Visualization of P-selectin glycoprotein ligand-1 as a highly extended molecule and mapping of protein epitopes for monoclonal antibodies.
P-selectin glycoprotein ligand-1 (PSGL-1), a sialomucin on human leukocytes, mediates rolling of leukocytes on P-selectin expressed by activated platelets or endothelial cells under shear forces. PSGL-1 requires both tyrosine sulfate and O-linked glycans to bind P-selectin. Electron microscopy of rotary-shadowed PSGL-1 purified from human neutrophils indicated that it is a highly extended molecule with an extracellular domain that is -50 nm long. Both individual PSGL-1 molecules and rosettes composed of several molecules presumably attached at their transmembrane segments were observed. The extracellular domain of PSGL-1 has 318 residues, including a signal peptide from residues 1-18 and a propeptide from residues 19-41. Using bacterially expressed fusion proteins and synthetic peptides derived from the extracellular domain, we mapped the epitopes for two IgG anti-PSGL-1 monoclonal antibodies, PL1 and PL2. PL2 bound to a region within residues 188-235 that is located in a series of decameric consensus repeats. PL1, which blocks binding of PSGL-1 to P-selectin, recognized an epitope spanning residues 49-62. This sequence overlaps the tyrosine sulfation sites at residues 46, 48, and 51 that have been implicated in binding of PSGL-1 to P-selectin. Our results demonstrate that PSGL-1 is a long, extended molecule and suggest that the P-selectin binding site is located near the N terminus, well above the membrane. This location may facilitate interactions of PSGL-1 with P-selectin under shear stress.
Li
F
F
Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City 73104, USA.
Erickson
H P
HP
James
J A
JA
Moore
K L
KL
Cummings
R D
RD
McEver
R P
RP
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D006224:Cricetinae; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics; D006801:Humans; D008024:Ligands; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000648:ultrastructure*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D019007:P-Selectin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008024:Ligands; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D011993:Recombinant Fusion Proteins