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Siglecs: sialic-acid-binding immunoglobulin-like lectins in cell–cell interactions and signalling
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Siglecs: sialic-acid-binding immunoglobulin-like lectins in cell–cell interactions and signalling Paul R Crocker Siglecs are sialic-acid-binding immunoglobulin-like lectins involved in cell–cell interactions and signalling functions in the haemopoietic, immune and nervous systems. Significant advances have been made in our understanding of the link between carbohydrate recognition and signalling for two well-characterised siglecs, CD22 and myelin-associated glycoprotein. Over the past few years, several novel siglecs have been discovered through genomics and functional screens. These ‘CD33-related’ siglecs have molecular features of inhibitory receptors and may be important in regulating leucocyte activation during immune and inflammatory responses. Addresses The Wellcome Trust Biocentre at Dundee, Division of Cell Biology and Immunology, School of Life Sciences, Dundee University, Dow Street, Dundee DD1 5EH, UK; e-mail: [email protected] Current Opinion in Structural Biology 2002, 12:609–615 0959-440X/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations BCR B-cell receptor Ig immunoglobulin ITIM immunoreceptor tyrosine-based inhibitory motif MAG myelin-associated glycoprotein p75NTR p75 neurotrophin receptor
Introduction The cell surface is richly decorated in glycans that are attached to proteins and lipids. The exposed termini of oligosaccharides are often capped with sialic acid — the term given to a diverse family of nine-carbon sugars derived from neuraminic acid (Neu) or keto-deoxynonulosonic acid (Kdn). Sialic acids are typically found attached to other sugars through α2,3 and α2,6 linkages, or to another sialic acid molecule through an α2,8 linkage. They therefore make an important contribution to the overall structural heterogeneity of glycans and are well suited to act as ligands in complex cellular recognition events, such as those that occur in the nervous and immune systems. Although for many years sialic acids have been known to act as host ligands for pathogen receptors, it is only relatively recently that endogenous receptors for binding sialic acid have been identified. The most prominent group among these receptors is the siglecs, which are members of the immunoglobulin (Ig) superfamily [1]. In this review, I discuss recent advances in our knowledge of the existence, structure and functions of siglecs.
The siglec family The founding members of the siglec family were sialoadhesin (siglec-1), a macrophage adhesion molecule [2];
CD22 (siglec-2), a B-cell inhibitory receptor [3]; CD33 (siglec-3), a marker of myeloid cells [4]; and myelinassociated glycoprotein (MAG or siglec-4), which is expressed by oligodendrocytes and Schwann cells in the nervous system [5]. These proteins share about 25–30% sequence similarity in their extracellular regions. In the past few years, several human, ape and mouse members of the siglec family have been identified through genomics and functional analyses (Table 1). All are highly related to CD33 and to each other, sharing about 50–80% sequence similarity. They are therefore described collectively as ‘CD33-related siglecs’ and can be considered as a separate subgroup, from both functional and evolutionary perspectives, to sialoadhesin, CD22 and MAG. The nomenclature for CD33-related siglecs is numerical in humans (e.g. hSiglec-5) and alphabetical in mice (e.g. mSiglec-E). This difference reflects current uncertainties in assigning murine orthologues to human CD33-related siglecs, as discussed below. All siglecs possess a characteristic N-terminal sialic-acidbinding V-set Ig domain and between 1 (CD33) and 16 (sialoadhesin) C2-set domains that project the sugarbinding site away from the plasma membrane. Each siglec has a distinct preference for specific types of sialic acid and also for specific types of linkage to subterminal sugars. These binding preferences are likely to be related to their biological functions; for example, CD22 on B cells binds specifically to α2,6-linked sialic acids in N-glycans that are co-expressed on the same cells and mediate biologically important CD22-dependent interactions [6,7••]. The cytosolic tails of siglecs vary in sequence and length, although most CD33-related siglecs share regions of sequence similarity surrounding two conserved tyrosine motifs that are implicated in signalling functions. The genes encoding human siglecs are on chromosome 20p (sialoadhesin) or 19q (all other siglecs), and those encoding putative murine orthologues are in syntenic regions of chromosomes 2 and 7, respectively.
Sialic acid recognition by siglecs: how is specificity achieved? As discussed in a recent review [8•], sialic acids may be evolutionarily ancient, but their expression in eukaryotes is primarily in the deuterostome lineage of animals (i.e. vertebrates and some higher invertebrates, such as starfish). On the basis of sequence alignments [9] and the absence of sigleclike sequences in the completed fly and worm genomes [10], it seems likely that siglecs evolved from a protein-binding Ig domain precursor that was adapted to sialic acid recognition.
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Table 1 Properties of human and mouse siglecs.
Human siglecs
Mouse siglecs
Number of Ig domains
Number of cytoplasmic tyrosine motifs
Sialic-acid-binding specificity*
Expression
17 0 2,32,62,8 Mac 7 6 2,6 B 2 2 or 0‡ 2,62,3 My-pro, Mon 5 1 2,32,6 Oligo, Schwann 4 2 2,3=2,62,8 Mon, N, B 3 2 STn B, Plac 3 2 2,82,62,3 Mon, NK 3 2 2,32,6 Eos, Baso, Mast †¶ mSiglec-E 3 2 2,3=2,6 Mon, N, NK, B mSiglec-G†¶ 5 3 2,3=2,6 Mon, NK 5 2 2,8 Mac †¶ mSiglec-H 2 0 ND ND *2,3, 2,6 and 2,8 refer to 2,3-, 2,6- and 2,8-glycosidic linkages of sialic acid to galactose (2,3 and 2,6) or sialic acid itself (2,8). †The sialic-acid-binding properties of these mouse ‘siglecs’ have not been determined. ‡hCD33 has two tyrosine-based motifs, whereas the postulated murine orthologue has none. §mSiglec-F is expressed on immature myeloid cells in the bone marrow and at lower levels on subsets of mature myeloid cells [40]. ¶The expression patterns of these mouse ‘siglecs’ have not been determined. B, B cells; Baso, basophils; Eos, eosinophils; Mac, macrophages; Mast, mast cells; Mon, monocytes; My-pro, myeloid progenitors; N, neutrophils; ND, not determined; NK, natural killer cells; Oligo, oligodendrocytes; Plac, placental trophoblasts; Schwann, Schwann cells. Sialoadhesin/hSiglec-1 CD22/hSiglec-2 CD33/hSiglec-3 MAG/hSiglec-4 hSiglec-5 hSiglec-6 hSiglec-7 hSiglec-8 hSiglec-9 hSiglec-10 hSiglec-11
Sialoadhesin/mSiglec-1 CD22/mSiglec-2 CD33/mSiglec-3† MAG/mSiglec-4 mSiglec-F§
Important molecular insights have been obtained from the crystal structure of the sialoadhesin V-set domain bound to 3′-sialyllactose at 1.85 Å resolution (Figure 1) [11]. This structure reveals the basic template for sialic acid recognition; this template is conserved among all siglecs and includes an invariant arginine on β strand F that makes a key salt bridge with the carboxylate of sialic acid. Specificity for different sialic acid linkages and extended glycan structures is likely to be provided by the highly variable loop regions between the β strands. Clear evidence for this has emerged from a study comparing hSiglec-7 and hSiglec-9 [12••]. Despite sharing about 76% sequence identity in the V-set domain, hSiglec-7 shows a striking preference for α2,8-linked disialic acids and for sialic acid with a α2,6 linkage to an internal N-acetylglucosamine [12••,13], whereas hSiglec-9 prefers terminal α2,3 and α2,6 linkages [10,12••,14]. Experiments using protein chimaeras of hSiglec-7 and hSiglec-9 have shown that the sialic acid linkage preference is determined by a sequence of six amino acids in the C–C′ loop of the V-set domain [12••]. Modelling suggests that a basic region close to the C–C′ loop can accommodate a second sialic acid in the α2,8-linked disialic acid motif (Figure 1). The significance of these sugar-binding preferences is currently unclear and will probably require an understanding of the biological functions of each molecule.
Trans and cis interactions: linking sialic acid recognition to signalling Ligand recognition by siglecs is complicated by the fact that these receptors interact with their sialic acid ligands both in cis and in trans. Cis interactions can result in masking of the sialic-acid-binding site at the cell surface, which can prevent trans interactions [15]. In the case of B cells, the binding site of CD22 can be unmasked by
treatment with sialidase or after cellular activation with anti-IgM and anti-CD40 [15]. Trans interactions and CD22 signalling
CD22 is a well-characterised B cell inhibitory receptor containing three immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that associates with the B-cell receptor (BCR) and inhibits cellular activation by recruiting the inhibitory tyrosine phosphatase SHP-1 (Figure 2a). B cells express high concentrations of α2,6-linked sialic acids, which results in the masking of CD22 [15]. A minor subset of B cells in mice has been shown to express unmasked CD22 [16] and it has been suggested that trans interactions between CD22 and sialylated ligands on bone marrow sinusoidal endothelial cells lead to the selective accumulation of these cells in the bone marrow [16,17]. Notably, trans interactions may occur even when CD22 is masked at the cell surface (Figure 2b): a series of elegant experiments has shown that, when B cells are activated in response to membrane-associated antigens, the co-expression of α2,6-linked sialic acids results in a CD22-dependent dampening of activation [18••]. Cis interactions and CD22 signalling
Evidence for the importance of cis interactions with α2,6-linked sialic acid in CD22-mediated inhibitory signalling has been provided by two recent studies [7••,19••]. The first study showed that a novel sialic-acid-based CD22specific inhibitor causes heightened activation of B cells in response to BCR cross-linking, which is accompanied by hypophosphorylation of CD22 and reduced recruitment of SHP-1 [7••]. The second study used CD22-deficient B-cell lines transfected with either the wild-type or mutant forms of CD22 in which the N-terminal domains had been removed or the sialic-acid-binding site had been inactivated by two point mutations. With both mutants of CD22, there
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Figure 1 Surface charge representations of the V-set domains of mouse sialoadhesin and hSiglec-7. (a) Structure of sialoadhesin complexed with 3′-sialyllactose. (b) Model of hSiglec-7 based on the solved sialoadhesin structure. Red indicates acidic regions and blue indicates basic regions. The hSiglec-7 model reveals the presence of a basic pocket (circled in yellow), located close to the C–C′ loop (outlined in green), that may favour interactions with the second sialic acid residue of the α2,8-linked disialic acid motif. The sequence-predicted binding site on hSiglec-7 for terminally positioned sialic acid is circled in white. Homology modelling was carried out using the program WHATIF and the diagrams were prepared using GRASP. For further information, see [12••].
(a)
(b)
C–C′ loop
Current Opinion in Structural Biology
was less reduction in inhibition of BCR triggering than with wild-type CD22 [19••]. Both studies concluded that the sialic-acid-dependent cis-interacting partners might be either the BCR itself or sequestering sialoglycoproteins such as the CD45 tyrosine phosphatase. The latter is thought to be important for activating the Lyn tyrosine kinase, which in turn phosphorylates CD22, leading to SHP-1 recruitment and activation (Figure 2a).
that MAG can mediate high-affinity, sialic-acid-independent interactions with the Nogo receptor — a glycosylphosphatidylinositol-linked protein that is important in neurite inhibition [26••,27••]. Taken together, these findings indicate that MAG-dependent inhibition of neurite outgrowth depends on a complex four-way molecular interaction between MAG, GT1b, p75NTR and Nogo receptor (Figure 2c).
MAG-dependent signalling and sialic acid recognition
Distinct repertoires of CD33-related siglecs in man and mouse
MAG (siglec-4) is expressed by oligodendrocytes and Schwann cells in the nervous system, and is important for maintaining the integrity of the myelin sheath [20]. MAG has also been studied extensively as an inhibitor of neurite outgrowth in postnatal neuronal cells [21]. Recently, significant advances have been made in our understanding of how sialic acid recognition by MAG triggers signalling events (see also the review by Allende and Proia [pp 587–592] in this issue). Vinson and co-workers [22•] proposed that ganglioside GT1b is an important MAG ligand on neuronal cells because antibodies against GT1b can mimic the inhibitory effect of MAG on neurite outgrowth. A subsequent study confirmed this hypothesis and also proposed that ganglioside GD1a is a functional MAG ligand [23•]. It has been postulated that the MAG-dependent clustering of GD1a and GT1b in lipid rafts activates a Rho-dependent signalling pathway that leads to the inhibition of neurite outgrowth [24]. Notably, a recent study has shown that GT1b associates with the p75 neurotrophin receptor (p75NTR) [25••]. Neurons from mice lacking p75NTR do not respond to a recombinant soluble form of MAG; addition of this protein to wild-type neurons stimulates Rho activation in a p75NTR-dependent manner [25••]. A further twist to this story has been provided by two recent studies that report
Seven new human siglecs and four new mouse siglecs have been discovered by genomics and in functional screens over the past few years [10,14,28–33,34•,35•,36–39,40••, 41•,42,43•,44–46]. Whereas the human genome contains eight CD33-related siglecs, one siglec-like gene and about sixteen siglec pseudogenes, the mouse genome contains only five CD33-related genes and two siglec pseudogenes [40••,46]. The genes encoding hSiglec-3 to hSiglec-10 are clustered within a 506 kb region on human chromosome 19q13.3–13.4; the gene encoding hSiglec-11 is located about 1 Mb upstream of this region but still within cytological band 19q13.3–13.4 [40••,46,47]. Apart from mSiglec-H, which is located on mouse chromosome 7B4, all murine CD33-related siglecs lie in a syntenic region of human chromosome 19q13.3–13.4 on mouse chromosome 7B2. Human chromosome 19 is unusually rich in genes and contains large families of Ig-related receptors, including the carcinoembryonic antigen family, killer inhibitory receptors and Ig-like transcripts, in addition to the CD33related siglecs (reviewed in [48]). The marked expansion of Ig-like families on chromosome 19q may be due, at least in part, to the presence of minisatellites specific to chromosome 19 that facilitated gene duplication by acting as nucleation sites for unequal crossovers during meiotic
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Figure 2 (a)
(b)
(c)
Inhibition of neurite outgrowth
Antigen-presenting cell
Rho kinase
Antigen
Rho p75NTR
CD45
CD22
BCR BCR SHP-1
–
SHP-1
SHP-1
Lyn
Ca2+
– Ca2+
MAG
– Ca2+ Myelin-forming cell
Setting B cell activation thresholds α2,6-linked sialic acid
Dampening B cell activation α2,6-linked sialic acid
GT1b
Nogo receptor
Current Opinion in Structural Biology
Sialic-acid-dependent signalling pathways mediated by CD22 and MAG. (a) Cis interactions between CD22 and either BCR or CD45 may result in activation of SHP-1 and inhibition of BCR-triggered B cell activation. For further details, see [7••,19••]. (b) Trans interactions of CD22 on B cells with α2,6-linked sialic acids co-expressed with an antigen dampen B cell activation. For more details, see [18••].
(c) Binding of MAG to GT1b on neuronal cells results in p75NTRdependent activation of Rho, leading to activation of Rho kinase and inhibition of neurite outgrowth. MAG can also bind the Nogo receptor in a sialic-acid-independent manner and is functionally important in MAG-dependent neurite inhibition. For additional information, see [22•,23•,24,25••–27••].
recombination [10,49]. In the case of CD33-related siglecs, frequent gene duplications have given rise to functional specialisation; for example, hSiglec-6 is expressed on placental trophoblasts, hSiglec-8 on eosinophils and hSiglec-11 on tissue macrophages (see below). None of these proteins has an obvious counterpart in the mouse proteome.
difficult to assign orthologues between the two species. This is in contrast to sialoadhesin, CD22 and MAG, which share about 25–30% identity and have clear-cut orthologues in all mammalian species examined so far. On the basis of gene structure, sequence alignments, relative chromosomal localisation and, where available, functional properties, it has been proposed that ‘mCD33’, mSiglec-E and mSiglec-F are orthologues of hCD33, hSiglec-9 and hSiglec-5, respectively [40••]. But mCD33 lacks ITIM-like motifs [4,51], and mSiglec-E and mSiglec-F share only about 50% identity with hSiglec-5 and hSiglec-9, respectively. By contrast, mSiglec-G is a clear orthologue of hSiglec-10 and shares about 60% sequence identity [40••]. The possible evolutionary origins of the CD33-related siglecs from a Siglec-10-like precursor have been discussed [46], but firm conclusions will require the completion of genome sequencing projects in a range of vertebrate and invertebrate species.
Human siglec-like molecule 1 (hSiglec-L1), also known as siglec with two V-set domains (S2V), is an unusual human siglec-like sequence, with two tandem V-set Ig domains that each lack the essential arginine required for recognition of sialic acid [41•,50•]. Surprisingly, one group has reported the sialic-acid-dependent binding of erythrocytes to hSiglec-L1 expressed in COS cells [41•], although this result is controversial as another laboratory has been unable to reproduce it [50•]. In addition, it has been shown that restoring the essential arginine in hSiglec-L1 leads to the recovery of sialic-acid-dependent binding in this normally ‘dead’ siglec-like molecule. Notably, the chimpanzee orthologue of hSiglec-L1 has retained the essential arginine in the N-terminal V-set domain and can mediate robust sialic-acid-dependent binding when expressed in COS cells [50•]. The sequence similarity shared between CD33-related siglecs in humans and mice (about 50–80%) makes it
CD33-related siglecs as inhibitory receptors of the innate immune system Collectively, the CD33-related siglecs are expressed broadly in the innate immune system, but are strikingly absent from most T lymphocytes (reviewed in [52•]; see Table 1). The presence of two conserved ITIM-like motifs in the cytoplasmic regions of all hCD33-related siglecs [53],
Siglecs Crocker
coupled with the differential expression of these proteins, suggests that they have a generic role in regulating the cellular activation of haemopoietic and immune cells. Detailed studies of several CD33-related siglecs have shown that these siglecs can recruit the inhibitory tyrosine phosphatases SHP-1 and SHP-2, but only after the cells are treated with pervanadate to inhibit endogenous tyrosine phosphatases [37,41•,43•,44,46,54,55]. It remains to be shown whether tyrosine phosphorylation can occur under physiological conditions. Mutagenesis studies have shown that the distal ITIM-like motif is dispensable for phosphatase binding, which presumably reflects its departure from the consensus ITIM sequence [37,41•,43•,44,54,55]. The ITIM-like motif is highly conserved among CD33-related siglecs, however, which suggests that it may be important for interacting with specific downstream signalling molecules. Notably, it is similar to a tyrosine-based motif in proteins related to SLAM (signalling lymphocyte activation molecule) that binds SAP — a single SH2 domain protein that inhibits the recruitment of SHP-2 and activates additional signalling pathways [56]. A recent report has also shown that the cytoplasmic tail of hCD33 can be also phosphorylated on serine residues by protein kinase C and that this can regulate sialic-acid-binding activity in transfected cells [57•]. Further studies are needed to examine serine/threonine phosphorylation in other CD33-related siglecs.
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Conclusions The siglec family has emerged as a principal subset of the Ig superfamily. On the one hand, siglecs can mediate cell–cell interactions, as exemplified by sialoadhesin on macrophages and MAG on oligodendrocytes and Schwann cells. On the other hand, siglecs are well suited for mediating cis interactions with sialic acids on the same plasma membrane and for modulating leucocyte activation, as demonstrated by CD22. Understanding how changes in glycosylation modulate the cis and trans interactions of siglecs with sialic acids, and elucidating the subsequent biological responses are interesting issues for the future. The marked expansion of CD33-related siglecs in the innate immune system suggests that these proteins have a fundamentally important role in host defence functions. Sialic acids are well-suited as markers of ‘self’, as they are absent from many pathogens but are expressed universally on the surfaces of host cells. Recent findings on CD22 show how trans interactions with sialic acids can dampen B cell activation. Similarly, CD33-related siglecs may regulate the autoreactivity of myeloid cells. The expanded repertoires of CD33-related siglecs in humans, compared with mice, illustrate how rapidly this gene family is evolving. The use of genetically modified mice and novel sialic-acidbased inhibitors is expected to give important insights into the functions of these intriguing molecules in the future.
Acknowledgements Evidence that siglecs can mediate inhibitory signals has been obtained in studies using antibodies to artificially cross-link hCD33 or mouse inhibitory siglec/mSiglec-E with an activating human receptor, FcγR1 on mononuclear phagocytes [37,55,58]. This results in reduced Ca2+ influx compared with cross-linking FcγRI alone. Likewise, clustering hSiglec-7 with a monoclonal antibody can inhibit the cytotoxicity of natural killer cells [31]. The addition of antibodies against hCD33 or against hSiglec-7 to haemopoietic cell cultures results in reduced cell growth and inhibition of dendritic cell development [59,60]. The physiological relevance of these findings remains unclear, because antibodies rather than natural ligands were used to cluster the siglecs. Similar to CD22, CD33-related siglecs are masked at the cell surface [61] and the extent to which they become unmasked on cellular activation is unknown. Therefore, although trans interactions may occur with sialic acids on other cells and dampen leucocyte activation in a manner similar to that shown for CD22 [18••], it is also possible that cis interactions with specific glycoproteins on the same plasma membrane could modulate the responses of leucocytes to activation. For the latter case, it will be important to identify cis-interacting partners for individual siglecs on different leucocyte populations and to investigate whether cellular activation leads to glycosylation changes that modulate the signalling functions of CD33-related siglecs.
Research in the author’s laboratory is supported by the Wellcome Trust, Biotechnology and Biological Sciences Research Council and the Consortium for Functional Glycomics. I am grateful to Magnus Alphey for help with preparing Figure 2, and Tony Avril and Ajit Varki for helpful comments on the manuscript.
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22. Vinson M, Strijbos PJ, Rowles A, Facci L, Moore SE, Simmons DL, • Walsh FS: Myelin-associated glycoprotein interacts with ganglioside GT1b. A mechanism for neurite outgrowth inhibition. J Biol Chem 2001, 276:20280-20285. See annotation to [23•].
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10. Angata T, Varki A: Cloning, characterization, and phylogenetic analysis of siglec-9, a new member of the CD33-related group of siglecs. Evidence for co-evolution with sialic acid synthesis pathways. J Biol Chem 2000, 275:22127-22135. 11. May AP, Robinson RC, Vinson M, Crocker PR, Jones EY: Crystal structure of the N-terminal domain of sialoadhesin in complex with 3′′ sialyllactose at 1.85 Å resolution. Mol Cell 1998, 1:719-728. 12. Yamaji T, Teranishi T, Alphey MS, Crocker PR, Hashimoto Y: A small •• region of the natural killer cell receptor, Siglec-7, is responsible for its preferred binding to α2,8-disialyl and branched α2,6-sialyl residues. A comparison with Siglec-9. J Biol Chem 2002, 277:6324-6332. By using multivalent glycoprobes carrying different oligosaccharides, the authors show that closely related hSiglec-7 and hSiglec-9 have distinct preferences for sialic acid linkages to other sugars, as well as for sialic acid with a α2,8 linkage to itself. The creation of chimaeric ‘swap’ mutants led to the identification of a sequence of six amino acids in the C–C′ loop of hSiglec-7 that confers hSiglec-7 binding specificity when transferred to hSiglec-9 and vice versa. 13. Ito A, Handa K, Withers DA, Satoh M, Hakomori S: Binding specificity of siglec7 to disialogangliosides of renal cell carcinoma: possible role of disialogangliosides in tumor progression. FEBS Lett 2001, 504:82-86. 14. Zhang JQ, Nicoll G, Jones C, Crocker PR: Siglec-9, a novel sialic acid binding member of the immunoglobulin superfamily expressed broadly on human blood leukocytes. J Biol Chem 2000, 275:22121-22126. 15. Razi N, Varki A: Masking and unmasking of the sialic acid-binding lectin activity of CD22 (Siglec-2) on B lymphocytes. Proc Natl Acad Sci USA 1998, 95:7469-7474. 16. Floyd H, Nitschke L, Crocker PR: A novel subset of murine B cells that expresses unmasked forms of CD22 is enriched in the bone marrow: implications for B-cell homing to the bone marrow. Immunology 2000, 101:342-347. 17.
Nitschke L, Floyd H, Ferguson DJ, Crocker PR: Identification of CD22 ligands on bone marrow sinusoidal endothelium implicated in CD22-dependent homing of recirculating B cells. J Exp Med 1999, 189:1513-1518.
18. Lanoue A, Batista FD, Stewart M, Neuberger MS: Interaction of •• CD22 with α2,6-linked sialoglycoconjugates: innate recognition of self to dampen B cell autoreactivity? Eur J Immunol 2002, 32:348-355. This study shows that CD22, even when masked at the B-cell surface, can interact in trans with α2,6 sialylated ligands on antigen-presenting cells and dampen activation of the B cells. These findings have important implications for the functions of siglecs in ‘self’/‘nonself’ discrimination. 19. Jin L, McLean PA, Neel BG, Wortis HH: Sialic acid binding domains •• of CD22 are required for negative regulation of B cell receptor signaling. J Exp Med 2002, 195:1199-1205. In these two studies [7••,19••], different experimental approaches are taken to demonstrate the importance of cis interactions with sialic acid in the inhibitory signalling functions of CD22. One study [7••] uses a novel sialicacid-based CD22 inhibitor and the other [19••] uses genetic complementation of CD22-deficient B-cell lines with wild-type CD22 or mutant forms of CD22 that cannot bind sialic acid. Addition of the inhibitor or expression of the mutant forms of CD22 result in a clear reduction in inhibitory signalling by CD22. Although the cis-interacting partners of CD22 are unknown, together with [18••], these are the first studies to link sialic acid recognition to signalling mediated by siglecs. 20. Schachner M, Bartsch U: Multiple functions of the myelin-associated glycoprotein MAG (siglec-4a) in formation and maintenance of myelin. Glia 2000, 29:154-165. 21. Tang S, Shen YJ, DeBellard ME, Mukhopadhyay G, Salzer JL, Crocker PR, Filbin MT: Myelin-associated glycoprotein interacts with neurons via a sialic acid binding site at Arg118 and a distinct neurite inhibition site. J Cell Biol 1997, 138:1355-1366.
24. McKerracher L: Ganglioside rafts as MAG receptors that mediate blockade of axon growth. Proc Natl Acad Sci USA 2002, 99:7811-7813. 25. Yamashita T, Higuchi H, Tohyama M: The p75 receptor transduces •• the signal from myelin-associated glycoprotein to Rho. J Cell Biol 2002, 157:565-570. This paper shows that neurons from mice lacking p75NTR do not undergo inhibition of neurite outgrowth in response to the addition of soluble MAG. In addition, MAG is shown to activate a signalling pathway involving p75NTRdependent Rho activation. p75NTR is tightly associated with GT1b and co-localised with MAG, consistent with the functionally important MAG–GT1b interactions described in [22•,23•]. 26. Domeniconi M, Cao Z, Spencer T, Sivasankaran R, Wang K, •• Nikulina E, Kimura N, Cai H, Deng K, Gao Y et al.: Myelin-associated glycoprotein interacts with the nogo66 receptor to inhibit neurite outgrowth. Neuron 2002, 35:283-290. See annotation to [27••]. 27. ••
Liu BP, Fournier A, GrandPre T, Strittmatter SM: Myelin-associated glycoprotein as a functional ligand for the Nogo-66 receptor. Science 2002, 297:1190-1193. These two studies [26••,27••] show that MAG can mediate high-affinity (10–20 nM) sialic-acid-independent interactions with the Nogo receptor — a glycosylphosphatidylinositol-anchored glycoprotein. Both studies provide convincing evidence that MAG–Nogo receptor interactions are required for MAG-dependent inhibition of neurite outgrowth. Together with [22•,23•,25••], these reports suggest that a complex four-way molecular interaction occurs between MAG, GT1b, p75NTR and Nogo receptor in the regulation of neurite outgrowth. 28. Takei Y, Sasaki S, Fujiwara T, Takahashi E, Muto T, Nakamura Y: Molecular cloning of a novel gene similar to myeloid antigen CD33 and its specific expression in placenta. Cytogenet Cell Genet 1997, 78:295-300. 29. Cornish AL, Freeman S, Forbes G, Ni J, Zhang M, Cepeda M, Gentz R, Augustus M, Carter KC, Crocker PR: Characterization of siglec-5, a novel glycoprotein expressed on myeloid cells related to CD33. Blood 1998, 92:2123-2132. 30. Patel N, Linden EC, Altmann SW, Gish K, Balasubramanian S, Timans JC, Peterson D, Bell MP, Bazan JF, Varki A et al.: OB-BP1/Siglec-6. A leptin- and sialic acid-binding protein of the immunoglobulin superfamily. J Biol Chem 1999, 274:22729-22738. 31. Falco M, Biassoni R, Bottino C, Vitale M, Sivori S, Augugliaro R, Moretta L, Moretta A: Identification and molecular cloning of p75/AIRM1, a novel member of the sialoadhesin family that functions as an inhibitory receptor in human natural killer cells. J Exp Med 1999, 190:793-802. 32. Nicoll G, Ni J, Liu D, Klenerman P, Munday J, Dubock S, Mattei MG, Crocker PR: Identification and characterization of a novel siglec, siglec-7, expressed by human natural killer cells and monocytes. J Biol Chem 1999, 274:34089-34095. 33. Foussias G, Yousef GM, Diamandis EP: Identification and molecular characterization of a novel member of the siglec family (SIGLEC9). Genomics 2000, 67:171-178. 34. Floyd H, Ni J, Cornish AL, Zeng Z, Liu D, Carter KC, Steel J, • Crocker PR: Siglec-8. A novel eosinophil-specific member of the immunoglobulin superfamily. J Biol Chem 2000, 275:861-866. See annotation to [35•].
Siglecs Crocker
35. Kikly KK, Bochner BS, Freeman SD, Tan KB, Gallagher KT, • D’Alessio KJ, Holmes SD, Abrahamson JA, Erickson-Miller CL, Murdock PR et al.: Identification of SAF-2, a novel siglec expressed on eosinophils, mast cells, and basophils. J Allergy Clin Immunol 2000, 105:1093-1100. These two papers [34•,35•] demonstrate the cell-type-restricted pattern of expression of siglecs that is a hallmark of many family members. By using specific monoclonal antibodies, hSiglec-8 is shown to be expressed predominantly on eosinophils in the circulation, but not on neutrophils. Although the cDNA characterised in these studies [34•,35•] does not include ITIM-like motifs, hSiglec-8 has been shown subsequently to also exist in an alternatively spliced form containing ITIM-like motifs [51]. 36. Angata T, Varki A: Siglec-7: a sialic acid-binding lectin of the immunoglobulin superfamily. Glycobiology 2000, 10:431-438. 37.
Ulyanova T, Shah DD, Thomas ML: Molecular cloning of MIS, a myeloid inhibitory siglec that binds tyrosine phosphatases SHP-1 and SHP-2. J Biol Chem 2001, 276:14451-14458.
38. Foussias G, Taylor SM, Yousef GM, Tropak MB, Ordon MH, Diamandis EP: Cloning and molecular characterization of two splice variants of a new putative member of the Siglec-3-like subgroup of Siglecs. Biochem Biophys Res Commun 2001, 284:887-899. 39. Li N, Zhang W, Wan T, Zhang J, Chen T, Yu Y, Wang J, Cao X: Cloning and characterization of Siglec-10, a novel sialic acid binding member of the Ig superfamily, from human dendritic cells. J Biol Chem 2001, 276:28106-28112. 40. Angata T, Hingorani R, Varki NM, Varki A: Cloning and •• characterization of a novel mouse Siglec, mSiglec-F: differential evolution of the mouse and human (CD33) Siglec-3-related gene clusters. J Biol Chem 2001, 276:45128-45136. The authors characterise a novel mouse CD33-related siglec, mSiglec-F, that is thought to be the orthologue of hSiglec-5, and then characterise the whole murine CD33-related siglec locus using the Celera Genomics database. This analysis reveals striking differences between the murine siglec locus and the human CD33-related siglec locus, and shows that, unlike the other siglecs (CD22, MAG and sialoadhesin), genes encoding the CD33-related siglecs underwent extensive duplications subsequent to mammalian speciation. 41. Yu Z, Lai CM, Maoui M, Banville D, Shen SH: Identification and • characterization of S2V, a novel putative siglec that contains two V set Ig-like domains and recruits protein-tyrosine phosphatases SHPs. J Biol Chem 2001, 276:23816-23824. See annotations to [43•,50•]. 42. Munday J, Kerr S, Ni J, Cornish AL, Zhang JQ, Nicoll G, Floyd H, Mattei MG, Moore P, Liu D et al.: Identification, characterization and leucocyte expression of Siglec-10, a novel human sialic acid-binding receptor. Biochem J 2001, 355:489-497. 43. Yu Z, Maoui M, Wu L, Banville D, Shen S-H: mSiglec-E, a novel • mouse CD33-related siglec (sialic acid binding immunoglobulinlike lectin) that recruits Src homology 2 (SH2)-domain containing protein tyrosine phosphatases SHP-1 and SHP-2. Biochem J 2001, 353:483-492. These studies [41•,43•] identify a novel murine siglec and a novel human siglec-like gene in a yeast two-hybrid screen using SHP-1 as a bait. A detailed characterisation of the interaction between the tyrosine-based motifs of these proteins and tyrosine phosphatases SHP-1 and SHP-2 is undertaken, and the membrane-proximal motif is found to be indispensable for binding in each protein. 44. Whitney G, Wang S, Chang H, Cheng KY, Lu P, Zhou XD, Yang WP, McKinnon M, Longphre M: A new siglec family member, siglec-10, is expressed in cells of the immune system and has signaling properties similar to CD33. Eur J Biochem 2001, 268:6083-6096. 45. Yousef GM, Ordon MH, Foussias G, Diamandis EP: Molecular characterization, tissue expression, and mapping of a novel Siglec-like gene (SLG2) with three splice variants. Biochem Biophys Res Commun 2001, 284:900-910. 46. Angata T, Kerr SC, Greaves DR, Varki NM, Crocker PR, Varki A: Cloning and characterization of human Siglec-11. A recently
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evolved signaling molecule that can interact with SHP-1 and SHP-2 and is expressed by tissue macrophages, including brain microglia. J Biol Chem 2002, 277:24466-24474. 47.
Yousef GM, Ordon MH, Foussias G, Diamandis EP: Genomic organization of the siglec gene locus on chromosome 19q13.4 and cloning of two new siglec pseudogenes. Gene 2002, 286:259-270.
48. Trowsdale J, Barten R, Haude A, Stewart CA, Beck S, Wilson MJ: The genomic context of natural killer receptor extended gene families. Immunol Rev 2001, 181:20-38. 49. Fitch DH, Bailey WJ, Tagle DA, Goodman M, Sieu L, Slightom JL: Duplication of the gamma-globin gene mediated by L1 long interspersed repetitive elements in an early ancestor of simian primates. Proc Natl Acad Sci USA 1991, 88:7396-7400. 50. Angata T, Varki NM, Varki A: A second uniquely human mutation • affecting sialic acid biology. J Biol Chem 2001, 276:40282-40287. These two groups [41•,50•] identify an unusual human siglec-like molecule, hSiglec-L1 or S2V that contains two tandem V-set domains. The essential arginine that is required for sialic acid binding is replaced by cysteine in the first V-set domain and by glutamine in the second. Although one group [41•] observed sialic-acid-dependent binding in transfected COS cells, the other group did not [50•], but instead showed that mutation of the cysteine to arginine results in robust sialic-acid-dependent binding. Notably, the chimpanzee orthologue has retained the arginine in the first V-set domain and can mediate sialic-acid-dependent binding of human erythrocytes [50•]. 51. Tchilian EZ, Beverley PC, Young BD, Watt SM: Molecular cloning of two isoforms of the murine homolog of the myeloid CD33 antigen. Blood 1994, 83:3188-3198. 52. Crocker PR, Varki A: Siglecs, sialic acids and innate immunity. • Trends Immunol 2001, 22:337-342. This review discusses potential links between sialic acid recognition and regulation of innate immunity in the context of newly discovered CD33related siglecs. 53. Aizawa H, Plitt J, Bochner BS: Human eosinophils express two Siglec-8 splice variants. J Allergy Clin Immunol 2002, 109:176. 54. Taylor VC, Buckley CD, Douglas M, Cody AJ, Simmons DL, Freeman SD: The myeloid-specific sialic acid-binding receptor, CD33, associates with the protein-tyrosine phosphatases, SHP-1 and SHP-2. J Biol Chem 1999, 274:11505-11512. 55. Paul SP, Taylor LS, Stansbury EK, McVicar DW: Myeloid specific human CD33 is an inhibitory receptor with differential ITIM function in recruiting the phosphatases SHP-1 and SHP-2. Blood 2000, 96:483-490. 56. Veillette A: The SAP family: a new class of adaptor-like molecules that regulates immune cell functions. Sci STKE 2002, 2002:E8. 57. •
Grobe K, Powell LD: Role of protein kinase C in the phosphorylation of CD33 (Siglec-3) and its effect on lectin activity. Blood 2002, 99:3188-3196. This is the first demonstration that a CD33-related siglec can be phosphorylated on serine residues in the cytoplasmic tail. Evidence is presented that phosphorylation results in altered sialic-acid-binding properties. 58. Ulyanova T, Blasioli J, Woodford-Thomas TA, Thomas ML: The sialoadhesin CD33 is a myeloid-specific inhibitory receptor. Eur J Immunol 1999, 29:3440-3449. 59. Vitale C, Romagnani C, Falco M, Ponte M, Vitale M, Moretta A, Bacigalupo A, Moretta L, Mingari MC: Engagement of p75/AIRM1 or CD33 inhibits the proliferation of normal or leukemic myeloid cells. Proc Natl Acad Sci USA 1999, 96:15091-15096. 60. Ferlazzo G, Spaggiari GM, Semino C, Melioli G, Moretta L: Engagement of CD33 surface molecules prevents the generation of dendritic cells from both monocytes and CD34+ myeloid precursors. Eur J Immunol 2000, 30:827-833. 61. Razi N, Varki A: Cryptic sialic acid binding lectins on human blood leukocytes can be unmasked by sialidase treatment or cellular activation. Glycobiology 1999, 9:1225-1234.
Siglecs: sialic-acid-binding immunoglobulin-like lectins in cell–cell interactions and signalling Paul R Crocker Siglecs are sialic-acid-binding immunoglobulin-like lectins involved in cell–cell interactions and signalling functions in the haemopoietic, immune and nervous systems. Significant advances have been made in our understanding of the link between carbohydrate recognition and signalling for two well-characterised siglecs, CD22 and myelin-associated glycoprotein. Over the past few years, several novel siglecs have been discovered through genomics and functional screens. These ‘CD33-related’ siglecs have molecular features of inhibitory receptors and may be important in regulating leucocyte activation during immune and inflammatory responses. Addresses The Wellcome Trust Biocentre at Dundee, Division of Cell Biology and Immunology, School of Life Sciences, Dundee University, Dow Street, Dundee DD1 5EH, UK; e-mail: [email protected] Current Opinion in Structural Biology 2002, 12:609–615 0959-440X/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations BCR B-cell receptor Ig immunoglobulin ITIM immunoreceptor tyrosine-based inhibitory motif MAG myelin-associated glycoprotein p75NTR p75 neurotrophin receptor
Introduction The cell surface is richly decorated in glycans that are attached to proteins and lipids. The exposed termini of oligosaccharides are often capped with sialic acid — the term given to a diverse family of nine-carbon sugars derived from neuraminic acid (Neu) or keto-deoxynonulosonic acid (Kdn). Sialic acids are typically found attached to other sugars through α2,3 and α2,6 linkages, or to another sialic acid molecule through an α2,8 linkage. They therefore make an important contribution to the overall structural heterogeneity of glycans and are well suited to act as ligands in complex cellular recognition events, such as those that occur in the nervous and immune systems. Although for many years sialic acids have been known to act as host ligands for pathogen receptors, it is only relatively recently that endogenous receptors for binding sialic acid have been identified. The most prominent group among these receptors is the siglecs, which are members of the immunoglobulin (Ig) superfamily [1]. In this review, I discuss recent advances in our knowledge of the existence, structure and functions of siglecs.
The siglec family The founding members of the siglec family were sialoadhesin (siglec-1), a macrophage adhesion molecule [2];
CD22 (siglec-2), a B-cell inhibitory receptor [3]; CD33 (siglec-3), a marker of myeloid cells [4]; and myelinassociated glycoprotein (MAG or siglec-4), which is expressed by oligodendrocytes and Schwann cells in the nervous system [5]. These proteins share about 25–30% sequence similarity in their extracellular regions. In the past few years, several human, ape and mouse members of the siglec family have been identified through genomics and functional analyses (Table 1). All are highly related to CD33 and to each other, sharing about 50–80% sequence similarity. They are therefore described collectively as ‘CD33-related siglecs’ and can be considered as a separate subgroup, from both functional and evolutionary perspectives, to sialoadhesin, CD22 and MAG. The nomenclature for CD33-related siglecs is numerical in humans (e.g. hSiglec-5) and alphabetical in mice (e.g. mSiglec-E). This difference reflects current uncertainties in assigning murine orthologues to human CD33-related siglecs, as discussed below. All siglecs possess a characteristic N-terminal sialic-acidbinding V-set Ig domain and between 1 (CD33) and 16 (sialoadhesin) C2-set domains that project the sugarbinding site away from the plasma membrane. Each siglec has a distinct preference for specific types of sialic acid and also for specific types of linkage to subterminal sugars. These binding preferences are likely to be related to their biological functions; for example, CD22 on B cells binds specifically to α2,6-linked sialic acids in N-glycans that are co-expressed on the same cells and mediate biologically important CD22-dependent interactions [6,7••]. The cytosolic tails of siglecs vary in sequence and length, although most CD33-related siglecs share regions of sequence similarity surrounding two conserved tyrosine motifs that are implicated in signalling functions. The genes encoding human siglecs are on chromosome 20p (sialoadhesin) or 19q (all other siglecs), and those encoding putative murine orthologues are in syntenic regions of chromosomes 2 and 7, respectively.
Sialic acid recognition by siglecs: how is specificity achieved? As discussed in a recent review [8•], sialic acids may be evolutionarily ancient, but their expression in eukaryotes is primarily in the deuterostome lineage of animals (i.e. vertebrates and some higher invertebrates, such as starfish). On the basis of sequence alignments [9] and the absence of sigleclike sequences in the completed fly and worm genomes [10], it seems likely that siglecs evolved from a protein-binding Ig domain precursor that was adapted to sialic acid recognition.
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Table 1 Properties of human and mouse siglecs.
Human siglecs
Mouse siglecs
Number of Ig domains
Number of cytoplasmic tyrosine motifs
Sialic-acid-binding specificity*
Expression
17 0 2,32,62,8 Mac 7 6 2,6 B 2 2 or 0‡ 2,62,3 My-pro, Mon 5 1 2,32,6 Oligo, Schwann 4 2 2,3=2,62,8 Mon, N, B 3 2 STn B, Plac 3 2 2,82,62,3 Mon, NK 3 2 2,32,6 Eos, Baso, Mast †¶ mSiglec-E 3 2 2,3=2,6 Mon, N, NK, B mSiglec-G†¶ 5 3 2,3=2,6 Mon, NK 5 2 2,8 Mac †¶ mSiglec-H 2 0 ND ND *2,3, 2,6 and 2,8 refer to 2,3-, 2,6- and 2,8-glycosidic linkages of sialic acid to galactose (2,3 and 2,6) or sialic acid itself (2,8). †The sialic-acid-binding properties of these mouse ‘siglecs’ have not been determined. ‡hCD33 has two tyrosine-based motifs, whereas the postulated murine orthologue has none. §mSiglec-F is expressed on immature myeloid cells in the bone marrow and at lower levels on subsets of mature myeloid cells [40]. ¶The expression patterns of these mouse ‘siglecs’ have not been determined. B, B cells; Baso, basophils; Eos, eosinophils; Mac, macrophages; Mast, mast cells; Mon, monocytes; My-pro, myeloid progenitors; N, neutrophils; ND, not determined; NK, natural killer cells; Oligo, oligodendrocytes; Plac, placental trophoblasts; Schwann, Schwann cells. Sialoadhesin/hSiglec-1 CD22/hSiglec-2 CD33/hSiglec-3 MAG/hSiglec-4 hSiglec-5 hSiglec-6 hSiglec-7 hSiglec-8 hSiglec-9 hSiglec-10 hSiglec-11
Sialoadhesin/mSiglec-1 CD22/mSiglec-2 CD33/mSiglec-3† MAG/mSiglec-4 mSiglec-F§
Important molecular insights have been obtained from the crystal structure of the sialoadhesin V-set domain bound to 3′-sialyllactose at 1.85 Å resolution (Figure 1) [11]. This structure reveals the basic template for sialic acid recognition; this template is conserved among all siglecs and includes an invariant arginine on β strand F that makes a key salt bridge with the carboxylate of sialic acid. Specificity for different sialic acid linkages and extended glycan structures is likely to be provided by the highly variable loop regions between the β strands. Clear evidence for this has emerged from a study comparing hSiglec-7 and hSiglec-9 [12••]. Despite sharing about 76% sequence identity in the V-set domain, hSiglec-7 shows a striking preference for α2,8-linked disialic acids and for sialic acid with a α2,6 linkage to an internal N-acetylglucosamine [12••,13], whereas hSiglec-9 prefers terminal α2,3 and α2,6 linkages [10,12••,14]. Experiments using protein chimaeras of hSiglec-7 and hSiglec-9 have shown that the sialic acid linkage preference is determined by a sequence of six amino acids in the C–C′ loop of the V-set domain [12••]. Modelling suggests that a basic region close to the C–C′ loop can accommodate a second sialic acid in the α2,8-linked disialic acid motif (Figure 1). The significance of these sugar-binding preferences is currently unclear and will probably require an understanding of the biological functions of each molecule.
Trans and cis interactions: linking sialic acid recognition to signalling Ligand recognition by siglecs is complicated by the fact that these receptors interact with their sialic acid ligands both in cis and in trans. Cis interactions can result in masking of the sialic-acid-binding site at the cell surface, which can prevent trans interactions [15]. In the case of B cells, the binding site of CD22 can be unmasked by
treatment with sialidase or after cellular activation with anti-IgM and anti-CD40 [15]. Trans interactions and CD22 signalling
CD22 is a well-characterised B cell inhibitory receptor containing three immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that associates with the B-cell receptor (BCR) and inhibits cellular activation by recruiting the inhibitory tyrosine phosphatase SHP-1 (Figure 2a). B cells express high concentrations of α2,6-linked sialic acids, which results in the masking of CD22 [15]. A minor subset of B cells in mice has been shown to express unmasked CD22 [16] and it has been suggested that trans interactions between CD22 and sialylated ligands on bone marrow sinusoidal endothelial cells lead to the selective accumulation of these cells in the bone marrow [16,17]. Notably, trans interactions may occur even when CD22 is masked at the cell surface (Figure 2b): a series of elegant experiments has shown that, when B cells are activated in response to membrane-associated antigens, the co-expression of α2,6-linked sialic acids results in a CD22-dependent dampening of activation [18••]. Cis interactions and CD22 signalling
Evidence for the importance of cis interactions with α2,6-linked sialic acid in CD22-mediated inhibitory signalling has been provided by two recent studies [7••,19••]. The first study showed that a novel sialic-acid-based CD22specific inhibitor causes heightened activation of B cells in response to BCR cross-linking, which is accompanied by hypophosphorylation of CD22 and reduced recruitment of SHP-1 [7••]. The second study used CD22-deficient B-cell lines transfected with either the wild-type or mutant forms of CD22 in which the N-terminal domains had been removed or the sialic-acid-binding site had been inactivated by two point mutations. With both mutants of CD22, there
Siglecs Crocker
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Figure 1 Surface charge representations of the V-set domains of mouse sialoadhesin and hSiglec-7. (a) Structure of sialoadhesin complexed with 3′-sialyllactose. (b) Model of hSiglec-7 based on the solved sialoadhesin structure. Red indicates acidic regions and blue indicates basic regions. The hSiglec-7 model reveals the presence of a basic pocket (circled in yellow), located close to the C–C′ loop (outlined in green), that may favour interactions with the second sialic acid residue of the α2,8-linked disialic acid motif. The sequence-predicted binding site on hSiglec-7 for terminally positioned sialic acid is circled in white. Homology modelling was carried out using the program WHATIF and the diagrams were prepared using GRASP. For further information, see [12••].
(a)
(b)
C–C′ loop
Current Opinion in Structural Biology
was less reduction in inhibition of BCR triggering than with wild-type CD22 [19••]. Both studies concluded that the sialic-acid-dependent cis-interacting partners might be either the BCR itself or sequestering sialoglycoproteins such as the CD45 tyrosine phosphatase. The latter is thought to be important for activating the Lyn tyrosine kinase, which in turn phosphorylates CD22, leading to SHP-1 recruitment and activation (Figure 2a).
that MAG can mediate high-affinity, sialic-acid-independent interactions with the Nogo receptor — a glycosylphosphatidylinositol-linked protein that is important in neurite inhibition [26••,27••]. Taken together, these findings indicate that MAG-dependent inhibition of neurite outgrowth depends on a complex four-way molecular interaction between MAG, GT1b, p75NTR and Nogo receptor (Figure 2c).
MAG-dependent signalling and sialic acid recognition
Distinct repertoires of CD33-related siglecs in man and mouse
MAG (siglec-4) is expressed by oligodendrocytes and Schwann cells in the nervous system, and is important for maintaining the integrity of the myelin sheath [20]. MAG has also been studied extensively as an inhibitor of neurite outgrowth in postnatal neuronal cells [21]. Recently, significant advances have been made in our understanding of how sialic acid recognition by MAG triggers signalling events (see also the review by Allende and Proia [pp 587–592] in this issue). Vinson and co-workers [22•] proposed that ganglioside GT1b is an important MAG ligand on neuronal cells because antibodies against GT1b can mimic the inhibitory effect of MAG on neurite outgrowth. A subsequent study confirmed this hypothesis and also proposed that ganglioside GD1a is a functional MAG ligand [23•]. It has been postulated that the MAG-dependent clustering of GD1a and GT1b in lipid rafts activates a Rho-dependent signalling pathway that leads to the inhibition of neurite outgrowth [24]. Notably, a recent study has shown that GT1b associates with the p75 neurotrophin receptor (p75NTR) [25••]. Neurons from mice lacking p75NTR do not respond to a recombinant soluble form of MAG; addition of this protein to wild-type neurons stimulates Rho activation in a p75NTR-dependent manner [25••]. A further twist to this story has been provided by two recent studies that report
Seven new human siglecs and four new mouse siglecs have been discovered by genomics and in functional screens over the past few years [10,14,28–33,34•,35•,36–39,40••, 41•,42,43•,44–46]. Whereas the human genome contains eight CD33-related siglecs, one siglec-like gene and about sixteen siglec pseudogenes, the mouse genome contains only five CD33-related genes and two siglec pseudogenes [40••,46]. The genes encoding hSiglec-3 to hSiglec-10 are clustered within a 506 kb region on human chromosome 19q13.3–13.4; the gene encoding hSiglec-11 is located about 1 Mb upstream of this region but still within cytological band 19q13.3–13.4 [40••,46,47]. Apart from mSiglec-H, which is located on mouse chromosome 7B4, all murine CD33-related siglecs lie in a syntenic region of human chromosome 19q13.3–13.4 on mouse chromosome 7B2. Human chromosome 19 is unusually rich in genes and contains large families of Ig-related receptors, including the carcinoembryonic antigen family, killer inhibitory receptors and Ig-like transcripts, in addition to the CD33related siglecs (reviewed in [48]). The marked expansion of Ig-like families on chromosome 19q may be due, at least in part, to the presence of minisatellites specific to chromosome 19 that facilitated gene duplication by acting as nucleation sites for unequal crossovers during meiotic
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Figure 2 (a)
(b)
(c)
Inhibition of neurite outgrowth
Antigen-presenting cell
Rho kinase
Antigen
Rho p75NTR
CD45
CD22
BCR BCR SHP-1
–
SHP-1
SHP-1
Lyn
Ca2+
– Ca2+
MAG
– Ca2+ Myelin-forming cell
Setting B cell activation thresholds α2,6-linked sialic acid
Dampening B cell activation α2,6-linked sialic acid
GT1b
Nogo receptor
Current Opinion in Structural Biology
Sialic-acid-dependent signalling pathways mediated by CD22 and MAG. (a) Cis interactions between CD22 and either BCR or CD45 may result in activation of SHP-1 and inhibition of BCR-triggered B cell activation. For further details, see [7••,19••]. (b) Trans interactions of CD22 on B cells with α2,6-linked sialic acids co-expressed with an antigen dampen B cell activation. For more details, see [18••].
(c) Binding of MAG to GT1b on neuronal cells results in p75NTRdependent activation of Rho, leading to activation of Rho kinase and inhibition of neurite outgrowth. MAG can also bind the Nogo receptor in a sialic-acid-independent manner and is functionally important in MAG-dependent neurite inhibition. For additional information, see [22•,23•,24,25••–27••].
recombination [10,49]. In the case of CD33-related siglecs, frequent gene duplications have given rise to functional specialisation; for example, hSiglec-6 is expressed on placental trophoblasts, hSiglec-8 on eosinophils and hSiglec-11 on tissue macrophages (see below). None of these proteins has an obvious counterpart in the mouse proteome.
difficult to assign orthologues between the two species. This is in contrast to sialoadhesin, CD22 and MAG, which share about 25–30% identity and have clear-cut orthologues in all mammalian species examined so far. On the basis of gene structure, sequence alignments, relative chromosomal localisation and, where available, functional properties, it has been proposed that ‘mCD33’, mSiglec-E and mSiglec-F are orthologues of hCD33, hSiglec-9 and hSiglec-5, respectively [40••]. But mCD33 lacks ITIM-like motifs [4,51], and mSiglec-E and mSiglec-F share only about 50% identity with hSiglec-5 and hSiglec-9, respectively. By contrast, mSiglec-G is a clear orthologue of hSiglec-10 and shares about 60% sequence identity [40••]. The possible evolutionary origins of the CD33-related siglecs from a Siglec-10-like precursor have been discussed [46], but firm conclusions will require the completion of genome sequencing projects in a range of vertebrate and invertebrate species.
Human siglec-like molecule 1 (hSiglec-L1), also known as siglec with two V-set domains (S2V), is an unusual human siglec-like sequence, with two tandem V-set Ig domains that each lack the essential arginine required for recognition of sialic acid [41•,50•]. Surprisingly, one group has reported the sialic-acid-dependent binding of erythrocytes to hSiglec-L1 expressed in COS cells [41•], although this result is controversial as another laboratory has been unable to reproduce it [50•]. In addition, it has been shown that restoring the essential arginine in hSiglec-L1 leads to the recovery of sialic-acid-dependent binding in this normally ‘dead’ siglec-like molecule. Notably, the chimpanzee orthologue of hSiglec-L1 has retained the essential arginine in the N-terminal V-set domain and can mediate robust sialic-acid-dependent binding when expressed in COS cells [50•]. The sequence similarity shared between CD33-related siglecs in humans and mice (about 50–80%) makes it
CD33-related siglecs as inhibitory receptors of the innate immune system Collectively, the CD33-related siglecs are expressed broadly in the innate immune system, but are strikingly absent from most T lymphocytes (reviewed in [52•]; see Table 1). The presence of two conserved ITIM-like motifs in the cytoplasmic regions of all hCD33-related siglecs [53],
Siglecs Crocker
coupled with the differential expression of these proteins, suggests that they have a generic role in regulating the cellular activation of haemopoietic and immune cells. Detailed studies of several CD33-related siglecs have shown that these siglecs can recruit the inhibitory tyrosine phosphatases SHP-1 and SHP-2, but only after the cells are treated with pervanadate to inhibit endogenous tyrosine phosphatases [37,41•,43•,44,46,54,55]. It remains to be shown whether tyrosine phosphorylation can occur under physiological conditions. Mutagenesis studies have shown that the distal ITIM-like motif is dispensable for phosphatase binding, which presumably reflects its departure from the consensus ITIM sequence [37,41•,43•,44,54,55]. The ITIM-like motif is highly conserved among CD33-related siglecs, however, which suggests that it may be important for interacting with specific downstream signalling molecules. Notably, it is similar to a tyrosine-based motif in proteins related to SLAM (signalling lymphocyte activation molecule) that binds SAP — a single SH2 domain protein that inhibits the recruitment of SHP-2 and activates additional signalling pathways [56]. A recent report has also shown that the cytoplasmic tail of hCD33 can be also phosphorylated on serine residues by protein kinase C and that this can regulate sialic-acid-binding activity in transfected cells [57•]. Further studies are needed to examine serine/threonine phosphorylation in other CD33-related siglecs.
613
Conclusions The siglec family has emerged as a principal subset of the Ig superfamily. On the one hand, siglecs can mediate cell–cell interactions, as exemplified by sialoadhesin on macrophages and MAG on oligodendrocytes and Schwann cells. On the other hand, siglecs are well suited for mediating cis interactions with sialic acids on the same plasma membrane and for modulating leucocyte activation, as demonstrated by CD22. Understanding how changes in glycosylation modulate the cis and trans interactions of siglecs with sialic acids, and elucidating the subsequent biological responses are interesting issues for the future. The marked expansion of CD33-related siglecs in the innate immune system suggests that these proteins have a fundamentally important role in host defence functions. Sialic acids are well-suited as markers of ‘self’, as they are absent from many pathogens but are expressed universally on the surfaces of host cells. Recent findings on CD22 show how trans interactions with sialic acids can dampen B cell activation. Similarly, CD33-related siglecs may regulate the autoreactivity of myeloid cells. The expanded repertoires of CD33-related siglecs in humans, compared with mice, illustrate how rapidly this gene family is evolving. The use of genetically modified mice and novel sialic-acidbased inhibitors is expected to give important insights into the functions of these intriguing molecules in the future.
Acknowledgements Evidence that siglecs can mediate inhibitory signals has been obtained in studies using antibodies to artificially cross-link hCD33 or mouse inhibitory siglec/mSiglec-E with an activating human receptor, FcγR1 on mononuclear phagocytes [37,55,58]. This results in reduced Ca2+ influx compared with cross-linking FcγRI alone. Likewise, clustering hSiglec-7 with a monoclonal antibody can inhibit the cytotoxicity of natural killer cells [31]. The addition of antibodies against hCD33 or against hSiglec-7 to haemopoietic cell cultures results in reduced cell growth and inhibition of dendritic cell development [59,60]. The physiological relevance of these findings remains unclear, because antibodies rather than natural ligands were used to cluster the siglecs. Similar to CD22, CD33-related siglecs are masked at the cell surface [61] and the extent to which they become unmasked on cellular activation is unknown. Therefore, although trans interactions may occur with sialic acids on other cells and dampen leucocyte activation in a manner similar to that shown for CD22 [18••], it is also possible that cis interactions with specific glycoproteins on the same plasma membrane could modulate the responses of leucocytes to activation. For the latter case, it will be important to identify cis-interacting partners for individual siglecs on different leucocyte populations and to investigate whether cellular activation leads to glycosylation changes that modulate the signalling functions of CD33-related siglecs.
Research in the author’s laboratory is supported by the Wellcome Trust, Biotechnology and Biological Sciences Research Council and the Consortium for Functional Glycomics. I am grateful to Magnus Alphey for help with preparing Figure 2, and Tony Avril and Ajit Varki for helpful comments on the manuscript.
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