Mapping the epitope of a functional P-selectin monoclonal antibody (LYP20) to a short complement-like repeat (SCR 4) domain: use of human-mouse chimaera and homologue-replacement mutagenesis.
P-selectin (CD62P), an adhesion molecule localized in platelet alpha-granules and endothelial cell Weibel-Palade bodies, is rapidly expressed 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 monoclonal antibody (mAb), LYP20. LYP20 recognizes human or rat, but not mouse, P-selectin. Human/mouse chimaeras and wild-type constructs, modified by homologue replacement mutagenesis, were expressed in COS cells. Blocking anti-(P-selectin) mAbs (G1, G3 or CLB-thromb/6) were observed, 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-like repeats (SCR domain 4). Homologue replacement mutagenesis of SCR domain 4 (region delineated by amino acid residues 359-457) identified three amino acids (Cys412-->Ser, Cys416-->Ser or Arg415-->Lys) as being implicated in the LYP20 epitope. Deleting the region bearing the 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. These results are the first to indicate (1) that a functional blocking anti-P-selectin mAb binds to SCR 4, a site other than the lectin-like/epidermal growth factor-like domain, and (2) that SCR domain 4 has a functional role in P-selectin-leucocyte interactions.
Ruchaud-Sparagano
M H
MH
INSERM U 331, Faculté de Médecine RTH Laennec, 8 rue Guillaume Paradin, 69372 Lyon Cedex 08, France.
Malaud
E
E
Gayet
O
O
Chignier
E
E
Buckland
R
R
McGregor
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D001792:Blood Platelets / Q000502:physiology; D016466:CHO Cells; D002448:Cell Adhesion / Q000235:genetics; D006224:Cricetinae; D018604:Epitope Mapping; D005434:Flow Cytometry; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D019007:P-Selectin / Q000737:chemistry* / Q000276:immunology; D011485:Protein Binding / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D012397:Rosette Formation; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D014162:Transfection / Q000235:genetics
D000911:Antibodies, Monoclonal; D019007:P-Selectin; D011993:Recombinant Fusion Proteins