BINDING OF SUGAR LIGANDS TO CA2-DEPENDENT ANIMAL LECTINS .2.. GENERATION OF HIGH-AFFINITY GALACTOSE BINDLNG BY SITE-DIRECTED MUTAGENESIS()

Changes have been introduced into the Ca2+-dependent carbohydrate-reco gnition domain (CRD) of rat serum mannose-binding protein by site-dire cted mutagenesis to model the binding sites of homologous galactose-bi nding CRDs. Binding assays reveal that galactose-binding activity near ly identical to that of the CRD from the asialoglycoprotein receptor c an be introduced into the mannose-binding site by 3 single amino acid changes and insertion of a segment of 5 amino acids. Separate changes are required to establish high-affinity binding to galactose and creat e high selectivity by exclusion of mannose from the binding site. The mutagenesis studies and NMR analysis of sugar CRD titrations demonstra te that an important component of high-affinity galactose binding is i nteraction between the B face of the sugar and tryptophan. The binding properties of the C-type CRD from the cartilage proteoglycan, aggreca n, can also be modeled based on the mannose-binding CRD framework. Thi s lower affinity binding site involves stacking of a phenylalanine res idue against the sugar ligand.