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http://purl.uniprot.org/citations/25869490http://www.w3.org/1999/02/22-rdf-syntax-ns#typehttp://purl.uniprot.org/core/Journal_Citation
http://purl.uniprot.org/citations/25869490http://www.w3.org/1999/02/22-rdf-syntax-ns#typehttp://purl.uniprot.org/core/Journal_Citation
http://purl.uniprot.org/citations/25869490http://www.w3.org/2000/01/rdf-schema#comment"

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 3.17×10(3) 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."xsd:string
http://purl.uniprot.org/citations/25869490http://purl.org/dc/terms/identifier"doi:10.1016/j.bbagen.2015.04.004"xsd:string
http://purl.uniprot.org/citations/25869490http://purl.org/dc/terms/identifier"doi:10.1016/j.bbagen.2015.04.004"xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/author"Goda S."xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/author"Goda S."xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/author"Hirabayashi J."xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/author"Hirabayashi J."xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/author"Tateno H."xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/author"Tateno H."xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/author"Unno H."xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/author"Unno H."xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/author"Hatakeyama T."xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/author"Hatakeyama T."xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/author"Moriuchi H."xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/author"Moriuchi H."xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/date"2015"xsd:gYear
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/date"2015"xsd:gYear
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/name"Biochim. Biophys. Acta"xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/name"Biochim Biophys Acta"xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/pages"1457-1465"xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/pages"1457-1465"xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/title"Mannose-recognition mutant of the galactose/N-acetylgalactosamine-specific C-type lectin CEL-I engineered by site-directed mutagenesis."xsd:string
http://purl.uniprot.org/citations/25869490http://purl.uniprot.org/core/title"Mannose-recognition mutant of the galactose/N-acetylgalactosamine-specific C-type lectin CEL-I engineered by site-directed mutagenesis."xsd:string
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