SITE-DIRECTED MUTAGENESIS STUDIES ON THE LIMA-BEAN LECTIN - ALTERED CARBOHYDRATE-BINDING SPECIFICITIES RESULT FROM SINGLE AMINO-ACID SUBSTITUTIONS

The wild-type seed lima bean lectin (LBL), and recombinant LBL express ed in Escherichia coli show specificity for the human blood group A im munodominant trisaccharide GalNAc alpha 1-3 [Fuc alpha 1-2]Gal beta 1- R. We have generated four site-specific mutants of LBL, two of which s how altered specificity for extended carbohydrate structures. Four mut ants, [C127Y]LBL, [H128P]LBL, [H128R]LBL and [W132F]LBL were expressed in E. coli. Two mutants show altered specificity for the substituent at the C2 hydroxy group of the penultimate Gal in the wild-type ligand which is alpha-L-fucose in the A trisaccharide. The mutant [C127Y]LBL showed specificity for the A disaccharide (GalNAc alpha 1-3Gal) and G alNAc alpha 1-4Gal, with free hydroxyl groups at the C2 position of Ga l. The mutant [H128P]LBL bound the Forssman disaccharide structure Gal NAc alpha 1-3GalNAc, in which the C2 hydroxyl group is substituted wit h an acetamido group. The third and fourth mutants, [H128]LBL and [W13 2F]LBL, exhibited wild-type specificities, both recognizing the A tris accharide. All of these mutant lectins bound the terminal GalNAc resid ues exposed on asialoovine submaxillary mucin, thus indicating that th e monosaccharide-binding site had not been altered. We also determined that all but one mutant ([C127Y]LBL) retained the high-affinity bindi ng site for N-6 derivatives of adenine, indicative of tetramer formati on; each mutant also expressed the low-affinity binding site for 8-ani linonaphthalene 1-sulfonate (1/monomer). Thus, by targeting two residu es in LBL, we have identified a region of the protein that is part of the extended carbohydrate-binding site and which is specifically invol ved in the binding/recognition of substituents at the C2 position of t he penultimate Gal of the A disaccharide. We have determined, by site- directed mutagenesis, that an essential Cys residue is involved in the specificity of LBL for the A trisaccharide.