Localization of multiple functional domains on human PECAM-1 (CD31) by monoclonal antibody epitope mapping.
PECAM-1, a cell adhesion molecule of the immunoglobulin gene (Ig) superfamily, has been implicated in white cell transmigration, integrin activation on lymphocytes, and cell-cell adhesion. The purpose of this investigation was to identify specific regions of the PECAM-1 extracellular domain mediating these functions by identifying the location of epitopes of bioactive anti-PECAM-1 monoclonal antibodies. The binding regions of mAbs important in PECAM-1-mediated leukocyte transmigration (Hec 7.2 and 3D2) were mapped to N-terminal Ig-like domains. The epitopes of monoclonal antibodies that activated integrin function on lymphocytes were dispersed over the entire extracellular region, but those that had the strongest activating effect were preferentially localized to the N-terminus of the molecule. The binding regions of mAbs that blocked PECAM-1-mediated heterophilic L-cell aggregation were located either in Ig-like domain 2 (NIH31.4) or Ig-like domain 6 (4G6 and 1.2). Site-directed mutagenesis further pinpointed the epitope of the 4G6 mAb to a hexapeptide, CAVNEG, within Ig-like domain 6. These results demonstrate that PECAM-1 contains multiple functional domains. Regions within N-terminal Ig-like domains appear to be required for transmigration. In contrast, two distinct regions were implicated in L-cell mediated heterophilic aggregation.
Yan
H C
HC
Department of Medicine, University of Pennsylvania Medical Center, Philadelphia 19104, USA.
Pilewski
J M
JM
Zhang
Q
Q
DeLisser
H M
HM
Romer
L
L
Albelda
S M
SM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015214:Antigens, Differentiation, Myelomonocytic / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D002465:Cell Movement; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000032:analysis*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016023:Integrins / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000276:immunology; D017384:Sequence Deletion / Q000502:physiology
D000911:Antibodies, Monoclonal; D015214:Antigens, Differentiation, Myelomonocytic; D015815:Cell Adhesion Molecules; D000939:Epitopes; D016023:Integrins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011993:Recombinant Fusion Proteins