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

Abstract

CEL-IV is a C-type lectin isolated from a sea cucumber, Cucumaria echinata. This lectin is composed of four identical C-type carbohydrate-recognition domains (CRDs). X-ray crystallographic analysis of CEL-IV revealed that its tetrameric structure was stabilized by multiple interchain disulfide bonds among the subunits. Although CEL-IV has the EPN motif in its carbohydrate-binding sites, which is known to be characteristic of mannose binding C-type CRDs, it showed preferential binding of galactose and N-acetylgalactosamine. Structural analyses of CEL-IV-melibiose and CEL-IV-raffinose complexes revealed that their galactose residues were recognized in an inverted orientation compared with mannose binding C-type CRDs containing the EPN motif, by the aid of a stacking interaction with the side chain of Trp-79. Changes in the environment of Trp-79 induced by binding to galactose were detected by changes in the intrinsic fluorescence and UV absorption spectra of WT CEL-IV and its site-directed mutants. The binding specificity of CEL-IV toward complex oligosaccharides was analyzed by frontal affinity chromatography using various pyridylamino sugars, and the results indicate preferential binding to oligosaccharides containing Gal␤1-3/4(Fuc␣1-3/4)GlcNAc structures. These findings suggest that the specificity for oligosaccharides may be largely affected by interactions with amino acid residues in the binding site other than those determining the monosaccharide specificity.

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

“…Typical CLRs that simply recognize sugars, such as DC-SIGNR and CEL-IV, exhibit remarkably low affinities (K d ∼mM) (32,33). They require multiple valencies of sugar ligands to mediate signaling.…”

The carbohydrate recognition domain of collectins

“…The carbonyls of Gln and Asp interact preferentially with an equatorial hydroxyl at C3 of the saccharide and an axial hydroxyl group at C4, as found in galactose. Although this prediction of saccharide specificity based solely on the EPN motif is a strong simplification and neglects other structural factors involved, it is still widely used and very often correct, although exceptions do exist [19]. Evidence for the utility of the EPN rule is provided in an elegant study by Drickamer [20], who was the first to show that a mutational change of the EPN motif into QPD completely changed the ligand specificity of MBP from mannose‐ to galactose‐binding.…”

Mannose-recognition mutant of the galactose/N-acetylgalactosamine-specific C-type lectin CEL-I engineered by site-directed mutagenesis

“…The QPD and EPN motifs are located in this long loop region constituting part of the carbohydrate-binding site and have been known to be important characteristics to predict the binding specificity for galactose-or mannose-related carbohydrates. These three amino acids are directly involved in the binding of both lectins CEL-III [36] and CEL-IV [37]. Interestingly, CEL-IV shows galactose specificity, although it contains an EPN motif in the carbohydrate-binding site.…”

“…Interestingly, CEL-IV shows galactose specificity, although it contains an EPN motif in the carbohydrate-binding site. This discrepancy was explained by the crystal structure of CEL-IV complexed with galactose-containing carbohydrates, in which the aromatic residue Trp79 specifies the orientation of the bound galactose by stacking interaction so that EPN can form proper hydrogen bonds with galactose [37].…”