CHARACTERIZATION OF THE SIALIC ACID-BINDING SITE IN SIALOADHESIN BY SITE-DIRECTED MUTAGENESIS

The sialoadhesins are a distinct subgroup of the immunoglobulin superf amily, comprising sialoadhesin, CD22, the myelin-associated glycoprote in, and CD33. They can all mediate sialic acid-dependent binding to ce lls with distinct specificities. Sialoadhesin is a murine macrophage-r estricted cell-surface molecule with 17 extracellular immunoglobulin-l ike 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 mutagenes is in an attempt to identify the binding site of sialoadhesin. A subse t 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 mod el predicts that these residues form a contiguous binding site on the GFCC'C '' beta-sheet of the V-set domain centered around an arginine i n the F strand. A conservative mutation of this arginine to lysine als o abolished binding, This amino acid is conserved among all members of the sialoadhesin family and is therefore likely to be a key residue i n mediating sialic acid-dependent binding of sialoadhesins to cells.