Site-Directed Mutagenesis Studies on the Lima Bean Lectin. Altered Carbohydrate-Binding Specificities Result from Single Amino Acid Substitutions

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Abstract
The wild-type seed lima bean lectin (LBL), and recombinant LBL expressed in Escherichia coli show specificity for the human blood group A immunodominant 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 show altered specificity for extended carbohydrate structures. Four mutants, [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 GalNAc alpha 1-4Gal, with free hydroxyl groups at the C2 position of Gal. The mutant [H128P]LBL bound the Forssman disaccharide structure GalNAc alpha 1-3GalNAc, in which the C2 hydroxyl group is substituted with an acetamido group. The third and fourth mutants, [H128R]LBL and [W132F]LBL, exhibited wild-type specificities, both recognizing the A trisaccharide. All of these mutant lectins bound the terminal GalNAc residues exposed on asialoovine submaxillary mucin, thus indicating that the monosaccharide-binding site had not been altered. We also determined that all but one mutant ([C127Y]LBL) retained the high-affinity binding site for N6 derivatives of adenine, indicative of tetramer formation; each mutant also expressed the low-affinity binding site for 8-anilinonaphthalene 1-sulfonate (1/monomer). Thus, by targeting two residues in LBL, we have identified a region of the protein that is part of the extended carbohydrate-binding site and which is specifically involved in the binding/recognition of substituents at the C2 position of the 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.