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先天免疫学糖生物学
先天免疫学糖生物学
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)
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore
Pte Ltd. 2022
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Contents
v
vi Contents
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
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.
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
1.3.1 Phagocytosis
1.3.2 Leukocytes
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.
1.3 Historical Profile of Defense Constituents and Progress in Innate Immune. . . 7
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
1.3.3 Neutrophils
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. 1.5 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].
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.
1.3 Historical Profile of Defense Constituents and Progress in Innate Immune. . . 11
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
12 1 Repertoire in Innate Immunity
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.
1.3 Historical Profile of Defense Constituents and Progress in Innate Immune. . . 13
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].
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
1.3 Historical Profile of Defense Constituents and Progress in Innate Immune. . . 15
[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].
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
16 1 Repertoire in Innate Immunity
complex and characterized its enzyme, and with the natural serum C1 esterase
inhibitor.
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
1.3 Historical Profile of Defense Constituents and Progress in Innate Immune. . . 17
1.3.8 Opsonization
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.
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
1.3 Historical Profile of Defense Constituents and Progress in Innate Immune. . . 21
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.
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
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.
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
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
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)
Tandem repeat
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].
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].
1.4 The Outline of Innate Immunity 29
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.
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
mTOR pathway
LC3
fusion
MHC-II compartment
(B) Endosome mediated autophagy in DCs
/Autolysosome
Bacteria
LPS binding to TLR receptor
Selecve autophagy
MHC-II compartment
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Chapter 2
Dendritic Cells (DCs) in Innate Immunity
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 37
C.-H. Kim, Glycobiology of Innate Immunology,
https://doi.org/10.1007/978-981-16-9081-5_2
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].
40 2 Dendritic Cells (DCs) in Innate Immunity
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+
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
42 2 Dendritic Cells (DCs) in Innate Immunity
Complement-binding protein
like domain
wG
EGF domain
j lG Lectin domain
jG
sTzG uG
lTzG
wTzG
l
h
p
h
p
p
a z
a z
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.
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].
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
2.2 Classification and Different Function of DCs 45
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.
2.2.3.1 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.
46 2 Dendritic Cells (DCs) in Innate Immunity
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
2.2 Classification and Different Function of DCs 47
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
48 2 Dendritic Cells (DCs) in Innate Immunity
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
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].
References 49
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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.
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 53
C.-H. Kim, Glycobiology of Innate Immunology,
https://doi.org/10.1007/978-981-16-9081-5_3
54 3 Glycan Biosynthesis in Eukaryotes
A) Monosaccahrides
B) Different N-glycan structures in different organisms
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
56 3 Glycan Biosynthesis in Eukaryotes
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
58 3 Glycan Biosynthesis in Eukaryotes
GSLs
High Hybrid Complex N/O Core 1 Core 2 Core 3 Core 4 GM3 GM2 GM1 GD3 GD2
mannose type
%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
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].
60 3 Glycan Biosynthesis in Eukaryotes
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
62 3 Glycan Biosynthesis in Eukaryotes
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.
3.6.1 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
66 3 Glycan Biosynthesis in Eukaryotes
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
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
68 3 Glycan Biosynthesis in Eukaryotes
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].
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
70 3 Glycan Biosynthesis in Eukaryotes
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,
3.8 Glycosaminoglycans (GAGs) 71
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
72 3 Glycan Biosynthesis in Eukaryotes
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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
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].
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
74 3 Glycan Biosynthesis in Eukaryotes
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 Glycosaminoglycans (GAGs) 75
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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
Cytosol
SQV-/-
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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
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.
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
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.
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.
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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].
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].
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
3.8 Glycosaminoglycans (GAGs) 87
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].
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
88 3 Glycan Biosynthesis in Eukaryotes
3.8.8.1 Aggrecan
3.8.8.2 Perlecan
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.
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Fig. 3.8 Basic components and structure of GPI anchor. (a) Organic components in GPI bridge. (b)
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
3.9 Glycosylphosphatidylinositols (GPIs) Anchor Glycosylation 91
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
92 3 Glycan Biosynthesis in Eukaryotes
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
3.9 Glycosylphosphatidylinositols (GPIs) Anchor Glycosylation 93
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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
94 3 Glycan Biosynthesis in Eukaryotes
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Fig. 3.10 A proposed mechanism on how CD52 carbohydrates recognize the proinflammatory B
box of HMGB1 for Siglec-10 engagement and functions of human T cells. https://en.wikipedia.org/
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.
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
96 3 Glycan Biosynthesis in Eukaryotes
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].
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
98 3 Glycan Biosynthesis in Eukaryotes
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?
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
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
102 3 Glycan Biosynthesis in Eukaryotes
proinflammatory cytokine expression. Parasite hemozoin binds DNA and the com-
plex recognizes TLR9.
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Chapter 4
Glycans in Glycoimmunology
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
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 115
C.-H. Kim, Glycobiology of Innate Immunology,
https://doi.org/10.1007/978-981-16-9081-5_4
116 4 Glycans in Glycoimmunology
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
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].
118 4 Glycans in Glycoimmunology
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
120 4 Glycans in Glycoimmunology
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
122 4 Glycans in Glycoimmunology
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.
124 4 Glycans in Glycoimmunology
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
4.5 Glycans Regulate T Cells 125
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
4.5 Glycans Regulate T Cells 127
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].
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.
4.5 Glycans Regulate T Cells 129
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
Galectin-1 LacNAc T cell apoptosis
Galectin-3 LacNAc T cell apoptosis
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
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].
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.
138 4 Glycans in Glycoimmunology
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
4.7 Glycan Regulation of NK Cell Receptors 139
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.
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.
4.7 Glycan Regulation of NK Cell Receptors 141
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
142 4 Glycans in Glycoimmunology
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.
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
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.
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
4.8 Carbohydrate Recognition of Target Antigens by DCs During Infection and. . . 147
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. 4.3 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
148 4 Glycans in Glycoimmunology
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
Fig. 4.4 Structures and synthetic enzymes of sialyl Lewisa (type I) and sialyl Lewisx (type II)
determinants. (a) Schematic structure. (b) Synthetic enzymes
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
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
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
Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→R
Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→R
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
NeuNAcα2→3Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→R
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
NeuNAcα2→3Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→R
NeuNAcα2→3Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→R
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
4.8 Carbohydrate Recognition of Target Antigens by DCs During Infection and. . . 151
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
152 4 Glycans in Glycoimmunology
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.
154 4 Glycans in Glycoimmunology
SLeA SLeX
SiaD2,3GalE1,4GlcNAcE1,3Gal1-R
SiaD2 ,3GalE1,3GlcNAcE1,3Gal1-R
&%
FucD1,4 FucD1,3
%JGOQCVVTCEVCPVU
Sialyl
LeX,A
%JGOQMKPG
%QORNGOGPV
2#( .6$
HQTO[NRGRVKFG
.WOGP
Fig. 4.7 Common trafficking molecules during DC recruitment during general inflammation and
migration step
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.”
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.
156 4 Glycans in Glycoimmunology
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
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
4.11.1 Chemokine
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].
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
160 4 Glycans in Glycoimmunology
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.
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.
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
4.11 Chemokine Receptors in DC Trafficking 163
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
164 4 Glycans in Glycoimmunology
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
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
166 4 Glycans in Glycoimmunology
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
D (-)
TNF-D
TNF-D (+)
Fig. 4.11 Tumor cell adhesion to endothelial cells stimulated with TNF-α
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
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 4 Core 2
Asn
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
4.12 Glycan Structure-Recognizing Selectins in DC-Endothelium Interaction. . . 169
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.
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.
4.12 Glycan Structure-Recognizing Selectins in DC-Endothelium Interaction. . . 171
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
172 4 Glycans in Glycoimmunology
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].
The developed GMI-1070 is a pan selectin antagonist. GMI-1070 has been synthe-
sized through convergent steps and large scale-up chemistry development
4.12 Glycan Structure-Recognizing Selectins in DC-Endothelium Interaction. . . 173
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
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
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 199
C.-H. Kim, Glycobiology of Innate Immunology,
https://doi.org/10.1007/978-981-16-9081-5_5
200 5 Pathogen-Host Infection Via Glycan Recognition and Interaction
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
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
202 5 Pathogen-Host Infection Via Glycan Recognition and Interaction
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)
204 5 Pathogen-Host Infection Via Glycan Recognition and Interaction
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
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).
206 5 Pathogen-Host Infection Via Glycan Recognition and Interaction
Table 5.7 Fungal SA-specific lectins. a Also binds GlcNAc. b Phytopathogenic fungus
Species Lectin SA-binding specificity
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
6KIJVDCTTKGTRTQVGEVUCICKPUVOKETQDKCNRCVJQIGPU
)GPGTCNTQWVGUQHKPHGEVKQP
6TCPUFHOOXODUW\SH 2CTCFHOOXODUW\SH
5CNOQPGNNC5JKIGNNC.KUVGTKC;GTUKPKC
8EJQNGTCG%LGLWPK2CGTWIKPQUC
Fig. 5.2 Microbial pathogens infect host cells through tight barrier interaction
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].
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
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
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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
5.3 How Do Hosts Interact with Pathogens? 213
① WZY-dependent pathway
② ABC transporter-dependent pathway
③ Synthase-dependent pathway
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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]
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
5.3 How Do Hosts Interact with Pathogens? 215
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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
Klebsiella pneumoniae
Lipopolysaccharide
(LPS; O antigen)
CPS Pathogenicity factor ugd atf
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Lipid chain - Fucose gnd Wzy (magA)
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Kdo wcaH wzb
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P44/42 hBD3
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MKP-1
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
5.3 How Do Hosts Interact with Pathogens? 217
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].
218 5 Pathogen-Host Infection Via Glycan Recognition and Interaction
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
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N-acetylglucosamine C3
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V2 Ig-like domain
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ITIM IPCNKPI
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cell surface protein
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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
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
Species Lectin SA-binding specificity
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
220 5 Pathogen-Host Infection Via Glycan Recognition and Interaction
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 Pathogen-Producing Lectins as Receptors to Bind to the Host Carbohydrates 221
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
222 5 Pathogen-Host Infection Via Glycan Recognition and Interaction
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].
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
224 5 Pathogen-Host Infection Via Glycan Recognition and Interaction
RBC α-N-Acetylgalatosidase
A
Agglutination Abolished
NO AGGLUTINATION
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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
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
5.4 Pathogen-Producing Lectins as Receptors to Bind to the Host Carbohydrates 227
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.
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
228 5 Pathogen-Host Infection Via Glycan Recognition and Interaction
[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.
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 Pathogen-Producing Lectins as Receptors to Bind to the Host Carbohydrates 229
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,
230 5 Pathogen-Host Infection Via Glycan Recognition and Interaction
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
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
232 5 Pathogen-Host Infection Via Glycan Recognition and Interaction
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
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
238 5 Pathogen-Host Infection Via Glycan Recognition and Interaction
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.
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
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 261
C.-H. Kim, Glycobiology of Innate Immunology,
https://doi.org/10.1007/978-981-16-9081-5_6
262 6 Innate Immunity Via Glycan-Binding Lectin Receptors
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.
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
264 6 Innate Immunity Via Glycan-Binding Lectin Receptors
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,
266 6 Innate Immunity Via Glycan-Binding Lectin Receptors
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].
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.
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
270 6 Innate Immunity Via Glycan-Binding Lectin Receptors
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
6.4 How Are Pathogenic Bacteria Recognized by Receptors of DCs of the Host. . . 273
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.
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).
274 6 Innate Immunity Via Glycan-Binding Lectin Receptors
6UF 3DN
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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].
276 6 Innate Immunity Via Glycan-Binding Lectin Receptors
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
6.4 How Are Pathogenic Bacteria Recognized by Receptors of DCs of the Host. . . 277
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].
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.
6.4 How Are Pathogenic Bacteria Recognized by Receptors of DCs of the Host. . . 279
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
6.4 How Are Pathogenic Bacteria Recognized by Receptors of DCs of the Host. . . 281
Protein
Gal Man P EtN
Fig. 6.3 The Plasmodium parasite of malaria protozoa is coated with a GPI anchor
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.
282 6 Innate Immunity Via Glycan-Binding Lectin Receptors
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.
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
284 6 Innate Immunity Via Glycan-Binding Lectin Receptors
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.
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-
286 6 Innate Immunity Via Glycan-Binding Lectin Receptors
Plasmodium
invasion Human
falciparum
(Neu5Gc-)
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
288 6 Innate Immunity Via Glycan-Binding Lectin Receptors
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
6.4 How Are Pathogenic Bacteria Recognized by Receptors of DCs of the Host. . . 289
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
6.4 How Are Pathogenic Bacteria Recognized by Receptors of DCs of the Host. . . 291
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.
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
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
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
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before leaving from the bone marrow. Host SAs can be regarded as “immunosup-
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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.
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 311
C.-H. Kim, Glycobiology of Innate Immunology,
https://doi.org/10.1007/978-981-16-9081-5_7
312 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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].
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
314 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
vo vo
ov v
_ jvvT
α YS_T
$ hou v
vo
ov _
ov v
jvvT
hou v vo
ov _
ov v
jvvT
hou
ov
v
pnG
w TzGh
hTG
mT Gy
wG
oG
w GzV{
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
ujht jht
w
z
h
l sS S
u
h
z
juz
5KINGE
1+
9VHWLPPXQRJOREXOLQGRPDLQ
6LDOLFDFLGELQGLQJVLWH
6.4
&VHWLPPXQRJOREXOLQGRPDLQ
+6#/
2
+6+/ 2
5*2
5*2
,7,0PRWLI7\URVLQHNLQDVHWDUJHWVLWH
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
318 7 Sialic Acid-Binding Ig-Like Lectins (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
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 - - - - )
CD33
Basic amino acid residue in transmembrane domain
0DFUR
˞VLDOLFDFLGV
1HX$F
α2-6
2 6HU7KU *OF1$F
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)
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].
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
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
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
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
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].
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
328 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
OLJDQG OLJDQG
$FWLYDWRU ,QKLELWRU
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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 Structure of Siglecs 329
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
330 7 Sialic Acid-Binding Ig-Like Lectins (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
(continued)
Table 7.2 (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
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)
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 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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].
338 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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
342 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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.
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,
7.9 Siglec-1 (CD169, Sialoadhesin/Sn) 345
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,
346 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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
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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
348 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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].
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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
352 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
Left: Right
Adjacent cells
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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
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3 3 3 3 3 3
3 3 3 3
3 3 3 3 3 3
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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
354 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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.
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
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
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
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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].
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
362 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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.
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
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.
364 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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.
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
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.
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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
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).
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].
374 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
ITIM like
COOH
376 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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
7.13 Siglec-3 (CD33)-Related Siglecs on DCs 377
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
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].
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.
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].
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
7.13 Siglec-3 (CD33)-Related Siglecs on DCs 383
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
384 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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
7.13 Siglec-3 (CD33)-Related Siglecs on DCs 385
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.
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
386 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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
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
388 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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
392 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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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
7.13 Siglec-3 (CD33)-Related Siglecs on DCs 393
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.
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
394 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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
7.13 Siglec-3 (CD33)-Related Siglecs on DCs 395
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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 Siglec-3 (CD33)-Related Siglecs on DCs 397
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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
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
+
SIGLEC8LSL eoCre SIGLEC8Eo (Express human Siglec-8)
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
6S
α3 β4 6’-S-Siayl-LacNAc Yes Yes
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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
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].
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].
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
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Fig. 7.32 Presumptive mechanism of CD200–CD200R1 interaction between immune cells and related cells. CD200–CD200R1 recognition and the CD200R
7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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]
7.13 Siglec-3 (CD33)-Related Siglecs on DCs 405
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
406 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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
7.13 Siglec-3 (CD33)-Related Siglecs on DCs 407
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.
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
410 7 Sialic Acid-Binding Ig-Like Lectins (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
412 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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,
7.13 Siglec-3 (CD33)-Related Siglecs on DCs 413
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).
414 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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].
Siglec-9
V-set
Ig domain Sig-E16
C-set
Sig E
Sig 16 : activating
ITIM
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
7.13 Siglec-3 (CD33)-Related Siglecs on DCs 417
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)
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
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.
5KCNKECEKFU
2CVJQIGP
5KINGE
Siglec-9 bind to sialic acids on pathogen
inhibit neutrophil activity
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]
0'6U
Innate immune
HQTOCVKQP
system ↑
Neutrophil
Siglec-9
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)
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-
7.13 Siglec-3 (CD33)-Related Siglecs on DCs 425
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.
426 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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
7.13 Siglec-3 (CD33)-Related Siglecs on DCs 427
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].
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
7.13 Siglec-3 (CD33)-Related Siglecs on DCs 429
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
430 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
%&(E
%&(E
TCR %&(E
Siglec-10
Cell membrane
2 ITIM motif
2
5*2 2
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
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.
432 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
40# 5GNHCPVKIGPU
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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–
7.13 Siglec-3 (CD33)-Related Siglecs on DCs 435
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.
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
438 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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.
K-capsule(D-2,8 glycan)
E.coli K1
E.coli K1
6LJOHF 6LJOHF
ITIM
ITAM
DAP12
,PPXQH5HVSRQVH
0$3.SDWKZD\
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
440 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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-
7.13 Siglec-3 (CD33)-Related Siglecs on DCs 441
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
Acvaon Inhibion
INFLAMMATION
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
444 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
cytosol
Monocyte
ITAM ITIM
Acvaon Inhibion
INFLAMMATION
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-
446 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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].
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
448 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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.
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
450 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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
7.14 Mouse CD33-Related Siglecs with ITIM-Like Domains 451
[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.
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
7.14 Mouse CD33-Related Siglecs with ITIM-Like Domains 453
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
7.14 Mouse CD33-Related Siglecs with ITIM-Like Domains 455
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.
7.14 Mouse CD33-Related Siglecs with ITIM-Like Domains 457
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,
458 7 Sialic Acid-Binding Ig-Like Lectins (Siglecs)
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Chapter 8
C-Type Lectin (C-Type Lectin Receptor)
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
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 497
C.-H. Kim, Glycobiology of Innate Immunology,
https://doi.org/10.1007/978-981-16-9081-5_8
498 8 C-Type Lectin (C-Type Lectin Receptor)
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.
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
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
502 8 C-Type Lectin (C-Type Lectin Receptor)
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
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.
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.
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.
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
508 8 C-Type Lectin (C-Type Lectin Receptor)
Glucosyl
residue
Acyl group
Glucosyl-diacylglycerol in
S.pneumoniae
Fatty acid chain
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,
8.4 Macrophage Inducible CTLR (Mincle, Clec4e, ClecSf9)/Macrophage CTL (MCL,. . . 509
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.
510 8 C-Type Lectin (C-Type Lectin Receptor)
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.
Extracellular regions
0DQQRVHUHFHSWRU&'
&5
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&\VWHLQHULFKGRPDLQ&5UHFRJQLVHVVXOSKDWHG
FDUERK\GUDWHVWHUPLQDWHGLQ62*DO RU62
*DO1$F7U\
)LEURQHFWLQW\SH,,GRPDLQ)&,,UHFRJQLVHVQDWLYHDQG
&7/'
GHQDWXUHGFROODJHQV,WELQGVFROODJHQ,,,,,,,9DQG9
&\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
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].
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.
516 8 C-Type Lectin (C-Type Lectin Receptor)
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 Mannose (or Mannan)-Binding Protein (MBP) and Mannose-Binding Lectin (MBL) 517
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
518 8 C-Type Lectin (C-Type Lectin Receptor)
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,
8.6 Mannose (or Mannan)-Binding Protein (MBP) and Mannose-Binding Lectin (MBL) 519
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
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
520 8 C-Type Lectin (C-Type Lectin Receptor)
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].
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.
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-
8.7 Fucose-Binding Lectin (FBL) and Ficolin 523
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].
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
8.7 Fucose-Binding Lectin (FBL) and Ficolin 525
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.
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
528 8 C-Type Lectin (C-Type Lectin Receptor)
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
530 8 C-Type Lectin (C-Type Lectin Receptor)
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.
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,
8.8 Dectin 1 (CLEC-7A in Human) 531
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.
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
532 8 C-Type Lectin (C-Type Lectin Receptor)
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].
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.
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
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
538 8 C-Type Lectin (C-Type Lectin Receptor)
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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XM. E3 ubiquitin ligase Cbl-b negatively regulates C-type lectin receptor-mediated antifungal
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Chapter 9
Galectins
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 557
C.-H. Kim, Glycobiology of Innate Immunology,
https://doi.org/10.1007/978-981-16-9081-5_9
558 9 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.
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
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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]
C type galectin: 2 CRDs fused with tandem repeat of Pro- and Gly-rich stretches. Galectin-4, 6, 8, 9 and 12.
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
β-galactosides
jG
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
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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
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
9.1 General and Structural Aspects of Galectins 565
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.
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
9.3.1 Galectin-3
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].
9.4.1 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
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
[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
9.4 Galectin-1 and -4 Bind to GM1, But Not GM3 573
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
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
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
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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.
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 585
C.-H. Kim, Glycobiology of Innate Immunology,
https://doi.org/10.1007/978-981-16-9081-5_10
586 10 DC-SIGNs
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).
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
Neck domain
Transmembrane domain
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
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
10.1 DC-Specific ICAM-3-Grabbing Non-integrin, DC-SIGNB (CD209) 589
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).
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
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 DC-Specific ICAM-3-Grabbing Non-integrin, DC-SIGNB (CD209) 591
Sialyllactose
HIV HIV
Ceramide
Ceramide
Ceramide
DCs
Viral glycoprotein (gp120)
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
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.
kunyTX
S S
ITAM
ITAM
P P
Syk
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).
10.2 Other DCs-Derived Receptors 595
Necrotic cell
F-actin
DNGR-1
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
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,
10.2 Other DCs-Derived Receptors 597
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
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
jsljTXYh
Inflammation
Uric acid crystal (MSU)
CLEC-12A
Reactive oxygen
O2
species
NADPH
oxidase
ITAM
ITIM
Src family kinase + P
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].
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
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 607
C.-H. Kim, Glycobiology of Innate Immunology,
https://doi.org/10.1007/978-981-16-9081-5_11
608 11 Toll-Like Receptors (TLRs)
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
j G{pyG
610 11 Toll-Like Receptors (TLRs)
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
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.
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.
614 11 Toll-Like Receptors (TLRs)
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)
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).
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
membrane
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
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.
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
620 11 Toll-Like Receptors (TLRs)
LBP
swz {sy[ {sy[
C
D syy syy
1 MD-
4 2
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
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).
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
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
622 11 Toll-Like Receptors (TLRs)
TRAF3
TBK1 TANK
IKKi
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
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.
624 11 Toll-Like Receptors (TLRs)
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
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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85. Shurin GV, Aalamian M, Pirtskhalaishvili G, Bykovskaia S, Huland E, Huland H, Shurin
MR. Human prostate cancer blocks the generation of dendritic cells from CD34+ hematopoietic
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effect on flagellin binding to toll-like receptor 5. J Biol Chem. 2005;280:9482–8.
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Chapter 12
CD33 and CD33-Related Siglecs
in Pathogen Recognition and Endocytosis
of DC in the Innate Immune System
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 631
C.-H. Kim, Glycobiology of Innate Immunology,
https://doi.org/10.1007/978-981-16-9081-5_12
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
Viral glycoproteins
Captured
CD4+
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)
634 12 CD33 and CD33-Related Siglecs in Pathogen Recognition and Endocytosis of. . .
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].
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.
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
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
638 12 CD33 and CD33-Related Siglecs in Pathogen Recognition and Endocytosis of. . .
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].
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.
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
640 12 CD33 and CD33-Related Siglecs in Pathogen Recognition and Endocytosis of. . .
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,
12.3 Pathogenic Suppression of the Pathogen-Specific Host Immune Response 641
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.
642 12 CD33 and CD33-Related Siglecs in Pathogen Recognition and Endocytosis of. . .
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
12.3.2.7 Herpesviruses
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.
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.
12.3.2.11 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
648 12 CD33 and CD33-Related Siglecs in Pathogen Recognition and Endocytosis of. . .
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.
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
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
650 12 CD33 and CD33-Related Siglecs in Pathogen Recognition and Endocytosis of. . .
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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