 and CD200R in CD200/-deficient KO mice activates microglial and
macrophage phenotypes, inducing inflammation such as uveoretinitis with macro-
phage infiltration [506]. Unlike most general inhibitory receptors, which have ITIMs
in the cytoplasmic tail of the protein for further relay of inhibitory signals, CD200R1
does not have an ITIM. CD200R1, instead, has three tyrosine residues in its
cytoplasmic tail, i.e., Y291/Y294/Y302 in humans (Y286/Y289/Y297 in mice).
Among these tyrosine residues, Y302 is located in the phosphor-Tyr-binding
(PTB) domain recognition motif (NPxY). CD200 as a substrate of the CD200
receptor (CD200R) family recognizes CD200R1, and, consequently, the Src kinase
adds phosphate to the three Tyr residues. Downstream of tyrosine kinase (Dok)2, the
adaptor protein recognizes these phosphorylated Tyr regions, especially, Y302/
Y291, and is associated with CD200R1 via the PTB domain. After that,
Ras-GTPase activating protein (RasGAP) recognizes Dok2 to inactivate the Ras
protein, leading to suppression of myeloid cell functions like cytokine production,
calcium release, proliferation, etc. [431].
CD200 and CD200R1 are also glycoproteins. CD200R1 consists of seven gly-
cosylation sites. Among them, three glycosylation sites have GlcNAc linkages and
four of them are putative regions. CD200 consists of eight glycosylation sites, which
also starts with GlcNAc. Many pathogens of bacteria and other parasites use CD200:
CD200R1 signaling for their survival and proliferation. Some of them modify the
expression of CD200 and CD200R1. Toxoplasma gondii increases microglial
CD200R1 and endothelial CD200 on blood vessels. Leishmania amazonensis and
N. meningitidis upregulate CD200 on bone marrow macrophages. The mechanism
modifying CD200 and CD200R1 expression is unclear. It is suggested that patho-
gens exploit or influence the enhancer region that regulates CD200 expression.
Many viruses also use the CD200:CD200R1 signaling pathway for their survival
or mortality. Mouse hepatitis viruses (MHVs) require functional CD200:CD200R1
signaling for their virus genomic replications. The influenza virus exhibits high
pathogenic outcomes with severe mortality on CD200/-deficient KO mice. How-
ever, there is a study that, unlike in the case of CD200-deficient KO mice, in
CD200R1/ KO mice, the virus pathogenic progression is decreased. There are
some virus orthologs of CD200. HHV8 has a vOX2 ortholog, RRV has R15, and the
Myxoma virus has a M141 ortholog [431]. In conclusion, the CD200:CD200R
pathway is involved in immunosuppression and causes blocked cytokine production,
immune responses, proliferation, etc. For some pathogens, induction of immuno-
suppression through CD200:CD200R1 is beneficial, sometimes necessary, for their
survival, proliferation, and pathogenesis. Therefore, such pathogens of bacteria,
parasites, and viruses use the CD200:CD200R1 pathway by modifying expression
or using orthologs of CD200 (Fig. 7.32).
CD200 also enhances graft survival [507], reduces fetal loss [508], and increases
tumor cell growth [509]. CD200 overexpression protects autoimmune inflammation
in EAE [485], autoimmune uveoretinitis (EAU) [510], CIA [511], and inflammatory
neurodegeneration [511]. For the functional analysis of CD200 in DSS-induced
colitis, doxycycline-reactive CD200tg mice and CD200KO/CD200R1KO mice
were used. CD200 enhancement protects colitis even in acute and chronic types
during DSS progression. In contrast, colitis is observed in CD200 or CD200R KO
mice [512]. CD200-deficient KO mice and CD200R1-deficient KO mice are highly
sensitive to the acute type of colitis, compared to wild-type animals, in the parameter
outcomes including body weight loss, macrophage–neutrophil infiltration and CD3+
cell infiltration, and macrophage-produced inflammatory cytokines. In contrast,
CD200tg mice are resistant to DSS, when compared to wild-type mice. In chronic
colitis models, Foxp3+ Treg infiltration is often observed in the CD200tg mice
colons than in those of wild-type mice. In addition, CD25-reactive mAb adminis-
tration inhibits protection. The CD200:CD200R axis immunoregulates in
DSS-induced mice colitis. Foxp3 + Treg cells maintain tolerance and suppress
inflammation but defects induce the IBD pathogenesis [512]. In mouse IBD models,
Tregs suppress disease when CCR4 is absent [513].
7.13.11.6 Cannabinoid (CB) Receptors Also Upregulate CD200–

CD200R Interaction and Expressions
Cannabinoids (CBs) including endocannabinoids (eCBs) and exogenous CBs regu-

late anti-inflammatory activities on macrophages and microglia through activation of
the immune inhibitory CD200 and CD200R. CD200R agonists like fusion protein
CD200Fc exhibit anti-inflammatory activities through suppressed macrophage and
microglial immune responses. CD200–CD200R interaction is upregulated by can-
nabinoids, where CBs promote the CD200–CD200R interaction. CBs are therapeu-
tic targets [514]. eCBs protect against neurodegenerative and inflammatory damage.
For example, an eCB, AEA, protects through the CB1/CB2 receptors, inducing the
expression of CD200/CD200R genes [515] and CD200–CD200R binding. This is a
new strategy to prevent inflammation and axonal demyelination [516]. In the last
two decades, cannabis plant extracts containing cannabinoids have been used as anti-
inflammatory agents with remyelination [517]. CBs are anti-inflammatory and
neuroprotective, promoting oligodendrocyte survival [518]. In signaling, CBs bind
to GPCRs, CB1 and CB2 receptors, and peroxisome proliferator-activated receptors
(PPAR)-γ and PPAR-α [519]. CB1 receptors are expressed on neurons [520] and
leukocytes [521], whereas CB2 receptors are expressed on immune cells, sharing
44% protein identity with CB1 receptors. Delta-9-tetrahydrocannabinol (THC) binds
to CB2 receptors [522], reducing inflammation through apoptosis of immune cells
and inhibition of macrophage’s antigen presentation to Th cells.
7.13.11.7 Siglec-9 in Neutrophil Function and T Cells
The sialic acid-based pathogenic evolution is detailed in a Japanese homepage

(www.glycoforum.gr.jp). CD33-related Siglecs are indeed PRRs with immune-
regulatory activity. Although Siglecs have a low expression on T cells, they bind
to various sialoglycan ligands that act as SAMPs and thus attenuate autoimmune
responses. PRRs including TLRs or NOD-like receptors recognize PAMPs or
DAMPs as pathogenic or dangerous signal information. Engagement of PRRs by
PAMPs or DAMPs activates immune cell responses. PRR binding to DAMPs and
PAMPs also provokes tumor progression and antitumor immune responses
[54]. Siglec-9 is a key CD33rSiglec and is mainly expressed on DCs macrophages,
monocytes, and neutrophils [523]. Siglec-9 binds to SAs in a cis-type interaction on
neutrophil surfaces to diminish immune responses. Siglec-9–TLR-4 interaction
induces septic pathology by the polarization of macrophages and inhibition of
neutrophil activation. TLR-4-mediated murine Siglec-E activation is performed
MyD88-dependently and this attenuates TLR-4 signaling upon treatment with
LPSs [245]. Siglec-E is also involved in the E. coli-induced endocytosis of TLR-4
in inflammation [156]. Siglec-E-lacking DCs fail to internalize TLR-4, causing high
levels of proinflammatory cytokine expression during infection of E. coli strains.
Siglec-E controls the septic response with TLR-4, maintaining a normal cytokine
balance. Siglecs regulate macrophage function in a manner such that Siglec-9 or
mouse counterpart Siglec-E inhibits proinflammatory cytokine production in mac-
rophages, when the cells are induced with TLR ligands [153, 483]. Mouse Siglec-G
also inhibits the macrophage antiviral potentials [524]. IL-4 elicits macrophage
effector function when IL-4 recognizes the IL-4 receptor, which binds SHP-1
through the ITIM; however, Siglec-9 strengthens the signaling pathway of the
MEK/ERK/PI-3 K/Akt axis in macrophages induced by IL-4 [487]. This indicates
that Siglecs are increasingly expressed on blood monocytes but are weakly
expressed on BM-derived macrophages.
Siglec-9 as an MHC-independent inhibitory receptor is exclusively present on
NK cells, without more information on its role in infectious diseases yet. Currently,
the regulatory function of Siglec-9 on NK cells in the pathogenesis is not well
understood. Sialoglycans act as SAMPs via binding to CD33rSiglecs that consist of
an ITIM [525]. In contrast to the conserved common Siglecs including Siglec-1, -2,
-4, and -15, the CD33rSiglecs family, such as Siglec-3, -5, -6, -7, -8, -9, -10, -11, -14,
and -16, is suggested to have rapidly evolved by pathogenic interactions through
sialyl-SAMPs and by interaction with inhibitory receptors, CD33rSiglecs, to escape
from immune surveillance [526]. T-cell-drived killing assay of tumor cells has been
developed, which use Catumaxomab specific for T cell CD3/Tumor cell EpCAM as
a T cell-bispecific antibody, tumor cell apoptosis is observed in the combined
treatment system of tumor cell + T cells + Catumaxomab by detection of apoptosis
marker (cleaved Caspase 3) [526]. They constructed HS9 mice which selectively
express the human Siglec-9 in cell produceing Cre recombinase. Then, the HS9/
CD4-Cre mice cross mating exhibit the selective Siglec-9 expression in CD4+ T
Siglec-9
V-set
Ig domain Sig-E16
C-set
Sig E
Sig 16 : activating
ITIM
Fig. 7.33 Human Siglec-9 as an inhibitory CD33rSiglec. Siglec-9 is composed of a SA-binding

domain, Ig domain, and an ITIM. The inhibitory signal is formed by the ITIM domain. It has a broad
binding spectrum. Siglec-9 binding sialosides are such Neu5Ac α2,6 or α2,3 glycosidic linkage to
Gal. It is expressed on monocytes, neutrophils, and DCs. The murine functional counterpart is
Siglec-E
Fig. 7.34 HS9 mice and

CD4-Cre mice cross-mating HS9 mice: selective expression of human Siglec-9 in
to selectively generate cells producing Cre recombinase
Siglec-9 in CD4+ and CD8+
CTL cells (strategy obtained
from Ref [527] Stanczak
MA et al., 2018. J Clin HS9 mice X CD4-Cre mice: selective expression of
Invest. 128(11):4912–4923)
Siglec-9 in CD4+ and CD8+ cells
cells and CD8+ CTLs (Fig. 7.33). Siglec-9 expression is enhanced on CD8+ tumor-
infiltrating lymphocytes (TILs) and Sig9 + CD8+ TILs coexpress PD-1highTIM-
3+LAG-3+, which are inhibitory receptors. Although in Sig9 + CD8+ TILs as the
intratumoral CD8+ T cells, SA-SAMPs suppress T cell–driven cytotoxic activity
against tumor cells, T cells in tumors are promoted by targeting Sia-SAMP/Siglec-9
interaction because sialyl SAMPs elevate immune escape and tumor growth
(Fig. 7.34).
Synthesis of sialoglycans on tumor cells and expression of Siglec-9 are both
related to immune suppression. Tumor cell glycosylation influences tumor pheno-
types and progression. Tumor sialoglycans allow immune evasion, tumor growth,
invasion, and metastasis through Siglec ligand–cis/trans-interaction. Siglec-14, -5,
and -9 are mainly present on neutrophils. SA-receptor coevolutionary adaptation
may create some adapted Siglecs to counteract the immune evasion of pathogens.
The SIGLEC-5 genes undergo genetic conversion events with the SIGLEC-14 gene,
as Siglec-14 is functionally similar to Siglec-5 but assembles with the DAP12
adaptor bearing an ITAM, not an ITIM. Siglec-14 is absent in humans due to the
fusion event of SIGLEC-5/-14 genes [528]. The paired receptor has activating and
inhibitory functions with a homologous glycan-binding motif [114, 529]. During
activation, the paired receptors trigger reversely directed signaling to maintain a

balanced immune response [117]. The conceptional explanation of paired receptors
has been accepted for two paired Siglecs, Siglec-5/-14 and Siglec-11/-16 pairs;
nonetheless, an actual demonstration with proven evidences has not yet been
obtained [117]. Siglec-14 functions in counterbalancing Siglec-5 suppression of
GBS β-protein-mediated activation on leukocytes. Siglecs are expressed on leuko-
cytes, and Siglec-5 and -14 are rarely expressed on the amniotic sac of a fetus. In
fact, gene polymorphisms in the human SIGLEC-14 and SIGLEC-5 affect amniotic
epithelial inflammation by a neonatal pathogen GBS passed through the fetal
placental membranes. Lack of Siglec-14 influences the risk of premature
delivery [108].
Upon recognition of SA ligands present on cell surfaces, Siglec-9/-E suppresses
immune responses of host cells. Siglec-9/-E modulates neutrophil functions such as
apoptosis, inhibition of cellular activation, migration attenuation, oxidative stress
regulation, and inflammatory cytokine production, although its natural sialylglycan
ligands are not well known. Cancer cells are frequently aberrantly glycosylated with
sialylation. For example, MUC1 is expressed on cancer cells with multiple sialylated
O-linked glycans. Cancer-specific MUC1 glycoform induces leukocytes to express
tumor-associated microenvironment remodeling and cancer progression via MUC1
interaction with Siglec-9. Sialyl MUC1 induces neighboring macrophages to enable
to a TAM-like phenotype change with an immune checkpoint inhibitor of PD-L1
expression. Sialyl MUC1 binding to Siglec-9 blocks SHP-1/-2 activation [467]. Sol-
uble Siglec-9 in mice functions as an antitumor molecule against MUC1-expressing
tumors, in a mechanism such that MUC1 binding to immune cell Siglec-9 is
competitively prevented and leads to retarded immunocontrol and blocked tumor-
associated MUC1 signaling [530].
Siglec-9 has a broad spectrum and the capacity to bind glycan ligands, which are
sialylated [530], positioning as a typical PRR for sialyl-SAMPs. Sialyl-SAMPs help
in tumor evasion of host immune responses through signaling of inhibitory Siglecs
of CD33rSiglecs such as Siglec-7/-9 [467, 531, 532] because Siglec-7 and -9
suppress NK cell-driven cytotoxic death of tumor cells [532]. Human Siglec-9 and
homologue Siglec-E of murine are directly related to myeloid cell-mediated cancer
progression [531]. A human polymorphism decreases Siglec-9–tumor interaction,
and this indicates the early survival in NSCLC patients. Macrophage Sigle-9 inter-
action with cancer-associated sialyl MUC1 induces a TAM phenotype to stimulate
tumor progression and immune evasion [532]. For tumor progression under septic
conditions, macrophage polarization is needed. Therefore, the macrophage pheno-
types in sepsis are important for sepsis and cancer treatment [533]. Siglec-9
enhances Arg1 induction by the MAPK/ERK signaling pathway during IL-4 induc-
tion [487]. Siglec-9 also enhances IL-4-elicted expression of CD200R and inhibits
LPS-stimulated CCR7 expression of human macrophages [534]. The strategic idea
on Siglec-E-targeting therapy of sepsis is potentiated.
Neutrophils occupy approximately 50–70% of leukocytes in vascular circulation
as a first line of defense against innate immune responses in the host defense system
[535]. Neutrophils transmigrate from the vascular stream to infection sites upon
occurrence of pathogenic bacterial or host-expressed chemoattractants, endothelial

adhesion molecules, and inflammatory mediators. As the lifespan of neutrophils is
known to be the shortest among circulating leukocytes, neutrophil lifespan prolon-
gation is needed for better host defense. Neutrophils capture microbes to kill as a
manner of phagocytosis, generate ROS, release antimicrobial agents, and produce
neutrophil extracellular traps (NETs) [531]. However, an overactive neutrophilic
function induces inflammatory host cell damages, destroying the homeostatic state.
Human inhibitory Siglec-9, which is CD33-related, is ubiquitously present on
neutrophils in humans [536, 537], whereas mouse neutrophils express its homo-
logue, mouse inhibitory Siglec-E [104]. Neutrophil Siglec-9 interaction with eryth-
rocyte sialic acids also regulates the neutrophil quiescent status in normal
environments. Erythrocytes are known to suppress neutrophil activation including
bacterial killing or induction of apoptosis. Sialic acids on glycophorin A of erythro-
cytes recognized by neutrophil Siglec-9 dampen neutrophil activation. This may
explain why neutrophils become activated upon isolation from blood. It has been
suggested that sialyl “SAMP” on erythrocytes keep neutrophils in a resting state in
the blood [538].
Siglec-9 is also an inflammation-inducible vascular adhesion protein-1 (VAP-1)
ligand. As a CAM, VAP-1 recognizes endothelial cells related to leukocyte or
monocyte transendothelial migration from the blood to the inflamed sites. VAP-1
translocates from intracellular deposits on the endothelial cell surface to aid in
leukocyte-endothelial adhesion [539]. As Siglec-9 is a VAP-1 ligand, the
radiolabeled oligopeptide 293-CARLSLSWRGLTLCPSK-297 of Siglec-9 has
been used in PET imaging of inflammation [540–543] for the detection of skin/
muscle inflammation in rats.
7.13.11.8 Siglec-9 Functions as a Leukocyte Ligand for Vascular

Adhesion Protein-1 (VAP-1)
Leukocyte surface Siglec-9 belongs to a counter-receptor to bind adhesin expressed

on endothelial cell surfaces, which is the human primary amine oxidase (hAOC3)
enzyme. Leukocyte homing to inflammation sites is mediated by endothelial adhe-
sion species including VAP-1. The adhesive protein VAP-1 undergoes an enzyme
activity to oxidize primary amines to produce hydrogen peroxide, aldehyde, ammo-
nium [544], and an adhesin. AOC3 is a member of the semicarbazied-sensitive
copper containing amine oxidase family and primarily oxidizes amines to aldehyde.
It interacts with leukocytes through sialylated carbohydrates found on its surface.
The hAOC3 structure is a heart-shaped homodimer, where each monomer has three
domains, D2, D3, and D4. The active site is buried in the D4 domain with catalytic
2,4,5-trihydroxyphenylalanine quinone (TPQ) residue, catalytic Asp, and three
His-coordinated copper ions. The enzymatic products are inflammatory mediators
and can elicit the expressions of P-/E-selectin and VCAM-1/ICAM-1
[545, 546]. VAP-1 binds to Siglec-9 in the catalytic groove in VAP-1 [540]. For
detection, positron emission tomography (PET) using the 68Gallium-labeled Siglec-
6WOQTGPXKTQPOGPV *KIJHWPEVKQPCN UWDUGV

2&JKIJ6+/ .#)
5KI %& 6+.

6WOQTEGNN
5KC5#/2JKIJ
+PJKDKVKQP UKIPCN
6 EGNN KPJKDKVKQP
5KC5#/25KI
6WOQTEGNN MKNNKPI FGETGCUG
KPVGTCEVKQP
6WOQTITQYVJ KPETGCUG
Fig. 7.35 Sia-SAMP/Siglec-9 pathway as a class of inhibitory immune checkpoints for T-cell
activation (derived from Ref [527] Stanczak MA et al. 2018. J Clin Invest. 128(11), 4912–4923)
9 peptide can recognize VAP-1 in vascular inflammation sites or cancerous sites.

Siglec-10 also acts as a ligand for VAP-1 [547], although it is present on B cells,
monocytes, and eosinophils but not on granulocytes. Endothelial VAP-1 (AOC3)
acts as a recognition candidate for inhibiting inflammation because it is translocated
from the cellular granule stores to the endothelial cell surfaces for delivery of
granulocytes, lymphocytes, and monocytes. The VAP–Siglec-9 or hAOC3–Siglec-
9 interaction can diagnose inflammation state and cancer, since the Siglec-9 C2
domain (C22), which is labeled, recognizes the inflammatory hAOC3 enzyme
[548]. Siglec-9 binding to the enzyme catalytic groove of hAOC3 is caused by
two arginines, Arg284 and Arg290, as binding sites. R3 occupies the hAOC3 active
cavity near TPQ and R9 binds to the N2 glycan and D4 domain of hAOC3
(Fig. 7.35). Siglec-9 peptide binding to the immobilized hAOC3 in the condition
of semicarbazide (SC) and imidazole inhibitors is influenced. N-glycosylated
hAOC3 binds to the Siglec-9 peptide. The Siglec-9 binding to hAOC3 is crucial
for therapeutic and diagnostic agents [549]. As the peptide
(CARLSLSWRGLTLCPSK) on Siglec-9 binds to hAOC3, it can be used as a tracer
in hAOC3-specific PET detection. The Siglec-9 peptide binding abolishes the
interaction between wild-type with the Siglec-9 peptide and hAOC3. Siglec-9
enhances the catalytic activity of hAOC3.
7.13.11.9 Siglec-9 Recognition with the Pathogenic Group B

Streptococcus (GBS) Sialic Acid Glycan Capsule and Group
a Streptococcus (GAS) Hyaluronan Capsule
CD33-related Siglecs on human leukocytes are a rapidly evolving family with an

ITIM and/or ITIM-like sequences for sialoglycan recognition to elicit inhibiting
Siglec-9
Cyclic
hAOC3 pepde
N2
TPQ
cofactor
Fig. 7.36 Siglec-9 interaction with the enzyme catalytic groove of hAOC3 by two arginines,
Arg284 and Arg290 (R3 and R9). Interaction sites: R3 occupies the active cavity of hAOC3 near
TPQ and R9 binds to the surface N2 glycans and the D4 domain of hAOC3. The interaction triggers
catalytic activity of hAOC3
signals. Endogenous sialoglycans or SAMPs dampen leukocyte activation. Recep-

tors of inhibitory CD33-related Siglecs (CD33rSiglecs) negatively modulate the host
immune responses upon engagement of their own Siglecs and own sialic acids on
host cell surfaces. Sialic acids expressed during sepsis are recognized by Siglecs.
Differentially expressed Siglec-9 mediates cytotoxic death responses of neutrophils
in septic shock conditions. Therefore, host-sialylated glycan mimicry allows micro-
bial pathogens to recognize Siglec-9 on neutrophils and dampen the immune
responses of innate immunity because Siglec-9 inhibits neutrophil responses and
pathogens mimic host sialic acids. When LPSs are recognized by TLR-4, Siglec-9 is
induced. Induced Siglec-9 negatively regulates TLR-4 responses. Moreover, Siglec-
9 helps internalize TLR-4, when cells are infected by E. coli. If TLR-4 is not
internalized, levels of proinflammatory cytokines increase. When neutrophils are
stimulated by LPSs, Siglec-9 inhibits neutrophil recruitment to the inflammation
sites. Neutrophils, which have immobilized multimerized Sia α2,3-Galβ1-4GlcNAc
units, interact in a trans-manner through Siglec-9, and bacterial mimicry of sialic
acid can engage neutrophil Siglec-9. GBS with similar sialylated glycans of neutro-
phils can evade neutrophils’ killing activity. In the survival of wild-type GBS, GBS
(-Sia), and group A Streptococcus, which are nonsialylated bacterium, Siglec-9
engagement by GBS reduces neutrophils’ killing activity. Neutrophils cannot rec-
ognize GBS because Sigleg-9 binds to GBS’s sialic acid, and neutrophils’ killing
activity is reduced (Fig. 7.36).
Neutrophils recognize pathogens in a trans-binding of Siglec-9 to SAs. Peripheral
blood neutrophils kill the invading pathogens as the first line of defense against
pathogens, by NETs along with extracellular phagocytosis. NETs are a specialized
killing system that helps neutrophils to eliminate pathogens. Neutrophil-mediated

cell death by NETs is called “NETosis.” Granular and chromatin proteins capture
pathogenic agents and express the agents against the pathogenic microbes to
develop NETs.
However, GBS sialic acids interact with Siglecs on the surface of leukocytes.
GBS SA recognition in human Siglecs inhibits leukocyte activation. As confirmed
by Siglec-E-deficient mice, GBS is engaged with Siglecs in infection [550]. In
pathogenic bacteria of GBS and P. aeruginosa, their surface sialic acids inhibit the
host Siglec-9 function, resulting in blocked neutrophil activation and consequent
bactericidal activity. The human bacterial pathogen GBS synthesizes SA mimicry in
polysaccharide capsules on their surfaces to engage Siglec-9 and mSiglec-E toward
suppression of neutrophil function. In human pathogens, including GBS,
H. influenzae, E. coli K1, N. gonorrhoeae, and T. cruzi, terminal sialylglycans
induce molecular mimicry of host sialylglycans, thus enabling to potentiate activa-
tion of inhibitory Siglecs, dampening of leukocyte activation, and suppressing of
immune clearance [551]. GBS with capsule serotype III binds to Siglec-9 expressed
on human neutrophils and, consequently, inhibits the oxidative bursting out, NET
secretion, and pathogenic bacterial death. The sialyl-LOS core of C. jejuni strains
recognizes Siglec-7 expressed on DCs and consequently suppresses the function of
T cells [322]. In addition to SA recognition, some GBS species also engage another
Siglec-5 via β-protein [552]. GBS β-protein-associated ITIM-having Siglec-5 moti-
vates the inhibiting SHP-2 signaling pathway to block activation and phagocytosis
of macrophages [350].
Some pathogenic bacteria utilize mimicry of SAs on their own surfaces
containing sialylglycans to evade immune surveillance of the host immune
responses via engagement of Siglecs. For example, by generating molecular mimicry
of SAs, SA-containing GBS pathogens bind inhibitory Siglec-9 and attenuate
activation of neutrophils and consequently evade host immunity and safely grow
in the infected hosts. Using an artificially created soluble form of Siglec-9, which is
the extracellular domain (ExD) of Siglec-9, it has been demonstrated that Siglec-9
upregulates the neutrophil immune response to induce an antibacterial activity
against GBS infection, as a mechanism that inhibits capsular polysaccharide binding
to Siglec-9 [553]. GBS produces terminally α2,3-SA-linked ligands in polysaccha-
ride capsules of bacterial cell surfaces, which is also a molecular mimicry to the
host SAs.
Apart from GBS, another pathogen, group A Streptococcus (GAS), produces a
high-molecular-weight hyaluronan capsule that also engages hSiglec-9, blocking
neutrophil activation. On a structural basis, Siglec-9 binds high-molecular-weight
hyaluronan of host glycans through the Ig-like V-set domain in the N-terminal
region different from the SA recognition region. Therefore, Siglec-9 recognition of
hyaluronan also restricts neutrophil activation, in a pattern similar to those of sialic
acids. Thus, hyaluronan is a modulator of neutrophil activation. Siglec-9 as a single
inhibitory lectin receptor recognizes two different glycan motifs as SAMPs to
regulate neutrophil function, indicating that pathogens have independently adapted
to express glycan mimicry to manage immunoinhibition.
Table 7.7 Siglecs that interact with pathogens [554]

Pathogens Siglecs Sialic acids of pathogens Responses References
N. meningitidis Sn/Siglec-1, LPS-SAs Binding, [6]
Siglec-5 phagocytosis
C. jejuni Sn/Siglec-1, LPS-SAs Th cell [7]
Siglec-7 differentiation
GBS type III Siglec-9 LPS-SAs Immune [10]
attenuation
P. aeruginosa Siglec-9 SAs (derived from human Immune [11]
fluid glycoproteins) suppression
T. cruzi Siglec-E SAs (derived from human Immune [12]
(mouse) glycoproteins) suppression
HIV Sn/Siglec-1 gp120-SAs Infection [13]
GBS surfaces are coated with a capsule. The cell capsule is an extremely large
structure of some prokaryotic bacterial cells. Among the polysaccharide layers
outside the bacterial cell walls, the capsules are present in both Gram-positive and
Gram-negative bacteria. They are different from LPSs and lipoproteins found only in
Gram-negative bacteria. The GBS surface capsule is characteristic of sialic acids that
bind to Siglecs on leukocytes. GBS surface polysaccharide capsules have an
antiphagocytic property, in terms of the mechanism in which sialic acids of the
capsule disturb the binding of the complement C3 component to the cell surface,
consequently blocking the alternative pathway. The transplacental delivery of
anticapsular IgG antibodies from a mother to her infant protects against bacterial
diseases. In general, the GBS type A of S. agalactiae is commensal as the human
microbiota colonizes the gastrointestinal and genitourinary tracts of healthy humans.
Another type, GBS type B (S. agalactiae), is the major cause of neonatal pneumonia,
septicemia, and meningitis. GBS vaccination against this Gram-positive encapsu-
lated bacterium includes polysaccharide-based vaccines, native polysaccharide vac-
cines, polysaccharide–protein conjugate vaccines, multivalent conjugate vaccines,
carrier proteins and adjuvants, and protein-based vaccines. Table 7.7 lists Siglecs
that interact with pathogens. Pathogens inhibit Siglec function to neutralize activated
Siglecs. The activated Siglecs recognize the pathogens, triggering immune responses
to survive. From the interaction between Siglecs and pathogens, immunity is
decreased because pathogens inhibit the Siglecs (Fig. 7.37). The bacteria synthesize
sialic acids to interact with the host Siglecs, utilize the host’s sialic acids, and
produce Siglec-interacting proteins.
7.13.11.10 Siglec-9 Recognition with Pathogenic Pseudomonas

aeruginosa Attenuates Innate Immune Responses
P. aeruginosa is a Gram-negative, rod-shaped, and opportunistic pathogenic bacte-

rium. It causes pneumonia and chronic lung diseases in immunocompromised hosts
[555]. P. aeruginosa, an opportunistic pathogen, uses sialic acids to evade host
5KCNKECEKFU
2CVJQIGP
5KINGE
Siglec-9 bind to sialic acids on pathogen
inhibit neutrophil activity
ROS, NET, granule proteins
0GWVTQRJKN
Fig. 7.37 Interaction of Siglec-9 and bacteria such as Pseudomonas aeruginosa (modified from
Liu YC et al., 2017. Front Immunol. 8:1601) [245]
immunosurveillance and neutrophil activity. Sialic acid–Siglec-9 binding in PA and

neutrophils potentiate to subvert immune responses in the host. This colonizing
pathogenic bacterium is involved in AIDS or immunosuppression in hosts, cancers,
nosocomial infections, chronic cystic fibrosis, and transplantation. The PA species
adsorb SAs in the cultures and use them to block complement C3 deposition on their
cell surfaces [556]. PA utilizes such sialic acids to interact with Siglecs on immune
cells, such as peripheral blood neutrophils, to subvert innate immunity which is the
first line of defense. A pathogen infection recruits neutrophils and the cells undergo
phagocytosis via ROS and ROI production as well as NETosis of NET formation by
ROS production, where NETs (neutrophil extracellular traps) dissociate to form
chromatin and proteins (Figs. 7.37 and 7.38).
PA+Sias bind to human Siglec-9. Consequently, the binding of PA surface
SAα2,3-glycans to Siglec-9 on neutrophils stimulates production of TGF-β and
IL-10 by neutrophils, although the underlying molecular mechanisms regarding
how PAs survive in host-defense environments have not yet been explained. Sialic
acid-producing PAs are associated with α2,3-linked Sia–Siglec-9 interaction in
neutrophils for survival by subverting innate immune responses. The sialic acids
on PA protect them from neutrophils, reducing ROS, protease secretion, and NET
formation [556].
When the host is infected with bacteria or viruses, the host innate immune cells
are recruited to the infected tissue [68, 554, 555]. Neutrophils are the first-to-be-
infiltrated immune cells of an infected tissue. Neutrophils produce ROS to burst
bacterial cells. Siglec-9 is expressed on neutrophil surfaces, inhibiting immune
activity when binding to the sialic acid ligand for the ITIM. Many opportunistic
pathogens, such as P. aeruginosa, have many SAs on their surfaces, thus exhibiting
Pseudomonas aeruginosa Siglec-9

(Sias negative) 415 ↑
0'6U
Innate immune
HQTOCVKQP
system ↑
Neutrophil
Siglec-9
Pseudomonas aeruginosa 415 X Siglec-9

Siglec-9 (Sias positive)
0'6U Anti-inflammatory cytokines
HQTOCVKQP: IL-10 , TGF β production ↑
Neutrophil
+.6)( β
(O)
Neutrophil
Sias (Sialic acid) PA
SURVIVE
Fig. 7.38 Interaction between Siglec-9 and bacteria such as Pseudomonas aeruginosa. The Siglec-
9 and Sias engagement leads to suppressed ROS and NET formation in neutrophils (modified from
[555] Khatua et al. 2012. J. Leukoc. Biol. 91, 641–655)
neutrophil activity; neutrophils construct extracellular fibrous networks to interact

with pathogens. As a result, morphological changes in neutrophils are observed.
Such a function is suppressed after the engagement of neutrophils with pathogenic
sialic acids through Siglec-9–sialic acid binding [245, 557].
7.13.12 Siglec-10 (Mouse Ortholog Siglec-G) in Humans

as a CD33-Related Siglec
7.13.12.1 General Aspects of Siglec-10 (Mouse Ortholog Siglec-G)
Siglec-10 is a member of the CD33rSiglecs family. As most of the Siglecs have their
specificities toward the terminal SAα-2,3 or SAα-2,6 positions, attached to the Gal
residue of the targeting glycans, Siglec-10 recognizes α2,3-SA- or α2,6-SA-linked
glycoconjugates. Siglec-10 is mainly present on human leukocytes of B cells,
eosinophils, macrophages, and monocytes. The role of human Siglec-10 on DCs
has been elucidated in innate and adaptive immunities. Among the two sialyl
linkages, α2,3-SA is the preferred residue to bind to Siglec-10 for bioactivity. This
indicates that α2,3-SA is the major SA–Gal linkage binding for Siglec-10. Siglec-G
is a mouse ortholog of human Siglec-10. They share a similar sequence homology
and chromosome locus [245, 558]. Siglec-10 has several extracellular Ig-like
domains, one TM domain, and cytosolic tail ITIMs. The N-terminal V-set domain
of Siglec-10 recognizes SA as a ligand. Human Siglec-10 and Siglec-G, which is a
mouse ortholog, are broadly expressed on DCs, macrophages, and B cells. Owing to
ethical issues and limitations in humans, functional and analytical studies of Siglec-
10 have been carried out with the mouse ortholog Siglec-G. Moreover, due to mouse
application, human Siglec-10 has been studied using mouse ortholog Siglec-G to
infer the human Siglec-10. This is also due to the structural and functional similar-
ities between Siglec-10 and Siglec-G.
In reality, to distinguish between the ‘self’ and ‘nonself’ in immune response, the
antigen-recognition and immune response-regulatory capacity of DCs are first deter-
mined. Siglec-10 expressed on DCs is used to discriminate self from nonself. Thus,
Siglec-10 is considered as one of the self- and nonself-recognizing mechanisms.
DAMPs including HSPs, HMGB1, IL-1a, IL-33, and calreticulin are known. Siglec-
10 is indeed a negative regulator of immune responses that occur by the cytoplasmic
ITIM-recruited SHP-1 signaling pathway, and the activity is expressed by DAMP
signaling. Siglec-10 can recognize host origins, DAMPs, which are induced by host
cell necrosis or other cell deaths. However, Siglec-10 cannot be activated by a
pathogen of foreign origin, PAMPs [301]. Siglec-10 only can regulate inflammation
against antigens of self-origin. It suppresses inflammation by promoting IL-10
expression and interacting with CD24. It reduces DC and T-cell interaction by
inhibiting MHC-I production. It also binds to the BCR and reduces B-cell responses.
The functions of binding of Siglec-10–CD24 sialyl ligands have been exploited by
treatment of bacterial sialidase in sepsis [559]. Moreover, some infectious pathogens
use the interaction between sialic acid and Siglec-10 to afford immune suppression
in the host [560]. In addition, the interaction between CD52 and Siglec-10 is one of
the mechanisms responsible for inactivation of autoantigen-specific T cells
[319]. Therefore, Siglec-10 could be a subject of related research studies on auto-
immune and inflammatory diseases.
Siglec-10 expression is found on DCs, B cells, macrophages, PBMCs, and even
in T cells, splenocytes, and the liver in humans. Like most Siglecs, Siglec-10 also has
ITIMs in its cytoplasmic tail with immune-regulatory functions. Siglec-10 was first
discovered on human DCs obtained from PBMCs in 2001 [561]. The Siglec-10
protein sequence has high homologies with Siglec-5 and Siglec-3. The Siglec-10
protein structure mostly has a higher similarity with those of Siglec-6, -7, and -9 than
with the others [562]. The well-known ligand for Siglec-10 is CD24, a
glycosylphosphatidylinositol (GPI)-anchored protein. Siglec-10 recognizes α2,3-
and α2,6-linked sialylglycans of CD24 [301]. As a receptor for sialosides on
CD24, Siglec-10 presents immune inhibitory functions through a cytoplasmic
ITIM signal, as observed in other Siglecs. Siglec-10 binding to CD24 activates
cytoplasmic ITIMs to be phosphorylated and to associate with SHP-1 or SHP-2.
Downstream signaling of SHP inhibits inflammatory signaling of PRRs like TLRs
[301]. As SHP-mediated inhibitory signaling is known as the acting signal of ITIM
containing Siglecs, Siglec-10 functions as an immune regulator. However, for better
understanding of Siglec-10, structural studies on the exact action mechanism of
Siglec-10 are anticipated.
7.13.12.2 Human Siglec-10 and Siglec-G as a Mouse Ortholog

in Immune Responses and Inflammatory Process
For Siglec-10 function, some analogical studies using the mouse homologue Siglec-
G have been conducted to infer the function of human Siglec-10 in inflammatory
disease models. In pathogenic inflammation, pathogens are recognized by PRRs of
host innate immune cells. In some cases, ‘self-’ molecules can elicit an inflammatory
response in even undesired conditions. The inflammatory “self” is called DAMPs,
and infectious pathogenic inflammatory “nonself-” molecules are called PAMPs.
Both DAMPs and PAMPs are recognized by host TLRs, indicating the host distinc-
tion between ‘self’ and ‘nonself.” First, in inflammation, the regulatory function of
the GPI-anchored protein CD24 is associated with Siglec-10 action (Fig. 7.39)
[301]. The CD24 structure is similar to that of CD52 [562] that binds to Siglec-10
by associating with DAMP proteins such as HMGB1 [301]. HMGB1 is known as a
mediator of inflammation. CD52 cDNA encodes only 12 amino-acid, lipid-anchored
membrane glycopeptides of lymphocytes. Similarly, mouse cDNA for a small-
surface glycopeptide as the mouse counterpart of CD52 was also found in lympho-
cytes [563]. CD52 sequences are not homologous to all mammals, but with only a
proline residue at the anchor attachment site in the C- and N-terminal N-glycosyl-
ation sites. However, the peptide sequences of other lipid-anchored glycoproteins of
CD24 and CD59 are limitedly similar between species. The short peptides function
as a scaffold for carbohydrate presentation, while the cell type-specific lipid and
carbohydrate moieties determine their fates.
1VJGTEGNNU
#WVQCPVKIGP
$KPFVQUKCNKECEKFU
$KPFVQUKCNKECEKFU
5KINGE

5KINGE)KP
OWTKPG
2JQURJQT[NCVKQP
4GETWKVKPI5*2
%C UKIPCNKPI
Conventional B cells
Fig. 7.39 Siglec-10 (Siglec-G in mouse) in the septic immune response. Siglec-10/-G reduces the
inflammation by DAMPs with the help of CD24. DAMPs, danger-associated molecular patterns
HMGB1 is a nuclear nonhistone DNA-binding protein and has transcriptional

activities. It is now known as a DAMP to induce immune-inflammatory states such
as sepsis [564]. HMGB1 consists of two regions, an anti-inflammatory region named
as Box-A and a proinflammatory region named as Box-B, and a tail region with an
acidic C-terminal region comprising amino acids. Upon cellular necrosis and
pyroptosis, it is actively released by both macrophages and monocytes. HMGB1 is
thus regularly found in the circulatory system [565] and is upregulated in inflam-
matory conditions, but it is not clear how it regulates immune-inflammatory homeo-
stasis. CD24 and Siglec-G block proinflammatory signals in sepsis [245]. HMGB1
produced by necrotic death cells binds to CD24. The CD24 glycoprotein expressed
on DCs recognizes the inhibitory Siglec-G expressed on the same DCs and sup-
presses HMGB1-triggered TLR induction. The inhibitory feedback function sup-
presses TLR signaling caused by DAMPs. Such inhibitory receptors in systemic
sepsis indicate that bacterial neuraminidases cause heavy inflammatory damages by
digesting the SAs linked to CD24 glycans, which are crucial for Siglec-G interaction
or Siglec-10 in humans. Therefore, neuraminidase inhibitors can block bacterial
neuraminidase from dampening the sepsis. Instead of modulation of the Siglec
ligands, pathogens also control the expression of Siglec itself. For example, RNA
viruses enhance the expression of Siglec-G on macrophages via RIG-I-proteasomal
degradation, consequently reducing the IFN level and viral spreading.
Siglec-G on DCs inhibits CD8 T-cell proliferation. Siglec-G recruits SHP-1 that
dephosphorylates NADPH oxidase in phagosomes, and CD24 binds to Siglec-G by
associating with DAMPs and negatively regulates the stimulatory activity. One of
the CD24 ligands is HMGB1, a representative DAMP. HMGB1 directly interacts
with CD24. AAP-treated CD24-deficient mice show enhanced hepatotoxicity, and
blocking HMGB1 reduces liver damage in AAP-treated mice. Thus, CD24 inhibits
liver inflammation through reducing immune responses to HMGB1, indicating that a
dampening immune response mechanism of HMGB1 is based on CD24–Siglec-10
binding. As CD24 is a ligand for Siglec-10, HMGB1/CD24/Siglec-10 interaction is
CD24-dependent2. This interaction regulates AAP-induced hepatotoxicity, as regu-
lated by DCs because DCs express CD24 and Siglec-10. In addition, stimulation
with PAMPs did not activate the CD24/Siglec-10 inhibitory signal on DCs. After
HMGB1 stimulation, the CD24/Siglec-10 inhibitory signal is activated, and the
following SHP-1 downstream pathways inhibit the NF-κB signaling pathway of
TLRs. Moreover, inflammation by HMGB1 (DAMPs) in CD24- or Siglec-10-
deficient mice is enhanced, but inflammation by LPSs (PAMPs) has no effect.
Thus, DAMP-mediated inhibition of the TLR-NF-κB signaling pathway through
Siglec-10 activation could be the ‘self/nonself’ distinction mechanism. Second,
DAMP-induced inflammation is suppressed by CD24/Siglec-10 interaction in a
mouse sepsis model [559]. Sepsis-causing bacteria produce microbial sialidases
and the enzymes eliminate sialic acid moiety on CD24, losing its binding capacity
to Siglec-10 [559]. CD24 expressed on DCs can inhibit inflammation by DAMPs
due to sepsis induction. However, elimination of sialic acids in CD24 on DCs by
microbial sialidase treatment exacerbates sepsis, and this effect is HMGB1-
independent. Microbial sialidases of bacteria interfere in the interaction between
CD24 and Siglec-10 and cause severe inflammation. Finally, Siglec-10 has been
known to lead DCs to become tolerogenic in a bacterial infection model
[560]. C. jejuni mainly causes gastroenteritis and is an intestinal infection model.
Some enteropathogens use various strategies to fight against the host immune
system. Siglec-10 is involved in the infection of C. jejuni and promotes an anti-
inflammatory response upon recognition of flagellin of C. jejuni. For example,
although the bacterial flagella are a main target for TLR-5, C. jejuni has TLR-5
evasion capacity, and so the infection with C. jejuni does not activate TLR-5
signaling. In addition, inducing a host anti-inflammatory IL-10 expression is a
major mechanism of many pathogens that leads to effective inflammation. C. jejuni
also uses this strategy. In particular, C. jejuni infection in human DCs induces higher
expression of IL-10 than other enteropathogens, but proinflammatory cytokine
expression was low [560]. That is, C. jejuni does not stimulate inflammatory DCs
but induces IL-10 expression and immune suppression. The key factor of this
mechanism is the flagella of C. jejuni. It has O-linked pseudaminic acid derivatives
in its flagella, which are known to interact with Siglec-10 expressed on DCs
[560]. The target organ of C. jejuni is the intestine, and the major subset of intestinal
DCs is a type of CD11c + CD103+ DCs. Both CD11c + CD103+ DCs and this
subset express Siglec-10 on their surfaces. Therefore, the bacterial pseudaminic acid
moiety can bind to Siglec-10 on intestinal DCs [560]. A direct interaction between
the pseudaminic acid derivative moiety and Siglec-10 leads to high IL-10 expression
on DCs. Downstream NF-κB and MAPK signaling regulates cytokine production of
DCs. In addition, C. jejuni infection activates p38, a protein involved in MAPK
signaling. It means that p38/MAPK-dependent signals induce IL-10 production of
DCs upon an interaction between the pseudaminic acid derivative moiety and
Siglec-10. The question of how Siglec-10 activation induces p38 activation is still
unanswered (Fig. 7.40). Siglec-10 upregulates the expression of IL-10 via MyD88/
p38 MAPK axis signaling [319]. Siglec-G induces an evasion of innate immune
responses during infection of RNA viruses, as these viruses upregulate Siglec-G
expression via NF-κB or RIG-I signaling in macrophages. Siglec-G recruits phos-
phatase SHP-2 and cCbl known as the E3-ubiquitin ligase to the RIG-I complex. It
consequently induces RIG-I cleavage through K48 ubiquitination at amino acid
Lys813 by the c-Cbl E3-ubiquitin ligase. Siglec-G helps protect against the infection
of RNA virus by immunosuppression derived from the inhibition of IFN-I produc-
tion [524]. In Siglec-G regulation, CD24 is involved in host protection from the
exaggerated inflammation in septic condition [566].
7.13.12.3 Siglec-10 in T Cells during Autoimmune Inflammation
The major Siglec-10-expressing cells are DCs, B cells, and macrophages. However,
activated T cells only limitedly express Siglec-10 [319]. In the homeostatic regula-
tion of an individual, excessive or prolonged inflammation is harmful. Naive T cells
can be activated by self-antigens. The activated naive T cells become autoreactive T
cell types and they induce autoimmune responses. However, Tregs inhibit activated
Fig. 7.40 Summary of the immunological role of Siglec-10 in DCs
T-cell functions. This indicates that Tregs are crucial for prevention of autoimmune
diseases. Thus, Tregs are suppressors of autoreactive T cells in autoimmune
responses and they reduce inflammation. As a small GPI-anchored protein, CD24
transduces costimulatory signaling to T cells. In septic conditions, CD24 cooperates
with DAMPs like HMGB1, Hsp70, and Hsp90 to downregulate the stimulation
potentials. In addition, CD24 with Siglec-G cooperation inhibits NF-κB transloca-
tion [301]. Addition of microbial neuraminidase with Siglec-G inhibits the signaling
of the CD24–Siglec-G complex and exacerbates inflammatory responses. Inhibitors
of neuraminidase protect sialyl pattern recognition and rescue mice from cecal sepsis
that occurs by the CD24–Siglec-G interaction [567, 568]. The septic pathogenesis
produces various mediators to induce inflammation, and the CD24–Siglec-G inter-
action regulates the production of mediators. Hence, specific inhibitors of neuramin-
idase inhibit the CD24–Siglec-G binding and these inhibitors can be regarded as
antisepsis candidates. Siglec-G expressed on DCs initiates T cell-mediated antigen
responses. Siglec-G also suppresses cross-presentation of extracellular antigens to
CD8 CTLs through inhibition of MHC-I–peptide complex formation. To do this,
Siglec-G recruits the phosphatase SHP-1 to dephosphorylate the p47phos, known as
a NADPH oxidase protein, to inhibit the NOX2 function in phagosomes [569].
Box B of HMGB1 activates a soluble CD52 binding to Siglec-10. A soluble
CD52 form generated by phospholipase C digestion also recognizes Siglec-10 and
consequently blocks the phosphorylase activity of the TCR-associated kinases of
Zap70 and Lck. T-cell activation is different from the normal function of T cells
[560]. Soluble CD52 requires HMGB1 box B to bind to Siglec-10. α2,3-SA in Sia
α2,3-Galβ1 is important for CD52–HMGB1 interaction. α2,3-sialylated CD52-Fc
glycan is the binding site of HMGB1. HMGB1-bound α2,3 sialyl-CD52-Fc sup-
presses the function (Fig. 7.41). CD52 rapidly induces Siglec-10 tyrosine
%&(E
HGMB1 Sialic acid
%&(E
TCR %&(E
Siglec-10
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2
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T cell activation
Fig. 7.41 Soluble CD52 associates with HMGB1 box B to bind to Siglec-10 and the HMGB1–
CD52–Siglec-10 complex recruits SHP-1 phosphatase and inhibits T-cell function via binding to
the TCR for T-cell inhibition. A soluble CD52–HMGB1 box B–Siglec-10 complex is formed. α2,3-
sialyated linkage of Sia α2,3-Galβ1 CD52-Fc binds to HMGB1. The HMGB1–CD52-Fc–Siglec-10
complex binds to the TCR and recruits SHP-1. This mechanism potentiates inflammation homeo-
stasis and the soluble protein form of CD52 for use as a therapeutic candidate. Other DAMPs do not
induce the binding of CD52 and Siglec-10. Linkage of SAα2,3-Galβ1 is required for CD52–
HMGB1 interaction
phosphorylation, as demonstrated using CD52-Fc, because CD52-Fc induces Siglec-

10 Tyr phosphorylation. Therefore, it is summarized that the CD52/Fc-HMGB1/
Siglec-10 triple complex recognizes the TCR. CD52 can be recovered when
HMGB1 is associated with CD52, Siglec-10, TCR, and phospho-SHP-1 present in
the cells because the CD52-Siglec-10-HMGB1 multiple complex recruits SHP-1 and
recognizes the TCR. HMGB1 box B together with soluble CD52 suppresses T-cell
function. Therefore, the proinflammatory Box B of HMGB1 is needed for CD52-Fc-
dependent suppression of T cells. However, the anti-inflammatory Box A is known
to block such suppressive effects. HMGB1 box B stimulates the interaction between
soluble CD52 and Siglec-10, indicating that Siglec-10 binds to CD52 only in the
presence of HMGB1 but not with other forms. Other DAMPs cannot promote the
interaction between CD52 and Siglec-10.
In a type 1 diabetes model for an autoimmune disease, Tregs suppress pancreatic
islet autoantigen production or GAD65-mediated autoimmune reaction [558]. Apart
from the Treg suppression of immune responses, another GPI-anchored protein,
CD52-mediated regulation of the hyperresponse has also been interested. To control
the so-called soluble CD52, a natural sialoglycopeptide or immune regulator inhibits
excess inflammatory responses. For CD52 regulation, soluble CD52 initially seques-
ters an inflammation mediator HMGB1, as HMGB1 promotes the interaction
between CD52 sialylglycan and inhibitory receptor Siglec-10 in activated T cells
and related immune cells. The soluble CD52 contributes to suppression of homeo-
stasis between immunity and inflammation [558]. This mechanism can explain the
homeostasis between immune responses and inflammatory responses. In addition,
this indicates that soluble CD52 can be used as a candidate for therapeutic drug
development.
CD52 as a GPI-AP is present on the hematopoietic lineage of B and T cells, DCs,
macrophages, monocytes, eosinophils, and NK cells [570], as well as on the
reproduction epithelial cells in males [558]. Human CD52 consists of only
12 amino-acid residues, but it has a scaffold function with a GPI-AP N-glycosylation
[570]. CD52-expressing CD4+ T cells are specific activation suppressors, because
they act with the released soluble CD52. Siglec-10 expressed on T cells acts as a self-
receptor for binding to soluble CD52. Siglec-10 interaction with soluble CD52
reduces phosphorylation levels of the TCR complex Tyr kinases such as Lck and
ZAP-70. This event inhibits the T-cell function [560], because Siglec-10 is an
inhibitory receptor. Cytoplasmic ITIMs suppress phosphorylation-based signaling
in some Siglecs. After Src family kinase-mediated phosphorylation, ITIMs associate
with SHP-1 and SHP-2 phosphatases as well as the SHIP inositol phosphatase
[117]. In cases of Siglec-7 and -9, the ITIM–SHP-1 binding contributes to
TCR-associated dephosphorylation. A soluble protein of CD52, termed “CD52-
Fc,” inhibits NF-κB transcriptional regulation in DCs, macrophages, and monocytes,
and depletes the Mcl-1 protein, which acts as a prosurvival inducer. This process
promotes apoptosis [571]. In an experimental model of a T-cell transferring system,
the depleted level of CD52-expressing cells induces autoimmune diabetes. In addi-
tion, in normal mice, CD52-Fc inhibits LPS-mediated inflammatory responses
[572]. Thus, CD52 can be applicable for T-cell immunity and cancer. The
immune-regulatory GPI-anchored protein, CD52, can also be applied to the repro-
duction tracts. The soluble CD52 [571] may suppress and block the rejecting
responses against the sperm allograft in females. In humans, a CD52-positive
T-cell clone was discovered in GAD65-specific CD4+ T-cell clones [319]. The
CD52-positive T-cell clone has immunosuppressive functions similar to those of
conventional Tregs, which have the phenotype of CD4+CD25+Foxp3+, but different
from Tregs [319]. In type 1 diabetes patients, blood samples show lower CD52-
positive T cells than in those of healthy people. When NOD mice were injected with
CD52-positive T cells, the diabetes onset in type 1 diabetes mice was slower than
that in normal NOD mice [319] because CD52-positive T cells suppress autoim-
mune responses in the type 1 diabetes model. Scientifically, soluble CD52 from
CD52-positive T cells binds to Siglec-10 expressed on autoreactive T cells. Conse-
quently, Siglec-10 signaling inhibits TCR activation signaling. Phospholipase
releases soluble CD52 from the GPI-anchored form on reactivated CD52-positive
cells. The released soluble CD52 directly binds to Siglec-10 on activated T cells and
inhibits TCR activation. As a result, the autoimmune response in human type
1 diabetes is inhibited by an interaction between soluble CD52 from suppressive
CD52-positive T cells and Siglec-10 on autoreactive T cells. From this, one hypoth-
esis could be made. Soluble CD52 from CD52-positive T cells has to bind to Siglec-
10 on antigen-presenting cells, like DCs, and thus induce immune suppression.
7.13.12.4 Comparison Between the Mouse Siglec-G Ortholog

and Human Siglec-10
Siglec-G is an important factor in autoimmune responses. It is related to CD33 in

mice [573]. Siglec-G exists mainly on the surfaces of DCs and B cells, particularly,
B1 cells. Different Siglec-G expression levels are observed. Siglec-G is present on B
cells but it is lowly present on myeloid lineage cells and DCs [574, 575]. Siglec-G
suppresses DC presentation by inhibiting the formation of the MHC-I–antigen
peptide complex [576]. Cross-presentation indicates that MHC-I class delivers
foreign antigen peptides to CD8 + -positive cytotoxic T lymphocytes (CTLs).
Another cross-presentation cell, DCs, are important in adaptive immune responses
[577], as known for splenic CD8α + DCs, CD103 + -positive nonlymphoid and
CD141hi DCs. Siglec-G is a controller of cross-presentation between CD8α + DCs
and CTLs in adaptive immune responses [578]. Using a Siglec-G KO mice model
(Siglec-G/) in C57BL/6 J (B6), the authors showed that a decreased MHC-I–
peptide complex level is important for cross-presentation. Siglec-G deficiency also
induced autoimmunity in mice. Siglec-G exists on DCs and B cells such as B1 cells
[574]. Siglec-G expression on B cells determines a BCR downregulation in inflam-
matory responses. Siglec-G recognizes the BCR of the B-cell surfaces via binding to
SAs and induces tolerogenic B cells [579]. B1a cells, which are Siglec-G-deficient,
exhibit an abnormal BCR behavior with a lowered spontaneous apoptotic death with
a lifespan expansion [580], indicating that Siglec-G downregulates the inflammatory
response via decrease in the number of B1a cell subsets, weak signaling of B1 cells,
and Ig expression shift. It also regulates the innate and adaptive immune responses
for anti-inflammation in septic conditions through IL-10, CD24 recognition, DC
presentation inhibition, and inhibition of B1 cell signaling. Siglec-10 also allows the
RNA virus to evade immune responses (Fig. 7.42). As Siglec-G is an inhibitory
receptor, it regulates B-cell expansion. For example, the B1 cells are enhanced in
number in Siglec-G-deficient mice. Siglec-G also interrupts the BCR and regulates B
cells via the BCR pathway [575]. CD22 as an inhibitory receptor also regulates
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Fig. 7.42 Siglec G-involved autoimmune system

B-cell proliferation. CD44 and Siglec-G double knockout induce autoimmune

responses in mice and generate tolerance of specific antigen B cells, which cause
autoimmune diseases [236]. BCR signaling induces B-cell activation and differen-
tiation, as well as initiates antigen–BCR binding and the TLR signaling pathway.
7.13.13 Human Siglec-11 as a CD33-Related Siglec
7.13.13.1 Siglec-11 Is a Paired Receptor with Siglec-16, Which Is

Caused by Gene Conversion in Primates
CD33rSiglecs are a large, rapidly evolving group of genes in mammals. The

coevolution events in CD33rSiglecs occur through an inverse duplication. This
process has been carried out to yield a primordial gene cluster of Siglec-coding
genes. It has been suggested that these events have been taking place for the last
180 million years [104]. These events contribute to the evolution of repeated gene
conversions in the primate lineage. Humans express more diverse groups of
CD33rSiglecs than do rodents. Most CD33rSiglecs have inhibitory motifs to carry
out signaling negatively. Paired receptors are known among leukocyte-associated
inhibitory receptors (LAIRs), killer cell Ig-like receptors (KIRs), leukocyte Ig-like
receptors (LILRs), Fc receptors, NKp46, paired Ig-like receptors (PIRs), and Siglecs
[581]. The cytosolic ITEM region of certain Siglecs interacts with SAMPs and
attenuate unwanted inflammatory responses to maintain homeostasis. Therefore,
ITEM-bearing Siglecs are inhibitory receptors. Siglecs without an ITEM domain,
including Siglec-14, Siglec-15, and Siglec-16, recruit DAP12. DAP12-recruited
Siglecs are thus classified as “activating” receptors. DAP12-coupled “activating”
CD33rSiglecs are found in Siglec-14 and -16 forms. These inhibitory receptors are
coupled between Siglec-5 and Siglec-11 and are known as paired receptors. More-
over, Siglec-11 and -16 are, as mentioned above, coupled receptors. Siglec-11 has an
ITIM that inhibits downstream signaling, whereas Siglec-16, due to a four base-pair
deletion in its gene, activates downstream signaling. Siglec-11 preferentially binds to
α2,8-linked SAs. Siglec-11 is present on many different cells including circulating
leukocytes and, most notably, on tissue macrophages.
Similar to other groups, the Siglec-11 cytosolic domain is Tyr phosphorylated
and consequently assembles with SHP-1 and SHP-2. The Siglec-11 V-set domain
mainly recognizes α2,8-linked SAs (Fig. 7.43). The expression of Siglec-11 is
specifically observed only on macrophage cell types including liver-resident Kupffer
cells and brain-resident microglial cells. The Siglec-11 expression is not found on
leukocytes of blood. The human Siglec-11 gene is loaded on chromosome 19 and the
location is different from those of genes encoding for other CD33/Siglec3rSiglecs.
There is no Siglec-11-related mouse ortholog. It is the last canonical type of human
Siglecs. Siglec-11 shows homology to Siglec-10 in its extracellular region sequence.
Siglec-11 is a chimera caused by duplication and recombination events, with a
closely related ancestral Siglec gene and a TM and cytoplasmic tail that originated
9 V-set
(Arg-rich for SA-binding)
2
Sialic Acid
Sialic Acid
C2-set
ITIM
Fig. 7.43 Siglec-11 and domain structure with V-set, C2-set, and ITIM. The Siglec-11 V-set
mainly recognizes α2,8-linked SAs
from another distinct ancestral Siglec [582]. As paired receptors display reverse
signaling, inhibitory receptors dampen cellular activation through self-associated
molecule recognition, whereas activating receptors are not clearly explained. For
example, Siglec-11 is functionally an inhibitory receptor, whereas Siglec-16 acts as
an activating receptor. The paired Siglecs of Siglec-11 and -16 are subjected to gene
conversions [583]. The nonfunctional SIGLEC16P alleles are easily generated rather
than SIGLEC-16 alleles in all human populations. In the microglia during human
evolution, the nonfunctional SIGLEC16P allele is generated through the converted
SIGLEC-11. Therefore, Siglec-16 is lost and Siglec-11 is gained. Siglec-11 is
specific to the primates and is specifically expressed on a human lineage. As the
specific Siglec is present on tissue macrophages of liver Kupffer and brain microglial
cells, Siglec-11 preferentially recognizes α2,8 sialic acids of three monomeric sugars
including oligosialic acid chains.
These activating receptors have evolved by pathogenic exploitation of inhibitory
Siglecs to allow the hosts to have alternative pathways to combat such pathogens. It
is interesting that the Siglec-5 extracellular region is homologous to that of Siglec-
14, because the gene conversion event occurs at the SIGLEC-5/SIGLEC-14 gene
loci. However, their intracellular regions exhibit opposing responses in action.
Inhibitory Siglecs regulate host hyperimmune responses, allowing negative modu-
lation of TLR signaling. In fact, Siglec-G negatively governs B1 cells and inhibits
inflammatory responses to the host’s DAMPs. Therefore, paired immune receptors
have behavioral roles with extracellular domains of ligand-binding regions and
intracellular sequences toward reverse signaling. Inhibitory receptors dampen cellu-
lar activation through recognition of SAMPs. Siglec-5 and -14 act as paired receptors
to regulate any response. However, activating receptors are not clearly explained.
Neutrophil-expressed Siglec-9 recognizes sialylated GBS and suppresses killing
responses, raising the pathogen exploitation of host CD33rSiglecs. For example,
the GBS bacteria attenuate host phagocytosis through inhibitory Siglec-5. GBS–
Siglec-14 binding induces activation of the MAP kinase pathway and rapidly clears
the pathogen [584]. In human SIGLEC-11 and SIGLEC-16 genes, the extracellular
domain-coding gene sequences are almost identical due to gene conversions. The
SA-binding potentials of human Siglec-11/-16 are almost similar because similar
extracellular regions are generated by the gene conversion events [36, 585]. However,
Siglec-11 and -16 bear intracellular regions capable of induction of reverse signals.
In detail, human SIGLEC-11 is nonfunctionally converted to the SIGLEC16P allele.
The SIGLEC-11 allele is set in humans. This process made a merit of
neuroprotective effects in neuronal microglia. However, the human SIGLEC-16
allele frequency is 0.22 and an inactive variant of the SIGLEC16P gene is found
with a four-nucleotide base deletion and a disrupted ORF. The evolution of
SIGLEC-11 and SIGLEC-16 is still unknown in other primates.
7.13.13.2 Human Siglec-11 and Mouse Siglec-E Function in Microglial

Cells
Gene conversion events are known to occur in human-specific genes. A conversion

event frequently induces alterations in the coding region for the Siglec-11 extracel-
lular domain with human microglial expression. The conversion of the SIGLEC-11
gene is human-specific and is based on an adjacent pseudogene location of
SIGLEC16P. This event yields a distinct human form in the brain, named Siglec-
11, especially in microglial cells [585]. As a family of CD33rSiglecs, Siglec-11
expression is only on microglial cells in the brain, indicating that brain evolution,
consequently, contributes to microglial modulation of neuronal survival. The
SIGLEC-11 transcript is found in the ovarian and adrenal cortex tissues. Moreover,
Siglec-11 is also present on stromal tumors of human ovary and polycystic syndrome
of human ovary [586]. Artificial engagement of human Siglec-11 in mouse
microglia, which is constructed using flag-specific antibodies, dampens the
LPS-stimulated inflammatory responses in the mouse microglia. This reduces the
phagocytic potential in the microglia and induces negative effects on pathology. A
PSA-bearing NCAM is a Siglec-11 ligand. Polysialylated glycans in NCAM
(CD56), CD36, or neuropilin-2 vary in their lengths between degree of polymeriza-
tion (DP)-10 to DP-200. Polysialic acids inhibit inflammatory phagocytosis [587].
Microglia, which are essentially brain macrophages, also have Siglec-11, and
Siglec-11 on the microglia inhibits microglia’s inflammatory response to prevent the
microglia from damaging its surroundings. Microglia are brain macrophages with
immune defense in the CNS and act as pathogen scavengers of infected and
unnecessary neurons. Therefore, Siglec-11 works as a “stop” signal to brain inflam-
mation, and it may have effects on many different neurodegenerative diseases like
Parkinson’s and Alzheimer’s. Low-molecular-weight polysialic acid (α2,8-linked as
Siglec-11 binds to α2,8-linked) binds to Siglec-11 and thereby inhibits downstream
signals. Low-molecular-weight polysialic acid successfully attenuates inflammation
by binding and activating Siglec-11. Therefore, it can be used in the future to
attenuate microglial inflammation thereby using it as a drug to potentially cure

Parkinson’s or Alzheimer’s.
Siglec-11 has two spliced variants in the brain tissue in humans. The alternatively
spliced second variant is ectopically expressed on murine microglial cells. Siglec-11
suppresses the LPS-promoted production of the proinflammatory IL-1β and NOS-2
in the microglia and reduces phagocytic death of apoptotic neuronal cells
[343]. Siglec-11 protects neuronal cells, depending on PSA residues expressed in
neurons, but independently in PSA expressed in microglial cells. Human Siglec-11
present on murine microglial cells binds to PSA expressed in neurons and reduces
expression levels of LPS-induced proinflammatory cytokines, suppresses phagocy-
tosis, and consequently alleviates microglial neurotoxicity. Therefore, it is consid-
ered that microglial cells express only Siglec-11 to emit immunosuppressive signals.
Siglec-11 suppresses other microglial receptors including PRRs of TLRs, NLRs, and
RAGE. As Aβ plaques are rupture-aggregated in AD by sialylglycoproteins and
gangliosides, sialyl plaques activate the immunosuppressive Siglec-11. This poten-
tiates to evade the host immune surveillance of the microglia [588]. In AD treatment,
the aggregates and apoptotic neural membranes should be eliminated without any
inflammatory response. The Fc receptors are known to perform this task in mam-
mals. Another case is the DAP12-linked receptors that perform the ITAM-Syk-
signaling. The ITAM-containing receptors present in microglial cells are reverse-
modulated by the ITIM because Siglecs inhibit the proinflammation process in the
microglia and phagocytosis of microglial cells through ITIM-mediated downstream
signaling. The level of neurotoxic cytotoxicity of microglial cells is increased by an
interaction between Siglec-11 and its SA ligands that occurs on the glycocalyx
present in neuronal tissues. Therefore, the ITAM receptors and ITIM receptors
separately control microglia phagocytic potentials and neuroinflammation
[589]. Microglial cells in the CNS transduce information signals using their own
recognition receptors. They either activate or inhibit the immune responses. If the
self is damaged, proinflammatory and phagocytic responses are suppressed in
microglial cells through inhibitory Siglecs. This event can inhibit the
phagocytosis-related oxidative burst. This results in neuroprotection. Siglec-E
blocks microglia phagocytosis and oxidation. Microglial Siglec-E and Siglec-11
prevent neurotoxicity via binding to SAs on the neuroglycocalyx [128]. Siglecs
interacting with the self-glycocalyx of adjacent cells silently keep the microglia in
homeostasis. Siglec-11 recognizes SAs of healthy neuronal cells and neighboring
glial cells to transduce signals via the ITIM to maintain the neuronal cells and
neighboring cells in homeostasis. Apart from Siglec-11, the conserved MAG
(Siglec-4) is present on Schwann cells and oligodendrocytes. Siglec-4 prevents
neuronal toxicity caused by neurotoxic conditions via SA binding on neuronal
SA-containing glycosphingolipids such as gangliosides. The second microglial
Siglec-11 inhibits neurotoxicity via α2,8-linked SA oligomer recognition on the
glycocalyx present in neuronal cells.
Mouse microglial Siglec-E type recognizes α2,8-SAs and α2,3-SAs in the
glycocalyx in neuronal and glia cells [590]. Oligosialic acids and PSAs of the
glycocalyx in neural and immune cells are α2,8 glycosidically linked. As Siglec-
11 is a primate lineage-specific receptor of human tissue macrophages and microglia,

soluble PSA interacts with Siglec-11 and acts anti-inflammatory on human THP-1
macrophages. Soluble PSA inhibits the LPS-induced TNF superfamily member
2. PSA attenuates the LPS-triggered phagocytosis of macrophages with prevention
of the oxidative burst of human macrophages caused by neural cell debris or
fibrillary Aβ1–42 plaque peptides. In the human macrophage–neuron coculture
system, PAS increases neurite formation reduced by fibrillary Aβ1–42 plaques
[591]. As an inhibitory receptor, Siglec-11 engages endogenous PSA to suppress
inflammation, and several pathogenic microbes have evolved by acquiring mimics to
recognize inhibitory receptors or repressors. Then, this acquired ability attenuates
the host immune responses. Therefore, pathogenic sialyl recognition of host inhib-
itory receptors indicates the acquisition of the evolutionary merit [592], as is
consistently observed in the paired receptor families. They also evolve rapidly to
complementarily recover from pathogen pressure [593, 594].
7.13.13.3 Human Siglec-11 and Mouse Siglec-E Function in Microglial

Cells
Host immune cells including innate and adaptive cells have sialic acid-binding
receptors called Siglecs. The Siglec family consists of many kinds of Siglecs. Siglecs
are composed of extracellular, transmembrane, and cytosolic domains. The extra-
cellular region which has a V-set domain and a C2 domain interacts with sialic acids.
Cytosolic ITIM or ITAM can induce a downstream cellular signaling cascade.
Human beings have been exposed to various pathogens including bacteria and
viruses. There are also many bacteria in the human intestine and other organs, and
they continuously communicate with the host cells. However, infections of some
bacteria and virus, which have pathogenic toxins, are harmful to humans. In humans,
the defense mechanism for pathogenic infection is regulated by immune cells.
Immune cells have distinct receptors. For example, B cells or T cells have BCRs
or TCRs, respectively, for nonantigen reacting receptors consisting of paratopes.
Otherwise, innate immune cells such as macrophages, neutrophils, and DCs also
have antigen receptors, but by molecular pattern recognition. The molecular inter-
action between receptors and patterns occurs not only in nonself-antigens but also in
self-antigens such as autoantigens, causing autoimmune diseases. However, inhib-
itory molecules expressed on self-cell surfaces serve as ligands for immune cells and
inhibit immune cell functions. This leads to protection of the host from immune
surveillance. In particular, the brain is an important organ and is composed of sialic
acid-expressing cells to evade immune surveillance.
To evade host immune surveillance, some bacteria have evolved with glycans
resembling the host sialic acids on glycan capsular lipopolysaccharides. Their
glycans can bind the host’s immune cells and inactivate immune cell activity through
ITIM activation. For example, the E. coli K1 strain induces neonatal meningitis and
urinary tract infection in humans. It has a PSA capsule, K capsule, mimicking human
sialic acid, and binds Siglec-11 to evade immune surveillance. The Siglec-E mutant
that is replaced in the C-terminus with Siglec-16 having the ITAM motif yields a
Siglec-E16 mutant receptor. This mutant receptor binds to the E. coli K strain
normally with a normal function showing IL-6 and IL-10 secretion [595]. Moreover,
Siglec-16 interacts with DAP12 and Siglec-E16 also interacts with DAP12. DAP12
is an adaptor protein used to activate the MAPK pathway, inducing inflammation
upon binding to Siglec-16E. Siglec-16E is expressed on the mice spleen and liver
with similar levels as those of mouse Siglec-E, indicating the interaction of Siglec-
16E with bacterial sialic acid. Siglec-16E-expressing mice show a reduced level of
bacterial growth and enhanced MAPK pathway with p38 phosphorylation. Finally,
Siglec-16E-expressing mice show a retarded bacterial growth in the blood, liver, and
spleen. Siglec-16E causes upregulation of cytokines IL-6/IL-12/MCP-1. Like
Siglec-11, Siglec-16 has a similar sequence of V-set domains in the extracellular
region. Siglec-16 could interact with the E. coli K strain, as confirmed by bacterial
binding to the Siglec-16 Fc antibody. Siglec-11 and Siglec-16 are paired receptors.
Bacterial glycans interact with Siglec-16. If bacteria are expressed by the
neuramidase gene, the Siglec-16-binding capacity is decreased, indicating that the
paired Siglec-16 and Siglec-11 are important for binding the E. coli K strain capsule
glycans.
The human-specific pathogen E. coli K1 utilizes the PSA capsule glycans as a
mimic to engage Siglec-11 to escape from the host phagocytic activity. However, the
activating receptor Siglec-16 eliminates bacterial pathogens. From the fact that mice
do not have paired Siglec receptors, humans survive under comprehensive circum-
stances (Fig. 7.44). Siglec-E16 stimulates synthesis of proinflammatory cytokines
and phagocytic cell death and consequently protects the host from bacterial attack
[595]. There is an extremely important evolutionary factor between bacteria and
their hosts. Sialic acid has two faces of host to prevent self tissue from inflammation
but bacteria mimic them to invade host’s immune surveillance. However, the host
does not defect to bacteria making counter part of Siglec-16. Hosts induce inflam-
mation via the ITAM upon binding to bacterial sialic acid although the self-tissue is
damaged. This indicates that it is probably not the end of the road in the battle
between parasites and their hosts. However, we do not know who will come out the
winner in the final stage.. However, it is important to retain this mechanism to cure
bacterial infection, as this will possibly help to develop a novel therapy using sialic
acids.
7.13.14 Siglec-14 in Humans as a CD33-Related Siglec

7.13.14.1 The Structure and Expression of Siglec-14 in Humans, Paired
with Siglec-5
Since CD33-related Siglecs have an ITIM or ITIM-like domains to signal inhibitory

direction in cells, the cytosolic and intracellular regions of human Siglec-5 and
Siglec-8 bear an ITIM and this ITIM acts to stop downstream signaling. Therefore,
K-capsule(D-2,8 glycan)
E.coli K1
E.coli K1
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ITIM
ITAM
DAP12
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Fig. 7.44 Siglec-11 and ITIM-mediated immunosuppressive activity against the human pathogen
E. coli K1, which contains the polySia capsular polysaccharides. The capsules act as molecular
mimics to engage Siglec-11 to escape from the host bactericidal activity. Comparison with Siglec-
16 is displayed. The Siglec-11 and -16 pair yields opposing inflammatory responses
in other words, the ITIM is a “tune-off” signaling maker, showing cellular inhibition
or apoptosis in cellular phenotypes. In contrast, Siglec-14 is unique in that it lacks an
inhibitory domain motif, and, instead, it modulates each cellular function, depending
on SA recognition using DAP12-based activation signaling upon recruitment of
DAP12 [596]. If the balance between “tune-on” and “tune-off” maker messages is
disrupted or broken, inflammation or immune tolerance is uncontrolled. This results
in host cell damage, pathogenic infection, and phenotype transformation. Human
Siglec-14 present on myeloid cells binds to pathogenic bacteria and diminishes the
inflammation process. Some Siglecs exhibit immunomodulatory functions upon
associating with sialylated glycoconjugates during inflammation or tumor progres-
sion. As a CD33-related Siglec, Siglec-14 was first identified in humans in
2006 [34].
With regard to the structural aspect, Siglec-5 and Siglec-14 share a high identity
in the first two Ig domain sequences, as the two receptors are found to be the first
glycan-binding paired receptors and both are expressed simultaneously in same
tissue. Therefore, Siglec-14 and Siglec-5 are similar regulators of the immune
system with a coupled activating receptor and inhibitory receptor with a sequence
similarity of more than 99% in their first two immunoglobulin regions [597]. The
Siglec-17-coding gene shows polymorphism. Counteraction between Siglec-5 and
Siglec-14 helps maintain a balance between cell activation and dampening. Siglec-
14 and Siglec-5 do not have murine homologues. Immune receptors having high
sequence identity exhibit antagonistic signaling characteristics for downstream
immune responses. The ligand recognition regions of Siglec-14 and -5 are identical
and they form a complex with DAP12 as an adaptor molecule bearing the ITAM in
the cytosolic region, instead of the ITIM. Siglec-14 consists of an Arg residue in its
transmembrane motif for binding to DAP12. The human Siglec-14 protein is three
Ig-like domain-bearing type I protein with a TM and a short cytosolic tail. Siglec-14
has 396 amino acids and the SIGLEC-14 gene is chromosomally located near the
Siglec-5 gene. A high sequence homology is observed between Siglec-14 and -5 in
the first two Ig-like regions. This indicates that favorable glycan recognition sites in
Siglec-14/-5 are quite similar. Moreover, the two Siglecs incorporate the same
activation adapter DAP12 protein. The expected signal peptides and the Ig-like
domain-1/-2 of Siglec-14/-5 are nearly similar to each other.
Siglec-14 as a TM and soluble protein is also found in human circulation. In a
recent report [598], it has found that the soluble Siglec-14 form is generated through
alternative mRNA variant splicing. A soluble form of the Siglec-14-coded spliced
variant contains intron-5, which has a terminating codon. This prevents the exon-6
translation that encodes the TM domain of Siglec-14. The translated intron-5 region
encodes a C-terminally distinct extension region of 7 amino-acid residues. The
Siglec-14 variant isoform bearing the C-terminal 7 amino-acid residues is detected
in human blood samples using a specific antibody. The variant form of soluble
Siglec-14 blocks myeloid proinflammatory responses. The soluble Siglec-14 inter-
cepts TLR-2 prior to TLR-2 recognition of normal Siglec-14, which is embedded in
the membrane, by interfering with binding of the embedded Siglec-14 to TLR-2. The
G-rich sequence of intron-5 is suggested to form a tertiary structure of RNA,
G-quadruplex, to increase the splicing level of intron-5.
The expression of Siglec-14 on lupus monocytes is associated with SLE. Siglec-
14 expression is enhanced on monocytes of SLE patients [599]. The increased
Siglec-14 level is directly correlated with a decreased C3 level in the patient
serum, indicating that SLE and Siglec-14 expression induce the inflammatory status
conferred by the alternative complement pathway but not by the lectin or classical
pathway [600]. The alternative pathway is activated by LPSs, as Siglec-14 is
upregulated on LPS-treated monocytes. The expression levels of Siglec-5/-14 are
also upregulated in patients with COPD [601]. Siglec-14 expression is
downregulated on nonclassical monocytic cells obtained from healthy individuals.
Siglec-14 as an activating receptor, on binding to glycan ligands, transduces activa-
tion signals by DAP12 adaptor protein. By a gene conversion event of the SIGLEC-5
gene, Siglec-14 evolves in response to pathogenic evasion of immune surveillance
[34, 104]. This is retrograde-evidenced by the result that SIGLEC-14 gene-deficient
fetuses are highly susceptible to prematurity during GBS infection than are wild-type
fetuses [602]. SLE symptoms are prevalent in individuals with the SIGLEC-14-
deficient genotype [528, 603]. In a cohort analysis of SLE patients, Siglec-14

expression on monocytes is increased. In addition, the Asian ethnicity of patients
exhibits the immunosuppressive Siglec-14/5 receptor, as the importance of this is
still emerging, requiring further immunomodulatory Siglec-14 function in
autoimmunity [599].
7.13.14.2 Role of Siglec-14 in Pathogenic Invasion
Microbial pathogens evolve in a timely manner to evade innate immune responses of

the host and phagocytosis for pathogenic clearance. Cell surfaces of pathogenic
microbes and hosts are coated with glycans, and, therefore, interactions between
glycans and their receptors trigger basic biological events including microbial
pattern recognition and manifestation of stimulating signals to raise regular immune
cell activity. Survival and life management of pathogens require glycans that mimic
host glycans and interfere with immune responses.
The currently known human pathogenic bacteria N. gonorrhoeae, E. coli K1,
GBS, H. influenza, and T. cruzi express or incorporate SAs into their produced
glycans, through either taking up forms directly from their host or synthesizing them.
So, what is GBS infection? Also known as group B streptococcal disease, it is an
infection caused by Streptococcus agalactiae found abundantly in newborns and in
the elderly. In terms of bacterial properties, it is a Gram-positive coccus, forms
chains, and is a β-hemolytic, catalase-negative, and facultative anaerobe. Group B
streptococcus (GBS) symptoms during pregnancy and in newborns are known. In
pregnancy, the symptoms are asymptomatic and generally cause no problems but
sometimes cause serious illness during gestation or after delivery. GBS infection in
mothers could cause chorioamnionitis or postpartum infections. Neutrophils, in
which Siglec 14 protein is deficient, exhibit high susceptibility to immune subver-
sion of GBS. Siglec-5 and -14, which are present on amniotic epithelial cells in
humans, may allow invasion of the fetus with GBS. In newborns with early-onset
(0–7 days) sepsis, pneumonia and meningitis are featured. Late-onset (7–90 days)
sepsis is specific for meningitis, cellulitis, osteoarthritis, and pneumonia. Gene
polymorphism can affect the prematurity risk in human fetuses of GBS-colonized
mothers.
In GBS infection, certain GBS strains bind to Siglec-5 by β-protein embedded in
the cell walls. β-Protein-producing GBS strains recognize both Siglec-5 and -14
present on neutrophils. GBS-produced β-protein recognition of ITIM-containing
Siglec-5 transduces SHP-2-dependent inhibitory downstream signaling to block
activation of macrophage cells and consequently reduces the killing activity of
phagocytes. GBS β-protein and Siglec-5 ITIM interaction elicits inhibitory SHP-2-
dependent macrophage inactivation. Therefore, pathogen SA mimicry or protein-
involved Siglec inhibition may coevolve to adapt the host SA or Siglec-recognition
property. Such evolutionary adaptation may induce host Siglecs to counteract
pathogen immune evasion. Pathogen’s carbohydrate SA mimicry engagement or
inhibitory Siglec engagement as protein forms develops evolutionary shifts in the
V set domain
(sialic acid binding site) Hsp70
Sias-Independent
Siglec-14 Siglec-5
C2 set domain
DAP12 extracellular
+
TM
cytosol
ITIM Monocyte
ITAM ITAM ITIM
Putative tyrosine based motif
Acvaon Inhibion
INFLAMMATION
Fig. 7.45 Structure of Siglec-14 as a CD33-related Siglec
Siglec-recognition and SA synthesis of hosts. In theory, certain evolutionary adap-

tations are emerging factors of activating Siglecs to counteract pathogen immune
evasion. The currently known primates show that the Siglec-5 genes undergo gene
conversion events even in the partial region with the neighboring Siglec-14 gene, as
the two Siglecs, Siglec-14 and Singlec-5, are highly identical, but the Siglec-14
cytosolic domain recruits adaptor DAP12 protein, which possesses the activating
ITAM instead of the inhibiting ITIM (Fig. 7.45). As evidenced, the currently known
primate SIGLEC-5 genes seem to progress in genetic conversions with the nearly
adjacent SIGLEC-14 gene. Siglec-14 was first identified in humans in 2006. Struc-
turally, Siglec-5 and -14 have almost identical sequences in the first two Ig domains.
They were found to be the first glycan-binding paired receptors expressed simulta-
neously on same tissue. Counteraction between Siglec-5 and -14 would balance cell
activation and dampening. The SIGLEC-14 gene sequence is highly homologous to
that of the SIGLEC-5 gene but it activates ITAM-bearing DAP12 adaptor protein
instead of the ITIM. Siglec-14 function is lacking in humans due to SIGLEC-5–
SIGLEC-14 gene conversion. The consequently fused SIGLEC-14/-5 gene sequence
is identical to that of SIGLEC-5. However, the SIGLEC-14 gene influences myelo-
cyte responses to the SIGLEC-5-binding β-protein-expressing GBS. Siglec-14 pro-
tein produced in humans consists of three Ig-like domains, and the Siglec-14-
encoding gene location of SIGLEC-14 is near the Siglec-5-encoding gene of
SIGLEC-5. As the human Siglec-14 protein is highly homologous to Siglec-5
protein in the first two Ig-like domain regions, the sialylglycan-recognizing speci-
ficity of Siglec-14 is also almost the same as that of human Siglec-5. A partial gene
conversion exists in the two genes, SIGLEC-14 and SIGLEC-5. The SIGLEC-14
and SIGLEC-5 genes of other primates are seen in genes with the same conversions,
sharing benefits in the evolutionary pathway [34]. Siglec-14 function is lacking in

certain individuals because of the fusion of the two Siglec genes SIGLEC-5 and -14
resulting in a converged gene, Siglec-14/-5. This gene is identical to Siglec-5 in the
coding region, except for the fact that it is expressed under a Siglec-14 transcrip-
tional promoter. This allows polymorphism in the human population. Individuals
with both Siglec-14 and Singlec-5 lack one or both alleles of Siglec-14 because of its
replacement by the Siglec-14/-5 gene.
Therefore, a question arises: how does Siglec-14 influence phagocytic immune
responses to the Siglec-5-binding β-protein-expressing GBS on bacterial surfaces?
Siglec-14 receptors in cultured macrophages isolated from human blood, which bear
different genotypes at the Siglec-14 locus, are studied [597]. Siglec-14 expressed on
THP-1 monocytes increases immune responses against GBS and LPS. The geno-
types of Siglec-5 and -14 influence the human cellular responses of primary mono-
cytes to GBS and LPS. Then, the question of how polymorphisms of the human
Siglec-14/-5 genes affect the primary monocytic responses of humans to infectious
agents is raised. Siglec-14 activates proinflammatory immune responses to GBS and
LPS via the AKT/p38 MAPK axis pathway. Therefore, the Siglec-5-generated
opposing power is operated, as the ITIM associates with the recruited SHP-1/-2 of
the Tyr phosphatases [597]. The Siglec-5 and Siglec-14 genotypes also affect human
neutrophil behavior toward GBS. Priming of TLR increases the inhibition level of
GBS against the responses of Siglec-14(/) but not Siglec-14(+/+) neutrophils.
GBS, which expresses surface β-protein, binds to blood neutrophils through recog-
nition of Siglec-5 and -14. The latter engagement counteracts GBS-induced host
immune suppression by activating p38 MAPK and AKT signaling pathways. In
individuals who have only Siglec-5- or Siglec-14-null neutrophils, and for whom
Siglec-14 is lacking, GBS immune subversion is more susceptible. The surface-
expressed Siglec-5 and -14 proteins in the amniotic epithelium are the initial binding
sites for attachment and invasion of GBS bacteria to the fetus. Therefore, the Siglec
gene polymorphisms affect the prematurity risk of fetuses of GBS-carrying mothers
in humans.
7.13.14.3 Expression and SA Specificity of Siglec-14
Siglec-14 is mainly present on the hematopoietic spleen, BM, and fetal liver, with a
minor presence on the lung and testis. Siglec-14 expression is similar in its pattern of
Siglec-5 mRNA, indicating that Siglec-5/-14 proteins are present simultaneously on
the same identical cells. For example, in human peripheral blood leukocytes, Siglec-
14 is present on monocytes, neutrophils, and granulocytes, whereas Siglec-5 is
expressed on B cells and granulocytes [528]. In humans, Siglec-5 consists of an
extracellular four Ig-like domains and a cytoplasmic inhibition motif. Furthermore,
Siglec-14 and -5 are the first coupled receptors with a high similar identity, indicat-
ing fragmentary gene modification in Siglec-14 and Siglec-5. Similar to the human
cases, Siglec-14 and Siglec-5, which are found in other primates, are also gene
modified within each lineage. Therefore, coevolved molecular binding of Siglec-14

and Siglec-5 to their ligands seems to have a common direction of animal adaptation.
Mammalian Siglec-14-recognizing sialyl glycans include Neu5Ac-Neu5Ac-Gal
of α2,8 and α2,3 linkages and Neu5Ac-GalNAc of α2,6 linkages. Siglec-14 and
Siglec-5 have highly similar homologue sequences. Siglec-14 with Siglec-5 is a
heterodimer receptor. The cytoplasmic domain of Siglec-14 lacks CD33-related
Siglecs’ common ITIM, whereas the transmembrane domain carries a positively
charged Arg362 residue. The Arg362 residue recognizes the ITAM associated with
adaptor proteins DAP10 and DAP12. Upon interaction with its ligands, Siglec-14
transduces intracellular signaling through DAP10/DAP12 that enhances protein-
phosphorylation pathways. Siglec-14 couples with Siglec-5 and suppresses cell
signaling to allow opposing roles in immune cells. There are still unresolved
questions regarding keeping the balance between inhibitory and activating signaling.
The binding capacity of Siglec-14 is stronger than that of Siglec-5 [603]. Therefore,
the Siglec-14 signaling intensity is stronger than the Siglec-5 inhibitory intensity.
Signaling motifs and transmembrane region interactions with DAP12 provide ITAM
signaling activity triggered by the recruited SYK. Hsp70 has been identified as sialic
acid-independent Siglec-5 ligands. Extracellular Hsp70 belongs to a DAMP when it
interacts with human monocytes and induces proinflammatory TNF-α secretion. The
functional activity of Hsp70 in inflammatory cytokine secretion is unknown. Ele-
vated levels of Hsp70 in bronchoalveolar lavages are associated with lung inflam-
mation. Colocalized Hsp70 with Siglec-5/-14 ligands is known in lung bronchioles
[604] and they directly interact. Hsp70 binds to the V-set domain of Siglec-5/-14,
inhibiting inflammation through Siglec-5 in THP-1 cells. Hsp70 augmentation for
inflammation is based on Siglec-14 in THP-1 cells because Hsp70 modulates human
monocytes in a dependent manner on the SIGLEC-14 genotype (Fig. 7.46). Thus, an
association between Siglec-14 and DAP12 occurs through its TM Arg residue.
Siglec-14 acts as an activating receptor to produce IL-8 and TNF-α in neutrophils
and monocytes. However, the inhibitory receptor has endogenous ligands, whereas
the activating receptor of the pair also recognizes the same ligand. The imbalanced
interaction of the ligand or excessive interaction with the activating receptors
induces hyperinflammation or autoimmune responses in the host.
A region of the Siglec-14 gene in the chimpanzee and baboon genomes indicates
a high sequence similarity with that of Siglec-5 in the respective species, indicating
that analogous gene conversions take place in other primates. This is also extended
to gorillas, orangutans, chimpanzees, humans, and baboons, as represented in the
molecular phylogenetic tree. The Siglec-14 gene in each species exhibits a sequence
similarity with a part of Siglec-5 in the respective species. Thus, one independent
gene conversion event may involve the Siglec-5 and Siglec-14 genes, and the event
takes place in the above-mentioned primates. Siglec-14 and Siglec-5 transmit their
signals in a reverse manner via ITIM- and ITAM-driven signaling, respectively. The
same Arg residue in the first Ig domain binds to the SA residue in both human
Siglec-14 and -5. The two proteins act in a complementary manner, with homeosta-
sis activation and inhibition signaling via SA recognition [605]. Moreover, gene
conversion in the 50 -flanking regions of the Siglec-14 and Siglec-5 promoters of
Fig. 7.46 Putative

correlation between Hsp70
SIGLEC-14 and Siglec-5 Sias-Independent
upon interaction of Siglec-14 Siglec-5
pathogens such as
H. influenzae and group B
Streptococcus. Siglec-5 and
-14 as polymorphic coupled
receptors regulate neutrophil
function during GBS DAP12 extracellular
infection
cytosol
Monocyte
ITAM ITIM
Acvaon Inhibion
INFLAMMATION
primates confers a coupled receptor status on SA recognition, although the scientific

reason why coupled receptors are formed (or with pairs) is questionable. The
activating receptor is believed to have initially evolved from an inhibitory region,
from alternative advantage in controlling of the virus.
7.13.15 Siglec-16 as a CD33-Related Siglec Is a Paired

Receptor with Siglec-11
SAs are the first evolved molecules in higher animals and later several microbial
pathogens acquired SAs, although opposing theories also exist [583]. The sole case
of the opposing theory is that Siglec-16 is an activating Siglec. The activating
Siglecs, herein Siglec-16, play a role in fighting the pathogenic utilization of the
inhibitory Siglecs of hosts and dampening of host immune responses [105]. Acti-
vated Siglec-16 engagement contributes to pathogenic bacterial elimination. For
example, E. coli K1 surface capsules recognize Siglec-11/-16 on cell surfaces.
Microglial cells CHME-5 transfected with Siglec-11 or Siglec-16 are bound to
E. coli K1, because Siglec-11- or Siglec-16-expressing microglial CHMR-5 cells
bind to the E. coli K1 surface capsules [36]. The role of Siglec-11 and Siglec-16 has
been suggested to protect them from pathogenic bacterial infection by innate
immune responses of the hosts. Siglec-11-expressing microglial cells are easily
infected with more bacterial population of E. coli K1, whereas extopic Siglec-16
expression exhibits a high bacterial cytotoxic activity. Inhibition of Siglec-11-
dependent cytotoxicity is not found in the same E. coli ΔneuDB strain, which is
negative for capsule production. The Siglec-16-carrying cells are also reported to kill
capsule-negative E. coli K1 strains due to some interactions of sialic acid
independence [36].
As described earlier, the Siglec-16 and Siglec-11 forms are also coupled receptors
generated by repeated gene conversions. The gene conversion events frequently take
place in the primate lineage during emerging genetic evolution. The existence of the
nonfunctional allele known as the converted SIGLEC-11 and SIGLEC16P allele is
implicated in the Siglec-16 loss and the new Siglec-11 gene genesis during the
evolution [104]. Gene conversion events among these chromosome loci take place in
a manner of tandem gene conversion starting from SIGLEC16P to SIGLEC-11,
resulting in a harmful and nonbeneficial short segment, which is interspaced in an
intervening manner. For a mechanistic explanation of the gene conversion event in
Siglec-11 and Siglec-16, unexpected pressure is generated due to pathogenic infec-
tion and immune escape that is intended to remove or eliminate the Siglec-16 gene
and its function during immune responses [595]. Therefore, Siglec-16 and Siglec-11
are coevolved by gene conversion events in the human primate lineage; however, the
evolutionary roles are not explained by any biological pathway. From the above-
mentioned situations, it is clear that human Siglec-16 was originally regarded as a
pseudogene. The SIGLEC-11 and SIGLEC-16 genes are loaded on the same human
genome at a distance of approximately 9 kb. The short distance observed in the
SIGLEC-11 and SIGLEC-16 gene locations is caused by an evolutionary gene
duplication event [36, 606]. The human SIGLEC-16 locus of the same population
contains different alleles for the nonfunctional allele and the functional allele
[36, 607]. The nonfunctional SIGLEC16P allele is specifically 4-bp deleted in
exon-2, resulting in a disrupted ORF in translation and a short protein, i.e.,
Siglec-16P. In humans, conversion of the SIGLEC16P gene to the SIGLEC-11
gene is estimated to be traced back to approximately one million years ago
[606]. In SIGLEC16P–SIGLEC-11 gene conversion, human brain microglia express
the human SIGLEC-11 gene [607]. The event of the SIGLEC16P–SIGLEC-11 gene
conversion is an example of a coding gene evolution of a nonfunctional allele or a
pseudogene. SIGLEC16P has been maintained for more than three million years in
the human population [583].
Mice are negative for the presence of the paired Siglec receptors. Therefore, to
observe a similar phenomenon in experimental animals, the inhibitory domain of
mouse Siglec-E is replaced by the activating domain of Siglec-16 [36]. The created
replaced clone, named Siglec-E16, promoted proinflammatory cytokine production
and consequent cytotoxic death of bacteria by macrophages and exhibited host
survival, protecting from pathogenic infection and invasion. Therefore, activating
Siglecs protect the hosts in vivo from pathogenic bacterial infection. In mice model,
the human-type paired Siglecs, which mouse Siglec-E is replaced by a chimeric
receptor of the Siglec-E extracellular domain and the human Siglec-16 TM region.
The native Siglec-E contains an ITIM as the inhibitory region. However, the
chimeric receptor Siglec-E16 displays inflammatory responses to protect the hosts
from pathogen infection [36].
7.14 Mouse CD33-Related Siglecs with ITIM-Like Domains 447
However, human Siglec-16 is present on various cell lines and tissues on the cell
surfaces with DAP12, but not with the FcR-gamma chain [105]. For example,
Siglec-16 is mainly expressed on CD14 (+)-positive tissue-resident macrophages.
Interestingly, human brain as well as esophagus and lung cancers also express
Siglec-16. Siglec-11 and -16 are mainly present on macrophages [36, 606]. The
specific point is that Siglec-16 is the first human Siglec with an activation motif as
well as a functional allele and a nonfunctional allele. The functional and
nonfunctional alleles are observed even in the human populations. Certain Siglecs,
like Siglec-11, contain some inhibitory ITIMs in the cytoplasmic tail and therefore
are called inhibitory receptors. The inhibitory Siglec receptors recognize sialic acids.
However, Siglec-16 does not bear signaling domains. Instead, Siglec-16 activates
cellular functions with an adaptor named ITAM [583]. Siglec-16 induces innate
immune responses and also inflammatory responses in the human microglia of the
brain. Therefore, Siglec-16 counteracts the neuroprotective potentials of Siglec-11.
Such an unexpected event of Siglec-16 in the human brain yielded the following
hypothesis because the Siglec-16 function is unusual in the brain. The hypothesis is
that the event contributes to the elimination of Siglec-16 in humans. Siglec-16 is
indeed the new type of activating CD33rSiglecs in humans and other primates.
Siglec-16 is no more a pseudogene but encodes a full ORF [36].
In proinflammatory responses in the gonococcal pathogenesis of N. gonorrhoeae,
Siglec-14 and Siglec-16 as activating receptors induce gene expression of
proinflammatory cytokines to potentiate clearance of infection [608]. However, in
N. gonorrhoeae, the pathogen endlessly strives to survive within myelocytes includ-
ing DCs, macrophages, and neutrophils [609]. In Namibian pastoralists, activating
Siglec-16 has been demonstrated to be associated with a low female gonorrhea level.
Siglec-16 is present on immune cells and cervical cells in the epithelium. Siglec-16 is
highly effective in gonococcal amplification rather than activating Siglec-14 found
only on neutrophils. Unsialylated N. gonorrhoeae efficiently recognizes Siglec-16
rather than sialylated N. gonorrhoeae. Neuraminidases present in the female genital
tract [610] efficiently cleave off SA residues present in gonococcal LOS and elevate
the infection level in males because asialo gonococci strains can be involved with the
asialoglycoprotein receptor (ASGPR) [611]. Siglec-16 response to desialylated
gonococci indicates an evolutionary adaptation of the host to gonococci, because
gonococci surfaces are desialylated by sialidases surrounded by microbial
flora [610].
7.14 Mouse CD33-Related Siglecs with ITIM-Like Domains
To date, mouse Siglec-E, -F, and -G belong to the CD33rSiglecs group, which have
ITIM-like domains. In the CD33rSiglecs subgroups of different biological species,
only humans have eight different CD33rSiglecs species, whereas mice have five
species [573]. From phylogenetic analysis, it can be observed that the eight CD33-
related Siglecs in humans originated from gene duplications that separate primate
from rodent lineages. Human Siglec-7, -8, and -9 belong to the three Ig-domain-
containing Siglecs. Human Siglec-10 and Siglec-11 belong to the five Ig-domain-
containing Siglecs. In the mice CD33rSiglecs family, one three-Ig-domain-
containing Siglec is found and it is called mSiglec-E/MIS [28, 612] and five Ig-
domain-containing Siglecs is called Siglec-G of mouse [573]. Siglec-E and Siglec-G
of mouse are the molecular orthologs of Siglec-9 and Siglec-10 of humans, respec-
tively [573]. The four Ig-domain-containing CD33rSiglecs in mice and humans are
named human Siglec-5 and mouse Siglec-F, respectively.
Mice CD33rSiglecs with ITIM-like domains are therefore Siglec-E, -F, and -G of
mouse, and the eight CD33rSiglecs of humans, which consist of the ITIM, are found.
Unfortunately, murine ITIM-containing CD33rSiglecs are not well known. Two
mouse Siglec-E and mouse Siglec-F as CD33rSiglecs are functionally known. The
mouse Siglec-F is present on immature types of BM-derived myeloid cells and
CD11b-positive myeloid cells residing in the spleen and thymus [573]. The mouse
Siglec-E is a myeloid cell inhibitory receptor [612]. Mouse Siglec-E and mouse
Siglec-F have been characterized previously [150]. The mouse Siglec-E is present on
mature innate immune cells. Mouse Siglec-F is present on mature and immature
blood eosinophils and bone marrow. Mouse Siglec-E has the combinatory features
of both human Siglecs-7 and human Siglec-9, whereas Siglec-F of mice is the
Siglec-8 ortholog of humans, where human Siglec-8 expression is restricted only
in eosinophils. Siglec-G was discovered as an inhibitory receptor of B1 cells. Siglec-
G inhibits BCR-driven calcium signaling. Siglec-G-deficient B1 cells induce cal-
cium signaling, showing Siglec G-driven downregulation in B1 cells. Therefore, the
abnormal B1 cell signaling seems to be caused by Siglec-G because B1 cells are
negatively regulated by Siglec-G dependency. Siglec-G-null mice exhibit large B1a
cell expansion and high production of IgM subtypes but not IgG autoantibodies.
7.14.1 mSiglec-E that Belongs to CD33-Related Siglecs
Siglec-E and its homologue Siglec-9 of humans regulate TLR-4-mediated cytokine

expression on macrophages and DCs. Siglec-E as a murine CD33rSiglec is also the
inhibitory receptor present on macrophages and neutrophils. Mice Siglec-E belong-
ing to the CD33-related Siglec family is dominantly present on leukocytes of the
spleen. Mouse Siglec-E is also found on mature NK cells, peritoneal cavity macro-
phages, and splenic neutrophils or DCs, whereas mSiglec-F is found specifically on
blood eosinophils. Hence, mSiglec-E regulates those cell functions. Mouse Siglec-E
is present on phagocytes and APCs such as neutrophils, macrophages, and DCs
[150, 613]. Mouse-specific mSiglec-E was identified by a yeast two-hybrid system
using the SHP-1 gene as the fifth mouse Siglec [28], The SHP-1 gene-targeted and
rapid amplification of cDNA ends (RACE)-PCR was successful. Siglec-E leads to
constitutive Tyr phosphorylation and consequently recruits SHP-1. mSiglec-E mod-
ulates cell signaling by controlling TLR signaling and the β2-integrin signaling
pathway. The mSiglec-E gene encodes 467 amino acids, with a V-set Ig domain to
bind SAs, two C2-set Ig domains, a TM region, and two ITIMs in its cytosolic tail.
mSiglec-E ITIMs recruit two phosphatases of SHP-1/-2, which are negative immune
regulators. Therefore, the mSiglec-E form regulates hematopoietic and immune
cells. The coding gene contains six exons. mSiglec-E is closely connected to
mouse CD33 (Siglec-3) at 118 kb downstream of mSiglec-E. COS-7 cell-expressing
mSiglec-E mediates SAs, depending on the interaction with human red blood cells.
The mouse Siglec-E sequence is highly identical to those of Siglec-7 and -9, which
are all human-expressed. Therefore, mSiglec-E is an ortholog of Siglec-7 and -9,
which are human forms. The gene for mSiglec-E is loaded on the same location of
mouse chromosome as mCD33. mSiglec-E or mCD33 does not have any relation-
ship with any human Siglecs.
As mSiglec-E is present on leukocytes residing in the mouse spleen, it inhibits
receptor signaling in immune and hematopoietic cells. Human Siglec-9 is found on
NK cells, monocytes, and neutrophils. mSiglec-E has a higher affinity to α2,8-di-SA
linkage than α2,3-SA or α2,6-SA linkage. Siglec-E expression is mainly observed on
blood neutrophils. Siglec-E and Siglec-9 are myeloid-specific Siglecs that regulate
TLR-4-mediated cytokine production in DCs and macrophages but have been less
well characterized. Siglec-9 engagement reduces TNF-α production and increases
IL-10 levels [153]. Although Siglec-E is present on myeloid cells, the mechanisms
are not yet demonstrated. TLRs regulate the inhibitory Siglec-E expression to
modulate the immune response to TLR ligands. Siglec-E is MyD88-dependently
expressed in TLR-depended NF-κB translocation. Siglec-E associates with the
negative TRIF-dependent signaling regulator of SHP-2. Siglec-E is also a negative
modulator of TRIF-driven signaling and inhibits TLR-induced IFN-β and RANTES
expression [551]. Siglec-9 engagement with sialylglycan ligands results in apoptosis
in human neutrophils. Siglec-E-specific antibody treatment abrogates the neutrophil
recruitment during neutrophil-involved pulmonary inflammation of mouse [614]. As
neutrophils are involved in inflammation events, Siglec-9 targeting will be beneficial
in asthma treatment or related pulmonary disorders characteristic of neutrophilia.
In murine BMDMs, antibody–Siglec-E interaction diminishes the synthesis of
cytokines TNF-α, IL-6, and RANTES when treated with LPS [483]. Enforced
Siglec-9 synthesis on human macrophage THP-1 cells and RAW264 mouse macro-
phage cells attenuates the expression of the above-mentioned cytokines even upon
LPS treatment [153]. Siglec-E suppresses expression of the inflammatory cytokines
in macrophages when a SA-containing glycan-expressing GBS strain is encountered
[154]. Murine macrophages treated with sialylglycans inhibit LPS-induced inflam-
mation [615]. Direct recognition of TLRs with Siglec-E is known [353]. The cis-
interaction of Siglec-E with TLR-4 leads to TLR-4 endocytosis during capture of
E. coli and downregulates TLR-4-mediated inflammation [156]. Siglec-E regulates
TLR-4 downstream through cis-recognition [157]. Although Siglec-E expression is
strongly increased by LPS with Tyr phosphorylation and SHP-1 recruitment, Siglec-
E affects macrophage differentiation when treated with LPS. Thus, Siglec-E does not
seem to affect the direct regulation of TLR-4-driven downstream signaling in DCs or
macrophages. LPS-induced Siglec-E expression regulates the macrophage pheno-
typic changes. Siglec-E KO mice increase S. enterica Typhimurium growth in the
mouse liver tissue. However, Siglec-E KO macrophages did not show any changes
such as altered uptake or bacterial killing. From the results obtained from several cell
types such as BMDCs, macrophages, and splenic DCs prepared from normal wild-
type mice and Siglec-E KO mice, Siglec-E has been shown to be unnecessary for
endocytotic internalization of TLR-4 upon E. coli infection and LPS treatment.
Splenic DCs express Siglec-E in an extremely week manner.
On the other hand, TLR functions positively in cytokine production and in the
removal of viral and bacterial invaders in immune cells. From a negative viewpoint,
TLR overproduction is harmful to cells. To avoid an inflammatory pathogenesis,
mSiglec-E expression is MyD88-dependently induced by TLRs and Tyr phosphor-
ylated during LPS stimulations, and finally TLR response is repressed [616]. Alter-
natively, in TLR immune response-regulating Siglec-E, Siglec-E is involved in
E. coli endocytotic internalization. Siglec-E-deficient DCs abrogate TLR-4 expres-
sion when the cells are infected with E. coli [156]. Siglec-E expression also
suppresses immune responses to a sialylated GBS. Direct recognition of TLRs and
Siglec-E is derived in the cis- and trans-modes. Cis-binding of Siglec-E to TLR-4
leads to endocytotic internalization of TLR-4 upon E. coli infection and
downregulates TLR-4-promoted inflammatory responses. Cis-binding regulates the
activation of B cells against antigens and consequently prevents autoimmune
responses. Although Siglec-E–TLR-4 binding occurs through a cis-type interaction,
a trans-type interaction is also observed during crosslinking with antibodies, SA–
macrophage interactions, or SA-bearing pathogen–macrophage interaction. Trans-
binding suppresses Siglec-E-dependent TLR signaling. The trans-binding to Siglec-
E enhances ITIM phosphorylation and regulator recruitment to control TLR signal-
ing. It is considered that activation of NF-κB and p38 MAPK signals mediated by
TLR-4 induces TNF-α and IL-6 levels. BM-derived DCs increase the ubiquitinated
TLR-4 and ubiquitin ligase binding to TLR-4. Thus, Siglec-E inhibits the TLR
activation through endocytosis to restrict TLR-induced cytokine expression. Thus,
Siglec-E acts as a keeper of TLR signaling-mediated immune responses in the
infection period.
In membrane signaling, mSiglec-E is a β2-integrin signaling mediator in immune
cells. Neutrophils are crucial for host defense when bacterial and fungal pathogens
invade. Siglec-E expressed on mouse myeloid cells acts as a suppressor of β2-
integrin-dependent neutrophil recruitment upon LPS exposure. Siglec-E induces
production of ROS in neutrophils, allowing CD11b 2-integrin binding to fibrinogen
in a SA-dependent manner [617]. Siglec-E-silenced neutrophils treated with fibrin-
ogen show a decreased phosphorylation level of Akt, and ROS production seems to
occur via Akt activation. Siglec-E stimulation of ROS is important for its functional
inhibition, while the NADPH oxidase inhibitor blocks neutrophils and ROS produc-
tion suppresses neutrophil recruitment of the Siglec-E. As an inhibitory receptor of
neutrophils, the inhibitory activity of Siglec-E is expressed through NADPH oxidase
activation and ROS production. Siglec-E KO mice also showed exaggerated neu-
trophil recruitment and this event is reversed by treatment with the β2-integrin
blocker, CD11b. Mouse Siglec-E is an inhibitory controller of recruitment of
neutrophils at the entry of the lung and β2-integrin-dependent signaling
[150]. Protozoan pathogen parasite, T. cruzi, strains produce sialyl ligands for
Siglec-E. T. cruzi binding to Siglec-E present on host cells rapidly mobilizes
Siglec-E to the T. cruzi surfaces. Siglec-E recognition regulates the APC function
to a blocked IL-12 production crucial for a Th1 response [618]. Siglec-E induces
LPS-elicited macrophage differentiation, without effect on TLR-4 signaling by
macrophages or DCs [157].
In an in silico analysis [619], the SIGLEC-12 gene protects the cells from SLE
onset in human populations. Two distinct missense mutations were detected in a
lupus B6.NZMSle1/Sle2/Sle3 (Sle1–3) mouse of Siglec-E homologous to human
SIGLEC-12. The missense mutations decreased the binding level of Siglec-E to
splenic cells. The Siglec-E/ mice had SLE phenotypes. They also exhibited
elevated glomerular antigen–antibody complex deposition, autoantibody release,
and kidney dysfunction like human SLE nephropathy. Thus, Siglec genes including
the SIGLEC-E gene induce resistance to SLE in humans and mice. The Siglec-E-
exerted protection of TLR signaling is well studied in the SLE pathology, because
Siglec-E binds to TLRs and downregulates TLR functions for TLR-2 and TLR-4
[353]. In lupus-prone animal models, TLR-2 and TLR-4 bind to bacterial cell walls.
TLR-2 and TLR-4 lead to dsDNA-reactive autoantibodies via HMGB1 function in
nucleosomes [620, 621]. SIGLEC-E gene mutation also increases the responses to
HMGB1 and thus the SIGLEC genes regulate the risk onset of SLE in macrophages.
7.14.2 Siglec-F (Human Paralog Siglec-8) as a CD33-Related

Siglec
In this chapter, Siglec-F-related biology is reviewed in various cells and mice.

Mouse Siglec-F is indeed an apoptotic receptor in eosinophils. Siglec-F is a func-
tional inhibitory and surface glycoprotein receptor on eosinophils discovered a
decade ago. Siglec-F is an eosinophil marker in mice [622]. Siglec-F is used to
monitor and identify murine eosinophils in combined assays with IL-5 receptor
alpha (IL5Rα). CD11b+ Siglec-F+ IL5Rα myeloid lineage cells, which are pre-
pared from the BM of the mouse C57BL/6 strain [623], reside in eosinophil-negative
PHIL mouse and the cells are not propagated even in the condition of IL-5
overexpression. The CD11b+ Siglec-F+ IL5Rα subpopulation propagates in the
BM of Myb mice from negative transcription of Siglec-F by Myb. Siglec-F+
IL5Rα cells exhibit granulocyte-macrophage progenitor (GMP)-like developmen-
tal potential. Both Myb and Siglec-F regulate GMP.
Human paralog Siglec-8 corresponds to Siglec-F as surface glycoproteins
expressed on eosinophils [396, 624]. mSiglec-F and hSiglec-8 are crosslinked and
this is confirmed by specific mAbs and carbohydrate ligands as well as the possi-
bility for apoptosis of eosinophils. These features can be applied for treating asthma.
Siglec-F is utilized in combination with IL-5R-α (IL5Rα) in mouse eosinophils.
Siglec-F and Siglec-8 interact with sialylglycans or antibodies, leading to eosinophil
death [625, 626]. Siglec-F recognizes natural tissue ligands such as mucin Muc5b-
derived glycans on tracheal epithelial cells [626]. Apart from eosinophils, Siglec-F is
also present on alveolar macrophages, peritoneal macrophages [627], DCs, mast
cells [628, 629], and epithelial cells of tuft and M in the intestines [630, 631].
Siglec-8 is also termed “Sd family 2,” known as SAF-2 protein, which is an I-type
TM protein. Human Siglec-8 was identified from eosinophils in hypereosinophilic
syndrome patients in 2000. Siglec-8 of MW 54 kDa with 431 amino-acid residues
has 49% homology to Siglec-3, 42% to Siglec-5, and 68% to Siglec-7 [397]. Similar
to known CD33rSiglecs, Siglec-8 contains two ITIMs resembling a conventional
ITIM (ILVxYxxLV sequence, where x indicates any amino acid) and an
immunoreceptor Tyr-based switch motif (ITSM) in the distal membrane, which
resembles a motif (TxYxxIV) located on the signaling lymphocyte activation mol-
ecule (SLAM). Human Siglec-8 is an eosinophil marker because it is present on mast
cells and eosinophils and also, weakly on basophils, indicating that Siglec-8 may be
the receptor of the three allergic effector cell lineages. As confirmed in human
CD34+ cells, Siglec-8 is a terminal biomarker for the differentiation of eosinophils
and mast cells. The SIGLEC-8 gene, similar to other known CD33rSiglec genes, is
encoded on the chromosome 19q13 centromere. As the current Siglecs recognize the
α2,3-SA and α2,6-SA linkages, Siglec-8 recognizes the α2,3-SAs linkage to
Galβ1,4-GlcNAc-R. Only a minor group binds to α2,8-linked SAs [23, 632]. From
the glycan array experiments, supplied by the Consortium for Functional Glycomics,
Siglec-8 was found to specifically bind to 60 -sulfated SLeX with the carbohydrate
structure of 60 -sulfo-SLeX/NeuAc-α2,3-Galβ1,4(Fucα1,3)(6-O-sulfo)
GlcNAcβ1-R. Human Siglec-8 is negative for recognition of the SLeX structure,
known as the ligands for three different selectins, namely, E, L, and P. Thus, the
C-60 -sulfate in the Gal residue is a prerequisite for Siglec-8 glycan recognition
[405]. Siglec-F also binds to 60 -sulferic sialyl LacNAc and multivalent sialyl
LacNAc structures in glycan arrays. Siglec-F recognizes the NeuAc-α2,3-Gal(6S)β
1,4GlcNac motif.
Combined glycans expressed in the mouse lung induce apoptosis of eosinophils
during allergic asthma in the lung tissue by Siglec-F present on the eosinophils.
Although Siglec-F can recognize the sialylated sulfated glycans, Siglec-F-recogniz-
ing glycoproteins or endogenous sialoside ligands are still unknown. Siglec-F and -8
are commonly characteristic of the membrane protein, with expressions in humans
and mice, belonging to the CD33-related Siglec family, and with the ITIM and
ITIM-like domains. As the engagement of Siglec-F induces apoptosis of eosinophils,
therapeutic application can be made to eosinophilic diseases. As Siglec-F is a protein
expressed on mice eosinophils, a combination of Siglec-F and its ligand induces
apoptosis, resulting in reducing eosinophilia and inducing eosinophil-borne disor-
ders during apoptosis. In fact, eosinophils contribute to various human diseases,
although its role is still not yet studied.
Eosinophils fight parasitic infections and are associated with the asthma patho-
genesis and allergy. IL-5 as a key regulator of eosinophil development regulates
proliferation, differentiation, and maturation. Mice expressing IL-5 bear ten-fold
more eosinophils in the hematopoietic system than wild-type mice. This expansion is
observed in Siglec-F-expressing eosinophils [633]. Eosinophils are accumulated on

exposure to allergens and parasitic worms. Siglec-F expressed on eosinophils
inhibits accumulation of eosinophils in the inflammation condition. Even though
the endogenous ligands are known for Siglec-F, the exact binding property is not yet
reported. For Siglec-F ligand synthesis, two sulfotransferases, keratan sulfate
(KS) Gal-6-O-sulfo-transferase (KSGal6ST) and chondroitin 6-O-sulfo-transferase
1 (C6ST-1), synthesize Gal-6-O-sulfate. Sulfotransferases synthesize the Siglec-F
ligand, as demonstrated using KSGal6ST- and C6ST-1-deficient mice. Although the
Siglec-F ligand is present in alveolar macrophages, eosinophils, and neutrophils, the
expression of the Siglec-F ligand is increased in the lung upon infection of parasitic
nematode, Nippostrongylus brasiliensis. By galactose 6-O-sulfotranferase, expres-
sion of the Siglec-F ligand is induced in various cell types during allergic lung
inflammation [634]. Siglec-F is a negative regulator of ovalbumin (OVA)-induced
eosinophilia. Moreover, an antibody for Siglec-F can reduce eosinophilic inflamma-
tion. Thus, for pharmacological application, combined sialylated ligands with
Siglec-F or Siglec-8 can induce eosinophil apoptosis because Siglec-F or Siglec-
8 functions as a negative regulator for eosinophilic inflammation [635]. As the role
of Siglec-F depends on allergen-caused airway inflammation, Siglec-F-deficient
mice can be used in functional study.
Mouse lung glycoprotein recognizes Siglec-F in a SA-dependent manner
[636]. Muc5b and Muc4 were discovered as Siglec-F-binding glycans. In the lung,
Muc5b is a glycoprotein ligand for Siglec-F and binds to eosinophils to induce cell
death. In Muc5b-deficient mice, eosinophilic inflammation was increased by IL-13.
Thus, for future prospects, Siglec-F-binding glycans can be developed to treat
eosinophilia in the lung. Accumulation of eosinophils by allergic inflammation in
allergen-induced lung tissues involves several steps to eosinophilia. Although a
trans-endothelial migration event of eosinophils is documented, a trans-epithelial
accumulation event is not scientifically accepted. In allergic inflammation, eosino-
phil migration is an end point, although eosinophil recruitment and sequential events
are not certain. Accumulation of eosinophils and gradual phenotype changes from
Siglec-FmedCD11c to Siglec-FhighCD11clow are also reported [637].
For the immunosuppressive status of the body, a recent study has clarified Siglec-
F engagement in tumor progression [638]. Glucocorticosteroids such as dexameth-
asone (Dex) regulate tumor-caused edema in patients by immunosuppression. Dex
blocks immune surveillance, contributing to lowered survival of patients. Dex
affects the cell surface sialylation pattern and Siglec-F recognition. Poor immuno-
genic glioma cells such as GL261 and SMA560 exhibit Dex-induced α2,8
sialylation, whereas the α2,3-linked SA levels are unchanged. Dex reversely affects
α2,6 sialylation in glioma cells in a Dox dose-dependent manner and binding of
Siglec-F to glioma cells. Dex reduces α-neuraminidase activity. Dex-caused changes
in sialylation and binding capacity of Siglecs may block the immune response
against tumor and affect glioma cell growth.
7.14.3 Human Siglec-10 and Mouse Ortholog Siglec-G

as CD33-Related Siglecs
The human ortholog of Siglec-G is Siglec-10. Siglec-10/Siglec-G belongs to CD33-

related Siglecs. Siglec-G, an inhibitory receptor of B cells such as B1 cells, is mainly
present on B cells, DCs, and eosinophils, suppressing BCR signaling on B1a cells.
However, Siglec-G’s coreceptor CD22 is expressed only on B cells with the
conventional B-cell functions, called B2 cells. Siglec-G blocks Ca2+ signaling,
regulating B1 cell expansion and antibody secretion. Thus, Siglec-G acts as a
BCR signaling modulator in B-cell populations. B cells produce Siglec-2 (CD22)
and Siglec-G. Siglec-2/Siglec-G inhibits signaling of B cells. Hence, they are the
inhibitory coreceptors for BCR-mediated signaling of B cells, which is operated
through suppression of calcium mobilization and cell function. Siglec-22/CD22
associates with SHP-1 via ITIMs, consequently blocking the BCR-elicited Ca2+
response in normal B cells. CD22 recognizes α2,6-linked SAs in a cis-manner to
regulate the CD22–BCR association and hence controls the CD22 inhibition role.
CD22–ligand trans-binding controls B-cell migration and BCR signaling [229]. Dis-
criminating self from nonself is crucial for maintaining tolerance of B cells. The
inhibitory coreceptors Siglec-G/CD22 in the BCR induce tolerance of B cells to self-
antigens. When autoreactive B cells encounter self-antigens, they are normally
restricted by an anergy state or apoptosis. The “two-signal” system explains the
B-cell tolerance if T cells or inflammation signals are not activated. However, the
signals are not crucial for the self-antigen response through T cell-independent type
2 (TI-2) multimers. The underlying mechanism is that the mature B cells avoid the
sialyl-self-antigenic TI-2 responses, as TI-2 antigens are sialylated and hence are
rarely immunogenic and induce tolerance. TI-2 antigens elicit innate immune
responses but the innate immunity to induce tolerance against host TI-2 antigens is
not understood. Then, the following questions are raised: how to induce such innate
immune tolerance? How to induce B-cell tolerance to TI-2 self-antigens? Addition-
ally, how do B cell-restricted Siglecs like Siglec-2 and Siglec-G contribute to
tolerance?
7.14.3.1 Mouse Siglec-G Functions in B-Cell Tolerance
Siglec-2 and Siglec-G induce B-cell tolerance and thus prevent plasma cell differ-
entiation. Siglecs discriminate B-cell self from nonself, by inhibiting self-antigen
responses while allowing “missed self-” responses to asialo glycan antigens
[639]. Although CD22-lacking mice increase calcium signaling in the conventional
B2-cell and normal B-cell maturations, Siglec-G-lacking mice increase Ca2+ mobi-
lization with expansion of B1 cells. CD22- and Siglec-G-lacking mice do not induce
autoimmunity. Siglec-G/CD22 double-KO mice increase Ca2+ responses in B1/B2
cells with elevated IgM production in the sera and increased B1 cell subsets. The
increased B1 cell levels are increased in Siglec-G KO mice. Siglec-G/Siglec-2
double-KO animals diminished immune responses to both different type II antigens

such as thymus-dependent and thymus-independent type II antigens. B cells pre-
pared from Siglec-G/CD22 double-KO mice are rapidly proliferated upon treatment
with TLR ligands. Aged Siglec-G/Siglec-2 double-KO animals produce anti-DNA
and antinuclear autoantibodies. Siglec-G and Siglec-2 are compensatory and main-
tain B-cell tolerance [640].
Siglec-G is indeed an inhibitory modulator of BCR function of B1a cells. The
number of B1a cells is increased in Siglec-G KO mice for unknown reasons. Siglec-
G KO B1a cells are rarely subjected to spontaneous apoptosis, allowing a prolonged
lifespan. The low apoptotic cell death is caused by high NFATc1 production in
Siglec-G- KO B1a cells, where the cells show an altered BCR repertoire. The B1a
cell population is upregulated in Siglec-G-null mice [641]. Two Siglecs, CD22 and
Siglec-G, inhibit the BCR signal by binding to SAs to elicit tolerance to self-
antigens. Sialylglycans are not present in microbes but present in vertebrates that
provide a tolerogenic signal. Siglec-G and Siglec-2 on B cells block BCR signaling.
CD22 is a dominant regulator of calcium signaling on conventional B2 cells and is a
minor regulator on restriction zone B cells. Siglec-G functions in B1 cells and affects
the lifespan and antibody genesis. It is involved in TLR signaling, regulating the host
immune responses of B cells [251]. Sialyl antigens induce tolerance.
B cells bear many cytoplasmic ITIMs such as CD22, FcγRIIB, FCRLs, CD72,
Siglec-G/-10, PECAM-1, PIR-B/LIRB1, and LIRB2PD-1. The phosphor-ITIMs
associate with adaptors such as SHP-1, SHP-2, SHIP1, and SHIP2 and activate the
receptor functions. These phosphatases then negatively regulate BCR signaling.
From inhibition of BCR signaling, the ITIM-possessing receptors are called inhib-
itory BCR coreceptors. Inhibitory coreceptor-negative mice showed that the inhib-
itory coreceptors modulate development of B cells, antibody production, and
autoimmune diseases. The ligands for inhibitory coreceptors also regulate their
inhibitory activity. They can control antibody production and autoimmune disease
progression [642]. They contain cytoplasmic ITIMs and recruit the SHP-1 to inhibit
downstream signaling. The two functional Siglecs, Siglec-G and -10, bear an ITIM
domain and an ITIM-like domain as well as one Grb2 binding site [23]. Information
on Siglec-G signaling is restricted. SHP-1/-2 is found as a Siglec-10 counterpart. For
example, the fused cytosolic region of Siglec-10-GST is phosphorylated by Tyr
kinases, as demonstrated by GST pulldown and SHP-1/SHP-2 immunoassay. Y667
is the key Tyr residue to adapt SHP-1/-2 [643].
Mouse Siglec-G exclusively regulates B1a cells, whereas CD22 acts as an
inhibitory receptor specifically on B2 cells. Then, how do B cells act when both
Siglec-G and CD22 lose their ability to bind SAs? CD22/Siglec-2 recognizes α2,6-
SA linkages on glycans in a cis- or trans-manner [644]. In contrast, Siglec-G
recognizes both α2,6-SA and α2,3-SA linkages in a cis- or trans-manner [645]. Of
interests, each ligand recognition regulates in a different mode they function in
B-cells. For the last two decades, information on BCR signaling, ligand distribution,
autoimmunity prevention in naive B-cell subsets is being accumulated. Apart from
Siglec-G, CD22–CD45 and CD22–galectin-9 interactions influence BCR signaling,
indicating CD22 as a central regulator in B-cell therapeutical approaches. Siglec-G
deficiency causes autoimmune diseases such as CIA and lupus-MRL/lpr in animals

[646]. However, a Siglec-G and CD22-double-KO mouse develops autoimmune
diseases like SLE [638], highlighting the importance of Siglecs in preventing
hyperactivity of B cells. The ligand binding of both Siglec-G and CD22 may prevent
autoimmunity. This is because sialic acids are SAMPs and therefore Siglec recog-
nition to SAs in a cis- or trans-manner potentially induces tolerance signals
[236]. To answer this, Siglec-G R120E-CD22-R130E-crossed mice were developed
to show Siglec-G and CD22 mutations, allowing defective ligand recognition
[647]. The results showed that different CD22-R130E and Siglec-G-R120E are
restricted to B cells by them. In addition, the animals did not show autoimmunity
in parameters of autoantibodies, BUN, and proteinuria, indicating that their ligand
recognition is not a major factor in B-cell tolerance development [647]. Using
Siglec-G R120E-CD22-crossed R130E mice, the number of mature B cells was
decreased in the BM region similar to that in mutated CD22 in mice. They increased
B1a cell number in the peritoneal area and a skewed BCR repertoire in peritoneal
B1a cells, similar to mice with mutated Siglec-G. Ca2+ mobilization was reduced in
B2 cells and was altered in peritoneal B1a cells, whereas B-cell survival was not
affected in B2 and B1a cells. Siglec binding to sialic acids as abundant self-ligands
cannot be a solid mechanism for Siglec-mediated B-cell tolerance [647]. B cells
enable B1a cells to express the IgM isotype. However, B1b cells prefer to potentiate
adaptive responses to express the IgM subtype. Typical B2 cells are involved in T
cell-dependent acquired immune responses [648]. Both Siglec-G and -10 are alter-
natively spliced for variants [649–651]. All variants have the same cytoplasmic
ITIM to interact with a negative regulator SHP-1 for B1 cell lineage. Siglec G is
synthesized during development of B cells. Siglec G KO mice exhibit an expanded
B1a cell lineage, through activation of NF-κB function. Mouse Siglec G restricts the
B1a cell expansion after postnatal development through suppression of NF-κB
function [574].
7.14.3.2 Mouse Siglec-G Functions in T-Cell Downregulation
Siglec-G and -10 bind to CD24 [301], as demonstrated in the result that Siglec G KO
mouse [652] resembles CD24 KO mouse in the susceptible phenotype to
acetaminophen-induced hepatitis. CD24 is a small GPI-anchored sialoprotein. A
targeted mutation of Siglec-G increases the natural IgM antibody production in B1
cells. Siglec-G ligand incorporation with T cell-dependent and T cell-independent
antigens decreases antibody expression and activates tolerance of B cells. Upon
CD24 recognition, Siglec-G inhibits inflammatory responses to DAMPs, such as
HSPs and HMGP-1, but not TLR ligands. Apart from CD24 binding, Siglec-G binds
to Cbl, degrades RA-inducible gene-1, and decreases IFN type I expression upon
infection of RNA viruses. The Siglec-G/-10 degradation function allows the host to
distinguish infective nonself from noninfective self [652].
Siglec-G also negatively regulates the T-cell immune responses, which can be
applied in graft-versus-host disease (GVHD) reduction during transplantation.
Siglec-G negatively regulates DAMP-mediated innate immune responses and T-cell

functions. Siglec-G adapts with CD24 through DAMPs of HMGB1, HSP70, and
HSP90 and consequently represses those DAMPs effects. CD24 and Siglec-G do not
influence the inflammatory response to LPSs or poly (I:C), known as TLR-4 and
TLR-3 agonists, respectively. CD24-Siglec-G/-10 distinguishes DAMPs and
PAMPs through selective repression of the DAMP-specific host responses [31]. In
allogeneic transplantation of hematopoietic stem cells (HCTs), the injured host
tissues are raised by immune rejection and consequently, produce proinflammatory
cytokines, DAMPs, and PAMPs. DAMPs and PAMPs activate APCs to provoke
acute GVHD. Siglec suppression of DAMP-induced responses on APCs is required
for the prevention of GVHD [653]. Recently, Siglec-G in the condition of DAMP
treatment suppressed T cell-mediated immune responses. T-cell modulation of
Siglec-G is applicable for targeting the T cell-driven cytotoxic pathology in immune
rejection of GVHD [654]. However, interaction of Siglec-G with its agonist CD24Fc
fusion protein in T cells ameliorates GVHD, retaining graft-versus-tumor (GVT)
effects.
Siglec-G has a distinct SA-recognition pattern in peritoneal B1 cells than in
spleen B cells. SAs are produced differentially in the two B-cell subsets; thus, cis-
ligand interaction is important in B1 cells. Knockin (KI) mice with a ligand recog-
nition region mutation of Siglec-G show increased numbers of Ca2+ signaling,
survival, and Ig repertoire in B1 cells. Such phenotypes resemble the Siglec-G-
lacking mice. The ligand recognition region mutation of Siglec-G decreases the
Siglec-G–IgM interaction on the surfaces of B cells. Therefore, the Siglec-G
SA-dependent interaction with the BCR is important for Siglec-G inhibition in a
B1 cell-restricted manner and is downregulated when compared to CD22/Siglec-2
expressed on B cells [655].
Siglec-G KO mice prematurely expand for polyclonal CD5+ B cells at the
peripheral region and spleen. Siglec-G KO-aged animals accumulate monoclonal
B cells and enhance the lymphoproliferative disorders of B cells. Lymphatic tumors
in Siglec-G KO-aged animals are monoclonal and are featured on follicular lym-
phoma and large B-cell lymphoma, not chronic lymphocytic leukemia (CLL)
[656]. The KO mice decreased expression of human ortholog Siglec-10 in B-cell
lymphoma and leukemia. Negative BCR regulators of Siglec-G and -10 induce
formation of B-cell lymphomas.
7.14.4 Siglec-H as a CD33-Related Siglec
Mouse Siglec-H is also one of the CD33-related Siglecs and a molecular signature
for microglia. Siglec-H is present in a precursor subset of DCs in the BM [657],
spleen marginal zone macrophages, and lymph node macrophages. However,
Siglec-H is not expressed on brain macrophages and brain-infiltrating monocytes.
Mouse Siglec-H is regarded as a microglia-specific marker because it is absent in
myeloid cells of blood monocytes and peripheral macrophages [658–660]. However,
its expression is not histologically confirmed in the nervous system. Recently,

Siglec-H has been stained in mouse microglia using an anti-Siglec-H antibody.
Siglec-H is costained with parenchymal microglia of a developmental- to-adult-
stage fetus. It is persistently present on activated microglial cells upon brain injury
inflammation [661]. Siglec-H is also a biomarker for pDCs [662].
Although many Siglec receptors contain inhibition motifs such as ITIMs, Siglec-
H does not contain such motifs, lacking the gene region, and, instead, it associates
with DAP12 on pDCs [662]. Siglec-H transduces signals via DAP12/TYROBP
[663]. Siglec-H gene and protein expression in mouse microglia is increased upon
interferon-γ treatment or M1 microglial polarization. Microglial Siglec-H is indeed a
phagocytosis receptor. The Siglec-H extracellular domain is found only on mouse
glioma lines, not in astrocytic cells or normal cells in mouse. Siglec-H-expressing
microglial cells internalize intact glioma cells. Siglec-H promotes phagocytosis of
glioma cells by activated microglia with a collaborative action of its adapter protein,
DAP12. M1-type microglial cells internalize glioma cells through a DAP12–Siglec–
H interaction [663]. Antibody–Siglec-H conjugation eliminates type I IFN produc-
tion and IFN-α generation in pDCs when CpG-containing oligonucleotide (TLR-9)
is administered in vitro and in vivo [662]. Siglec-H-deficient pDCs induce IFN-α
expression upon TLR induction in vitro [664]. Siglec-H controls the response of type
I IFNs when mouse CMV is infected and can be used in the prevention of viral
induction of development of autoimmune diseases, which systemically emerge
[665]. Siglec-H is a mouse-specific antigen; however, IFN-α-producing pDCs of
SLE patients and normal individuals produce blood DC antigen 2 (BDCA-2) and
BDCA-4 [666]. Siglec H-positive pDCs regulate inflammation and
autoimmunity [667].
In the mice clone backcrossed, which the BDCA-2–DTR gene is transgened to
congenic B6.Nba2 mice, Siglec H–positive pDCs have been examined for the lupus-
like symptom development [668]. Lupus-like symptoms such as SLE, with
hypergammaglobulinemia, antinuclear autoantibodies (ANAs), lymphadenopathy,
splenomegaly, IgG–antigen complex formation, glomerulonephritis, and IFN-α
expression were detected in female B6.Nba2 mice via IFN-α signaling [669–
673]. In B6.Nba2.BDCA-2–DTR-Tg mice, ablation of the Siglec H-positive sub-
population of pDCs in young female B6.Nba2 mice reduced IFN-α expression with
the inhibition of disease development [669].
In the interaction between sialylated N-glycans and Siglec-H in the mouse brain,
N-glycan A2G’2F of the structure of Galβ1,3GlcNAcβ1,2Manα16
(Galβ1,3GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAc-as a
sialylated form is known [674]. The branched form of sialylated A2G’2F is a
major N-glycan structure in the mouse brain. Sialylated A2G’2F contains a type
1 antennary structure (Galβ1–3GlcNAc-R) with two types of α2,6-sialylated
N-glycans of Neu5Ac-α2,6-Gal-R and a type 1 antennary structure of Galβ1–3
[Neu5Acα2–6]GlcNAc-R. The branched structure Galβ1–3[Neu5Acα2–6]
GlcNAc-R is 6-sialyl-Lewis C (6SLeC). The level of 6SLeC-bearing branched
sialylated A2G’2F is upregulated during development [675, 676] because 6SLeC-
containing branched sialylated A2G’2F is involved in the development of the mouse
References 459
brain. However, its function is not known. Recently, as an A2G’2F carrier,

calreticulin has been discovered in the dendritic spines of cultured cortical neurons.
The branched sialylated A2G’2F glycan binds to Siglec-H and interacts with den-
dritic spine microglia in synaptosome. Calreticulin as a chaperone protein of the ER
is observed in the dendritic shafts of cultured rat hippocampal neurons [677] and also
in the ER and the plasma membrane as well as in the PSD-95-positive spines
[674]. Branched sialylated A2G’2F has been suggested to be transported to the
synaptic cellular membrane of neurons by LAMP, calreticulin, and neurotrimin as
carriers. The synthesized 6SLeC binds to Siglec-H [674]. Microglia also express
Siglec-E, and Siglec-E recognizes both SAα2,3 and SAα2,8 glycans together with
6SLeC [678]. Cell surface calreticulin on target cells functions as an ‘eat-me’ signal
to promote phagocytosis [679]. Thus, calreticulin used as an eat-me signal will
induce synapse clearance. Branched sialylated A2G’2F on synaptosomal proteins
in the mouse brain may be involved in synaptic pruning via binding to Siglec-H and
Siglec-E. Sialylated N-glycans and Siglec recognitions form synapse and afford
surveillance [680]. Although an ortholog of Siglec-H is not found in humans, human
Siglec-L2 was found as a possible ortholog having 42% identity with mouse Siglec-
H [681]. The signal transduction between synapses and microglia may occur via the
recognition of branched sialylated N-glycans with Siglecs.
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Chapter 8
C-Type Lectin (C-Type Lectin Receptor)
8.1 Evolutionary Diversity of C-Type Lectins
All organisms have their own specialized patterns like face appearances. The
molecular patterns are grasped by specific pattern-understanding molecules called
pattern recognition receptors (PRRs). Likewise, pathogen-related stimuli are recog-
nized through the specific transmembrane receptors of the host cells, PRRs, present
on the cell surfaces of adaptive and innate immune systems in hosts. PRRs are
responsible for sensing the incidence of any cell damage as well as the presence of
infecting microorganisms. So, they recognize endogenous molecules derived from
cellular damages or internal injuries, called DAMPs, as well as evolution-based
structures surfaced on microbes, termed PAMPs. CTL or CLR is a pathogen and
antigen receptor on DCs. DCs specially recognize pathogens to regulate immune
responses and protect the host from external environments. DCs are also engaged in
homoeostatic regulation, recognizing self-antigens to allow tolerance from the tissue
environment. Therefore, one question is how the nature of the recognized antigens
takes the balance between immunity and tolerance. CLRs expressed on DCs sub-
stantially recognize and bind glycosylated self-antigens and pathogens. It is cur-
rently becoming deciphered that the CLRs serve as antigen receptors potentiating
internalization to present antigens and as recognition receptors of glycosylated self-
antigens to function as adhesion and signaling molecules. Thus, C-type lectins are
accurately expressed depending on maturation stimuli. One of the well-known CLRs
represent DC-SIGN that recognizes high-mannose types and Lewis antigens of Lex,
LeY, LeB, and LeA. Another CLR known as macrophage Gal and GalNAc-specific
C-type lectin (MGL) recognizes GalNAc residue. From the fact that the glycan
structures of antigenic epitope enhance MHC-I responses and the antigen-reactive
CTLs’ functions, it is considered that glycosylated antigen specifically interacts with
C-type lectin to induce antigen-specific T-cell functions. Consequently, C-type
lectin-recognized antigens can regulate T-cell polarization. The CLRs stimulate
DCs signaling events with TLR activation. Understanding the CTLs on DCs and
498 8 C-Type Lectin (C-Type Lectin Receptor)
the carbohydrate-specific recognition structures of DCs will greatly help us to

comprehend pathogen recognition in diseases. In addition, because tissue tumors
frequently express LeX LeY and GalNAc glycan structures, the information can also
help to understand how DCs regulate cellular tumor immunity through cellular
interactions.
Receptors expressed in human cells are largely divided into two families: Ig
superfamily including a classical MHC-I or killer cell Ig-like receptor that binds to
nonclassical HLA-G and CLR-like family. CLR also includes NKG2 receptors that
bind to nonclassical HLA E or stress cells, transformed cells, or other distant
molecules that are upregulated in infected cells. This is because NKG2 receptors is
a family of lectin-like proteins. NKG2 receptors include two distinct groups of
inhibitory receptor called NKG2A and activating receptors called NKG2C and
NKG2D [1]. Among them, two subclasses of NKG2-A and -C receptors recognize
the HLA-E that is nonclassic HLA-I. However, NKG2D receptor recognizes the
MHC-I-like A (MICA) and MICB as well as UL16-binding protein (ULBP).
Therefore, the interaction between NKG2D ligands and NKG2D receptor transduces
to hosts. In tumor cells, they express NKG2D ligands, like HLA-E expression,
elevating graft versus tumor. Because DCs are the specialized components in
distinguishing “self” from “non-self,” DCs easily uptake Ags and viruses in periph-
eral tissue and deliver them to lymphatic organs for induction of T cell functions.
Apart from antigen presentation, DCs trigger antigen-based T cells education
through binding of antigen-loaded MHC-II molecules and TCRs. This allocates
the clonal expansion of TCR-matched T cells. Also, T cells have inflammatory
functions through cytokine productions upon TCR-launched signaling. DCs also
prevent immune reaction against self-antigens by the mode of tolerogenic DCs to
secrete immune-suppressive IL-10 and induce regulatory T cells (Tregs) to inhibit T
cells-driven immune responses. DCs contribute to the T cell clonal deletion with
TCRs reacted against self-antigens, creating a word of “central tolerance.” There-
fore, specific DCs concept is raised for each specific position, as known in some DCs
which exhibit bifunctional immunogenic and tolerogenic characters. Because
immune system’s functional balance is regulated by inhibitory and activating recep-
tors in leukocytes and nonimmune cells, inhibitory receptor families are associated
with specific signaling mechanisms.
With respect to molecular features of CLRs or CTLRs, they are present on cell
surfaces of most cell types of macrophages and DCs as PRR which recognizes a
variety of glycan structures. CLRs use their CRDs in their extracellular portion.
CLRs crucially recognize and bind exogenous PAMPs, because the patterns are
mostly common to fungi, viruses, and helminths. In addition, endogenous ligands of
self dead cells or self glycans are also recognized by CLRs. Particularly, CLRs
expressed on DCs are essential for binding and endocytosis of glycosylantigens into
intracellular compartments present in DCs, leading to intracellular digestion and
processing of antigens, and eventually presentation of the antigen fragments on the
surface receptors of MHC-I and MHC-II. Ligand binding of CLRs on the surface of
the cells often induces intracellular signaling events. The cell surface ligand-binding
molecules refer to CRD of proteins of host cell surfaces, which is known as a lectin.
8.2 Ca2+-Dependent Glycan-Binding CTLs 499
When the ligands are bound to CRD of CLRs on the surface, intracellular signaling
events are induced. Through intracellular signaling, several transcriptions of target
genes are increased by functional stimulation and translocation of downstream
factors including AP-1, NFAT, and NF-κB into nuclear region. These signals
promote the expression, production, and secretion of proinflammatory IL-1β/IL-6/
TNF-α cytokines. On the contrary, anti-inflammatory IL-1ra and IL-10 cytokines are
also increased to control inflammation progression.
8.2 Ca2+-Dependent Glycan-Binding CTLs
A CLR as a domain type of glycan-binding proteins or lectins Ca2+-dependently

bind to glycans. The term of C-type lectin commenced with distinguishment of the
lectin proteins having Ca2+ dependent carbohydrate-binding specificities from the
regular animal lectins. Types of CLRs are mostly expressed on DCs and the lectin
function of these CLRs is found in the conserved CRDs. CLR domain-bearing
proteins have a wide scope of roles in cellular attachment, cell–cell adhesion, cell–
cell interaction, cell endocytosis, cell migration, pathogen-specific responses of
immune cells, and apoptotic cell death. The carbohydrate-recognition and binding
properties of CLR or C-type lectin are within the spatial sequence region with a
characteristic structural pocket designated as C-type lectin domain or type-C CRD.
Sequence homology allowed the identification of type-C CRD from candidate pro-
teins, including those not bound to calcium Ca2+ ion or carbohydrates. Therefore,
solid contradiction is deduced from the structural basis of the type-C CRD. But this
discrepancy is resolved by introducing the “CTL-like domain” (CTLD) as a new
terminology. The CTLD is therefore just the protein sequences with unique struc-
tural motifs, which are available from protein databases. Therefore, the terminology
of type C lectin is generally accepted for certain protein structures with one or more
units of CTLDs, regardless of the presence of calcium or carbohydrate. More
specifically, some cases of CTLDs are independent of calcium Ca2+ ion or carbo-
hydrate binding capacity. The terminology of C-type lectin was derived from the
primary capacity to bind calcium ion in order to facilitate carbohydrate binding.
Although C-type lectin domains have a wide scope of 1,000 more proteins as the
largest lectin family, not all CLRs require Ca2+ ions in order to facilitate the
carbohydrate recognition and binding capacity. In addition, many CLRs can bind
non-carbohydrate ligands including proteins and lipids. CLRs exhibit diverse func-
tions in cellular processes including cell–cell attachment and adhesion, innate
immune cell interaction to pathogenic invaders, clearance of pathogens, and apo-
ptosis of injured selfs. These types of receptors consist at least one of CTLD, a
distinct folding domain arranged with S-S disulfide bonds in Cys residues-conserved
regions [1]. The C-type lectin family proteins have many roles in adhesion, natural
killer cell regulation, complement cascade activation, tissue reorganization, platelet
hemostasis, thrombosis, ligand-receptor endocytosis, pathogen phagocytosis in
innate immunity with C-type lectins of 1000 members more. CLRs are classified
C-type lectin
Type Ϩ
Type ϩ
DEC-205
S
S
MR DC-SIGN Langerin
S DCIR
S CLEC-1 Dectin-1
Soluble form : MBL
CRD
Dectin-2
Carbohydrate recognition domains (CRD)
DLEC
Tyrosine-based motif for targeting to
coated pits and internalization
P I Triad of acidic amino acids

I
P T
P I
T Fibronectin type II repeat
A
M
M Di-leucine motif
Tandem repeat
Fig. 8.1 Types of C-type lectin receptor and signaling cascade. Activation receptors includes
Dectin-1/-2, Mincle, and MCL, which are mainly mediated by Syk kinase and Raf-1. Inhibitory
receptors like MICL are mediated by SHP-1 Tyr- phosphatase known as an attenuator [6]
into 17 groups with membrane anchored or secreted soluble proteins [2], while
CLRs are classified into 14 groups based on the arrangement of CTLDs. Type V
receptors include a lectin-like NKG2 family, which are distinct receptors present in
endothelial cells and myeloid cells with binding capacity to specific ligands of
β-glucan-binding receptor and lectin-mimic receptor of oxLDL-1, which is also
alternatively called LOX-1. The β-glucan receptors are active phagocytic receptors
of β-glucan expressed in microbial pathogens such as fungi and yeast.
CLRs are directly related to innate immunity. For example, during leukocyte
homing event, a CLR endothelial selectin binds leukocytic sialyl-lewis antigens of
surface glycoproteins to potentiate migration activity of leukocytes to inflamed sites
[3]. MBL as a collectin type is involved in complement activation upon microbial
infection [4]. In innate immunity of myelocytes, DC-SIGN is a representative CLR
[5], and other subgroups of CLRs include Dectin-1 and Dectin-2, macrophage CTL
(MCL), myeloid inhibitory CTL (MICL), macrophage inducible CTLs (Mincle), and
the macrophage MR (Fig. 8.1). They are structurally in homology with a typical
carbohydrate-binding C-type lectin, which is Ca2+-dependent. However, type V
receptors lack Ca2+ coordinating amino acid residues. For antigen presentation of
C-type lectins, CLR’s cytoplasmic motif has an internalization motif and some
C-type lectins are specific for antigen presentation to CD4+ and CD8+ together.
The glycan specificities of C-type lectins of Langerin, DC-specific DC-SIGN, and
MGL are well studied. For example, the DC-SIGN ligands are known for
8.2 Ca2+-Dependent Glycan-Binding CTLs 501
non-sialylated Lewis (Le) glycans of Lex, Ley, Leb, and Lea as wells as LDN-F and
high-mannose. For Langerin, the binding ligands are Ley, Leb, GlcNAc, LDN-F,
GalNAc, and high-mannose. On the other hand, the MGL ligands are LDN-F and
GalNAc. These are all involved in pattern recognition, antigenic presentation,
cellular adhesion, and signaling-immunomodulation.
DCs also functions as a key displayer of Th1-type CD4+ T cell responses to
protect the host from eukaryotic pathogens of fungal infections. Th1-specific cyto-
kine, IFN-γ is important for protection against fungi because Th1 CD4+ T cells-
driven immunity induces the protection immune responses. If CLRs are stimulated
and activated by interaction with ligands, proinflammatory responses are started to
protect the host cells from fungal microbial pathogenic infections. To date, the
activation mechanism of CLRs is quite informative, while the mechanism of how
the activated CLRs are negatively controlled is little informative.
DCs can largely be grouped into the two subsets of plasmacytoid DCs (pDCs) and
conventional DCs (cDCs). The pDCs subsets are present as rarely existing cell types,
occupying up to 0.8% of the total peripheral blood mononuclear cells (PBMCs), and
they protect from viral infections, through antiviral type-I IFNs. pDCs subsets
mainly express type-I IFNs and are regarded as type-I IFN-expressing cells to protect
from viral infection [7]. Because carbohydrate recognition is the first step of the
innate immune responses, free carbohydrates such as mucus expressed in body fluids
can prevent pathogenic attachment to host cell surfaces [8].
In fungal infections, pDCs are also suggested to involve in antifungal immunity
of Aspergillus fumigatus hyphae and conidia [9]. In virus attachment to host cells,
various surface proteins of hosts are activated to elicit their innate immune
responses. The known surface molecules include DC-SIGN, MBP, surfactant pro-
tein (SF)-A and -D, liver/lymph node-SIGN (L-SIGN), and the ficolins. They bear
CRDs responsible for molecular recognition of microbial pathogens-derived carbo-
hydrates on LPS and glycoproteins [10, 11]. Ebola and Marburg viruses are deco-
rated with carbohydrates. Glycoprotein spikes of the filovirus Ebola and Marburg as
well as Venezuelan equine encephalitis virus (VEEV) recognize attachment to host
cells. Inversely, CRD present in the DC-SIGN of DCs and L-SIGN on lymphotic
cells recognize the oligo-Man saccharides attached O-glycans on GP1-anchored
proteins. L-SIGN is also expressed in certain hepatic cells and endothelial cells
[12]. More precisely, the DC-SIGN CRDs recognize the Man9GlcNAc2 carbohy-
drates via a high affinity, compared to Man residue. However, MBP blocks the
DC-SIGN binding to DCs through recognition to the GP1 glycoprotein decorated on
Marburg virus and Ebola virus [13]. The Marburg virus and Ebola virus recognize
many different cells through the folic acid receptor-α (FR-α) and folic acid can block
the entry into the host cells [14]. Experimental attachment of lentivirus bearing
Ebola glycoproteins to Hela cells has been blocked by the multivalently constructed
oligo-Man glycodendrimers or α-methyl-D-Man, which are known for filovirus
inhibitors. Their minimum inhibitory concentrations were calculated to be IC50
337 nM and 1.27 mM, respectively. The alphavirus of VEEV infects equine with
transmission to humans through mosquitoes. The VEEV glycoprotein E2 com-
mences with adherence to host cells. Glycoprotein E2-specific receptor has been
suggested to be laminin-binding protein or heparin present on the mosquito cells or

Chinese hamster ovary cells (CHO) [15]. Several lectins such as Con-A, WGA, and
soybean agglutinin inhibit the viral attachment to murin macrophages. Therefore,
VEEV binds to GlcNAc, α-linked Man, and GalNAc as the terminal sugars.
8.3 Myeloid CTL-Like Receptor or Myeloid-Suppressive

or Inhibitory CLR (MICL), CLEC 12A
The bacterial, mycose and viral signatures of PAMPs are comprised of structural
carbohydrates or glycans, because glycans or carbohydrates are the predominant
constituents of cell walls located on extracellular regions of bacterial, fungi, plant,
and mycobacteria as well as viral spiked coat proteins. High Man-type carbohydrate
structures are frequently found in several fungi, bacteria, and even in viruses. Fuc
residue-linked structures are also detected on the surfaces of parasitic helminths and
several bacterial pathogens. Currently known C-type lectins bind to glycans for host
defense. Interestingly, some lectins, which cannot bind carbohydrates, can also bind
to pathogenic agents for host defense. This group of lectins includes soluble defensin
proteins of RegIIIγ that is released from the intestines, which directly kill bacteria
through its bactericidal activity, and collectin of MBP, which has a capacity of the
complement activation cascade when MBP binds to cell surfaces of bacteria. Several
CTLD-carrying CLRs as transmembrane proteins recognize pathogenic agents and
activate signal transduction pathways to induce inflammatory or bactericidal pro-
cess. The CLRs are markedly found in BM-derived differentiated cells for cellular
survival functions. Some CLRs play important roles in exertion of their PRRs to
initiate the defense mechanism using innate and adaptive immunity upon infection
(Fig. 8.1). Some CLRs attenuate and alter the myeloid cell activation with modula-
tion and regulation of intracellular signaling in immune cells. C-type lectin (CTL) is
cleaved on the CTLD sequence and domain configuration to give signaling
properties.
In ITIM signaling of CLR, inhibitory ITIM motif is featured for suppressive
phenotype upon pathogenic stimulation. For example, a DC inhibitory receptor
(DCIR) is known as one of the ITIM-containing CLRs. DCIR is also found in
certain immune DCs, B cells, monocytic cells and macrophages in human. Upon
crosslinking with specific antibodies, DCIR diminishes the induction and release of
TLR8-driven IL-12/TNF-α and TLR9-driven IFN-α release by pDCs [16]. In mice,
4 DCIR homologs including mDCIR, mDCIR-2, mDCIR-3, and mDCIR-4 are
discovered. However, only mDCIR and mDCIR-2, which is termed 33D1, have
specific ITIM region, although their functions are unknown. However, mDCIR-
deficient mice result in significant expansion of autoimmune and DC
compartments [17].
Myelosuppressive CTLs or myeloid inhibitory CTL (MICL) is the myeloid-
expressed CLR which bears ITIM in its cytoplasmic tail. MICL has four different
8.3 Myeloid CTL-Like Receptor or Myeloid-Suppressive or Inhibitory CLR (MICL),. . . 503
types of CLEC12A, CLL-1, DCAL-2, and KLRL-1. It is regulated during inflam-

matory processes as pattern recognition receptor and NK cell cytotoxic agent. MICL
is a monomeric glycoprotein with a structure similar to Dectin-1. MICL is a myeloid
CLR carrying an ITIM in cytoplasmic tail for inhibitory intracellular signaling via
SHP-1/SHP-2 dephosphates [18]. More specifically, MICL belongs to the ITIM-
bearing receptor, which is structurally similar to Dectin-1 and LOX-1. MICL is
expressed in granulocytes, immature DCs, and monocytes [18]. During pathogenic
stimulation, MICL recruits specific phosphatase enzymes of SHP-1 and SHP-2.
Altered Tyr- and Ser- phosphorylation of downstream signaling kinases of ERK
and p38 MAPK are found after interaction between MICL and immature DCs. Role
of self-tolerant MICL is unknown in detail. MICL as an ITIM-containing CLR
inhibits the IL-12 response during treatment with LPS or zymosan. How CLRs
with ITIM inhibit TLR signaling without the phospho-Tyr cascade is also unknown.
ITIM signals such as Syk binding receptors synergize with TLR signaling. Inhibitory
cell function involves ITAM-bearing receptors. For example, blood DC antigen
2 protein (BDCA2) transduces its downstream signaling through several adaptor
molecules such as Syk, BLNK, and PLCγ2 in pDC to suppress type-I IFN expres-
sion and TRAIL secretion, which are elicited by interaction with TLR agonists. Dual
roles of ITAM signaling are known in cellular activation and inhibition events. The
dual roles regulate CLR activity using Tyr residues [19, 20]. In pDCs, BDCA2
recruits spleen Tyr kinase (SYK) to the phospho-ITAM associated with the paired
signaling adaptor of Fc receptor (FcR)-chain. The SYK stimulates a complexed form
of phospholipase C2 (PLC-2), B cell linker (BLNK) and Bruton's tyrosine kinase
(BTK), which elevates Ca2+ ion mobilization. The precise mechanism(s) underlying
the downstream signaling of the complexed receptors is unknown. However, the
complex signaling downregulates TLR-9-activated synthesis of IFN-γ, TNF-α, and
IL-6 in the pDCs. The Ca2+ mobilization inhibits the association of a key recruited
adaptor molecule, MYD88, and hence prevents the TLR-driven cytokine releases.
On the other hand, DCIR activation contributes to its endocytosis into endosomes,
where TLR-8 and TLR-9 are present. The ITIM phosphorylation recruits the SHP1
or SHP2, which activates their signaling toward the modulated TLR-8-elicited IL-12
and TNF-α expression or TLR-9-elicited IFN-γ and TNF-α expression in both
mDCs and pDCs, respectively. Myeloid DCs signaling with MICL also contributes
to the ITIM phosphorylation with additional recruitment of the SHP1 or SHP2.
MICL also activate ERK, although ERK activation and downregulation of TLR-4-
drivn IL-12 expression are not correlated.
Human MICL gene is alternatively spliced to form variants with three isoforms
(α, β, and γ). Human MICL is found on certain myeloid cells such as DCs,
granulocytes, macrophages, and monocytes, whereas mouse BM NK cells, B cells,
and CD8+ CTLs are reported to express mouse MICL [17]. A remarkable aspect of
MICL expression is profound in acute myeloid leukemia cells as a biomarker and
target of antibody-based immunotherapies and murine MICL is a marker for a subset
of CD8α-DCs [21]. MICL, as an inhibitory receptor, inhibits the activation signals of
PRRs. MICL suppresses cytotoxic killing activity of NK-cells and also
downregulates responses of DCs in production of cytokine IL-12 [20]. Recently,
MICL is regulated by ATG16L1 (autophagy-related protein 16-like 1)-dependency,
giving an antibacterial autophagy. MICL can bind endogenous ligands such as dead
cells and uric acid [17, 22]. MICL suppresses the inflammatory reactions in human
leukocytes [23], controlling injured inflammation and autoimmune diseases such as
RA. Autoantibodies to MICL seem to be related with a mouse RA subset, and
dysregulation of MICL may be hyperinflammatory.
8.4 Macrophage Inducible CTLR (Mincle, Clec4e,

ClecSf9)/Macrophage CTL (MCL, CLEC4d, ClecSf8)
8.4.1 Expression and Ligand-Binding Specificity of Mincle,

Clec4e, ClecSf9, and MCL
Mincle (macrophage inducible CTLR) and MCL (macrophage CTL) have the
ITAM. Mincle or Mincle receptor is highly homologous to Dectin-2 in its structure
of proteins sequence. Mincle is initially found during the strong induction in
macrophages during inflammation by treatments with IL-6, IFN-γ, LPS, and
TNF-α. In humans and murines, Mincle expression is found in various immune-
related cells including certain types of B cell subsets, myeloid DCs, macrophages,
monocytes, and neutrophils [24]. In contrast, the Mincle is not present in pDCs, NK
cells, and T cells. Mincle reciprocally express in neutrophils and monocytes. Mincle
belongs to a type II TM protein and Mincle is basically expressed in DCs and
activated macrophages [25]. As activating receptor like the Dectin-2 cluster, Mincle
binds to the FcRγ via a positive Arg residue in the CTLR transmembrane region.
Mincle–FcRγ complex elicits receptor signaling via CARD9-Bcl10-Malt1/MAPK/
PKCδ/Syk axis pathway for expressions and releases of the target cytokines and
chemokines such as keratinocyte-generated chemokine (KC or CXCL1), macro-
phage inflammatory protein 2 (MIP-2 or CXCL2), IL-6, and TNF-α. Mincle-
responses are independent of MyD88 but Mincle and the TLRs cooperate for
inflammatory cytokine release and regulation of respiratory burst by complement
receptor-3 (CR-3) [26]. Mincle bears a typical CTLD region, which has an EPN
motif sequence, and binds certain carbohydrates such as α-Man-glycans. Fungal,
mycobacterial, and necrotic cells are Mincle ligands towards antimicrobial immunity
and homeostasis. C. albicans wall components are ligands, and Mincle protects host
immunities of inflammatory cytokine releases, fungal death, and phagocytosis from
pathogen [24].
Like to Dectin-2 type, Mincle also contains one CTLD in the extracellular region
with a short tail in the cytoplasmic region. Mincle is subclassified into Clec4e and
ClecSf9. Mincle and MCL are TM germline-encoded PRRs, specific for the innate
immune responses. MCL and Mincle are transmembrane PRRs to recognize DAMPs
and PAMPs from a bacteria or fungi. Mincle as one of the CLRs recognizes various
exogenous stimulants including mycobacterial strains and certain fungal strains. In
addition, the Mincle also recognizes endogenous stimulants such as necrotic cells of
self. Human monocytes express Mincle for inflammatory cytokine and inversely for
8.4 Macrophage Inducible CTLR (Mincle, Clec4e, ClecSf9)/Macrophage CTL (MCL,. . . 505
fungal uptake and killing. Neutrophils’ Mincle expression activates killing functions
[24]. Ligand binding to Mincle phosphorylates ITAM of FcRγ and recruits the Syk.
Syk recruitment by FcRγ activates NF-kB through Card9-Bcl10-MALT1 signaling
for immune responses and developments of Th-1 and Th-17 subtypes. Mincle
expression is induced by inflammatory stimulants of LPS, IL-6, IFN-γ, and
TNF-α. MCL and Mincle genes are loaded on chromosome 12 and chromosome
6 of humans and mouse, respectively. The gene locations on chromosomes corre-
spond to the telomere gene region of the natural killer complex. In the Mincle-MCL
hetero complex signaling pathway, the two independent receptors of Mincle and
MCL form a pair with the adaptor signaling molecule FcRγ. This association with
FcRγ is enforced by a positively charged Arg residue present in the TM region of
Mincle. However, the Arg residue in MCL is absent. During interaction with PAMPs
like fungi or bacteria and DAMPs, ITAM phosphorylation recruits Syk to trigger its
downstream signaling via the complex of Card9/Bcl10/MALT1. For example,
MCL–Mincle heteromeric complex induces sensing signaling of the well-known
wall lipid trehalose-6,6’-dimycolate (TDM). The complexed Mincle binds to the
headgroup part of carbohydrate, whereas the MCL part binds to the lipid tail part.
The Mincle has a bridge role for a MCL–Mincle–FcRγ complex formation.
8.4.2 Pathogenic PAMPs-Recognition of Mincle and MCL
It recognizes DAMPs and PAMPs including fungi, mycobacteria, glycolipid, and

damaged cell. Mincle recognizes Fonsecaea pedrosoi of chromoblastomycosis and
co-stimulates CTLR responses [27]. Mincle also binds to Malassezia to induce
certain cytokine and chemokine expressions. In addition, Mincle also invite immune
cells, which are related to inflammatory responses. Mincle binds to TDM as a
mycobacterial cord factor or an immunostimulatory glycolipid for immune response
against mycobacteria and adjuvant action to elicit cell-mediated immunity
[28]. Their ITAM upregulates inflammation responses as PRRs of DAMP and
PAMPs of fungi, Mycobacteria, glycolipid, damaged cells, or spliceosome-
associated protein (SAP130) as an endogenous carbohydrate ligand (SAP130).
Mincle also acts in the immune responses to damaged self-cells. Dead self-cells
stimulate the host self cells to express Mincle. SAP130 recognizes Mincle in the
Ca2+- independent condition. Interestingly, EPN motif-defective mutations in
Mincle do not influence its binding capacity and SAP130 recognizes the specific
site of CRD. SAP130 acts as DAMPs and provides an alarm signal for dysregulated
cell death. Mincle expression is increased upon irradiation, and Mincle-specific
antibodies inhibit neutrophil infiltration.
Fungal α-Man and mycobacteria-membrane glycolipids such as TDM are exog-
enous ligands for Mincle. Mincle binds to various endogenous and exogenous
ligands in necrotic death cells, mycobacterial surfaces, and some fungal PAMPs.
FcRγ chain and Mincle complex also mediates the TDM-associated signaling
responses when the chemically synthesized analog trehalose dibehenate has been
treated. Consequently, multiple immune responses are observed for inflammatory

cytokine expression, inflammatory mediator nitric oxide (NO) generation, granu-
loma formation, NLRP3 inflammasome formation, and T cell differentiation to Th1
and Th17 subsets. Mincle responds to mycobacteria [28]. Mincle responds to
necrotic cell death for inflammatory MIP-2 and TNF-α responses for neutrophil
recruitment to the damaged sites. Fungal strains represent Candida, Saccharomyces,
and Malassezia with preference to the latter fungal species [29, 30]. Mincle induces
various cytokines of KC, IL-6, IL-10, MIP-2, and TNF-α expression. Mincle KO
mice are susceptible to infectious Candida invasion. Antibody-treated Mincle
blocking in vivo diminishes recruitment of neutrophil and expression of inflamma-
tory cytokines [29, 30]. Glycolipids of mycobacterial cell walls are the strong
antigens to bind to MCL and Mincle during infection of mycobacteria. TDM is an
essential glycolipid produced by mycobacteria. Mincle is important for the immune
response of granulomatous formation to TDM, indicating Mincle as a TDM receptor.
Neutrophil and monocyte MCL also triggers the Syk-mediated cellular activation.
The Mincle ligands include fungal-produced α-mannan, mycobacterial TDM or
trehalose-dibehenate (TDB) as a cord factor, and small nuclear ribonucleoprotein,
termed SAP130 [28, 29, 31]. From the Mincle-mediated immune responses to
inflammatory TDM and TDB, TDM acts as a mediator of tuberculosis pathogenesis.
The less toxic TDB is then used for adjuvants for mycobacterial vaccine adjuvant in
the protective Th1 and Th17 responses. Exogenous ligands, which can be recog-
nized by Mincle, are known for fungal α-Man residue, and TDM, which is a
glycolipid produced by Mycobacterial strains. Mincle also recognizes damaged
and injured self cells and senses them for signaling. Therefore, Mincle can easily
recognize the endogenously occurring DAMPs from necrotic cells. Mincle also
recognizes injured or damaged cells by using the endogenous ligands for DAMPs
from necrotic cells. Mincle binds to the Fc common receptor, Fc-Rγ, for cellular
signal transduction via the Syk and consequently stimulation of the downstream
CARD9-dependent NF-κB axis activity. Syk activates the intracellular Ca2+ mobi-
lization and the calcineurin-NFAT activation. Mincle interacts with the FcRγ as
adaptor to exert signaling via the Syk/CARD9 axis [29]. They make a heterotrimeric
complex with the ITAM-carrying FcRγ at the cell surface [32–34]. The FcRγ
adaptor associates with Mincle by a positive Arg residue in the TM domain to
stimulate the Syk, PKC-δ, and CARD9-Bcl10-Malt1, as well as MAPK signaling
and NF-κB [35]. This adaptor also interacts with MCL in a special mode without any
cooperation of charged amino acids [36]. Like the Syk-linked CLRs, Mincle can
trigger Th17 responses and contribute to the immune reaction in fungal infection.
These receptors bind to DAMPS and initiate damaged self-sensing, and PAMPs,
which include mycobacterial TDM as cord factor during immune response to
invasive bacterial and fungal infections [37–39]. PAMP recognition is mediated
by Mincle via the carbohydrate-binding site in the CRD in the CLR extracellular
domain. In contrast, DAMP binding occurs via the CRD. The CLRs-involved
signaling pathway occurs through the adaptor molecule FcR-γ with ITAM as
activation motif. ITAM phosphorylation of FcR-γ and Syk recruitment occurs
upon binding of ligand to Mincle. Subsequently, Syk recruitment via FcR-γ leads to
B cell activation via NF-kB activation through Card9-Bcl10-MALT1 signaling axis,
which are the modulators between innate and adaptive immunities. Mincle expres-
sion is promoted by TDM with an increased level of Mincle protein, as demonstrated
in BMDCs. Enhanced expression of lung Mincle is detected upon TDM injection in
mice. In contrast, the expression of Mincle was not detected under normal
conditions.
8.4.3 Th1/Th17 Activation and T Cell Development in Mincle

or MCL Interaction with Host
The MCL–Mincle interaction is mediated by the stalk region. The receptors are
expressed in naive and inflammatory status [33, 34]. The CTLD of MCL regulates
surface expression. Mincle expression of the innate immune cells including neutro-
phils, macrophages, and granulocytes is highly increased during S. pneumonia
infection. As a Mincle and MCL complex, they are found in myeloid lineages of
macrophages, monocytes, neutrophils, and DC. In addition, they are also found in
other B cells and leukocytes [32, 33, 40], upregulating in inflammatory condition of
LPS. In case of Mincle, MyD88- and CCAAT-C/EBPβ-dependent expression is
suggested [33, 41]. Mincle and MCL activate endocytosis, phagocytosis, respiratory
burst, formation of NET and Nlrp3 inflammasome, and proinflammatory cytokines
and chemokines. Various regulatory molecules including MIP-2, TNF, IL-6, IL-1β,
MIP-1α, G-CSF, and KC (known as keratinocyte-derived chemokine) are reported
as the cases [32, 34–36, 42]. Both receptors induce adaptive immunity by Th1 and
Th17 activation [28, 32, 36]. Mincle suppresses Dectin-1-driven IL-12 expression to
promote Th2 cell type. Mincle CTLD consists of a Man-binding EPN motif and
Mincle recognizes microbial glycolipids [42–44]. Specific microbial ligands such as
mycobacterial cord factor, TDM, TDB, glycerol monomycolate, fungal
glyceroglycolipid, and mannitol-linked mannosyl fatty acids are known [41] as
Mincle’s CTLD can bind sugar and fatty acids [44] and MCL’s CTLD recognizes
TDM [45]. Like Mincle, the MCL’s CTLD interacts with sugar and fatty acid
moieties of glycolipids [21] in antimycobacterial immunity (Fig. 8.2).
In S. pneumoniae infection, Mincle acts as receptor of glucosyl-diacylglycerol
(Glc-DAG) of S. pneumoniae to protect pneumococcal pneumonia
[45]. S. pneumoniae infection in the lungs induces the Mincle expression by
means of the fact that Mincle recognizes glycolipids and Glc-diacylglyarol
(Glc-DAG) species produced by the S. pneumoniae and senses the bacterial com-
ponents to the signaling molecules. In Glc-DAG, two acyl groups are bound to the
glycosyl residue, and long fatty acid binds to acyl groups such as TDM. Trehalose is
esterified to two mycolic acid residues in the sixth carbon of each monosaccharide.
Mincle expression is high in mice lungs and humans during infection with
S. pneumoniae. The mycolic acid fraction of S. pneumoniae promotes Mincle
Glucosyl
residue
Acyl group
Glucosyl-diacylglycerol in
S.pneumoniae
Fatty acid chain
Trehalose-6,6’-dimycolate 2 Mycolic acids

in t G
G
Trehalose
Fig. 8.2 Mincle ligands of glycolipid moieties in glucosyl-diacylglycerol in S. pneumoniae and

trehalose-6,60 -dimycolate of Mycobacterium tuberculosis species. Mincle ligand glycolipid moiety,
trehalose-6,60 -dimycolate is found in the M. tuberculosis cell walls. It contains a disaccharide
trehalose, and the trehalose is linked to two mycolic acids via its esterification. One of the two
mycolic acids is attached to each sixth carbon of monosaccharides
expression, but not innate receptors of TLR2 and TLR4 signal. Glc-DAG is a better
inducer of Mincle expression than other lipid-glycerol conjugates, as shown in the
fact that Glc-DAG induces TNFα and IL-1ra expression in WT macrophages or WT
mice, but not in Mincle KO cells or untreated cells or animals. Thus Glc-DAG is a
ligand for Mincle. In animal studies, low survival is observed in Mincle-deficient
mice. In Mincle KO mice, inflammatory tissue damages are progressed by cytokines
specific for inflammatory responses and exhibit the suppressed generation of anti-
inflammatory cytokines. In chimeric mice experiment, Mincle expression on alveo-
lar immigrating leukocytes is indicative of lung protective immune determinant for
the protection of the host from S. pneumoniae-caused pneumococcal pneumonia.
Thus, Mincle-Glc-DAG is a protector line from lung infection of S. pneumoniae.
Mincle mediates the expression of inflammatory cytokines and NO, granuloma
formation, and Th1 and Th17 responses upon binding to mycobacterial ligands
[28, 32, 40]. Thus, Mincle has the adjuvant activities in complete Freund’s adjuvant
[32, 46]. MCL also has the adjuvant activity of TDM. Defected MCL indicates
defective in innate immune responses, such as inflammation and granuloma forma-
tion, and acquired immune reaction of T-cell. Mincle directly recognizes intact
mycobacteria. MCL in myeloid cells recognizes the nonopsonic mycobacteria and
defaulted MCL induces extracellular mycobacterial burdens for neutrophilic inflam-
mation. Importantly, human polymorphism of MCL increases in susceptibility to
tuberculosis [47]. Both Mincle and MCL can recognize several bacterial strains of
Klebsiella pneumoniae, as MCL KO mice showed increased susceptibility to infec-
tion of K. pneumoniae with increased inflammatory responses of neutrophils,
bacterial burdens, and inflammatory lung tissues [48]. Thus, Mincle specifically
controls K. pneumoniae infection [42], as the first characterized receptor for fungal
strains and yeasts such as C. albicans. Mincle is an immune protector in phagocy-
tosis, fungal killing, and inflammatory cytokine production [49]. Mincle expression
on leukocytes and monocytes reduces fungal uptake and induces fungal killing with
inflammatory cytokine production. MCL KO mice are susceptible to C. albicans,
giving fungal burdens. Mincle recognizes Fonsecaea pedrosoi and Fonsecaea
monophora [50]. Mincle reduces the expression of Dectin-1-induced IL-12, which
is essential for immune responses of Th2 cells. Mincle also inhibits Dectin-2-
induced Th17 cell differentiation. In binding of endogenous ligands, Mincle recog-
nizes SAP 130 of necrotic cells as a ligand. However, the ligand-recognition site of
the CTLD is diverse. SAP130 recognition of Mincle elicits the expression of
inflammatory cytokines of TNF-α/MIP-2 and accumulation of neutrophils on
inflammation sites [32]. Mincle recognizes cholesterol and is also involved in RA,
stroke, and brain injury and obesity-induced inflammation and fibrosis [32, 51].
MCL is subclassified into Clec4d and ClecSf8 types. MCL (Dectin-3 or
CLECSF8) is structurally similar to the Dectin-2. Clec4d, known as a MCL type,
possesses a TM region. MCL belongs to a type II TM protein, sharing high
homology with Mincle and contains a VEGQW sequence within its CRD. MCL
recognizes the cord factor as a TDM receptor. Although Mincle functions as an
FcR-γ-coupled receptor, MCL is present in the immune cell surfaces of the host only
in the presence of FcR-γ, implying that a complex of MCL with FcR-γg is formed.
Association between endogenous MCL and FcR-g observed in a DC line and
BMDCs indicate that FcR-g is a pre-requisite subunit of MCL. MCL functions as
an FcR-g-coupled activating receptor. Defect in the innate immunity system is found
in MCL-deficient mice. In Clec4d/ cells, the production of the cytokines is
impaired. The lethal systemic inflammation was elicited in response to TDM,
whereas Clec4d/ and Clec4e/ mice showed resistance. TDM-evoked lung
inflammations were reduced in Clec4d/ mice. MCL is needed for
TDM-mediated innate immunity [52]. The presence of MCL and Mincle is involved
in evoking the innate immune responses and there may be two possible explanations
for their required presence. First, Mincle and MCL may synergistically operate for
downstream signaling and/or secondly, priority relationship between the two may
exist where MCL regulates Mincle expression. In order to examine the synergistic
relationship between the two receptors, they were co-expressed in reporter cells, but
it was concluded that no synergistic relationship was detected between the two
systems. Mincle was dispensable for MCL expression and MCL is critically
involved in TDM-mediated innate immune responses by initiating Mincle expres-
sion. Molecular mechanisms for MCL–Mincle complex formation is interaction
between the two receptors through the stalk region in the MCL side, resulting in
enhanced expression of Mincle protein and simultaneous increase of TDM-induced
responses [53]. Mincle gene is indeed a stress-inducible gene where Mincle expres-
sion is enforced during cellular stimulation, and the reversed behavioral expression
is therefore observed in MCL-deficient mice. Mincle expression is severely
impaired. Within the CRD, the C-terminal end mediates the strong binding force.
Overall, the stalk region found in mouse MCL is essential for the binding of MCL to
mouse Mincle. Information of DAMPs and PAMPs which stimulate Mincle function
will uncovers the importance of MCL CLRs involved in an auxiliary CLR
heterodimers with Mincle.
In summary, MCL and Mincle induce development of T cells. After binding to
their cognate ligands, naive CD4+ T cells start to undergo the differentiation to the
effector CD4+ T cells for active immunities. The Th cells’ fate is decided by the
pattern of cytokines and antigen recognition by the TCR. The Th17 cells are defined
and characterized by IL-17 expression, which activates downstream inflammation
signaling with the recruited monocytes and neutrophils to make a clearance of
infected tissue damages. Th1 cells express a key cytokine, IFNγ, as an NK cell
and macrophage activator. Mincle is activated by TDM. Mincle binds to two glucose
residues within TDM and glucose monomycolate is indeed a glycolipid antigen
when the glycolipids are accountered to T cells by cooperation of CD1b. Mycobac-
terial cell wall component, glycerol monomycolate, acts as an antigen ligand for
human Mincle; however, the same mycolic acid species is not the ligand of the
mouse Mincle, indicating the structure difference between human and mouse
Mincles. For CRD structures of MCL and Mincle, the CRD of human MCL consists
of a globular fold structure with two independent α-helical structures around a β-core
strand structure and a Ca2+ interaction. The structures of human and bovine Mincle
are equally featured with a large fold, and two ions of Ca2+. Interestingly, the bovine
Mincle consists of another Na+ ion. Mincle-trehalose complex exhibits that trehalose
pyranose ring recognizes Ca2+ ion via O-3 and O-4 of Glc residue. In pathogenic
fungal infection, MCL and Mincle protect hosts from their pathogens through
clearance. The two receptors induce both Th1 and Th17 cells-mediated complex
immune responses, and fungal cells are growth-restricted and phagocytically
cleared. The downstream pathway via the signaling complex of Syk-Card9-Bcl10-
MALT1 axis activates NF-kB function for cytokines IL-6/IL-23/IL-1 synthesis.
These cytokines elevate Th17 cell differentiation. Apart from mycobacterial ligand
TDM, another fungal example of Mincle ligand antigens includes gentiobiosyl
diacylglycerides of Malessezia pachydermatis, which are known ligands of mouse
Mincle. All isomers activate Mincle, less potent than TDM.
8.5 Mannose Receptor (MR) as CLR and Macrophage

Mannose Receptor
8.5.1 Structural Basis and Functions of MR
The MR (CD 206) as a type I TM protein belongs to the membrane glycoprotein

having a 165 kDa, comprising of a cytosolic domain, 45 amino acids region and
3 extracellular regions. The extracellular regions carry a Cys-rich domain in the
N-terminal region, fibronectin type II (FN-II) domain repeats and 8 CRD-bearing
8.5 Mannose Receptor (MR) as CLR and Macrophage Mannose Receptor 511
Mannose receptor (MR)

Cytoplasmic
regions
Extracellular regions
HOOC TM 8 CRDs 1 1 R-type LDs NH2

FNϩ
CRD: C-type Lectin Domain

FN II: fibronectin type II repeat
R-type LD: R-type Lectin Domain
0DQQRVHUHFHSWRU&'
&5
)1,,
&\VWHLQHULFKGRPDLQ&5UHFRJQLVHVVXOSKDWHG
FDUERK\GUDWHVWHUPLQDWHGLQ62*DO RU62
*DO1$F7U\
)LEURQHFWLQW\SH,,GRPDLQ)&,,UHFRJQLVHVQDWLYHDQG
&7/'
GHQDWXUHGFROODJHQV,WELQGVFROODJHQ,,,,,,,9DQG9
&W\SHOHFWLQOLNHGRPDLQ&7/'ELQGVFRPSOH[

FDUERK\GUDWHVWHUPLQDWHGLQ0DQ)XF RU*OF1$F,W
UHTXLUHV&D DQGQHXWUDOS+
&\WRSODVPLFWDLOD 7\UEDVHGPRWLILQWKHF\WRSODVPLFWDLO
Fig. 8.3 The structure of MR (CD206) as a type I membrane protein in DCs and macrophages. The
MR is an endocytic receptor. It has an ability to recognize molecules of endogenous and microbial
origin. It has a major role in homeostasis and immunity. It has three distinct ligand-binding domains
in the N-terminal region. Among them, CR domain is a specific receptor for sulfated glycans as
ligand and CTLD domain is for endogenous and exogenous ligands. The FNII domain is a collagen
receptor [65] as a type I membrane glycoprotein, which contains three domains of the extracellular
domain in N-terminal region, TM, and cytoplasmic region in C-termina region. The MR extracel-
lular region is composed of 8 CRDs, FN-II, and an N-terminal R-type lectin domain
CTLDs [54], as shown in Fig. 8.3. The MR is a functional endocytic receptor to

interact with self’s and microbial ligands [55]. In addition, the MR is also involved
in intracellular signaling to activate cell functions upon recognition to many ligands.
However, signal-activating function of the MR is not well known, because the
receptors do not have signaling activation motifs or domains in their cytoplasmic
regions. Therefore, the questions how to activate the signaling activator are future’s
target to study [56]. The MR is present on macrophages, endothelium of the liver and
lymph vessels and immature DCs, but also on skin cells such as human skin
fibroblasts and keratinocytes [56–59]. MR contains a glycosylated domain in the
extracellular region and a Cys-rich domain, an FN-II, and 8 CTLDs (Fig. 8.3)
[56, 59]. The MR bears 8 CTLDs in the extracellular region and a classic signaling
motif-lacking cytosolic tail. Most MR forms are normally expressed on intracellular
region. Upon the endocytic pathway, a soluble MR is also shed into the serum. Two
types of the extended MR form and a compact “bent” form are known. The MR is an
endocytic receptor. It is the first family member of endocytic receptors including
Endo 180 (CD280), M type PLA 2 R, and DEC-205 (CD205). The MR is present in
macrophages, certain DC types, and hepatic and lymphatic endothelium-derived
cells and tissues [60, 61]. The MR recognizes the terminal Man, Fuc, or GlcNAc
residues on glycans found on the microorganisms for both innate and adaptive
immune systems. Therefore, it recognizes diverse ligands, which are endogenously
expressed and exogenously surrounded, such as bacterial, fungal, and viral patho-
genic agents of C. neoformans, C. albicans, and Pneumocystis carinii [62].
8.5.2 MR Expression in Immune Systems and Interaction

with Helminth Flatworm Trematodes
MR is present on human myeloid lineaged cells, mouse DCs, and macrophages as

well as nonvascular endothelium with large population. The MR induces helminth-
induced immune response [56]. The Th2-expressed IL-13, IL-10, and IL-4 cytokines
with arachidonic metabolites of PGE1 and PGE2 induce the macrophage expression
of MR in mouse [62]. IL-4 stimulates MR expression [63]. In monocytes, the MR is
expressed during maturation stage [56]. Subpopulations of monocytes exhibit the
MR expression dominantly increased in asymptomatic filarial infected patients,
where TGF-β, IL-10, and suppressor of cytokine signaling-1 (SOCS-1) are
co-expressed [64]. In the stimulation experiment with Trichuris suis extracts,
human monocytes are differentiated into activated macrophage subpopulations,
with concomitant MR and IL-10 expressions [65]. Macrophage activated with
helminth flatworm trematode of F. hepatica extracts increases in MR expression
as well as PD-L1, Arg-1, IL-10 and TGF-β expression levels. Moreover, the
macrophages are not responsive to LPS-mediated activation [65, 66]. Furthermore,
the MR blocking by an MR-specific antibody and hapten-like mannans results in
inflammatory responses in mice, indicating disruption of microphage phenotype
functions. For example, mice intraperitoneally injected with mannan before
F. hepatica infection exhibited such phenomenon [66]. The CLRs such as the MR
in human DCs can bind to T. suis membrane components [67, 68]. Endothelial cells
treatment with T. suis extracts increases in motility but decreases in events of trans-
endothelial migration. The MR-specific antibody with blocking activity suppresses
8.5 Mannose Receptor (MR) as CLR and Macrophage Mannose Receptor 513
the T. suis-mediated monocyte function and rescues the T. suis-reduced monocyte

trans-endothelial migration. MR also induces the PKC-mediated downstream sig-
naling in monocytes [67]. Thus, MR is a monocyte modulator.
The MR induces various responses in cells, although the intracellular signaling
mechanisms are not clear yet. The MR recognition of fungal carbohydrate ligands
occur in the phagosome and produces multiple IL-12/IL-8/IL-6/TNF-α/IL-1β/GM-
CSF cytokines, while the MR inhibits the TNF-α expression [61]. The MR also
display for cellular event related to the host homeostasis during microbiam infection,
although the MR functions in antimicrobial immunity for host homeostasis is unclear
yet. Understanding the acting mechanisms of the MR, wich are quite different from
other PRRs, will help to use for the innate immunity. MR activates T-cell tolerance
through CD45 inhibition and CTLA-4 activation. MR expression is enhanced in
peritoneal DCs in endometriosis. Further functions include clearance of glycopro-
teins from the circulation, including sulfated glycoprotein hormones and glycopro-
teins released by pathological events. The mannose receptor continuously
recirculates in a clathrin-dependent manner between the plasma membrane and
endosomes. Expression in macrophages and DCs is regulated by microbial products
and cytokines. Mannose receptor constantly recirculates through the cellular PM and
the early endocytotic compartments. Mannose receptor involves in homeostasis and
immunity because Mannose receptor recognizes endogenous and microbial origin
molecules.
The MR is located intracellularly in DCs, macrophages, and endothelial cells
[56, 69], depending on IL-4. MR is a biomarker for macrophages activated [70]. The
MR extracellular domain (MR-Ed) structure is digested to a functional soluble form
(sMR) by metalloproteinases through Dectin-1 signaling [71]. The Cys-rich motif in
MR-Ed recognizes the sulfated glycans and the FN domain recognizes collagens. In
contrast, the CTLD 4-8 Ca2+-dependently bind to terminal Man-, Fuc-linked glycan
structures, and GlcNAc residues [56]. The MR recognizes CpG
oligodeoxynucleotides [72] in both homeostasis and antimicrobial immunity. MR
recognizes viruses, helminths, trypanosomes, fungi, and bacteria [56] to induce
endocytosis, phagocytosis, antigen cross-presentation, cytokine production, and
adaptive immunity [73, 74]. MR is a phagocytic receptor, collaborating with other
receptors such as Dectin-1 and TLRs upon fungal infection such as C. albicans and
Paracoccidioides brasiliensis to express IL-17 in Th17 and Tc17 cells
[73, 75]. MR-deficient KO mouse is highly susceptible to infection of microbial
pathogens, when compared with normal MR-bearing mouse.
MR-mediated endocytosis is regulated by a model molecule of ADAMTS13 in
DCs. The MR molecular structure consists of an N-terminal Cys-rich domain
(CR) which binds to D-Man, glycoprotein terminating in L-Fuc and N type of
CLR like CRD (CTLD 1–8). The CTLD of MR can bind to glycoconjugate
terminated with Man, Fuc, or Glc. It has been shown that 4-7 CTLDs bind to
ADAMTS13. Once the MR-ADAMTS13 complex is internalized it is transported
to endosomal pathways including early endosomal organella, late endosomal
organella, and lysosomes. In the early endosome, endocytic ADAMTS 13 is disso-
ciated from MR and can be loaded onto MHC-I for CD8 CTL activation, called
cross-presentation. Other studies have demonstrated that certain soluble protein

antigens incorporated via the MR pathway can target LE and LS for proteolysis.
The antigenic peptides are then presented to the MHC-II molecule. Then, the loaded
antigens are presented to CD4 T cells. Endocytosis ADAMTS 13 is primarily
detected in the early endosomes of iDC suggesting that other pathways for endocy-
tosis of ADAMTS 13 may be necessary to present antigenic peptides to CD4 cells
[76, 77]. In inducing iDC maturation, it is possible that the antigen can be
transported to the MHC II molecule, which is called CD4 cell presentation.
Macrophages and epithelial cell MR bind foreign molecules and carbohydrates
on host molecules. The tandemly arranged C-type lectin domain promotes
carbohydrate-dependent macrophage uptake of infectious organisms and the CR
binds to sulfated glycoproteins including pituitary hormones. MR is a molecular
weight 180 kD type I TM protein containing a CR, a domain containing type II
fibronectin FN II, 8 tandem CLR CRD, and short intracellular domain. The MR is an
endocytic receptor and belongs to the CLR family. MR is present in a certain DC
subpopulation and macrophages as well as vascular endothelial cells. Despite the
presence of 8 CTLDs, only CTLD4 is involved in carbohydrate binding. In addition,
MR can bind to sulfated carbohydrates via its CR and bind to collagen via the FN II
domain. In addition to these subsets, DCs are further classified by a parameter of the
surface protein expressions of cells. CD83 and other maturation markers are
expressed in DCs that capture pathogens and acquired antigen presenting capacity.
Type C lectin receptors, which include Man-receptor (MR; CD206), DEC
205 (CD 205), and CD 209 (DC-SIGN), are responsible for pathogen recognition
and uptake. CD163 is classified to the scavenger receptor (SR)-Cys-rich (SRCR)
family class B, which is known to play the role of eliminating haptoglobin–hemo-
globin and regulating immune tolerance.
8.5.3 Recognition of Pathogenic Microbes by MR
MR as a CLR type is predominantly present in DCs and macrophages. MR recog-

nizes some pathogenic microbes such as Pneumocystis carinii, C. albicans, and
C. neoformans as well as some virus groups [78]. The receptor binds Man, Fru,
GlcNAc, and Glc residues. Upon ligand binding, the MR receptor involves in the
internalization of the pathogen to kill in the intracellular region. The details in fungal
phagocytotic roles are taken up from the previous outcomes. However, the func-
tional activity of MR, which involves phagocytosis, is still unanswered because the
MR does not have any signaling motif in the cytoplasmic region. The MR-activating
downstream signaling is still unknown. However, MR mediates various cellular
events through NF-κB stimulation and promotes gene expression of multiple pro-
tective cytokines. Cross-linking of MR with MR-specific antibody is known to elicit
the IL-10/IL-1Ra expressions in DCs. In addition, the cross-linking upregulates
costimulatory receptors to polarize Th2 phenotypes. In the mouse model, MR
binding by C. albicans induces expression of the GM-CSF/IL-16/IL-1β cytokines,
8.6 Mannose (or Mannan)-Binding Protein (MBP) and Mannose-Binding Lectin (MBL) 515
which are acting for inflammatory initiation. Deficiency of MR in model animals

induces the releases of MCP-1/TNF-α when C. albicans is infected. MR binds to the
yeast C. albicans via the branched α-Man sugar structures and N-glycosidic man-
nans. MR protects the host from C. albicans infection. Intraperitoneal administration
by C. albicans injection increases the burden levels of the fungus in MR-/- deficient
mice, even though it does not affect mortality. MR recognizes N-linked mannan
produced from C. albicans and preferentially recognizes α-linked oligomannose.
CTL-domain family member A (CLEC 5 A) is a viral recognition receptor
instead of MR and DC—SIGN of macrophages. Thus, in case of Dengue viral
infection, the viral infection is mediated by interactive recognition of MR and
CLEC5A as a multiple heterogeneous complex on macrophage [79]. Dengue virus
recognizes human MR and DC-SIGN on macrophages. After CLEC5A interacts
with dengue viruses and transduces its signaling to macrophages. However, the
exact molecular recognition of MR and DC-SIGN and CLEC5A is unclear yet.
Dengue virus penetrates macrophages through CLEC 5 A and induces a host
cytokine-dependent immune response. MR/DC-SIGN and CLEC5A on the macro-
phage lead to antiviral antibody production and other cytokine-dependent immune
responses.
8.6 Mannose (or Mannan)-Binding Protein (MBP)

and Mannose-Binding Lectin (MBL)
Lectins are diverse group of non-Ig molecules with high avidity for carbohydrates
present on glycoprotein and/or glycolipids, without functional activity as enzymes.
Immune responses to pathogenic viruses are started by virus recognition of innate
immune cells. The most cases in immune responses via molecular recognition are
performed by the lectin families as PRRs. PRRs recognize many PAMPs such as
viral PAMPs, composed of glycoproteins. Both the soluble type PPRs and mem-
brane type PRRs confer innate immunity against virus pathogens. The known CLRs
are the ficolins, Man-binding lectin (MBL), and the membrane type CD209 present
in DCs. MBL is generated in the liver and vascularly secreted into the cascular
vessel. Complement cascades of classical and alternative pathways are initiated
though antibody–antigen complex and binding to foreign PAMPs. MBL is an
oligomeric, calcium-dependent lectin of the collectin subfamily. MBL, as a part of
innate immunity, functions as an opsonic antiviral protein through cellular immunity
in DCs. MBL overaction and resulting inflammation are harmful to the host.
8.6.1 Structural Basis and Glycan Ligand Binding Specificity

of MBL
MBL is also called mannose binding protein (MBP), and an oligomeric, soluble,
calcium-dependent lectin of the collectin subfamily. It is functionally a pattern-
recognition molecule with a broad range of nonself ligands and altered self in
some cases. The MBL belongs to the collectin subfamily and CLRs with collagen
domain, because this family member possesses collagen regions and lectin domains.
In humans, two major collectin types, the SP-A and SP-D are present in lungs.
Among the three proteins of MBL, SP-A/-D proteins are relatively large in their
molecular sizes. Among them, two proteins, MBL and SP-A, are featured with a
bouquet-like and C1q-like structures.
The collectin genes are loaded to the clustered site at q21-24 of chromosome 10 in
humans. The MBL gene MBL2 is sited on chromosome 10q11.2–10q21 [80] of
humans with the two MBL genes. Among the two genes, MBL-1 gene is a
nonfunctional pseudogene, while MBL-2 gene encodes a functional form termed
MBL. MBL-2 gene structure contains four exons. Among the exons, the exon
No. 1 encodes a signal peptide, a Cys-rich motif and Gly collagen region. Exon-2
and exon-3 encode the remaining collagen region and “neck” region having α-helix
structure, respectively. The exon 4 encodes the CRD for lectin activity. MBL is
rather an acute phase protein produced in infection and trauma from liver tissue.
MBL deficiency in human is caused by point mutations occurred at three single
52/54/57 codons, known as the variants D, B, and C, respectively, in exon 1 in the
MBL gene [81]. The three referred variant types are termed D, B, and C, respec-
tively. Among them, the variant A form is a wild-type. The MBL exon 1 gene
mutation defaults for oligomerization and biological function. Apart from the struc-
tural gene mutations, polymorphisms are known in the promoter sequence of the
MBL gene. Exon 1 mutation on one chromosome and the LXP promoter polymor-
phism on the other chromosome reduce in MBL levels in individuals.
MBL exists as oligomeric forms, ranging from dimeric forms to hexameric forms.
MBL monomer has a molecular weight of 32 kDa and holds a typical structure of
collectin having four distinct regions of N-terminal 20 Cys-rich sequence motifs, a
neck region, 20 Gly-Xaa-Yaa tandem sequence repeats that are characterized as
collagen-like domain (CLD), a C-terminal Ca2+-binding domain, and ligand-binding
CRD [82]. The oligomers are composed of subunits comprising three identical
peptides, where each chain has a CRD on lectin, a hydrophobic neck domain, a
collagen domain, and an N-terminal Cys-rich domain. The three collagen regions
bind to yield a classical triple helix complex. Each neck region form located on
chains keeps a coil structure and the lectin domains in the carboxy-terminal end,
forming the globular protein forms. Each domain recognizes a Ca2+ ion, known to
form the coordinating bonds to the 3-OH/4-OH groups attached to saccharides of
GlcNAc, Man, ManNAc, Fuc, and Glc residues. The chain structure patterns of
saccharide residues found on microbial surfaces are suggested to be target molecules
for MBL recognition because the three domains of lectin are clustered with each
subunit.
8.6.2 Immunoprotective Activity of MBL
For function of MBL, MBP or MBL is regarded as C-type lectin, as Ca2+ dependent
in serum, functions in innate immunity via lectin pathway. Recognition and inter-
action with diverse bacterial and viral pathogens are cooperated with CRD with
binding specificity for Man, Fuc, GlcNAc, Glc and their derivatives. However, Gal
and Sialic acid are not recognized by MBP or MBL. MBL recognizes glycan patterns
expressed on the outmost surfaces of pathogenic agents to activate the intracellular
signaling or lectin pathway of the complement cascades. MBL distinguishes the self-
carbohydrates in host and carbohydrate patterns of pathogenic non-self surface.
Therefore, host immune cells having defaulted MBL function are susceptible to
pathogenic infections. Normal MBL can stimulate inflammatory response during
bacterial, viral, fungal and parasitic infections.
MBP recognizes cancer cells of human primary colorectal carcinoma, as MBP
ligands was easily detected by immunohistochemistry on primary tissues of human
colorectal region. MBP discriminates cancerous regions from adjacent noncancerous
regions because lignads of MBP are expressed on cancer tissues of human colorectal
carcinoma mucosae. MBP stains cancerous mucosae but not noncancerous mucosae
in the presence of a Ca2+, indicating the interaction is occurred by the CRD part of
MBP. From the patient tissues with adenocarcinomas or mucinous carcinomas, MBP
staining was around 40%. Interestingly, Fuc residues are associated with tumor
recognition by MBP in primary colorectal carcinoma cells. As Fuc residue-MBP
interaction can be detected using a Fuc-inding lectin AAL, it was demonstrated that
MBP recognizes colorectal tumor-derived Leb+ glycans and α1,2-fucosylated type-1
Le (Leb –type) glycans [83]. In tumor regression, it was also reported that MBP
specifically binds to diverse human colorectal cell lines with potent growth inhibi-
tion, as confirmed in colon cancer SW1116 cells [84]. MBP ligand oligosaccharides
prepared from oligosaccharide digests of colon cancer SW1116 cells were identified
as fucosylated polylactosamine-type structures having Leb structure of Fucα1-
2Galβ1-3(Fucα1-4)GlcNAc and Lea structure of Galβ1-3(Fucα1-4)GlcNAc. As the
endogenous oligosaccharides of tumor cells with high binding affinity, the MBP can
distinguish cancer tissues from noncancerous regions in cancer patient. Lewis
glycans as the tetrasaccharide carbohydrate are mostly linked and attached to
O-glycans on cell surfaces, but to a lesser amount to N-glycan or glycolipids.
ABO blood group antigens also contain the Lewis antigens on glycolipids. The
angenic structures of tumor-associated anti-Leb+ glycans are distinctly different from
the conventional blood group Leb antigens. CA19-9 (α2,3-sialyl-Lea) as MBP ligand
is abundantly expressed in colorectal carcinoma patients and MBP recognizes
preferably α1,2-fucosyl Leb. If the terminal Fuc residue attached to Leb epitope is
replaced by α2,3-SA, the MBL binding capacity is blocked. Endogenous MBP is
physiologically important in colorectal carcinomas and exogenous MBP is a novel
tool for diagnosis or prognosis of colorectal carcinomas in order to clarify tumor
specific carbohydrate-mediated interaction between endogenous lectin MBP and
human primary colorectal carcinoma tissues. A possible role for endogenous
MBP, as well as its usage as a potent diagnostic or prognosis marker for colon
carcinomas. MBL reduces poly(I:C)-stimulated expression of pro-inflammatory
IL-6/TNF-α cytokine genes in DCs and monocytes [84]. Instead, MBL could
suppress cytokine IL-12 and TNF-α responses in immature mDCs and monocytes
during stimulation with LPS [85], inhibiting the dsRNA receptor, TLR3 signaling
pathway without affecting to TLR3 expression. MBL accelerates LPS-induced
TLR4 activation [86] and MBL inhibits TLR3 signaling pathway by colocalization
with TLR3. MBL and CR1 interaction as well as trafficking into phagosomes
modulates the TLR3 activation. Thus, MBL inhibits TLR3-ligand poly(I:C)-induced
innate immune responses. This response is modulated by the downregulation of
innate immune cytokines of TNF-α, IL-6 and INF-γ. Therefore, MBL is believed to
exhibit anti-inflammatory capacity during viral infection and antiviral immune
response. MBL specifically recognizes adjacent monosaccharide 3-/4-OH groups,
attached to terminal saccharides of Man, GlcNAc, ManNAc, and L-Fuc carbohy-
drate oligomers [87]. MBL also recognizes nucleic acids and phospholipids, clearing
for necrotic tissue [88, 89].
MBL binds to a wide spectrum of microorganisms, but the interactions between
MBL and individual microorganisms are not well defined yet. The organisms with
surface sialylated carbohydrates are resistant to MBL recognition and binding,
whereas de-sialylation in organisms enables to susceptible and vulnerable. The
sialylated lipooligosaccharides are crucial for MBL recognition. For example, in
Salmonella enterica serovar Typhimurium, the enrichment of O antigen abrogated
the MBL recognition and binding. The truncated LPS structures facilitated MBL
recognition and binding. MBL does not easily bind to sialylated organisms but bind
to de-sialylated organisms. The capsule presence or absence is less crucial than LOS
sialylation. The O antigen addition blocks the MBL binding and the truncated LPS
structures potentiate MBL binding. The terminal LPS carbohydrate is not always a
predictor of MBL binding. The three-dimensional structure of the LPS is important
for MBL binding to the organism, and certain LPS structures act as bacterial
virulence factors by decreasing MBL binding capacities.
Thus, LPS structures function as virulence factors of pathogenic bacteria by
decreasing MBL recognition and binding levels. LPS acts as an opsonin and
stimulates the lectin pathway elicited by the specific MBL-associated serine pro-
teases (MASPs) in lectin complement cascade. The lectin complement cascade is
mediated through interaction of microbial carbohydrates with each binding lectin,
which stimulates lectin-interacted MASPs and proteins (MAP-1) (Fig. 8.4). For the
MBL-associated serine proteases in complement activation pathway, the MASP1
gene alternatively splices to the three products of MASP-1/-3 and MAP-1 [90]. How-
ever, MASP-2 gene encodes the sMAP and MASP2 enzymes. The two MASP-1 and
-2 enzymes catalyse the cleavages of the complement C3 and C4 components to
activated forms, respectively. While, the MASP-1 and MASP-2 enzymes cleave
complement C2 component [91]. MASP-2 makes the C3 convertase termed C4bC2a
stimulate complement system (Fig. 8.4). Ligand–lectin interaction induces self-
conformational changes to expose MASP Ser protease domains by cleaving an
Arg-ILe bond in the Ser protease domain [92]. To activate MASP-2 function,
Mannose-binding lectin pathway

C4b2a is a C3 convertase. It leads to the
generaon of C5 convertase
MASP1
MBL
Fig. 8.4 Complement activation by lectin pathway. C4b2a is a C3 convertase, which generates C5
convertase. MBL’s conformational shifts occur by MBL-glycoprotein recognition. The binding
event that occurs via MBL–glycan Man interaction stimulates MASP-1, followed by MASP-2, and
consequently initiates cascade reaction of complement factor cleavage. The process leads to
opsonization, inflammation, and lytic death of pathogens and infected cells
MASP-1 is necessary because MASP-1 autoactivates and transactivates the MASP-2

proenzyme by heterodimeric complex formation of MASP complexes, named the
MBL/MASP-1/-2 complex or the MBL–MASP complex [93]. The sMAP form and
MAP-1 form are the truncated forms, which were derived from the original MASP
proteins and thus they lack serine protease domains but have modulation activities of
MASP-involved activation of complement [90]. Especially, MAP-1 blocks the
MASP-2 activation through destruction of the co-complex between inter-MASP
and lectin-MASP [93].
8.6.3 MBL Function in Diseases
For the potential role of MBL in diseases, it has been initially suggested that MBL
deficiency causes for a functional opsonic defect during pathogenic infections. MBL
plays multiple roles in many diseases in humans. The major function of the MBL is
to protect in an antigen nonspecific manner from microbial infections. Hence, the
MBL involves in the first-line defense to protect from pathogenic attacks before
production of pathogen-specific antibodies commences with immune response. This
function of fist-line defenses is referred to as the pre-antibody-like character of MBL.
Lack of the MBL protein in host increases in susceptibility to various infectious
pathology and particularly acute respiratory tract infections. However, MBL protects
from infectious diseases derived from intracellular parasites like Leishmania [94],
because such intracellular parasites in humans utilize complement opsonin C3 and
C3 receptors to enter host cells but reducing the activation process of complemen-
tation function of the host. The parasite-infected patients exhibit higher level of
serum MBL than normal healthy individuals. The component deficiency of the
classical complement leads to the increased susceptibility to the increased level of
autoimmune diseases. An association similar to the complementation defect is also
observed in the MBL-deficient hosts. MBL is also involved in downregulation of
disease severities occurring in pathogen infection and autoimmune disease. MBL
modulation of autoimmune diseases is still unclarified. Therefore, the present ques-
tion how MBL elicits immune modulatory responses in the many infectious and
autoimmune diseases remains unclear. One clue for the answer may be in the fact
that the MBL-MASP pathway induces the initiation of the complement
activation [95].
During the events of MBL recognition of viruses, it has been known that MBL
binds to certain viruses in the condition of Ca2+-dependent binding. MBL recognizes
HIV-1 via high Man-type of N-glycosylated gp120 enveloped protein [96]. HIV-1
effectively evades immune responses in human via the glycan shielding process. If
Asn sites of the gp120 glycosylation are mutated, the binding capacity of the
premade HIV-1-neutralizing antibodies is prevented. However, cell receptor binding
of the antibodies is maintained [97], and also MBL can bind and neutralize HIV-1. In
addition, MBL has no capacity to neutralize HIV-1 via its complement activation
[98], but MBL directly prevents the infection in a complementation-independent
manner by eliciting MBL opsonization and consequently leading to DCs or macro-
phage phargocytosis events [99]. Recognition between MBL and gp120 can also
prevent the interaction between HIV-1 and inhibitors of virus entry to cells, conse-
quently preventing trans-viral infection raised by direct prevention of binding. The
complement-dependent and -independent mechanisms caused by the glycoproteins
coated on other viruses lead to MBL-mediated neutralization of such viruses, as
reported for the influenza A virus [100], hepatitis C virus [101], coronaviral respi-
ratory syndrome with acute onset [102], West Nile virus and Dengue virus infection
in vitro [103].
8.7 Fucose-Binding Lectin (FBL) and Ficolin
8.7.1 Fucose-Binding Lectin (FBL) Diversity of F-Lectin

Repertoires
Glycan-binding specificity of F-lectin is determined by Tandem F-CRDs, which are

composed of multiple isoforms made of recombination, alternative splicing, and
other factors. F-lectins have oligomeric quaternary structures. CRDs are distinct
from different lectins such as F-, C-, H-, and pentraxin. F-lectins involve in many
biological roles including opsonization of microbial pathogens, fertilization, and cell
adhesion. FBL binds to microbial ligands including L-Fuc, L-Gal, 2-acetoamico
L-Fuc, 3-deoxy L-Fuc (colitose), and L-rhamnose (6-deoxy-L-Man). Also, F-lectin
binds to blood group H antigen and Lea oligosaccharides [104]. For example, an
8.7 Fucose-Binding Lectin (FBL) and Ficolin 521
FBL, MsFBP-32, isolated from Morone saxatilis (Striped Bass) binds L-Fuc and has
subunit of Mr 32.5 kDa. FBP-32 found in the liver is released to plasma when
induced by infection or inflammation. MsFBP32 structure is a binary CRD lectin and
trimeric CRD topology is characteristic with 1–4 CRDs per subunit [105]. Discoidin
II is an F- and H-fused hybrid lectin and involves in cell adhesion. F-type binding
involves in fertilization and has a structural diversity through recombination and
alternatively splicing [106]. F-type eel lectin isoforms are also diverse in glycan
recognition. The eel lectin subunits are trimeric structures with sequence variability
among eel isolectins. Multiple isolectins are present in eel. The highest sequence
variability is localized to loops that encircle the carbohydrate-binding site, and
amino acid substitutions in CRDs make isolectins in carbohydrate specificity.
8.7.2 Specificity of Ficolins or FBL
The increasing evidences are prospective for ficolins in a plethora of pathogenic

infections; malfunctional mutations increase pathogen susceptibility to infection.
Ficolins are involved in fungal diseases, with beneficial effects to antifungal immune
responses. Currently, human ficolins include three M-, L-, and H-type proteins. They
are also redesignated as ficolin-1, -2, and -3 to logical naming. Information of the
two ficolin M and H forms is available, while the information of L-ficolin is limited.
Therefore, M-ficolin and H-ficolin proteins are desired in antifungal immunity and
innate immunity. Ficolins are a family of PRRs and resemble the collectin, MBL,
and surfactant proteins. Human ficolins have been discovered but three types
including H-ficolin, L-ficolin and M-ficolin are charaterized. Protein structure-
based ortholos are also identified in many different animals, indicating the common
ancestral origins evolved from ancient ficolin lectins. The ancestral similarities in the
ficolin lectins are commonly observed from the quite restricted organisms such as
chickens, invertebrates, pigs and nonhuman primates. The human ficolins are dif-
ferentially featured in many parameters including each localization in organ and
tissue as well as each capacity to elicit an immune response in human.
However, mice bear only two ficolins of A and B types as ortholog forms of L-
and M-forms, respectively. Mouse form of H-ficolin is found as a pseudogene type
[107]. The H-ficolin, M- and L- polypeptides are encoded on the genes of FCN-3,
FCN-1 and FCN-2, respectively. The proteins contains amino acids of 326, 313, and
299 as well as signal peptides [108]. Among them, the two genes of FCN-1 and
FCN-2 are present on the chromosome 9q34 in humans, while the FCN-3 gene exists
in a different location on the chromosome 1p36.11 position of humans. The two
genes of FCN-2 and FCN-3 bear a long genome structure with eight exons, while
FCN-1 gene contains nine exons. The SNP structures are present due to the high
polymorphic properties of FCN genes [109]. The high polymorphic structures
present in the transcriptional 5’-flanking region of promoter of each ficolin-coding
sequence influence regulatory properties of gene expression and protein production.
However, polymorphisms in the coded regions influence the stability, modification
levels, folding levels, and activities of ficolin proteins, thereby altering functions of
picolin protein. Nonsynonymous substitutions at certain amino acids on protein
structures alter activities of proteins; however, nonsynonymous mutations possibly
affect mRNA splicing and maturation as well as expression levels of proteins.
Each ficolin monomer consists of several functional regions. N-terminal region
carries two functional Cys residues and CLD consists of three amino acids unit of
Gly-Xaa-Yaa repeats. Ficolins form trimers through the CLDs and assemble to
bouquet structured oligomer. C-terminal region contains a globular fibrinogen-like
domain [108]. Compared to other CRDs, the FBG binds to specific PAMP of
pathogen-loaded carbohydrates and the CLD transduces intracellular signaling to
elicit immune response through MASP proteins [110]. Similar to collectins, ficolins
have an N-terminal Cys-rich domain, a CLD, and a neck domain. While, the CRD
regions of collectin structures are replaced by a FN-like domain as a FBG in the
C-terminal region. The ficolin FBG has multiple binding sites to distinguish non-self
PAMPs from selfish structures. Therefore, ficolins are essential to opsonize foreign
pathogens through recognition of the microbial cell-surfaced ligands of PAMPS. For
example, DCs M-ficolin recognizes particularly 9-O-acetyl SA through the FBG
[111]. M-ficolin recognizes a variety of acetyl-carbohydrates such as GlcNAc,
GalNAc, LacNAc, N-acetylcysteine (CysNAc), and acetyl-albumin of serum
[112]. In glycolipid and protein recognitions of M-ficolin, gangliosides and
sialylated biantennary N-glycoproteins are specifically bound by M-ficolin
[113]. M-ficolin binds to sialylglycoprotein CD43 expressed on neutrophils to elicit
cell adhesion, aggregation, polarization, and complement activation [114]. The
M-ficolin has been well studied for its recognition domain. The structure of a
ligand-binding site consists of the Asp282-Cys283 sequence by a regular trans-
conformation, which is different from the cis-conformation. Amino acid residue at
the His-284 protonation induces the trans to cis shifts for the GlcNAc
recognition [115].
L-ficolin recognizes a broad range of PAMP antigens on microorganisms and
L-ficolin, and in addition to GlcNAc and GalNAc it has a broad binding spectrum to
recognize hemagglutinin (HA), lipoteichoic acid (LTA), β1,3-D-glucan, N-glycans,
and neuraminidase [116, 117]. Apart from S1 site, L-ficolin bears three inner sites for
recognition, termed S2-S4 site, having structure plasticity. Therefore, the sites can
receive Ca2+-dependently or Ca2+-independently diverse structural molecules,
where ligands include phosphocholine parts of teichoic acids and acetyl derivative
[118, 119]. The S2 site is the Gal and N-acetylcysteine-binding site but S3 and S4
collaboratively recognize β1,3-D-glucans [118]. L-ficolin contains the S2-S4 binding
site rather than the S1 binding site present in M- and H-ficolins, which recognizes
GlcNAc residues. The L-ficolin S2-S4 site has a Phe residue, indicating that a
GlcNAc-interacting Tyr residue is replaced [120]. The S2 site recognizes GlcNAc,
CysNAc, and Gal residues and the S3 site recognizes various N-acetyl-ligands and
the S3-S4 sites recognize β1,3-D-glucans [120].
H-ficolin recognizes GalNAc residue and D-Fuc residue but not Man residue and
Lac disaccaharide [118]. Especially, M-ficolin recognizes SA [121]. The difference
in ligand preferences is based on amino acid differences on the recognition site-S1-
near the Ca2+-recognizing fibrinogen site [118]. H-ficolin has a common binding
property like L- and M-ficolins as they bind to the acetylated carbohydrates GlcNAc
and GalNAc as well as D-Fuc and Gal residues. The H-ficolin structure resembles
L-ficolin in the cis-conformation of the peptides Asp282-Cys283 linkage
[17]. H-ficolin S1 site recognizes both D-Fuc and Gal residues. H-ficolin also
recognizes in Ca2+-dependent mode N-acetylcystein (CysNAc), acetylsalicylic
acid (SaAc) and N-acetylglycine (GlyNAc) residues.
Active forms of L- or M-ficolin have dodecamer structures, which four
homotrimer subunits are comprised for a “bouquet” structure, while H-ficolin is an
octadecameric form. Similar to MBL, stable ficolin homotrimers are formed through
hydrophobic amino acid residues in the CLDs [122]. The ficolin forms oligomers
through monomeric and trimeric S-S bonds between the Cys residues in N-terms
[123]. Hepatocytes mainly express and secrete both L- and H-ficolins [124]. Specif-
ically, H-ficolin is found in epithelial cells resident in type II alveolar and also in
bronchial tract. Although M-ficolin expression is not high in lung tissue and blood,
the M-ficolin expression is also observed in the peripheral blood leukocyte
surfaces [121].
8.7.3 Ficolin Functions in the Immune Response
Ficolins as PRR involve in the immune response of innate immunity to a variety of

infectious pathogens in vascular vessels, tissues, and organs. The roles of ficolins in
innate immunity are expressed through the recognition of microbial PAMPs. Among
acetylated carbohydrates present on microbial pathogens, GlcNAc and GalNAc are
specialized common ligands of the ficolins. Ficolins also recognize bacterial pepti-
doglycan, LPS, fungal β1,3-D-glucan, and SA [125]. After the recognition of cell
surface glycans of infected pathogens, ficolins act as opsonins to recruit leukocytes
and potentiate lung epithelial cells [126]. In the opsonizing process of pathogen
removal through binding of the cell surface carbohydrates or the alternative lectin
complementation, C3b opsonin is accumulated to recruit innate immune cells for
phagocytic cell lysis. Ficolins recognize microbial and viral invaders.
Similar to MBL, ficolins activate lectin complementation through MASP activa-
tion in an indirect manner to elicit phagocytic cell death by opsonin activation. Also,
they promote the synthesis of pro-inflammation inducers and mediators including
cytokines and NO in DCs or macrophages [127]. Ficolin binding to phagocytic cells
is mediated by the Lys residue in the CLD. The Lys residues at amino acid number
57 and 47 are located on the L-ficolin and H-ficolin, respectively. The Lys residue
recognizes calreticulin (CRT) molecule present on the surfaces of phagocytic cells.
The Lys residue also binds to MASP to enable the phagocytosis and complementa-
tion of L-ficolin. L-ficolin can remove apoptosed cells and necrosed dead cells via
the apoptosis-related ligand recognition [128]. All ficolins induce the typical path-
way of lectin-complement activation during the MASPs and collectin-11 interaction
[129]. Ficolins interact with the three enzymatic MASP-1, -2, and -3 forms as well as
the non-enzyme forms of two MAp19 and MAp44 proteins. The consequently
formed complex of Ficolin-MASP cleave C2 and C4 and the C4bC2a as a C3
convertase is generated [130]. The opsonic factor C3b is accumulated on C4bC2a
factor. In parallel, C2a generates the C5 convertase enzyme that generates C5a and
C5b. The C3b functions as an opsonin and C5b makes the membrane attack complex
(MAC) which is associated C6, 7, 8, and 9 on pathogenic membrane [131]. In
addition, L-ficolin effectively prevents viral attachment and entry to host cells
[132]. The pathogen clearance activity of ficolin is interested in view of their binding
roles.
The H-ficolin exhibits defense activity against viral infection and invasion. For
application of the H-ficolin function, recombinant H-ficolin form, serum H-ficolin
form, and bronchoalveolar H-ficolin form are therapeutically used for viral clear-
ance. The above three forms of ficolins bind to influenza A virus (IAV) such as
mouse PR-8 H1N1 and pandemic H1N1 strain. This H-ficolin-PTX3 binding
inhibits HA activity and IAV infectivity. H-ficolin suppresses the IAV propagation
and pandemic amplication by virus uptaked capture and dampening expression of
monocytic TNF-α [133]. In viral clearance, L-ficolin bins to viruses through
N-glycans of envelope proteins [134]. L- and M-ficolin recognize IAV to inhibit
them [135]. L-ficolin binds to HA and neuraminidase through its FBG and blocks the
IAV invasion of kidney cells [114]. Chimeric lectins combined with L-ficolin
collagen-like domain and MBL defend the IAV and the Ebola virus
[136, 137]. H-ficolin also inhibits the pandemic infection of the H1N1 strain.
M-ficolin recognizes acute phase proteins to elicit the immune response. M-ficolin
Ca2+ dependently recognizes the long pentraxin, pentraxin 3 (PTX3). However,
GlcNAc inhibits the M-ficolin-PTX3 binding, and SA enhances the binding and
the activation of the lectin-complementation cascade [138]. The M-ficolin-PTX3
binding reduces the IA infectivity [135]. Unfortunately, rare cases of M-ficolin
recognition to pathogens are known, although the complementation induction
[121] is observed in IAV infection to block [135]. M-ficolin recognizes the mucin-
like domain in glycoprotein produced by the previously evoked Zaire Ebola virus.
This is rather known to increase the viral infection [139]. L-ficolin concentration in
sera of chronic HCV carriers is upregulated and binds to envelope N-glycoproteins
E1 and E2 and activates the lectin-complement cascade. L-ficolin also blocks
hepatitis C virus (HCV) attachment and entry to host cells. Apolipoprotein E3 is
known to inhibit the entry event of HCV [140]. L-ficolin-HCV recognition elicits
viral infected-host cell death through complement C4 accumulation but L-ficolin
binding is blocked if the glycoprotein E2 is deglycosylated [134]. Oligomeric
L-ficolin with virus-binding activity neutralizes dose-dependent HCV attachment
and entry to host cells by E2 recognition prevention with surface lipoprotein receptor
of host cells. Scavenger receptor B1 required for HCV attachment to host cells is also
prevented in a similar fashion. Monomer form of L-ficolin stimulates complemen-
tation but does not block HCV attachment and entry to host cells [132]. After
induction of L-ficolin complementation, binding to HIV-1 gp120 is made
[141]. The M-ficolin is mainly present in monocyte and granulocyte membranes,
although they lack TM domain but form sialyl membrane microdomain through its
fibrinogen domain [142]. An M-ficolin receptor is so-called GPCR-43 protein. When

M-ficolin recognizes pathogens, it indirectly stimulates IL-8 expression [143].
8.7.4 Ficolin Interaction with Microorganisms
Ficolin-A interaction with microorganisms is not well known. Ficolin-A recognizes

a plethora of microbia; pathogens including Gram-negative/-positive bacteria like
pathogenic E. coli O157:H7 strain and S. aureus strain. Ficolin-A binds to LPS
derived from P. aeruginosa and E. coli. However, their LPS binding does not protect
inflammation [144]. Ficolin-A is also associated with the lectin-complement cascade
through MASPs. Ficolin A binding to fibrinogen and thrombin stimulates comple-
mentation cascade on S. aureus. M-ficolin responds to bacterial pathogens. H-ficolin
agglutinates human erythrocytes via LPS recognition, present in E. coli O11,
S. minnesota, and S. typhimurium. H-ficolin binds to PSA of Aerococcus viridans.
Therefore, H-ficolin can directly interact with bacteria. However, H-ficolin does not
interact with pathogenic bacteria such as S. aureus, S. agalacticae, S. mitis, or
S. pneumoniae strains [145]. Moreover, H-ficolin also does not recognize other
K. pneumoniae, L. monocytogenes, and P. aeruginosa strains. However, H-ficolin
binds to enteroaggregative and enteropathogenic and E. coli strains as well as
Pasteurella pneumotropica strain [146]. The Ag85 antigen of Mycobacterium
tuberculosis is a key antigenic ligand for H-ficolin. The Ag antigen influences the
mycobacterial binding to the ECM [147]. The well-studied L-ficolin enhances the
opsonin-derived phagocytosis of S. typhimurium through complement cascade.
L-ficolin binds to the LPS O-specific polysaccharide-deficient R strain of
S. typhimurium, not to the smooth (S) LPS-producing S. typhimurium strain.
L-ficolin also recognizes LTA species produced by serotypes of S. aureus and
S. pneumoniae through the alternative lectin complementation cascade [116] due
to binding to the choline-binding protein (Cbp) virulence factor on pneumococcal
surface by the FBG. L-ficolin binds to O-acetyl-epitopes [148] and the PCho residue
in teichoic acid [149]. Also, L-ficolin stimulates the lectin complement pathway
through interaction with pneumolysin toxin of S. pneumoniae [150].
Serum M-ficolin form recognizes the terminal SA-attached capsular polysaccha-
ride (CPS) of Streptococcus agalactiae serotype VI but not to the noncapsulated
strains [151]. M-ficolin in serum recognizes SA on Streptococcus agalactiae capsule
lipopolysaccharides through its fibrinogen and consequently stimulates complemen-
tation, but L- and H-ficolin do not bind to the bacteria [152]. L-ficolin binds to Group
B streptococci (GBS) capsular polysaccharide (CPS) and elicits response to opsonin-
phagocytosis axis and C3b deposition through the alternative lectin complement
cascade [153]. The L-ficolin-GBS binding is irrespective of LTA or GBS CPS
content with SA [154]. L-ficolin cooperates with CPS-specific IgG to enhance
opsonin-phagocytic killing. L-ficolin recognizes several bacteria such as Pasteurella
pneumotropica, Listeria monocytogenes, Leptospira biflexa, E. coli, and Enterococ-
cus faecalis. In addition, L-ficolin binds to C-reactive protein (CRP) and enhances
the C3-deposited P. aeruginosa level [155]. L-ficolin acts as a defense molecule

produced in liver to prevent sepsis [156]. M-ficolin binds to M. bovis BCG,
M. kansasii, and M. tuberculosis leading to C3b deposition. C3b deposition can
increase in entry to macrophages for the virulence of M. bovis BCG. L-ficolin
recognizes efficiently the virulent strain of M. tuberculosis H37Rv rather than
nonvirulent strains of M. bovis BCG and M. smegmatis [157]. GlcNAc or neuramin-
idase treatment abolishes the binding property of M-ficolin. Recombinant M-ficolin
also binds to serotype VI streptococci and activates complement cascade
[151]. M-ficolin acts as an opsonin in U937 cells’ phagocytosis of E. coli, and M-
ficolin-specific antibodies abolish the phagocytic activity.
In fungi, M-ficolin can recognize β1,3-glucans and chitin of fungal cell wall
substances towards complement cascade and IL-8 function of a lung epithelial cells
[158]. Leukocytes in peripheral circulation and type II alveolar cells express
M-ficolin for antifungal immune response. L-ficolin recognizes the pathogenic
fungi including Aspergillus fumigatus and activates the lectin-complement cascade.
L-ficolin-PTX3 complex enhances complement cascade. The classical and
lectin-complement pathways cooperate. L-ficolin FBG recognizes A. fumigatus,
and consequently A. fumigatus associates to phagocytes including epithelial cells,
neutrophils, and monocyte-derived macrophages (MDM) [126, 159], hence the
fungal cells are killed. Also, inflammatory cytokine IL-8 is expressed by epithelial
cells but the expression levels of inflammatory IL-6/IL-8/IL-1β/TNF-α cytokines are
reduced in the MDM and neutrophils. L-ficolin concentration is increased in the
bronchoalveolar lavage fluid with fungal infections as an antifungal defense
[159]. Ficolin A binds to the Cryptococcus neoformans as a pathogenic yeast.
Parasites also bind to L-ficolin via the glycoproteins present in Giardia
intestinalis and T. cruzi cell surfaces. They trigger activation of complement cascade
[160, 161]. T. cruzi strain rather utilizes the L-ficolin and thus the L-ficolin is a
virulence agent for the bacteria whereby T. cruzi CRT binds to L-ficolin to block
alternative lectin complement cascade [162]. Ficolin-A also defends against para-
sites. It enhances the immune protection capacity of the surface protein-1 19 kDa of
Plasmodium berghei merozoite form, contributing to invasion blocking and survival
enhancement [163]. Ficolin-A is upregulated in mice macrophages infected with
tropical pulmonary eosinophilia-causing parasites [164].
8.8 Dectin 1 (CLEC-7A in Human)
8.8.1 Basic Function and Structure of Dectin 1
Dectin 1 (CLEC7A in human) belongs to the CTL family, and it mediates antifungal
innate immunity. Dectin 1 is primarily located in cell surface of DCs, monocytes,
macrophages, neutrophils, mast cells, subsets of T lymphocytes, and B cells. Dectin
has a few distinct structures compared to other members of C-type lectins. One is
that, it lacks cysteine residue in stalk region, which makes it impossible to dimerize.
8.8 Dectin 1 (CLEC-7A in Human) 527
The other type has an ITAM-like motif in the cytosolic tail region. The Dectin-1
ligand is a β1,3-glucan-containing polysaccharide, and Dectin-1-ligand binding
induces phagocytosis and produces ROS and pro-inflammatory cytokines. Dectin
1 belongs to a type II TM, NK-cell-receptor-like CTL, which has a single CRD. This
CRD domain recognizes β-glucans, T cells, and other ligands. After recognizing,
CRD is dislocated by stalk region, creating functional isoforms. The cytosolic tail
region contains ITAM-like motif for intracellular signaling.
Dectin-1 is predominantly expressed in myeloid cells. Dectin-1 ITAM
upregulates inflammation responses. It binds to β-1,3-glucan-containing carbohy-
drates and confers antimycobacterial and antifungal immunity. It induces intracel-
lular signaling through Syk/CARD9 and Raf1 towards induction of Th1 and Th17
antifungal responses. Since 2000 years, CLRs have increasingly been studied with
respect to the innate immunity in mammals during pathogenic infections. Dectin-1 is
also a group of a type II-TM protein present in myeloid lineage neutrophils,
macrophages, monocytes, and DCs. Dectin-1 also appears in certain lymphocyte
subpopulations including T cells and B cells [165]. Dectin-1 is called a CTL domain
family 7 member A, termed CLEC7A, as a glycoprotein and type II membrane
receptor. Dectin-1 is one of the well-studied CLRs among the myeloid cells-derived
CLRs, as it is found in myeloid-lineage cells [166]. The expression of Dectin-1 is
increased during inflammation upon pathogenic entry to portal cells. Dectin-1 can be
expressed in mucose environments of epithelial region [167]. Dectin-1 is composed
of a stalk-linked CTLD in extracellular domain and TM domain linked to a tail in the
C-terminal region, having an ITAM-like motif, which is named a hemi-ITAM.
Therefore, Dectin-1 consists of a signal ITAM-like motif or hemi-ITAM motif
with tri-acidic amino acids-bearing motif in the intracellular cytosolic tail region, a
TM domain, a stalk region, and the single extracellular CTLD as a CRD in the
extracellular domain. It is also one of typical PRRs.
8.8.2 Dectin-1 Recognizes β1,3/β1,6-glycans in Fungi, Plants,

Bacteria, and House Dust Mite
As a type-2 TM protein, human Dectin 1 is coded by the CLEC7A gene and belongs
to the CTL-CTLD superfamily. For CTLs, calcium potentiates the interaction of
carbohydrate ligand with Dectin-1, triggering cell death, cell attachment, and
immune response. The CTLD in PRR of Dectin-1 binds to the β1,3glycan-
containing carbohydrates and β1,6-containing carbohydrates synthesized in fungi,
plants, bacteria, and house dust mite (HDM), functioning in innate immunity.
Dectin-1 in CD11b+ DCs senses HDM molecules to display allergic airway inflam-
mation following activation of chemokine/chemokine receptors in DCs. In allergic
hypersensitivity, Dectin-I on CD11b+ DCs displays HDM-induced allergic hyper-
sensitivity [168]. HDM causes asthma, atopy, dermatitis and rhinitis and are used in
the experimental allergic asthma. Clec7a KO mice suppressed the Th2 and Th17
cells-based allergic hypersensitivity. The adaptive T cell responses are stimulated by

Dectin-1 of DCs. Among many DCs subsets, the lung CD11b+ DCs express Dectin-
I with the increased expression level of HDM-induced leukocyte chemokine receptor
CCR7, as migration level is decreased in the Dectin-I-deficient CD11b+ DCs to
lymph nodes. This also failed to induce Th2 and Th17 differentiation.
Because glycans with β-1,3/β-1,6-glycosidic linkages are frequently presented in
fungal cell walls, Dectin 1 exerts an antifungal immunity against Aspergillus,
Candida, Coccidioides, and Pneumocystis [169]. Dectin-1 binds to mycobacterial
strains and also certain ligands endogenously expressed in T-cells. However, the
detailed structures of the endogenous ligands are not clarified yet [165]. It also
recognizes the endogenous ligands of DCs and the bacterial ligands [170]. The
binding of Dectin-1 and ligands leads to intracellular signal transduction via
Dectin-1 phosphorylation by the nonreceptor tyrosinase kinase Src, where Syk
regulates the protein-9-containing caspase recruitment domain (CARD9)-Bcl10-
Malt1 complex. As an adapter molecule of CARD9 in innate immunity, CARD9
activates Dectin-1-dependent NF-kB behavior. CARD-caspase complex formation
regulates apoptosis of target cells, as NF-kB expresses proinflammatory TNF, IL-6,
and IL-2 [171]. During inflammation response, Dectin-1 is not present in epithelial
cells [172]. Dectin-1 is expressed in two alternative and spliced isoforms with or
without stalk region, depending on cell types, which possess functional differences
[165]. Dectin-1-triggered signaling activates the Syk to transduce the downstream
NF-kB, NFAT, and AP-1 known as transcription factors to activate T cells, during
inflammasome stimulation. Dectin-1 as a group V class of the CLR family interacts
with β-glucan ligand, resulting in that two signaling pathways of the Syk-dependent
and Syk-independent stream activate the tyrosine phosphatase SHP-2 to form with
Dectin-1. Dectin-1/Syk-dependent signaling pathway phosphorylates tyrosine resi-
dues in the ITAM-like motif, PKC-δ and complex of the CARD9-Bcl10-Malt1. The
consequent phosphorylation leads to the NF-kB activation as well as interferon
regulatory factor 1 (IRF1) and IRF5 activation. Dectin-1 activates Raf-1 kinase for
the downstream Syk-dependent signaling. Dectin-1-mediated phagocytic event in
macrophage is Syk-independent with Vav-1 and BTK [173]. During the actin-
mediated phagocytosis, the initial stage covers the actin-motived phagosome/
inflammasome formation, respiratory burst, neutrophil extracellular traps (NET),
DC differentiation, and DC maturation. Dectin-1 facilitates IL-1β expression by
the pyrin domain-containing 3 (Nlrp3), known as an NRL family, with noncanonical
inflammasome associated with caspase-8 by CARD9 and Malt1, nonreceptor tyro-
sine kinase Tec [174]. Dectin-1 also induces CR3 (or Mac-1) by Vav1, Vav3, and
PLC-gamma to produce phagocytic ROS [175] via microdomain formation with
lipid rafts and Syk-Jun N-terminal kinase–AP1 pathway. Likely to TLR-4, β-glucan-
Dectin-1 binding activates adapted immune reaction for Th1 and Th17 cell immu-
nity, as IL-17 is expressed in Tregs CD25+Foxp3+ [176]. During Th-1 systemic
inflammation by pathogenic infection, Dectin-1 activates Th17 responses to sup-
press the immune reaction.
In the case of a human polymorphic receptor, a Y238X polymorphism shows
homozygous Dectin-1-deficient phenotype and is susceptible to superficial fungal
infections [177], but not to yeast Candida infection [178]. In addition, Dectin-1
recognizes mycobacteria [179, 180]. In C. albicans recognition by Dectin-1,
C. albicans is bound by TLRs. TLR 2 and 4 could induce proinflammatory signals,
whereas TRIF could start Th1 responses through IRAKs, TRAF6 and TAK1 which
are protein kinases. As to be more specific about dectin-1, TLR 2 signaling is also
manifested by dectin-1 and galectin-3, making stronger proinflammatory effects. In
Dectin-1 induction of APCs, general fungal cell wall consists of layers of
mannosylated proteins, chitins, and β-glucans. APC cells such as macrophages,
DCs, and monocytes engage with fungi to activate host responses via Dectin-1
and TLRs. Dectin-1 itself generates two variants through alternative splicing, and
the two functional variants recognize branched β1,3-glucans, causing production of
cytokines. CLR receptors of Mincle, Dectin-2, Mcl, or Dectin-1 contain ITAM FcRγ
signaling chain or hemITAM in their cytoplasmic tail complex. Upon ligand recog-
nition, ITAM is phosphorylated and recruits Syk. When it recruits Syk, PLCγ,
PKCδ, and Card9/ Malt1/ Bcl10 containing complex, signaling is initiated for
NFĸB and AP-1 via MAPK pathway. β-glucan in Dectin-1-Syk-NFĸB Pathway
induces expression of miR-146a, which suppresses NFĸB pathway through Dectin-1
signaling [181].
Dectin-1 was firstly discovered as a T-cell costimulatory molecule upon endog-
enous ligand binding, and it crosses the primed CD8+ T-Cell. CARD9 as a Dectin-1-
induced CTL primer gives an anti-tumor CD8 T-Cell cross-priming to allow an
antitumor immune reaction [182]. Curdlan polysaccharide as a β-1,3-Glucan type
functions as an agonist of Dectin-1 and suppresses the expression of costimulatory
CD80, IFN-γ, and IL-1β in CARD KO DCs, and CARD9 KO mice have reduced
CD8+ T-Cells numbers. In tumor-injected curdlan vaccinated wild-type mice, tumor
growth was diminished, whereas no effect was observed in CARD9 KO mice. Thus,
Dectin-1-induced T-cell cross-priming is efficient for tumor cytotoxic immunity and
immunostimulating efficiency. Dectin-1 mediates the IgA-antigen complex
transcytosis in intestinal cells [183]. Dectin-1 also interacts with FcgammaRIIB to
inhibit complement-mediated inflammation in the IgG1, by binding to Gal residue in
IgG and FcgammaRIIB with galectin-3, increasing the anti-inflammatory DCs
behavior [184]. Dectin-1 also exhibits antitumor immunity by Dectin-1 binding to
tumor-specific N-glycans and NK cell cytotoxicity and TLR-4 [185].
Different from the Dectin-1, there are other forms of Dectin-1 species, which are
mainly present in myeloid lineage cells [186, 187]. CLEC-2 like other CLRs such as
CLEC-1 (CLEC1A), CLEC9A, CLEC12B, Dectin-1 (CLEC7A), Lox-1 (CLEC8A),
and MICL (CLEC12A) also belongs to the Dectin-1 cluster family. Among them, the
three CLEC-2, CLEC9A, and Dectin-1 forms consist of cytoplasmic half-ITAM and
undergo Syk pathway. Dectin-1 recognition of β-glucans on DCs, monocytes, and
macrophages elicits IL-2 release through the conserved YxxL motif phosphorylation
and adaptor protein, Syk kinase [188]. CELC9A as a Dectin-1 cluster is present in
BDCA3+ DCs or various tissues. When receptor is clustered, inflammatory cyto-
kines are released through the Syk [189]. CLEC12B and MICL bear an ITIM motif
of the conserved VxYxxL sequence and inhibit signalings through the SHP-1 and
SHP-2 Tyr phosphatase [187]. NK gene complex (NKC)-encoded NKp80 in NK
cells elicits cell killing during the ligand AICL linking [190]. NKp80 leads to cell
killing via the mono-YxxL motif and Syk [191]. DC-SIGN in DCs is an YxxL-
bearing family of the CLR superfamily on chromosome 19 [192]. DC-SIGN trans-
duces PLCγ2 signaling in DCs [193]. But DC-SIGN activates signaling without
YxxL and Syk. The mono-YxxL preceding sequence is the DEDG motif present in
the CLEC-2 and Dectin-1. The mono-YxxL sequence is the DEER motif in NKp80
and also the EEEI motif in CLEC9A [189]. The mono-YxxL preceding sequence is
QTRG in DC-SIGN. Therefore, D (or E) ExxYxxL sequence is an essential motif to
activate the downstream signaling with Syk dependency.
8.8.3 Dectin-1 Cluster Includes CTL-Like Receptor

2 (CLEC-2)
A dectin-1 cluster, CLEC-2, belongs to a CTL superfamily and new type of Tyr
kinase-dependent platelet activation receptor of sialoglycoprotein podoplanin pre-
sent certain tumor cells. CLEC-2–podoplanin binding involves in hematogenous
metastasis of tumor cells, but there is no direct data to support the CLEC-2 role in
tumor growth and hematogenous metastasis. Platelets release the ADP and TxA2 as
secondary effectors during activation. The ADP and TxA2 secondary mediators
mediate feedback regulation on agonistic inducer activation of platelets. The ADP
and TxA2 secondary mediators stimulate the collagen receptor known (CR) as
integrin α2β1 and consequently collagen is recognized by α2β1 integrin. Thus, the
ADP and TxA2 secondary mediators are essential for collagen-elicited platelet
aggregation activation. Collagen is a macromolecule, and therefore collagen and
α2β1 integrin adhesion receptor binding is essential for the activation receptor,
glycoprotein (GP)-VI and collagen interaction. GPVI agonists-mediated platelet-
activated aggregations, by small molecules of collagen-related peptide (CRP) or
convulsin, are weakly depended on the ADP and TxA2 secondary mediators.
Rhodocytin-elicited platelet activation aggregation is dependent on the ADP and
TxA2 secondary mediators TxA2. The ADP and TxA2 mediators induce polymer-
ization of actin filaments during activation of CLEC-2-elicited platelet.
CLEC-2 identification commences with binding of the snake venom rhodocytin,
which activates platelets [194]. CLEC-2 induces activation signaling of platelets
through downstream molecules such as Src kinase, Syk kinase, and PLC-γ2. The
CLEC-2-adaptor molecule complex resembles the CP GP-VI and FcRγ complex.
The GSL-enriched membrane microdomain (GEM) is directly associated with
CLEC-2 signaling. Different from GP-VI ITAM receptor, hemi-ITAM receptors
also include the dectin-1. Dectin-1 transduces via cholesterol-rich membrane
domains, lipid-raft GEMs, associating with the Src kinases and the adapters such
as LAT [195].
Historically, CLEC-2 has been reported to engage in snake venom-mediated
platelet aggregation in humans. Snake venoms consist of protein-targeting toxins,
acting on the vasculature. The snake venom rhodocytin or aggretin isolated from
Calloselasma rhodostoma venom [196] elicits platelet activation as aggregation
phenotype. This event is not related to the typical known protein activation system,
named collagen receptor GPVI-FcRγ complex. GPVI elicits platelet-activated aggre-
gation through the Src kinases in mouse. However, rhodocytin-elicited platelet
aggregation is also controlled by the Src family kinases [197]. This suggests that
Src kinases-involved platelet activation can be controlled by receptors that are
different from the collagen receptor GPVI-FcRγ complex. Bow, Rhodocytin-elicited
platelet-activated aggregation is undergone through CLEC-2-rhodocytin binding.
Thus, rhodocytin receptor is CLEC-2 [194]. CLEC-2 transports to GEMs to undergo
Tyr phosphorylation during rhodocytin binding, as confirmed by the cholesterol-
depleting reagent MβCD.
8.8.4 CLEC Structures and Ligand Recognition
CLRs are a receptor superfamily having one or more CLTDs. CLRs are classified as
“classical” and “nonclassical” forms, depending on glycans and non-glycan ligands,
respectively. CLEC-2 consists of a 1 CLTD as the non-classical form, bearing a
CLTD region similar to the general CRD but lacks the glycan-binding region and
calcium-binding region [198]. For example, a CLEC-2 ligand, rhodocytin known as
a snake venom, does not bear glycosylation. Also, podoplanin protein belongs to a
type I-TM glycoprotein as a sialomucin-like type. Podoplanin acts as the CLEC-2-
binding ligand. Podoponin-CLEC-2 interaction needs SA species linked to the
podoplanin O-glycans, even as non-classical CLR. CLEC-2 is also glycosylated to
yield the MW 32-kDa and 40-kDa protein forms expressed in platelets by two
different N-glycosylation sites at the amino acids 120 and 134 sites. Furthermore,
nascent type CLEC-2, which is not glycosylated, can recognize rhodocytin [199],
glycans in CLEC-2 is not involved in rhodocytin recognition but difference between
CLEC-2-Src-Syk-Cγ2 complex and GPVI-FcRγ complex is found. The GPVI-FcRγ
complex elicits activation of platelets via the ITAM motif with the tandem YxxL
sequence, whereas CLEC-2 complex elicits activation of platelets via a hemi-ITAM
motif with the YxxL sequence because CLEC-2 bears a cytoplasmic YxxL motif.
Interestingly, This YxxL motif is similar to the YxxL-(X)10-12-YxxL motif con-
served on ITAM sequences featured with 2 YxxL motifs. Generally, the mono-YxxL
motif identified on CLEC-2 is referred to the atypical ITAM, half-ITAM or
hemi-ITAM. Regarding the ITAM, ITAM has been earlier described in detail for
immune receptors including TCR. Thus, additional case of ITAM is the collagen
receptor GPVI-FcRγ-chain complex expressed on platelets and the GPVI-FcRγ
complex is well understood. GPVI binding elicits Tyr phosphorylation of ITAM
domain located on the cytoplasmic FcRγ-chain bound to GPVI via the two known
Fyn and Lyn, which are Src kinases. The ITAM phosphorylation consequently
potentiates the binding of the phosphorylated ITAM to tandem SH2 domain of the
Syk, activating Syk. Syk activation triggers downstream events including the final
phosphorylation of each Tyr residue located on SLP-76, Vav1/3 and LAT mole-
cules. In addition, the activation stimulates each enzyme of Btk, Rac/Cdc42, PLC-γ2
and PI3-K [200]. In the CLEC-2, Syk phosphorylates the half-ITAM and a Syk
inhibitor R406 inhibits rhodocytin-induced CLEC-2 half-ITAM phosphorylation,
but not the collagen-induced GPVI ITAM phosphorylation [201]. Another inhibitor
PP2 of Src kinase can inhibit phosphorylation of CLEC-2 half-ITAM protein,
specifying platelet Syk and Src kinase regulation in humans. Intracellular signaling
of CLEC-2-Syk axis is quite similar to the GPVI/LAT/SLP-76Vav1/3 axis signaling
with Btk and PLC-γ2. The PLC-γ2 and Syk are essential for the CLEC-2-mediated
downstream signaling, depending in part on SLP-76 or LAT. The ITIM motif bears
the commonly conserved sequence of the -L/I/V/S)xYxx(L/V-tandem sequence.
Phosphoryl form of ITIM recognizes tandem SH2 domain-bearing Tyr phosphatases
of SHP1 and SHP2, inhibiting ITAM receptor signaling. Platelets consist of 4 recep-
tors with different ITIMs of G6B-b, CEACAM1, PECAM-1 and triggering receptor
expressed on myeloid cell (TREM)-like transcript-1, as an Ig superfamily [202]. The
three receptors of CEACAM1, G6B-b, and PECAM-1-deficient cells exhibit the
suppressed signalings of both GPVI and CLEC-2 [202–204]. CLEC-3 has a phago-
cytosis activity via release of cytokines. In experimental animals, CLEC-2 is found
in peripheral neutrophils. In neutrophils, mouse CLEC-2 involves in phagocytic
death of cells. Neutrophils activated by rhodocytin release proinflammatory TNF-α
via the YITL of CLEC-2 [205]. In tumor progression, tumor podoplanin and
neutrophil CLEC-2 interaction induces tumor metastasis. Macrophage RAW264.7
cells produce TNF-α and IL-6 during aggretin (rhodocytin) treatment through
MAPK and NF-κB [206]. The human monocytic THP-1 cells express CLEC-2
and cytokines during aggretin (rhodocytin) treatment.
The CLEC-2 ligand is an endogenous podoplanin found on tumor cells’ surface,
and it elicits platelet activation-based metastasis [207]. From CLEC-2-deficient
model animals, CLEC-2. Podoplanin protein is produced by several cell types of
type I alveolar cells, kidney podocytes or endothelial cells in lymphoids. However,
CLEC-2 in humans is present in platelets and megakaryocytes. Contrary to humans,
mouse CLEC-2 is present in certain DCs, B cells, macrophages, ML cells, and
neutrophils in immune response [208], in addition to platelets and megakaryocytes.
CLEC-2 induces thrombosis and hemostasis [209, 210]. However, vascular endo-
thelial cells do not express the podoplanin. As lymphpoid sac is formed in the
cardinal vein, the podoplanin-CLEC-2 binding elicits platelet activation associated
with lymphatic endothelial cells. The podoplanin-CLEC-2 binding leads to separa-
tion of blood from lymphatic vessel. CLEC-2 also stabilize thrombus via homophilic
recognition. In HIV transmission, DC-SIGN and CLEC-2 expressed in platelets
involve in HIC capture and transfer to the host T cells, potentiating dissemination of
HIV-1 virus in human. Thus, DC-SIGN recognizes HIV-1 through the envelope
glycoprotein present in HIV-1 and CLEC-2 recognizes the HIV-1 virus. Podoplanin
was demonstrated to be incorporated into HIV-1 produced in experimental
HEK-293T cells in order to bind to CLEC-2. Thus, DC-SIGN and CLEC-2 act
differently to recognize HIV-1. DC-SIGN recognizes the envelope protein of HIV-1,
while CLEC-2 binds to podoplanin in HIV-1 particles incorporated [211]. In fact,
8.9 DC-Associated CTL-2 (Dectin-2) Family or CLEC4n 533
podoplanin is not present in T-cells; viral spread does not occur in vivo. However,
HIV-1 released in PBMCs that podoplanin is not expressed CELC-2 dependently
infects host T-cells. Therefore, the host T-cells express the CLEC-2 ligand [211].
8.9 DC-Associated CTL-2 (Dectin-2) Family or CLEC4n
8.9.1 Structural Basis and Function of Dectin-2
Dectin-2, which is DC-associated CTL-2, is highly similar to Dectin-1 in its struc-

ture. The last two decades have provided and exploited insights into the behaviors of
the Dectin-2 as a CTL. The Dectin-2 cluster binds to various pathogens and PAMPs,
triggering signalings for profound benefits on immune responses in antimicrobial
responses. They recognize endogenous ligands and maintain homeostasis and auto-
immunity. It is still surprising that we know only a part of their roles but knowledge
will be undoubtedly increased in near future. The CLRs display key cellular roles in
responses of innate immunity and immune homeostasis. The NK gene cluster,
termed NK gene complex (NKC), is loaded on human chromosome 12 and mice
chromosome 6. The NK gene cluster encodes many CLRs genes and NKC-encoded
CLRs are mainly present in NK cells. Non-NK cells-expressed CLRs are expressed
from the NKC complex. Both the myeloid lineage and nonmyeloid lineage cells
produce various CLR families. The CLRs have a commonly conserved CTL-like
domain in the extracellular region, which mediates direct intracellular signaling via
signal domains and indirect with adaptor proteins. These CLRs recognize endoge-
nous and exogenous ligands to act as pathogen-specific PRRs. In fact, pathogens
such as bacterial, fungi, and parasites engage the host immunities. Meanwhile, CTRs
include over 1000 receptor proteins in the forms of PM-anchored proteins and
extracellular-soluble proteins [2]. Certain receptors trigger signaling directly via
domains or indirectly via adaptor molecules-recruiting. They contain at least one
or more CRD, or CTLD folded through S-S bonds. Initially termed C-type indicates
its requirement for Ca2+ binding with carbohydrates. However, all CTLRs are not
always Ca2+-dependent, recognizing various ligands. CTLRs are multivalently
bound to ligands.
Among CLRs, Dectin-2 family includes Dectin-2, blood DC antigen 2 (BDCA-
2), CTL superfamily 8 (CLECSF8), Mincle, DC immunoreceptor (DCIR), and DC
immunoactivating receptor (DCAR). The genes of Dectin-2 cluster family of
Mincle, BDCA-2, CLECSF8, DCAR, and DCIR are all located on the chromosome
6 telomere area in the mouse NK cells and the same location of chromosome 12 of
human. The CTRs belong to the type II TM protein receptors. The receptor bears a
CTLD and short cytoplasmic tails. They, except for DCIR, elicit indirect signaling.
DCIR bears a long tail in the cytoplasmic region for an integrated inhibitory
signaling motif [212].
Among Dectin-2 family, BDCA-2 is a marker for human pDCs but a murine
homolog is not known [213, 214]. Alternative names of human BDCA-2 are CD303,
CLEC4C, CLECSF1, CLECSF7, CLECSF11, DLEC, and HECL. BDCA-2 ligands

include the envelope protein gp120 of HIV-1, asialo-Gal-oligosaccharides, having
the β1,3-Gal and β1,4-Gal residues in terminal sugar linkages and HCV-E2 glyco-
protein. Human BDCA-2 decreases the IFN-γ/IL-6/TNF-α release levels but
increases IL-10 release. Signaling motifs deficiency in cytoplasmic tail of human
BDCA indicates its roles via TM domain with the ITAM-bearing signal adaptor,
FcRγ. This elicits intracellular signaling in BCR fashion via Syk and downstream
signaling molecules [215]. Unlike ITAM-coupled receptors, BDCA-2 signaling is
negative in NF-κB activation. BDCA-2 recognition on pDCs blocks the transloca-
tion of NF-κB and cytokine releases and type I-IFNs upon interaction with ligands
specific for TLR9. BDCA-2 inhibits the pDC secretion of TRAIL [216]. BDCA-2-
ligand binding elicits phosphorylation of cytoskeletal proteins such as actin, clathrin
heavy chain, and tubulin, causing BDCA-2 endocytosis. This event commences with
the specific motif present in the late endosomal sorting in the BDCA-2 cytosolic
region. Antibody-binding to BDCA-2 causes the antibody-BDCA-2 complex endo-
cytosis and T cell presentation, due to BDCA-2 antigen capturing and presenting
role [217]. BDCA-2 also blocks the capture and processing of antigens by pDCs and
peptide antigen presentation to T cells [218] through BDCA-2-recognition. In
addition, BDCA-2 inhibits the expressions of CD40 and CD86 costimulatory recep-
tors in pDCs treated with the TLR9 CpG ligand, but not in CD40 ligand-treated pDC
activation, giving a specificity of BDCA-2 ligand preference [219, 220]. BDCA-2
consists of an EPN motif on the CTLD sequence as Man-binding domain and
BDCA-2 binds to asialo-Gal-oligosaccharides, which terminally have the β1,4-Gal
and β1,3-Gal residues [221]. BDCA-2-glycan binding induces the recognition of
CD14+ monocytes, monocyte-derived DCs, or certain tumour cells. BDCA-2 rec-
ognizes the gp120 envelope protein of HIV-1 and the HCV glycoprotein E2,
blocking IFN expression in pDCs [213].
Regarding DCAR, information of DCAR and DCAR1 are limited and not present
in humans. DCAR and DCAR1 are activating receptors. Only mouse DCAR
(mDCAR) transcripts are found in BM-derived DCs, lung tissue, lymph node tissue,
spleen tissue, and skin [222]. mDCAR1 is expressed in a tissue-dependent manner in
spleen and thymus CD8+ DCs and CD11b+ myeloid lineage cell subpopulations in
BM and spleen but DCAR1 is not present in lymphatic nodes or peripheral bloods
[223]. Only DCAR recruits the FcRγ chain, through an Arg residue present in the
CTLR TM domain. Antibody–DCAR1 binding causes endocytosis, trafficking to
lysosomal endosomes, and elicits humoral immune responses as well as immunity
elicited through CD4+ and CD8+ CTLs. DCAR1 regulates the function of DCs via
IL-12 expression inhibition and blocking of the IL-10 release.
In the DCIR, humans have a single DCIR gene, while mouse has four DCIR-like
proteins of mouse DCIR-1 to mouse DICR-4 [223]. The B cells, CD14+ monocytic
cells, CD15+ granulocytic cells, two pDCs and mDCs subsets express human DCIR.
DCIR expression is also increased in HIV-infected CD4+ T cells. Expression
patterns of mouse and human DCIR1 forms are quite similar, while mouse form of
DCIR2 is a distinct biomarker for a subset of CD8-DCs which are resident in splenic
red pulp and marginal zone. Unlike other Dectin-2 families, the DCIR cytoplasmic
8.9 DC-Associated CTL-2 (Dectin-2) Family or CLEC4n 535
tail is long. hDCIR, mDCIR1, and mDCIR2 have classical ITIMs [224]. Mouse
DCIR3 and DCIR4 are deficient for consensus ITIMs but have conserved Tyr
residues. The ITIMs in the human DCIR and mouse DCIR1 mediate inhibitory
signaling via SHP-1 and SHP-2. HCV or antibody-activated hDCIR on DCs or
pDCs inhibit TLR8-driven TNF-α and IL-12 release, and TLR9-driven synthesis of
IFN-α, respectively [225]. Antibody-DCIR recognition elicits the receptor endocy-
tosis, which is clathrin-dependent, transporting to endosomal lysosomes and
presenting peptide antigens [226]. The human DCIR recognition also elicits the
antigen presentation of DCs to activate CD8+ T cells [227, 228]. The mouse DCIR2
recognition prefers MHC-II presentation and CD4+ T-cell and FoxP3+ Treg cell
stimulation. The mouse DCIR2 recognition elicits extrafollicular B cell function to
stimulate antigen-dependent T-cells. The DCIR CTLD bears consensus motifs such
as an EPS motif to bind carbohydrates of Fuc and Man [229]. DCIR is a HIV
attachment factor on DCs and HCV glycoprotein E2 binding site on pDCs. DCIR-
binding of HIV elicits HIV-1 infection to the host cells of CD4+ T cells [227]. DCIR
is also associated with development of autoimmunity.
8.9.2 Langerhans Cell-Specific Expression of Dectin-2

and Interaction with Fungal High-Man Glycans
Dectin-2 recognizes high-Man polysaccharides to induce the host innate immune

responses to pathogens such as fungi. Dectin-2 activates DCs upon interaction with
ligands. Dectin-2 is initially a Langerhans cell-specific CTLR but Dectin-2 expres-
sion is detected in myeloid lineage cells such as certain DCs subsets like pDCs,
neutrophils, and tissue macrophages [230–235]. Dectin-2 expression is also found in
DCs, lymphocytes and blood monocytes with upregulation during inflammation.
Like other CTLRs, Dectin-2 interacts with the FcRγ, where Arg residue in the Dectin
2 TM region is not involved but a proximal membrane region in the cytosolic tail
region is involved [236]. Dectin-2 transduces its signaling via the complexed
Syk/PKCδ/CARD9-Bcl10-Malt1 axis pathway. CARD-9Bc110-Malt1 indicates
the roles of B-cell lymphoma-10/caspase recruitment domain 9/mucosa-associated
lymphoma translocation gene-1 in lymphoid tissues. Chemokines and cytokines of
IL-2, -6, -10, -12, -23, IL-1β and TNF-α are is stimulated. Dectin-2 signals mediate
PLC-γ2 and MAPKs [237] as well as the NF-κB c-Rel subunit to release Th17-
specific cytokines of IL-23/IL-1β. Dectin-2 also elicits the respiratory burst and
Nlrp3 inflammasome as well as arachidonic metabolites to activate Th2 response
[238]. In structure, Dectin-2 has a typical CTLD region bearing a Man-recognition
EPN motif capable of high Man glycan recognition [237] as PRR. Dectin-2 recog-
nizes pathogens of Histoplasma capsulatum, A. fumigatus, M. tuberculosis,
C. albicans, non-encapsulated Cryptococcus neoformans, Microsporum audouinii,
Paracoccidioides brasiliensis, Saccharomyces cerevisiae, Schistosoma mansoni,
Trichophyton rubrum, and dust allergens [236, 238]. The C. albicans-Dectin-2
binding protects host immunity [237] to release host inflammatory cytokines and
develop adaptive immunity such as Th17 and Th1 immune responses. The Dectin-2-
deficient mouse increases susceptibility of fungal burdens to death. Dectin-2 also
regulates the UV-irradiated immune-suppression.
The detailed functions of Dectin-2 are highly related to Dectin-1 functions. Its
ITAM also upregulates inflammation responses via recognition of high mannose-
based structures to induce antifungal immunity. It induces Nlrp3 inflammsome,
IL-1β and NF-kB signaling (CARD9, BCL10 or MALT1), IL-23 and IL-12 expres-
sion inTh17 or Th1 immunity. Dectin-2 belongs to a type II TM receptor expressed
mainly in tissue type of macrophages, monocytes, and DCs [239]. The Dectin-2
receptor consists of a CTLD which recognizes the high Man-type carbohydrates in a
Ca2+ dependency. Dectin-2 structure is also similar to that of Dectin-1. A difference
between Dectin-1 and -2 is that Dectin-2 lacks the recognition signaling motifs in the
short cytoplasmic tail [213]. Dectin-2 recognizes many high-Man-typed pathogens
such as Cryptococcus neoformans, C. albicans, Histoplasma capsulatum,
M. tuberculosis, Microsporum audounii, Paracoccidioides brasiliensis, Saccharo-
myces cerevisiae, and Trichophyton rubrum [240, 241]. Dectin-2 recognizes high-
mannose-based structures in pathogenic bacteria, nematodes, and pathogenic HDM
allergens via carbohydrate-binding domain, CTLD, which has an EPN motif.
Dectin-2 induces cytokine expressions of IL-1β/IL-23/TNF-α/IL-6 when DCs were
stimulated with fungal ligands. The Syk/CARD9 signaling of the Dectin-2-down-
stream is essential for the Th1 immune response and Th17 immune response elicited
by infection of yeast C. albicans strain [242].
Dectin-2 upon fungal α-mannans recognition exhibits resistance to infection with
C. albicans [243]. Dectin-2 and Dectin-1 commonly share with the Syk/CARD9-
dependent signaling induced by fungal pathogens [244]. Dectin-2 can recognize
various allergens including house dust mites and fungal products, displaying for
UV-raised tolerance by interaction with endogenous ligands on CD4+ CD25+
T-cells [233]. Dectin-2 interacts with the ITAM-bearing FcRγ adaptor molecule to
lead to downstream signaling via the Syk-CARD9 axis [243, 244]. However, to
facilitate the similar intracellular signaling, a membrane-proximal domain in its
intracellular tail of Dectin-2 unusually interacts with the ITAM-bearing FcRγ adap-
tor molecule. Considering the role of transmembrane arginine residue in Dectin-1,
the ITAM motif of Dectin-2 is unusual, as associate with the Syk, PKC-δ, CARD9-
Bcl10-Malt1 pathway and PLC-γ2 [35, 245]. As Dectin-2 is found in several
myeloid lineage cells of DCs, neutrophils, monocytes and macrophages, and highly
expressed in inflammation, mycobacterial Man-type lipoarabinomannan upon
Dectin-2 binding elicits antimycobacterial immunity [246] and Dectin-2 is mostly
impacted in antifungal immunity to protect fungal invasion of Candida species
[247]. Like Dectin-1, Dectin-2 responds to microbial stimulations to stimulate
adaptive immunity, as it induces expression of proinflammatory IL-12, TNF-α,
and IL-6 cytokines with Th17 and Th1 immunities. Dectin-2 elevates the production
of Th17-specific cytokines of IL-1β/IL-23 by Malt1 and NF-κB c-Rel.
Dectin-2 can bind to an O-di-Man-rich glycoprotein present in Blastomyces
dermatitidis, C, neoformans, A. fumigatus, and Fonsecaea pedrosoi
8.10 Dectin-3 (Clec4D, Clecsf8, MCL, Macrophage CTL) 537
[248, 249]. Dectin-2 recognizes several endogenous ligands to modulate

UV-induced immunosuppression. Dectin-2 regulates essential neutrophil IL-17
autocrine during fungal infection [248] upon recognition of fungi. Dectin-2 activates
respiratory burst, cysteinyl leukotrienes, inflammasome Nlrp3, and extracellular trap
formation in house dust mite-derived airway inflammation in DCs to stimulate the
Th2 development [33, 242]. In mouse HDM-mediated pulmonary inflammation,
Dectin-2 stimulates eosinophils and neutrophils via Th17 and Th2 immunity
[250]. Dectin-2 can associate with MCL (Dectin-3) to form heterodimeric
complex [33].
Dectin-2 recognizes the fungal strain if C. neoformans through the fungal cell
wall polysaccharide as a Dectin-2 ligand. In Dectin-2 recognition of C. neoformans
capsule polysaccharides, the polysaccharides as a virulent factor interferes host
phagocytosis of macrophage and protects the fungus. The known capsule poly-
saccharides include glucuronoxylomannan (GXM) with a huge MW 7 MDa and
galactoxylomannan (GalXM) with a MW of 100 kDa. Cell wall mannoproteins are
present as heavy N-glycans and O-glycans with decorated with Man residues
[233]. CLRs-mediated CARD9 contributes to the Th1-driven defense mechanism
upon infection of C. neoformans. Although Dectin-1 cannot clear the infected
C. neoformans and induce Th1-involved cytokines during the fungal infection,
Dectin-2 contributes to the induction of the cytokines such as IL-12 and TNF-α as
well as activation of DCs to induce co-stimulatory molecules. Dectin-2-deficient
mice exhibit enhanced Th2 induction and IL-4-elicited mucin synthesis in lungs
when the deficient mice are infected with Dectin-2-deficient C. neoformans [251].
CTL superfamily 8 (CLECSF8) is limited in its CTLR function of the Dectin-2
cluster. It was initially a mouse macrophage-specific receptor [251]. The human
CLECSF8 is widely expressed on neutrophils of blood, CD14+CD16– classic
monocytic cells and DCs [252]. A specific point different from other Dectin-2
cluster, the CLECSF8 CTLD is deficient for common amino acid motifs involved
in glycan ligand recognition. As an endocytic receptor [253], CLECSF8 transduces
via the Syk for phagocytosis, respiratory burst and inflammatory cytokine release.
CLECSF8 requires adaptor proteins on myeloid cells but not DAP10, DAP12 or
FcR-γ due to the defect of charged amino acids in the cytosolic region and TM
CLECSF8 domain. CLECSF8-/mice respond to necrotic cell death, peritonitis,
autoimmune arthritis, and uveoretinitis [252]. Moreover, these mice are resistant
against pathogenic infections of pathogenic bacteria including C. albicans,
S. aureus, L. monocytogenes, and Nippostrongylus brasiliensis [252]. Functionally,
CLECSF8 is an activating receptor [253].
8.10 Dectin-3 (Clec4D, Clecsf8, MCL, Macrophage CTL)
Dectin-3, also called Clec4d, Clecsf8, or MCL, binds to mycobacterial TDM as well
as fungal cell wall α-mannans. As previously described, Dectin-1 binds to β1,3-
glucans found on the cell surfaces of fungal A. fumigatus conidia or the yeast
C. albicans. Dectin-2 binds to α1,2-mannan glycans present on the hyphae surfaces

of fungi like A. fumigatus hyphae or C. albicans yeast, lipophilic and hydrophilic
molecules of Malassezia, mycobacterial Man-lipoarabinomannan and noncapsular
polysaccharides, to elicit anti-inflammatory responses of Th1, Th17, and Th2
cytokines [254].
Microbial eliciting in Mincle expression involves MyD88 signaling, which is not
related to MCL. MCL heterodimers are ubiquitously present during Mincle induc-
tion. PRRs regulate homoeostasis and defense during pathogenic attacks in innate
immune cells. TLRs and CLRs are the representative PRRs to realize PAMPs.
Dectin-3, called MCL, CLECSF8, or Clec4d, binds to α-mannan chains, too
[255]. Dectin-3 is comprised of a signaling motif-deficient cytoplasmic tail region
and recruits an adaptor CARD9 and functions as a mediator of antifungal defense
immunity [256]. To date, the known fungal-specific CLRs include the three Dectin
forms: Dectin-1, -2, and -3 as well as a Mincle [257]. Therefore, the fungal infection-
sensing PRRs of Dectin-1, -2, and -3 as well as Mincle are directly involved. Fungal
infections are surveyed by host immune system via Dectin-3. They recruit phos-
phorylated Tyr residues of the Syk/CARD9/NF-kB axis signaling to trigger reactive
oxygen species production and phagocytic death with the assistance of
pro-inflammatory cytokines and immune responses [258].
Dectin-3 is an antifungal immune actor and the role of Dectin-3 has limitedly
been investigated in infection of such pulmonary Cryptococcus neoformans strain.
Dectin-3 functions to elicit immune responses against Cryptococcus neoformans
pulmonary cryptococcosis. C. neoformans and C. gattii are representative for
meningoencephalitis or pulmonary diseases. The Cryptococcus strains produce the
capsular glucuronoxylomannan (GXM) carbohydrates which are used as the sero-
type markers of A and D as well as AD hybrid serotypes of C. neoformans.
Cryptococcal capsule GXM comprises over 90% of the total capsule mass
[254]. The GXM contains a linear α1,3-D-mannan carrying β-D-xylosyl (Xyl), β-D-
glucopyranosyluronic acid, and 6-O-acetyl additives [259]. The O-acetyl groups are
the key antigenic moieties. The GXM activates the TLR-derived innate immune
responses in C. neoformans infections. Distinct GXM structures bind to TLR2 [260],
TLR2-TLR1, and TLR2-TLR6 [261]. Thus, Dectin-3 encoded by Clec4d gene is a
receptor for GXM. Dectin-3 deficiency does not influence on mortality and also
anti-Cryptococcus activities of tissue and blood phagocytes in mice subjected to
pulmonary Blastomyces dermatitidis, C. neoformans, Candida spp., and Fonsecaea
pedrosoi infections. In order to recognize mannan, Dectin-2 as well as other known
receptors such as MR (or termed CD206), Dectin-3 and DC-SIGN are involved.
Dectin-3 KO (Clec4d/) mice lack its fungal infection-sensing CLR and are more
susceptible to experimental agent of dextran sodium sulfate (DSS)-elicited colitis
[262]. In Dectin-3-deficient mice, infectious susceptibility to Crytococcus
neoformans of the KO mice as well as recruitment of pulmonary leukocytes and
cytokine expression are not altered. No changes in C. neoformans uptake and anti-
cryptococcal activity are detected in the Dectin-3-deficient DCs and macrophages
[257]. Dectin-3-deficient pDCs exhibit the defected pathogen capture with the
suppressed pDC growth. Thus, deficiency in Dectin-3 contributes to the impaired
8.10 Dectin-3 (Clec4D, Clecsf8, MCL, Macrophage CTL) 539
phagocytosis and fungal immune responses of macrophages, as shown in

C. tropicalis.
Like Dectin-1 and -2, Dectin-3 recognizes the β-glucan structures of cell wall
components in pathogenic microorganisms, especially of fungal glucan antigens to
provide antifungal immunity by the CRD and then induce intracellular signaling via
ITAMs or ITAM-adaptor FcRγ proteins. During fungal immunity in DCs and
macrophages, Dectin-1 and -2 stimulate the downstream Syk-CARD9-dependent
signal transduction to activate translocation of NF-kB to the nucleus and upregulated
expression of inflammatory cytokine genes. In addition, the Dectin-1 and -2 signal-
ing induces the differentiation of Th-1 and Th-17 cells. Th-1 and Th1-7 cells-
produced cytokines stimulate macrophages and neutrophils, thereby killing such
infected fungi [263]. The Dectin-2 family expressed by the NK includes several
different CLRs of Dectin-2, Dectin-3, BDCA-2, DCIR, and Mincle [263]. All CLRs
as type II TM receptors have extracellular CTL-like domains and regions of stalk and
TM. They all carry an ITIM, but not only in Dectin-2, DCAR, DCIR, Mincle and
BDCA-2 require FcR-γ chains as adaptorcontaining ITAM. Dectin-3 has a unique
feature; containing a cytoplasmic tail with short length and lacking for signaling
motif. More specifically, upon interaction with ligands such as α-mannans, Dectin-3
recruits the tyrosine kinase Syk through the ITAM-carrying FcR-γ as an adaptor to
form the Dectin-3 machinery complex. Resultantly, Syk- and CARD9-downstream
signal information are transmitted to transcriptional factors such as NF-kB. Dectin-2
and Dectin-3 are known to form heterodimeric complex during α-mannan interac-
tion. Understanding the Dectin series as host defense line will enforces the mecha-
nistic and pathogenic infection and invasion.
In specific recognition and prevention of fungal infectious pathogens, Dectin-3
(Clecsf8, Clec4d or MCL) recognizes α-Man type of glycans expressed in the cell
surface of fungi as well as to a less extent, mycobacterium-expressed TDM. How-
ever, the heterodimeric mycobacterial receptors include macrophage CTL, of MCL
and Mincle but the regulation mechanisms are not clarified yet. CLR Dectin-2
recognizes mycobacterial TDM through MCL and Mincle or
Man-lipoarabinomannan through Dectin-2 [264] by engagement of surface adaptor
FcRγ. In particular, Dectin-3 or MCL is important in a nonredundant manner in a
lung TB [265], where the CARD9-Bcl1-MALT1-drived NF-κB p65 signaling is
linked in Mincle expression [266]. The MCL as a constitutive receptor is upregulated
during lung infection with M. bovis BCG but as a heterodimer form of MCL and
Mincle. Of Dectin-2 family of CLRs in mycobacterial infection, Dectin-2, MCL
and Mincle bind to mycobacterial antigens with MCL [265]. MCL type is
heterodimerized with Mincle for a heterodimer [267] and mouse MCL is associated
with Mincle and FcRγ. Cell surfaced Mincle expression is correlated to MCL
expression upon microbial stimulation. MCL expression in constitutive but Mincle
expression is inductive upon microbial treatment. Mincle induction triggers the
MyD88 signaling through TLR2, TLR4, and TLR9 in mycobacterial immune
response [268].
In the fungal Dectin-2 and Dectin-3 stimulation, degradation and ubiquitination
events are observed in a Syk-dependent mode [175]. E3 ubiquitin ligase C, which is
a B-lineage lymphoma protein b (Cbl-b), degrades the targets through the Dectin-2/-
3 ubiquitination adapted with FcR-γ and tyrosine kinase Syk. Finally, the Dectin-2
and Dectin-3 degraded through ubiquitination are translocated and localized in
lysosomes and degraded. Syk carries both tandem SH2 domain in the N-terminal
region and C-terminal SH2 domain as well as a C-terminal kinase domain, where the
SH2 domains recognize the pentaamino acids with the Tyr-X-X-Ile/Leu residues,
which is phosphorylated in the ITAM. Then Syk is consequently activated by a Tyr
phosphatase SHP-2 [269, 270]. Therefore, the Dectin-3 machinery complex con-
tribute to phosphorylation and activation of Syk. Because the Syk phosphorylation
and activation are under cascade process, the Syk stimulates the downstream
signaling-enzymes including PLC-γ2 and PKC-δ, and the 2 enzymes lead similarly
to phosphorylation of the adapter CARD9 to form the multiple protein complex with
mucosa-associated lymphoid tissue-1, B cell leukemic-lymphoma 10 (Bcl10), and
CARD9 [25]. The CARD9-Bcl10-Malt1 machinery complex now stimulates the
TAK1–IKK–NF-κB pathway [269, 271], and this stimulates the cytokine/chemo-
kine receptor-driven upregulation of IL-23, IL-1β, TNF-α, IL-12, IL-6, CXCL1,
CXCL2, and CCL3 [263].
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Chapter 9
Galectins
9.1 General and Structural Aspects of Galectins
Immune response is regulated by multiple pathways with activation and tolerance of

immune cells. Galectins are one of the most ancient mammalian lectins and galectins
found in all metazoans. Galectins are soluble proteins belonging to the family of
animal lectins. Galectins are first found in 1994 as β-galactoside-binding lectins that
agglutinate cells [1, 2]. Galectins belong to an animal lectin family that has affinity
for β-galactoside present in the extracellular milieu and that share similar amino acid
sequences. They have well-conserved CRDs with about 130 amino acids. Therefore,
galectins are defined as a carbohydrate-binding protein family with an affinity for
β-galactoside sugars and sharing a conserved sequence motif within their CRDs. The
CRDs are composed of 5- or 6-stranded antiparallel β-sheets in α-helix forms lacking
β-sandwich/jelly-roll configurations. To date, 15 mammalian galectins, encoded by
the LGALS genes, are known. Galectins are lectins with carbohydrate-binding
capacity. Among them, Galectins-1, -2, -3, -4, -7, -8, -9, -10, -12, and -13 are
found in human. They bind glycosylated receptors. Many galectin types recognize
their ligands in either bivalent or multivalent binding manner when they bind to the
carbohydrates and cause the clustering of multiple multivalent glycoconjugates
resulting in a lattice formation. Galectins are bound with cells in a homotype or
heterotype. The CRDs of galectins are known to be binding regions for LacNAc
residues [3]. CRD and N-terminal domain mediate self-aggregation and multiple
identical subunits formation. Galectins are also found in eels, chicks, and calves.
Galectins are therefore defined as proteins with affinity to β-Gal-bearing carbohy-
drates such as lactose. CRD present in galectins have several common characteristics
in Ca2+-independent activity, a specificity for β-galactosides, a blocked N-terminus,
lack of a signal peptide and carbohydrate, and predominantly cytoplasmic location.
Galectin-type CRD has a highly conserved protein sequence motif with
β-galactoside binding affinity.
558 9 Galectins
9.1.1 Biological Roles of Galectins
The interactions at the cell surface between galectins and their ligands are pivotal to
cell signaling. Pathogens and parasites have evolved their surfaced glycans to mimic
their host cells. They recognize invading pathogens. Although the biological impor-
tant roles of galectins are conserved in organisms, organisms lost galectin and other
innate immune recognition systems to acquire adaptive immunity. Although adap-
tive immune system responds to various invasive pathogenic glycans, immune
tolerance decreases responses to “self,” and instead, galectins become responsible
to pathogenic microbes. Galectins also elicit innate immune responses against
altered blood group glycans. As blood group in individuals lost blood group-
responsible B cells, the resultant autoimmune responses are lost. Thus, galectins
produced by host cells kill blood group-expressing microbial organisms.
Galectins as PRRs play a key role in self-recognition vs. non-self recognition.
Galectins recognize exogenous ligands in innate immunology. Galectins recognize
non-self-related determinants. Galectins involve in host development and immune
responses. Their interactions affect cellular behaviors. For example, in cancers,
galectins are involved in the progression and cancerous galectins regulate invasion,
migration, metastasis, and progression in tumor microenvironments. Among them,
galectin-9 has been interested in cancer-targeted therapy. Although the fundamental
galectins specifically recognize glycoconjugates at the molecular level, they involve
in multiple physiological actions such as developmental fate determination, differ-
entiating forces, morphogenic induction, apoptosis, immune responses, and meta-
static invasion of malignant cells. In addition, oxidized galectin-1 without glycan-
binding capacity functions as a biomodulator for nerve cell growth. The multiple and
diverse physiological activities and structures of galectins make the relationship
between structure and activity and their evolution histories.
As the Janeway and Medzhitov hypothesis directs that PRRs recognize pathogens
via microbe-associated molecular patterns (MAMPs) due to their absence in the host.
The paradox that galectins bind to similar “self” and “non-self” patterns implies
there should be limited knowledge on galectin conformation and tertiary structures.
Galectins-4 and -8 may be a different form of innate immunity. Galectins-4 and -8
bind to MAMPs as innate immune factors. Galectin-4 and -8 strongly bind to blood
group antigens but the precise explanation how their abilities to bind to microbial
blood group antigens are not well known. Gal-4 and Gal-8 recognize pathogenic
microorganisms with specific antigenic determinants. For example, Gal-4 and Gal-8
bind to E. coli BG B+ strain to kill the E. coli BG B+ strain. Galectins bind to the
surfaced glycans of viruses, bacteria, fungi, and parasites. For example, they bind to
complex N-linked glycans of the HIV gp120, N. meningitidis LPS, N. gonorrhoeae
LPS, H. influenzae LPS, H. pylori LPS O-antigen, S. pneumoniae polysaccharide
type XIV, Leishmania lipophosphoglycan (LPG), Trichomonas vaginalis LPG,
Schistosoma mansoni LacdiNAc (LDN), and Candida albicans oligomannan [4].
In the galectin–ligand interaction, galectins recognize the core β-Gal structures.
The recognition of galectins with the endogenous ligands occurs at the cell surface,
9.1 General and Structural Aspects of Galectins 559
because the GSLs expressed on cell surfaces interact with lectins of cell surface
receptor proteins to modulate signal transduction. For β-galactoside interaction, the
galectins are flexible for binding to broad and naturally occurring oligosaccharides.
Gangliosides play roles in galectin-dependent adhesion and growth control of cells
as well as signal transduction. For example, GM3 is well known to colocalize with
transduction molecules in lipid rafts of B16 melanoma cells, interacting with
galectin-8 in lipid rafts microdomains known as GSL-enriched membrane. Cellular
galectins bind to the lacto- and neolacto-series GSLs and gangliosides. Lac-N-
neotetraose (LNnT) and Lac-N-tetraose (LNT) as tetrasaccharides are also included.
The LNnT and LNT form complexes with the core components of neolacto-series
and lacto-series of GSLs, respectively. Lacto-series GSL and neolacto-series GSL
are used for synthesis of glycan antigens including ABH blood group antigens,
HNK-1 antigen, and Lewis type of histo group antigens. They are also frequently
found in haematopoietic cells, while some structures modified from the Lacto/
neolacto-series GSLs are found in some cancer cells. Lacto and neolacto-series
also regulate the immune responses. For example, lacto/neolacto-series-deficient
mice (B3gnt5-negative) exhibit B cell hyperactivation and hyperproliferation, indi-
cating the galectin involvement.
From the structures of co-crystals of galectins-LacNAc or -Lac complex, the
highly conserved amino acid residues were identified on the carbohydrate interaction
sites, which are formed via the interaction between hydrogen bonds. Trp residue is
the conserved amino acid residue in the galectins, playing a crucial role in tacked
binding to B face of the Gal residue. Also, the Arg residue in certain galectin types
like galectins-2 and -3 is conserved in humans, its side chain does not interact with
such galectins. The multiple galectin number is considered to be generated by
adaptive evolution, and this consequently provides advantages and merits for the
biodefense innate immune system. Gal-1 and Gal-9 regulate predominantly immu-
nosuppressive responses. In contrast, Gal-3 and Gal-8 exhibit dual positive or
negative regulation of inflammation depending on cellular microenvironment.
9.1.2 Immunological Roles of Galectins
Galectins do not directly influence membrane integrity of host cells but bind to
diverse host cell surface glycan ligands. Galectins are specifically expressed in
immune cells in a cell type expression pattern (Table 9.1). Galectins-1, -3, -8,
and -9 are highly expressed in endothelial cells, while galectins-2, 4, and -12
expressions are very low. Galectins increase cancer metastasis via enhancement of
tumor cell-matrix, tumor cell to cell, and tumor-endothelial recognition. Galectins
enhance tumorigenesis and tumor survival through regulation of tumor develop-
ment, growth, apoptosis, and cell-cycle progression. In tumor metastatic stages,
galectins potentiate adhesion, migration, and angiogenesis of cancer cells as well
as the inflammatory response and avoidance from host immunity. For example,
galectin-3 importantly mediates tumor immune responses through modulation of
560 9 Galectins
Table 9.1 Immune cell type expression of several galectins

Galectin 1: ImmuneCD4+ and CD8+ T cells, B cell, macrophage
Galectin-3: CD4+ and CD8+ T cells, B cell, macrophage, myeloid, Langerhans cell, DCs,
monocyte/macrophage, mast cell, neutrophil, eosinophil
Galectin-9: T cell, PBMC, eosinophil
Galectin-10: Eosinophil, basophil
Galectin-12: PBMC, neutrophil, myeloid cell lines
homeostasis of T cells. Galectins control various events including activation, apo-

ptosis, signalling, cytokine production, and Treg function of T cells. More specifi-
cally, galectins regulate thymic T-cell functions. Galectins-1, -3, -8, and -9 elicit
CD4–/CD8– or CD4+/CD8+ thymocytic apoptosis towards immune central toler-
ance. Galectin 3 potentiates TCR clustering via N-glycan lattice formation. Galectin-
3 binding to N-glycan ligand prevents the association of TCR, CD4, and LCK
tyrosine kinase to ganglioside-anchored lipid raft microdomains. This is the mech-
anism(s) underlying how TCR activation is prevented in the condition of galectin-3
ligand absence.
Upon activation of T cells, several galectins-1/-2/-3/-4/-9 recognize glycosylated
T cell-associated receptors such as T-cell Ig domain, Tim3, CD3, CD7, CD29,
CD43, CD45, and CD71. Consequently, apoptotic cell death of T cells is induced.
The specific N- and O-glycan structures are bound to galectin-1/-9 to elicit the
differential apoptotic cell death of Th1, Th2, and Th17 subpopulations.
Galectins-1, -2, -3, -4, -8, and -9 specifically modulate T cell activation and cytokine
expression. Thus, changes in glycosylation pattern modulate galectin-host sugar
recognitions by galectins. Also, the two galectins galectin-1 and -10 inhibit the
CD4 + CD25+ Treg cells. Galectin-1 suppresses early TCR-elicited activation and
keeps the naive T cell survival in the peripheral tissues. Galectins induce apoptosis in
specific CD4+ or CD8+ thymocytes after activation. In the periphery, galectin-4 and
-1 promote T cells to express IL-6 and IL-10, respectively (Fig. 9.1).
Tumor immune responses are mainly associated with siglecs and galectins.
Tumor cell glycan epitopes potentiate cell binding to microenvironment via
glycan-binding lectins and binding to immune cell receptors [5]. Tumor transforma-
tion involves in galectin-1 and -3 expressions. They recognize oncogenic Ras and
consequently control apoptosis. If galectin is added to tumor cells or if galectin gene
is enforcedly expressed in tumor cells, the tumor cells are resistant to apoptosis via
galectin-3 Ser-6 phosphorylation. Galectins also modulate tumorigenesis and growth
by cell cycle genes. Cyclin-E/-A expressions are suppressed. The expressions of
p21, p27, and cyclin D1 genes are increased. Galectin-3-β-catenin binding promotes
cyclin D1. Galectin-1 acts as an autocrine cell growth suppressor. In carcinogenesis,
certain lectins bind to β1,6-glycans, but binding affinity to β-galactosides is
prevented because of ST6Gal-1-produced α2,6-SA linkage. Consequently,
sialylation blocks the galectin-induced apoptosis of cancer cells with the cancer
cell survival [6]. For example, galectin-3-N-glycan binding induces anergy status of
tumor-specific CTLs. Galectin-3-N-glycan binding on CTLA-4 prevents the
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Fig. 9.1 Galectins regulate T-cell homeostasis. Galectins induce apoptosis in specific CD4+ or
CD8+ thymocytes after activation. In the periphery, galectin 4 activates T cells and production of
IL-6. Gal-1 induces Treg cell and suppresses T-cell activation. Gal-2 induces CD8+ T cell
apoptosis. Gal-3 induces T cell apoptosis and inhibits T cell differentiation. Gal-3 suppresses
Treg cell differentiation. Gal-4 induces mucosal T cells apoptosis. Gal-8 induces Treg cell and T
cell activation. Gal-9 induces Treg cell expansion and T cell apoptosis [30]
A type galectin: 1 CRD. Galectin-1, 2, 5, 7, 10, 11, 13, 14 and 15)
B type galectin: 1 CRD connected with a linker peptide. Galectin-3.
C type galectin: 2 CRDs fused with tandem repeat of Pro- and Gly-rich stretches. Galectin-4, 6, 8, 9 and 12.
Fig. 9.2 Three types based on CRD structure of galectins
CTLA-4 endocytosis to prolong arrest signals [7]. Moreover, galectin-3 binding to T

antigen on cancer-associated MUC1 and to CD146 (MCAM/MUC18) enhances
tumor metastasis [8]. Pathogenic microbes produce self-similar glycan antigens,
and tolerance blocks the adoptive immune response against self-like structures.
Thus, the galectin-immune system interaction protects against molecular mimicry
through innate immunity. The microbial carbohydrates prevent immune recognition
and removal as a microbial strategy to avoid immunological surveillance of host. In
fact, certain bacteria synthesize host glycans-like carbohydrates [9].
9.1.3 Classification of Galectins
Galectins are classified by the discovery order but also classified into A, B, and C
types through their CRD properties. A type galectin has a 1 CRD, and B type has a
1 CRD connected with a linker. C type is featured of 2 CDRs fused with tandem
repeat of Pro- and Gly-rich stretches (Fig. 9.2). They are also structurally
562 9 Galectins
Prototype Tandem repeat type Chimera type

One CRD Two non-identical CRDs One CRD and N-terminal extension
Galectin-1, -2, -5, -7, -10, -13, Galectin-4, -6, -8, -9, -12 Galectin-3
-14. -15
β-galactosides
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Fig. 9.3 The structure and classification of the galectin family members. The three major types of
galectin families. The three primary classes are prototypical (left), chimeric (right), and tandem
repeat (center). Prototypic galectins bear a CRD and are homodimerized. Tandem-repeated-type
galectins have two specific CRDs linked up to 70 amino acids. Gal-3 is a chimera galectin type for
intracellular and extracellular interactions
subclassified to three different types: proto-, chimera-, and tandem-repeat type.

CRDs’ number and structure are also classifying parameters. Galectin classification
gives three groups: (i) prototype galectins, which comprise of galectins-1, -2, -5, -7,
-10, -11, -13, -14, and -15, and this group contains one dimer-forming CRD,
(ii) tandem repeat-type galectins, which are comprised of tandemly repeated form
and are galectins-4, -6, -8, -9, and -12, this group consists of two identically
tandemed CRDs, and (iii) galectin-3is a chimeric form, which consists of a lectin
oligomer-forming CRD in the N-terminal non-lectin domain [10, 11]. The tandem-
repeat structured galectins carry two specific CRDs, but genetically encoding for a
single polypeptide. Each can exhibit distinct binding preferences. Galectin-3 mono-
mer and oligomers are linked to its N-terminus in the presence of its binding ligands.
The galectin-3 forms a pentameric lattice, resulting in modulation of intracellular
signaling and cell-to-cell and ECM–cell interactions (Figs. 9.3 and 9.4).
The prototype galectins have two identical homodimeric CRDs. Tandem-
repeated galectins contain two or more CRDs linked by a single linker. The
galectin-3 is a single chimeric form. Galectins-1/-2/-3/-4/-7/-8/-9/-10/-12/-13 are
found in humans. Galectins-5/-6 are expressed in rodents. Galectins-11/-14/-15 are
present in goats and sheep [11]. Galectin genes have alternatively spliced isoform
variants. Human galectin-8 has seven variant isoforms and galectin-9 has three
variant isoforms [11]. Galectins mediate homotypic and heterotypic interactions to
control cell signaling [12], as Galectins-1, -2, -3, -7, -8, -9, and -12 elicit apoptotic
cell death in blood-resident cells [13].
Galectins as an old protein family and an ancient protein type expressed in
eukaryotic cells are present in all metazoans ranging from fungi and sponges to
animals including vertebrates and invertebrates [14, 15]. Galectins are involved in
diverse functions of cellular homeostatic functions including growth, cell cycle
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Fig. 9.4 The galectins modulate intracellular signaling, cell–cell and ECM–cell bindings.
Galectin-3 forms a pentameric lattice, resulting in modulation of intracellular signaling and cell-
to-cell and ECM–cell interactions
progression, differentiation, apoptosis, cell adhesion, chemoattraction, cell migra-

tion, angiogenesis, and pathological events of disease states such as cancer, cell
tumor progression, inflammation, and pathogenic microbial infections [4, 16,
17]. Galectins are β-galactoside-binding lectins produced by mammals which bind
to Galβ1-4GlcNAc oligosaccharids produced on cell surfaces [4].
To date, the primary structures of more than 130 galectins have been determined.
By the CRDs, galectins are reclassified into three CRD types of prototype, chimera,
and tandem-repeat, based on the CRD structures [18], indicating existence of two
galectin-types of CRDs on a polypeptide and dimerization. This is the reason why
galectins have multiple sugar-binding sites [19] and are diverse proteins with
different functions. Prototype galectin forms a noncovalent dimer consisting of
two identical CRDs. Prototype galectins representatively include galectin-1 which
carries a single CRD, as monomer forms or homodimer forms. On the other hand,
the chimera-type has two specific domains: an N-terminal collagen-like domain and
galectin CRD in the C-terminal region. Chimera-type galectin representatively
includes galectin-3 with an N-terminal non-lectin domain, and this lectin is linked
to the CRD through a Pro-, Gly-, and Tyr-rich repeated sequence. The tandem
repeat-type has two homologous but different sugar-specific CRDs on a single
polypeptide chain [20]. For multiple splicing, different splicing variants make
various isoforms of galectins.
As galactose binding proteins, galectins are expressed in various human tissues.
Because galectins are the causative factors for a diverse type of pathologies, the
function of the galectins has been related to immunomodulation, immunotolerance,
564 9 Galectins
and metabolic responses [21]. Immunomodulating aspects are frequently observed

in the galectin family because they are found on all immune cells and expressed in
conditioned immune cells like inflammatory macrophages, NK cells, and activated
B/T cells [22]. Galectin-9 is observed in low levels in healthy normal individuals.
However, some disease status leads to the elevation in galectin-9 expression,
indicating that galectin-9 may serve as a negative modulator of antiviral responses
of host immune system. Galectins modulate apoptosis, proliferation, and cell divi-
sion cycle. Galectins are also implicated in tumor microenvironment due to their
fundamental participation in the interaction between cell and tissue surfaces, as they
are expressed in cancer tissues. The roles and functions of galectins-1, -3, -4, -7, -8,
and -9 are documented in tumors. For example, galectins-2, -4, and -8 are recognized
as cancer prognosis biomarker and circulated in blood systems, allowing efficient
cancer detection and prediction, because expression levels of such types increase in
the metastatic conditions of colon cancers [23]. Galectins are also implicated in the
regulation of immune system. In inflammation, diverse cells of DCs, eosinophils,
mast cells, macrophages, monocytes, neutrophils, activated T/B cells, and Tregs
express the galectin isotypes, depending on cell type. Gangliosides also have the
reduction activity of the IL-15/IL-12/TNF-α synthesis in DCs [24–28]. Currently,
there is a question whether ganglio GSLs influence the expression of Ag-processing
machinery (APM) components. Upon Singlec-7 binding, gangliosides are also
known to modulate immune cells such as natural killer (NK) cells’ cytotoxicity.
Many lower vertebrates possess galectins in the skin mucus. Additionally, many
galectins possess cysteine residues as free thiols, giving unstable oxidization capa-
bility [29]. Galectins function as both innate and adaptive immunity factors. For
example, galectins modulate the activity of the complement receptor 3, which is the
macrophage membrane receptor for complement C3b and iC3b.
9.1.4 Galectin Ligands in Proteins and Gangliosides
Galectins are not surface membrane receptors, but they are soluble immunomodu-
latory proteins. However, CTLs and Siglecs belong to types of cell surface receptors.
Therefore, galectins function in two different ways of intracellular signaling pathway
or extracellular interaction [31]. Galectins basically recognize LacNAc (Galβ1,
3GlcNAc or Galβ1,4GlcNAc) on O-/N-linked glycans. Cell surface glycans regulate
the galectin repertoire through the specific glycan structure recognition. Galectins as
soluble receptors mediate immune cell function, fetus-maternal tolerance, cell sig-
naling, recognitions of host cells with microbes, homeostasis of Th cells, and
autoimmunity suppression as well as immunosuppressive tumor microenviron-
ments. Surprisingly, galectins can also bind proteins of non-carbohydrate ligands
in the small organella compartments via protein–protein interactions. Galectin–
ligand interaction is dependent on the changes in N- and O-glycosylation of ligands,
altering galectin endocytosis and clustering. Galectins can bind and crosslink to
glycan ligands on other receptors to form supramolecular interactions or lattices on
multiple receptor–ligand interactions [32, 33]. For example, such multiple receptor
interactions are well explained in signaling of cytokine receptor, TCR synapse, and
B cell receptor (BCR) [32]. Some galectins such as galectin-1 and -3 exhibit a wide
distribution in tissue and the remaining other galectins are tissue-specific for distri-
bution. For example, galectin-7 and galectin-12 are expressed in skin and adipose
tissue, respectively. Interestingly, only galectin-10 is specifically expressed in cer-
tain immune cells like eosinophils and Treg cells [34]. Among the galectins, the
well-defined galectin-3 and galectin-9 are PRRs in innate immune responses upon
Leishmania species infection. Mice disrupted for the galectin-1 or galectin-9 gene
are characteristic of the increased Th1 and Th17 responses, augmenting inflamma-
tory responses [35, 36]. Galectin-3 KO mice are not inflammatory in multiple
sclerosis and arthritis disease status [37, 38]. These different phenotype changes in
the galectin delete functions indicate each distinct role in regulating immune
homeostasis.
Recently, glycosphingolipids such as gangliosides biologically important ligands
have also been known to be bound to some galectin family. The glycosphingolipids
in mammals are diversely abundant and involved in cell–cell communications by
lectins and receptors [39, 40]. The GSLs are divided into four major groups namely,
ganglio-, globo-, lacto-, and neolacto-series GSL [40]. The LNT and LNnT as
tetrasaccharide structures constitute the structural core units of lacto-series GSLs
and neolacto-series GSLs, respectively. Lacto and neolacto-series GSLs function as
carbohydrate antigens. The examples are HNK-1 antigen, ABH antigen, and Le
blood group antigens, as they are expressed in innate immune cells like leukocytes
[40, 41] and on some cancer cells [42]. Galectins are highly homologous in ligand
affinities for β-galactosidic glycans [10]. Galectins bear a CRD in all subtypes of the
three galectin types namely, proto-, chimera-, and tandem-repeat types [15]. A
prototype Galectin-1 has only a single CRD in the form of monomers or
homodimers. In contrast, tandem-repeated galectins bear two different CRDs linked
by a specialized linker as a monomer. Galectin-3 as a chimera-type has a non-lectin
domain located on N-terminal region domain of the CRD through a repeated Pro-,
Gly-, and Tyr-rich domain [43]. Galectins recognize the β-Gal residues, and
galectin-3 and galectin-9 can recognize long poly-LacNAc chains [18], indicating
the similarity between ligands of galectin-3 and galectin-9. In some tumor cells such
as B16 melanoma cells, GM3 is reported to colocalize with signaling receptors
[44]. Galectins can bind to GM1 as the pentasaccharide form that bears the core
GM3 backbone [18, 45, 46]. Galectin-3 binds GM3 and forms galectin-3-GM3
complex. Galectin-3 can recognize and bind to the trisaccharide moiety of GM3.
9.1.5 Galectins in Lower Organisms such as Zebrafish

or Marine Oyster
Host galectins prevent parasitic infection, as known with Zebrafish (Danio rerio) or
marine oyster (Crassostrea virginica) galectin repertoires. All three types galectins,
566 9 Galectins
proto, chimera, and tandem repeat, are expressed in zebrafish but their galectin
repertoire is simpler than that of vertebrates. The zebrafish galectins are found as
isoforms, most likely by gene duplications in the genome level [47]. For example,
zebrafish galectins are secreted forms and bind to viral, bacterial, and fungal
pathogens and parasites. Galectins are associated in viral infection. In the model
organism Zebrafish, the proto-typical galectin Drgal1-L2 and the chimeric-type
galectin Drgal3-L1 directly bind to the LacNAc residue of envelope protein of
IHNV to block viral attachment of a hematopoietic necrosis virus (IHNV) of a
rhabdovirus family. The galectins are the sites of IHNV attachment to the zebrafish
epithelial cells [48].
For example, oysters of Crassostrea virginica species are known as efficient
filter-feeders. Oyster hemocytes as phagocytes phagocytose its parasite, Perkinsus
marinus trophozoites through galectin–lignad recognition and engulfment
[49]. CvGal galectin of oyster hemocytes is “stolen” by the P. marinus to invade
the oyster host. P. marinus species is similar to a dinoflagellate and known as a key
pathogen of oysters. The disease is called dermo or perkinsosis due to the degrada-
tion of oyster tissues. C. virginica 4-tandem CRD-bearing galectin CvGal-2 binds to
β-galactoside motif in bacteria, algae, and Perkinsus spp. P. marinus escapes from
intracellular killing of the host cells. In granulocytes, CvGal spreads to the periphery.
Blocking hemocyte CvGals by its specific antibody leads to Perkinsus spp. survival
due to phagocytosis prevention of host cells [50]. CvGal-1 has a binding specificity
to the blood group A tetrasaccharide and related structures, while CvGal-2 has its
specificity to recognize both blood group A and B tetrasaccharides and related
carbohydrates.
9.2 Galectin-2
Gal-2 is expressed in monocytes and regulates mucosal immunity on epithelial cells

in the gastrointestinal tract [51]. Gal-2 induces monocytes and macrophages polar-
ization to a proinflammatory M1 state, inhibiting M2 differentiation [52]. In contact
allergenic response, Gal-22 eliminates activated CD8+ T cells [53]. Gal-2 binding to
β1 integrin (CD29) leads to proapoptotic function [51]. Like Gal-1, Gal-2 activates
neutrophils through induction of phosphatidylserine exposure [54]. Thus, Gal-2 acts
similarly with Gal-1 to T cells and neutrophils but influences differently to
macrophages.
9.3 Galectin-3 and -8 Recognize GM3, But Not Galectin-4 567
9.3 Galectin-3 and -8 Recognize GM3, But Not Galectin-4
9.3.1 Galectin-3
Galectin-3 has a MW 32- to 35-kDa of β-galactoside-binding lectins. Gal-3, known

as a chimera-type, regulates cellular activation, trafficking, survival, and differenti-
ation in myeloid cells. Gal-3 effectively recruits DCs to draining lymph nodes after
maturation signals and enhances motility of mast cells to fibronectin but inhibits
antigen-induced chemotaxis [55]. Thus, Gal-3 has dual capacities of positive and
negative regulation of myeloid cell migration. Also, Gal-3 functions as a paracrine
TLR-4 ligand in the CNS. Gal-3-driven TLR4 induction activates microglial cells for
the inflammatory response in the neural region through remyelination and oligoden-
drocytes differentiation [56, 57]. Intercellular Gal-3 also activates DCs’ function.
During Leishmania major infection, endogenous Gal-3 potentiates the Leishmania
parasite susceptibility by regulating Jagged1/Notch signaling in DCs and polariza-
tion of Th cell response [58]. In addition, Gal-3 regulates cytokine mediators in
innate immune response. Gal-3 reduction affects proinflammatory cytokine produc-
tion by moDCs and consequently inhibits Th2/Th17 differentiation [59].
By its carbohydrate-binding function, it regulates cell growth, differentiation, and
inflammation through its expression in epithelial cells, endothelial cells, and mac-
rophages. An increase in galectin-3 is also associated with fibrosis of tissues with
increased risks of progressive renal impairment and cardiovascular mortality. Thus,
galectin-3 regulates numerous biological processes through its CRD [4, 16]. Cyto-
plasmic galectin-3 is secreted to extracellular fluid or serum, also shuttles into the
nucleus. Secreted galectin-3 interacts between epithelial cells and extracellular
matrix [17], while cytoplasmic galectin-3 is associated with cell survival via
blocking the apoptosis, and nuclear galectin-3 induces cell proliferation [4, 16].
Galectin-3 binds to poly-LacNAc chains in glycolipids and N-/O-glycans of
glycoproteins [60, 61]. Galectin-3 activates T cells by interaction with poly-
LacNAc-bearing glycans on the T-cell receptors [62]. In eosinophils, galectin-3
inhibits the IL-5 expression, although galectin-3 activates mast cells, neutrophils,
lymphocytes, and monocytes [63]. Interestingly, galectin-3 activates phagocytosis of
macrophages and promotes airway inflammation of the mast cells [45]. Galectin-3-
mediated apoptosis is associated with glycoprotein receptors located on cell surfaces
to trigger cytotoxic T cell killing and intracellular overexpression of galectin-3
shows anti-apoptosis [1]. Galectin-3 defect indicates reduced mast cell function,
liver fibrosis, age-dependent glomerular lesions, fatty liver disease, and lung fibrosis
in mice [1].
Gal-3 has two CRDs and is the only chimera-type galectin. Galectin-3 comprises
of an extended carbohydrate-binding site, capable of binding to broad ligand glycans
extended from the β-Gal O3 position. Loss of Gal-3 in cells indicates no interaction
with epithelial tissue. Galectin-3 can bind to apparently ordered lacto and neolacto-
series GSLs including ABH(O) blood group and HNK-1 antigens. Binding of
galectin-3 with glycosphingolipids can give us information to identify galectin-3’s
568 9 Galectins
binding receptors which may be related to specific diseases. The lacto and neolacto-
series also form the lipid rafts of immune cells. For example, mice deficiency for
biosynthesis of the lacto- and neolacto-series GSLs, as in the B3GnT-5-KO mice,
showed the enhanced activation and hyperproliperation of B cells, because the cells
lack for receptors for any galectins [41]. Poly-N-acetyllactosaminylated Core-2
O-glycans promote metastasis [64] on bladder tumor cells. Recognition of poly-
LacNAc-added N-/O-glycans by galectin-3 facilitates lung metastasis of melanoma
cells [65] and metastasis of bladder tumor cells [39]. Moreover, binding of galectin-3
with poly-N-acetyllactosaminylated Core-2 O-glycans at the tumour cell surface is a
key point in evasion [66]. Masking using poly-N-acetyl-lactosamine-linked Core-2
types of O-glycan on the MHC-I-related chain A glycoprotein is an escape strategy
to avoid tumors from surveillance and cytotoxic killing of NK cells. Endogenous
receptors of galectin-3 have been suggested to be the lacto- and neolacto-series
GSLs. In addition, from the recent study [67], core structural components of
tetrasaccharides such as ganglio-series, lacto-N-tetraose series, and lacto-N-
neotetraose series of GSLs are also known to be potent endogenous receptors. The
study on CRD crystal structures of human wild-type galectin-3 and a mutant
(K176L) complexed with lacto-N-tetraose, lacto-N-neotetraose and GM3
(α2,3-sialyllactose) has been made. Galectin-3-GSL core structure interaction pro-
vides an understanding of the capacity of galectin–receptor interaction, which
facilitates some diseases [67]. Galectin-3, which is similar to galectin-9, exhibits
increased affinities for the extended poly-LacNAc chains; however, galectin-1 does
not exhibit affinity to the increased chain length. Affinity profiles of Galectin-3 and
-9 are quite similar The glycan-recognition domain in the N-terminal region of
galectin-9 internally recognizes LacNAc dimer (LN2) and LacNAc trimer (LN3),
and also binds to a GlcNAcβ1,3Galβ1,4GlcNAc carbohydrate epitope located on the
poly-LacNAc chains. Galectin-3 recognizes the poly-LacNAc chains linked to the
N-glycans, O-glycoproteins, and glycolipids. Galectin-3 recognizes poly-LacNAc
-modified N and O-glycans and facilitates melanoma tumor cells metastasis to lung
tissues and metastasis of bladder tumor cells, respectively. Core-2 O-glycans linked
by poly-LacNAc are known as metastasis-accelerating O-glycans with their
increased expression on the MHC-I-related chain. A glycoprotein of bladder cancer
cells helps the evasion of the host immunity that masks the tumor cells from
surveillance by NK cells, with the interaction of galectin-3 at the cell surfaces of
tumors. Using X-ray diffraction of the crystallized CRD of the galectin-3, which was
complexed with LNT, LNnT, and ganglioside GM3, the glycan recognition has been
suggested. From the human wild-type CRD of galectin-3 complexed with LNT,
LNnT, or GM3, Lys176, the carbohydrate recognizing site of galectin-3 has been
known to be important. Trp residue recognizes hydrogen bonds via the Gal residue
O4 and O6 positions.
It was demonstrated that the galectin-3 recognizes the core β-galactoside residue
of the core oligosaccharide epitope of GM3. The lacto- and neolacto-GSLs were
found to be in distinct orientation with each terminal Gal residue in the complex
structures of galectin-3-LNT and galectin-LNnT. If the galactose residue of
sialyllactose is embedded into longer sugar chain gangliosides such as GM1,
9.3 Galectin-3 and -8 Recognize GM3, But Not Galectin-4 569
GM2, GD2, and GT2, the galectin-3 binding is suggested not to be ensured.
Although the galectins can generally bind to diverse longer sugar chain gangliosides,
such bindings are assumed to observe only with terminally located galactose residue
as galectin recognition ligand epitope on those gangliosides. The assumption has
been based on the data obtained from X-ray diffraction of the crystal galectin-3-GM1
complex structure, where the galectin-3 binds to the terminally located
Galβ1,3GalNAc carbohydrate epitope [68], and on the interaction results that
galectin-3 recognizes GM1 and GM3, but not GM2 that the galactose residue is
embedded in long sugar chain [69]. From the X-ray crystalline structures of the CRD
in human galectin-3 complexed crystals [67], the galectin-3 was demonstrated to
bind to tetrasaccharides of LNT and LNnT as well as GM3. Structurally, GM1 has
the Galβ1,3GalNAc carbohydrate epitope, which galectin-3 CRD recognizes; how-
ever, GM2 has the extended sugar residue, GalNAc and consequently, galectin-3-
CRD cannot recognize it. This is the reason why galectin-3-CRD does not bind
GM2, although it recognizes GM3. These reports suggested that GSLs could also be
functionally essential ligands for certain galectin family.
Galectin-3 is also linked to the viral and bacterial infection and pathogenesis,
autoimmunity, innate immunity, and inflammatory responses [70, 71]. In bacterial
infection, galectin-3 binds to LPS to suppress LPS-induced inflammative response
[72]. In acute tissue damage, for instance, it is an essential component in the host
protection from microbes such as S. pneumoniae [73]. Thus, the serum levels of
galectin-3 can be used in monitoring their infections. However, galectin-3 also
involves in the chronic inflammatory response [14] and is secreted by activated
macrophages in cardiac pro-inflammation and pro-fibrosis [74], indicating that
galectin-3 is a regulatory molecule in acute and chronic inflammation. Therefore,
galectin-3 regulates cell growth, proliferation, differentiation, and inflammation.
Galectin-3 promotes tumor progression by binding to ligands of cancer cell. In
mice transplanted with Lewis lung carcinoma cells, high galectin-3 expression
induces the migrative potentials of myeloid lineage suppressor cells to the TAMs,
contributing to immune-suppressive status of tumor microenvironment
[75]. Galectin-3 is also heterogeneously expressed in tumor cells. Galectin-3 expres-
sion is increased in brain cancer, acute myeloid leukemia, and colorectal cancer with
poor survival and prognosis. Circulating serum galectin-3 level is a poor prognostic
indicator and monitoring therapeutic biomarker in cancer patients and secreted and
circulated galectins are useful metastatic biomarkers, as galectin-3 level in sera is
upregulated in cancer patients [76]. Galectin-3 can be a prognostic marker, as
galectin-3 is expressed in nucleus and the galectin-3 expressed in nuclear region is
a prediction marker of NSCLC recurrence [77]. The galectin-3 expression is
increased in normal lung cells, bronchial epithelial cells, interstitial fibroblasts,
bronchial cartilage chondrocytic cells, alveolar wall pneumocytes, and alveolar
macrophages [1].
570 9 Galectins
9.3.2 Galectin-8
On the other hand, galectin-8 also interacts with the GM3, and the GM3-bound
galectin-8 forms caveolae-lipid rafts, leading to downstream signaling
[44, 69]. Galectin-8 as one of the galectin family consists of CRDs with two tandem
repeats. Galectin-8 binds to glycosphingolipids [69]. Galectin-8 is expressed on
several tumors including human prostate carcinoma tumor [78, 79] and squamous
cancer cells [79] as a key regulator of carcinogenesis or metastasis. The level of
galectin-8 expression is significant in certain tissues of liver, muscles, and kidneys of
rat but low in other tissues such as rat fat, intestine, lungs, testis, and thymus
[80]. Recently, the GSL-recognition specificity of galectin-8 has been demonstrated
using enzyme-linked immune assay and surface plasmon resonance method
[69]. More specifically, galectin-8 has high affinity for negative charged carbohy-
drates such as 30 -O-sialyl Lac or 30 -O-sulfated Lac. Galectin-8 can bind to more
diverse classes of binding ligands. For example, galectin-8 recognizes GSLs includ-
ing GM3, GD1a, sialyl-Lc4Cer, sialyl-nLc4Cer, SB1a, sulfatide of SM4s, and
sulfatide SM3. Therefore, galectin-8 specifically binds to a 3-O-sulfated Gal residue
and 3-O-sialyl-Gal residue. The substituted sulfate group on the Gal C-3 position in
Lac or NAcLac increases in the enhances the binding capacity of galectin-8 rather
than sialic acid substitution. Galectin-8 rcognizes certsin sulfated GSLs and sialyl-
GSLs, where amino acid residue at Gln-47 specifically recognize the Neu5Ac(SA)α
2,3-Gal residue or sulphonic SO3 3-Gal residue. In fact, recognition capacity of
galectin-8 to membrane-embedded GM3 is also demonstrated in the controlled
experiments in vitro using CHO cells.
Gal-8 exists widely in various tissues and has a tandem-repeat type with
α2,3-sialylated glycans preference. In particular, Gal-8 regulates proliferation, dif-
ferentiation, and survival of immune cells. In recent studies, Gal-8 promotes differ-
entiation of regulatory T cells through TGF-β signaling and inducing IL-2R
signaling [81]. Also, Gal-8 blunts production of pathogenic T helper type (Th) 1
and Th17 response [82]. However, Gal-8 acts as an immune stimulator molecule to
upregulate the adaptive immune response in bone marrow-derived DCs by stimu-
lating antigen-specific T cells and proinflammatory cytokines IL-6 and IFN –γ [83].
9.4 Galectin-1 and -4 Bind to GM1, But Not GM3
9.4.1 Galectin-4
Galectin-4 expression is mainly found in rat colon, intestine, and stomach, which is
different from the previous galectin-8 [84]. It was reported that galectin-4 binds to
certain glycosphingolipids [85]. Galectin-4 has its specificity of carbohydrate rec-
ognition to the core 1 structure of 30 -O-sulfated Galβ1,3GalNAc-. Galectin-4 recog-
nizes the 3-O-sulfated Gal residue linked to GSLs. Galectin-4 binds strongly to
9.4 Galectin-1 and -4 Bind to GM1, But Not GM3 571
SB1a, SM3, SM4s, and SB2, and weakly to GM1, SM2a, and GD1a. However,
GSLs with a 3-O-sialyl-Gal, including sLc4Cer, snLc4Cer, GM2, GM3, and GM4,
are not good ligands for galectin-4. Thus, galectin-4 does not recognize and not bind
to GalCer, LacCer, GM3, GM2, GM4, sLc4Cer, and snLc4Cer’. Therefore, galectin-
4 recognizes the GSLs containing 3-O-sulfated Gal residue, because this moiety is
common among SB1a, SM3, SM4s, SB2, and SM2a [86]. However, galectin-4 binds
to GM1 because galectin-4 has a weak affinity for the Galβ1 ! 3GalNAc structure
[87]. Gal-4, discovered in epithelial cells in gastrointestinal tract, is similar to Gal-2.
It reduces intestinal inflammation through mucosal T cell apoptosis and
proinflammatory cytokine secretion [88, 89]. Gal-4 triggers inflammatory response
in intestine through IL-6 production in T cells by PKCϕ-dependent pathway [90].
9.4.2 Galectin-1
Galecin-1 has a diverse range of immunomodulatory activities during apoptosis of

activated T cells, B cell development in BM, and modulation of inflammatory
cascade. Galectin-1 is a family of a homobivalent endogenous lectin group and
binds to GM1 found on the cell surfaces. Galectin-1 is a homodimeric prototype of
the galectins with a single CRD. Galectin-1 is described as one of the first galectins
and featured prototype of galectin. The structure of galectin-1 is a classic prototype
of the galectins [91]. In pathogenic infection, bacterial pathogenic infection induces
the highest expression level of galectin-1 among the known galectins [92], and the
expressed galectin-1 directly downregulates microbial infections and manifests. If
host cells are infected, galectin-1 functions to induce apoptotic cell death of host
cells [93], as was reported in the report that galectin-1 inhibits infection of dengue
virus type-1 [94]. Gal-1 also modulates DCs function, migration, and differentiation.
Gal-1 binding to CD43 and CD45 triggers ECM migration by Syk and protein kinase
on mo-DCs. The major Gal-1 receptor on DCs is the mucin CD43. CD43 core
2 O-glycans contribute to different effects of Gal-1 on migration between immuno-
genic and tolerogenic DCs [95]. Gal-1 binds to the Galβ1,4GlcNAc extended on
core 2 O-glycans (Fig. 9.5) when Tn antigen is β1,3-galactosylated by core 1 Gal-T
enzyme to the T antigen. The T-antigen is further sialylated by ST3Gal-1 to generate
sialyl 3 T antigen as well as by ST6GalNac-IV, ST6GalNac-I and ST6GalNac-II to
generate disialyl T-antigen. The SAα2,3Gal in the sialyl 3 T antigen is recognized by
a plant lectin MAL-II. The SAα2,3Gal and SAα2,6Gal in the disialyl T antigen are
recognized by lectins of MAL-II and SNA, respectively (Table 9.2).
Gal-1 suppresses innate and adaptive immune response for destruction of T-cell
activation, differentiation and survival, inhibition of neutrophil recruitment, polari-
zation of macrophages toward an M2 phenotype, and induction of tolerogenic DCs.
In fact, Gal-1 activates differentiation of IL-27-positive tolerogenic DCs, which
induce IL-10-secreting Foxp3-regulatory T (Tr1) cells during autoimmune response
and cancer growth [96]. Pathogenic bacterium Yersinia entercolitica and protozoan
parasite Trypanosoma cruzi trigger Gal-1-mediated immune tolerance
572 9 Galectins
D2,3
PNA
MAL-II D2,3 Core 2 E1,6 GlcNAcT
E1,3 ST3Gal-I E1,4
(Core2-synthase) Galectin-1
Sialyl 3T D
E1,3 Thr/Ser
ST6GalNAc-IV, I, II Thr/Ser Core 2
T antigen
D2,3
D2,6
SNA
MAL-II Core 1 Gal-T
D
Disialyl T
Sialic acid (SA) Thr/Ser

Galactose (Gal) Tn antigen Thr/Ser
Core 2
Fig. 9.5 Galectin-1-binding galactose residues in O-glycan
Table 9.2 O-glycan-based Gal and SA-specific lectins

Maackia amurensis and MAL and Neu5Acα2,3Galß1,4GlcNAc and Neu5Acα2,3Galß1,3
M. amurensis MAH [Neu5Acα2,6]GalNAc, respectively
Sambucus nigra and SNA and Neu5Acα2,6Gal
S. sieboldiana SSA
Trichosanthes japonica TJA Neu5Acα2,6Galβ1,4GlcNAc
Viscum album and ML-I and Neu5Acα2,6Galβ1,4GlcNAc and Neu5Acα2,6/
Saraca indica saracin 3Galβ1,4GlcNAc, respectively
Artocarpus integrifolia Jacalin Gal and Man > Neu5Ac
Triticum vulgaris and WGA and Internal GlcNAc > Neu5Ac and Neu5Gc, respectively
Morus alba MLL
Arachis hypogaea PNA Galβ1,3GalNAc
[97, 98]. Gal-1 inhibits cell surface CD20-utilized therapy, where CD20 is expressed
in non-Hodgkin lymphoma and used as a therapeutic target [99]. Thus, galectin-1
plays crucial roles in anti-inflammatory signaling and eliciting tolerogenic response
of DCs. These events of galectin-1 can stimulate Tregs and IL-10 expression in order
to protect pregnancy status [100].
It is known that GM1-reactive antibodies inhibit binding of galectin-1 to neuro-
blastoma cells [101, 102]. Cell surface gangliosides are constituents of
microdomains within the plasma membrane and as contact site for carbohydrate
receptors (lectins) such as the pentameric lectin part of the cholera toxin (Ctx)
[103]. GM1, the Ctx counter-receptor, is a physiological target for human
adhesion/growth-regulatory galectins, a tissue lectin family with the β-sandwich
fold and a central tryptophan residue for ligand contact [104–106]. The
pentasaccharide of GM1 is a branched structure. GM1 binding to galectins is
different from the Ctx-bound structure [107, 108]. Ganglioside GM1 is located on
plasma or nuclear membranes [109] and Gal-1 is localized in cell nuclei
[110, 111]. Of note, galectins are present as a network in vivo, with the possibility
for functional competition [22, 28]. The interaction of ganglioside GM1 with the
homodimeric (prototype) endogenous lectin galectin-1 induces growth regulation in
tumor and activated effector T cells. The pentameric lectin part of the cholera toxin is
another type of GM1-specific lectin. Gal-1 exerts growth control via GM1 binding to
SK-N-MC cells of human neuroblastoma and GM1 present on activated T cells
in vitro [112–116].
Galectin-1 does not bind to internal β-galactose epitopes, because it has not
affinity to larger poly-N-acetyllactosamine chains in the Gal binding site. GM1 binds
to the galectins. GM1 suppresses the extent of autoimmune diseases through GM1
cross-linking to B subunit of cholera toxin AB5 (CTX-B). Because GM1 is a
recognition ligand for the endogenous lectin, galectin-1, symptoms found in certain
autoimmune disorders are ameliorated [117]. Gal-1 and GM1 interaction elicits
responses of immunosuppression. On murine experimental autoimmune encephalo-
myelitis (EAE), CTX-B and galectin-1 indicates suppressive effects, where GM1
enhances susceptibility to EAE. In the in vitro experiment, polyclonal stimulation of
murine Treg cells causes the enhanced galectin-1 expression. Stimulation of CD4+ T
cells and CD8+ CTL effector cells elevates GM1 and GD1a in mouse. Galectin-1
stimulation of T effector cells also enhance TRPC5 channels for Ca2+ influx during
GM1 cross-linking by galectin-1 or CTX-B. This process includes membrane-
associated events such as co-cross-linking of heterodimeric integrins, α4β1, and
α5β1 glycoproteins with GM1. Therefore, GM1 ganglioside present in CD4+
effector and CD8+ CTL effector cells is the basic binding site of galectin-1 of
Treg cells contributing to the autoimmune suppression. In both cases, GM1 is
made available enzymatically by a sialidase from its precursor GD1a. As reported
for bacterial AB5 toxin and their host enterocytes [117, 118], GM1-galectin-1
interaction triggers rapid internalization of Gal-1 in T leukemic (Jurkat) cells
[119]. For the immunoregulatory function, galectin-1 also elicits apoptotic cell
death of activated T-cells. T cell death elicited by Galectin-1 needs Lck, Src family
tyrosine kinase. Nonreceptor tyrosine kinase, Lck is localized in the synapse of
tumor cell-T-cell. Cell surface presentation of Gal-1 on the effector (tumor) cells
phosphorylates the negative regulatory tyrosine residue, Tyr505, while receptor
tyrosine phosphatase, CD45 is also related to this event. Gal-1-binding glycolipid
GM1 regulates Lck localization and segregation of Lck, and Gal-1 induces apopto-
sis, although it is diminished when GM1 is expressed in T cells. Therefore, regula-
tion of Lck by CD45 and GM1 determines the apoptotic response to Gal-1 only upon
effector (Gal-1 expressing) cell-T-cell attachment [120].
On the other hand, bacterial AB5 toxin-GM1 binding also mimics such autoim-
mune disorders in animal models [121–123]. Human galectin-1 lectin is indeed a
growth regulator for the neuroblastoma cells [124]. It seems to be some differences
between bacterial AB5 toxin-GM1 and galectin-1-GM1 bindings as recent topic
[103]. In the recent study [125], Galectin-1 as a type of cross-linking lectins trans-
lates its roles, as galectin-1 metabolically convert GD1a to GM1 ganglioside through
sialidase involvement into axonal growth in neuronal region. Galectin-1 through
cross-linking with glycoprotein α5β1-integrin and GM1 functions as an axonogenic
effector in cerebellar granule neurons (CGNs) and in vitro NG108–15 cells.
574 9 Galectins
GM1-deficient CGNs were defect in axonogenesis, indicating Gal-1 ligand GM1 as

functional receptor of axon development. GD1a/b and GT1b can be enzymatically
desialylated to generate GM1 by a certain type of neuraminidase such as Neu-3 and
the event can be explained to be a possible molecular switch for on/off in neuronal
differentiation during development and neurite outgrowth through axon elongation
[126–128]. In artificial state, CTX-B-GM1-elicited cross-linking to the α5β1-
integrin activates focal adhesion kinase (FAK) autophosphorylation as well as
consequent PLCγ and PI-3 K activation [129]. The study on the bacterial sugar
receptor raised the question of how such endogenous lectin can also exert such
function? Studies to date concluded that galectin-1 lectin is a potent axon regenerator
[130, 131]. Further, because GM1 is a precursor of GD1a and GT1b interacted with
Siglec-4 known as MAG, the capability in axon outgrowth by siglec-4-GD1a/GT1b
interaction is suspected when sialidases generate GM1 from GD1a-GT1b [132]. In
this regard, the interaction between galectin-1 and GM1 should be revisited for its
functions in multicellular system.
Galectin-1 is an endogenous immunoregulatory lectin-type protein and
overexpressed in tumor-associated capillary endothelial cells. Galectin-1 is a key
molecule in antitumor immune surveillance, providing a possibility to use for its
administration in autoimmune diseases. The most crucial function of galectin-1 is to
inhibit the differentiation and cytokine production in the two distinct Th-1 and Th-17
cells. In addition, galectin-1 induces apoptotic death of activated T-cells. Suppres-
sion of galectin-1 production and responsiveness induce systemic lupus
erythematosus (SLE) T-cells, since lupus displays abnormalities in T-cell activation,
differentiation, and viability [133]. T-cell signaling processes is influenced by
galectin-1 because galectin-1 interacts with GM1 ganglioside in lipid raft aggrega-
tion, early activation signalling steps involving p56Lck, the exchange of the CD3
ζ-ZAP-70 to the FcRγ-Syk pathway, defective MAPK pathway activation, impaired
regulatory T-cell function, the failure to suppress the activity of IL-17-expressing
T-cells, and decreased suppression of the PI3K-mTOR pathway by phosphatase and
tensin homolog (PTEN). Thus, galectin-1 is pathogenic molecules in SLE.
As the first discovered galectin, Galectin-1 is indeed a monocytic regulator in the
innte immune responses by collaboration with IL-10 and G-CSF. Dimeric form of
galectin-1 links T cells in CD7, CD29, and CD43-positive T cells to interact.
Moreover, galectin-1 increases the levels of anti-inflammatory IL-5/IL-10 cytokines,
and in parallel decreases the IFN-γ level, leading to inflammation inhibition.
Galectin-1 expression is also increased in most tumor cells and tumor tissues. The
known cancers including lymphoma, astrocytoma, melanoma, colon, bladder, liver,
oral, ovary, and pancreatic cancers are known to express the Galectin-1. Such
aberrant galectin-1 expression in cancer represents the metastatic aggressiveness of
cancer, as a prognostic marker in early stage cancer. Galectin-1 excelerate tumor
progression by Galβ1-4NAcGlc binding to ligands of cancer cell and activates p38
MAPK, ERK, and COX-2 to sustain survival. They also inhibit functions of T cells
to make better TAMs. Galectin-1 and cancer progression are correlated in cancer
patients and increased expression of galectin-1 is related to poor survival
[134]. Galectin-1 regenerates axon and galectin-1 deficiency induces in neuron
Gal-1
1
2
3
4
18 putative N-glycosylation sites at 46,
5 65, 96, 143, 156, 245, 318, 374, 395,511,
6 523, 580,613, 619, 631, 675, 704 and
721-Asn have the terminal Gal resieues
7
VEGFR2
Endothelial cells
P P
Tyrosine kinase
domains
P P
Sialic acid (SA)

Galactose (Gal)
Activation of VEGFR2 N-Acetylgalactosamine (GalNAc)
and signaling
Fig. 9.6 VEGFR2 binding of galectin-1 and VEGF-like signaling by Gal-1 lectin. High levels of
β1,6-GlcNAc-branches by GlcNAcT-V and decreased terminal α2,6sialyl residues by ST6Gal-1
increases in the Gal residues usable for Gal-1 ligand
mal-development. In Hodgkin lymphoma cells, the level of circulated galectin-1 is

related to poorer prognosis of tumor patients [135].
The Gal-1 regulates endothelial functions. The neuronal receptor neuropilin-1
(NRP1) mediates growth in endothelial cells as a co-receptor with VEGFR. Gal-1 is
overexpressed in the tumor-associated endothelial cells of carcinoma cells. Gal-1
enhances the growth and adhesion of endothelial cells, and, consequently,
upregulates cancer cell migration. Gal-1 CRD binds to NRP1 but does not bind
VEGFR-1, VEGFR-2, or VEGFR-3. The Gal-1–NRP1 interaction allows for endo-
thelial cell migration and adhesion. The Gal-1–NRP1 binding stimulates VEGFR-2
phosphorylation and MAP kinases SAPK1/JNK [136]. In addition, galectin-1
directly recognizes the Asn-linked N-glycans on VEGFR-2. Galectin-1 functions
as a survival factor in the corpus luteum through VEGFR-2 N-glycan interaction.
High levels of β1,6-GlcNAc-branches produced by GlcNAcT-V and decreased level
of terminal α2,6sialyl residues synthesized by ST6Gal-1 increases in the Gal resi-
dues usable for Gal-1 ligand. The corpus luteum is an endocrine gland that produces
progesterone. Galectin-1 expressed on luteal steroidogenic cells binds to the glycans
on VEGFR-2 [137]. VEGFR-2 protein modified by multi-antennary glycan struc-
tures is recognized by galectin-1 (Fig. 9.6).
576 9 Galectins
9.5 Galactine-9 and Galelctin-10
Galectin-9 (Gal-9) exerts immunosuppressive effects through induction of apoptotic

cell death of T cells, which express specific cytokines of IFN-γ and IL-17 [138]. In
addition, Gal-9 triggers the signal transduction of p38 MAPK/ERK1/2 pathway in
DCs. Gal-9 plays a role innate immunity as well as adaptive immunity through
induction of DCs maturation and induction of the Th-1 immune response
[139]. Aldehyde dehydrogenase activity is upregulated by Gal9 exposed
monocyte-derived DCs through fructo-oligosaccharides [140]. Gal9-Tm3 interac-
tion induces antitumor immunity between Tim-3+ DCs and CD8+ T cells
[141]. Gal-10 is a prototype of galectin family and acts as an intracellular immuno-
suppression mediator of eosinophils and Treg cells [142, 143]. Gal-13/-14-16 forms
are suggested to function as immune tolerance regulators in placental tissues [144].
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Chapter 10
DC-SIGNs
10.1 DC-Specific ICAM-3-Grabbing Non-integrin,

DC-SIGNB (CD209)
10.1.1 Molecular Characteristics of DC-SIGN
Among the PRRs, the DC-SIGN (CD209) directly involves in diverse molecular
events in the interaction of DCs with some pathogens. DC-SIGN as a TM protein is a
CTL with a calcium-dependency and exists on the cell surfaces of both DCs and
macrophages. DC-SIGN is, therefore, a pathogenic microbe- and antigen-binding
receptor present in DCs. Human DC-SIGN as one of CTLs is specifically present in
DCs with capacity of a Ca2+-dependent glycan interaction with CRD. DC-SIGN
belongs to an adhesion receptor and recognizes ICAM-2 of endothelial cells and
mediates to tether and migrate transendothelium of DCs. In addition, it mediates the
formation of the clustered DCs with CD4+ T cells by DC-SIGN interaction with
ICAM-3. DC-SIGN recognizes and binds carbohydrate structure including fucose
containing blood group antigens and mannose containing glycoconjugates. The
membrane-bound C type lectin and innate immune DC-SIGN receptor is also called
CD209 and discovered over two decades ago. It recognizes a broad ligand structure
of pathogen-derived patterns and self-glycoproteins. DC-SIGN functions for
intercellular adhesion, antigen uptake, and signaling, crucial for DCs, although
most studies on DC-SIGN come from in vitro results. For the roles of DC-SIGN,
DC-SIGN-related protein (DC-SIGNR) known as CD209L and L-SIGN is the
homolog of DC-SIGN, which are located on human chromosome 19p13.3 locus
and belong to a subfamily in the lectin gene cluster along with the CD23 and
LSECtin [1]. DC-SIGN is present in the mature DCs’ surfaces in the lymph node
as well as immature monocyte-derived and interstitial DCs in the placenta, cervical
mucosa, uterus, and colon, while DC-SIGNR appears in endothelial cells located on
the placenta, liver, and lymph nodes. DC-SIGNR shares 70% higher amino acid
sequence homology with DC-SIGN.
586 10 DC-SIGNs
Fig. 10.1 General feature of DC-SIGN as DC-specific CTL form
DC-SIGN has several features that contribute to the role of DCs due to its
property as a DC-specific CLR and cell adhesion receptor, as shown in the general
structure of DC-specific C-type adhesion receptor in Fig. 10.1. More specifically,
DC-SIGN is a tetramer on the cell membrane and is organized as clustered patches.
The formation of DC-SIGN tetramer is imaginable way of increasing binding
avidity-affinity of ligands containing sugar moieties. As a transmembrane protein,
the molecular structures of the CLR, DC-SIGN consist of several domain structures
of an intracellular, a TM, and an extracellular region. The domains carry out
pathogen recognition and cell adhesion functions. Extracellular domain is composed
of a CRD and a neck region, having repeated sequences with 23 amino acid residues
and forms an α-helical region, which stabilizes the tetramer of the CRD domain in
DC-SIGN. The N-terminal tail region of DC-SIGN is forwarded to the cytoplasm
and is responsible for signaling for the phagocytosis, binding, and intracellular
trafficking of ligand molecules. Those N-terminal tails contain various endocytosis
motifs like a Tyr-based motif and a di-Leu-motif as well as a tri-acidic amino acids
cluster (Fig. 10.2).
10.1.2 General Signaling of DC-SIGN
DC-SIGN downstream signaling pathway initiates with the Raf-1 kinase as the direct
upstream mediator. For the activation of Raf-1, the active Ras form should be bound
to receptor. This interaction elicits a conformational shift of Raf-1. A G protein
signaling molecule, Ras, mediates its signaling through recognition of GTP and
hydrolyzes the GTP. The Ras form bound to GTP is indeed an activated form, so
capable of interacting with Raf-1. Meanwhile, the form-bound by GDP is not active
for function. The DC-SIGN interaction with Man-LAM elicits the GTP-bound Ras
10.1 DC-Specific ICAM-3-Grabbing Non-integrin, DC-SIGNB (CD209) 587
Fig. 10.2 DC-SIGN

structure
Carbohydrate recognition domain
(CRD)
Neck domain
Transmembrane domain
Tyrosine based motif
Triacidic cluster
Di-leucine-based motif
active form via the known Ras guanine exchange factor (GEF). However, interaction
between GTP-bound Ras and Raf-1 is insufficient to activate Raf-1. Therefore, an
additional Raf-1 phosphorylation event is needed. In fact, multiple sites for addi-
tional phosphorylation are identified in the Raf-1 protein and the phosphorylation
regulates Raf-1 kinase activity. Those two phosphorylation sites are serine
338 (Ser338) and tyrosines 340/341 (Tyr340/341). Raf-1 Tyr340/Tyr341 phosphor-
ylation is carried out by Src kinases of tyrosine kinases, likewise, Hck, Fgr, or Lyn,
which are abundant Src kinases in dendritic cells. Ser338 phosphorylation of Raf-1
is catalyzed via the Paks known as the p21-activated kinases. Certain molecules of
GTPases from Rho GTPase family, Cdc42 or Rac1, activate the Paks. Therefore,
DC-SIGN activates one of these three pathways to stimulate the Raf-1 (Fig. 10.3).
After nuclear NF-κB translocation via TLR-activation, DC-SIGN-induced Raf-1
induction contributes to the phosphorylation at the amino acid Ser276 on NF-κB
submit p65, which contributes to p65 acetylation. The p65 acetylation increases and
prolongs transcription of IL-10 gene and IL-10 production (Fig. 10.3).
The in vivo DC-SIGN study seems to be difficult because eight genetic homologs
are found in mice without clear DC-SIGN ortholog or homolog. This receptor DC-
SIGN also recognizes “self” glycans present in human cells and also “non-self
foreign” glycan ligands expressed on bacterial or parasitic pathogens. DC-SIGN
facilitates intracellular DC trafficking of high-mannose-type structures to promote
their antigen presentation. DC-SIGN recognizes both mannosyl and fucosyl glycan
ligands. Protein–carbohydrate interactions usually exhibit weak binding affinities
588 10 DC-SIGNs
Fig. 10.3 DC-SIGN

signaling in regular
transduction pathway HIV-1
M. tuberculosis
DC-SIGN
Src Pak
Ras
Tyr340/341 Ser338
cytosol
Raf-1
Ser276
nucleus
NF-NB
(mM range). Nature uses multivalency to overcome this weak interaction problem.
To overcome such limitations, multiple binding sites on the lectin molecule to
recognize multiple ligands are formed. For example, to facilitate the DC-SIGN
biding capacity, a multiple number of mannose or fucose residues is designed for
strong affinity.
DC-SIGN has a role in “host defense” and “immune homeostasis,” forming
tetramers via neck repeats. DC-SIGN recognizes Lewis-type and high Man-type
glycans. CLRs like DC-SIGN are crucial for homeostatic control of hosts. DC-SIGN
as a type II TM receptor equipped through a Ca2+-dependent Man-specific CRD with
specificity for antigens decorated with high-mannose or Lewis-type structures.
Contrary to other antigen-uptake receptors, DC-SIGN binding to pathogens often
rather cause the increased potentials of immune escape. DC-SIGN mediates DC
migration and T cell activation. Internalization domain in the DC-SIGN cytosolic tail
functions as an endocytosis receptor. DCs-expressed DC-SIGN is quickly internal-
ized during interaction with soluble forms of ligand. Complexed form of DC-SIGN
and ligand is trafficking to late endosomes or late lysosomes. Interaction between
DC-SIGN and its ligands on immune cells mediates adhesion and favors communi-
cation during cognate interactions. DC-SIGN ligands on nonimmunological tissues,
such as the tumor-associated antigen of epithelial carcinoembryonic antigen (CEA),
and CEACAM-1 can enhance the DCs’ cytokine responses to the LPS as a repre-
sentative TLR4 ligand by synergistic enhancement of the LPS-induced IL-10
release. Therefore, DC-SIGN is regarded as a homeostasis-maintaining receptor,
which is easily subverted by invaders or cancer cells through altering the glycosyl-
ation structures.
DC-SIGN-endocytosed ligands are further progressed to antigen processing and
final presention to the CD4+ T cells [2] because human DC-SIGN is appeared on
DCs and macrophages for the immunological response [3–5]. DC-SIGN+ DCs
interact with B cells or some related cells in order to regulate the immune response
elicited by T cells and B cells [6]. DC-SIGN expression is induced during monocyte
differentiation when IL-4 and M-CSF are present [7]. DC-SIGN-expressing macro-
phages suppress T cell growth, as confirmed in a model animal of solid organ
transplantation. DC-SIGN recognizes glycans, which contain Man or Fuc residues
of Lewis glycans expressed by self and non-self-antigens [8, 9]. The human
DC-SIGN induces the IL-10 secretion [10]. Because DC-SIGN expressed in mac-
rophages induces IL-10 secretion during Fuc ligand engagement and the generation
of Tregs [11], DC-SIGN maintains the immunosuppressive tissue environment
[12]. Indeed, DC-SIGN binding with non-immune cells, more precisely with path-
ogens and tumor cells, results in immune escape from the surveillance [13]. There-
fore, tumor cells and pathogens may evolve to target DC-SIGN to escape
immunesurveillance, eventually to survive from the environment. Actually, a report
clearly suggested that the CD169+ macrophage depleted condition or silencing of
DC-SIGN decreases tumor growth [14]. Thus, we can mention that DC-SIGN+
macrophages may participate in the tumor progression. For the biding ligands of
DC-SIGN, fucosylated glycans have been suggested to serve as ligands of DC-SIGN
on the macrophages. The main LeX synthetic FUTs are FUT-IV and FUT-IX
[15]. Fucosyltransferase IX attaches terminal fucose residue as the key Lex structure
to CEA and CEACAM1, by the reaction manner that the FUT-IX enzyme catalyzes
the fucosylation of the distal GlcNAc residue of the poly-LacNAc units, while the
FUT-IV enzyme catalyzes the fucosylation of the internal GlcNAc residue. There-
fore, the FUT-IX enzyme efficiently synthesizes the glycan structure of LeX anti-
genic epitope. The Lex epitopes are expressed as SSEA-I in mouse embryos and
human adults’ myeloid, nervous, digestive, urinary, and cervix uteri cells. For
example, some autoantigens such as myelin oligodendrocyte glycoprotein (MOG)
with fucosylated N-glycans are recognized by DC-SIGN on APCs within the human
brain, including microglia and DCs. This interaction between DC-SIGN and MOG
results in the transmission of a tolerogenic signal with secretion of IL-10 and
defaulted T cell growth through DC-SIGN- and glycosylation process. The exposure
of oligodendrocytes to proinflammatory factors resulted in the downregulation of
fucosyltransferase expression and myelin glycosylation can control immune homeo-
stasis through the MOG, DC-SIGN-dependent regulatory axis [16]. Therefore, the
fucose residues on myelin are crucial for DC-SIGN-dependent homeostatic control
and Th17 differentiation (Fig. 10.4).
10.1.3 α2,6 Sialyl IgG Fc Function by DC-SIGN Receptor
Serum IgG has α2,6–linked Sias at the terminus of its N-glycan and functions as an
inhibitory marker through the human DC-SIGN receptor and upregulates FcRγ-IIB
expression level in macrophages. Consequently, it triggers to dampen the immune
responses [17–19]. This action is well observed in human intravenous pooled IgG
(IVIG), which is applied for suppressive activities of immune responses. The
590 10 DC-SIGNs
Fig. 10.4 DC-SIGN as a homeostatic receptor regulates T cell responses. MOG, Myelin oligo-
dendrocyte glycoprotein
responsible sialyltransferase (ST6Gal-I) is subjected to be regulated by

inflammation [20].
10.1.4 DC-SIGN Binds to Pathogens, Antigen, and Glycans
DCs recognize several pathogens through PRRs. Those surface receptors elicit
specific intracellular signaling to leading to the elicited pathogen-specific immune
responses. Among them, C-type lectins play an important role in recognizing
pathogens. In particular, DC-SIGN acts as a key player in the initiation of immune
activation against various pathogenic infections through modulation of TLR-derived
stimulation. Raf-1 kinase, a Ser/Thr kinase, is a key mediator of the DC-SIGN
signaling pathways. However, some pathogens utilize the DC-SIGN to modulate
host immune responses. DC-SIGN enables macrophages or dendritic cells to capture
numerous antigens for presentation and processing, including HCV, Dengue virus,
human HIV-1, Ebola virus, Lassa virus, SARS-CoV, A. fumigatus, C. albicans,
H. pylori, M. tuberculosis, N. meningitides, Schistosoma mansoni, and Leishmania
parasites. In addition, some pathogens target DC-SIGN to regulate TLR signaling in
human DCs. DC-SIGN specifically binds to the glycan ligands and antigens
expressed on pathogens.
10.1.5 DC-SIGN Role in DC-Mediated Viral Transmission by

HIV-1
A well-designated example of intracellular DC delivery of DC-SIGN is its strong

binding to highly mannosylated HIV gp120, and this interaction promotes virus
transposition from mucosal surface to lymphoid cells to potentiate the HIV transfer
to host T-cells. In the virus case, DC-SIGN is also used as the main receptor for
HIV-1 uptake in DCs. The DC-SIGN as a CLR expressed largely on iDCs recog-
nizes the HIV surface gp120 glycoprotein. Therefore, DC-SIGN receptor is the
acting capture molecule for HIV-1 on iDCs (Fig. 10.5). Binding of DC-SIGN to
HIV-1 downregulates TLR induction by Raf-1-associated downstream signaling.
For example, DC-SIGN-induced signaling during HIV-1 interaction increases
TLR-driven IL-10 expression and also prevents TLR-elicited formation of dendrite
synapses of DCs and T cell growth. DCs capture HIV-1 viruses via DC-SIGN
recognition of the gp120 glycoprotein of HIV-1 with a high affinity. Consequently,
DCs are the intermediate transmitting cells of HIV-1 to the real host T cells, by the
infectious synapse formation with HIV-1-infected DCs and CD4+ T cells. It is
Sialyllactose
HIV HIV
Ceramide
Ceramide
Ceramide
GM1 GM2 GM3

Viral
Gangliosides
glycoprotein
(gp120)
Siglec-1
DC-
SIGN
DCs
Viral glycoprotein (gp120)
DC-SIGN Sialic acid (SA)

Galactose (Gal)
Gangliosides
Siglec-1 Glucose (Glc)
Sialyllactose
Fig. 10.5 HIV recognizes DC receptors, DC-SIGN, and Siglec-1. HIV recognizes DC-SIGN
through the envelope glycoprotein gp120. DCs Sigelc-1 can capture sialyllactose-based ganglio-
sides in the HIV
592 10 DC-SIGNs
reported that DC-SIGN recognition with HIV-1 activates LARG-driven GTPase

Rho A function, which is important for synapse formation of HIV- infection.
In the DC-SIGN ligands of virus, the HIV-1 and HIV-2’s gp120 and its high-m
captured HIV-1 mannose are known. DC-SIGN functions as an intermediate infec-
tion receptor of DCs for HCV and HIV-1, and a pathogen receptor. DCs using
DC-SIGN present the HIV to CD8+ T cells. Thus, DCs let HIV transmit T cells
through the process that DC-SIGN of DCs is used as HIV infection tool to T cells.
Langerhans cells (LCs) as a DC type are also involved in HIV transmission, where
LCs are antigen presenting cells in mucosal tissues. In phenotype, LCs are CD1a+
and E-Cadherin+ (DC-SIGN negative). Instead, it has a Langerin, a C-type lectin
specific for Man residue, Fuc residue, and GlcNAc residue. Langerin therefore has a
capacity to bind to HIV-1. LCs is to protect the HIV-1 infection, inhibiting HIV-1
infection. The other viral ligands are GP of Ebola virus and its high-mannose,
Cytomegalo virus’s gB, Hepatitis C virus’s E1/E1, and Dengue virus’s gE. In the
pathogenic bacteria, the glycan ligands of Helicobater pylori’s LPS and its Lewis X,
Klebsiella pneumoniae’s LPS and Mannose, and also Mycobacteria tuberculosis’s
ManLAM and di-mannose and tri-mannose are known. For the yeast, Candida
albicans is used as the ligand. On the other hand, in the parasites, Leishmania
pitanoi’s LPS and its high-mannose, and Schitosoma mansoni’s SEA and its Lex
are the DC-SIGN ligands.
10.1.6 DC-Mediated Immunosuppression by Mycobacteria
Mycobacterial strains target DC-SIGN to modulate TLR4-induced immune response

by enhancing IL-10 production and impairing dendritic cell-maturation. Likewise,
the binding between DC-SIGN and the Lewis antigens present on LPS from
H. pylori inhibits Th1 polarization but induces IL-10 production. In contrast, the
binding between DC-SIGN and specific Lactobacilli species provokes regulatory T
cell differentiation. As binding between different pathogens and DC-SIGN results in
distinct immunological outcomes, this finding supports the significant role of
DC-SIGN as an immunomodulator. M. tuberculosis interacts with DCs via diverse
membrane receptors such as CTLs and TLRs. Mycobacterial TLR-activation acti-
vates maturation of DCs and the expression of specific cytokines. Additionally,
mycobacterial strains also recognize DC-SIGN of DCs. The interaction of ManLAM
present in M. tuberculosis with DC-SIGN of DCs activates the IL-10 production,
which is one of the immune-suppressive cytokines.
10.2 Other DCs-Derived Receptors 593
10.1.7 DC-SIGN Recognizes Lewis Antigens Expressed

in PMN
DCs-induced immune response is modulated by PMN, and the PMN cells are
composed of 5% CD16 negative eosinophils +95% CD16+-positive neutrophils.
Among them, DC’s DC-SIGN binds to 160 kDa ligands on neutrophils, and the
DC-SIGN also binds to Lewis X on CEACAM-1 of neutrophils. DC-SIGN binds
glycans of CEA and CEACAM1. DC-SIGN interacts with the Lex residues of
CEACAM1 and CEA as the human Lex-binding glycan receptor. Certain human
macrophage subsets express DC-SIGN and thus Lex-containing CEACAMs can
regulate the immune responses in normal status, likely in the placenta of humans or
malignant tumor tissues such as hepatoma, colon, brain, or metastatic carcinomas.
The family of CEACAMs belonging to the Ig superfamily are expressed in most
normal and malignant human tissues. CEACAM1 and CEACAM5 (CEA) express
Lewisx (Lex) structures, where CEACAM1 carries at least seven Lex residues. The
Lex epitope is synthesized by α1,3-fucosyltransferases (FUTs). DC’s DC-SIGN does
not bind to Mac-1 and CEACAM-1 of other cells because of the absence of Lews-X.
DC’s DC-SIGN binds to breast and colon adenocarcinoma cells. In cancer cells,
DC-SIGN’s ligands are CEA and CEACAM-1 on colon carcinoma cells, and it binds
to the CEA of colon tumor cells. DC’s DC-SIGN binds to ligands of Lex, LeA, LeB,
and LeY on CEA and CEACAM-1 of colon carcinoma.
10.2 Other DCs-Derived Receptors

10.2.1 Dendritic Cell NK Lectin Group Receptor (DNGR-1;
CLEC9A)
Conventional DCs are general activators of immune responses, developing as a sort

of the myeloid lineaged cells of hematopoietic stem cells. DCs, but not other
leukocytes, express DNGR-1/CLEC9A. Necrotic or dying cells release endogenous
molecules, as called DAMPs and recognized by PRRs in immune cells. 2 main PRRs
of TLRs and CLRs are present in immune DC and macrophage cells. The exogenous
antigenic molecules are cross-presented in DCs because DCs are professional APCs,
as easily take up foreign antigens present in the peripheral circulation and transmi-
grate to the lymphoid nodes. The captured antigens are presented to naive T cells.
Cross-presentation events evolutionarily ensure in terms of DCs presentation of
foreign agents such as virus-derived antigenic epitopes to CD8+ CTLs to prime
CTLs. Antigen processing and presentation on MHC-I are the key steps to elicit
CD8+ CTLs responses to invaders such as viruses. In the classical pathway, cyto-
solic peptides loaded to MHC-I molecules in the ER region are delivered to the cell
surface upon viral infection. In contrast, in case of uninfected cells, they present viral
antigens in a manner of “cross-presentation,” which uses viral antigens released from
594 10 DC-SIGNs
kunyTX
S S
ITAM
ITAM
P P
Syk
Fig. 10.6 Structure of DNGR-1 and signaling
infected cells. Representatively, the example is the debris of lysed cells performing
endocytosis or phagocytosis to the MHC-I pathway. Cross-presentation of antigens
derived from the phagocytosed antigens is carried out by the CLR, DNGR-1, or
CLEC9A. DNGR-1 is present on CD103+ and CD8α + DCs and their human
equivalents [21]. Clec9a or DNGR-1 is involved in sensing necrotic cells. Sensing
of necrotic cells by DNGR-1 elicits inflammatory response (Fig. 10.6).
DNGR-1 specifically binds F-actin exposed by dying cells upon membrane
integrity dysfunction. By F-actin recognition, DNGR-1 signals to prevent
phagosomal maturation and preserve antigens to allow the cross-presentation, and
thus, it favors CD8+ T-cells’ behavior against cell debris antigens [22]. DNGR-1 is
involved in recognizing necrotic cells and is expressed mainly by the CD8α + DCs
subset. DNGR-1 internalizes antigens by endocytosis and guiding the endosomal
vesicles for cross-presentation of DAMPS by CD8α + DCs. DNGR-1 also belongs to
a type II TM CLR, having an extracellular CTR-like domain and a cytoplasmic
hemi-ITAM motif that enables the Syk recruitment and the Syk pathway activation
[23]. DNGR-1 mostly appears in the CD8α + DCs subsets (CD8α + DCs) as a
receptor for necrotic cells, which favors cross-priming of cytotoxic T lymphocytes to
DAMPS in mice, even though the receptor is not essential for antigen particle
capture through phagocytic event. This signaling is demonstrated from the result
that CD8α + DCs promoted increased IL-10 production when DNGR-1 is absent that
is dependent on IL-10 expression from CD8α + DC [24] (Fig. 10.7).
Necrotic cell
F-actin
DNGR-1
Src family kinase

IL-10 production
Antigen internalinzation by endocytosis
Dead cell associated antigen presentation

NFκB
DC activation
Fig. 10.7 DNGR-1 recognizing necrotic cells suppress DC activation and decrease IL-10 produc-
tion by cross-presenting dead cell-associated antigen. DC NK lectin group receptor-1 (DNGR-1)
recognizes necrotic cells and is mainly expressed by the CD8α + DCs subset. DNGR-1 internalizes
antigens by endocytosis to the endosomal vesicles to participate in cross-presentation of the deaded
or damaged cell antigens to CD8α + DCs subsets
10.2.2 CTL-Like Receptor-1 (CLEC-1)
The orphan CTL-like receptor-1 (CLEC-1) belongs to the subfamily of CTL-like

receptors. CLEC-1-coding gene is located on the human NK gene complex and
contains several PRRs for innate immunity [25]. The human CLEC-1 has not been
well studied to date. CLEC-1 is present in myeloid lineage cells or endothelial cells.
CLEC-1 is an N-linked glycoprotein to form dimeric forms. However, expression of
the CLEC-1 gene is elicited by TGF-β. CLEC-1 is detected intracellularly with ER
localization proteins in human. The CTLRs lack the amino acid residues required for
carbohydrate-binding ligands that need Ca2+ and bind to a wide class of binding
ligands such as lipids and proteins, as DCs represent general APCs for connecting
the innate and adaptive immunities and linking responses of T cells.
CLEC-1 is poorly understood among the Dectin-1 clusters of CLRs. CLEC-1 is
appeared on endothelial cells, DCs and some myeloid lineaged cells present in
tissues. However, CLEC-1 receptor is not expressed in blood granulocytes,
monocytes, B, T cells or NK cells present in peripheral circulation [26]. As the
type II TM receptor, CLEC-1 resembles Dectin-1, but without any canonical sig-
naling motifs [26]. The cytoplasmic domain contains one Tyr residue. CLEC-1
expression is suppressed by inflammatory LPS or IFNγ, while induced by immuno-
suppressive IL-10 and TGF-β [27]. CLEC-1 mediates IL-17 expression at the lower
596 10 DC-SIGNs
extent and during the occurrence of the CD4 + CD25+ Tregs. Expression of the
CLEC-1 gene is upregulated in endothelial cells during binding to CD4 + CD25+
Treg cells. Therefore, the lowered expression of CLEC-1 from DCs using siRNA
develops onset of Th17 cells, while Foxp3 expression is decreased [27]. CLEC-1
regulates the Th17 cells’ development. DCs express the CLEC-1 and CLEC-1 in
vitro inhibits downstream signaling of Th17 functional activation.
Effector Th cells are divided into Th1 and Th2 cells. Each of them largely
produces IFN-γ and IL-4, respectively. Another type of effector Th cells, Th17
cells, as a third subpopulation of effector Th cells was discovered. The Th-17 cells
are characteristic of the IL-17 expression. The Th17 cell differentiation event needs
several cytokines and factors. For example, several regulating cytokines of IL-1β/
TGF-β/IL-6/IL-21/IL-23, and also transcriptional factors such as RAR-related
orphan nuclear receptor (ROR)-α and ROR-γt are involved [25]. The cytokines of
IL-21 and IL22 are also released from the Th17 cells to induce many inflammatory
mediators and anti-microbial immune responses in myeloid-lineaged cells and
epithelial cells. Th17-related responses are consequently regarded in immunological
protection against some bacterial and fungal pathogens. Unfortunately, such inflam-
matory responses are implicated in the autoimmune diseases. The subset of Th17
cells are the recently discovered types of Th cells characteristic of the IL-17
expression. The Th17 cells are specific for immune responses against microbial
pathogens and also involve in manefest of autoimmune diseases. Recently, the CLRs
to regulate the balance between Th1 and Th17 immune activities are required. Such
Th1 and Th17 response-balancing receptors can recognize fungi and related patho-
gens, displaying the anti-microbial immunity. The C-type lectins that regulate the
Th17 type responses in human anti-fungal immunity are reported [28].
The CLRs as PPR receptors involve in fungal and bacterial recognition, eliciting
innate adaptive immune responses. These archetype PRRs generally refers to the
TLR family. The CLRs implicated in Th17 immunity induces adaptive immune
response during anti-fungal immune reaction. The CLRs contain one or more
CTLDs, which are involved in glycan ligand binding and recognition through
protein folding with an S-S bond. The CLRs are present as forms of soluble and
membrane protein. As the characterized carbohydrate binding proteins, they bind to
diverse exogenously found PAMPs and endogenously expressed ligands. Membrane
form receptors can trigger intracellular signaling via cytoplasmic motifs with adap-
tors including the Fcγ chain [29]. These receptors can inhibit cell function vis ITIMs
or can trigger cell function activation via ITAMs. Depending on the phylogeny and
domain organization, CLRs are classified to 17 families [30]. Among them, the type
II, V and VI receptors are involved in Th17-related immune responses. Such
receptors include CD161, DC-SIGN, CLEC-1, Mincle and Dectin-1/ 2
(Table 10.1). These receptors are implicated in anti-microbial immunity.
In human DCs, CLEC-1 is expressed on the CD16-negative DC surfaces of blood
and in mDCs. Inflammatory mediators or agents downreguate CLEC-1 expression
on moDCs and TGF-β induces the expression [31]. CLEC-1 as a receptor of human
mDCs suppresses Th17 responses without affecting the splenic Tyr kinase-depended
canonical NF-κB signaling transduction. Down-regulated CLEC1A expression,
Table 10.1 Ligands of C-type lectin receptor (CTLR) and function in the Th17 responses
CRR Pathogen ligands Function in Th7 cell response
Dectin-1 Fungal or bacterial β1,3-glucans Th17 response to Mycobacteria
T-cell ligand and fungi
Dectin-2 Fungal or bacterial high-Man structures Th17 responses to fungi
T-cell ligand
Mannose Fungal terminal Man, Fuc, GlcNAc, sul- Th17 response suppression to
receptor fated sugars Mycobacteria
Glycoprotein receptors Induction to Candida
Mincle Mycobacterial cord factor and fungal Th17 responses to Mycobacteria
α-mannan and fungi
DC-SIGN Fungal high-Man and Fucosyl structures Th17 response suppression
ICAM-2, 3, Lex
CLEC-1 T-cell ligand Th17 response suppression
CD161 PILAR, CLEC2D Th17 marker
which is known in lung transplants, is prognostic or predictive of the chronic

rejection responses in human and this is related to IL17A expression. CLEC-1 is
also an inhibitory receptor expressed in DCs and dampen effector Th responses
including activation and signaling. The CLEC-1 is also a candidate target to regulate
immune responses of T cells [32]. Following activation, CLRs of DCs downregulate
NF-κB translocation via the SYK pathway and activate or inhibit cell function, and
downstream Th1 and Th17 responses (Fig. 10.8).
10.2.3 CTL-Like Receptor, CLEC12A, Known as Myeloid

Inhibitory CTL-Like Receptor (MICL), CTL-Like
Molecule-1 (CLL-1), DC-Associated CTL 2 (DCAL-2),
and CD371
Antitumor immunotherapies are being tried to raise host immunity and control
disease in cancer patients using DCs. Because DCs are powerful APCs to
reinvigorate T cell activation responses, effective Ag processing and presentation
to stimulate T cell can be obtained by in vivo tumor Ag delivery through natural DC
subsets. Therefore, for such purposes, endocytic C-type lectin receptors are required
as targeting molecules. The objective receptor is the CLEC12A, which delivers
tumor antigens into DC subsets of human, consequently effectively inducing
immune responses by CD4+ T cells and CD8+ CTLs. CLEC12A is expressed in
myeloid cells of the mDC subsets and the pDCs subset. Such DC subsets exclusively
internalize CLEC12A and quick trafficking to the early endosomes and lysosomes.
The TM receptor of CTL family 12 is a member A of CLEC12A. The expression
of CLEC12A lectin is abundant in circulatory neutrophils and monocyte cells.
CLEC12A is regarded as a biomarker of AML. CLEC12A also peripherally appears
in CD45lowSSClowCD14-CD123+ basophils in blood system. The receptor binds
598 10 DC-SIGNs
jsljTX
Unknown ligand
CLEC-1
TGFβ
naive
Pro-inflammatory stimuli CD4 T
cell
moDC
IL-12p40
Th1/17
cell
IFNγ ↓
IL-17A ↓
Fig. 10.8 CLEC-1 modulate IL-12p40 production by moDC, suppressing Th1 and Th17
responses. CLEC-1 expressed in DCs inhibits downstream signaling of Th17. In mDCs, the
CLEC-1 expression is suppressed by inflammatory stimulation but activated by TGF-γ stimulation.
CLEC-1 receptor in human moDCs does not regulate the Syk-dependent canonical NF-kB and
represses Th17 responses
to unknown endogenous ligands and is widely present in innate myeloid immune

cells including neutrophils, monocytes, DCs, and macrophages [33]. The ITIM of
CLEC12A recruits the Tyr phosphatases of SHP-1 and -2 to suppress Syk down-
stream signals. This Clec12a senses dead cells. Uric acid crystals as monosodium
urate (MSU) is specific ligands of Clec12a [34]. MSU binds to CLEC-12A,
inhibiting inflammatory responses induced by cell death to MSU in vitro. Therefore,
Clec12a is a receptor for dead cells through the recognition of uric acid crystals,
allowing the damp responses.
CLEC12A is alternatively called DCs-associated CTL-2 (DCAL-2), myeloid
inhibitory CTL-like receptor (MICL), CTL-like molecule-1 (CLL-1), and CD371.
CLEC12A expression is widely present in all human DC subsets [35]. The function
of CLEC12A is not well understood, but CLEC12A is involved in the sterile
inflammation control by neutrophils. Because inhibitory receptors control cellular
activation, they regulate homeostasis and immunity. As an inhibitory receptor of
human, CTL-like receptor, CLEC12A (MICL) is a hyper-glycosylated monomer
molecule. The murine MICL homolog, called mMICL, resembles the human
ortholog, called hMICL. mMICL is a dimer form, but not a heavily glycosylated
form. The receptor recruits inhibitory phosphatases upon activation. Recently,
CLEC12A is involved in microbial defense in myeloid cells in bacterial autophagy
[36], suggesting that CLEC12A is involved early during bacterial autophagy upon
jsljTXYh
Inflammation
Uric acid crystal (MSU)
CLEC-12A
Reactive oxygen
O2
species
NADPH
oxidase
ITAM
ITIM
Src family kinase + P
Recruit / Activation Inhibitory role
Syk
Fig. 10.9 CLEC-12A suppress the activating pathway of NADPH oxidase by inhibiting the
function of Syk. (1) CLEC-12A receptor binds to unknown endogenous ligands and is widely
present in innate immune cells like DCs, macrophages, monocytes, and neutrophils. Its ITIM
recruits the phosphatase SHP-1 and -2 to downregulate the Syk downstream signaling. Clec12a
senses dead cells and MSU are specific ligands of Clec12a. MSU binds to CLEC-12A, inhibiting
inflammatory responses. It activates Syk signaling in DCs via lipid membrane recognition, inducing
ITAM cross-linking or through the CD16 and CD11b. MSU induces pro-inflammatory responses
via NLRP3 and Syk at the innate immune cells through ITAM-bearing transmembrane adapters
on DCs
pathogen recognition and membrane damage. Chen et al. [37] showed that binding
of human monocyte-derived DCs (MoDCs) with αCLEC12A Ab-controlled
TLR-mediated maturation. CLEC12A can facilitate Ag cross-presentation by
human DC subsets. CLEC12A can potentiate antigen delivery into human DCs to
stimulate immune cells such as CD4+ and CD8+ CTLs. CLEC12A initially trans-
locates to the endosomes. Therefore, CLEC12A is a target-binding receptor in vivo
for tumor antigenic translocation into mDC and pDC subsets to raise tumor-reactive
immune responses in cancer patients. CLRs shape immune responses via the sig-
naling motifs or via the adaptor molecules. CLEC12A consists of an inhibitory ITIM
motif in the cytoplasmic tail. It activates Syk signaling in dendritic cells via a
membrane interaction and ITAM receptor or the receptors CD16 and CD11b.
MSU activates inflammatory responses by NLRP3 and activation of Syk of innate
immune cells through ITAM-containing transmembrane adapters on dendritic cells
(Fig. 10.9).
CLEC12A is endocytosed via the caveolin pathway. To internalize, CLEC12A is
targeted for Ag delivery and presentation via MHC-I and MHC-II. Receptor/Ag
delivery to the lysosomes results in loading onto MHC-II and subsequent CD4+ T
600 10 DC-SIGNs
cell activation [38]. CLEC12A and DEC205 are colocalized with the early
endosomes, trafficking to the lysosomes. CLEC12A colocalizes with the early
endosomes. CLEC12A provides an endosomal escape of targeted Ag to facilitate
cross-presentation. CLEC12A, when the cells were triggered by uric acid crystals,
blocks synthesis of the Syk-dependent reactive oxygen species (ROS), limiting
infiltration of immune cells to the damaged site. Human mDCs and pDCs can
cross-present natural tumor Ags delivered via CLEC12A, activating the tumor-
specific CD8+ CTLs. Humoral and cellular immune responses are induced after
CLEC12A-mediated OVA delivery into APCs. CLEC9A and DEC205 are more
effective targets than CLEC12A. The human BDCA1+ mDCs, BDCA3+ mDCs,
pDCs, and ex vivo-generated MoDCs express CLEC12A. CLEC12A is efficiently
internalized via a clathrin-independent mechanism and is translocated from the early
endosomes to the lysosomes. CLEC12A-targeted Ag delivery results in potent
activation of CD4+ T cell responses and highly effective cross-presentation to
HA-1–reactive CD8+ T cells [39]. In conclusion, CLEC12A is the specialized
receptor for in vivo Ag delivery into mDCs and pDCs to boost tumor-reactive T
cell immunity in cancer patients [40].
10.2.4 CD161 (NKR-P1A)
One of CLRs of NK cells, CD161 (NKRP1a, gene KLRB1), belongs to a type II TM

receptor, possessing a CTLD in the extracellular region and a cytosolic tail domain
that consists of a nontypical ITIM-like motif [41]. Ligand of CTL-like CD161 is the
lectin-like transcript 1 (LLT1) expressed from the gene CLEC2D [42]. The four
different structures of human LLT1 are present in the monomeric and dimeric forms
deglycosylated after the first GlcNAc unit from GlcNAc2Man5 structure in two
forms as well as hexameric form attached with homogeneous GlcNAc2Man5 struc-
ture. The dimeric form has been made through the classical dimerization way of
human CD69. The hexameric form of glycosylated LLT1 contains three classical
dimers. The hexameric form formation indicates recognition of CTL-like proteins as
the glycosylated form. Therefore, LLT1 exists in different monomeric forms, called
LLT1-monomer and LLT1-dimer. When they are deglycosylated at the the first
GlcNAc unit, they are called LLT1-D1 and LLT1-D2. In addition, if glycosylated
dimeric form is packed with hexamers of homogeneous GlcNAc2Man5 residues, it
is called LLT1-glyco.
LLT1 has initially been discovered as a typical ligand of CD161 (NKR-P1), and
LLT1 is the only human NKR-P1 subfamily (CD161) [43]. LLT1 appears in
activated lymphocytes including B cell, T cells, NK cells, and APCs including
macrophages and DCs [44]. The CLEC2D gene is alternatively spliced to six
different transcripts. LLT1 isoform 1 is the sole interactable transcript with
NKR-P1 [45]. The NK cell NKR-P1 recognizes LLT1 on the target cells, contrib-
uting to inhibition of cytotoxicity of NK cells and IFNγ expression [43] and giving
NK self-tolerance likely to the rodent case [46]. Human glioblastoma regulates the
LL1 surface expression to escape the immune response [47]. LLT1 is upregulated by
pathological infections of microbes and viruses. Activation of NKR-P1-positive T
cells stimulates their growth and cytokine production [48]. Thus, LLT1–NKR-P1
interaction induces recognition of pathogenic patterns and lymphocyte stimulation,
as the innate and adaptive immunities. Human LLT1 also belongs to a type II TM
glycoprotein with a cytoplasmic chain on N-terminal region, TM and stalk regions
and a CTL domain on C-terminal region with two N-glycosylation sites.
The CD161 receptor is present in NK T cells, NK cells, and T cell subsets. DCs
and monocytes also express CD161 [41, 49]. CD161 binds to many carbohydrate
ligands such as proliferation-induced lymphocyte-associated receptor (PILAR),
CLEC2D, and lectin-like transcript-1 called LLT1 [43, 50, 51]. Although CD161
function is not clear yet, the regulatory functions of CD161 including trans-
endothelial migration, lysis of NK cells, and the immature thymocyte growth of
NKT cells and T cells are clarified [43, 49, 50]. CD161, a CTL-like receptor, is
mainly present in the NK cells. Although the CD161 function in NK cells has not
been well understood, CD161 expression defines an innate functional phenotype for
T cell populations and NK cells. CD161 marks NK cells towards innate differenti-
ation. Such NK cells, which are pro-inflammatory, are located in the lamina propria
region, which is inflamed, with integrin CD103 [52]. Thus, CD161 expression
implies that NK cells in the state of inflammatory pathogenesis and an innate
immune reaction in NK cells/T cells.
NK cells are a group of the most classical subset of innate immune cells as
lymphoid lineage that can recognize and elicit the lysis of tumor target cells, viral
infected cells, or stressed cells without prior antigen sensitization. The NK cell
receptors are structurally classified into two distinct Ig and CTL superfamilies.
The CTL receptor group of NK cells lacks carbohydrate-binding capacity, but
instead has protein ligand-binding capacity [53]. While some CTL receptors bind
to the MHC-I-like folds such as NKG2D, CD94-NKG2A, and Ly49 receptors of
murine [54], the NKR-P1 receptor subfamily cannot. Apart from direct cytotoxicity
mediated by cells, NK cells can activate the responses of adaptive immunity.
Functions of NK cells are regulated by surface-stimulating and surface-suppressive
receptors with ligands expressed in the cell surfaces. The stimulation of NK cells and
target cell-specific cytotoxicity are induced by cellular markers of MHC-I-like
glycoproteins with the lowered inhibitory NK receptors in a missing-self-binding
manner or by the activated NK receptors-stimulating ligands, in virus-infected,
malignant, or stressed cells in an induced-self manner. NK cells express a wide
repertoire of germline-expressing receptors to distinguish stressed or infected cells
from normal cells [55]. Among the NK cell receptors, CD161 is an early biomarker
in maturation of NK cells from precursors CD34+ hematopoietic stem cells
[56]. CD161 synthesis exerts the cell lysis role of CD16+ NK cells [57], and
CD161 binding to its ligand LLT1 suppresses cytotoxic NK cell potentials
[58]. CD161 expressed in early stage of NK cell development allows cross-talk
between the NK cell precursors and resident cells in the BM, releasing CXCL8
[59]. In the peripheral area, CD161 cross-linking increases in NK cell cytotoxicity
suppression and IFNγ secretion [60].
602 10 DC-SIGNs
The receptor modulates the synthesis of IL-4, IL-1β, TNF-α, IFN-γ, and IL-12
cytokines in CD161+ lymphoid cells and myeloid cells [49, 61]. The question how
CD161 affects such efficacy is raised. Only two facts are elucidated that CD161
effect on T cell proliferation is based on CD161 interaction with PILAR and CD161-
mediated NK cell signaling is depended on acid sphingomyelinase [51, 62]. CD161
is also a biomarker of human T cells rather than IL-17, as known in the human CD4+
T cells and CD8+ CTL subsets, where CD161 expression is increased in the
IL-17-expressing T cells, the precursor type present in the newborn thymus and
umbilical cords [63]. Thus, CD161 as a marker for Th-17 cells is expressed in all
IL-17-expressing T cell subpopulations in human, which is found even in acute and
chronic inflammation of intestinal area, as elicited by pathogenic virus infection
[63, 64]. But the CD161 function is not explained. Mouse homolog, called NK1.1, is
not identified in IL-17-expressing T cells. Thus, murine Th17 cells may be not the
same cells of humans. From the fact that Th-17 cells recognize specific cells via
chemokine receptors of CCR6, CD161 is suggested to function in the transmigration
of the Th17 cells to the endothelium [63, 65].
CD161 expression is modulated in NK cells upon virus infections. In fact, CD161
reduces the HCV infectivity and potentiates viral clearance and increases in inflam-
mation of liver. Similarly, in HIV-infected patients, CD161+ NK cells are depleted.
Overall, despite the CD161 expression in several viral infections, the CD161 roles of
NK cells are not fully understood.
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Chapter 11
Toll-Like Receptors (TLRs)
Host immune system selects the targets through immune recognition. The target-
recognition is primary in innate immunity through the innate immune cell
populations of myeloid lineage NK cells, innate lymphoid, and non-immune cells
in certain circumstances and also the ancient humoral complements. Innate immune
system expresses distinct receptors known as PRRs, which directly recognize
PAMPs. Complement receptors and Toll receptors bind to PAMP recognition
products. Receptors as the first defense line alarm the pathogenic presence. Although
innate immunity eliminates most pathogens, certain pathogens are not eliminated
due to the pathogens produced virulence factors. Innate immune responses are not
specific originally but adapted. Because of the nonspecificity of innate immune
responses, alternative complementation cooperates with the PRRs. The PRR recep-
tors recognize specific pathogenic components such as glycans because glycans face
the outmost world with diversity in the glycan structures and patterns as the self and
non-self recognition basis.
During microbial infection, host innate or acquired immune defense involves the
systemic changes in host glycosylation in cell surfaces. In the side of pathogens, they
evolve to modulate host immune defenses by modulating its glycosylation. The
“self” and “non-self” glycans are distinguished by lectins. SAs on cell surfaces in
prokaryotes are the targets for attack but they are eukaryotes are SAMPs. In innate
immune system, the PRRs recognize the foreign stimulating agents such as bacteria,
virus, and fungus. Such invasive agents are regarded as PAMPs. Many receptors are
found to be pathogen-recognizing receptors in innate immune cells, including TLRs,
C-type lectins, ML, and Siglecs. Most TLRs are localized to the cell surfaces as
immune receptors and certain types of TLRs are also intracellularly located as
cytosolic compartments like the endosome. TLRs recognize PAMPs and DAMPs
to afford immune responses in innate immunity. TLRs are interacted with PAMPs or
DAMPs, allowing affordable recruiting of TIR domain-carrying adaptor proteins.
Currently, the well-defined TIR-adaptor protein is MyD88, which mediates diverse
signal transduction pathways in order to protect them from the microbial infection.
However, if TLRs are not sufficiently regulated or negatively regulated in the
608 11 Toll-Like Receptors (TLRs)
condition of excessive immune responses, abnormal immune responses are

displayed. The resulting diseases include autoimmunity and inflammation.
To recognize such molecular patterns, TLRs as one of the PRRs discriminate each
type of PAMPS. In inflammatory responses inflamed and performed by innate
immune cells like monocytes, TLR signals are kye players to activate the responses.
To date, several PRRs are well known in immunity [1]; TLRs 1-10 (human), the MR,
NOD-like receptors (NLRs), and RLRs as well as dectin 1 and 2. Ruslan Medzhitov,
a biochemist from Uzbekistan who received the Shaw Prize in Life Science and
Medicine 2011 together with Bruce Beutler and Jules Hoffmann, outlined the
concept of PRRs. Richard Ulevitch, a biochemist from the USA has focused on
the signaling pathways of PRRs. From the pattern recognizing issues, the concept of
the Toll receptor has initially been utilized in receptor identification for Drosophila
dorsoventral pattern formation during embryo development, because the Drosophila
Toll receptor responded to fungal infection [2]. Then, TLRs are recognized during
the innate immune responses [3] and the concept of Toll receptors have been
expanded to mammals. “Toll” means literally amazing, bodacious, cool, corky,
crazy, frantic, furious, great, like blazes, mad, madcap, screaming, super, wild, or
wow. What is a Toll receptor? A woman scientist, Dr. Christiane Nüsslein-Volhard
was a Nobel laureate (Medicine, 1995) with her discovery of the Toll receptor. She
as German biologist and geneticist published in 1985 the first article on the Toll gene
product; She was the 1995 Nobel Prize recipient in Physiology or Medicine for her
discoveries. For her hypothesis historically in Malaria study, Toll receptors impor-
tantly function in the malaria pathogenesis through the hypothesis of hemozoin
recognition, where hemozoin is a potential candidate for a proinflammatory sub-
stance made by malaria. The protein Toll was first identified from Drosophila
melanogaster, and it generally exhibits host defense roles with its distinct mecha-
nisms, as the TLRs are mostly found in DCs, eosinophils, basophils, and mast cells,
which are innate immune cells, as well as some specific epithelial cells. Toll as a
type-I transmembrnae (TM) receptor was isolated by Schneider and Anderson
[4]. Toll has a high homology to the cytoplasm portion of the IL-1R, TIR domain.
The observation that Toll has a domain in common with the IL-1R led to the
hypothesis that IL-1 was involved in innate immunity. Jules Hoffmann, born in
Echternach (Luxemburg), is a chemist and biologist. Together with Bruno Lemaitre
he made groundbreaking contributions to the function of Toll receptors in antifungal
immune response. The dorsoventral gene with regulation cassette spätzle-Toll-
cactus potentially modulates the anti-fungi immune responses in adult
Drosophila [5].
11.1 TLR Molecular Structure, Subtypes, and Recognition Ligand 609
11.1 TLR Molecular Structure, Subtypes, and Recognition

Ligand
TLRs belong to the type 1 transmembrane receptors that are comprised of extracel-
lular ligand-recognition domains, TM domain, and a TIR domain in the cytoplasmic
region (Fig. 11.1). In mammals, 11 TLR family (TLR 1-11) was cloned, each
expressed on the cell surfaces of innate immune response of macrophage [6] in
terms of evolution of vertebrate TLRs. The first homolog of the Drosophila Toll
receptor was called hToll, which was found in 1997, and it was retermed TLR4 by
Medzhitov et al. [7]. Thereafter, several other TLRs, which are structurally related to
TLR4, were discovered. In humans and mice, 10 TLR1-10 members and 12 TLR1-9
and TLR11-13 members were discovered, respectively. On the other side, Toll
receptor-independent microbial recognition has also been found. Innate immune
response is the virtue of a recognition via nonclonal system. The mammalian TLR
family carries 13 family members with specific capacity of patterns recognition of
microorganism surfaced components, known as PAMPs to induce the innate immu-
nity of hosts and consequently to activate antigen-specific acquired immunity. In
structure, the cytoplasmic region of TLRs resembles the IL-1Rr family, calling to the
TIR domain. In a large scale, the TLR-associated signaling pathways involve in the
named MyD88 and TRIF signalings [8]. The specific recognition specificities of
TLRs with their ligands in the microbial recognition are characterized, as each TLR
interacts with microbial pattern components (Table 11.1). As the endosomal mem-
brane-anchored TLRs, several forms of TLR7, TLR3, TLR8, and TLR9 are known.
TLRs can be divided into several types of TLR-1 to TLR-13 and each TLR has
different localization and ligand (Fig. 11.2). TLR 1, 2, 4–6 and 11 are expressed on
plasma membrane but TLR 3, 7–9 are prominently present on intracellular
endosome-like vesicles like ER and endosomes [9]. The TLR extracellular domains
contain short tandem Leu-rich repeats (LRRs) which can bind to ligand. The TLRs
are monomeric usually but form homo- or hetero-dimers when they recognize and
bind to the ligand. TLR 2 and 6 or 1 and 2 can bind to various diacyl- or triacyl-
lipoproteins and lipopeptides including the component of gram-positive bacteria and
form heterodimer respectively (Fig. 11.2). On the other hand, TLR 4 can recognize
Fig. 11.1 The TLR

molecular structure. TLRs
have three domains
including ligand-binding lGTG
domains in the extracellular
region, transmembrane
domain, and a TIR domain
in the cytoplasmic region
Cell membrane {GG
j G{pyG
Table 11.1 TLRs and their ligand patterns. The monoclonal antibodies are all commercially
purchasable
TLR Patterns as ligands Innate recognition
TLR1 Triacyl lipopeptides Cells derived from TLR1-efficient KO
mice exhibited a regular inflammatory
response to diacyl lipopeptides and TLR1
recognizes two different ligands of triacyl
lipopeptides and diacyl lipopeptides. TLR1
recognizes the outer surface lipoprotein of
Borrelia burgdorferi [10]
TLR2 Peptidoglycan, lipopeptides, zymosan, TLR2 recognizes many microbial patho-
glycolipids, lipoteichoic acid, genic components of lipoproteins from
lipoarabinomannan, GPI-anchors, phe- Gram-negative bacteria, Borrelia
nol-soluble modulin burgdorferi, Mycoplasma fermentans, and
Treponema pallidum. TLR2 also binds to
Gram-positive bacterial lipoteichoic acid
and peptidoglycan. It binds to T. cruzi,
GPI-anchors, mycobacterial
lipoarabinomannan, T. maltophilum glyco-
lipids, S. epidermis phenol-soluble
modulin and fungal zymosan. A primary
ligands of TLR2 recognition are peptido-
glycans and lipoproteins. TLR2 collabo-
rates with the related receptors of TLR1
and TLR6 [11]
TLR3 dsRNA TLR3 has a specific signaling cascade.
TLR3 recognizes dsRNA. Because dsRNA
is produced by viral replication, dsRNA
induces the type I IFN-α and β synthesis
and IFN-α/β activate antiviral and
immunostimulatory functions through reg-
ulation of IFN-elicited genes and DCs
maturation. TLR3 promotes cross-
presentation of virus dsRNA and viral
infected cells-captured DCs to T cells [12]
TLR4 LPS, envelope protein of MMTV, Taxol, From the C3H/HeJ strain of mice, the
RSV fusion protein, endogenous ligands LPS-hyporesponsive gene has been identi-
of HSP, fibronectin and hyaluronic acid fied as TLR [13]. LPS associates in a close
proximity with CD14 and TLR4
[10]. MD-2 protein is also complexed as
LPS receptor with the TLR4 extracellular
domain [14]. In LPS recognition of B cells,
the third protein RP105 binds to TLR4.
RP105 negatively regulates LPS responses
in macrophages [15]. Taxol, an anti-tumor
agent, has LPS-like activity due to TLR4-
elicited LPS-mimetic to Taxol [16]. TLR4
and CD14 bind to the respiratory syncytial
virus (RSV) fusion protein [17]. In addi-
tion, the mouse mammary tumor virus
(MMTV) envelope protein stimulates B
cells through TLR4 binding [18]
(continued)
11.1 TLR Molecular Structure, Subtypes, and Recognition Ligand 611

TLR5 Flagellin Human TLR5 responds to monomeric fla-
gellin of flagella in bacteria [19]. TLR5 is
present in the basolateral site of epithelial
cells in the mucosal surfaces of intestine. A
polymorphism with stop codon in the
ligand-recognition domain is involved in
pneumonia susceptibility by the flagellin
type Legionella pneumophila bacteria in
TLR5. TLR5 also appears in CD11c cells
in the lamina propria of intestines,
responding to intestinal infection of
S. typhimurium via TLR5-mediated
inflammation and maturation of intestinal
DCs [20]
TLR6 Diacyl lipopeptides TLR6 associates with TLR2. TLR6 recog-
nizes di-acyl lipopeptides produced by
mycoplasma. Macrophages derived from
TLR6 KO mice are not responsive to
inflammation induced by diacyl
lipopeptides produced by mycoplasma
strains. In contrast, the cells are responsive
to triacyl lipopeptide forms produced by
Gram-negative bacteria
TLR7 ssRNA, imidazoquinolines TLR7 responds to imidazoquinoline and
recognizes guanosine (G)- or uridine (U)-
enriched ssRNA viruses like HIV, stoma-
titis virus, and influenza virus [21]. ssRNA
produced within host is not recognized by
TLR7 because self-derived ssRNA is not
translocated to the TLR-7-produced
endosomal membrane. TLR-7 binds to
FcR-derived endocytosed ssRNA in the
pDCs endosome. In alternative pathway,
ssRNA engages the RNA-recognizing
BCR of autoreactive B cells, inducing
autoantibody production [22]. Autoim-
mune diseases are observed in such
Sjögren’s syndrome and systemic lupus
erythematosus (SLE)
TLR8 ssRNA, imidazoquinolines of human TLR8 and TLR7 are homologous and X
chromosome-encoded. Human TLR8 binds
to the imidazoquinolines and ssRNA
known as the TLR7 ligands. However,
mouse TLR8 could not bind to the TLR7
ligands. Mouse TLR8 simultaneously pro-
mote Tregs with the imidazoquinolines and
poly(T) oligodeoxynucleotides
[23]. Human TLR8 appears in Tregs and its
activation blocks Treg function [24]
(continued)

TLR9 CpG DNA TLR9 recognizes the bacterial and viral
CpG motifs. Functions of CpG motifs are
suppressed due to the Cys methylation of
the CpG motifs in vertebrates, preventing
immunostimulation. BCR or FcR-derived
endocytosis of the immune complex is
caused from TLR9-derived host CpG DNA
recognition in chromatins
TLR10 Non-identified Human TLR10 was discovered as a TLR1
and TLR6-related form. The TLR10 ligand
is not identified. TLR10 structure resem-
bles TLR1 and TLR6. TLR10 discrimi-
nates the various ligands of TLR2. Mouse
TLR10 homolog is not found yet
TLR11 Profilin, Flagellin Human TLR11 is not present due to a stop
codon insertional inactivation in the
genome of TLR11 gene. TLR11 of mouse
present in bladder epithelial cells exerts
resistance to uropathogenic bacterial
infection [25]. A parasite-derived profilin
[26] and flagellin of Salmonella
typhimurium [27] are TLT11 ligands
TLR12 Profilin TLR12 is homologous to TLR11 and rec-
ognizes the profilin of Toxoplasma gondii
[28]
TLR13 Bacterial 23S ribosomal RNA TLR13 recognizes the conserved
CGGAAAGACC motif of bacterial 23S
ribosomal RNA [29]
endotoxin LPS of Gram-negative bacteria with MD-2 protein and CD14. TLR5 can
bind to a highly conserved site of monomeric flagellin of bacteria. TLRs on
intracellular vesicles can recognize the viral nucleic acids involved in sensing the
virus. TLR-3, -7, -8, and -9 can recognize ssRNA, dsRNA, and CpG DNA.
11.2 Signal Initiation and Transduction of TLRs
Activating TLRs by binding stimuli to the receptor extracellular domain and induc-
ing the dimerization of receptors can make the cytosolic TIR-TIR domains interac-
tion acting as a scaffold for signal transduction downstream [30]. The cytosolic TIR
domain has three highly conserved regions among all family members, called box
1, 2, and 3. These boxes are thought to provide the binding site for intracellular
adaptors which participate in signaling pathway. The several adaptor proteins also
have TIR domains and participate in TLRs signaling pathway by binding to TIR
domain of TLRs (Fig. 11.3). The Myddosome complex containing a key adaptor
11.2 Signal Initiation and Transduction of TLRs 613
k {
m swz

{syGY {syG] {syGX {syG\ {syG[
{syGZ
{syG^
{syG_
yuh {syG`
yuh
yuh

jn kuh
Fig. 11.2 TLR subtypes and localization. TLRs 1, 2, 4, 5, and 6 found on the PM bind to the
extracellular pathogen molecules. TLR 3, 7, 8, and 9 are located on the intracellular membranes
such as ER and recognize the microbial components such as RNA or DNA
protein MYD88 can stimulate the NF-κB which is well known for transcription
factor of pro-inflammatory related gene expression and all TLRs pathway except
TLR 3 make this complex. On the other hand, the Triffosome complex containing a
key adaptor TRIF can activate the IFN-regulatory factors (IRFs) and TLR 1-4 and
6 pathway can make this complex. Each TLR has a little different mechanism during
signaling pathway but the overall schemes are very similar [31].
In the first pathway as in Fig. 11.3a, the TIR domain of dimerized TLRs can bind
to other TIR domains of MyD88 and MyD88 adaptor-like (MAL), and MyD88 can
recruit and activate the IL-1R-associated kinase 1 and 4 (IRAK1 and IRAK4) which
are serine-threonine protein kinases. This complex can recruit TRAF-6 and TRICA1,
which are E3 and E2 ligases, and generate a scaffold by making polyubiquitin chains
on TRAF- 6, followed by recruiting of the TAK1 kinase. The activated TAK1 kinase
can activate the IkB kinase (IKK) which can degrade the IkB by phosphorylating.
This response can release NF-κB and also elevate the several kinds of IL-6/IL-1β/
TNF-α pro-inflammatory cytokines, through translocation into nucleus. Another
way is shown in Fig. 11.3b, where nucleic acid-sensing TLR recuites a key molecule
TRIF as downstream signalin molecule of endosomal TLRs. TRIF can recruit
TRAF3 and activate IKKε and TBK1, which are kinases. These can phosphorylate
IRF3, and activated IRF3 can induce the expression of type 1 interferon genes.
hP {sy iP {sy
t k__SGths {ypm
{yhmZ
pyhrGXS[ {hrX
{yhmT]
|ijXZSG|Xh
p rr p rrȿ u lt v
{irX
umi pi
pyhmZ
pGGGpmuG
pGGG G
w
Fig. 11.3 TLRs activate NF-kB and IRF transcription factors to perform the pro-inflammatory
function. NF-kB activating pathway through Myddosome (a) and type 1 IFN activating pathway
through Triffosome (b)
11.3 Glycosylation of TLRs
These days, it has been revealed that glycosylation of TLRs is required for their
maturation or trafficking. For example, TLR 5, 7, and 9 should be modified through
oligosaccharide transferase complex (OSTC) which can mediate TLR
co-translational glycosylation [32, 33]. Without these molecules, the expression of
TLR 5 would be retarded or abolished. Furthermore, N-glycosylation sites in the
many TLRs are present in the extracellular domains. For example, TLR2, 3, and
4 cannot do their activity well without sialidase Neu 1, which can cleave sialic acid
residues from the part of TLRs’ glycosylation (Fig. 11.4).
11.4 General TLR Functions as Pathogen and Antigen

Receptors on DCs
In innate immunity, DCs are activated by TLRs, as this event is crucial for host
defense [11]. As type I membrane-spanning receptors, the TLRs are dominantly
present in APCs like DCs and macrophages. Therefore, the role of TLRs in DCs and
innate immune cells is to bind to PAMPs of bacterial pathogens and viral agents.
11.4 General TLR Functions as Pathogen and Antigen Receptors on DCs 615
Fig. 11.4 N-linked

glycosylation of TLR
4. TLR 4 can induce its {sy[
downstream signaling
z
pathway after modification
of glycosylation parts with
Neuraminidase 1 (Neu 1)
Neu 1
membrane
Generally, immunological patterns of Gram-negative bacteria are LPS, while those

of Gram-positive bacteria are LTA and ds- or ssRNA from viruses. Thus, TLRs
recognize these patterns as dangerous signals and they transmit these “danger
signals” to the innate immune cells of the immune system [21, 34–36]. Binding of
those pattern molecules to TLRs leads to a typical complex formation with each
TLR. Such complexed forms of TLRs are homodimers, heterodimers, and larger
aggregates in cell surfaces. Consequently, TLR-pattern ligand interaction allows its
movement to lipid rafts or microdomain regions [37] with various adaptor proteins.
The adaptor proteins are MyD88, Toll-IL-1R domain-bearing adaptor-inducing
IFN-β, Toll-IL-1R domain-bearing adaptor protein, and/or Toll-IL-1R domain-
bearing adaptor-inducing IFN-related adaptor molecule. During downstream signal-
ing, TLR signaling recruits the adaptor proteins such as IRAK-1, IRAK-4, and
TRAF6. They phosphorylate NF-κB, and the pNF-κB form translocates to the
nucleus [38], resulting in the TLR signaling-mediated increases in specific target
gene and protein expressions. The target candidates are proinflammatory cytokines
such as IL-6/TNF-α/IL-12 and costimulating receptors such as CD80 and CD86
proteins [39–41]. On the other hand, there are also the inhibitory molecules of TLR
signaling, which are single Ig IL-1R-related protein receptor, ST2, inactive kinase
IRAK-M, E3 ubiquitin-protein ligase Triad3, and SOCS1 [42–46].
As a TLR ligand, a small leucine-rich proteoglycan (SLRP), biglycan is normally
arrayed in the extracellular matrix (ECM) [47]. Upon inflammatory response and
tissue damage, soluble biglycan form is proteolytically liberated from the ECM
[48]. Apart from the ECM, biglycan is alternatively synthesized by inflammatory
macrophages, and the liberated soluble biglycan acts as a ligand of the TLR-2 and
TLR-4, as Gram positive bacteria interact with TLR-2 and Gram-negative bacteria
interact with TLR-4 [48, 49]. By binding to TLR2/4 with biglycan, ERK1/2 and
MAPKs-mediated NF-κB activation directs IL-1β and TNF-α synthesis for the
inflammatory reaction. There are two adaptor molecules essential for the TLR
signaling: the MyD88 and TRIF. While signaling through TLR4 involves both
MyD88 and TRIF adaptors, the TLR2 signaling pathway exclusively requires
MyD88 for NF-κB activation [48]. Biglycan induces TLR2/TLR4/MyD88 and
TLR4/TRIF pathways. By selective engagement of TLRs and their adaptor mole-
cules, biglycan tightly regulates inflammatory outcome [49, 50]. The interactions
between biglycan and different receptors orchestrate the recruitment of macrophages

to inflamed tissues.
11.5 TLR-9 as a CpG DNA Receptor
Professor Shizuo Akira is a Japanese scientist who conducted research on microbial

ligands of pattern recognition receptors, such as the identification of TLR-9 as a CpG
DNA receptor. Among the two different CpG DNA types CpG-A and CpG-B,
CpG-B belongs to a type of conventional activator of expression of IL-12 and
TNF-α, which are proinflammatory cytokines. CpG-A strongly stimulates IFN-α
release in pDCs but weakly stimulates IL-12 release [51]. Differentially induced
cytokine production by CpG DNAs is linked to the CpG-A retention in the endo-
some of pDCs, which activates the MyD88-IRF7 signaling for IFN-α elicit
[52]. Likely to TLR7, TLR9 induces autoimmune disorders [53]. Auto-reactive B
cell secretion of rheumatoid factor is derived by IgG2a-chromatin complex by the
BCR and TLR9 [54]. The IgG2a binds and internalizes through the BCR and
chromatin as well as hypomethylated CpG motifs via TLR9 engagement and
consequently produces rheumatoid factors.
TLR-9 involves in innate immune responses elicited by the hemozoin pigment
produced by malaria [55]. During malaria parasites infection within red blood cells,
the parasites degrade hemoglobin proteins of hosts to a hydrophobic form of heme
polymer termed hemozoin (HZ). The generated HZ is released into the blood and
subsequently captured in the reticulo-endothelial system. HZ immunologically acti-
vates the innate immune system to synthesize cytokines, chemokines, and
costimulatory molecules. HZ is a non-DNA ligand for TLR-9 and stimulates an
antimalarial chloroquine (CQ) drug-sensitive, TLR9-mediated, and MyD88-
dependent innate immune activation. Hemozoin functions to traffic DNA into an
intracellular compartment to which TLR9 can be recruited. But, most malarial DNAs
are AT-rich because the malarial genome contains the motif ATTTTTAC over 6000
times. AT-rich DNA seems to mimic malarial DNA. For example, transfection of
AT-rich DNA drives a variety of promoter constructs like native DNA and activates
cytokines including TNFα, IL-1β, IL-6, and IFNβ. Six AT-rich motifs have been
studied (AT1-AT6), where AT-2 has a GCACACATTTTTACTAAAAC. Then, a
question, “can malaria DNA actually stimulate cells (presumably via TLR9)?” is
raised. Then, the question, “is the DNA on hemozoin human or malarial?” was
answered as, “the cytokine inducing component of hemozoin is DNA and PCR
analysis of hemozoin showed that most of its DNA is malarial.” Hemozoin/DNA
rapidly activates IFN-β production from PBMC. Human PBMC produces IFNβ in
response to AT-rich DNA. AT-rich DNA activates type I interferons independently
of TLRs but dependently on IRF1. For inflammation, malarial AT-rich DNA
activates the inflammasome, as the IL-1β roles in causing fever is well-established
and AT-rich motifs may be a major way in which malaria causes fever. How does the
DNA on the surface of hemozoin move from the phagosome to the cytosol?
11.6 TLR-3 as a dsRNA Receptor 617
Hemozoin is internalized into the phagolysosome. TLR9 can be recruited to the

phagosome, thus producing proinflammatory cytokines like TNFα (which causes
fever). Later, its DNA is liberated from the crystal surface by the proteases and other
enzymes. The innovative finding of Shizuo Akira was that hemozoin constitutes the
first non-nucleotide ligand for TLR9” from the results that natural hemozoin acti-
vates cytokine production via TLR9/MyD88; however, the stimulatory activity of
hemozoin was destroyed by DNase. Now the CpG rich DNA is recognized by
TLR-9. The malaria genome contains 269 CpG repeats. Does malaria DNA activate
innate immune responses? Malaria DNA is stimulatory for DCs only when intro-
duced into cells directly with the transfection reagent DOTAP (endosomal compart-
ment). Malaria DNA is also stimulatory for pDCs only when introduced into cells
directly with the transfection reagent DOTAP. TLR9 resides in the ER and TLR9
translocates to the endosomal compartments to bind its ligand (CpG-rich DNA).
11.6 TLR-3 as a dsRNA Receptor
TLRs have emerging roles in control of homeostasis, injury, and wound repair.
TLRs as a family of innate immune receptors bind to microbial PAMPs and elicit
immune responses for antimicrobial activity [56, 57]. The dsRNA is a viral
infection-related molecular pattern. The dsRNA is released from most viruses during
viral replication (Table 11.2) [58]. TLR3 essentially involves in the modulation of
innate immunity against viral infections. TLR3 and its polymorphisms play an
integral role in various viral infections of the nervous system. TLR3 activation
induces both protective and detrimental responses [59].
The dsRNA-sensing receptor, TLR3, has been particularly implicated in such
processes in different tissues such as intestine, skin, and liver, as well as in the
control of reparative mechanisms in the brain, heart, and kidneys, following ische-
mia reperfusion injury [60]. TLR3 interacts with the ribose-phosphate backbone of
dsRNA and has no specific sequence requirements. Given the absence of long
dsRNA under physiological conditions, TLR3 should be inactive in the absence of
an infection. Still, a number of studies proposed recognition of endogenous dsRNA
by TLR3 in the conditions of sterile tissue injured damage, but the specific ligand is
not well defined [61]. TLR3 belongs to a type I protein as an integral membrane form
with the 904 amino acid length. The TLR-3 extracellular region has a horseshoe-
shaped solenoid. The C- and N-terminal regions are linked to a Cys-rich Cap domain
and the concaved surface has N-glycosylation sites with heavy glycosylation on
Table 11.2 PAMP and DAMP ligands of TLR-3

Signal
Immune cell expression PAMPs DAMPs Adaptor Production
Endosomes of B cells, T dsRNA (poly(I:C)) mRNA, TRIF Inflammatory cyto-
cells, NK, and DCs tRNA, siRNA tRNA kines, type 1 IFN
Asn247 and Asn413. LRR12 and LRR20 are nontypical LRR motifs. Ligands are
bound at the glycan-free region in LRR20. The cytoplasmic domain consists of the
linker domain at amino acids 730-756 and TIR domain. Ala795 is a conserved
residue and involved in the binding of TRIF [62].
For the TLR3 signaling and downstream cellular responses, TLR3 interacts
directly with TRIF to initiate signaling. This may relate to the conserved Pro residue
deficiency, present in the BB-loop of other TLRs (Alanine 795 in TLR3). TRIF
signaling induces IFN-β expression via TBK-1 and IRF3, whilst epithelial cell-
specific IRF6 inhibits this response. TRIF also interacts with RIPK1 to drive
NF-κB activation and inflammatory gene expression of IL-6 and TNF-α. RIPK1
also acts as a signaling hub for control of TLR3-dependent cell survival, apoptosis,
and necroptosis [59, 63].
As a dsRNA receptor, TLR3 is classified into the endosome-functional receptor,
as known in cases of TLR7/8, TLR9 and TLR13. The signaling in endosome of
TLR-3 is to recognize the ligand dsRNA and transfer signal by cooperative signaling
molecules of TRIF and TRAF3/6. TLR3 signaling induces expression of
pro-inflammatory cytokines and IFNα/β by stimulation of transcriptional NF-κB
and IRF factors. MBL can calcium-dependently bind TLR-3 ligand, poly (I:C), and
peptidoglycan ligand. From the interaction between MBL and TLR3 ligand, the
interacted complex can lead to phosphorylation of NF-κB p65 with the decreased
phosphorylation of IRF3 and IRF7, although the TLR3 expression was not affected
in monocytes [64]. Because the interaction between MBL and poly (I:C) is calcium-
dependent, EDTA easily blocks the binding. MBL CRD recognizes the ligand poly
(I:C), and the MBL-poly (I:C) binding is inhibited by mannan. For the possibility of
TLR3 interaction with exogenously internalized MBL, the internalized MBL was
associated with TLR3 to colocalize with TLR3 in monocytes or DCs. Thus, it is
speculated that MBL may suppress complement receptor and phagosome-related
TLR3 immune reaction, as complement receptor (CR)-1 acts as MBL receptor
[64]. CR-1 acts as a collaborator of MBL-regulated immune reaction in TLR3
pathway. Experimental disruption of phagosome–lysosome fusion MBL enhanced
poly (I:C)-mediated TNF-α production, while Bafilomycin A1 reversed the effect.
TLR3 signaling is also inhibited by MBL, as MBL trafficks into cells to colocalize
with TLR3. MBL/CR1 interaction and MBL trafficking into phagosomes inhibit the
TLR3 activation.
11.7 TLR-4 as the LPS Receptor
As TLR is expressed in innate immune cell surfaces, they recognize specific

structural motifs expressed on bacteria, virus, or fungi and are activated, therefore
conferring the terminology of PRR family. Ligand-stimulated activation of TLRs
induces secretory releases of pro-inflammatory cytokine or chemokines of innate
immune system to provoke the functional maturation of antigen presenting cells.
Among them, TLR4 mainly recognizes the outer membranes of most Gram-negative
11.7 TLR-4 as the LPS Receptor 619
bacteria, in terms of LPS to regulate inflammation responses [65]. TLR4 is a

transmembrane signaling receptor protein as one of toll-like receptor family.
A gram-negative bacterial cell wall component, LPS interacts with LPS receptor
complex to initiate inflammatory response [66]. As a mammalian receptor for
bacterial LPS, TLR4 plays an essential role in protecting from bacterial infections
and induces abnormal inflammation and sepsis [67]. TLR4 is a member of TLR
family proteins to interact with LPS in mammals. TLR4 as the unique receptor can
stimulate MyD88 receptor and TRIF signaling pathway. This pathway results in
effective gene expression responsible for proinflammatory cytokines, chemokines,
and type I IFN [67]. The LPS receptor complex is comprised of three proteins:
CD14, TLR4, and MD-2. CD14 is a GPI-AP present in leukocytes [68]. CD14 helps
LPS presentation to TLR4 [6]. Binding of LPS with TLR4 activates the biosynthesis
of the proinflammatory cytokines via NF-κB signaling pathway. TLR4 signaling is
downregulated by anti-CD33 mAb, because CD33 positively activates the TLR4
signaling. Therefore, CD14 is suggested to be an endogenous cis ligand for CD33.
The interaction of CD14 with CD33 stimulates the LPS presentation of CD14 to the
cell surface TLR4. Thus, CD33 belongs to the positive modulators of LPS-NF-κB
signaling to regulate TLR4 signaling. TLR4 regulates LPS-elicited NF-κB signaling,
which downregulates inflammatory genes, upregulates anti-inflammatory genes, and
induces the apoptosis of leukocytes [69]. When cells are transfected with human
TLR4 cDNA, NF-kappaB-dependent stimulation is made by LPS and cell-surface
co-receptor CD14 complexes. The activated NF-κB enhances the proinflammatory
cytokine expression for the IL-6/IL-1β/TNF-α/IL-8 cytokines [70]. E5531, an LPS
antagonist, blocks TLR4-mediated signaling [71]. LPS, an endotoxin, embedded in
the Gram-negative bacterial membrane, is a potential inducer of Gram-negative
sepsis. Suppressing aberrant inflammatory responses through TLR4-NF-κB pathway
can prevent acute inflammation and improve the clinical sepsis symptom.
LPS-responsive cells are representatively monocytes and macrophages. LPS recog-
nizes LPS-interacting protein and CD14, a GPI-anchored protein [71]. LPS recog-
nizes serum LPS-binding protein (LBP) and consequently formed LPS-bound form
is complexed with CD14, which is a GPI-AP expressed in macrophages, monocytes,
and neutrophils. TLR4 recognizes heat shock proteins (HSP60, HSP70) as “danger
signals.” ECM components of fibronectins and oligosaccharides stimulate the innate
immune cells through TLR4.
11.7.1 Ligands of TRL4 Recognition
Human TLR4 is a TM receptor protein with 839 amino acids, considting of 624
amino acid extracellular domain, 34 amino acid TM domain, 180 amino acid
intracellular domain. Extracellular domain has 21 LRR to bind LPS [72]. To recog-
nize LPS, TLR4 needs several accessory proteins and is a complex process. LBP is
the soluble shuttle protein in serum and binds to LPS. To stimulate the LPS-CD14
association, LPS is transferred to CD14 that is a soluble form of
LBP
swz {sy[ {sy[
C
D syy syy
1 MD-
4 2
pG {py {py
TIR containing
adapter protein
p G
Fig. 11.5 LPS recognition of TLR4. TLR4/MD-2 complex recognizes LPS through LBP and
CD14 to activate the inflammation response
glycosylphosphatidylinositol-anchored membrane protein and has a strong affinity

toward LPS–LBP complex. CD14 helps to transfer LPS to MD-2 protein, where
MD-2 recognizes TLR4’s extracellular domain. LPS induces the TLR4/MD-2
dimerization (Fig. 11.5). Once LPS-dependent TLR4 oligomerization has occurred,
TIR domain-bearing intracellular adaptor protein is associated to TLR4 and the
recruited protein complex interacts with TLR4’s intracellular domain TIR domain
to induce inflammation response. TLR4’s TIR domain binds to TIR domain-bearing
MyD88/TRAM/TIRAP/TRIF adaptor proteins to regulate signaling. In this event,
MyD88-mediated MyD88-dependent pathway and TRIF-mediated MyD88-inde-
pendent pathway independently take place [65, 73]. TLR4 also acts as an autophagy
sensor upon innate immune response by MyD88- and TRIF-associated signaling
mechanism, as autophagy is also a part of the innate immunity. During
proinflammatory response, Lectin-like oxLDL receptor-1 (LOX-1) and TLR4 are
collaborated in the development of autophagy [74], which degrades long-lived
proteins and dysfunctional organelles. LOX-1 knocks down cells showed the
suppressed TLR4 (Myd88 and TRIF), p-p38 MAPK, and autophagy levels as well
as the inhibited levels of TLR4-related and autophagy-related proteins. LC3-positive
autophagosome was decreased.
11.7 TLR-4 as the LPS Receptor 621
11.7.2 MyD88-Dependent Pathway of TLR4
MyD88 dependent pathway needs MyD88 and TIRAP 2 as adaptor proteins. When
LPS activates TLR4, MyD88 is recruited to TIR domain and activates IRAK-4
kinase. The activated IRAK-4 recruits and activates IRAK-1, and it also activates the
adaptor protein TRAF6, which is crucial for MyD88 pathway. TRAF6 is complexed
with UBC13 and UEV1A and then activates TAK1. Such activated TAK1 regulates
IKK pathway and MAPK pathway. Transcription factor NF-kB regulates IL-12 as
proinflammatory cytokine and immune-related gene. Activated MAPK pathway
stimulates MAPK including ERK, p38, and JNK as well as transcription factor
AP-1 to upregulate proinflammatory cytokine gene expression (Fig. 11.6).
11.7.3 MyD88-Independent Pathway of TLR4
The adaptor proteins TRIF and TRAM are important for MyD88-independent
pathway. TRIF is essential for the activation of transcriptional factor IRF3 and
late-step induction of MAPK and NF-kB signaling [75]. TRIF activated by TLR4
dimerization recruits TRAF3 and TRAF3 induces IRF3’s dimerization through
TANK, TBK1, and IKKi complex as well as nuclear translocation. Activated IRF3
pGG
{py {py
MyD88
TIRAP
IRAK-4
IRAK-1
TRAF6
TAK1
IKK pathway MAPK pathway
NF-kB AP1 wp G

Gj
Fig. 11.6 MyD88-dependent pathway. LPS-induced TLR4 activation recruits MyD88 and
resulting signaling transduction activates NF-kB and AP1 transcriptional factors to induce expres-
sion of proinflammatory cytokine genes
Fig. 11.7 MyD88-

independent pathway. The pG G
activated TRIF activates
transcription factor IRF3 to
induce the type 1 Interferon
gene expression through TRIF
TRAM
TRAF3
TRAF3
TBK1 TANK
IKKi
IRF3 IRF3 { GXGp G
induces the mRNA transcription of targeting genes like type 1 IFN gene (Fig. 11.7).
TRIF’s C-terminal region can bind to RIP1, and RIP1 activates TNF-a mediated
NF-kB signaling, but not the LPS-induced IRF3activation.
11.8 TLR11
The innate immune system is a defense mechanism in organisms against a pathogen

that can produce disease by infecting the host organisms. In this system, many types
of receptors called PRRs encoded at the germ-line recognize the PAMPs, pathogen-
derived molecules not found in the host and activate the immune system by
recruiting phagocytic cells. Various types of PRRs are involved in the innate
immune system and each PRR recognizes a different PAMP as its ligand. Although
all PRRs are essential for host defense mechanisms, the TLR family is the most-
understood receptor in the immune system. TLRs are type I TM proteins with three
distinct domains; ligand binding LRR domain, TM domain, and intracellular signal-
ing TIR domain. TLRs are located on plasma membrane and endosome, and their
location is considered to be closely related to the type of binding ligand. TLRs have
ligand specificities. In TLR family, TLRs 1–13 are found in both mice and humans,
among which TLR11, TLR12, and TLR13 are found only in mice. Several types of
PAMPs are known as ligands that specifically bind to TLRs, such as cell wall
components of bacteria and fungi, lipoproteins, microbial proteins, and bacterial
and viral nucleic acids. In this chapter, a specific interest is in the activation of
signaling pathways when TLR11 (present in mouse only) interacts with its ligands of
microbial proteins (Fig. 11.8).
11.8 TLR11 623
Fig. 11.8 Structure of TLR-11. LRR (Leucine-rich-repeat domain) is an extracellular and

N-terminal 19–25 tandem repeat motif. The horseshoe-shaped ligand binding domain recognizes
PAMP ligands. TIR (Toll/IL-1 receptor domain): As TIRs three subgroups, IL receptors with
extracellular Ig domain in macrophages, monocytes, and DCs bind directly and indirectly to
PAMPs. Adaptor proteins are solely present in the cytosol for TIR1 and 2
11.8.1 Three Major Domains and Binding Ligand of TLR11
TLRs including TLR11 consist of three major structures of domains. The LRR motif
is present in N-terminal extracellular domain and this is required for ligand recog-
nition. The C-terminal and cytoplasmic TIR domain is homologous to IL-1R
signaling domain for intracellular signal transduction. LRR domain has 19-25
tandem repeats conserved LRR motif. TLRs can also be classified by the subtype
of TIRs, the C-terminus signaling domain. TIRs have three subgroups of interleukin
receptors with extracellular Ig domain. The typical TIR type binds directly and
indirectly to PAMPs, consisting of adaptor protein found in the cytosol that mediates
signaling from TIR [26, 76].
Mouse-specifically expressed TLR11 is highly present in epithelial cells in many
organs like intestine, lung, and skin in mice. The role of the TLR11 starts from
recognizing flagellin (FliC) of uropathogenic E. coli, Salmonella, and T. gondii
profilin known as apicomplexan parasite. Flagellin constitutes flagella, which is
present mainly in Gram-negative bacteria of Salmonella. Especially the very con-
served regions of the N, C-terminus of flagellin is recognized by PRR, where acidic
conditions are essential for TLR11 to recognize flagellin. Both N-/C-terminal
regions of TLR11 can interact with FliC (flagellin). Profilin is an actin-recognition
protein found in all the eukaryotes that function in turnover and restructuring of the
actin-cytoskeleton structure essential for cell shape change. Toxoplasma gondii
profilin (TRRF) also acts as a PAMP. Unlike flagellin, profilin recognition can
occur in acidic and neutral conditions. And according to a PLOS ONE 2016 journal,
both N- and C-terminus of TLR11 motif are needed for TRRF recognition, and
especially the Y39, C43 in LRR N-terminal and LRR24-C terminal ECD region are
important.
11.8.2 TLR11 Intracellular Signal Transduction
The activation of TLR signal transduction needs that MyD88 TIR domain recog-
nizes TIR domain of TLR. MyD88 and TLR binding activates downstream kinases
of IL-1 receptor-associated kinases (IRAKs)-1, -2, and - 4. IRAK4 is a first kinase to
form a complex and phosphorylates IRAK1. Ligand binding activates TLR11-TIR
domain to interact with TIR domain in TLR. In order to exert IRAK4 activation and
IRAK1 phosphorylation MyD88 interacts with MyD88 death domain. Phosphory-
lated IRAK1 phosphorylates TRAF6 and IKKs. NF-κb translocation is then
followed to the nucleus and increases the expression levels of pro-inflammatory
cytokines. For activating TLR signal transduction, MyD88 TIR domain interacts
with TIR domain in TLR. MyD88-TLR interaction allows downstream kinases
activation like IRAKs 1, 2, and 4. IRAK4 is a first kinase to form a complex and
phosphorylates IRAK1. After the kinases are activated, they interact with MyD88
death domains activated TRAF6 [77]. Finally, the activation of downstream kinase
cascade phosphorylates inhibitor of NF-κB (IκB) kinases (IKKs) to let IκB release,
allowing NF-κB move into the nuclear region result in increased gene expression of
inflammatory cytokine, provoke pro-inflammatory responses.
The adaptor protein MyD88 plays a decisive role in TLR signaling. MyD88 can
be called as a key adaptor protein. Looking deeply at the structure of the MyD88
protein, it has three main domains encoded by exons. The first domain is a death
domain, which mediates interactions between downstream signal cascade proteins
like IRAK kinase family, the second domain is interdomain, and the last domain is a
TIR domain, which makes an interaction between TLR and MyD88. MyD88 may
act as a negative regulator. If MyD88s–MyD88 form heterodimers, the downstream
molecule IRAK1 still can be recruited, through a death-domain-MyD88s interaction,
but MyD88s inhibits IRAK4 and blocks the phosphorylation of IRAK1. Therefore,
the kinase cascade cannot be activated by receptors and thus MyD88 acts as a
negative regulator in negative-feedback regulation (Fig. 11.9).
Fig. 11.9 MyD88 structure. MyD88 is a key adaptor protein which has three main domains: death
domain (mediates interactions between downstream signal cascade proteins like IRAK kinase
family), interdomain, and TIR domain (makes an interaction between TLR-MyD88). MyD88
functions as a negative modulator. When MyD88s–MyD88 forms, it inhibits IRAK4 and blocks
the phosphorylation of IRAK1 and therefore the kinase cascade cannot be activated
11.9 Inhibition of TLRs by Gangliosides 625
11.9 Inhibition of TLRs by Gangliosides
Some gangliosides are reported to block processing and presentation of antigens

[78, 79], lymphocyte growth [80], Th cell differentiation [81], and mast cell activa-
tion [82]. In addition, gangliosides are also known to inhibit differentiation and
activation of DCs and monocytes upon stimulation with LPS [79, 83–85]. In tumor
microenvironment, the tumor-shed gangliosides are frequently immunosuppressive,
coupling with the fact that some gangliosides inhibit both LPS-induced DCs and
monocytes activation [83, 86]. Tumor cells release immunosuppressive membrane
gangliosides to inhibit normal APC function. During LPS-induced maturation of
DCs, where immature DCs of human peripheral blood monocytes were exposed for
50–72 h to ganglioside G1a and LPS is added to effect maturation, GD1a induces the
LPS-induced maturation of the DCs. Ganglioside GD1a pretreatment of DC inhibits
the allostimulatory capacity and key characteristic of LPS-activated DCs [83]. For
more narrow meaning, some gangliosides are well known to suppress bacterial
flagellin-induced TLR-5 signaling in innate immune cells such as DCs and macro-
phages [87]. Consequently, ganglioside-induced immunosuppression is directly
related to the functional defect of the TLR signaling pathway as a mechanism.
Gangliosides including GM1, GD1a, and GD1b, but not GM3, suppress the TLR
signaling. Those suppressive gangliosides inhibited ligand-induced TLR signaling
activation and TLR-induced proinflammatory cytokine production in human mono-
cytes. For example, such gangliosides elicit immune tolerances to TLR signaling. In
the TAMs, tumor-shed gangliosides in APC PMs potentiate the APC cells to bypass
in the TLR signaling response [88]. In the previous report [88], when human
monocytes were incubated with GM2, the differentiation of DC was inhibited.
However, the adherence and spreading of cells are enhanced and MHC-II and
costimulatory proteins are reduced. In contrast, in the case of GM3, there were no
such effects [89]. In disialyl GD3, it inhibited the LPS-stimulated maturation of bone
marrow-derived DC, while monosialyl GM3 had lesser effects than the disialyl GD3
[90]. For downstream signaling, two different gangliosides of GM1 and GD1a
equally increased the IRAK-M expression in protein and mRNA levels. However,
when monocytes are preincubated with the gangliosides, cytokine releases and
NF-κB activation through nuclear translocation are not induced. However, the
genesis of the endogenous TLR signaling blocker, IRAK-M, is enhanced with the
similar level of expression obtained with LPS treatment [88]. Actually, GM1
enhanced the IRAK-M expression and this effect was quite similar to the effects
obtained with the known TLR ligands. However, the difference was found between
activation of downstream signaling of NF-κB and release of proinflammatory
cytokines. In case of bacterial flagellin, TLR5-flagellin interaction is active upon
presence of gangliosides [87].
Gangliosides expressed by tumors shed into the circulation. Although DCs
generate anti-tumor immune responses, tumor cell gangliosides inhibit DCs function
in the TAM. Ganglioside-enriched myeloid cells inhibits TLR- or IL-1R-induced
pro-inflammation and consequent cellular immunity. For example, tumor-generated
GD2 in neuroblastoma as well as GD1b and GD3 in glioma are prognostic markers.
IFN-γ induces PD-L1 while IFN-γ pro-inflammatory responses are inhibited by
ganglioside enrichment of DCs. Thus, tumor gangliosides immunosuppress and
implicate tumor progression. IFN-γ stimulates antigen-induced immune responses
in the TAM. How such immunosuppression is caused? Ganglioside-elicited DC
dysfunction contributes to downregulation of APC function and TLR-4 function,
and eventually phenotype shift from Th1 T-cells to Th2 phenotype polarization [91].
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Chapter 12
CD33 and CD33-Related Siglecs
in Pathogen Recognition and Endocytosis
of DC in the Innate Immune System
12.1 CD33 (Siglec-3)
12.1.1 General Biology of CD33
CD33 is a myeloid differentiation marker antigen with immune functions associated

with anti-inflammatory signaling, cell adhesion, and endocytosis. CD33 is expressed
by the earliest myeloid progenitors and present during myelomonocytic differenti-
ation. Thus, it is an immune function protein with anti-inflammatory signaling, cell
adhesion, and endocytosis functions with sialic acid-modified proteins or lipids as
ligands. Despite CD33’s importance in hematological cancers, the information is
limited to the CD33 expression. CD33 (Siglec-3) is also a type I TM glycoprotein
with 2 Ig-like extracellular domains [1, 2], as the smallest group of the siglecs with a
67 kDa transmembrane glycoprotein and recognizes both α2,3-SA and α2,6-SAs
[3, 4]. For the protein sequence and structure of Siglec-3, the Ig-like-V-type region is
suggested to be located on amino acid sequence No. 19–135. In the Ig-like-V-type
region, amino acid No. 119, Arginine, is suggested to be the sialic acid-binding site.
The region of amino acids 145–228 consists of Ig-like-C2 type region. The trans-
membrane region is located within amino acids 260–282. ITIM motif is in amino
acids 338–343 region and ITIM-like motif is in 356–361 region, respectively
(Fig. 12.1).
CD33 functions as an endocytic receptor required in the removal of sialyl
antigens or infectious agents having sialylated glycoconjugates. Siglecs specially
bind to SA-containing glycans and also involve in self-recognition. Infectious
pathogens express sialic acids or the sialic acid derivatives to escape the host
phagocytosis on cell walls or cell surfaces, mimicking the face molecules of hosts.
Consequently, the pathogenic sialic acids behave likely to the hosts, interfering with
DC functions. Then, pathogen clearance by phogocytosis is not possible. In this
circumstance, DCs express pathogen-uptaking receptors such as siglecs. The repre-
sentative is CD33 and later many CD33-like siglecs have been isolated, named
632 12 CD33 and CD33-Related Siglecs in Pathogen Recognition and Endocytosis of. . .
Fig. 12.1 Amino acid sequence and structure of Siglec-3. Ig-like-V-type region is located within
amino acids sequence No. 19–135. In the Ig-like-V-type region, amino acid No 119, Arginine, is the
sialic acid-binding site. Amino acids 145–228 constitute the Ig-like-C2 type region. The transmem-
brane region is located within amino acids 260–282. ITIM motif is in amino acids 338–343 region
and ITIM-like motif is in 356–361 region, respectively
Fig. 12.2 Inhibition of proinflammatory cytokines by Siglec-3. Genesis of IL-8, IL-1β, and TNF-α
cytokines is inhibited by CD33 in monocytes
CD33-related siglecs. The merit what host cells express CD33 or its related Siglecs
on the cell surfaces is to help for DCs to internalize and present pathogen’s antigens
(Fig. 12.2).
DCs can identify target microbial patterns, previously known as PAMPs. DCs
can also distinguish bacterial and viral unmethylated CpG DNA, bacterial flagellin,
and/or peptides containing N-formylmethionine residues. As glycan-containing
PAMPs, several molecules such as LPS, GlcNAc, and peptidoglycan are known.
PAMPs are recognized by PRRs expressed by DCs. PRRS are those of scavenger
receptors, Nod-like receptors, CLRs, and TLRs. TLRs contain a family of 12 evolu-
tionarily conserved PRRs consisting of type 1 integral membrane glycoprotein with
recognizing role in the microbial patterns. TLR recognition induces intracellular
signaling, consequent expression of antigen presenting molecules (MHC-II),
co-stimulatory molecules (CD86/80, CD40) as well as inflammatory cytokines and
chemokines. C-type lectins CLRs recognize glycan structure and pathogen-
associated glycans or glycosylated self-antigen. In DCs, DC-SIGN, CD207/
Langerin, or selectin family functionally bind glycan structures expressed by
12.1 CD33 (Siglec-3) 633
Fig. 12.3 Siglec-1 of HI

surfaced DC cells binds with
terminal sialic acid residues V
on surfaced gangliosides or
glycoproteins of HIV and
mediates internalization of
Gangliosides
viral particles. The viral
surface sialic acids are also
used as gateway-receptors Siglec-1
for entrance to CD4+ Helper
Th2 cells Bound
Trasnfered
Tra
Viral glycoproteins
Captured
CD4+
Monocyte- CD4+ T cells

derived DC (Helper)
mammalian cells, and also internalize pathogens without DC maturation. DCs can
also recognize opsonins in opsonized microbes by complement receptors and Fc
receptors. The antigens internalized by hosts are then fragmented or digested to
present to the immune system of hosts, called as APCs [5]. Reversely in pathogens,
using the expressed sialic acids, the pathogens can modulate DCs’ functions and or
rather utilize the DCs as hosts, delivery vehicles, or vectors. For example, HIV
utilizes Siglec-1 as a gateway-receptor for entrance to host cell. Th2 cells [6]
(Fig. 12.3).
In respect to sialic acid-recognizing endocytosis of DC, likely mammalian cells,
DCs are also sialylated. DCs sialylation is for the endocytosis of foreign invaders.
Intestinal bacteria have sialyltransferases for sialyl glycoconjugate biosynthesis and
consequently they acquire masking capacity to escape immune surveillance carried
out by innate immune system. From the sialidase-treated results on
macropinocytosis and phagocytosis in moDCs and bone marrow-derived DCs
(BMDCs) of sialyltransferase KO mice, the biological function of sialic acids have
been demonstrated. In the sialyltransferase KO-BMDCs, DCs lack for
macropinocytosis ability against ovalbumin used as an allergic agent. In contrast,
phagocytosis ability was not changed. The decrease in macropinocytosis ability is
caused by desialylation of the DC cells. Mature DCs have constant abilities of
phagocytosis and receptor-mediated endocytosis, while immature DCs are weak in
the abilities. Thus, the sialic acids of immature DCs are thought to be factors of better
differentiation specialized for the phagocytosis level.
During phagocytosis process, the DC cytoskeleton is arranged to fit the phago-
cytosis level. In the sialidase treatment with DCs, a cytoskeleton disorganization is
observed in DCs. The decreased activities of two Rho GTPases (Rac1 and Cdc42)
function for the actin-filament-linked macropinocytosis and phagocytosis. This

downregulated status means cytoskeleton disorganization and decreased
macropinocytosis. Desialylated DCs treated with sialidase exhibit enhanced level
of phagocytosis against foreign pathogens such as E. coli. Surely, this status is not
related to maturation. The DC phagocytosis ability is dependent on the SA species
produced by bacteria by means of host Siglecs. As a sialic acid ligand, sialylated
CD14 interacts with CD33 and the binding suppresses TLR-4 signaling [7]. Because
the uptake level of LPS is reduced in TLR/CD33-expressing cells, CD14 is an
endogenous ligand for CD33. Binding of CD33 with CD14 modulates the LPS
presentation from CD14 to TLR4, leading to the downregulation of TLR4-mediated
signaling. The sialic acid-modified ligands that activate CD33 signaling is required
to identify, because those Sialyl ligands might target the CD33 reactions and CD33
is a large family of Siglecs. TLR-4 receptor capacity is dependent on the membrane
sialidase activity prior to LPS-induced activation. For example, DCs phagocytosis
level is activated by desialylation of surface receptors, indicating the self sialic acid-
masking of TLR-4. Thus, treatment with sialidases such as Neu1 or Neu3 activates
phagocytosis by macrophages and DCs. Siglecs and TLRs are receptors with both
strong activating- and suppressive-inducing properties in regulating immune
responses after sialidase treatment.
On the other hand, DCs also induce immunological tolerance, through which
CD33 and its related siglecs function as immune tolerance events in DCs [8]. CD33
and CD33-related human Siglecs are specialized to bear the ITIMs in the cytosolic
tail region and the ITIMs are well observed in the siglec forms expressed on
hematopoietic system. The ITIMS are specialized rather for the inhibitory signaling
on stimulation with self antigens, allowing immune tolerance. CD33 functions as an
inhibitory receptor through recruiting the dephosphatases of SHP-1 and -2. How-
ever, it remains largely unknown whether CD33 really acts as an inhibiting receptor
as well as the ligand(s) recognized on their cellular targets. As most Siglecs on
immune cells possess the ITIM sequence in the cytosolic tails [9], CD33 stimulation
by ligands leads to ITIM phosphorylation and this inhibits signaling of receptors
with ITAM. CD33 internalization and breakdown is mediated by the SOCS-3
molecule, as SOCS-3 expression is increased upon inflammation. Clearly, it is
mentioned that CD33 recruits SHP1, SHP2, and SOCS3. SOCS-3 binds to the
activated ITIM domains of CD33 and consequently induce ubiquitination of this
complex [10–12]. It was reported that CD33 levels are decreased in monocytes from
diabetic patients, but SOCS-3 level is increased. The levels of CD33 and SOCS-3 are
similarly observed in high glucose conditioned monocytes culture [10], suggesting
that inflamed monocytes exhibit decreased CD33 level, which is controlled by
SOCS-3. To simply say, CD33 interacts with SOCS3 and consequently, SOCS3
inhibits the effect of CD33 engagement on cytokine-induced proliferation. SOCS3
accelerates proteasomal degradation of CD33. The inflammation activates the mono-
cytes with increased levels of CD33, but proinflammatory IFN-γ does no influence
the CD33 mRNA expression.
For the putative functions of CD33 in inflammatory cytokine synthesis, CD33
effectively blocks the synthesis of IL-1β/TNF-α/IL-8 of proinflammatory cytokines
12.1 CD33 (Siglec-3) 635
of monocytes (Fig. 12.2). However, interruption of SA binding or reduction of cell

surfaced CD33 via CD33-specific RNAi application leads to increasing of p38
MAPK activity, enhancing of cytokine secretion and cytokine-induced cellular
proliferation in monocytes.
12.1.2 CD33 (Siglec-3)-Targeting of Acute Myeloid

Leukemia (AML)
The AML is one of heterogeneous clonal disorders accumulated with

undifferentiated myeloid blasts [13]. As a heterogeneous form of human diseases,
AML is characteristically featured with abnormal occurrence of blastic cells at the
different maturation stages of the BM without regular hematopoiesis. There is no
effective agent to cure acute myeloid leukemia (AML). CD33 is mainly expressed on
myeloid progenitors. Although CD33-recognizing monoclonal antibodies
(e.g. gemtuzumab ozogamicin (GO)) improved AML-therapeutic effects, GO’s
license was withdrawn due to toxicity. Two different anti-CD33 therapies were
made. GO is a humanized MAb against CD33 conjugated to a toxin. Lintuzumab
is a humanized anti-CD33 monoclonal antibody. Although current therapy of AML
uses a combination of cytarabine and anthracyclines, the disease recurrence is
problematic due to leukemia-forming cells or leukemic stem cells, requiring a
novel therapeutic agent. Cell surfaced receptors of leukemic cells have been attracted
for their roles [14, 15]. Among them, the CD33 and CD123 antigens are targets.
CD33 expression is restricted to a certain stage of hematopoietic cells [16], but its
expression is absent in normal hematopoietic stem cells [17–19], suggesting it as a
potential target molecule for effective AML therapy. Then, the immunoconjugate
formed by CD33-reactive antibody gemtuzumab and ozogamicin (GO) was
approved for the phase II trial in the United States [20]. However, commercial
availability of GO was not made due to side effects [21]. Moreover, resistance
mechanisms against multidrugs are frequently detected during response to GO
administration [22, 23].
On the other hand, the leukemic hierarchy is continuously replenished by leuke-
mic stem cells (LSCs) [24]. LSCs are characterized by self-renewal as well as
clonogenic leukemic progenitors [25]. AML LSCs are quiescent or slow-dividing,
whereas clonogenic progenitors are rapidly proliferating [26]. A permanent cure for
AML requires the elimination of LSCs. A therapeutic approach using an antibody
against the adhesion molecule CD44 reduced leukemic repopulation. CD44 is a key
regulator of AML LSCs. CD44 is a highly glycosylated receptor on CSCs’ surface.
Using antibodies that target CD44 is one of the therapeutic approaches against
CSCs. Jin et al. (2006) reported that anti-CD44 antibody eliminated AML CSCs,
and CD44 is required for the maintenance of AML LSCs [27]. CD44 mediates
adhesive cell–cell as well as cell–ECM recognitions through recognition of the main
ligand, hyaluronan, a glycosaminoglycan highly concentrated in the endosteal
region [27]. Other ligands include osteopontin, fibronectin, and selectin, all of which
are involved in cell trafficking and lodgement. Beyond its adhesion function, CD44
can also transduce multiple intracellular signal transduction pathways when ligated
with hyaluronan or specific function-activating mAbs [22]. In vitro binding of CD44
with mAbs inhibit proliferation and stimulate apoptosis [24, 25].
12.1.3 CD33 (Siglec-3)-Targeting Treatment of Alzheimer’s

Disease (AD)
On the other hand, there are alternatively spliced variants in CD33 gene. For
example, the A allele in the single-nucleotide polymorphism (SNP), rs3865444, in
the CD33 gene is linked to the reduced AD level. rs3865444 alleles were identified
with AD pathology. The relationship between CD33 alternative splicing and human
diseases such as dementia and AML is found. SNPs analysis raised the CD33 exon
2 abnormality in neural tissues. From leukocytes isolated from AML patients, a
CD33 splice variant which the CD33 exon 2 was skipped, instead of CD33 intron 1,
was retained [30]. CD33 inhibitors including humanized CD33 antibodies (for
example, lintuzumab) are not effective in AML treatment. Lintuzumab inhibits
CD33 expression in phorbol-ester differentiated U937 cells at 10 ng/ml. Increased
CD33 mRNA was observed in the increased AD pathology in cortex brain. CD33
was largely expressed in the activated microglia [31]. Treatment of microglia with
amyloid-beta peptide or inflammatory agents reduced CD33 mRNA and protein
levels. Increased CD33 expression by microglia reduces Aβ peptide phagocytosis as
microglia lacking CD33 can phagocytize significantly greater amounts of Aβ peptide
than microglia expressing CD33 at normal levels [32]. Therefore, in Alzheimer’s
disease, CD33 antagonists have been suggested to reduce AD risk. Inhibition of
CD33 expression indicates inhibition of abnormal RNA splicing having CD33 splice
variant, and this consequently inhibits the CD33-associated AD risk. It suggests that
anti-CD33 antibody or antagonist functions as the AD-relevant drug.
12.2 CD33-Related Siglecs (CD33rSiglecs)
Primate CD33rSiglecs are divided into two groups: 1) inhibitory Siglecs, which
include certain classes of Siglec-5, -7, 9, and -10, and 2) activating Siglecs, which
include Siglec-14 and Siglec-16. In the regard to the protection from auto-reaction or
self antigens, and also to the immune tolerance to the self-recognition, it is consid-
ered that such CD33-like siglecs are diverse in the aspect of homology. The well-
studied case is CD22, which is expressed on the B-cells [33–35]. In fact, there are
many different CD33-related siglecs in innate immune cells of humans. The
sequence homologies of CD33 and CD33-related Siglecs of humans are well
12.2 CD33-Related Siglecs (CD33rSiglecs) 637
conserved in the proximal and distal regions of polypeptides. CD33-related Siglecs

also activate innate immune cells. For example, human monocytes produce CD33
and Siglec-5, -7, -9, and -10 [36–39]. The most interesting point is that each CD33-
related Siglec is specifically expressed on each hematopoietic cell, indicating a
property of immune cells for differentiation-based specialized CD33-like siglecs.
Siglec-9, a CD33 related Siglec is present in monocytes, neutrophils, NK cell
subsets, B cells, and a certain CD8+ CTLs [39, 40]. Other CD33-related Siglecs
are known to have some restricted expression. Siglec-8 is present in circulating
eosinophils and basophils [41]. Siglec-6 appears in placental cyto-trophoblasts or
syncytio-trophoblasts and B cells [42].
12.2.1 Inhibitory CD33rSiglecs in Escape from Tumor and

Bacterial Immunosurveillance
The inhibitory siglecs involve in the immune responses of hosts. In fact, most
CD33rSiglecs negatively regulate TLR signaling pathway. For example, Siglec-G
is a negative regulator of B1 cells and suppresses inflammatory immune responses
against DAMPs of hosts. The inhibitory CD33rSiglecs group includes Siglec-5, -7,
-9, and -10. This inhibitory receptor group transmits inhibitory signals through
cellular ITIMs or intracellular ITIM-like motifs. Various immune cell stimulations
can be regulated through a Ligand-CD33rSiglec signaling in cancer progression.
Thus, cancer cells can exhibit evasion of the host immunosurveillance by
upregulation of the ligand-recognition. Sialyl ligands can inhibit cancer cell death
by neutrophils even by partial inhibition of CD33rSiglecs. There is some question
that NK cells as immune cells are suppressed by sialylated ligands. CD33rSiglecs are
evolved to keep self-interaction event by self innate immune cells and to escape from
pathogenic infections [43]. In fact, pathogenic GBS coat them with SAs or Siglec-
binding ligands. The coated SAs recognize the Siglec-5 and -9 expressed in myeloid
lineage cells and this binding subsequently supresses the bacterial phagocytosis
[44, 45]. The CD33rSiglecs also downregulate host immune responses against
cancer cells. Because Siglec ligands in cancers are upregulated, the TAG ligands
that interact with inhibitory siglecs of CD33rSiglecs are regarded as immune ther-
apeutic targets for cancers. Metastatic tumor cells synthesize sialylated Siglec
ligands and thus suppress immune cell stimulation [46, 47]. A certain subpopulation
of NK cells produces Siglec-9 and the NK cells-expressed Siglec-9 enables to
evasion of host immune response. Tumor cell-produced ligands of Siglec-9 suppress
the host immune responses elicited by myelo lineage monocytes [47]. Thus, Siglec-
9-positive NK cells or Siglec-9-positive myelomonocytic cells bind to sialylated
tumor-associated ligands in human cancer. Lectin Gal-binding soluble 3 binding
protein (LGALS3BP, named Mac-2 binding protein) was identified from tumor
extracts as a SAs-bearing ligand, specific for Siglec-9 of human. In addition, other
immune-modulatory Siglecs were also found in tumor cells. Representatively, they
are Siglec-5 and -10 [48]. LGALS3BP inhibits activation of neutrophils in a SAs-
and Siglec-specific ways, indicating an immunoinhibitory function for evasion of
tumor cells of host immunity during tumor proliferation and invasion.
Until now, several cancer-associated CD33rSiglecs ligands are known: They are
MUC-1 and MUC-16 [49–53]. Tumor-associated O-glycan mucins bind to Siglec-9.
As Siglec-9-binding non-sialial ligand, VAP-1 is identified by phage display tools
[54]. Similarly, prohibitin-1 and prohibitin-2 also interact SA-independently with
Siglec-9 [55]. Siglec-5 is a predominant form in myeloid lineage macrophages,
monocytes, and neutrophils. Siglec-10 appears in B cells [37]. Siglec-10 functions
with glycosyl-CD52 form and thus inhibits activation of T cells [55]. Siglec-9 is
present in CD8 T cells [56]. Therefore, Siglec-10-binding sialyl ligands can activate
T cells to fight tumors and reduce the antitumoral Th1 immune response. If they are
bound with Siglec-7, the major inhibitory CD33rSiglec on NK cells, they modulate
the antitumor NK cell responseands in vitro [57].
12.2.2 Activating CD33rSiglecs
CD33rSiglecs mostly bear immune receptor tyrosine-based inhibitory motifs and

consequently negatively signal. Exceptional activating CD33rSiglecs are also
known. Activating Siglecs of Siglec-14/5 and Siglec-16/11 are the coupled recep-
tors. Interestingly, new DAP-12-coupled, so-called activating CD33rSiglecs are
discovered. This group includes Siglec-14 and Siglec-16. They are paired with the
inhibitory CD33rSiglec receptors of Siglec-5 and Siglec-11, respectively. This
activating group activates cells via DAP-12 recruitment. The above activating
CD33rSiglecs receptors are evolutionarily generated through, even in partial expla-
nation, exploitation of pathogens by inhibitory siglecs. This mechanism explains the
possibility that the infected hosts combat the pathogens by alternative pathways. The
Siglec-16 CD33rSiglec has a 4-bp deleted sequence in the second exon region,
encoding the first N-terminal Ig-like domain, and therefore, is nonfunctional and
called pseudogene. Interestingly, UK Caucasians have the functional Siglec-16 gene
with a complete ORF [58]. The UK population exhibits about 50% split between the
wild-type allele and the 4-bp deletion allele. Siglecs-11 and -16 share 99% homol-
ogy in their first three extracellular Ig superfamily domains [59]. Siglec-11
CD33rSiglec has neutral amino acids in the TM region as found in TM receptors.
This is compared to Siglec-16 CD33rSiglec. Siglec-16 CD33rSiglec has a positive
amino acid Lys and the Lys residue binds to the ITAM-containing DAP12. Siglec-16
also has a negative amino acid Glu residue around the Lys ( 4 position) of Lys)
[58]. The ITAM located in the cytoplasmic region of DAP12 recruits Tyr kinases
Syk. A difference between mouse CD33 and human CD33 is the presence of charged
amino acids such as Lys residue in the TM domain of mouse CD33. The mouse
CD33 can recruit DAP12 as an adaptor, although the related downstream inflam-
matory responses are not clearly evidenced.
12.3 Pathogenic Suppression of the Pathogen-Specific Host Immune Response 639
The first evolved molecule is the SA species in animals, and some pathogens
acquire SAs via multiple pathways. Alternatively, activating Siglecs are generated in
order to combat pathogenic microbes via inhibitory Siglecs of hosts and dampening
of innate immune responses [58]. However, the undesired overactivation of the
immune response is harmful. Activating Siglecs recognize the same ligand as the
counterpart inhibitory Siglecs, which are generated by the deselection of the newly
formed activating siglecs. For example, Siglec-11 is a neuroprotector because it is an
inhibitor of pro-inflammatory mediator synthesis including IL-1β and NOS-2 as well
as it is also a phagocytosis inhibitor in brain microglia. Brain Siglec-16 CD33rSiglec
engagement needs the same Siglec-11 CD33rSiglec ligand and triggers undesired
responses. Besides Siglec-16 CD33rSiglec, other three Siglecs interact with DAP12.
They have charged amino acid residues in the TM domains. They are human Siglec-
14 CD33rSiglec, human and mouse Siglec-15 CD33rSiglec and rodent Siglec-H
CD33rSiglec. Siglec-15 CD33rSiglec is distinct in the meaning of its wide distribu-
tion from mammals to fish. Rodent Siglec-H CD33rSiglec expressed on pDCs is a
biomarker for pDCs. Siglec-H CD33rSiglec has a TM Lys residue and recruits
DAP12. However, Siglec-H CD33rSiglec does not recognize SAs and inhibit
IFN-α synthesis.
12.3 Pathogenic Suppression of the Pathogen-Specific Host

Immune Response
Pathogens and hosts have been evolved to survive and defeat each other over the
host’s immune system. Macrophage activation is prerequisite in defense and protec-
tion against pathogenic agents such as bacterial or virus but regulation is required to
prevent tissue damage in hosts. Activated macrophages respond to infection and
injury for phagocytosis and inflammation in killing and tissue repair. However,
uncontrolled host responses provoke inflammatory damages. Pathogen PRRs
include TLRs and NOD-like receptors to mediate “innate immune activation” of
macrophage after recognition of microbial PAMPs or endogenous “danger” DAMPs
[60]. Phagocytic PRRs include scavenger receptors (SRs) and CLRs. They activate
innate immunity by cooperation with TLRs and NLRs as well as by direct macro-
phage regulation for adhesion and migration. In addition to innate activation, Th-1
and Th-2-derived cytokines IL-4 and IFN-γ induce distinct “classical” [61] and
“alternative” [62] macrophage activation, respectively. Pathogens desire to suppress
the pathogen-targeting host immune response. Conversely, the host immune
response is to prevent and remove pathogens. Immune cells express receptors of
TLRs and nucleotide-binding oligomerization domain-like receptors to bind and
respond to pathogens, inducing antivirulence genes and inflammatory mediators.
Simultaneously, the cells express inhibitory receptors to limit the amplitude of the
over-response. As a protective measure against tissue damage, host macrophages
adaptively modify chromatin to allow them to become unresponsive to repetitive or
persistent signaling by TLRs (TLR4 and LPS tolerance) [c], decreasing

proinflammatory signaling. Certain antipathogen molecules, however, are not damp-
ened after prolonged TLR signaling because chromatin modification allows
antipathogen genes to remain responsive to TLR4 in the presence of ongoing
infection [63]. Pathogens also employ various strategies to engage downregulatory
mechanisms to suppress host defenses.
Uncontrolled regulation of PRRs, IFN-γ, and IL-4 pathways is involved in
diverse inflammatory disorders. Therefore, the concept of self-protective host regu-
lation limits undesired and excessive inflammation, called “negative regulators of
inflammation” [64]. Many inhibitory receptors are reported in myeloid cells
[65]. The inhibitory receptors limit the amplitude of proinflammatory responses
[9]. Inhibitory receptors are targeted by pathogens. From the structure of the
extracellular domains, the inhibitory receptor superfamily has two major classes of
i) the Ig superfamily and ii) the calcium-dependent carbohydrate-binding (C-type)
lectin family. They have affiliated activating receptors, which contain charged amino
acids in the TM region, denoted by a plus sign. Inhibitory receptors include
CD200R, leukocyte inhibitory receptor (LIR), paired Ig-like receptor (PIR), and
signal-regulatory protein (SIRP).
How pathogens regulate CD200 expression is unclear. However, constitutive
CD200 expression is regulated by the transcription factor CCAAT/EBPβ)
[66]. Three enhancer sites (cis-elements) upstream of the CD200 transcriptional
start site, a NF-κB binding site, an IFNγ-activation site (GAS), and an
IFN-stimulatory response element-2 induce CD200 expression. NF-κB, STAT1,
and IFN regulator factor-1 bind to these enhancer elements, respectively. Further-
more, the NF-κB c-Rel subunit upregulates TLR-induced CD200 synthesis via
interaction with TLRs [67]. Perhaps pathogens utilize these enhancer sites and
transcription factors to induce CD200 expression following TLR recognition.
CD200 is also a target of p53 and is upregulated on apoptotic cells to decrease
responsiveness to self-antigen. Inducible expression of CD200R1 is regulated by
C/EBPβ [68]. Microglial cells exhibit a dramatic decrease in CD200R1 mRNA and
protein expression following stimulation with LPS, a TLR4 ligand. C/EBPβ directly
binds to the CD200R1 promoter to inhibit expression in LPS-treated cells. Further-
more, it was found that histone deacetylase 1 interacts with C/EBPβ to downregulate
CD200R1 expression.
12.3.1 Inhibitory Receptor CD200R and CD200:CD200R1

Signaling
CD200 and its receptor CD200R is one of the well-characterized immune inhibitory
ligand-receptor pairs [69, 70]. CD200 and CD200R are both cell-surface transmem-
brane glycoproteins consisting of two Ig superfamily domains. CD200 expression is
observed in the cell surfaces of neurons, endothelial, epithelial, fibroblastic,
lymphoid, and astrocytic cells [71, 72]. The regulation of CD200R1 signaling can
occur via PTM, namely, Tyr phosphorylation in the cytoplasmic tail region of
CD200R1 or the induction of downregulation of either CD200R1 or CD200 expres-
sion. Each of these mechanisms can ultimately be exploited by pathogens. CD200 is
expressed on many cell types, whereas CD200R is restricted in leukocytes and
myeloid cells. Interaction between CD200 and CD200R in Ig domains transduces
an inhibitory signal to the myeloid cells through CD200R that has three tyrosine
residues. CD200 KO or CD200R KO mice show increased susceptibility to autoim-
mune and inflammatory diseases [73, 74]. Thus, CD200–CD200R interaction
induces myeloid-specific inhibitory function. Chronic autoimmune diseases driven
by antigen-specific lymphocytes, lymphocyte hypersensitivity, and hyperactivated
Mϕ subsets-dominated inflammation can be therapeutically applicable using the
CD200–CD200R interaction-mediated myeloid-specific inhibition of inflammation
or autoimmune diseases.
Although most inhibitory receptors contain an ITIM in their cytoplasmic region
to recruit adaptor proteins upon activation, CD200R does not consist of an ITIM.
CD200R1 cytoplasmic region contains three tyrosine residues for adaptor protein
interactions upon phosphorylation. This feature is unlike most immune inhibitory
receptors. Instead, human CD200R1 consists of three Tyr residues, Y291, Y294, and
Y302 (Y286, Y289, and Y297 in the mouse) in the cytoplasmic region. Y302/Y297
is found in a phospho-Tyr binding (PTB) domain recognition motif (NPxY). Stim-
ulation by CD200 leads to these Tyr residues phosphorylation by Src kinases, which
recruit the tyrosine kinase (Dok) 2 adaptor proteins through its PTB domain
[75]. Y302/Y297 and Y291/Y286 residues are the main Tyr residues for
CD200R1 associated with Dok2 [76]. Dok2 is the main initiator of signaling through
CD200R1, beginning with recognition of Ras-GTPase activating protein (RasGAP)
and is required for CD200R1 function. This is completely different from the ITIM
inhibitory receptors, which recruit SHPs and SHIP-1, which are the initiator proteins
and Dok proteins as second modulators [76].
Pathogens have evolved to exploit the CD200:CD200R1 signaling pathway by
altering expression of either CD200 or CD200R1, or by expressing a CD200 mimic
to engage the host CD200R1. The greatest threat to the host is the excessive
inflammation in response to the infectious organism. In these cases, the disruption
of the CD200:CD200R1 axis leads to the death of the host. CD200:CD200 receptor
(CD200R) interaction contributes to host immunosuppression and autoimmunity
inhibition. Simply to say, CD200:CD200R1 inhibitory signaling pathway limits
various inflammations. Due to the regulatory role played by CD200R parasitic,
bacterial, and viral pathogens exploit suppressive pathway against host defenses.
CD200R present in the myeloid cell surfaces is an immune inhibitory ligand-
receptor pair. CD200R1can also be induced in certain T-cell subsets
[71]. CD200R1 interacts with CD200 to downregulate myeloid cell functions.
12.3.2 Pathogenic Decoy Ligands Neutralize Host Immunity

Through Eliciting Host CD200-CD200R1 Inhibitory
Signaling
Pathogens use host inhibitory receptors to modulate inflammatory responses. Infec-

tious pathogens such as virus and bacteria and hosts have evolved to modulate
receptor signaling. Certain pathogen proteins bind to many inhibitory receptors and
consequently distinguish self from non-self. From such regulations, they avoid
recognition of the host surveillance and escape from host immunity, towards acqui-
sition of persistence in the host. CD200R1 is an inhibitory receptor of Ig superfam-
ily. CD200 is a ligand of CD200R family. CD200R1 has seven glycosylated regions.
Three of them have GlcNAc. CD200 has eight glycosylated regions with GlcNAc.
CD200R1 does not bear ITI, instead has three Tyr residues at Y291, Y294, and Y302
in humans in its cytoplasmic tail, while mice have Y286, Y289, and Y297. Among
them, Y302 is found within a phosphor-Tyr binding domain recognition motif
(NPxY). CD200 is also a member of Ig superfamily. When CD200 binds to
CD200R1, Src kinase phosphorylates the three Tyr residues. Downstream of Tyr
kinase-2 (Dok2) adaptor protein binds to the phosphor-Tyr residues of Y302 and
Y291, and then binds to CD200R1 through phosphor-Tyr binding domain. Then,
Ras-GTPase activating protein (RasGAP) binds to Dok2, contributing to ras inacti-
vation and inhibition of myeloid cell functions such as cytokine and Ca2+ release.
How CD200R regulates inflammatory responses in myeloid cells and T-cells?
Inhibitory receptor multifunction and proinflammatory signaling are con-functional,
although CD200R has no ITIM, but an alternative domain in the cytoplasmic tail
modulates anti- or proinflammatory signals [77]. From this property, CD200-specific
antibodies can block CD200R signaling [78], allowing clinical trials cancer
(ClinicalTrials.gov identifier: NCT00648739). Interestingly, the antibody drug can
also prevent pathogenic infections via CD200-CD200R signaling. For viral thera-
peutic targeting of viral CD200 orthologs, viral clearance is obtained by blocking the
signaling through targeting the host CD200 or CD200R. This virus-specific targeting
method would allow normal host immune response regulation, avoiding potential
immunopathology and blocking the viral replication. This CD200-CD200R axis is
also applicable to transplantation tolerance [79]. However, it is thought that
disrupting this relationship in posttransplantation or immunosuppressed patients
raises short-term benefit if viral infections occur, as viral infections lead clinically
to failures in renal transplantation patients.
Therefore, CD200–CD200R1 binding elicits immunosuppression. Certain path-
ogens utilize CD200–CD200R1 binding to suppress host immune response. Thus,
pathogens evade the host immune response through host regulation of CD200,
CD200R1, or CD200 ortholog expression. In fact, bacteria and parasites utilize
CD200–CD200R1 inhibitory signaling for their survivals through CD200 and
CD200R1 modulation. For example, Toxoplasma gondii induces CD200R1 level
on microglia and CD200 level on endothelial cells. Leishmania amazonensis and
N. meningitidis also increase CD200 level on macrophages. Pathogens influence
transcriptional enhancer region of CD200 gene. Mouse hepatitis corona virus

(MHV) also increase CD200–CD200R1 signaling for its vital replication, survival,
and mortality. However, influenza virus increased pathogenesis and mortality in
CD200 KO mice. Compared to CD200 KO mice, CD200R1 KO mice exhibit
decreased viral pathogenesis during influenza infection. Many viral orthologs of
CD200 including vOX2 of HHV8, R15 of RRV, and M141 of Myxoma virus are
known.
Viruses are known to be commonly related with transplantation tolerance, as in
the cases of CMV, HSV-1, HHV8, Epstein–Barr, Varicella Zoster, BK, and PC
viruses [80]. In the example that certain viruses have the CD200 orthologs similar to
other inhibitory receptors. Then, such viruses directly use the inhibitory signaling of
the CD200–CD200R recognition to acquire the survival from the host. Herpesvi-
ruses and poxviruses are representative cases to incorporate or evolve orthologs of
the host CD200. Herpesviruses and poxviruses are exceptionally organized to avoid
or interrupt host immune responses. From the viral pathogenic genomes evolved to
utilize CD200 and CD200R expression as well as the CD200 mimics, the CD200–
CD200R reaction is the primary position for the host–pathogen interactions and
pathogen survival. How the pathogens use the CD200–CD200R axis to undergo host
defenses? The answers may give is clues in the therapeutic designation [77]. The
K14 gene of Kaposi’s sarcoma-associated herpesvirus and human herpesvirus
8 (HHV8)is well characterized, encoding a viral ortholog CD200 (vOX2). The
vOX2 is 36–40% homologous to human CD200 and found in the lytic phase on
the virus-infected cell surfaces [81]. vOX2 and CD200 bind to CD200R
[81, 82]. vOX2 decreases the production of TNFα, GCSF, and monocyte
chemoattractant protein-1 from IFNγ and LPS-stimulated macrophages [81]. The
CD200 and vOX2 function in the APCs (native HLA-A2 and HLA-B8-positive) to
express either CD200 or vOX2 suppressed T-cell IFNγ secretion, ERK1/2 and AKT
phosphorylation, and CD107a mobilization. CD200 and vOX2 induce the
homeostastic antigen-targeting T-cell responses in vivo by negatively regulating
their activity likely to CTLA-4 and PDL-1/2 [82]. Human herpesviruses 6 and
7 also express CD200 orthologs to bind human CD200R1 [83]. Human CD200
and HHV8 also inhibit basophil property. Basophils highly express CD200R in
human peripheral blood via FcεR1 signaling, as demonstrated by the results that
CD11b expression and histamine release are suppressed through cross-linking of
CD200R-human CD200 or vOX2 [83]. This specifies for CD200 inhibitory func-
tion. Rhesus rhadino virus (RRV) is a HHV8-like γ-herpesvirus. RRV seemingly
expresses a viral CD200 protein, R15, and this is present on the infected cell surfaces
to release into the medium [84]. Like the other viral CD200 orthologs, R15 decreases
TNFα expression from PMA-activated THP-1 macrophages as well as primary
rhesus monocytes/macrophages, to human CD200 levels. The myxoma virus
CD200 ortholog M141 is also the type with a single Ig-like domain similar to the
human CD200 in the N-terminal region [85] and inhibits macrophage and lympho-
cyte function to propagate viral replication [85, 86]. Rat CMV expresses an e127
CD200-like protein. With 50% high identity to the host CD200 protein, e127 binds
to CD200R [87]. In summary, pathogenic CD200 mimics provide an evolutionary

advantage to a variety of pathogens.
12.3.2.1 Murine Cytomegalovirus (MCMV)
Murine cytomegalovirus (MCMV) m157 protein is a structural homolog of MHC

class I proteins, and it recognizes the inhibitory receptor Ly49I in MCMV-infectious
mice (or susceptible strain) to prevent NK-mediated killing [88]. MCMV m144 is
also a MHC class I mimic required for viral replication in vivo [89]. The mouse Ly49
family proteins expressed on NK cells recognize the α1 and α2 subunits of H-2D
MHC-I molecules. Interestingly, non-infectious MCMV mouse strains (or resistant
strain) express the activating receptor Ly49H, affordable binding to m157 and NK
killing of the infected cells [90].
12.3.2.2 Human Cytomegalovirus (HCMV)
Human cytomegalovirus (HCMV) protein UL18 is a homolog of MHC class I

antigens [91]. Both UL18 and mouse m144 form the three α domains similar to
MHC class I and bind to β2M [m]. UL18 binds to both CD94/NKG2 and leukocyte
inhibitory receptor (LIR)-1. m144 also seems to interact similarly. The receptor
CD94/NKG2 binds to the nonclassical MHC-I human HLA-E and mouse Qa1
[92]. Human LIR-1 recognizes epitopes through interactions with the α3 and β2M
domains [93], as most MHC class I recognize the same epitope. In fact, LIR-1 binds
to UL18 more tightly than host class I molecules, indicating that this receptor has
evolved specifically to bind to UL18. Furthermore, the UL40 protein of HCMV is
homogenous to the MHC-I, HLA-E associated peptide HLA-Cw03. CD94/NKG2A
binds to HCMV-infected cells likely to the HLA-E-like peptide presentation and
does not kill them [94].
12.3.2.3 Rat Cytomegalovirus (RCMV)
The rat cytomegalovirus (RCMV)-produced CTL-like gene (rctl) is an early gene

and the structure is closely homologous to the mouse- and rat CTL-related protein
Clr-b [95]. The inhibitory receptor NKR-P1B on NK cells recognizes Clr-b as a
ligand, expressed on hematopoietic cells [95]. RCMV infection rapidly induces
RCTL expression to counteract Clr-b downregulation by host cells. Through a
non-MHC class I recognition mechanism, RCTL inhibits NK cell-mediated lysis
by directly interacting with NKR-P1B. Furthermore, RCTL-negative virus shows
lower virulence with rapid clearance from the host by NK cells. Interestingly, the
activation receptor NKR-P1A also recognizes RCTL. This indicates that host
defenses have evolved to counteract the RCTL function to escape the host
recognition.
12.3.2.4 Poxvirus
Poxviruses express a CD47 mimic [96], and the mimic proteins interact with signal-
regulatory protein (SIRP)α to undermine myeloid cell functions. SIRPα present in
myeloid cells and neurons [97] binds to CD47 to induce leukocyte chemotaxis and
proinflammatory cytokine production [96]. The myxoma virus CD47 mimic,
M128L, has a lethal infection in rabbits by controlling of macrophage activation
and recruitment [96]. The activating receptor SIRPβ does not bind to a ligand CD47,
but has evolved to counteract pathogen infections and recognize pathogen-infected
cells [98].
12.3.2.5 Coronaviruses
Coronavirus infections require a functional CD200: CD200R1 signaling interaction

to limit type I IFN production. CD200R1-silenced signaling in CD200 KO mice
increases in inflammatory signaling, specifically in type-I IFN signaling during
binding to TLR7-specific ligands in mouse hepatitis corona virus (MHV). MHV
causes the severe acute respiratory syndrome coronavirus. CD200 KO mice
exhibited decreased viral replication and viral amplification [99]. Levels of IFNα
was increased in MHC-infected CD200 KO mice.
12.3.2.6 Influenza Virus
CD200 KO mice are highly susceptible to the inflammation in response to pulmo-

nary influenza A infection, showing weight loss and mortality upon influenza A
infection rather than WT mice, although viral clearance was similar in both WT and
KO mice. CD200 KO mice upregulate NO production in lung and proteins such as
IL-6, TNFα, IFNγ, and macrophage inflammatory protein 1α. Administration of
CD200-Fc or anti-CD200R1 agonist reverses the phenotype of CD200 KO mice
[100]. In WT mice, alveolar macrophages show increased expression of CD200R1
for suppression of inflammatory responses to the virus. The role of the CD200:
CD200R1 axis protects the host. Influenza-infected CD200R1 KO mice are resistant
to bacterial infection with decreased pathogenesis and mortality than WT mice
following S. pneumoniae superinfection [101]. CD200R1 KO mice exhibit
decreased viral pathogenesis and pathology in response to influenza infection. The
increased inflammatory response seen in CD200R1 KO mice provides protection to
the host in terms of a bacterial superinfection.
12.3.2.7 Herpesviruses
Herpes simplex virus (HSV)-1-mediated keratitis (stromal keratitis) is a chronic

infection that causes an influx of CD200R1-expressing cells into the cornea, leading
to inflammatory lesions and blindness [102]. Various cell types such as myeloid cells
upregulate CD200R1 on their surface following ocular HSV-1 infection. CD200-Fc
treatment of ocular HSV-1-infected mice caused decreased CD11b + immune cells
in the cornea, decreased inflammatory lesions, and decreased angiogenesis. These
mice exhibited decreased IFNγ-expressing T-cells and increased FoxP3+ Tregs both
in lymphoid tissue and the cornea. CD200:CD200R1 signaling modulates inflam-
mation during a viral infection, decreasing the inflammatory milieu following a viral
infection for unwanted immunopathology. In the role of the CD200:CD200R1 axis
in the mouse model of HSV-1 encephalitis [102], significant morbidity and mortality
are from cytokines and chemokines releases by the interaction of HSV-1 with
macrophages and resident microglial cells through TLR2 [103]. Therefore, there
was increased morbidity and mortality in CD200R1 KO mice upon HSV-1 infection.
However, CD200R1 KO mice are resistant from infection and decrease in viral titers
and HSV-1 glycoprotein expression in the brain. In the interaction of HSV-1 with
thioglycollate-induced peritoneal macrophages, relationship between CD200R1 and
cell signaling by TLR2 is seen.
12.3.2.8 Bacterial Decoy Ligands
Certain bacteria, S. aureus and E. coli, can bind to the mouse paired Ig-like receptors
(PIRs) of PIR-B and PIR-A1 as well as human LIR-1 to suppress macrophage
proinflammatory responses [104]. The murine PIRs are structural homologs of the
human LIRs and bind MHC-I molecules [105]. The PIRs are expressed in macro-
phages, DCs, mast cells, and B cells.
12.3.2.9 Neisseria Meningitidis
During infection with the Gram-negative N. meningitidis, CD200 is induced but

CD200R is downregulated in macrophages, depending on Neisserial LPS, TLR-4,
and MyD88 pathways, not depending on a Neisserial receptor of SR-A. CD200 KO
mice are susceptible to bacterial infection with Neisseria meningitidis rather than
WT mice. CD200 KO mice had higher systemic IL-6 and TNFα levels, higher
numbers of F4/80 + CD11b + macrophages, and expressed higher levels of MHC
class II molecules on macrophages [106]. Among the host PRRs, NOD2 and
NACHT-LRR protein 3 (NALP3) also induce CD200. Furthermore, CD200 expres-
sion is upregulated in bone marrow macrophages following infection with
N. meningitidis. CD200 KO mice show lethal phenotype upon experimental menin-
gococcal septicemia, proinflammatory cytokines induction, and activated leukocytes
recruitment [106]. Thus, this is due to recognition of Neisserial LPS by TLR4, since
TLR ligation can increase CD200 surface expression in macrophages. CD200
expression is increased by TLR-, NOD2-, and NALP3-signalins to protect the host
from excessive inflammation. In WT mice, CD200:CD200R1 signaling involves in
regulating the response to N. meningitidis but does not necessarily affect the survival
of the pathogen. Therefore, increased mortality in this model is mediated by
uncontrolled inflammation, not uncontrolled pathogen replication.
12.3.2.10 Toxoplasma gondii
In WT mice, Toxoplasma gondii increases surface expression of CD200R1 and

CD200 in microglia and vascular endothelial cells, respectively. In CD200 KO mice,
microglial cells were increased in proliferation and activation with higher expression
of MHC II, TNFα, and iNOS during infection in chronic T. gondii encephalitis.
CD200 KO mice also exhibited decreased parasite burden and decreased mortality
compared to WT mice following chronic infection. This is due to CD200 KO mice-
increased inflammatory phenotype in response to the TLR ligands, including IL-6
and TNFα release and IκBα phosphorylation [72]. T. gondii stimulation of mouse
TLR-11 induces IL-12, which is key for the survival of the host [107]. TLR-2 and -4
are also implicated in the inflammatory response to T. gondii [108]. Thus, in the case
of Toxoplasmosis, increased inflammatory responses through TLR signaling are
detrimental to the pathogen.
12.3.2.11 Leishmania
Leishmania amazonensis, not all this species, causes severe disease in both humans
and mice, via induction of CD200 mRNA and protein expression in bone marrow
macrophages. CD200 is essential for replication and development of systemic
Leishmaniasis as L. amazonensis replication and virulence are significantly
decreased in CD200 KO mice. Virulence of L. amazonensis can be restored by
soluble CD200-Fc treatment. However, CD200-Fc treatment in L. major-infected
WT mice shifts its virulence to that of L. amazonensis [33]. L. amazonensis has
evolved to utilize CD200 expression as a mechanism for inhibiting both NO
production and induction of iNOS during infection. This was confirmed by treatment
of macrophages with an iNOS inhibitor, leading to increased replication of L. major.
Interestingly, L. amazonensis increased CD200 expression on macrophages. Mac-
rophages express CD200R1, which can then interact with nonmyeloid cells
expressing CD200. In the case of L. amazonensis, macrophages can inhibit neigh-
boring macrophages by expressing both CD200R1 and CD200. Macrophages
infected with intracellular pathogens can release exosomes, small vesicles
containing various membrane proteins, which can provide signals to naiïve macro-
phages [109]. These exosomes contain CD200, which can then bind to CD200R1 on
nearby macrophages. Alternatively, macrophages expressing CD200 may interact

with activated T-cells expressing CD200R1.
12.3.2.12 Schistosomes and Salmonella
Both CD200 and CD200R1 are upregulated and coexpressed in chronically acti-
vated CD4 T-cells from mice infected with Schistosoma mansoni and Salmonella
enterica. These cells also lost the ability to generate TNFα and exhibited increased
IL-4 secretion. Furthermore, in patients chronically infected with Schistosoma
haematobium, there was a correlation between CD200R1 expression and parasite
load and almost all IL-4 secreting CD4 T-cells were CD200R1 positive. This
suggests that chronic infections lead to increased expression of CD200 and
CD200R1 and subsequently a decrease in antipathogenic mediators, allowing path-
ogen persistence.
12.4 DCs Tumor Immunotherapy Through Sialyl Binding

of DCs to T Cells
The immunotherapy using the DCs is limited in its clinical aspect due to the
complexed DC property. Various DCs known in the skin tissues are subcategorized
into Langerhans cells (LC) in epidermis, mDCs, and pDCs. The TAMs inhibit the
activity of mDCs, whereas LCs exhibit potent immune stimulating activities. The
ultimate function of DCs is the DCs-T cells interaction and then presenting antigen
information. Immunological synapses involve glycoprotein receptor-mediated pro-
cess. DCs’ sialoglycoconjugates negatively influence T cell priming, and DCs’
sialoglycans interfere with MHC-2 antigen presentation and co-stimulation. This
fact is elucidated through sialidase-treated moDCs, which successfully prime T cells
and also induce proliferation. Endogenous sialidase in moDCs also promotes cyto-
kine production. The level of sialidase Neu3 expression is increased in the process of
DC differentiation. Reversely, tolerogenic and immature DCs express high level of
sialic acid production. Because induction of host tolerance in DCs is mediated by
Siglecs, DCs sialylation has capacity to interact with the T cells, inducing the
immunological/tolerogenic balance. DCs also identify specific tumor cells and
present the information of cancer antigens to T cells.
By recognition and binding to tumor-specific antigen (TSA) and exclusive tumor-
associated antigens (TAAs), DCs undergo their maturation. In the side of tumors,
they also create tolerance-inducing and immunosuppressant microenvironments as
well as secrete inhibitory factors against DCs function. As a key way to meet the
above tumor capacities, aberrant glycosylation as a hallmark of cancer cells is
known. Tumor cells with glycans can be shed into the host environment or normal
body fluids. Tumor-associated carbohydrates (TAC) aggressively help tumor cells to
12.4 DCs Tumor Immunotherapy Through Sialyl Binding of DCs to T Cells 649
Fig. 12.4 Sialic acids Isolation of PBMC from human

modulate DC functions and In vitro Differentiation by cytokines
sialic acid-containing Monocyte-derived DC
glycans cell adhesion during Pulsed exposure to tumor-associated antigens such as peptides
migration and homing as and glycans
well as tumor cell and Activated and primed Monocyte-derived DC
microbial recognition
Injection of DC to the patient
invade metastasize or evade immune system. Thus, the immune events such as
eliciting immune tolerance, T cell deletion, anergy and Treg activation are caused
by sialic acids of DCs. CLRs, macrophage Gal lectins-1 (MGL-1), and DC-SIGN
recognize tumor or induce tolerance. Sialic acids deliver intracellular inhibitory
signals from their ITIMs in DCs, keeping the tolerance state.
Sialic acids prevent differentiation and maturation and increase anti-inflammatory
cytokine and decrease proinflammatory cytokines. DCs become tolerogenic after
recognition of TAC (Fig. 12.4). Roles in DCs are important for better understanding
of the carcinoma and cancer immunotherapy [110]. For the present understanding of
DCs in tumor therapy, development of cytokine-driven expanding and differentiat-
ing of DCs ex vivo is important. DCs derived from precursor cells isolated and
extracted from cancer patients are also fundamental. Nevertheless, intranodal injec-
tion is regarded as a crucial technology, required for equipment and experience
(Fig. 12.4). Ex vivo upload of DCs with antigen and vaccination of cancer patients
with DCs loaded with tumor antigens are considered. Using specific antibodies
targeting DC endocytic receptors, receptor–ligand interaction can be blocked, and
targets of DNA vaccines encoding for antigen are also considered. At present, DCs
immunotherapy is experimentally applicable for the treatment of cutaneous carci-
noma [111]. For example, human patient DCs are routinely isolated and also derived
from peripheral blood CD14+ monocytes as monocyte-derived DC. Those isolated
patient’s monocytes can be incubated ex vivo with cytokines to differentiate into
immature mo-DCs. Those activated cells from mDCs are differentiated to matured
DCs by coculture with stimulating agents or factors. Before maturation, mo-DCs are
loaded with specific antigens and administered to the same patient and regression of
tumors are anticipated.
The immune functions of DCs allow cancer immunotherapy as ideal candidates in
an antigen-specific manner and attempts to eradicate tumors have been tried through
the body’s own innate immunities [111]. For example, in order to stimulate imma-
ture DCs such as human PBMCs, sialic acid derivatives can be used as the stimulator
in DCs immunotherapy. For example, because of the complexed characteristics DCs
in skin cancer patients, different DCs lineages of epidermal Langerhans cells (LC),
dermal mDC and pDC were isolated from the skin. In the skin tumor microenviron-
ment, mDCs are functionally suppressed, but LCs have still immunity. Thus,
promising trials for skin cancer treatment have been made using LCs for the DCs
immunotherapy. DCs tumor presentation is an essential step in the generation of
antitumor immunity. The problem is the poor antigen presentation of tumor cells
themselves, as discussed previously. Accordingly, various strategies of DCs
vaccination have been designed so far for tumor-specific effector T cell responses. If
it is successful, tumor cell mass will be reduced, generating immunological memory
and consequently eliminating tumor cell propagation in bodies [112]. However,
nevertheless the current development of diverse technologies to improve the DCs
immune therapeutic potentials, there is not scientifically explanable approaches to
DC vaccination with the effective treatment of human neoplasms. Then, in order to
better understand the DCs biology for tumor vaccination with effective immuno-
therapies, surfaced glycan-linked glycoimmunological approaches should be made.
Moreover, to enhance and strengthen an in depth glycan function of DCs
glycobiology, and to highlight the various therapeutic strategies, DCs
glycoimmunotherapy is required. To overcome the current limit in DCs therapy, a
stimulative strategy of human PBMCs has been tried by treatment of the sialic acid
precursor, N-propanoylmannosamine (ManNProp) [113], since PBMCs metabolize
ManNProp to N-propanoylneuraminic acid. PBMCs stimulated with ManNProp
resulted in an increased growth and expression of activation markers of CD25 and
CD71. Furthermore, PBMC secreted IL-2 upon activation with ManNProp. Since
sialic acid is a major constituent of lymphocyte cell surface components [114],
treatment of lymphocytes with ManNProp exhibited efficient regression tumors.
Such trials using sialic acid precursors were made by Bertozzi group. They intro-
duced functional groups in the N-acyl side chain of N-acyl-D-mannosamine, syn-
thesizing sialic acid derivatives [115]. Also, Yarema group introduced butyrate
residues to increase the uptake of N-acyl-D-mannosamines [116].
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先天免疫学糖生物学

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Cheorl-Ho

ISBN 978-981-16-9080-8 ISBN 978-981-16-9081-5 (eBook)

1 Repertoire in Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2.2.2 DCs Induce Tolerance State . . . . . . . . . . . . . . . . . . . 44

4 Glycans in Glycoimmunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

4.10.1 sLex-PSGL-1 Glycans in DC Trafﬁcking . . . . . . . . . . 154

5.6 Pathogenic Glycans to Trigger Innate Immune Enhancement . . . 238

7.2 Sialic Acid Recognition by Siglecs for Self-

7.12 Siglec-15, Non-CD33-Related Siglecs in Humans . . . . . . . . . . 370

8.4.1 Expression and Ligand-Binding Speciﬁcity

9.1.5 Galectins in Lower Organisms such as Zebraﬁsh

11.5 TLR-9 as a CpG DNA Receptor . . . . . . . . . . . . . . . . . . . . . . . 616

AhR Aryl hydrocarbon receptor

CRD Carbohydrate recognition domain

MMP Matrix metalloproteinase

ST3Gal-1 β-Gal α2,3-SA-transferase 1

1.1 Historical Expansion of Defense System

1.2 Columbus Era to Modern Revolution in Immunological

The Columbian Exchange includes the multiple inﬂuences through exchanges of

1.3 Historical Proﬁle of Defense Constituents and Progress

Johann August Ephraim Goeze (1731–1793) is recognized to be the ﬁrst researcher

In 1843, Gabriel Andral (1797–1878) and William Addison (1802–1881) described

Fig. 1.1 Johann August

Fig. 1.3 Florence Rena

In the concept of neutrophils, James Gerald Hirsch (1922–1987), an American

Fig. 1.4 James Gerald

1.3.5 Monocytes and Macrophages

Macrophage was initially identiﬁed as immune cells to phagocytosize infectious

Fig. 1.7 Siamon Gordon.

Fig. 1.8 Ralph Steinman.

1.3.6 Dendritic Cells

Fig. 1.9 Bernard Babior.

Fig. 1.10 David Lambeth.

1.3.7 Complement System

Complement is a thioester-containing protein (TEPs including C3 and C4), which is

Fig. 1.11 Jules Bordet. The

1.3.7.1 Alternative Complement System

1.3.7.2 Lectin Pathway

hepatic uptake protein of glycoproteins with terminally glycosylated

For opsonic phenomenon, Almroth Wright (1861–1947), Sir Almroth Edward

Fig. 1.13 Almroth Wright.

1.3.9 Lysozyme and Salvarsan

For lysozyme, Alexander Fleming (1881–1955), later respected to Sir Alexander

Fig. 1.14 Alexander

Fig. 1.15 Sahachiro Hata.

modern chemotherapeutic agents [76]. He was a prominent Japanese young scientist

1.3.10 Progress in Innate Immune Response Since Historic

In influenza virus, Spanish flu (1918–1919) was pandemic, and an influenza pan-

in modern history. Hong Kong ﬂu (1968–1969) caused by H3N2 inﬂuenza virus

Fig. 1.16 Christiane

Fig. 1.17 Jules Hoffmann.

appearance of the receptors, Jules Hoffmann, born in Echternach, Luxemburg, as a

1.4 The Outline of Innate Immunity

The concept of neutrophils, leukocytes, dendrites, monocytes, and macrophages has

Fig. 1.18 Antibody-mediated events of innate immune cells in mammals

1.4.1 Concept of Immune Receptors

Innate immune responses are displayed by distinct receptors, frequently deﬁned

PAMPs Example of PAMPs

As a ﬁrst step, metazoan organisms recognize microbial PAMP structures. CTLs

Tyrosine-based motif for

Fig 1.21 The representative C-type lectins (CTLs) in humans

hemocyte encapsulation. CfLec-1 from C. farreri opsonizes with hemocytes against

Pathogen (Vibrio anguillarum)

C-type lectin (CfLec)

s i

B) N/O glycans drawn on a membrane protein and GSLs 1)N[ECPU

GRCTCPUWNHCVG -GTCVCPUWNHCVG &GTOCVCPUWNHCVG [CNWTQPCP