Structural and functional relationships of PECAM-1 (platelet-endothelial cell adhesion molecule-1 or CD31)

PECAM-1 is a cell adhesion molecule of the immunoglobulin gene (Ig) superfamily expressed on the surface of cells of the hematopoietic and cardiovascular systems and has been implicated in a number of diverse cellular adhesive and developmental events within these systems. The purpose of this thesis was (1) to study the structural and functional relationships of PECAM-1 by identifying specific regions of the huPECAM-1 extracellular domain involved in mediating its adhesive function by mAb epitope mapping; (2) molecular cloning and functional characterization of muPECAM-1 isoforms; (3) to identify a critical region within the cytoplasmic domain that modulates the switch of extracellular domain binding specificity, and its potential operating mechanism. By using domain deletion mutations, the binding domains of a panel of anti-human PECAM-1 mAbs were mapped to distinct regions in the extracellular domain and the binding domain for two bioactive mAbs, 4G6 and 1.2, was located to the extracellular 6th Ig-like loop. Using human/mouse PECAM-1 chimeric constructs and site-directed mutagenesis further pinpointed the epitope of the 4G6 mAb to a six amino acid region within loop 6 (amino acids 496-501, CAVNEG). Correlating the distribution of bioactive mAb binding domains and their functions suggests that the N-terminal Ig-like loops may be more important in regulating the leukocyte functions and the C-terminus of the extracellular domain may play an important role in regulating PECAM-1 mediated heterophilic aggregation. A reverse transcription-polymerase chain reaction (RT-PCR) shot-gun cloning strategy using mRNA extracted from day 12-staged mouse embryos yielded a full-length and five novel cytoplasmic domain alternatively spliced isoforms of muPECAM-1. Functional characterization by L-cell aggregation mediated by each isoform revealed a distinct functional profile. Isoforms without exon 14 mediated calcium independent, heparin and anti-muPECAM-l mAb 390 insensitive, homophilic binding. However, full-length muPECAM-1 and other isoforms behaved like full-length huPECAM-1 and mediated calcium-dependent heterophilic aggregation that was inhibited by heparin and mAb 390. These results indicate a critical role of exon 14 in regulating the ligand binding specificity of muPECAM-1. In addition, deglycosylation of these isoforms with N-Glycosidase F indicated a change in the N-glycosylation profile of isoforms missing exon 14. In summary, the diverse functions of PECAM-1 are determined by its structure that harbors multiple functional antigenic epitopes on its extracellular domain. In addition, the functions of PECAM-1 are also regulated by alternative splicing of its cytoplasmic domain that may probably affect the folding of the newly translated PECAM-1 molecule and result in different degrees of N-glycosylation of the molecule