Biotinylation of glycan chains in beta2 glycoprotein I induces dimerization of the molecule and its detection by the human autoimmune anti-cardiolipin antibody EY2C9.

Binding of beta2GPI (beta2 glycoprotein I), a human plasma protein, to AnPLs (anionic phospholipids) plays a key role in the formation of antiphospholipid antibodies involved in autoimmune diseases like antiphospholipid syndrome or systemic lupus erythematosus. We recently showed that binding of beta2GPI to AnPLs was enhanced by biotinylation of its glycan chains with biotin-hydrazide. In the present study, we investigated why this chemical modification of beta2GPI increased both its affinity for AnPLs and its recognition by anti-cardiolipin antibodies. Electrophoretic analysis showed that: (i) high molecular mass beta2GPI (dimers and other oligomers) covalently coupled by imine bonds, were present in variable amounts in oxidized beta2GPI and in beta2GPI-bh (beta2GPI-biotin-hydrazide), but were absent in native beta2GPI; (ii) binding of beta2GPI-bh to phosphatidylserine-coated microtitre plates generated high molecular mass polymers in a time-dependent manner. Native beta2GPI did not polymerize in these conditions. These polymers did not bind more strongly to AnPLs than the monomer beta2GPI. However, in solution at 1 microM beta2GPI-bh essentially appeared as a dimer as revealed by light-scattering analysis. SPR (surface plasmon resonance) analysis showed that the increased affinity of beta2GPI-bh for AnPL monolayers was due to a lower dissociation rate constant compared with native beta2GPI. Finally, the monoclonal human aCL (auto-immune anti-cardiolipin antibody) EY2C9 bound to beta2GPI-bh but did not bind to monomeric native and oxidized beta2GPI. It is likely that the dimeric quaternary structure of beta2GPI-bh is in fact responsible for the appearance of the epitopes targeted by the EY2C9 antibody.

Oxidation and biotinylation of beta 2 glycoprotein I glycan chains induce an increase in its affinity for anionic phospholipids similar to that obtained by the addition of anti-beta 2 glycoprotein I or anti-cardiolipin antibodies.

Binding of beta 2 glycoprotein I (beta2GPI) to apoptotic cells plays a key role in the opsonization of apoptotic bodies and the formation of antiphospholipids antibodies. Here, we describe the binding of beta2GPI to apoptotic cells using beta2GPI labelled with biotin-hydrazide (beta2GPI-bh) after oxidation of its glycan chains. Flow cytometry analyses and confocal microscopy showed that beta2GPI-bh, contrary to native beta(GPI, bound to apoptotic cells, either permeable or non-permeable to propidium iodide (PI), as did annexin-V-FITC. But, in the absence of divalent ions, beta2GPI-bh, contrary to annexin V, was still able to bind to apoptotic cells. Binding equilibrium studies, performed on solid-state anionic phospholipids (AnPL), revealed that beta2GPI-bh had a greater apparent affinity for AnPL than native beta2GPI. In presence of the anti-beta2GPI mAb 8C3, the ability of native beta2GPI to bind to AnPL was increased and binding to apoptotic PI+ and PI- CEM cells was observed whereas binding of beta2GPI-bh was barely affected by the addition of 8C3. However, the 8C3-enhanced ability of native beta2GPI to bind to AnPL was still weaker than that of beta2GPI-bh. It is not clear why the oxidation and biotinylation of glycan chains of beta2GPI increases its affinity for AnPL, but it seems that if such oxidative process occurs naturally, it could participate in enhancing antiphospholipid formation.