Supplementary Material The glycobiology of the cd system: a dictionary for translating marker designa­tions into glycan/lectin structure and function

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This integrin subunit (_M) is structurally composed of three sections (Figure 2): the in­serted (I) domain (a module prominently acting in contact building to extracellular ma­trix glycoproteins, the intercellular adhesion molecules (ICAMs)-1/-2, fibrinogen, the platelet glycoprotein GPIb or the opsonic complement component inactivated (i)C3b), a region binding divalent cations and the lectin-like domain (proximal to the membrane) specific for -N-acetylglucosamine residues presented by N-glycans or for -glucans [S1]. In complex with CD18 (₂-integrin), CD11b forms the leukocyte complement recep­tor 3 (or Mac-1 antigen). Engagement of its lectin activity lets the integrin become a sen­sor for iC3b-coated cells and an effector for platelet clearance. Among its binding part­ners, also called counter-receptors, is CD23.

This type II transmembrane glycoprotein of about 45 kDa with a C-type CRD is a low-affinity IgE receptor (F_cRII). Lectin activity is attributed to this CRD, and the localization of its gene in a cluster with other sequences of C-type lectins such as CD209 (please see below) indicates its origin by gene divergence [S2, S3]. Extracellularly, the CRD is con­nected with a stalk region for oligomerization so that the distal lectin sites can form clusters (Figure 2). Key interaction partners are IgE, CD21 and CD11b/CD18. Capacity for signaling leading to pro-inflammatory cytokine responses allows cell surface-pre­sented and soluble (after ADAM10-dependent cleavage) CD23 to be classified as an im­mune regulator.

The platelet endothelial cell adhesion molecule-1 (PECAM-1) belongs to the Ig superfa­mily with its six extracellular C2-type domains. These versatile modules afford the struc­tural basis for homophilic and also for heterophilic interactions with proteins such as CD38 (ADP-ribosyl cyclase). The C2-type domains are followed by the transmembrane section and the cytoplasmic tail (118 amino acids) [S4, S5]. That each domain has dis­tinct features broadens the spectrum of in situ binding partners to include glycosamino­glycans. In detail, whereas homophilic events critically depend on Ig-like domain 1, high-affinity binding sites for heparin/heparan sulfate are located in Ig-like domains 2 and 3 [S6].

This rather ubiquitous type I transmembrane glycoprotein is known for its occurrence in a wide variety of isoforms through alternative splicing. A single link module at the N-terminus (residues 32-124), also called a link protein homology region, conveys binding capacity to hyaluronic acid to CD44 isoforms, as it does for the hyaladherins LYVE-1 and the product of tumor necrosis factor--stimulated gene-6 (TSG-6), for the hyaluronan receptor for endocytosis (HARE) and the extracellular matrix glycoproteins aggrecan, brevican, neurocan and versican, which are also known as lecticans due to the presence of a C-type CRD [S7, S8].

The neural cell adhesion molecule-1 (NCAM-1), another member of the Ig superfamily, is encoded by a single gene and, like CD44, present in many (up to 30) isoforms due to en­suing processing. The molecular weights and mode of membrane anchoring can differ among isoforms. Its extracellular region is composed of five Ig-like C2-type domains (two stacked -sheets cross-linked by a disulfide bond) and two fibronectin type III-like modules, which are proximal to the membrane [S9, S10]. Typical features of its N-glycosylation are the presence of 2,8-linked polysialic acid, oligomannosidic structures and the HNK-1 epitope (CD57, Figure 1) [S9, S10]. As noted for CD31, individual Ig-like modules have acquired particular binding properties: the fourth Ig-like domain can ac­commodate oligomannosidic glycans, and the second module binds heparan sulfates [S11].

That lymphocytes from distinct lymphoid sites were able to find their way back to their original home after re-injection into animals was interpreted as evidence for tissue-spe­cific adhesion mechanisms [S12]. As outlined in the introductory section, monoclonal antibodies against three different cell surface glycoproteins were crucial for the identifi­cation of the protein side of the assumed recognition. The three selectins, present in en­dothelial (E) cells, lymphocytes (L) or platelets (P), share the modular display with the N-terminal C-type CRD followed by an epidermal growth factor (EGF)-like domain, two (CD62L) to nine (CD62P) short consensus repeats known from complement-regulatory proteins (also called sushi domain), then completed by the about 25-amino-acid-long transmembrane section and the C-terminal cytoplasmic tail (Figure 2) [S13]. Summarizin­g this domain composition, the descriptive term LEC-CAM (Lectin-Egf-Com­plement Cell Adhesion Molecule) had been used synonymously. CD15s (Figure 1) is among the pan-selectin binders; the glycan-lectin association is driven by the entropic gain (TS: 23 kJ mol^-1) [S14, S15]. Preformed complementarity between the contact sur­faces leading to directional polar interactions accounts for a fast on-rate (>10⁶ M^-1s^-1) in selectin binding, which is essential to allow anchoring of cells flowing by in the blood [S16]. Tyrosine sulfation can serve as non-glycan part of the docking site, e.g. in P-se­lectin glycoprotein ligand-1 (PSGL-1) (Tys7/Tys10) [S16] or the glycoprotein T cell immunoglobulin and mucin domain 1 (TIM-1) [S17]. Of interest, TIM-1 recognition by CD62E,P is operative without sialylation, which is a key determinant in binding PSGL-1 or CD44 [S17]. In addition to acting as a natural braking system to slow down leukocytes on an endothelial surface, the selectin (CD62)-counterreceptor recognition underlies the phenomenon of rolling of cells by formation of catch bonds. Their lifetime increases un­der shear stress, likely involving re-orientation of the C-type CRD against the EGF-like domain [S18]. The selectin-dependent phase is the prerequisite for the transition from rolling to firm adhesion mediated by integrins.

This antigen is expressed on NK cells. Activating receptors can make NK cells prone to also attack self targets, leading to auto-aggression. In order to counterbalance respective signaling, inhibitory mechanisms have developed based on three receptor classes. Among MHC class I receptors, the C-type lectin CD94 becomes disulfide bridged to a sig­naling companion of the NKG2 family, building a heterodimer (Figure 2). NKG2 genes, coding for type II transmembrane proteins with an extracellular C-type lectin-like do­main, belong to the NK complex on the short arm of human chromosome 12 [S19, S20]. Having first (expectedly) been detected by monoclonal antibodies [S21], CD94 is now that the presence of the CRD is known referred to as another member of the family of C-type lectins. Its own cytoplasmic portion is restricted in length to only seven amino acids [S22]. Consequently, CD94 makes use of the signaling motif of the NKG2 protein (A, B, C, E or H). Association with NKG2A/B brings their consensus ITIM (Immunoreceptor Tyro­sine-based Inhibitory Motif) sequence into the complex, engendering the negative effect on NK cell activity (please see CD170 and CD328 for the role of ITIMs in siglecs). Attest­ing to its physiological role, antibody blocking of CD94 reduced NK cell-mediated cyto­toxicity against human melanoma cells with a high-level sialyl Le^x (CD15s) presentation, as did a neoglycoconjugate with this epitope (inert carrier with custom-made chemical glycosylation as bioactive part [S23]). These experiments provided first information of CD94’s lectin activity [S24]. Tri- and tetraantennary N-glycans with 2,3-sialylation (of the human ₁-acid glycoprotein) and heparin (conjugated to bovine serum albumin) were later described to bind to a fusion protein with the C-type lectin CRD of CD94 as sensor, as they did to similarly engineered NKG2D [S25, S26].

Thrombomodulin is a predominantly endothelial glycoprotein that has anticoagulant activity, through inhibition of the pro-coagulant thrombin and by serving as cofactor for thrombin-catalyzed activation of the anti-coagulant protein C [S27]. It controls multiple biological processes in inflammation and vascular integrity, therefore looking at its modular design is a step toward unraveling structure-activity relationships: following a short cytoplasmic tail and the transmembrane region, the extracellular portion com­prises a serine/threonine-rich section with a chondroitin sulfate chain (relevant for an­ticoagulation; for further information on glycosaminoglycans/proteoglycans, please see [S7, S28]), six EGF-like repeats, a hydrophobic region and the C-type CRD (Figure 2). The lectin module is a receptor for Le^y determinants (CD174, Figure 1) on lipopolysaccha­ride, connected to CD141’s activity to confine tissue damage, and on cellular glycopro­teins such as the EGF receptor, to block its angiogenic activity [S29, S30].

This 185-kDa member of the Ig-like family (a type I transmembrane glycoprotein with its CRD in the distal V-set Ig-like domain followed by 16 extracellular C2-type modules to give the CRD excellent spatial accessibility, Figure 2) was the first Ig-like receptor found to have binding specificity for sialylated glycans. It was thus termed siglec (sialic acid-binding Ig-like lectin). Its original name ‘sheep erythrocyte receptor’ was based on results of experiments studying adhesion activities on resident bone marrow macro­phages, whose association to unopsonized erythrocytes was reduced by 3’-sialyllactose and ganglioside GD1a [S31]. The SER-4 blocking antibody, raised against murine serum-induced peritoneal macrophages, was applied in affinity chromatography. This method facilitated the purification of the lectin (now named sialoadhesin or siglec-1), which is known to be a marker for macrophages in transition regions. Siglec-1 has affinity to 2,3-sialylated Thomsen-Friedenreich (TF) disaccharide (CD176s) in sialoglycoproteins (human glycophorin) and to the glycan chains of certain gangliosides (GT1b, GD1a and GM3) [S32]. The exceptionally length of its extracellular section (17 Ig-like domains compared to two to seven in other siglecs) makes it ideally suited for trans-interactions. A preferential role of siglec-1 as an active player in mediating interactions with other cells is further supported by the absence of an intracellular ITIM or any other tyrosine-harboring putative signaling motif. Siglec-1 can bridge lymphocytes/tumor cells and macrophages, via recognition of sialomucins leukosialin (CD43) on T cells or MUC-1 on breast cancer cells, as glycans of the siglec, in addition, become sites for associating cells by a C-type macrophage lectin (CD301) or by CD206. Of note, surface epitopes of human pathogens, i.e. sialylated lipooligosaccharides from Campylobacter jejuni and Neisseria meningitidis, are targets of siglec-1 in host defence [S33, S34] (for information on bacte­rial glycosylation, please see [S35]). On the chromosomal level, the genes for human and murine siglec-1 are not part of the gene cluster for the reminder of the siglec family on human chromosome 19q or mouse chromosome 7 but located on chromosomes 20 (human) or 2 (mouse) [S36].

Siglec-5 was the first member of this lectin family to be tracked down by a computa­tional homology search, using the CD33 (siglec-3) sequence and a database containing more than one million expressed sequence tags [S37]. It contains one V-set and three C2-set Ig-like domains that have specificity toward sialic acid irrespective of its linkage to the rest of the glycan. As for all eight human CD33-related siglecs, CD170 has a mem­brane-proximal ITIM sequence and a distal ITIM-like motif in its cytoplasmic tail. Tyro­sine phosphorylation recruits the protein-tyrosine phosphatases SHP-1/-2 (two Src ho­mology (SH)-1/-2 domain-containing enzymes) for inhibitory signaling [S38]. Despite its initial classification as OB-BP2 (binding protein for the product of the putative obesity gene, which codes for leptin, causing extreme obesity) its low level of reactivity was con­sidered “unlikely to be physiologically relevant” [S39]. Expression was detected in granulocytes and B lymphocytes, its paired activating receptor siglec-14, a product of concerted evolution, is present on monocytes instead of B cells, illustrating that there can be unique features even between very closely related family members [S40]. Special among siglecs, it is engaged in a protein-dependent (sialic acid-independent) interaction with the cell wall-anchored -protein of group B Streptococcus, which subverts the ITIM-based signaling of siglec-5 to dampen innate defense reactions [S41].

The macrophage (tandem-repeat-type) mannose receptor was first detected as endo­cytic entry site for glycoproteins with mannose/N-acetylglucosamine-terminated N-gly­cans on rat Kupffer cells [S42] (for further information on N-glycosylation, please see [S9, S43]), later targeted with clinical benefit in enzyme replacement therapy [S44]. Its modular design as type I transmembrane glycoprotein is composed of a cysteine-rich (-trefoil) domain, a fibronectin type II module with collagen (I-IV) reactivity, eight C-type lectin/lectin-like domains and the cytoplasmic tail with signals for delivery to and recy­cling from early endosomes (Figure 2) [S45, S46]. As lectin, CD206 is thus bifunctional via two structurally different sites: CRD no. 4 binds mannose, N-acetylglucosamine and fucose, the segment of domains 4-8 is reactive with multivalent sugar ligands, and the -trefoil domain has affinity for SO₄-4-GalNAc1,4GlcNAc termini of N-glycans of glyco­protein hormones [S47]. This capacity for glycan binding through two structurally dis­tinct sites is unique within the group of the four mammalian endocytic receptors of this type (not shared by the M-type phospholipase A2 receptor, urokinase-type plasminogen activator receptor-associated protein Endo180 (CD280; discussed later) and the den­dritic cell receptor DEC205 (CD205)). To give examples of glycan ligands, cell-specific glycoforms of CD45 and CD169 are reactive with the -trefoil domain. As ligand for a lectin, glycans of CD206 appear to associate with CD62L so that contact of lymphocytes and lymphatic endothelium is mediated. Hereby, a role in immune cell trafficking is added to the lectin’s activity for efficient glycoprotein endocytosis, with participation also in antigen presentation [S48, S49].

Langerin is so named because the detecting monoclonal antibody (DCGM4) selectively stained (via this 40 kDa antigen) Langerhans cells, a subset of dendritic cells residing in skin epidermis and mucosal epithelium, [S50]. Like CD206, albeit type II, it is a glycopro­tein that is active as endocytic receptor, but it has only one C-type CRD, not a tandem-repeat display (Figure 2). Binding to oligovalent ligands is made possible by an ex­tracellular neck region for trimerization stabilized by a coiled-coil of -helices as in CD23. Uniquely for a C-type lectin with the mannose-binding tripeptide motif (Glu-Pro-Asn), its CRD can also accommodate 6-sulfated galactose (e.g. the terminal sugar in the glycosaminoglycan keratan sulfate) [S51]. Single nucleotide polymorphisms in the CRD (K313I, N288D) act as an off switch for this specificity, a case of a direct effect of single-site mutations on glycan binding [S52]. Alternatively, long-range consequences of such sequence alterations are known to occur, e.g. in a lectin of a different family [S53]. In addition, Ca²⁺-independent binding of heparan-sulfate-type glycosaminoglycans at the trimeric neck region (involving Arg187) has been reported [S54]. Its endocytic capacity, relevant for formation of Birbeck granules typical for Langerhans cells, and its reactivity to fungal surfaces resemble the activity profile of related C-type lectins on dendritic cells and macrophages. Thus, a group of cooperating C-type lectins, to which the next lectin discussed (CD209) belongs, have distinct glycan-binding features to cover a broad range of pathogenic glycan signatures [S55, S56].

The genes for this dendritic C-type lectin and the closely related liver/lymph node-spe­cific CD209L/CD299 (also called DC-SIGNR- or L-SIGN; please see below) are part of a cluster on chromosome 19p13.3, along with the gene for CD23 [S57]. It was originally detected as contact partner for the glycoprotein gp120 of the human immunodeficiency virus by expression cloning using a placental cDNA library [S58]. Later, it was shown to connect resting T cells that present ICAM-3 to dendritic cells, explaining its name as dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) [S59]. The 44 kDa glyco­protein is a type II transmembrane receptor with a 40-amino-acid intracellular section harboring at least three intracellular sorting motifs, a neck for tetramer formation and the C-type CRD (Figure 2). The CRD has affinity for mannose and for Le epitopes (similar to CD207), the further affinity for Le^y (CD174) shared by CD141 [S60, S61]. The lectin is found in immature (periphery) and mature (lymphoid sites) dendritic cells (but not plasmacytoid/follicular dendritic cells) and macrophages (M2, CD14⁺). In addition to roles in cell adhesion and antigen presentation, DC-SIGN helps shape immune responses as the pathogen sensor of a signalosome (containing the three scaffold proteins LPS-1, KSR-1 and DNK and the kinase Raf-1) [S62] (for comment on relevance of elucidating in vivo functions in murine knock-out models, please see section on CD299). Acting as docking site for viral glycans, CD209 counterintuitively promotes infection (as similarly seen for CD169, CD206, CD294, CD301-303: for recent review, please see [S63]), a clini­cally relevant lesson in how a defence line can be exploited by viral glycosylation.

The Ca²⁺-independent lectin property of this 300 kDa dimeric type I transmembrane glycoprotein is assigned to a domain that is reactive with mannose-6-phosphate, thus it is referred to as P-type CRD. The unusual ability to bind phosphomannosyl residues, which is shared only by a second (cation-dependent) lectin, was delineated from studies on fibroblasts of patients with the lysosomal storage disorder mucolipidosis-II (I-cell disease), together with the discovery that this type of sugar is a marker (routing signal) for lysosomal en­zymes [S64]. Sequence alignments later uncovered homologies to three proteins in the endoplasmic reticulum (erlectin (XTP3-B), OS-9 (upregulated in osteosarcomas) and the 55 kDa non-catalytic -subunit of -glucosidase-II) as well as the -subunit of the Golgi GlcNAc-phosphotransferase. As consequence, the P-type CRD is now a part of the man­nose-6-phosphate receptor homology (MRH) family. This CRD present in two P-type lectins is responsible for uptake of cognate glycoproteins and their intracellular traf­ficking [S65]. Similar to CD206, its extracellular domains, here a total of 15, are arranged in a tandem-repeat orientation (Figure 2). Carbohydrate binding was localized to two high-affinity sites at domains 3 and 9 [S64]. Of note, domain 11 interacts with insulin-like growth factor type 2 (IGF-2) [S64]. Thus, the receptor exhibits a dual functionality to bind sugar and protein at different domains, and is thus referred to as P-type lectin/IGF-2 receptor. Also, plasminogen, the precursor of the central enzyme of fibrinolysis, inter­acts with domain 1, of potential relevance for its conversion to the active serine protease plasmin, and retinoic acid is also a non-glycan binding partner [S64]. Obviously, the P-type domain is subject to diversification with respect to molecular interactions beyond sugars, as seen above for C- and I-type lectins. Three separate internalization sequences guide the lectin’s intracellular routing. Loss of heterozygosity at the locus of this gene occurs in human cancer, pointing to a role of a deficiency in this receptor for enhance­ment of tumorigenicity in established tumor cells [S66].

The quest to identify new members of the C-type lectin family led Wu et al. [S67] to search the expressed sequence tag cDNA data base for homologues of the sequence mo­tif for the CRD of CD62E. Although the sequence hit only reached a degree of “low ho­mology (~ 23 %)” in total, it had the same amino acids at positions that are conserved among C-type lectins, and cloning yielded a sequence with a remarkable degree of iden­tity (32.5-34%) to the known members of the mannose receptor (CD206) family [S67]. Independently, CD280, at that time referred to as glycoprotein (p180), had been de­scribed as constitutively recycling surface antigen (Endo180) in human fibroblasts [S68] and as urokinase plasminogen activator receptor-associated protein (uPARAP) [S69]. Similar to CD206, Endo180 is also a collagen receptor via its fibronectin type II region; in contrast to CD206, the cysteine-rich domain is not a lectin. Besides the common protein-collagen contact, the second C-type lectin domain of this receptor is also capable of in­teracting with O-glycosylated collagen, which CD206 cannot do [S70]. The nearly com­plete abrogation of collagen endocytosis, diminished initial adhesion to collagens and impaired migration of murine fibroblasts deficient in this receptor intimate CD280’s importance for cellular collagen interactions [S71].

The sinusoidal endothelial cell receptor DC-SIGNR (L-SIGN, CD209L) shares its modular display with the dendritic cell DC-SIGN (CD209), with 77 % amino acid sequence iden­tity (Figure 2). Two features, besides the cellular expression profile, appear different by comparison: i) Val351 in DC-SIGN, which creates a hydrophobic pocket for accommo­dating 1,3/4-fucosylated Le epitopes and building van der Waals contacts with the 2’-OH group of fucose, is substituted by Ser363 in CD299, making the interaction impossi­ble in CD299 [S72] and ii) minor sequence variations in the neck domain for tetramerization, which has 23-amino-acid repeats, account for the significantly en­hanced stability of CD299 aggregates compared to CD209 tetramers [S73]. In gauging the potential of mouse models to illuminate the physiological significance of these C-type lectins, the occurrence of a recent, independent divergence of the murine gene family leading to a total of seven expressed genes and a pseudogene, six proteins proven to be lectins, is worth noting [S74].

Three separate approaches all converged to the identification, cloning and characteriza­tion of expression of this CD marker: immunization of mice with human NK cell clones, resulting in an antibody specific for adhesion inhibitory receptor molecule-1 (AIRM1/p75), screening of a human primary dendritic cell cDNA library for clones with sequence similarity with the CD33 (siglec-3) gene and homology searches in the dbEST division of the GenBank database [S75-77]. The structure of CD328 is composed of one V-set and two C2-set Ig-like modules, which led to its classificiation as siglec-7, and common inhibitory signaling motifs (Figure 2). It is related to siglec-5 (CD170) and iden­tical in modular design to siglecs-6, 8, and 9, produced by NK, dendritic and CD8⁺ T cell, respectively. Ligand engagement (including 2,8-linked sialosides) negatively impacts NK cell cytotoxicity, as shown in response to recognition of ganglioside GD3 and the disialosyl globopentaosylceramide DSGb5 [S78].

The natural cytotoxicity receptors NKp46 (CD335) and NKp30 (CD337) are glycopro­teins with two C2-set (CD335) or one V-set (CD337) Ig-like domain(s) (Figure 2). They exert their trigger capacity via association with signaling proteins that have immunore­ceptor tyrosine-based activating motifs (ITAMs), mirroring how CD94 teams up with ITIM-containing proteins to dampen NK cell responses [S79, S80]. Similar to CD94, heparan sulfate-derived oligosaccharides are binding partners, as are N-glycans with 2,3-sialylation or sLe^x (CD15s) determinants [S81].

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