High Affinity Interaction between a Bivalve C-type Lectin and a Biantennary Complex-type N-Glycan Revealed by Crystallography and Microcalorimetry

Abstract

Codakine is an abundant 14-kDa mannose-binding C-type lectin isolated from the gills of the sea bivalve Codakia orbicularis. Binding studies using inhibition of hemagglutination indicated specificity for mannose and fucose monosaccharides. Further experiments using a glycan array demonstrated, however, a very fine specificity for N-linked biantennary complex-type glycans. An unusually high affinity was measured by titration microcalorimetry performed with a biantennary Asn-linked nonasaccharide. The crystal structure of the native lectin at 1.3 Å resolution revealed a new type of disulfide-bridged homodimer. Each monomer displays three intramolecular disulfide bridges and contains only one calcium ion located in the canonical binding site that is occupied by a glycerol molecule. The structure of the complex between Asn-linked nonasaccharide and codakine has been solved at 1.7 Å resolution. All residues could be located in the electron density map, except for the capping ␤1-4-linked galactosides. The ␣1-6-linked mannose binds to calcium by coordinating the O3 and O4 hydroxyl groups. The GlcNAc moiety of the ␣1,6 arm engages in several hydrogen bonds with the protein, whereas the GlcNAc on the other antenna is stacked against Trp 108 , forming an extended binding site. This is the first structural report for a bivalve lectin.Lectins are multivalent carbohydrate-binding proteins that play important roles in the social life of cells. A growing repertoire of lectins has been identified in invertebrates (1), where these molecules are involved in self/nonself recognition (2). For example, lectins play a role in aggregation mechanisms in corals and sponges (3) or in sperm-egg recognition in oysters (4). Lectin mediation of symbiosis with algae or bacteria has been observed in coral (5) and nematodes (6). Nevertheless, the most common function assessed for lectins in marine invertebrates is their role in innate immunity by specific binding of polysaccharide-coated pathogenic bacteria (7,8).Different lectins have been identified in bivalves and they most frequently belong to the C-type lectin family. Proteins from this group of calcium-dependent lectins have been reported in oysters (9, 10), scallops (11), and clams (12, 13). C-type lectins are characterized by a carbohydrate recognition domain (CRD) 3 with a conserved fold and the involvement of a calcium ion in carbohydrate binding (14). The crystal structure of mannose-binding protein was the first one to be described (15). The CRD belongs to a larger family sharing a common fold and is referred to as the C-type lectin-like domain (16).Codakine is a 14-kDa C-type lectin purified from the gill of the tropical clam Codakia orbicularis (Linné 1758) by affinity chromatography on a mannose-agarose column (17). It forms homodimers and heterodimers with isoforms 1 (NCBI accession number AAX19697) and 2 (NCBI accession number ABQ40396) (13). A 19-amino acid peptide signal suggests that the lectin travels through a secretory pathway. The 129-amino acid sequence of the mature ...

Structural analysis for glycolipid recognition by the C-type lectins Mincle and MCL

“…S7, clearly showing that citric acid inhibits glycolipid binding to Mincle, whereas acetic acid does not. Notably, another mannose-binding C-type lectin, codakine, bound similar positions of oxygens of glycerol and glycan in a Ca 2+ ion-mediated manner (26). Citric acid is likely accommodated at this position to block the ligands, and hydroxyl groups are likely used following the coordination of Ca 2+ ions generally observed in CLRs.…”

Antibody WN1 222-5 mimics Toll-like receptor 4 binding in the recognition of LPS

“…The first Glc and the branching Gal residues of the outer core bind via Arg H52 and Asn H53, which explains the enhanced affinity observed for oligosaccharides containing an outer core and a branching substitution at position 6 of the first hexose (30). These residues are bound independently of their stereochemistry of ring hydroxyls, which opens the possibility for WN1 222-5 recognition of LPS with other outer core types.…”

“…Remarkably, WN1 222-5 binds its LPS core epitope even in the presence of O-PS. By using whole LPS and a number of neoglycoconjugates containing core-OS from all E. coli core types, S. enterica, and the mutant strain E. coli J-5 in ELISA binding studies, we previously identified parts of an inner core epitope accessible to high-affinity binding of mAb WN1 222-5 (27), with an affinity orders of magnitude higher than that determined for mAb Se155-4 against Salmonella group B O-PS and most other carbohydrate binding proteins (30).…”

“…The antigen is forced to adopt a limited number of conformations by the relatively large number of intramolecular hydrogen bonds and steric crowding within the core region (30,33) that are seen in the antibody and in the TLR4-MD-2 complexes (3). Kdo II forms only a weak hydrogen bond to Tyr H99 (which also forms two other hydrogen bonds to the antigen), so its importance to antigen recognition probably comes as a steric impediment to conformational rearrangement of the inner core.…”

Galactose Recognition by a Tetrameric C-type Lectin, CEL-IV, Containing the EPN Carbohydrate Recognition Motif

“…1A, the four carbohydrate-binding sites of the CEL-IV tetramer are oriented apart from one another. Because (23). Although the fold of the CEL-IV protomer resembles the other C-type CRDs, as representatively shown for C. echinata lectin CEL-I (6), tunicate (Polyandrocarpa misakiensis) lectin TC14 (11), and rat mannose-binding lectin MBL (MBP-A) (24) (Fig.…”