alpha-Galactosyl-mediated activation of porcine endothelial cells - Studies on CD31 and VE-cadherin in adhesion and signaling

Background. Ligation of alpha-galactosyl epitopes on endothelial cells by n aturally occurring human antibodies causes hyperacute rejection in porcine- to-human xenotransplantation. The alpha-galactosyl-specific lectin Bandeira ea simplicifolia isolectin B4 (IB4) has been reported to trigger endothelia l "gap" formation and tyrosine phosphorylation of an unidentified 130-kDa p rotein (1,2). We have studied two 130-kDa junctional adhesion molecules, CD 31 and VE-cadherin, in porcine aortic endothelial cells (PAECs) during IB4- mediated activation. The cellular distribution of these molecules, their su sceptibility to tyrosine phosphorylation, and their capacity to bind IB4 or natural human antibodies have been determined. Methods. Porcine CD31 and VE-cadherin were cloned. Recombinant proteins and monoclonal antibodies were prepared. The distribution and phosphorylation of CD31 and VE cadherin in confluent PAECs activated with IB4 or human seru m were studied by confocal microscopy and Western blotting, respectively. Results. IB4 caused rapid redistribution of CD31 and VE-cadherin away from cell-junctions and tyrosine-phosphorylation of CD31 but not VE-cadherin. A monoclonal antibody to CD31 also triggered tyrosine phosphorylation of this molecule, but brief exposure of PAECs to normal human serum did not. Tyros ine-phosphorylated CD31 complexed with SHP2 and other unidentified phosphop roteins. Both IB4 and natural human antibodies bound to porcine CD31 but no t to VE-cadherin, Cell adhesion tests showed that porcine and human CD31 ar e functionally incompatible. Conclusions. Endothelial cell retraction during IB4-mediated activation of PAECs is associated with rapid loss of CD31 and VE-cadherin from cell junct ions. CD31 becomes strongly tyrosine-phosphorylated and forms a cell signal ing complex, which may have a significant role in the response of the xenog raft vascular endothelium.