Human p2-Glycoprotein I Binds To Endothelial Cells Through A Cluster of Lysihe Residues that Are Critical for Anionic Phospholipid Binding and Offers Epitopes for Anti- p2-Glycoprotein I Antibodies

摘要

Beta 2 glycoprotein I (p2GPI) is a phospholipid binding protein recognized by serum autoantibodies from the anti-phospholipid syndrome (APS) both in cardiolipin (CL) and P2GPI-coated plates. We found that : a) recombinant wild-type p2GPI bound to human umbilical cord vein endothelial cells (HUVEC) and was recognized by both human monoclonal IgM and affinity purified polyclonal IgG anti-p2GPI APS antibodies and b) a single amino acid change from Lys to Glu significantly reduced endothelial adhesion. Double and triple mutants (from Lys~(284,287) to Glu~(284,287), from Lys~(286,287) to Glu~(286,287) and from Lys~(284,286,287) to Clu~(284,286,287)) completely abolished endothelial binding. A synthetic peptide (PI) spanning the sequence Glu~(274)-Cys~(288) of the p2GPT fifth domain still displayed endothelial adhesion. Another peptide (P8), identical with PI except that Cys~(281)and Cys~(288) were substituted with serine residues, did not bind to HUVEC. Anti-p2GPI antibodies, once bound to PI adhered to HUVEC, induced E-Selectin expression and up-regulated Interleukin 6 (IL-6) secretion. Control experiments carried out with irrelevant antibodies as well as with the P8 peptide, did not show any endothelial antibody binding nor E-Selectin and IL-6 modulation. Our results suggest that: a) p2GPI binds to endothelial cells through its fifth domain, b) the major phospholipid binding site that mediates the binding to anionic phospholipids is also involved in endothelial binding, c) HUVEC provide a suitable surface for P2GPI binding comparable to that displayed by anionic phospholipids dried on microtitre wells and d) the formation of the complex between p2GPI and the specific antibodies leads to endothelial activation in vitro.