Characterization of the sialic acid-binding site in sialoadhesin by site-directed mutagenesis.

Journal article

The sialoadhesins are a distinct subgroup of the immunoglobulin superfamily, comprising sialoadhesin, CD22, the myelin-associated glycoprotein, and CD33. They can all mediate sialic acid-dependent binding to cells with distinct specificities. Sialoadhesin is a murine macrophage-restricted cell-surface molecule with 17 extracellular immunoglobulin-like domains that recognizes NeuAc alpha 2-3Gal in N- and O-glycans and interacts preferentially with cells of the granulocytic lineage. Its sialic acid-binding site is located within the NH2-terminal (membrane-distal) V-set domain. Here we have carried out site-directed mutagenesis in an attempt to identify the binding site of sialoadhesin. A subset of nonconservative mutations disrupted sialic acid-dependent binding without affecting binding of three monoclonal antibodies directed to two distinct epitopes of sialoadhesin. A CD8 alpha-based molecular model predicts that these residues form a contiguous binding site on the GFCC'C" beta-sheet of the V-set domain centered around an arginine in the F strand. A conservative mutation of this arginine to lysine also abolished binding. This amino acid is conserved among all members of the sialoadhesin family and is therefore likely to be a key residue in mediating sialic acid-dependent binding of sialoadhesins to cells.

Authors: Vinson, M; van der Merwe, P; Kelm, S; May, A; Jones, E

• Publication status: Published
• Journal: Journal of biological chemistry
• Volume: 271
• Issue: 16
• Pages: 9267-9272
• Publication date: 1996-04-01
• DOI: 10.1074/jbc.271.16.9267
• EISSN: 1083-351X
• ISSN: 0021-9258
• Language: English
• Keywords: Recombinant Proteins; Binding Sites; Transfection; Amino Acid Sequence; Antibodies, Monoclonal; Molecular Sequence Data; Membrane Glycoproteins; Protein Structure, Secondary; Carbohydrate Conformation; Conserved Sequence; Carbohydrate Sequence; Models, Structural; Epitopes; Polymerase Chain Reaction; Mice; Cell Adhesion Molecules; Mutagenesis, Site-Directed; Receptors, Immunologic; Point Mutation; Sialic Acids; Cell Line; Erythrocytes; Arginine; Animals; Humans; Polysaccharides; Antigens, CD8; Sequence Homology, Amino Acid