Binding of sugar ligands to Ca(2+)-dependent animal lectins. II. Generation of high-affinity galactose binding by site-directed mutagenesis.
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.
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Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism*; D007700:Kinetics; D037102:Lectins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008358:Mannose / Q000378:metabolism*; D037241:Mannose-Binding Lectins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D037102:Lectins; D037241:Mannose-Binding Lectins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D008358:Mannose; D002118:Calcium; D005690:Galactose