Proteolytic cleavage of β2-glycoprotein I: reduction of antigenicity and the structural relationship

Abstract

Binding of beta(2)-glycoprotein I (beta(2)-GPI)-dependent anticardiolipin antibodies (aCL) derived from antiphospholipid syndrome (APS) is significantly reduced in aCL ELISA due to loss of the phospholipid (PL) binding property of beta(2)-GPI by plasmin treatment. In the present study, the treatment generated a nicked form of beta(2)-GPI and resulted in loss of antigenicity for the autoantibodies detected in ELISA, using an beta(2)-GPI directly adsorbed polyoxygenated carboxylated plate, the assay system of which was not related to PL binding. The nicked form bound to neither Cu(2+)-oxidized low-density lipoprotein (oxLDL) nor to beta(2)-GPI-specific lipid ligands isolated from oxLDL, the result being a complete loss of subsequent binding of anti-beta(2)-GPI autoantibodies. The conformational change in the nicked domain V was predicted from its intact structure determined by an X-ray analysis (implemented in Protein Data Bank: 1C1Z), molecular modeling and epitope mapping of a monoclonal anti-beta(2)-GPI antibody, i.e. Cof-18, which recognizes the related structure. The analysis revealed that novel hydrophobic and electrostatic interactions appeared in domain V after the cleavage, thereby affecting the PL binding of beta(2)-GPI. Such a conformational change may have important implications for exposure of cryptic epitopes located in the domains such as domain IV.

The Orientation of β2GPI on the Plate is Important for the Binding of Anti-β2GPI Autoantibodies by ELISA

“…In this report we have sought to resolve some of these discrepant findings. We show that the orientation of adsorbed 2GPI on the plastic surface of an ELISA plate is important for the binding of autoantibodies, as originally proposed by Matsuura et al [24].…”

Nicked 2-glycoprotein I: a marker of cerebral infarct and a novel role in the negative feedback pathway of extrinsic fibrinolysis

“…According to the conformation of the nicked domain V, as predicted from the x-ray structure of the intact domain V and confirmed by heteronuclear magnetic resonance, the nicked C-terminal loop is tightly fixed by electrostatic interaction with enhanced stability, the result being neutralization of the positive charge of the lysine cluster. 26,30 Glu-plasminogen, a full-length protein, is the naturally circulating form of plasminogen. Kringle 5 of Glu-plasminogen has a higher affinity for intact fibrin.…”

“…␤ 2 -GPI was purified from human plasma, as described. 25 Nicked ␤ 2 -GPI was prepared as reported 26 with slight modifications that included an additional purification step; ␤ 2 -GPI was treated with human plasmin (Calbiochem Novabiochem, La Jolla, CA) at 37°C for 3 hours, at a molar ratio of ␤ 2 -GPI/plasmin of 8:1. Plasmin-treated ␤ 2 -GPI was first purified on a Cof 22-Sepharose column and subsequently on a heparinSepharose column.…”

Binding of β2–glycoprotein I to anionic phospholipids facilitates processing and presentation of a cryptic epitope that activates pathogenic autoreactive T cells

“…13 Nicked and reduced ␤ 2 GPI was prepared by treating ␤ 2 GPI with plasmin 14 and dithiothreitol, 5 respectively. Fusion recombinant maltosebinding proteins (MalBPs) expressed in Escherichia coli included GP-F and GP3, which encoded the entire amino acid sequence (amino acids 1-326) and domains IV and V (amino acids 182-326), respectively, of human ␤ 2 GPI.…”