Tailoring GalNAc alpha 1-3Gal beta -specific lectins from a multi-specific fungal galectin: dramatic change of carbohydrate specificity by a single amino-acid substitution.

摘要

Galectins exhibit multiple roles through recognition of diverse structures of beta -galactosides. However, this broad specificity often hinders their practical use as probes. In the present study we report a dramatic improvement in the carbohydrate specificity of a multi-specific fungal galectin from the mushroom Agrocybe cylindricea, which binds not only to simple beta -galactosides, but also to their derivatives. Site-directed mutagenesis targeting five residues involved in beta -galactose binding revealed that replacement of Asn46 with alanine (N46A) increased the binding to GalNAc alpha 1-3Gal beta -containing glycans, while eliminating binding to all other beta -galactosides, as shown by glycoconjugate microarray analysis. Quantitative analysis by frontal affinity chromatography showed that the mutant N46A had enhanced affinity towards blood group A tetraose (type 2), A hexaose (type 1) and Forssman pentasaccharide with dissociation constants of 5.0x10-6 M, 3.8x10-6 M and 1.0x10-5 M respectively. Surprisingly, all the other mutants generated by saturation mutagenesis of Asn46 exhibited essentially the same specificity as N46A. Moreover, alanine substitution for Pro45, which forms the cis-conformation upon beta -galactose binding, exhibited the same specificity as N46A. From a practical viewpoint, the derived N46A mutant proved to be unique as a specific probe to detect GalNAc alpha 1-3Gal beta -containing glycans by methods such as flow cytometry, cell staining and lectin microarray.