A functional epitope on P-selectin that supports binding of P-selectin to P-selectin glycoprotein ligand-1 but not to sialyl Lewis X oligosaccharides.
P-selectin mediates the adhesion of leukocytes to activated platelets and endothelial cells. To characterize the functional domains of P-selectin for ligand recognition, we established nine hybridoma cell lines secreting anti-rat P-selectin mAb. Among them, the mAb C215 bound both rat and human P-selectins, and inhibited binding of rat and human P-selectins to P-selectin glycoprotein ligand-1 (PSGL-1) from HL-60 cells. In contrast, mAb C215 failed to inhibit the binding of rat and human P-selectin-IgG to sialyl Lewis X (sLe(x)) oligosaccharides. Epitope mapping of mAb C215 using synthetic decapeptides revealed that mAb C215 binds specifically to an eight-residue epitope that spans amino acids 76-83 of rat P-selectin, a region completely conserved by human P-selectin. Synthetic peptides containing the mAb C215 epitope inhibited binding of P-selectin to PSGL-1, but not to sLe(x) oligosaccharides, suggesting that the C215 epitope on P-selectin may directly interact with a particular site on the PSGL-1 core protein essential for interaction with P-selectin, such as sulfated tyrosine residues. Our results suggest the presence of two ligand recognition sites on P-selectin necessary for binding to PSGL-1--one recognizes sLe(x), while the other recognises the PSGL-1 core protein.
Hirose
M
M
Department of Bioregulation, Biomedical Research Center, Osaka University Medical School, Suita, Japan.
Kawashima
H
H
Miyasaka
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002448:Cell Adhesion; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D066298:In Vitro Techniques; D007983:Lewis Blood Group Antigens; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D019007:P-Selectin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000080506:Sialyl Lewis X Antigen
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007074:Immunoglobulin G; D007983:Lewis Blood Group Antigens; D008024:Ligands; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D019007:P-Selectin; C084882:P-selectin ligand protein; D011993:Recombinant Fusion Proteins; D000080506:Sialyl Lewis X Antigen