1. Mannose-recognition mutant of the galactose/N-acetylgalactosamine-specific C-type lectin CEL-I engineered by site-directed mutagenesis
- Author
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Hiromi Moriuchi, Hiroaki Tateno, Jun Hirabayashi, Tomomitsu Hatakeyama, Hideaki Unno, and Shuichiro Goda
- Subjects
Models, Molecular ,Carbohydrate ,Acetylgalactosamine ,Stereochemistry ,Glycoconjugate ,Amino Acid Motifs ,Biophysics ,Mannose ,Calorimetry ,Crystallography, X-Ray ,Protein Engineering ,Binding, Competitive ,Biochemistry ,Chromatography, Affinity ,N-Acetylgalactosamine ,chemistry.chemical_compound ,C-type lectin ,Animals ,Lectins, C-Type ,Amino Acid Sequence ,Site-directed mutagenesis ,Molecular Biology ,X-ray crystallography ,chemistry.chemical_classification ,Binding Sites ,biology ,Chemistry ,Circular Dichroism ,Galactose ,Lectin ,Isothermal titration calorimetry ,Cucumaria ,Protein tertiary structure ,Protein Structure, Tertiary ,Amino Acid Substitution ,Mutation ,Mutagenesis, Site-Directed ,biology.protein ,Protein Binding - Abstract
Background CEL-I is a galactose/N-acetylgalactosamine-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. Its carbohydrate-binding site contains a QPD (Gln-Pro-Asp) motif, which is generally recognized as the galactose specificity-determining motif in the C-type lectins. In our previous study, replacement of the QPD motif by an EPN (Glu-Pro-Asn) motif led to a weak binding affinity for mannose. Therefore, we examined the effects of an additional mutation in the carbohydrate-binding site on the specificity of the lectin. Methods Trp105 of EPN-CEL-I was replaced by a histidine residue using site-directed mutagenesis, and the binding affinity of the resulting mutant, EPNH-CEL-I, was examined by sugar-polyamidoamine dendrimer assay, isothermal titration calorimetry, and glycoconjugate microarray analysis. Tertiary structure of the EPNH-CEL-I/mannose complex was determined by X-ray crystallographic analysis. Results Sugar-polyamidoamine dendrimer assay and glycoconjugate microarray analysis revealed a drastic change in the specificity of EPNH-CEL-I from galactose/N-acetylgalactosamine to mannose. The association constant of EPNH-CEL-I for mannose was determined to be 3.17 × 103 M- 1 at 25 °C. Mannose specificity of EPNH-CEL-I was achieved by stabilization of the binding of mannose in a correct orientation, in which the EPN motif can form proper hydrogen bonds with 3- and 4-hydroxy groups of the bound mannose. Conclusions Specificity of CEL-I can be engineered by mutating a limited number of amino acid residues in addition to the QPD/EPN motifs. General significance Versatility of the C-type carbohydrate-recognition domain structure in the recognition of various carbohydrate chains could become a promising platform to develop novel molecular recognition proteins., Biochimica et Biophysica Acta (BBA) - General Subjects, 1850(7), pp.1457-1465; 2015
- Published
- 2015