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Mammalian glycosylation in immunity

Jamey D. Marth and Prabhjit K. Grewal Abstract | Glycosylation produces a diverse and abundant repertoire of , which are collectively known as the glycome. Glycans are one of the four fundamental macromolecular components of all cells, and are highly regulated in the . Their diversity reflects their multiple biological functions that encompass ligands for proteinaceous receptors known as . Since the discovery that and their ligands are important for the regulation of leukocyte trafficking, it has been shown that additional features of the immune system are also controlled by endogenous cellular glycosylation. This Review focuses on the emerging immunological roles of the mammalian glycome.

Glycome Glycosylation is the enzymatic process that produces that is formed by Ogt, secretory glycosylation in the The biological repertoire of glycosidic linkages of saccharides to other saccharides, ER and Golgi produces a large structural repertoire, glycan structures. and lipids, and is probably as ancient as life including oligomeric glycan linkages that are presented itself. Unicellular and multicellular organisms depend at the surface and in extracellular compartments. on glycosylation to produce monomeric and multimeric Intracellular glycosylation has been reviewed elsewhere glycan linkages that are essential for cell viability and and is not discussed further in this Review12. normal function1–4. The resulting glycome encompasses a Glycosylation can substantially modify the structure diverse and abundant repertoire of glycans, which are one and function of proteins by steric influences involving of the four fundamental macromolecular com ponents of intermolecular and intramolecular interactions and all cells (together with nucleic acids, proteins and lipids) can mediate the production of glycan ligands for lectins (FIG. 1). Glycans have important biological functions (FIG. 2). Lectins are proteins with glycan-binding activ- in maturation and turnover, and ity that were first described in plants but subsequently trafficking, and binding and activation5–8. have been found in all cells, from microorganisms to Glycosylation is prominent in the lumen of the endo- humans13–15. –glycan binding is a means of molec- plasmic reticulum (ER) and in the . The ular recognition, which organisms can use to identify cellular repertoire of glycans that are produced by gly- and decode the biological information that is present in cosylation in these organelles of the secretory pathway their own cellular glycome as well as the glycomes of reflects the combinatorial expression of subsets of glyco- other organisms. syltransferase and glycosidase enzymes, of which there are The extracellular positioning of the secretory gly- more than 200 in the mammalian genome. The formation come and its phylogenetic variation between organisms and breakdown of glycans are regulated at several levels in (Box 1) allows glycans to be involved in pathogen–host the cell. One of the mechanisms involves transcriptional interactions16. Glycan linkages of the mammalian host regulation of the that encode these enzymes, but are an abundant and attractive target for pathogens to others include access to substrates and molecular inter- establish an infection through lectin binding. actions that alter enzyme localization in the lumen of have developed a phyloglycomic recognition system, the ER and Golgi2,9,10. Changes in the glycome can occur which includes a subset of Toll-like receptors (TLRs) Department of Cellular and in response to environmental and genetic stimuli, and and C-type lectins, to detect the glycans of lower Molecular Medicine and the are frequently associated with the acquisition of altered organisms as a means of non-self discrimination and Howard Hughes Medical 1–10 17 Institute, 9500 Gilman Drive- cellular pheno types . immunological activation . These pattern-recognition 0625, University of California Glycosylation also occurs among proteins in the receptors and their responses to non-vertebrate glycans San Diego, La Jolla, California cytoplasm and nucleus through the actions of the are not considered further in this Review. 92093, USA. Ogt , which produces a reversible The development and function of the mammalian Correspondence to J.D.M. O-linked β-N-acetylglucosamine (O-GlcNAc) post- immune system are associated with restructuring of the e-mail: [email protected] 11 doi:10.1038/nri2417 translational modification . O-GlcNAc linkages are cellular glycome. The immune-cell glycome is altered Published online important in numerous physiological processes and during cell differentiation, activation and , and 10 october 2008 disease. In contrast to this intracellular glycosidic bond these alterations are being linked to both homeostatic

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Secretory pathway α3 β4 α3 α3 α6 β4 α3 α6 α3 β3 α6 α3 β4 α3 α2 α2 α2 β4 β4 β4 β4 β4 β4 β4 β4 β3 β3 α2 α6 β4 β4 β4 α3 β2 β2 β2 β2 β6 β2 β2 β6 α3 α6 α3 α6 α3 α6 α3 α6 β3 β4 β4 β4 β4 β4 β4 α6 β4 α6 β4 α6 β4 α6 β3 Asn Asn Asn Asn β4 High Bi-antennary Tri-antennary Tetra-antennary N-glycans Hyaluronan

α3 α3 α3 β4 α3 β4 6S β3 β3 β4 β4 α3 α3 α3 α3 β3 β3 α3 α3 β4 β4 β4 β4 β4 β4 α3 β2 α3 β3 β3 β6 β3 β3 β6 β3

Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr Core 1Core 2Core 3Core 4 O-mannose O-fucose O-glucose O-glycans β4 6S β3 β4 6S 6S β4 β4 β3 β4 6S β4 α3 β3 β4 6S NS 6S 4S 6S β3 α4 β4 β4 β4 α3 α3 β4 6S 6S 6S α4 β4 β4 β3 2S Et α4 β3 α3 β4 NS 6S 4S β3 P α4 β4 β4 β4 α3 β4 α3 β3 α2 β3 β3 β3 β3 β4 α6 β3 β3 β3 β3 β6 β4 α4 β4 β4 β4 β4 NH2 Ser Ser Ser Ser PI Heparan Chondroitin DermatanKeratan

Glycolipids GPI anchor Glycosaminoglycans

Intracellular Fucose Glucuronic acid Mannose Iduronic acid Ser/Thr N-acetylglucosamine Glucose Ceramide O-GIcNAc N-acetylgalactosamine Xylose Figure 1 | The types of mammalian glycan. Glycan structures of the six classes of secretory glycan (N-glycans, hyaluronan, O-glycans, , glycosylphosphatidylinositol (GPI) anchor and glycosaminoglycans) and the single intracellular glycan O-linked β-N-acetylglucosamine (O-GlcNAc) are shown. Representative examplesNature Re ofvie eachws | Immunologtype are y indicated using the symbol nomenclature for monosaccharides (see key). Multiple and multi-antennary examples of the predominant mature, complex-type N-glycans are shown, including the high-mannose N-glycan that is found attached to some . Examples of core 1–4 O-glycans are depicted, as well as O-mannose, O-fucose and O-glucose structures. Core 5–7 O-glycans are not shown. The GPI anchor and examples of the glycosaminoglycans and glycolipids are also depicted. In all examples, glycan linkages are identified by the anomeric configuration (α or β) of the donor saccharide following its linkage to the ring position (1–6) of the glycan acceptor. Et–P denotes a phosphoethanolamine linkage. NS, 2S, 4S and 6S denote the sulphation positions of the glycosaminoglycan chains. Asn, asparagine; Ser, serine; Thr, threonine.

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a Lectin binding subsequent firm binding by the and the transmi- Homotypic cis-interaction Homotypic trans-interaction gration of leukocytes through the and into the surrounding tissue24–26. This mechanism of cell traf- ficking is responsible for the colonization of lymphoid organs by during development and allows leukocytes to migrate into lymphoid organs or inflamed tissues during an . Each binds Extracellular a particular glycan ligand, such as sialyl-Lewis-X (sLex) Intracellular for E- and P-selectin, and 6-sulpho-sLex for L-selectin. The binding interactions between selectins and their Heterotypic cis-interaction Heterotypic trans-interaction ligands can occur between many different cell types, including between lymphoid and non-lymphoid, as well as non-immune, cells26,27.

Regulation of selectins and their ligands. various cellular mechanisms regulate selectin function. selectin expres- sion, for example, is normally restricted to endothelial cells, and leukocytes: E- and P-selectin expres- sion is induced on the vascular endothelium during , P-selectin expression is also induced on b Steric interactions platelets and L-selectin is expressed on the surface of leu- Intramolecular folding Intermolecular inhibition kocytes28. In addition, expression of the glycoproteins that ‘carry’ (that is, are scaffolds for the construction of ) the selectin ligands sLex and 6-sulpho-sLex can be similarly regulated, thereby altering the abundance and cellular localization of sLex and 6-sulpho-sLex. The glycoproteins that carry selectin ligands continue to be discovered and include P-selectin ligand 1 (PsGL1; also known as sELPG), E-selectin ligand 1 (EsL1), CD34, CD44, glycosylation-dependent cell-adhesion mol- α β 23,29,30 ecule 1 (GlyCAM1) and M 2- . Another Protein Glycan Lectin domain Multivalent lectin mechanism that controls selectin function involves the combinatorial enzymatic activities of different Golgi- resident and sulphotransferases Figure 2 | Basic mechanisms of glycan function in the immune system. Glycans can x function as ligands for lectins (a) and as steric elements that alter molecular interactions that contribute to the synthesis of sLe and 6-sulpho- x at the cell surface and between extracellular compartments Na(b).tur Interactionse Reviews | Immunologcan involvey sLe . These enzymes are tightly regulated in different the same cell (cis) or different cells (trans), and can be either homotypic or heterotypic, cell types and can therefore contribute to the diverse and intramolecular or intermolecular. It is often difficult to distinguish steric effects of functions of selectins in the immune system. glycans from alterations in lectin–glycan binding. This figure is idealized and not drawn The synthesis of selectin ligands requires a set of to scale. enzymes, including fucosyltransferases, sialyltrans- ferases, galactosyltransferases, glucosaminyltransferases and sulphotransferases, that act in a specific order on and disease mechanisms of immune-cell functions, an underlying glycan chain as it transits through the including immune-cell trafficking and differentia- Golgi apparatus. The expression of these enzymes can tion, - and -receptor activation, auto- be regulated in response to intracellular signalling and immunity, and the induction of leukocyte apoptosis18–21. transcription-factor induction31–33. The sLex and 6-sulpho- This Review discusses the roles of mammalian secretory sLex glycans exist in vivo mainly on the O-glycan chains glycosylation in the immune system as established by of glycoproteins, and their production depends on the in vivo studies that combine knowledge of the glycan expression of the fucosyltransferases Fut4 and Fut7. biosynthetic pathways with genetic and cellular research The absence of Fut7 or Fut4, and in particular of both approaches. enzymes together, markedly attenuates homing to peripheral lymph nodes and myeloid-cell Cell trafficking trafficking in inflammation34–36. Of the immunological functions that have been ascribed The α2,3-sialyltransferases sT3Gal4 and sT3Gal6 to mammalian glycosylation, perhaps the best under- seem to participate in the formation of selectin ligands stood involves the glycan ligands of the lectins E-selectin, in vivo. studies of mice that lack sT3Gal4 have revealed L-selectin and P-selectin22,23. The selectins and their a partial deficiency in selectin ligands, with a marked ligands form a cell–cell adhesion system that operates effect on leukocyte homing and trafficking in inflamma- mainly between leukocytes and endothelial cells. This tion, which was not observed in mice that lack sT3Gal1, system is essential for the tethering and rolling of circulat- sT3Gal2 or sT3Gal3 (ReFs 37–39). The in vivo effect of ing leukocytes on the vascular cell wall, which promotes sT3Gal6 deficiency has yet to be reported.

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Box 1 | The extracellular glycome is phylogenetically distinct

α3 α2 α3 α3 α6 α2α2 α3 α2 α6 α2 α2 α2 α6 α6 α3 α6 α6 α6 High-mannose N-glycan β4 Hybrid N-glycans Complex N-glycan β4

Common Common Common precursor precursor precursor α2 α3 α3 α2 α2 α2 α2 α3 α6 α3 α6 β4 ER β4 Nucleus

Yeast and fungi Sialic acid Fucose Galactose Glucose Invertebrates N-acetylglucosamine Mannose

Vertebrates

The glycome, which is the repertoire of glycans that bear glycosidic linkages, is produced by the enzymatic process of glycosylation. The glycome of a cell at any given time reflects the cell’s ‘portfolio’ of functioningNa turglycosyltransferasese Reviews | Immunolog and y glycan-modifying enzymes. The combinatorial and sequential function of glycosyltransferases and glycosidases in the secretory pathway contribute to the diversity of cellular glycans and provide a means for the regulation of glycan formation and turnover. In the lumen of the (ER) and Golgi apparatus, resident glycosyltransferases participate in the stepwise incorporation of saccharides into glycan chains and branches. As proteins and lipids transit through the secretory pathway, they are differentially modified by glycosyltransferases and glycosidases, which add and remodel glycan linkages, thereby promoting glycan diversification. Other enzymes, including sulphotransferases, can further modify these nascent glycan linkages. The resulting ‘mature’ glycans exist alone or attached to proteins and lipids, and are mainly transported to the cell surface and to extracellular compartments. Glycan complexity has increased with evolution (see figure), such that there has been a decrease in the use of mannose linkages and the addition of new glycosidic bonds that bear different saccharides, including sialic acid. This is particularly evident when comparing the N-glycan biosynthetic pathway in lower eukaryotes (yeast and fungi), invertebrates and . The increased complexity of the glycome through phylogeny has led to the formation of glycan-dependent that distinguish stem cells, different cell lineages and cellular activation states, and that can be diagnostic of inherited genetic diseases and prognostic in cancer174–176.

The contribution of O-glycans in carrying selectin homing41,42. The finding that GCNT1 deficiency did ligands in vivo has been investigated in mice that lack not severely affect L-selectin ligands and lymphoid core 2 N-acetylglucosaminyltransferase 1 (GCNT1) trafficking was addressed by the discovery of an endog- activity. GCNT1 is one of three glycosyltransferases enous β1,3-glucosaminyltransferase. In endothelial that construct the core 2 O-glycan branch, which is a cells, this enzyme compensates for the lack of core 2 type of O-glycan structure that is commonly found on O-glycan branches and extends the core 1 O-glycan O-glycoproteins. GCNT1 deficiency strongly attenu- branch to provide a scaffold on which L-selectin lig- ated E- and P-selectin-mediated inflammation involv- ands can also be produced43. Moreover, with loss of both ing myeloid cells, but had relatively little effect on the this core-1-extension enzyme and GCNT1, L-selectin lymphoid system, including the formation of secondary ligands were observed on N-glycan chains, which seem lymphoid organs and adaptive immune responses40. to contribute to a robust lymphocyte homing response subsequent studies of GCNT1-deficient mice detected in the absence of these two glycosyltransferases44. a subtle but significant defect in the formation of Compensation was also observed between the glycan L-selectin ligands in cells that form the high endothe- sulphotransferases that are required for 6-sulpho-sLex lial venules of some lymph nodes, which was associ- synthesis, namely GlcNAc6sT1 and GlcNAc6sT2. In ated with decreased kinetics of peripheral lymphocyte contrast to deficiency of a single enzyme, deficiency

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Experimental autoimmune of both sulphotransferases led to almost complete loss TCR signalling. Glycosylation is involved in the cellular encephalomyelitis of L-selectin-ligand formation on the high endothe- mechanisms that control the threshold of TCR activa- An experimental model of lial venules of lymph nodes, together with a marked tion, and can therefore have profound effects on the multiple sclerosis. Autoimmune decrease in lymphocyte homing and immune func- function of the . The GnT5 disease is induced in animals 45,46 by immunization with myelin or tion . The different defects of innate and adaptive glycosyltransferase is encoded by Mgat5 (mannoside derived from myelin. immune responses that are associated with these pro- acetylglucosaminyl transferase 5) and initiates forma- The animals develop a files of selectin-ligand deficiency reflect the combina- tion of the β1,6 N-glycan-branch structure on various paralytic disease with torial and regulated expression of glycosyltransferases glycoproteins, including the TCR. This N-glycan branch inflammation and and glycan sulphotransferases that construct the E-, commonly includes polylactosamine, which consists demyelination in the brain and spinal cord. L- and P-selectin ligands. This modulation of ligand- of N-acetyllactosamine disaccharide repeats and is a expression profiles has progressed our understanding of ligand for various lectins of the family57. In the complexity of selectin function in the mammalian studies of mice that lack GnT5 function, TCR cluster- immune response. ing is significantly increased, which coincides with a decreased threshold of T-cell activation (FIG. 3a). Selectin-targeted therapy. selectin-mediated binding GnT5-deficient mice have increased delayed-type promotes cell–cell adhesion that induces intracellular hypersensitivity responses, increased susceptibility to signalling and could therefore be exploited in therapeu- experimental autoimmune encephalomyelitis (EAE) and tic approaches. Once engaged by their cognate glycan increased glomerulonephritis that is associated with ligands, the selectins recruit tyrosine kinases, such immune-complex deposition58. The phenotype that as FyN and spleen tyrosine kinase (syk), to their is conferred by GnT5 deficiency also includes a TCR cytoplasmic domains, thereby inducing intracellular trafficking abnormality, and the resulting hyperimmune tyrosine phosphorylation, activating -activated response might be a contributing factor in some human protein kinases and promoting integrin activation47–50. autoimmune diseases59. Pharmacological intervention that is focused on Endogenous lectins have been implicated in the for- inhibiting the interactions between leukocytes and mation of homotypic and heterotypic TCR interactions endothelial cells might therefore block the intracel- that modulate immunological signalling. Galectin-3 is lular signalling pathways that are induced by selectin chemically crosslinked with the TCR on naive resting binding. In some disease contexts, selectin-mediated cellular trafficking might contribute to tumour-cell adhesion in and leukocyte infiltration in a GnT5 activity GnT5 deficiency . Indeed, blocking selectin bind- MHC Antigen ing or inhibiting the synthesis of selectin ligands can attenuate the pathogenesis of inflammatory diseases Lectin and decrease the tumorigenicity of cells51–55. In other contexts, the induction of selectin ligands can be beneficial. Glycan remodelling of the mesenchymal TCR Glycan stem-cell surface in vitro to express selectin ligands CD3 complex increased cell tropism for the after trans- 56 Immune plantation and resulted in increased bone formation . signalling Pharmacological alteration of the production of selec- tin ligands and modulation of the binding of selectins b MHC in vivo are active areas of research and development class I molecule that could ultimately lead to new treatments for the diseases of the immune system that are linked with CD8 leukocyte trafficking and activation.

Adaptive immunity and autoimmune disease α β During their maturation, the antigen receptors of b and T cells are post-translationally modified with N- and Immune O-glycan chains. The addition of different N-glycan signalling linkages to the b-cell receptor (bCR) and T-cell recep- tor (TCR) by various glycosyltransferases in the Golgi Figure 3 | glycans in T‑cell‑receptor activation. apparatus seems to alter the molecular interactions at a | Co-clustering of αβ T-cell receptorsNature Re (TCRs)views decreases| Immunolog iny the cell surface that regulate receptor trafficking, asso- the presence of N-glycan branching on the TCR through lectin binding involving one or more of the . Loss ciation with other glycoproteins on the cell surface, of GnT5 glycosyltransferase activity, which mediates and receptor internalization by N-glycan branching, and therefore lectin binding, increases endocytosis. Moreover, glycans and endogenous multi- the propensity for TCR and co-receptor co-clustering and valent lectins that bind to them might, in some cases, be decreases the threshold of TCR activation. b | Lectin engaged in a precise spatial and geometrically predict- binding might also control TCR signalling in cytotoxic able array on the (often termed a lattice) T cells by inhibiting the association of the TCR with the that could control glycoprotein function (see later). CD8 co-receptor.

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Immunoreceptor T cells in a GnT5-dependent manner, and disruption galactose residue of the N-acetyllactosamine disac- tyrosine-based inhibitory of this interaction with glycan-ligand mimetics of charide65. Although this glycan ligand for CD22 is motif galectin-3 also results in increased TCR clustering and present on many glycoproteins, in addition to CD22 (ITIM). A short amino-acid a decreased threshold of activation58. by contrast, in itself, CD22 tends to bind to the glycan ligands of sequence (the consensus sequence of which is post-activated mature cytotoxic T cells, a decrease in neighbouring CD22 molecules, thereby forming 66 V/IxYxxV/L, in which x denotes effector activity, such as occurs during anergy, can be homotypic multimers . In the absence of ligands for any ) that is found reversed by ex vivo treatment with glycan-ligand mimet- CD22 owing to sT6Gal1 deficiency, bCR activation in the cytoplasmic tail of ics of galectin-3. Decreased effector activity is associated and humoral immunity are markedly decreased, and inhibitory receptors. ITIMs are with decreased co-localization of the TCR and CD8, and this coincides with increased co-localization of the thought to mediate inhibitory signalling by recruiting decreased binding of the T cells to HLA– tetra- bCR with CD22, constitutive recruitment of sHP1 60 phosphatases such as sHP1 mers (FIG. 3b). studies of galectin deficiency in mice to CD22, decreased protein phosphotyrosine signal- (sRC-homology-2-domain- also indicate that galectin-1 modulates the threshold ling, increased bCR trafficking to clathrin-enriched containing protein tyrosine of TCR signalling during thymocyte development61. microdomains and increased bCR endocytosis65,67,68. The phosphatase 1). Interestingly, treatment of cells or mice with high absence of both CD22 and sT6Gal1 restored normal Clathrin-enriched concentrations of GlcNAc increases GnT5-mediated bCR signalling, microdomain association and rate microdomain N-glycan branching and inhibits TCR activation and of endocytosis. Measurements of intracellular signal A region of the plasma autoimmune responses in mouse models of EAE and transduction in response to bCR stimulation in the membrane that is associated type 1 diabetes62. This is thought to be the result of a absence of the sT6Gal1-dependent ligands of CD22 with clathrin-dependent endocytic mechanisms of higher concentration of the cellular donor UDP (uridine show dominance of negative signalling that suppresses protein trafficking and 5′-diphosphate)–GlcNAc in the Golgi apparatus, which b-cell activation. This negative signalling complex turnover. might increase the capacity of GnT5 and other closely prevents the development of autoimmune disease and related glycosyltransferases to produce GlcNAc linkages markedly improves survival in LyN-tyrosine-kinase- IgA nephropathy 68 An inflammatory disease of the that contribute to N-glycan-branch formation. deficient mice . These findings are consistent with a kidney that is associated with model of bCR function in which -dependent the glomerular deposition of BCR signalling. The bCR forms complexes with binding of CD22 to the bCR dictates the threshold of IgA. immunoregulatory glycoproteins that modulate the immunological activation (FIG. 4). threshold of bCR activation, including the lectin CD22 can bind ligands that are presented in trans CD22 (also known as siglec-2), a member of the and this might modulate glycoprotein complex assem- siglec (sialic-acid-binding immunoglobulin-like bly and its localization at the b-cell surface177. Moreover, lectin) family63 that is expressed exclusively on the induced co-localization of CD22 with the bCR b cells. The structure of CD22 includes an extracel- in cis seems to occur in a siglec-independent manner lular lectin domain at the amino terminus of several that might involve protein–protein interactions that are immunoglobulin-like domains, and an intracellular mediated by the other extracellular (immunoglobulin- immunoreceptor tyrosine-based inhibitory motif repeat, like) domains of CD22. such a mechanism might explain which has an inhibitory role as it recruits the phos- how binding of dendritic cells to CD22 in a siglec- phatase sHP1 (sRC-homology-2-domain-containing independent manner inhibits b-cell proliferation69. The protein tyrosine phosphatase 1) and thereby dampens biology of CD22 is complex and there is potential for bCR signalling64. synthesis of the glycan ligands for many siglec-independent functions, some of which could CD22 requires the sT6Gal1 sialyltransferase, which be influenced by the siglec-binding activity of CD22. adds sialic acid in an α2,6 linkage to the underlying The glycan-dependent immune modulation that has been defined for sT6Gal1 in b cells and GnT5 in ST6Gal1 activityST6Gal1 deficiency T cells indicates that crucial glycoprotein-selective Antigen Glycan Lectin functions can be modulated by single glycosyltrans- BCR ferase genes that contribute to the cellular mechanisms CD22 of antigen-receptor activation and the pathogenesis of autoimmune disease.

Antibody function. The secreted immunoglobulins that are produced following b-cell activation and CD79 complex Clathrin-enriched immunoglobulin class switching are glycoproteins with microdomain Endocytosis of the BCR; 70 Immune termination of immune characteristic glycan linkages . The glycans of IgG are signalling signalling important for binding to Fc receptors and for other effector functions, including binding to complement Figure 4 | glycans in B‑cell‑receptor activation. The threshold of B-cell receptor component 1q (C1q) and complement activation71. (BCR) activation is modulated by ST6Gal1 and CD22, a member of the Siglec (sialic- Alterations of endogenous IgG glycosylation have been acid-binding immunoglobulin-like lectin) family that has a negativeNature Re rolevie wsin BCR| Immunolog y signalling. CD22 Siglec binding to its glycan ligand promotes homotypic CD22–CD22 reported in various immune diseases, including rheu- interactions, thereby decreasing the frequency of interaction of CD22 with the IgM BCR matoid arthritis and IgA nephropathy. In patients with on mature naive B cells. Loss of CD22 Siglec ligands owing to ST6Gal1 sialyltransferase , the N-glycans that are attached to deficiency markedly increases the co-localization of CD22 with the BCR, thereby IgG often lack terminal galactose and sialic-acid link- increasing the threshold of immune signalling and promoting endocytosis of the ages and therefore bear exposed N-acetylglucosamine72. BCR in a CD22-dependent manner. This terminal linkage is common to non-mammalian

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vertebrate species as well as various invertebrates and resulting in the formation of NeuGc-specific antibodies83. lower organisms, and is a ligand for some endogenous specific for glycans are also induced during mammalian immune receptors and lectins that might human Tn syndrome, which is a haematological stem- promote inflammatory responses73,74. In patients with cell disorder that is associated with thrombocytopaenia IgA nephropathy, the O-glycans that are attached to IgA and leukocytopaenia and is caused by a mutation of are similarly truncated to expose N-acetylglucosamine, COSMC (also known as C1GALT1C1). This nor- which correlates with deposition in the mally encodes a protein with chaperone function that inflamed kidney75. Deficiency of the β1,4GalT1 gly- is required for β1,3 galactosyltransferase activity in cosyltransferase (one of several galactosyltransferases) O-glycan biosynthesis84. Mutation of COSMC leads to recapitulates the pathology of IgA nephropathy in a truncated O-glycan structure on haematopoietic cells mice76. by contrast, the extent of IgG sialylation corre- and immunoglobulins (known as the Tn antigen) that lates with decreased inflammation following functions as an for antibody production and binding77. This seems to be due to the α2,6 sialic acids cell lysis and might contribute to IgA nephropathy on IgG that are contributed by sT6Gal1 and are linked and Henoch–schönlein purpura84. to the anti-inflammatory effects of intravenous IgG The aberrant synthesis of endogenous glycans can when used as a therapy for autoimmune diseases78. result in the exposure of cryptic epitopes that are per- ceived by the immune system as non-self and induce Cytokine receptors. The core 1,6 fucose linkage is found an inflammatory condition in the absence of infection on most N-glycans owing to the activity of Fut8 fucosyl- that is linked to the pathogenesis of autoimmune dis- transferase, and deficiency of this enzyme results in an ease. This explains how α-mannosidase-II (αM-II; also emphysema-like disease in mice. Remarkably, this syn- known as MAN2A1) deficiency causes an autoimmune drome was linked to impaired binding of the receptor syndrome in mice that is remarkably similar to systemic for transforming growth factor β1 (TGFβ1) to its ligand, lupus erythematosus. Disease pathogenesis in αM-II and could be attenuated by increasing the concentration deficiency is non-haematopoietic in origin (unlike other of TGFβ1 (ReFs 79,80). An inherited or sporadic gene autoimmune disease models), is independent of both the mutation that introduces new glycosylation sites into a adaptive immune system and C3 complement activation, protein can also produce receptors with ligand-binding can be mitigated by intravenous IgG administration and defects81. In this study, an N-glycosylation site that was is linked to chronic activation of the innate immune sys- found at a new position in the protein sequence of the tem85. The block in N-glycan maturation in the Golgi -g receptor 2 (IFNgR2) resulted in the forma- that is caused by αM-II deficiency increases the expres- tion of an N-glycan at this location that disrupted the sion of phylogenetically primitive hybrid N-glycans at ability of the receptor to bind its ligand. such pathologi- the cell surface and among extracellular glycoproteins. cal changes to protein glycosylation represent another The exposed mannose linkages of these primitive hybrid mechanism by which glycosylation can contribute to N-glycans on multiple glycoproteins are ligands for protein dysfunction and disease aetiology. It seems endogenous mannose-binding lectin receptors of the that glycosylation in the Golgi apparatus can in some innate immune system, and this interaction results in the cases directly affect the binding of immunomodulatory chronic induction of a pro-inflammatory response that receptors to cognate ligand. can recruit and activate macrophages. The requirement for complex N-glycan branching to safeguard against Innate immunity and autoimmune disease this immunological recognition of cryptic mannose Glycans might have been used to sense the presence ligands reveals a distinct mechanism in the pathogen- of non-self since the beginning of multicellular life. esis of autoimmune disease that can be termed epitope Glycomic differences in phylogeny and between spe- modification or neo-ligand formation. Mechanisms that cies can allow for this type of molecular discrimination impair N-glycan branching could therefore contribute to between self and non-self, and thus glycan structures are the pathogenesis of various autoimmune syndromes of activators of the innate immune system and pathogenic presently unknown origin. triggers for autoimmune diseases. Unlike new-world pri- The mechanisms that establish and maintain immuno- mates and other vertebrates, old-word primates lack the logical tolerance also involve lectin–glycan ligand α1,3 galactosidase (α1,3Gal) galactose-terminated glycan interactions that suppress immune-cell activation. For branch linkage owing to a germline mutation of the α1,3 example, galectin-1 is becoming increasingly impor- galactosyltransferase. This results in a substantial fraction tant in the study of T-cell and dendritic-cell biology of circulating immunoglobulins in old-world primates and in the pathogenesis of immunological diseases86. (including humans) that bind the α1,3Gal epitope and Galectins are involved in attenuating graft-versus-host

Old-world primates contribute to the hyperacute rejection of xenotransplants disease, collagen-induced arthritis, type 1 diabetes and 82 18,86–91 A grouping of primates, which from other mammals . Another emerging example T-cell-mediated tumour rejection . Galectin-1 includes humans, that are concerns endogenous antibodies that are specific for the seems to bridge innate and adaptive immunity in part native to Africa and Asia. sialic-acid variant N-glycolylneuraminic acid (NeuGc). by regulating the Fc receptor- and MHC-dependent Humans seem to be unique in their lack of the enzyme immune functions of monocytes and macrophages Henoch–Schönlein purpura An inflammatory disease that that converts N-acetylneuraminic acid (NeuAc) to through an extracellular-signal-regulated kinase 91 involves the blood vessels and NeuGc, and the consumption of NeuGc in the diet could (ERk) signalling pathway . The immunomodulatory causes vasculitis. lead to its incorporation into endogenous glycoproteins, activity of galectin-1 extends to its involvement in

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fetal–maternal tolerance during pregnancy. Deficiency collagen101. Fate decisions in thymic T-cell development of galectin-1 increases the loss of fetuses that have been can be modulated by galectin-1, which promotes negative conceived by the mating of allogeneic mice. In addition, selection through ERk signalling100. Altered glycosylation treatment of galectin-1-deficient mice with exogenous in glycosyltransferase-deficient animals, for example, galectin-1 restores normal fecundity, and this coincides may influence the formation of the TCR repertoire and with the induction of tolerogenic dendritic cells and thereby have an effect in immunity. The repertoire of the proliferation of interleukin-10 (IL-10)-producing T cells in an intact , which is determined by regulatory T cells92. self-, might therefore reflect, in Normal and abnormal catabolism of glycans can part, the portfolio of endogenous glycopeptides and their also provide signals of immunological importance. presentation by MHC molecules. This possibility could Hyaluronan is a glycan that consists of a disaccharide also involve intracellular O-GlcNAc glycan linkages that repeat that modulates innate immunity by interacting are attached to many cytoplasmic and nuclear proteins102. with TLRs. This glycan is synthesized as a linear chain similarly, glycolipids might be involved in development unattached to either a protein or a lipid and its size seems of the natural killer T (NkT)-cell lineage; the presenta- to be important. In response to tissue injury, endogenous tion of an endogenous structure by the CD1 hyaluronidases cleave extracellular hyaluronan into molecule might promote the development of a functional small fragments that induce inflammatory gene expres- NkT-cell population103. sion by binding TLR2 and TLR4 (ReFs 93,94). some pathogens express hyaluronidase as a virulence factor T-cell repertoire development. Expression of the glyco- that may cleave hyaluronan into fragments that do not proteins CD4 and CD8 as TCR co-receptors confers bind TLRs and thereby subvert the immune response. MHC class II and class I restriction of TCR activation, The enzymatic regulation of hyaluronan synthesis and respectively, during T-cell development in the . degradation is increasingly recognized as having an The sialylation state of glycans of the CD8–αβTCR important role in innate immunity and the response to heterodimer affects co-receptor binding to MHC class I infection95. molecules. A low level of O-glycoprotein sialylation in CD4+CD8+ thymocytes is associated with binding to Immune-cell differentiation MHC class I tetramers, whereas the increased abundance Glycoproteins, such as antigen receptors and MHC of sialic-acid linkages on differentiated CD8+ thymic molecules, are important regulators of immune-cell T cells decreases the binding avidity of CD8 for MHC differentiation and must be properly folded to function. class I molecules, thereby decreasing T-cell adhesion to without correct folding and maturation in the ER and MHC class I tetramers104–106. This decrease in binding Golgi apparatus, the expression of improperly folded to MHC class I molecules is in part mediated by the sialic and dysfunctional glycoproteins on the cell surface and acids that are produced by the sT3Gal1 sialyltransferase, in extracellular compartments can cause defects in the as deficiency of this enzyme in mice increases the bind- signalling pathways that promote immune-cell differen- ing of MHC tetramers to single-positive CD8+ thymic tiation. In addition, acquisition of secondary determi- T cells and alters the repertoire of the TCR β-chain vari- nants of the differentiated immune-cell phenotype can able region (vβ)105. This mechanism seems to involve a also be controlled by glycan-dependent mechanisms. For change in the CD8–αβTCR heterodimer conformation example, glycosaminoglycan sulphation by the sulpho- that promotes MHC class I binding in the presence of transferase NDsT2 (N-deacetylase/N-sulphotransferase unsialylated core 1 O-glycans, which lack the steric hin- (heparan glucosaminyl) 2) promotes the development of drance and negative charge that are associated with sialic mast-cell secretory granules, which are essential for the acid105,107. However, another study that used a different function of these immune cells96,97. MHC–peptide complex showed no significant change in tetramer binding of sT3Gal1-deficient CD8+ thymic T-cell ontogeny and endogenous glycans. The range T cells108. The reasons for this discrepancy are not clear, and structures of peptides that are presented by MHC but they might relate to the intrinsic binding affinities molecules could be influenced by glycan linkages on of the TCR transgenes and MHC tetramer probes that native glycoproteins and thus could contribute to the were used in each study. Nevertheless, the expression immunological definition of self 98. Although peptides and deficiency of sT3Gal1 alter the TCR vβ repertoire that are produced in the cell for presentation on MHC in an opposing manner, which indicates that sT3Gal1 class I molecules are typically deglycosylated during has a role in the MHC-restricted thymic differentiation in the , some glycan linkages per- of CD8+ T cells105,109. sist on various peptides and are recognized by cytotoxic T cells99. Peptides that arise from external sources can Notch ligands and immune-cell development. The bind- retain a broader range of glycan linkages, which might ing of Notch ligands to Notch receptors provides another influence peptide proteolysis and loading on MHC example of how glycosyltransferases can influence class II molecules for binding by αβTCRs. For example, intracellular signal transduction and thereby govern the some TCRs from T cells that have been isolated from development and function of immune cells. Notch sig- mice with collagen-induced arthritis recognize an nalling, which controls cell-fate determination in many MHC class II-associated peptide of type II collagen that organisms, dictates lineage commitment in the mam- contains galactose linked to hydroxylysine residues of malian lymphoid system and has recently been shown

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to be involved in myelopoiesis110. The closely related responses to various stimuli. It seems that mammalian Fringe glycosyltransferases (Manic, Radical and Lunatic glycosylation has a role in the emergence and phenotypic

in the mammalian genome) were discovered as genes that profiles of these TH-cell subsets, in part by its effect on the establish cell-boundary fields in embryogenesis and were production of ligands of various lectins. The Delta-like 1

later found to intersect with the functions of Notch recep- and Delta-like 4 glycan ligands of Notch promote TH1- tors in early development111–115. The Fringe glycosyltrans- cell differentiation of CD4+ T cells in part by suppressing ferases are β1,3 N-acetylglucosaminyltransferases that IL-4-mediated signalling, which would otherwise induce

operate on the O-linked fucose glycans. These glycans TH2-cell differentiation and promote TH2-cell immune are found in the extracellular epidermal growth factor responses137,138. by contrast, the GnT5 glycosyltransferase

(EGF)-like repeats of Notch receptors in the secretory promotes TH2-cell immune responses by inducing dif- pathway and control cell-surface Notch signalling116,117. ferentiation signals that inhibit IFNg production139. The

The O-fucose linkage that is the substrate of the Fringe induced deficiency of galectin-1 restores the TH1–TH2- glycosyltransferases is first generated by Pofut1 (protein cell balance of T-cell infiltrates in classical Hodgkin’s O-fucosyltransferase). The glycan products of the Fringe lymphoma, apparently by modulating the of β glycosyltransferases are then substrates of 1,3 galacto- TH2-cell and the proliferation of regulatory 140 syltransferases. These glycans provide a physiological T cells . Differential glycosylation of TH-cell subsets has mechanism to modulate Notch binding and activation been linked with their susceptibility to death by galectin- by the extracellular glycoprotein ligands Jagged/serrate induced apoptosis, which provides an explanation for 118–123 and Delta . In addition to generating the O-fucose the increased TH1- and TH17-cell responses that have linkage that is the substrate of the Fringe glycosyltrans- been observed in galectin-1-deficient mice141. A related ferases, Pofut1 provides a chaperone activity that is inde- mechanism might operate during parasitic infection pendent of its glycosyltransferase activity and promotes by which helminth glycans skew CD4+ T-cell immune 124 142 Notch maturation . More recently, Notch signalling has responses towards the TH2-cell phenotype . been shown to be also influenced by the Rumi glycosyl- transferase, which generates O-glucose linkages on the Apoptosis EGF-like repeats of Notch125,126. Cell death by apoptosis is an important mechanism of signalling through the Notch axis has profound immune-system homeostasis during lymphoid develop- effects on lymphocyte development and activation in ment and in the clearance of effector cells after immune mammals. For example, a constitutively active form of activation143. Mammalian glycosylation contributes to Notch can promote the development of CD8+ T cells in this process by generating ligands for endogenous lectins, the absence of MHC class I molecules127. The effect of including galectins57,144,145. These lectins bind to glycans Fringe glycosyltransferases on lymphoid-cell fate includes that contain the disaccharide and to polylactos- the suppression of b-cell development from lymphoid amine, which are present in some cellular glycoproteins progenitors in the thymus128. However, Fringe activity and glycolipids. promotes increased Notch binding to Delta-like ligands, The T-cell lineage is sensitive to both the endogenous which can lead to a block in T-cell development. This expression and exogenous administration of galectin, might be due to an effect on the access of progenitor cells and most studies have examined the effects of galectin-1 to intrathymic niches or a direct effect on the induction and galectin-3. Exogenously administered galectin-1 was of T-cell differentiation signals129,130. Moreover, Notch first reported to induce apoptosis in thymocytes, T-cell activation leads to the formation of marginal-zone b cells lines and activated T cells146. The in vitro methodology in the spleen and promotes immunoglobulin secretion by used was thought to contribute in part to this result, as a a signalling mechanism that involves Notch binding to subsequent study reported that activated T cells do not the Delta-like 1 ligand131–133. Dendritic- and myeloid-cell normally undergo apoptosis in response to galectin-1, differentiation also seem to be modulated by Notch sig- but become apoptotic after administration of galectin-3 nalling in response to the binding of Notch ligands, and (ReFs 147,148). In multiple cell types, including T cells the activation of Nk-cell cytotoxicity is promoted by the and , binding of galectins -1, -2, -3, -4 or -8 Notch ligand Jagged2 (ReFs 134,135). Immune responses to cellular glycans was shown to induce phosphatidyl- can also be inhibited by Notch binding to Fringe- serine exposure on the outer leaflet of the plasma mem- dependent glycan ligands; for example, Delta-like-1 brane, which is associated with cellular apoptosis with ligand seems to inhibit Notch activation, which skews phagocytic uptake in vivo, and normal cell viability was the outcome in airway inflammation from a T helper 2 retained in vitro147–150. Galectins bind to glycan ligands 136 (TH2)- to TH1-cell response . The functional relation- that are present on various glycoproteins, such as CD2, ships between Notch receptors and their glycan ligands CD3, CD7 and CD45, thereby forming glycoprotein are only partly understood, but already a remarkable complexes that induce intracellular signalling cascades complexity in glycan-mediated and Notch-dependent and inhibit IFNg and IL-2 production and IL-2-mediated Hodgkin’s lymphoma immune-cell differentiation is indicated. signalling148,151–154. Among the glycan ligands of the A type of lymphoma that is galectins, the polylactosamine moieties that are found in characterized by the presence T -cell polarization. Activated CD4+ effector T cells can O-glycans are sometimes present on the core 2 branch. of Reed–sternberg cells H (derived from B cells) and that differentiate into distinct subsets (TH1, TH2, TH17 and T-lymphoma cells that are deficient in core 2 O-glycans spreads between the lymph regulatory T cells), each of which produces a unique as a result of GCNT1 mutation or haploinsufficiency are nodes. profile of cytokines that differentially govern immune resistant to galectin-1-induced apoptosis155. In addition,

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some galectins seem to oppose the functions of others. among glycoproteins and lectins at the cell surface161. Galectin-3 binds different glycans and glycoproteins to Further study could resolve whether galectins normally those that are recognized by galectin-1, and galectin-3 operate in vivo to construct a repetitive and spatially can inhibit galectin-1- but not galectin-9-mediated apop- precise ultrastructure (a lattice) that thereby modulates tosis of some human T-cell lines. by contrast, galectin-9 the presentation, interaction, function and turnover of induces T-cell apoptosis that can be inhibited by b-cell glycoproteins161–166. There is increasing evidence that lymphoma 2 (bCL-2) (ReFs 156–158). mammalian glycosylation controls the rate of receptor The sT3Gal1 sialyltransferase seems to control the endocytosis as a means to modulate cellular responses largest and most synchronous apoptotic response in through the interaction of lectins and their ligands167–169. adult mammals, that of eliminating the increase in It seems possible that what is presently considered basal cytotoxic CD8+ T cells that results from an immune protein-turnover activity at the cell surface is instead a response to infection by various microorganisms and highly orchestrated process that controls glycoprotein . More than 90% of the clonally expanded CD8+ fate and function. Furthermore, it remains probable that T-cell population must be rapidly cleared by apoptosis more lectins without canonical lectin-like domains exist, during the resolution of the immune response so that which are identifiable as proteins that bind their targets the body is not overcome by . sT3Gal1 and function in a glycan-dependent manner. It follows is involved in this process by linking sialic acid in an that glycan-dependent regulation of glycoprotein and α2,3 linkage to the core 1 O-glycan of one or more glycolipid interactions might contribute in many con- O-glycoproteins. Attenuation of sT3Gal1 function by a texts and in presently unknown ways to the mechanisms post-translational mechanism occurs during the resolu- that govern the immune responses of cells to various tion phase of the immune response, and the resulting extracellular stimuli. unsialylated core 1 O-glycan structure is linked to the The evolutionary success of the selectins and their gly- onset of post-activated CD8+ T-cell apoptosis by a can ligands in regulating immune function by controlling caspase-dependent mechanism that seems to dominate cell–cell adhesion and signalling makes it unlikely that the effects of the intracellular apoptotic modulators they are the only lectin–ligand system that has evolved to bCL-2 and bIM109,159. Remodelling of the glycome in direct immune-cell trafficking. Other glycan structures, activated CD8+ T cells by downmodulation of sT3Gal1 such as Lewis X and Lewis y, for example, have recently function induces a signalling pathway that ensures been found to participate in the cell–cell recognition that apoptotic cell death in the absence of TCR stimulation modulates dendritic-cell adhesion and signalling170,171. and of factors that induce memory-cell differentiation. Moreover, hyaluronan has been recently found to modu- The lectin (or lectins) that induces post-activated CD8+ late adhesion in liver sinusoids172. The extent T-cell apoptosis in vivo, and the glycoprotein counter- to which glycosylation contributes to immune-cell recog- receptor (or receptors) that transmits the apoptotic signal nition is uncertain, but the regulated changes that occur following binding to the unsialylated core 1 O-glycan in glycan formation during cell maturation and activation ligand, are yet to be identified. are profound and may alter the production of various lec- tin ligands that might engage various molecules and cell Summary and future directions types173. Even the glycosyltransferases that participate in A rapid expansion of our knowledge of the immuno- selectin-ligand synthesis have yet to be fully identified, logical roles of mammalian glycosylation has resulted and the regulatory events that control the formation of from interdisciplinary studies that combine biochemi- selectin ligands are incompletely understood. In addition, cal, enzymatic, structural and genetic techniques. the mechanisms by which selectin ligands differentially Establishing the relationships between glycan linkages, affect the immune response, including their ability to the enzymes that produce them, and the glycoproteins modulate intracellular signalling, are just beginning that are modulated by them remains at the forefront of to be studied. research (TABLe 1). studies that bridge immunology and Glycosylation in mammals constructs a glycome glycobiology, and which link the enzymes of glycosyla- that can be distinguished phylogenetically and immu- tion with lectin recognition, will continue to provide nologically from those of lower and divergent organ- new insights into the immune system and will reveal isms, and encompasses glycan linkages with profound new approaches to therapeutic intervention in disease. immunoregulatory functions. Improved experimental studies of glycosylation by the sT3Gal4 sialyltransferase, techniques that can rapidly define glycan structure and for example, recently revealed that the Ashwell–Morell can image distinct cellular glycoprotein interactions are receptor of hepatocytes — the main lectin expressed increasingly relevant. Antibodies that specifically bind by hepatocytes — mitigates the lethal coagulopathy of the synthetic and degradative enzymes of glycosylation sepsis160, exposing an unexpected pathogen–host rela- are for the most part not available today, which makes tionship that might reflect an ancient arm of the innate it difficult to detect the molecular interactions that immune system. constitute the regulatory machinery of cellular glyco- The mechanisms by which glycans and lectins sylation. Determining the elements of the mammalian contribute to immunity are only now beginning to be glycome and the mechanisms by which the glycome is understood. Among the potential mechanisms, the constructed and regulated will provide integrative and nature of multivalent lectin–ligand binding as observed profound immunological insights into the biological in vitro indicates that higher-order structures might exist roles of mammalian glycosylation.

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Table 1 | Mammalian glycan structures that are linked to immune functions glycan* Enzymes‡ Function Recognition§ slex ST3Gal4, ST3Gal6, β1,4GalT1, Trafficking, adhesion and signalling E- and P-selectin Fut4 and Fut7 Asn α3 β4α3

Ser/Thr α3 β3 β4 α3 6‑sulpho‑slex ST3Gal4, GlcNAc6ST1, Lymphocyte homing L-selectin GlcNAc6ST2, Fut4 and Fut7 Asn α3 β4 α3 6S

Ser/Thr β3 α3 β4 α3 6S

Core 1 o‑glycan α3 ST3Gal1 CD8+ T-cell apoptosis Unknown β3 Ser/Thr

Terminal glcnAc β2 GnTs IgA nephropathy and complement Integrins activation Asn β4 β6 β2

Ser/Thr β6

N‑acetyl‑lactosamine β1,4GalTs and β1,3GnTs T-cell-mediated tumour rejection, Galectins and polylactosamine Asn fetal–maternal tolerance and DC function β4 β3 β4 Ser/Thr

β4 β3 β4 β3 β4 β1,6 n‑glycan branch GnT5 TCR activation threshold Galectin-3 Asn β6

Sia6lacnAc α6 ST6Gal1 BCR activation threshold CD22

Asn

Hybrid n‑glycan αM-II Deficiency causes SLE-like Mannose-binding Asn syndrome in mice lectin (or lectins) α3 α6

O‑fucose glycan β3 β1,3GlcNAcTs and Pofut1 Lymphocyte development and Notch Ser/Thr activation, myeloid and DC development, and NK-cell toxicity O‑glucose glycan Ser/Thr O-glucosyltransferase (Rumi) Notch signalling Notch

Hyaluronan β4 β3 β4 β3 β4 β3 β4 Hyaluronan synthases Innate immunity, response to TLR2 and TLR4 infection n‑glycan Fut8 TGFβ1 receptor ligand binding and TGFβ1 Asn signalling α6

*Glycan structure is indicated using the symbol nomenclature for monosaccharides; see FIG. 1 for further information. ‡Glycosyltransferase and glycosidase enzymes that are known to be responsible for the presence of the indicated glycan linkages, whether owing to their regulated expression or lack of expression. §Recognition proteins that include classically defined lectins, which function by binding to the highlighted glycan structures. αM-II, α-mannosidase-II; β1,4GalT, β1,4-galactosyl- transferase; β1,3GnT, β1,3-glucosaminyltransferase; BCR, B-cell receptor; DC, ; Fut, fucosyltransferase; GCNT1, core 2 β1,4-glucosaminyltransferase 1; GlcNAc, β-N-acetylglucosamine; GlcNAc6ST, glycan sulphotransferase; GnT, N-acetylglucosaminyltransferase; NK, natural killer; Pofut1, protein O-fucosyltransferase; SLE, systemic lupus erythematosus; sLex, sialyl-Lewis-X; ST3Gal, α2,3-sialyltransferase, TCR, T-cell receptor; TGFβ1, transforming growth factor β1; TLR, Toll-like receptor.

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1. Paulson, J. C., Blixt, O. & Collins, B. E. Sweet spots in 24. McEver, R. P. Leukocyte interactions mediated by 48. Simon, S. I., Hu, Y., Vestweber, D. & Smith, C. W. functional glycomics. Nature Chem. Biol. 2, 238–248 selectins. Thromb. Haemost. 66, 80–87 (1991). Neutrophil tethering on E-selectin activates β2 (2006). 25. Lasky, L. A. Selectins: interpreters of cell-specific integrin binding to ICAM-1 through a mitogen- This publication provides an introduction to the carbohydrate information during inflammation. activated protein kinase signal transduction pathway. definition and concept of the glycome and describes Science 258, 964–969 (1992). J. Immunol. 164, 4348–4358 (2000). new approaches to research in this field. 26. Ley, K. & Kansas, G. S. Selectins in T-cell recruitment 49. Urzainqui, A. et al. ITAM-based interaction of ERM 2. Ohtsubo, K. & Marth, J. D. Glycosylation in cellular to non-lymphoid tissues and sites of inflammation. proteins with Syk mediates signaling by the leukocyte mechanisms of health and disease. Cell 126, Nature Rev. Immunol. 4, 325–335 (2004). adhesion receptor PSGL-1. Immunity 17, 401–412 855–867 (2006). 27. Rossi, F. M. V. et al. Recruitment of adult thymic (2002). This article reviews the process of glycosylation progenitors is regulated by P-selectin and its ligand 50. Zarbock, A., Lowell, C. A. & Ley, K. Spleen tyrosine and the mechanisms by which glycans contribute PSGL-1. Nature Immunol. 6, 626–634 (2005). kinase Syk is necessary for E-selectin-induced

to molecular interactions that govern health and 28. McEver, R. Selectin–carbohydrate interactions during αLβ2 integrin-mediated rolling on intercellular disease. inflammation and metastasis. Glycoconj. J. 14, adhesion molecule-1. Immunity 26, 773–783 3. Raman, R., Raguram, S., Venkataraman, G., Paulson, 585–591 (1997). (2007). J. C. & Sasisekharan, R. Glycomics: an integrated 29. McEver, R. P. & Cummings, R. D. Role of PSGL-1 This study links selectin binding to signal systems approach to structure–function relationships binding to selectins in leukocyte recruitment. transduction by tyrosine kinases. of glycans. Nature Methods 2, 817–824 (2005). J. Clin. Invest. 100, S97–S103 (1997). 51. Kim, Y. J., Borsig, L., Varki, N. M. & Varki, A. 4. Haltiwanger, R. S. & Lowe, J. B. Role of glycosylation in 30. Hidalgo, A., Peired, A. J., Wild, M. K., Vestweber, D. & P-selectin deficiency attenuates tumor growth and development. Annu. Rev. Biochem. 73, 491–537 Frenette, P. S. Complete identification of E-selectin metastasis. Proc. Natl Acad. Sci. USA 95, (2004). ligands on neutrophils reveals distinct functions of 9325–9380 (1998). This article reviews the developmental biology of PSGL-1, ESL-1, and CD44. Immunity 26, 477–489 52. Chen, S. & Fukuda, M. Cell type-specific roles of glycosylation and the mechanisms by which glycans (2007). carbohydrates in tumor metastasis. Methods Enzymol. control ontogeny. 31. Zisoulis, D. G. & Kansas, G. S. H-Ras and 416, 371–380 (2006). 5. Caramelo, J. J. & Parodi, A. J. How sugars convey phosphoinositide 3-kinase co-operate to induce 53. Romano, S. J. Selectin antagonists: therapeutic information on protein conformation in the α(1,3)-fucosyltransferase VII expression in Jurkat potential in and COPD. Treat Respir. Med. endoplasmic reticulum. Semin. Cell Dev. Biol. T cells. J. Biol. Chem. 279, 39495–39504 (2004). 4, 85–94 (2005). 18, 732–742 (2007). 32. Underhill, G. H. et al. A crucial role for T-bet in selectin 54. Uchimura, K. & Rosen, S. D. Sulfated L-selectin ligands 6. Varki, A. Nothing in glycobiology makes sense, except ligand expression in T helper 1 (Th1) cells. Blood 106, as a therapeutic target in chronic inflammation. Trends in the of evolution. Cell 126, 841–845 (2006). 3867–3873 (2005). Immunol. 27, 559–565 (2006). This review provides a perspective on how glycan 33. Kang, R. et al. Transcriptional regulation of the 55. Woollard, K. J. & Chin-Dusting, J. Therapeutic function arose and continues to change in the N-acetylglucosaminyltransferase V gene in human bile targeting of P-selectin in atherosclerosis. Inflamm. evolution of organisms. duct carcinoma cells (HuCC-T1) is mediated by Ets-1. Drug Targets. 6, 69–74 (2007). 7. Helenius, A. & Aebi, M. Roles of N-linked glycans in J. Biol. Chem. 271, 26706–26712 (1996). 56. Sackstein, R. et al. Ex vivo glycan engineering of CD44 the endoplasmic reticulum. Annu. Rev. Biochem. 73, 34. Maly, P. et al. The α(1, 3)fucosyltransferase Fuc-TVII programs human multipotent mesenchymal stromal 1019–1049 (2004). controls leukocyte trafficking through an essential role cell trafficking to bone. Nature Med. 14, 181–187 8. Lowe, J. B. & Marth, J. D. A genetic approach to in L-, E-, and P-selectin ligand biosynthesis. Cell 86, (2008). mammalian glycan function. Annu. Rev. Biochem. 72, 643–653 (1996). 57. Rapoport, E. M., Kurmyshkina, O. V. & Bovin, N. V. 643–691 (2003). 35. Homeister, J. W. et al. The α(1, 3)fucosyltransferases Mammalian galectins: structure, carbohydrate 9. Comelli, E. M. et al. A focused microarray approach to FucT-IV and FucT-VII exert collaborative control over specificity, and functions. Biochemistry (Mosc) 73, functional glycomics: transcriptional regulation of the selectin-dependent leukocyte recruitment and 393–405 (2008). glycome. Glycobiology 16, 117–131 (2005). lymphocyte homing. Immunity 15, 115–126 (2001). 58. Demetriou, M., Granovsky, M., Quaggin, S. & Dennis, 10. Wu, X. et al. Mutation of the COG complex subunit 36. Smith, P. L. et al. Conditional control of selectin ligand J. W. Negative regulation of T-cell activation and COG7 causes a lethal congenital disorder. Nature expression and global fucosylation events in mice with autoimmunity by Mgat5 N-glycosylation. Nature 409, Med. 10, 518–523 (2004). a targeted mutation at the FX locus. J. Cell Biol. 158, 733–739 (2001). 11. Hart, G. W. Dynamic O-linked glycosylation of nuclear 801–815 (2002). 59. Lee, S.-U. et al. N-glycan processing deficiency and cytoskeletal proteins. Annu. Rev. Biochem. 66, 37. Ellies, L. G. et al. Sialyltransferase specificity in promotes spontaneous inflammatory demyelination 315–335 (1997). selectin ligand formation. Blood 100, 3618–3425 and neurodegeneration. J. Biol. Chem. 282, 12. Dias, W. B. & Hart, G. W. O-GlcNAc modification in (2002). 33725–33734 (2007). diabetes and Alzheimer’s disease. Mol. Biosyst. 3, 38. Sperandio, M. et al. α2,3-sialyltransferase-IV is 60. Demotte, N. et al. Restoring the association of the 766–772 (2007). essential for L-selectin ligand function in inflammation. receptor with CD8 reverses anergy in human 13. Sharon, N. Lectins: carbohydrate-specific reagents and Eur. J. Immunol. 36, 3207–3215 (2006). tumor-infiltrating lymphocytes. Immunity 28, biological recognition molecules. J. Biol. Chem. 282, 39. Frommhold et al. Sialyltransferase ST3Gal-IV controls 414–424 (2008). 2753–2764 (2007). CXCR2-mediated firm leukocyte arrest during 61. Liu, S. D. et al. Endogenous galectin-1 enforces This publication describes the origins and activities inflammation. J. Exp. Med. 205, 1435–1446 (2008). class I-restricted TCR functional fate decisions in of lectins as proteinaceous receptors that bind 40. Ellies, L. G. et al. Core 2 biosynthesis thymocytes. Blood 112, 120–130 (2008). glycans. distinguishes between selectin ligands essential for 62. Grigorian, A. et al. Control of T cell-mediated 14. Taylor, M. E. & Drickamer, K. Paradigms for glycan- leukocyte homing and inflammation. Immunity 9, autoimmunity by metabolite flux to N-glycan binding receptors in cell adhesion. Curr. Opin. Cell 881–890 (1998). biosynthesis. J. Biol. Chem. 282, 20027–20035 Biol. 19, 572–577 (2007). 41. Sperandio, M. et al. Differential requirements for (2007). This article describes the various lectins and their core2 glucosaminyltransferase for endothelial 63. Crocker, P. R., Paulson, J. C. & Varki, A. and binding activities in adhesive functions between L-selectin ligand function in vivo. J. Immunol. 167, their roles in the immune system. Nature Rev. cells. 2268–2274 (2001). Immunol. 7, 255–266 (2007). 15. Goldstein, I. J. Lectin structure-activity: the story is 42. Gauguet, J.-M., Rosen, S. D., Marth, J. D. & This review covers the structure and activities of never over. J. Agric. Food Chem. 50, 6583–6585 von Andrian, U. H. Core 2 branching β1,6-N-acetyl- the Siglec family of lectins and their immunological (2002). glucosaminyltransferase and high endothelial cell functions. This publication describes the extent of origins and N-acetylglucosamine-6-sulfotransferase exert 64. Tedder, T. F., Tuscano, J., Sato, S. & Kehrl, J. H. activities of lectins. differential control over B- and T-lymphocyte homing CD22, a B lymphocyte-specific adhesion molecule 16. Gagneux, P. & Varki, A. Evolutionary considerations in to peripheral lymph nodes. Blood 104, that regulates antigen receptor signaling. Annu. Rev. relating oligosaccharide diversity to biological 4104–4112 (2004). Immunol. 15, 481–504 (1997). function. Glycobiology 9, 747–755 (1999). 43. Yeh, J.-C. et al. Novel sulfated lymphocyte homing 65. Hennet, T., Chui, D., Paulson, J. C. & Marth, J. D. 17. Medzhitov, R. & Janeway, J. C. The Toll receptor family receptors and their control by a core1 extension β1, Immune regulation by the ST6Gal sialyltransferase. and microbial recognition. Trends Microbiol. 8, 3-N-acetylglucosaminyltransferase. Cell 105, Proc. Natl Acad. Sci. USA 95, 4504–4509 (1998). 452–456 (2000). 957–969 (2001). 66. Han, S., Collins, B. E., Bengtson, P. & Paulson, J. C. 18. van Kooyk, Y. & Rabinovich, G. A. Protein–glycan 44. Mitoma, J. et al. Critical functions of N-glycans in Homo-multimeric complexes of CD22 in B cells interactions in the control of innate and adaptive L-selectin-mediated lymphocyte homing and revealed by protein–glycan cross-linking. Nature immune responses. Nature Immunol. 9, 593–601 recruitment. Nature Immunol. 8, 409–418 (2007). Chem. Biol. 1, 93–97 (2005). (2008). 45. Kawashima, H. et al. N-acetylglucosamine- 67. Collins, B. E., Smith, B. A., Bengtson, P. & Paulson, J. C. 19. Baum, L. G. Developing a taste for sweets. Immunity 6-O-sulfotransferases 1 and 2 cooperatively control Ablation of CD22 in ligand-deficient mice restores 16, 5–8 (2002). lymphocyte homing through L-selectin ligand receptor signaling. Nature Immunol. 7, 20. Lowe, J. B. Glycosylation, immunity, and autoimmunity. biosynthesis in high endothelial venules. Nature 199–206 (2006). Cell 104, 809–812 (2001). Immunol. 6, 1096–1164 (2005). 68. Grewal, P. K. et al. ST6Gal-I restrains CD22-dependent 21. Rudd, P. M., Elliott, T., Cresswell, P., Wilson, I. A. & 46. Uchimura, K. et al. A major class of L-selectin ligands antigen receptor endocytosis and Shp-1 recruitment in Dwek, R. A. Glycosylation and the immune system. is eliminated in mice deficient in two sulfotransferases normal and pathogenic immune signaling. Mol. Cell. Science 291, 2370–2374 (2001). expressed in high endothelial venules. Nature Biol. 26, 4970–4981 (2006). 22. Lowe, J. B. Glycosyltransferases and glycan structures Immunol. 6, 1105–1113 (2005). 69. Santos, L. et al. Dendritic cell-dependent inhibition of contributing to the adhesive activities of L-, E- and 47. Hidari, K. I., Weyrich, A. S., Zimmerman, G. A. & B cell proliferation requires CD22. J. Immunol. 180, P-selectin counter-receptors. Biochem. Soc. Symp. McEver, R. P. Engagement of P-selectin glycoprotein 4561–4569 (2008). 69, 33–45 (2002). ligand-1 enhances tyrosine phosphorylation and 70. Kobata, A. The N-linked sugar chains of human 23. Rosen, S. D. Ligands for L-selectin: homing, activates mitogen-activated protein kinases in human immunoglobulin G: their unique pattern, and their inflammation, and beyond. Annu. Rev. Immunol. neutrophils. J. Biol. Chem. 272, 28750–28756 functional roles. Biochim. Biophys. Acta 1780, 22, 129–156 (2004). (1997). 472–478 (2008).

NATURE REvIEws | immunology vOLUME 8 | NOvEMbER 2008 | 885 REVIEWS

71. Wright, A. & Morrison, S. L. Effect of glycosylation on 95. Girish, K. S. & Kemparaju, K. The magic glue 1 and Fringe. J. Biol. Chem. 278, 42340–42345 antibody function: implications for genetic engineering. hyaluronan and its eraser hyaluronidase: a biological (2003). Trends Biotech. 15, 26–32 (1997). overview. Life Sci. 80, 1921–1943 (2007). 122. Shi, S. & Stanley, P. Protein O-fucosyltransferase 1 is 72. Parekh, R. B. et al. Association of rheumatoid arthritis 96. Forsberg, E. et al. Abnormal mast cells in mice an essential component of Notch signaling pathways. and primary osteoarthritis with changes in the deficient in a heparin-synthesizing enzyme. Nature Proc. Natl Acad. Sci. USA 100, 5234–5239 (2003). glycosylation pattern of total serum IgG. Nature 400, 773–776 (1999). 123. Yang, L.-T. et al. Fringe glycosyltransferases 316, 452–457 (1985). 97. Humphries, D. E. et al. Heparin is essential for the differentially modulate Notch1 proteolysis induced by 73. Hoffmeister, K. M. et al. Glycosylation restores survival storage of specific granule proteases in mast cells. Delta1 and Jagged1. Mol. Biol. Cell 16, 927–942 of chilled blood platelets. Science 301, 1531–1534 Nature 400, 769–772 (1999). (2005). (2003). 98. Carbone, F. R. & Gleeson, P. A. Carbohydrates and 124. Okajima, T., Xu, A., Lei, L. & Irvine, K. D. Chaperone 74. Jones, A. J. S. et al. Selective clearance of glycoforms antigen recognition by T cells. Glycobiology 7, activity of protein O-fucosyltransferase 1 promotes of a complex glycoprotein pharmaceutical caused by 725–730 (1997). Notch receptor folding. Science 307, 1599–1603 terminal N-acetylglucosamine is similar in humans and 99. Zhao, X. J. & Cheung, N. K. GD2 oligosaccharide: (2005). cynomolgus monkeys. Glycobiology 17, 529–540 target for cytotoxic T lymphocytes. J. Exp. Med. 125. Acar, M. et al. Rumi is a CAP10 domain (2007). 182, 67–74 (1995). glycosyltransferase that modifies Notch and is required 75. Hiki, Y. et al. Mass spectrometry proves 100. Liu, S. D. et al. Endogenous galectin-1 enforces for Notch signaling. Cell 132, 247–258 (2008). under-O-glycosylation of glomerular IgA1 in IgA class I-restricted TCR functional fate decisions in 126. Stanley, P. Glucose: a novel regulator of Notch nephropathy. Kidney Int. 59, 1077–1085 (2001). thymocytes. Blood 112, 120–130 (2008). signaling. ACS Chem. Biol. 3, 210–213 (2008). 76. Nishie, T. et al. Development of immunoglobulin A 101. Corthay, A. et al. Epitope glycosylation plays a critical 127. Robey, E. et al. An activated form of Notch influences nephropathy-like disease in β-1,4-galactosyl- role for T cell recognition of type II collagen in the choice between CD4 and CD8 T cell lineages. Cell transferase-I-deficient mice. Am. J. Pathol. 170, collagen-induced arthritis. Eur. J. Immunol. 28, 87, 483–492 (1996). 447–456 (2007). 2580–2590 (1998). 128. Koch, U. et al. Subversion of the T/B lineage decision 77. Kaneko, Y., Nimmerjahn, F. & Ravetch, J. V. Anti- 102. Wells, L., Vosseller, K. & Hart, G. W. Glycosylation of in the thymus by lunatic fringe-mediated inhibition of inflammatory activity of immunoglobulin G resulting nucleocytoplasmic proteins: signal transduction and Notch-1. Immunity 15, 225–234 (2001). from Fc sialylation. Science 313, 670–673 (2006). O-GlcNAc. Science 291, 2376–2378 (2001). 129. Visan, I. et al. Regulation of T lymphopoiesis by 78. Anthony, R. M. et al. Racapitulation of IVIG anti- 103. Zajonc, D. M. & Kronenberg, M. CD1 mediated T cell Notch1 and Lunatic fringe-mediated competition for inflammatory activity with a recombinant IgG Fc. recognition of glycolipids. Curr. Opin. Struct. Biol. 17, intrathymic niches. Nature Immunol. 7, 634–643 Science 320, 373–376 (2008). 521–529 (2007). (2006). 79. Wang, X. et al. Dysregulation of TGF-β1 receptor 104. Daniels, M. A. et al. CD8 binding to MHC class I 130. Jenkinson, E. J., Jenkinson, W. E., Rossi, S. W. & activation leads to abnormal lung development and molecules is influenced by T cell maturation and Anderson, G. The thymus and T-cell commitment: the emphysema-like phenotype in core fucose-deficient glycosylation. Immunity 15, 1051–1061 (2001). right niche for Notch? Nature Rev. Immunol. 6, mice. Proc. Natl Acad. Sci. USA 102, 15791–15794 105. Moody, A. M. et al. Developmentally regulated 551–555 (2006). (2005). glycosylation of the CD8αβ coreceptor stalk modulates 131. Tanagaki, K. et al. Notch–RBP-J signaling in involved 80. Wang, X. et al. Phenotype changes of fut8 knockout ligand binding. Cell 107, 501–512 (2001). in cell fate determination of marginal zone B cells. mouse: core fucosylation is crucial for the function of 106. Kao, C., Daniels, M. A. & Jameson, S. C. Loss of CD8 Nature Immunol. 3, 443–450 (2002). growth factor receptor(s). Methods Enzymol. 417, and TCR binding to class I MHC ligands following T cell 132. Hozumi, K. et al. Delta-like 1 is necessary for the 11–22 (2006). activation. Int. Immunol. 17, 1607–1617 (2005). generation of marginal zone B cells but not T cells 81. Vogt, G. et al. Gains of glycosylation comprise an 107. Moody, A. M. et al. Sialic acid capping of CD8β in vivo. Nature Immunol. 5, 638–644 (2004). unexpectedly large group of pathogenic mutations. core 1-O-glycans controls thymocyte-major 133. Santos, M. A. et al. Notch1 engagement by Nature Genet. 37, 692–700 (2005). complex class I interaction. Delta-like-1 promotes differentiation of B lymphocytes 82. Macher, B. A. & Galili, U. The Galα1, 3Galβ1, J. Biol. Chem. 278, 7240–7246 (2003). to antibody-secreting cells. Proc. Natl Acad. Sci. USA 4GlcNAc-R. (α-Gal) epitope: a carbohydrate of unique 108. Kao, C., Sandau, M. M., Daniels, M. A. & Jameson, 104, 15454–15459 (2007). evolution and clinical relevance. Biochim. Biophys. S. C. The sialyltransferase ST3Gal-I is not required for 134. Cheng, P. & Gabrilovich, D. Notch signaling in Acta 1780, 75–88 (2007). regulation of CD8-class I MHC binding during T cell differentiation and function of dendritic cells. Immunol. 82. Padler-Karavani, V. et al. Diversity in specificity, development. J. Immunol. 176, 7421–7430 (2006). Res. 41, 1–14 (2008). abundance and composition of anti-Neu5Gc 109. Van Dyken, S. J., Green, R. S. & Marth, J. D. Structural 135. Kijima et al. Dendritic cell-mediated NK cell activation antibodies in normal humans: potential implications and mechanistic features of protein O glycosylation is controlled by Jagged2–Notch interaction. Proc. Natl for disease. Glycobiology 31 July 2008 linked to CD8+ T-cell apoptosis. Mol. Cell. Biol. 27, Acad. Sci. USA 105, 7010–7015 (2008). (doi:10.1093/glycob/cwn072). 1096–1111 (2007). 136. Okamoto et al. Essential role of Notch signaling 84. Ju, T. & Cummings, R. D. Protein glycosylation: 110. Zhou, L. et al. Notch-dependent control of in effector memory CD8+ T cell-mediated airway chaperone mutation in Tn syndrome. Nature 437, myelopoiesis is regulated by fucosylation. Blood 112, hyperresponsiveness and inflammation. J. Exp. Med. 1252 (2005). 308–319 (2008). 205, 1087–1097 (2008). 85. Green, R. S. et al. Mammalian N-glycan branching 111. Irvine, K. D. & Wieschaus, E. Fringe, a boundary- 137. Schaller, M. A. et al. Notch ligand Delta-like 4 protects against innate immune self-recognition and specific signaling molecule, mediates interactions regulates disease pathogenesis during respiratory inflammation in autoimmune disease pathogenesis. between dorsal and ventral cells during Drosophila viral infections by modulating Th2 cytokines. J. Exp. Immunity 27, 308–320 (2007). wing development. Cell 79, 595–606 (1994). Med. 204, 2925–2934 (2007). 86. Bianco, G. A., Toscano, M. A., Ilarregui, J. M. & 112. Wu, J. Y., Wen, L., Zhang, W.-J. & Rao, Y. The secreted 138. Sun, J., Krawczyk, C. J. & Pearce, E. J. Suppression of Rabinovich, G. A. Impact of protein–glycan interactions product of Xenopus gene lunatic fringe, a vertebrate Th2 cell development by Notch ligands Delta1 and in the regulation of autoimmunity and chronic signaling molecule. Science 273, 355–358 (1996). Delta4. J. Immunol. 180, 1655–1661 (2008). inflammation. Autoimmunity Rev. 5, 349–356 (2006). 113. Goode, S. & Perrimon, N. Brainiac and fringe are 139. Morgan, R. et al. N-acetylglucosaminyltransferase V 87. Baum, L. G. et al. Amelioration of graft versus host similar pioneer proteins that impart specificity to (Mgat5)-mediated N-glycosylation negatively regulates disease by galectin-1. Clin. Immunol. 109, 295–307 notch signaling during Drosophila development. Th1 cytokine production by T cells. J. Immunol. 173, (2003). Cold Spring Harb. Symp. Quant. Biol. 62, 177–184 7200–7208 (2004). 88. Rubinstein, N. et al. Targeted inhibition of galectin-1 (1997). 140. Juszczynski, P. et al. The AP1-dependent secretion of in tumor cells results in heightened 114. Johnston, S. H. et al. A family of mammalian Fringe galectin-1 by Reed–Sternberg cells fosters immune T cell-mediated rejection: a potential mechanism of genes implicated in boundary determination and the privilege in classical . Proc. Natl tumor-immune privilege. Cancer Cell 5, 241–251 Notch pathway. Development 124, 2245–2254 Acad. Sci. USA 104, 13134–13139 (2007).

(2004). (1997). 141. Toscano, M. A. et al. Differential glycosylation of TH1,

89. Perone, M. J. et al. Dendritic cells expressing 115. Panin, V. M., Papayannopoulos, V., Wilson, R. & Irvine, TH2, and TH-17 effector cells selectively regulates transgenic galectin-1 delay onset of autoimmune K. D. Fringe modulates Notch–ligand interactions. susceptibility to cell death. Nature Immunol. 8, diabetes in mice. J. Immunol. 177, 5278–5289 Nature 387, 908–912 (1997). 825–834 (2007). (2006). 116. Brückner, K., Perez, L., Clausen, H. & Cohen, S. 142. Thomas, P. G. & Harn, D. A. Immune biasing by 90. Seki, M. et al. Galectin-9 suppresses the generation Glycosyltransferase activity of Fringe modulates helminth glycans. Cell. Microbiol. 6, 13–22 (2004). of Th17, promotes the induction of regulatory T cells, Notch–Delta interactions. Nature 406, 411–415 143. Opferman, J. T. & Korsmeyer, S. J. Apoptosis in the and regulates experimental autoimmune arthritis. (2000). development and maintenance of the immune system. Clin. Immunol. 127, 78–88 (2008). 117. Moloney, D. J. et al. Fringe is a glycosyltransferase Nature Immunol. 4, 410–415 (2003). 91. Barrionuevo, P. et al. A novel function for galectin-1 that modifies Notch. Nature 406, 369–375 (2000). 144. Hernandez, J. D. & Baum, L. G. Ah, sweet mystery of at the crossroad of innate and adaptive immunity: 118. Chen, J., Moloney, D. J. & Stanley, P. Fringe death! Galectins and control of cell fate. Glycobiology galectin-1 regulates monocyte/macrophage physiology modulation of Jagged1-induced Notch signaling 12, 127R–136R (2002). through a nonapoptotic ERK-dependent pathway. requires the action of β4galactosyltransferase-1. This publication reviews galectins in immune-cell J. Immunol. 178, 436–445 (2007). Proc. Natl Acad. Sci. USA 98, 13716–13721 (2001). apoptosis. 92. Blois, S. M. et al. A pivotal role for galectin-1 in 119. Wang, Y. et al. Modification of epidermal growth 145. Hsu, D. K., Yang, R. Y. & Liu, F. T. Galectins in fetomaternal tolerance. Nature Med. 13, factor-like repeats with O-fucose. Molecular cloning apoptosis. Methods Enzymol. 417, 256–273 1450–1457 (2007). and expression of a novel GDP-fucose protein (2006). 93. Taylor, K. R. et al. Recognition of hyaluronan released O-fucosyltransferase. J. Biol. Chem. 276, 146. Perillo, N. L., Pace, K. E., Seilhamer, J. J. & Baum, in sterile injury involves a unique receptor complex 40338–40345 (2001). L. G. Apoptosis of T cells mediated by galectin-1. dependent on Toll-like receptor 4, CD44 and MD-2. 120. Okajima, T. & Irvine, K. D. Regulation of Notch Nature 378, 736–739 (1995). J. Biol. Chem. 282, 18265–18275 (2007). signaling by O-linked fucose. Cell 111, 893–904 147. Stowell, S. R. et al. Human galectin-1, -2, and -4 94. Jiang, D., Liang, J. & Noble, P. W. Hyaluronan in tissue (2002). induce surface expression of phosphatidylserine in injury and repair. Annu. Rev. Cell Develop. Biol. 23, 121. Okajima, T., Xu, A. & Irvine, K. D. Modulation of activated human neutrophils but not in activated 435–461 (2007). Notch-ligand binding by protein O-fucosyltransferase T cells. Blood 109, 219–227 (2008).

886 | NOvEMbER 2008 | vOLUME 8 www.nature.com/reviews/immunol REVIEWS

148. Stowell, S. R. et al. Differential roles of galectin-1 and lectin-saccharide-mediated cellular interactions. antigen expressed on ICAM-2. Mol. Immunol. 45, galectin-3 in regulating leukcoyte viability and Curr. Opin. Struct. Biol. 12, 616–623 (2002). 2359–2369 (2008). cytokine secretion. J. Immunol. 180, 3091–3102 This article reviews the hypothesis that galectins 172. McDonald, B. et al. Interaction of CD44 and (2008). generate a spatially precise, repetitive structure hyaluronan is the dominant mechanism for neutrophil 149. Stowell, S. R. et al. Dimeric galectin-8 induces (lattice) that controls glycoprotein trafficking and sequestration in inflamed liver sinusoids. J. Exp. Med. phosphatidylserine exposure in leukocytes through function on the cell surface. 205, 915–927 (2008). polylactosamine recognition by the carboxyl terminal 162. Nieminen, J., Kuno, A., Hirabayashi, J. & Sato, S. 173. Bax, M. et al. Dendritic cell maturation results in domain. J. Biol. Chem. 283, 20547–20559 (2008). Visualization of galectin-3 oligomerization on the pronounced changes in glycan expression affecting 150. Dias-Baruffi, M. et al. Dimeric galectin-1 induces surface of neutrophils and endothelial cells using recognition by siglecs and galectins. J. Immunol. 179, surface exposure of phosphatidylserine and fluorescence resonance energy transfer. J. Biol. 8216–8224 (2007). phagocytic recognition of leukocytes without inducing Chem. 282, 1374–1383 (2007). 174. Hakomori, S. Glycosylation defining cancer apoptosis. J. Biol. Chem. 278, 41282–41293 (2003). 163. Rabinovich, G. A., Toscano, M. A., Jackson, S. S. & : new wine in an old bottle. Proc. Natl 151. Vespa, G. N. et al. Galectin-1 specifically modulates Vasta, G. R. Functions of cell surface galectin– Acad. Sci. USA 99, 10231–10233 (2002). TCR signals to enhance TCR apoptosis but inhibits IL-2 glycoprotein lattices. Curr. Opin. Struct. Biol. 175. Freeze, H. H. Genetic defects in the human glycome. production and proliferation. J. Immunol. 162, 17, 513–520 (2007). Nature Rev. Genet. 7, 537–551 (2006). 799–806 (1999). This publication reviews findings regarding the A review of the human genomic data that are 152. Pace, K. E., Hahn, H. P., Pang, M., Nguyen, J. T. & galectins and their potential to form various lattices known to contribute to genetic disease by altered Baum, L. G. CD7 delivers a pro-apoptotic signal during that can define functional units and membrane glycosylation. galectin-1-induced T cell death. J. Immunol. 165, domains. 176. Brooks, S. A. et al. Altered glycosylation of proteins 2331–2334 (2000). 164. Hernandez, J. D. et al. Galectin-1 binds different in cancer: what is the potential for new anti-tumour 153. Waizel, H., Blach, M., Hirabayashi, J., Kasai, K. I. & CD43 glycoforms to cluster CD43 and regulate T cell strategies. Anticancer Agents Med. Chem. 8, 2–21 Brock, J. Involvement of CD2 and CD3 in galectin-1 death. J. Immunol. 177, 5328–5336 (2006). (2008). induced signaling in human Jurkat T-cells. 165. Chen, I.-J., Chen, H.-L. & Demetriou, M. Lateral 177. Collins, B. t. et al. Masking of CD22 by cis ligands Glycobiology 10, 131–140 (2000). compartmentalization of T cell receptor versus CD45 does not prevent redistribution of CD22 to sites of cell 154. Fuertes, M. B. et al. Regulated expression of by galectin-N-glycan binding and microfilaments contact. Proc. Natl Acad, Sci. USA. 282, 6104–6109 galectin-1 during T-cell activation involves Lck and Fyn coordinate basal and activation signaling. J. Biol. (2004). kinases and signaling through MEK1/ERK, p38 MAP Chem. 282, 35361–35372 (2007). kinase and p70S6 kinase. Mol. Cell. Biochem. 267, 166. Lajoie, P. et al. Plasma membrane domain Acknowledgements 177–185 (2004). organization regulates EGFR signaling in tumor cells. We apologize to the colleagues and researchers who have 155. Cabrera, P. V. et al. Haploinsufficiency of C2GnT-I J. Cell Biol. 179, 341–356 (2007). made contributions that were not referenced owing to space glycosyltransferase renders T lymphoma cells resistant 167. Partridge, E. A. et al. Regulation of cytokine receptors limitations. J.D.M. is supported by the Howard Hughes to cell death. Blood 108, 2399–2406 (2006). by Golgi N-glycan processing and endocytosis. Science Medical Institute and grants from the National Institutes of 156. Hahn, H. P. et al. Galectin-1 induces nuclear 306, 120–124 (2004). Health, USA (HL57345, HL78784 and GM62116). translocation of endonuclease G in caspase- and 168. Ohtsubo, K., Takamatsu, S., Minowa, M. T., Yoshida, cytochrome c-independent T cell death. Cell Death A. & Marth, J. D. Dietary and genetic control of Diff. 11, 1277–1286 (2004). glucose transporter-2 glycosylation promotes insulin DATABASES 157. Stillman, B. N. et al. Galectin-2 and galectin-1 bind secretion in suppressing diabetes. Cell 123, Gene: http://www.ncbi.nlm.nih.gov/entrez/query. distinct cell surface glycoprotein receptors to induce 1307–1321 (2005). fcgi?db=gene T cell death. J. Immunol. 176, 778–789 (2006). 169. Lau, K. S. et al. Complex N-glycan number and αM-II | CD22 | COSMC | E-selectin | galectin-1 | GCNT1 | 158. Bi, S., Earl, L. A., Jacobs, L. & Baum, L. G. Structural degree of branching co-operate to regulate cell L-selectin | Mgat5 | Notch | P-selectin | PSGL1 | ST3Gal4 | features of galectin-9 and galectin-1 that determine proliferation and differentiation. Cell 129, 123–134 ST3Gal6 distinct T cell death pathways. J. Biol. Chem. 283, (2007). OMIM: http://www.ncbi.nlm.nih.gov/entrez/query. 12248–12258 (2008). 170. van Gisbergen K. P., Sanchez-Hernandez, M., fcgi?db=OMIM 159. Priatel, J. J. et al. The ST3Gal-I sialyltransferase Geijtenbeek, T. B. & van Kooyk, Y. Neutrophils rheumatoid arthritis | systemic lupus erythematosus | controls CD8+ T cell homeostasis by modulating medicate immune modulation of dendritic cells Tn syndrome O-glycan biosynthesis. Immunity 12, 273–283 (2000). through glycosylation-dependent interactions 160. Grewal, P. K. et al. The Ashwell receptor mitigates the between Mac-1 and DC-SIGN. J. Exp. Med. 201, FURTHER INFORMATION lethal coagulopathy of sepsis. Nature Med. 14, 1281–1292 (2005). Jamey D. Marth’s homepage: http://www.hhmi.org/ 648–655 (2008). 171. Garcia-Vallejo, J. J. et al. DC-SIGN mediates adhesion research/investigators/marth_bio.html 161. Brewer, C. F., Miceli, M. C. & Baum, L. G. Clusters, and rolling of dendritic cells on primary human All linkS ARE ACTivE in THE onlinE pdF bundles, arrays, and lattices: novel mechanisms for umbilical vein endothelial cells through Lewis(Y)

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