Siglecs in the immune system
Year: 2001
Type: review
Abstract: Siglecs1 (sialic acid binding Ig-like lectins) are I-type (Ig-type) lectins2 characterized by an N-terminal V-set Ig domain that mediates sialic acid binding,3 followed by varying numbers of C2-set Ig domains (Fig. 1). The initial discovery of this lectin family came about through independent studies on sialoadhesin (Siglec-1/CD169), a macrophage lectin-like adhesion molecule,4 and CD22 (Siglec-2), a B-cell restricted member of the Ig superfamily (IgSF)5 that plays an important role in regulating B-cell activation. Both molecules were found to mediate cell–cell interactions in vitro via recognition of sialylated glycoconjugates.6-10 The cloning of sialoadhesin11 revealed striking sequence similarities to CD22 and led to the demonstration that two other related IgSF proteins, myelin-associated glycoprotein (MAG/Siglec-4) and CD33 (Siglec-3), which were not previously known to bind sialic acids, were also members of the Siglec family (Table 1).12, 13 Structural features of Siglecs. (a) Siglecs are type I membrane proteins with an extracellular region containing a sialic acid binding V-set Ig-like domain at the N-terminus and 1–16 C2-set Ig-like domains. The cytoplasmic tails of all Siglecs apart from sialoadhesin have one or more tyrosine residues within potential signalling motifs. A crystal structure of the sialoadhesin N-terminal domain complexed to 3′-sialyllactose3 revealed some key features of sialic acid recognition by Siglecs, including the presence of an essential arginine (Arg97) on the F strand (conserved in the other Siglecs) which forms a salt bridge with the carboxylate of sialic acid (b). The V-set and adjacent C2-set domains of Siglecs contain an unusual arrangement of conserved cysteines that give rise to an intrasheet disulphide (rather than the more usual intersheet disulphide) within the V-set domain and a disulphide between the domains. The conserved intrasheet disulphide in sialoadhesin results in widening of the Ig β-sandwich and exposure of two tryptophans (Trp2 and Trp106) on the A and G strands that form hydrophobic contacts with the N-acetyl and glycerol side groups of NeuAc, respectively (see 3for further details). Six additional human Siglecs (Siglecs 5–10) have been identified and characterized over the last 3 years. These previously unknown molecules share a high degree of sequence similarity with CD33 in their extracellular and intracellular regions, and are hence collectively referred to as 'CD33-related Siglecs'. A striking feature of the CD33-related Siglecs is their differential expression pattern amongst the cell lineages of the haemopoietic system (see Table 1). This, together with the presence of two conserved immunoreceptor tyrosine-based inhibition motif (ITIM)-like motifs in their cytoplasmic tails, suggests that, like CD22, CD33-related Siglecs are involved in regulating cellular activation within the immune system. Here we discuss how sialic acid recognition by Siglecs might contribute to the regulation of immune functions. Sialic acid is a generic term for a large family of 9-carbon sugars that are all derivatives of neuraminic acid (Neu) or keto-deoxynonulosonic acid (KDN). They are typically found at the exposed, non-reducing ends of oligosaccharide chains attached to a wide variety of proteins and lipids. Many different types of sialic acid exist in mammals and each can occur in a variety of glycosidic linkages. Thus, in addition to their well-known roles in preventing cell–cell interactions through charge-repulsion effects and in masking subterminal sugars, sialic acids are also well situated to act as ligands for mediating selective cell–cell interactions (reviewed in 14-16). With the exception of Siglec-6, which shows restricted specificity for the sialyl Tn antigen,17 all of the known Siglecs recognize forms and linkages of sialic acid that are commonly found at cell surfaces and in the extracellular environment. One consequence is that the Siglec binding site can be masked by cis interactions with sialic acids on the same cell surface, thereby preventing them from mediating cell–cell interactions (Fig. 2). Diagram illustrating how the interaction of Siglecs with cells or sialylated pathogens could be affected by cis interactions with sialylated glycoconjugates. (a) (i) Some Siglecs such as sialoadhesin appear to be unmasked and able to mediate cell–cell interactions constitutively whereas (ii) the majority of Siglecs appear to be naturally masked due to cis interactions with adjacent sialic acids. Cis interactions may be important for signalling functions; for example CD22 is closely associated with the B-cell receptor complex and provides inhibitory signals that raise B-cell activation thresholds. (iii) Unmasking of Siglecs can occur in some cases by cellular activation or by exposure to sialidases. The unmasked Siglec may now be capable of stronger interactions with ligands on other cells. This could result in increased cellular interactions and/or signalling. (iv) In another scenario, a virally infected cell expressing a viral sialidase (neuraminidase) on the cell surface would result in loss of Siglec ligands and reduced interactions with unmasked Siglecs on cells of the innate immune system. In this situation, unmasking of Siglecs could also occur by close cell–cell contact with the virally infected cell. (b) Some pathogens can express sialylated glycoconjugates (see text for details) which may interact with Siglecs. In the case of sialoadhesin, this may lead to increased uptake of pathogens by macrophages and therefore be detrimental to the pathogen. In the case of CD22- and CD33-related Siglecs, interactions with sialylated pathogens may lead to subversion of the normal signalling functions of Siglecs (e.g. reduced activation) which could result in increased pathogen survival. This phenomenon of Siglec masking has been systematically studied with regard to CD22. When human or mouse CD22 was expressed in COS or CHO cells, high levels of sialic acid-dependent binding were observed.5, 12,18-21 However, both these cell types lack ST6Gal-I, the sialyltransferase that creates the sialylated sequence recognized by CD22·22 When ST6Gal-I was coexpressed with CD22, all CD22 binding activity was lost, but could be restored by sialidase treatment of the transfected cells.23, 24 Since B cells normally express ST6Gal-I and therefore carry CD22 ligands, these observations raised the issue of whether CD22 expressed naturally on B cells can mediate binding to exogenous ligands. Using an α2-6-sialyllactose-based synthetic probe that binds strongly to recombinant CD22, there was indeed no detectable binding to human peripheral blood B lymphocytes25 and to most mouse B cells26 due to masking of the sialic acid binding site (see below). However, activation of human B cells or sialidase treatment led to unmasking of CD22 and readily detectable probe binding.25 Similar findings were obtained using human peripheral blood leucocytes which collectively express all of the CD33-related Siglecs27 (Table 1). These studies indicate that most Siglecs exist on resting cells in a masked form, but that unmasking may occur during cellular activation. Sialoadhesin appears to be an exception to this general theme since it is able to mediate binding to other cells when expressed naturally on stromal tissue macrophages4, 7 (Fig. 2a). Cis interactions with sialic acids in the same plasma membrane could potentially regulate Siglec functions in a variety of different ways. For example, they could prevent cell–cell interactions until they are needed or facilitate specific interactions with other proteins on the same cell surface. Conversely, they could be preventing specific protein–protein interactions by non-specifically engaging the Siglecs to other sialylated molecules. Regardless of which of these mechanisms is operative in a given cell type, the actual 'unmasking' of the Siglec could be mediated by a variety of possible mechanisms, including a cell surface sialidase, via altered glycosylation, or by reorganization of plasma membrane domains (Fig. 2a). Sialoadhesin differs from other Siglecs expressed in the haemopoietic system in several significant ways. First, it has 17 Ig-like domains (compared with a maximum of seven found in other Siglecs; see Table 1). Secondly, unlike other Siglecs, it is able to mediate sialic acid-dependent binding to other cells when expressed in its native environment (i.e. macrophages).4, 7 Thirdly, it lacks tyrosine-based motifs in the cytoplasmic tail. It would therefore appear that sialoadhesin has been selected during evolution to mediate extracellular rather than intracellular functions. This is supported by the recent cloning of the human sialoadhesin orthologue28 which also has 17 Ig-like domains, and shares high sequence similarity (72% identity) with its mouse counterpart in the extracellular domains, but only low homology (30% identity) in the cytoplasmic tail. Sialoadhesin is expressed on resident macrophages in many tissues and also by inflammatory macrophages such those found in rheumatoid arthritis.28 Tumour-infiltrating macrophages in breast cancer also express high levels of sialoadhesin, and these macrophages were shown to make intimate contact with carcinoma cells expressing MUC-1, a membrane mucin which can be bound specifically by sialoadhesin.29 Sialoadhesin has also been implicated in functionally important interactions with T lymphocytes that lead to generation of cytotoxic T cells in a graft-vs.-leukaemia model.30 CD43, a cell surface mucin-like molecule on T cells, has been identified as a potentially important counter-receptor for sialoadhesin.31 The widespread expression of ligands for this Siglec on cell surfaces and the extracellular matrix and its ability to mediate cellular interactions, even in the presence of 100% plasma,4 suggest that sialoadhesin may be involved in diverse cell-cell and cell–matrix interactions of macrophages. Despite the fact that CD22 is generally masked on B cells, recent studies suggest that CD22 plays a role in homing of B cells to the bone marrow through interactions with sialylated ligands expressed on bone marrow sinusoidal endothelium.26, 32,33 CD22-deficient mice were found to lack mature (IgDhi) B cells in the bone marrow but not in the spleen.32 Adoptive transfer of CD22-deficient B cells into RAG–/– mice resulted in reduced entry into the bone marrow when compared with normal B cells.32 Staining of mouse bone marrow cryostat sections with recombinant mCD22-Fc chimaeras revealed high levels of CD22 ligands on bone marrow endothelium that were absent from endothelial cells in other tissues.33 In addition, injected recombinant CD22-Fc was able to bind in vivo to marrow endothelium and reduced the number of mature B cells in the bone marrow by around 50%. These findings prompted a careful re-examination of the masking status of CD22 on B cells from bone marrow, spleen and lymph node.26 Although the majority of B cells in all tissues expressed the typical masked forms of CD22, the number of B cells with unmasked CD22 was enriched 2–5-fold in the bone marrow, when compared with those from the spleen or mesenteric lymph nodes. One interpretation is that the subset of transiently circulating B cells with unmasked CD22 is able to bind bone marrow endothelial cell ligands, leading to transmigration into the marrow parenchyma and local enrichment of these cells. Although the biological significance of CD22-dependent lymphocyte homing is not well understood, the bone marrow is a major site of Ig production.34 CD22-deficient mice have reduced numbers of IgM-secreting plasma cells in bone marrow, indicating that some plasma cell precursors use a CD22-dependent pathway to enter the bone marrow.33 Interestingly, TNF-α and IL-1-β were shown to cause an up-regulation of CD22 ligands on endothelial cells, suggesting a possible effect on B-cell trafficking in conditions of inflammation.20 The importance of balancing positive and negative signals is an emerging theme in the immune system (see 35 for a recent excellent review). When cellular activation is triggered by receptors with immunoreceptor tyrosine-based activation motifs (ITAMs), counteracting inhibitory signals can be delivered through receptors bearing ITIMs. Following phosphorylation by Src family kinases, ITIMs can recruit phosphatases, either the inositol phosphatase SHIP, or more commonly the protein tyrosine phosphatases SHP-1 and SHP-2. These phosphatases inhibit signalling pathways through distinct mechanisms, resulting in raised activation thresholds. CD22 is a well-characterized inhibitory receptor for B-cell receptor (BCR) signalling. This has been most clearly demonstrated using B cells from CD22-deficient mice32, 36-38 which show an enhanced and prolonged Ca influx after BCR stimulation in comparison to normal B cells. B cells from CD22-deficient mice also exhibit a mildly activated phenotype, respond more robustly to LPS and have a tendency to generate autoantibodies with increasing age.39 However, B-cell responses to T-dependent antigens appear not to be grossly affected, as shown by normal germinal centre formation and switched Ig isotypes in CD22-deficient mice.32, 36-38 CD22 is constitutively associated with the BCR complex.40, 41 Following BCR engagement it becomes tyrosine phosphorylated in its cytoplasmic tail by Lyn.42 This leads to recruitment and activation of SHP-1 through binding to three ITIMs (reviewed in 43). Phosphorylated CD22 can also bind other signalling molecules such as Syk, PLCγ, PI3 kinase, Grb-2 and Shc, suggesting a potential role in positive as well as negative signalling.44, 45 Sequestration of CD22 away from the BCR using anti-CD22 coated beads lowered the activation threshold by a factor of 100, and resulted in a strong activation of the MAP kinase pathway in comparison to unligated CD22.46, 47 It was proposed that interactions with other lymphocytes expressing high levels of α2–6 sialylated glycans could result in a similar sequestration, thus providing a molecular switch that may bias signalling through BCR to anatomical sites rich in lymphoid tissues. However, key unresolved issues are whether sequestration can occur physiologically, whether sialylation of the BCR is important for its association with CD22 and whether alterations in sialylation that are known to occur on B-cell activation influence the association of CD22 with the BCR and subsequent activation response to antigens. Interestingly, ST6Gal-I-deficient mice which lack CD22 ligands display a B-cell immunodeficiency phenotype somewhat unlike that of CD22-deficient mice.22 These findings suggest a more general role of ST6Gal-I in regulating B-cell responsiveness, perhaps also involving interactions with other Siglecs expressed on B cells (see Table 1). In the final analysis, the precise biological role of the sialic acid binding activity of CD22 remains unresolved. Human Siglecs 5, 7, 8 and 10 were identified in expressed sequence tag (EST) databases as being homologous to CD33, and cloning of full-length cDNAs showed that they possess the key features required for sialic acid recognition (Fig. 1).48-53 Siglec-6 was isolated in a screen for leptin-binding proteins17 and Siglec-7 was independently identified as a NK cell inhibitory receptor in a redirected killing assay and designated p75/AIRM-1.54 Siglec-9, a close homologue of Siglec-7, was identified as a Siglec-like gene55 and also during characterization of Siglec-like cDNAs.56, 57 Most recently, a novel mouse Siglec with three Ig-like domains and two ITIM-like motifs, similar to hSiglecs 7, 8 and 9, was identified by one group as a CD33-like EST sequence (MIS)58 and by another as a binding partner for SHP-1 in a yeast two-hybrid screen (mSiglec-E).59 Human CD33-related Siglecs contain between two and five Ig-like domains and share a high degree of sequence similarity (∼50–80%). The genes encoding them are all clustered on chromosome 19q13.3–13.460 and seem to have evolved by gene duplication. Until recently, the only published murine CD33-related Siglec was a CD33-like protein with two Ig domains which, however, lacked the cytoplasmic ITIM-like motifs.61 Phylogeny analyses revealed that this molecule does not form an exclusive clade with hCD33, being instead loosely associated with several human CD33-related Siglecs.56 The eventual classification of CD33-related Siglec orthologues in mice will require the identification of all candidates from the mouse genome project, and the detailed characterization of each with regard to expression patterns, binding specificities and functions. It remains to be seen if all of the human CD33-like Siglecs share a corresponding mouse orthologue and vice versa. Monoclonal antibodies recognizing each of the new human Siglecs have allowed a detailed analysis of their expression patterns on blood leucocytes (Table 1). Some are expressed quite broadly; e.g. Siglec-9 is found on neutrophils, monocytes and a substantial fraction of NK cells and B cells.57 Others are much more restricted in their expression, notably Siglec-8 which is found only on circulating eosinophils50, 51 and at very low levels on basophils.51 On the other hand, several Siglecs can be present on the same cell type (e.g. monocytes express CD33 and Siglecs 5, 7, 9 and 1048, 49, 53, 56, 57, 62), suggesting some degree of functional redundancy at the cellular level. Regardless, each CD33-related Siglec exhibits a unique expression pattern amongst haemopoietic cells, indicative of specific functions. Siglec-6 is also prominently expressed by cyto- and syncytiotrophoblasts of the placenta, in addition to B cells.17 Limited information is currently available on the expression of the other CD33-related Siglecs on cells outside the haemopoietic system. The presence of two conserved ITIM-like motifs in the cytoplasmic regions of CD33-related Siglecs and the differential expression of these proteins on different types of leucocytes suggests a role in regulating cellular activation. Siglec-8, which was originally thought to lack ITIM-like motifs,50, 51 has now been found to exist in alternatively spliced forms containing such sequences.53, 63 While the membrane proximal motif of all the CD33-related Siglecs fits the consensus ITIM sequence (Ile/Val/Leu/Ser)-X-Tyr-X-X-(Leu/Val), the membrane distal motif does not. Following treatment of cells with pervanadate to inhibit tyrosine phosphatases, hCD33, Siglec-7 and MIS/mSiglec-E became tyrosine phosphorylated.54, 58,59, 64-66 Under these conditions, Siglec-7 recruited only SHP-1, whereas CD33 and MIS/mSiglec-E bound both SHP-1 and SHP-2. Mutation of the tyrosine in the distal motif did not significantly affect recruitment of SHP-1 and SHP-2. In contrast, an equivalent mutation in the proximal motif had a major effect.58, 59,64, 65 The much weaker binding of the distal motif to SHP-1 and SHP-2 presumably reflects its departure from the consensus ITIM sequence.59, 64 However, the distal motif is highly conserved in all CD33-related Siglecs, suggesting that it may be important for interacting with other regulatory molecules. In this regard, for some CD33-related Siglecs it is similar to a motif [Thr-Ile-Tyr-X-X-(Val/Ile)] found in signalling lymphocyte activation molecule (SLAM) and SLAM-related proteins that bind SLAM associated protein (SAP), a T-cell specific molecule that is known to inhibit SHP-2 recruitment to SLAM.67, 68 Functional evidence indicating that Siglecs can mediate inhibitory signals has been obtained using antibodies to cocross-link CD3365, 66 or MIS/mSiglec-E58 with an activating human receptor, FcγRI. This resulted in reduced Ca influx compared with cross-linking FcγRI alone. Likewise, Siglec-7 was identified as an inhibitory NK cell receptor in a redirected killing assay in which anti-Siglec-7 antibodies were used to cluster Siglec-7 at the NK cell–target cell interface.54 The physiological relevance of these interesting findings remains unclear, since antibodies rather than natural ligands were used to cluster the Siglecs. In other functional studies the addition of intact anti-CD33 or Siglec-7 monoclonal antibodies to haemopoietic cell cultures resulted in reduced cell growth62 and prevention of dendritic cell development.69 However, it is not clear if the intact antibodies used in these studies could also have interacted with myeloid cell Fc receptors, giving rise to 'confused signals' not necessarily relevant to Siglec functions. Clearly, additional experimental approaches are needed to elucidate the functions of CD33-related Siglecs. Many of the recently characterized ITIM-containing receptors are expressed by myeloid cells of the innate immune system (reviewed in 35). An emerging theme is that these inhibitory receptors can interact with broadly expressed 'self' ligands and thus regulate myeloid cell activation. This could be an important mechanism for preventing inappropriate during immune For example, I molecules are on most cell types and as ligands for both NK cell inhibitory receptors, and also for the Ig-like and inhibitory receptors found on myeloid an inhibitory receptor of myeloid and cells was shown to bind the expressed molecule When were injected into normal mice they were by unlike normal Thus, may act as a of 'self' that inappropriate macrophage by interacting with the inhibitory receptor inhibitory receptor of the Ig superfamily is the receptor expressed specifically by macrophages and three tyrosine-based motifs and with a similar molecule that lacks signalling motifs but is expressed In the major phenotype was associated with the myeloid cells that express with an and increased numbers of myeloid cells in lymphoid mice also a more and to experimental In a similar we suggest that sialic acids might act as broadly expressed 'self' ligands that interact with CD33-related Siglecs on myeloid cells, thereby preventing inappropriate most other commonly found sugars, sialic acids are thought to have in and therefore be absent from the majority of potential recent of and to encoding known of the sialic acid The interaction of Siglecs with sialic acids is to be affected by a number of different On the one hand, a Siglec on an cell that becomes unmasked activation might if there is an adjacent normal cell with cell surface sialic acids (Fig. 2a). On the other hand, if the cell is infected with a (e.g. A or or has been to a sialidase, its cell surface would be of sialic acids, and hence could not the unmasked In these the of the unmasked Siglec the lack could affect its and thus the phosphorylation of the cytoplasmic ITIM motifs, the activation of the cell in the most of these be in in vitro Despite the of sialic acids in it is striking that many known pathogens have independently evolved the to express sialic For example, such as groups B and and group B their sialic acids. In contrast, and transfer sialic acids from glycoconjugates using a Sialic acids can also be from acid by a cell surface e.g. by In some the of the sialic acids is e.g. pathogens such as and With several of these the expression of sialic acid has been shown to be essential for and within the the sialic acids on these pathogens are to be important for prevention of of and charge-repulsion an additional is that they interact with Siglecs and thus the of the (Fig. On the one hand, sialylated molecules on pathogens could interact with unmasked Siglecs and activation leading to increased within the On the other hand, interactions with sialoadhesin expressed on macrophages might lead to increased uptake and therefore be detrimental to the pathogen. Clearly, many For example, can resting leucocytes with masked Siglecs sialic acids on pathogens or are activation and unmasking required (Fig. Most Siglecs seem to recognize only a sialic in with its to the Thus, the regulation of Siglec formation is to be by the differential expression of in different cell However, in some the structure of the can also affect For example, the addition of a to sialylated chains can binding by most Interestingly, this also the structure a well-known motif for recognition by the family of cell adhesion the for this are also highly in their expression amongst haemopoietic cell in Siglec from the fact that natural of sialic acids can or For example, the addition of a group to or sialic acids can recognition by or and such are found in potentially important such as the spleen and bone In another example, the presence of an instead of an N-acetyl group at the of sialic acids can binding by and recognition by mouse In this regard, a mutation in the that the group might a different of macrophages found between and Siglecs are expressed on many cells of the immune system and are therefore to be important in both innate and immune Sialoadhesin is a molecule that has the potential to interact with a wide variety of ligands on other cells and in the extracellular CD22 is a well-characterized inhibitory receptor of B cells that may also mediate cell–cell interactions unmasking of its sialic acid binding CD33 is a of myeloid with the potential to as an inhibitory The discovery of new CD33-related haemopoietic Siglecs with features of inhibitory receptors that sialic acid recognition plays a more important role in regulation of the immune system than had been Although at the present we can only about how this might the with CD22 and other ITIM-containing receptors of the haemopoietic system a for It is that the generation and characterization of mice Siglecs and Siglec ligands or expressing Siglecs will on the roles of Siglecs and their sialic in the immune system. would like to for and for in the of in the is supported by the and and
Source: Immunology
Authors: Paul R. Crocker, Ajit Varki
Institutions: University of Dundee, University of California San Diego
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2148932927