Biotinylation of glycan chains in β2 glycoprotein I induces dimerization of the molecule and its detection by the human autoimmune anti-cardiolipin antibody EY2C9

Abstract

Binding of β2GPI (β2 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 β2GPI to AnPLs was enhanced by biotinylation of its glycan chains with biotin-hydrazide. In the present study, we investigated why this chemical modification of β2GPI increased both its affinity for AnPLs and its recognition by anti-cardiolipin antibodies. Electrophoretic analysis showed that: (i) high molecular mass β2GPI (dimers and other oligomers) covalently coupled by imine bonds, were present in variable amounts in oxidized β2GPI and in β2GPI-bh (β2GPI-biotin-hydrazide), but were absent in native β2GPI; (ii) binding of β2GPI-bh to phosphatidylserine-coated microtitre plates generated high molecular mass polymers in a time-dependent manner. Native β2GPI did not polymerize in these conditions. These polymers did not bind more strongly to AnPLs than the monomer β2GPI. However, in solution at 1 μM β2GPI-bh essentially appeared as a dimer as revealed by light-scattering analysis. SPR (surface plasmon resonance) analysis showed that the increased affinity of β2GPI-bh for AnPL monolayers was due to a lower dissociation rate constant compared with native β2GPI. Finally, the monoclonal human aCL (auto-immune anti-cardiolipin antibody) EY2C9 bound to β2GPI-bh but did not bind to monomeric native and oxidized β2GPI. It is likely that the dimeric quaternary structure of β2GPI-bh is in fact responsible for the appearance of the epitopes targeted by the EY2C9 antibody.