Alpha-galactosyl-mediated activation of porcine endothelial cells: studies on CD31 and VE-cadherin in adhesion and signaling.

Abstract

Ligation of alpha-galactosyl epitopes on endothelial cells by naturally occurring human antibodies causes hyperacute rejection in porcine-to-human xenotransplantation. The alpha-galactosyl-specific lectin Bandeiraea simplicifolia isolectin B4 (IB4) has been reported to trigger endothelial "gap" formation and tyrosine phosphorylation of an unidentified 130-kDa protein. We have studied two 130-kDa junctional adhesion molecules, CD31 and VE-cadherin, in porcine aortic endothelial cells (PAECs) during IB4-mediated activation. The cellular distribution of these molecules, their susceptibility to tyrosine phosphorylation, and their capacity to bind IB4 or natural human antibodies have been determined. 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 serum were studied by confocal microscopy and Western blotting, respectively. 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. Tyrosine-phosphorylated CD31 complexed with SHP2 and other unidentified phosphoproteins. Both IB4 and natural human antibodies bound to porcine CD31 but not to VE-cadherin. Cell adhesion tests showed that porcine and human CD31 are functionally incompatible. Endothelial cell retraction during IB4-mediated activation of PAECs is associated with rapid loss of CD31 and VE-cadherin from cell junctions. CD31 becomes strongly tyrosine-phosphorylated and forms a cell signaling complex, which may have a significant role in the response of the xenograft vascular endothelium.