Mutational analysis at Asn-73 in Agaricus bisporus lectin, a residue involved in T-antigen binding

ISSOGLIO, FEDERICO M.; IRAZOQUI, FERNANDO J.; CURTINO, JUAN A.; CARRIZO, MARÍA E.

Rosario, Santa Fe, Argentina

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2006

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

The lectin from the common edible mushroom Agaricus bisporus (ABL), has potent antiproliferative effects on human epithelial cancer cells without any apparent cytotoxicity. The three-dimensional structure of the lectin has been recently resolved by X-ray crystallography (Carrizo, M.E. et al., J. Biol. Chem., 280, 10614-23, 2005). ABL is a tetramer, each monomer presenting a novel fold with two â sheets connected by a helix-loop-helix motif. These studies showed that Galâ1-3GalNAc, the disaccharide moiety of Thomsen-Friedenreich antigen (T-antigen), binds at a shallow depression on the surface of the molecule. On the contrary, GlcNAc binds at a different site on the opposite side of the helix-loop-helix motif. Among the contacts involved in providing the specificity toward T-antigen is the interaction between the side chain of Asn-73 and the acetamido group of GalNAc. Site-directed mutational changes were introduced at this residue with the objective of probing the role of this residue in T-antigen binding and possibly engineering an altered species with increased specificity for T-antigen. The binding activity was analyzed by competitive-enzyme-lectin assays (CELA). The mutants N73E and N73Q showed similar binding affinity for T-antigen, while the last one is less potent for recognition of GalNAc. Results involving other changes are also described.