Transformation of Agrocybe cylindracea Galectin into αGalNAc-Specific Lectin.

A multi-specific fungal galectin from the mushroom Agrocybe cylindracea (ACG) binds a broad range of β-galactosides, as well as their derivative GalNAcα1-3Gal. Site-directed mutagenesis of the hydrophilic residues His, Asn, Arg, and Glu, involved in carbohydrate recognition, abolished the binding affinity of the derived mutants to β-galactosides, whereas only N46A caused increased affinity to GalNAcα1-3Gal-containing oligosaccharides and loss of β-galactoside-binding activity. Detailed structural analysis revealed that Pro45, the preceding residue of Asn46 of the wild-type ACG, takes the cis imide conformation to tether Asn46 onto a loop region to make new hydrogen bonds with β-galactosides and to compensate for the lack of evolutionarily conserved Asn. In contrast, in the N46A mutant, Pro45 takes the more stable trans conformation, resulting in "switched" specificity to αGalNAc. Such an altered recognition system in the binding specificity of galectins can be observed in other lectin molecules not only in nature but will also be observed in those engineered in the future.