Characterization of the sialic acid-binding site in sialoadhesin by site-directed mutagenesis.
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.
Vinson
M
M
Imperial Cancer Research Fund Laboratories, University of Oxford, United Kingdom.
van der Merwe
P A
PA
Kelm
S
S
May
A
A
Jones
E Y
EY
Crocker
P R
PR
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001120:Arginine; D001665:Binding Sites; D016827:CD8 Antigens / Q000737:chemistry; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D017124:Conserved Sequence; D000939:Epitopes / Q000032:analysis; D004912:Erythrocytes / Q000502:physiology; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011134:Polysaccharides; D017433:Protein Structure, Secondary; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D063267:Sialic Acid Binding Ig-like Lectin 1; D012794:Sialic Acids / Q000378:metabolism*; D014162:Transfection
D000911:Antibodies, Monoclonal; D016827:CD8 Antigens; D015815:Cell Adhesion Molecules; D000939:Epitopes; D008562:Membrane Glycoproteins; D011134:Polysaccharides; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; C571166:SIGLEC1 protein, human; D063267:Sialic Acid Binding Ig-like Lectin 1; D012794:Sialic Acids; C571167:Siglec1 protein, mouse; D001120:Arginine