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 1.3A 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 1.7A resolution. All residues could be located in the electron density map, except for the capping beta1-4-linked galactosides. The alpha1-6-linked mannose binds to calcium by coordinating the O3 and O4 hydroxyl groups. The GlcNAc moiety of the alpha1,6 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.
Highlights
Tin mediation of symbiosis with algae or bacteria has been observed in coral [5] and nematodes [6]
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]
Been observed between codakine and mermaid nematodes [18]. These results point to the probable role for the gill-located codakine in either antibacterial protection or in the recognition of sulfur-oxidizing bacteria, symbionts that are needed for the survival of C. orbicularis in sandy anaerobic environments [19]
Summary
Protein Purification—The previously described protein purification protocol [13] was used with minor modifications. After a 10-min centrifugation at 10,000 rpm, the supernatant was dialyzed overnight at 4 °C against fresh T-buffer four times. Hemagglutination Assays—Hemagglutination tests were performed using microtiter plates with U-bottom wells by the 2-fold serial dilution method. 25 l of rabbit erythrocytes (2% in NaCl; Biomerieux) were mixed with serially diluted codakine in T-buffer (described above), starting with a concentration of 1 mg/ml. Inhibition of hemagglutination was assayed by 2-fold serial dilutions of the following sugars in T-buffer: D-mannose, D-galactose, D-glucose, N-acetylglucosamine, L-fucose, N-acetylneuraminic acid, and D-rhamnose, each starting at a concentration of 100 mM. The purification of Alexa-labeled codakine was performed by mannose-agarose chromatography, as described above. Titration of codakine binding was performed in a cell with a volume of 1.447 ml by 30 injections of 10 l of ligand with 5-min intervals while stirring at 310 rpm.
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