Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells.
P-selectin (CD62, GMP-140, PADGEM), a Ca(2+)-dependent lectin on activated platelets and endothelium, functions as a receptor for myeloid cells by interacting with sialylated, fucosylated lactosaminoglycans. P-selectin binds to a limited number of protease-sensitive sites on myeloid cells, but the protein(s) that carry the glycans recognized by P-selectin are unknown. Blotting of neutrophil or HL-60 cell membrane extracts with [125I]P-selectin and affinity chromatography of [3H]glucosamine-labeled HL-60 cell extracts were used to identify P-selectin ligands. A major ligand was identified with an approximately 250,000 M(r) under nonreducing conditions and approximately 120,000 under reducing conditions. Binding of P-selectin to the ligand was Ca2+ dependent and was blocked by mAbs to P-selectin. Brief sialidase digestion of the ligand increased its apparent molecular weight; however, prolonged digestion abolished binding of P-selectin. Peptide:N-glycosidase F treatment reduced the apparent molecular weight of the ligand by approximately 3,000 but did not affect P-selectin binding. Western blot and immunodepletion experiments indicated that the ligand was not lamp-1, lamp-2, or L-selectin, which carry sialyl Le(x), nor was it leukosialin, a heavily sialylated glycoprotein of similar molecular weight. The preferential interaction of the ligand with P-selectin suggests that it may play a role in adhesion of myeloid cells to activated platelets and endothelial cells.
Moore
K L
KL
Department of Medicine, St. Francis Medical Research Institute, University of Oklahoma Health Sciences Center, Oklahoma City 73104.
Stults
N L
NL
Diaz
S
S
Smith
D F
DF
Cummings
R D
RD
Varki
A
A
McEver
R P
RP
D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000378:metabolism*; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005944:Glucosamine / Q000378:metabolism; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute; D008024:Ligands; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009504:Neutrophils / Q000378:metabolism*; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015815:Cell Adhesion Molecules; D008024:Ligands; D008562:Membrane Glycoproteins; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D005944:Glucosamine