title: Tailoring GalNAcα1-3Galβ-specific lectins from a multi-specific fungal galectin: dramatic change of carbohydrate specificity by a single amino-acid substitution.
abstract: Galectins exhibit multiple roles through recognition of diverse structures of β-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 β-galactosides, but also to their derivatives. Site-directed mutagenesis targeting five residues involved in β-galactose binding revealed that replacement of Asn46 with alanine (N46A) increased the binding to GalNAcα1-3Galβ-containing glycans, while eliminating binding to all other β-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 5.0 × 10⁻⁶ M, 3.8 × 10⁻⁶ M and 1.0 × 10⁻⁵ 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 β-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 GalNAcα1-3Galβ-containing glycans by methods such as flow cytometry, cell staining and lectin microarray.
authors.affiliation: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology, Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Dan
authors.initials: D
authors.lastname: Hu
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tateno
authors.forename: Takashi
authors.initials: T
authors.lastname: Sato
authors.forename: Hisashi
authors.initials: H
authors.lastname: Narimatsu
authors.forename: Jun
authors.initials: J
authors.lastname: Hirabayashi
mesh_terms: D000116:Acetylgalactosamine / Q000378:metabolism*; D000363:Agaricales / Q000378:metabolism*; D019943:Amino Acid Substitution; D001483:Base Sequence; D002241:Carbohydrates / Q000737:chemistry*; D017931:DNA Primers; D037161:Galectins / Q000378:metabolism*; D016133:Polymerase Chain Reaction
chemical_list: D002241:Carbohydrates; D017931:DNA Primers; D037161:Galectins; D000116:Acetylgalactosamine