VISUALIZATION OF P-SELECTIN GLYCOPROTEIN LIGAND-1 AS A HIGHLY EXTENDED MOLECULE AND MAPPING OF PROTEIN EPITOPES FOR MONOCLONAL-ANTIBODIES

P-selectin glycoprotein ligand-l (PSGL-1), a sialomucin on human leuko cytes, mediates rolling of leukocytes on P-selectin expressed by activ ated platelets or endothelial cells under shear forces, PSGL-1 require s both tyrosine sulfate and O-linked glycans to bind P-selectin. Elect ron microscopy of rotary-shadowed PSGL-1 purified from human neutrophi ls indicated that it is a highly extended molecule with an extracellul ar domain that is approximate to 50 nm long. Both individual PSGL-1 mo lecules and rosettes composed of several molecules presumably attached at their transmembrane segments were observed, The extracellular doma in of PSGL-1 has 318 residues, including a signal peptide from residue s 1-18 and a propeptide from residues 19-41, Using bacterially express ed fusion proteins and synthetic peptides derived from the extracellul ar domain, we mapped the epitopes for two IgG anti-PSGL-1 monoclonal a ntibodies, 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 spannin g residues 49-62, This sequence overlaps the tyrosine sulfation sites at residues 46, 48, and 51 that have been implicated in binding of PSG L-1 to P-selectin, Our results demonstrate that PSGL-1 is a long, exte nded molecule and suggest that the P-selectin binding site is located near the N terminus, well above the membrane, This location may facili tate interactions of PSGL-1 with P-selectin under shear stress.