MAPPING THE EPITOPE OF A FUNCTIONAL P-SELECTIN MONOCLONAL-ANTIBODY (LYP20) TO A SHORT COMPLEMENT-LIKE REPEAT (SCR-4) DOMAIN - USE OF HUMAN-MOUSE CHIMERA AND HOMOLOG-REPLACEMENT MUTAGENESIS

P-selectin (CD62P), an adhesion molecule localized in platelet alpha-g ranules and endothelial cell Weibel-Palade bodies, is rapidly expresse d on the surface of activated cells. This adhesion molecule, a member of the selectin family, mediates leucocyte interactions with activated platelets or endothelial cells. The aim of this study was to identify and characterize the epitope of a functional blocking P-selectin mono clonal antibody (mAb), LYP20. LYP20 recognizes human or rat, but not m ouse, P-selectin. Human/mouse chimaeras and wild-type constructs, modi fied by homologue replacement mutagenesis, were expressed in COS cells . Blocking anti-(P-selectin) mAbs (G1, G3 or CLB-thromb/6) were observ ed, by flow cytometry, to bind to the lectin-like domain. In contrast, LYP20 was found to bind to one of the P-selectin short complement-lik e repeats (SCR domain 4). Homologue replacement mutagenesis of SCR dom ain 4 (region delineated by amino acid residues 359-457) identified th ree amino acids (Cys(412) --> Ser, Cys(416)-->Ser or Arg(415)-->Lys) a s being implicated in the LYP20 epitope. Deleting the region bearing t he LYP20 epitope, from a wild-type CD62P construct, showed a decrease in polymorphonuclear leucocyte (PMNL) binding to transfected COS cells . In addition, mutation of one of the three amino acids, implicated in the LYP20 epitope, markedly affected PMNL binding to transfected COS cells but did not affect the binding of mAbs G1 and CLB-thromb/6. Thes e results are the first to indicate (1) that a functional blocking ant i-P-selectin mAb binds to SCR 4, a site other than the lectin-like/epi dermal growth factor-like domain, and (2) that SCR domain 4 has a func tional role in P-selectin-leucocyte interactions.