Binding of sugar ligands to Ca(2+)-dependent animal lectins. II. Generation of high-affinity galactose binding by site-directed mutagenesis

• Published in: The Journal of biological chemistry 1994-06, Vol.269 (22), p.15512-15519
• Main Authors: Iobst, S T, Drickamer, K
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Calcium - metabolism; Carrier Proteins - biosynthesis; Carrier Proteins - chemistry; Carrier Proteins - metabolism; Galactose - metabolism; Kinetics; Lectins - metabolism; Magnetic Resonance Spectroscopy; Mannose - metabolism; Mannose-Binding Lectins; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Protein Conformation; Rats; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Restriction Mapping; Sequence Homology, Amino Acid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)40709-5

Changes have been introduced into the Ca(2+)-dependent carbohydrate-recognition domain (CRD) of rat serum mannose-binding protein by site-directed mutagenesis to model the binding sites of homologous galactose-binding CRDs. Binding assays reveal that galactose-binding activity nearly identical to that of the CRD from the asialoglycoprotein receptor can 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 create high selectivity by exclusion of mannose from the binding site. The mutagenesis studies and NMR analysis of sugar-CRD titrations demonstrate that an important component of high-affinity galactose binding is interaction between the B face of the sugar and tryptophan. The binding properties of the C-type CRD from the cartilage proteoglycan, aggrecan, can also be modeled based on the mannose-binding CRD frame-work. This lower affinity binding site involves stacking of a phenylalanine residue against the sugar ligand.