Biotinylation of glycan chains in β(2) glycoprotein I induces dimerization of the molecule and its detection by the human autoimmune anti-cardiolipin antibody EY2C9

Binding of β(2)GPI (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 β(2)GPI to AnPLs was enhanced by biotinylation of its glycan chains with biotin-hydrazide. In the present study, we investigated why this chemical modification of β(2)GPI increased both its affinity for AnPLs and its recognition by anti-cardiolipin antibodies. Electrophoretic analysis showed that: (i) high molecular mass β(2)GPI (dimers and other oligomers) covalently coupled by imine bonds, were present in variable amounts in oxidized β(2)GPI and in β(2)GPI-bh (β(2)GPI-biotin-hydrazide), but were absent in native β(2)GPI; (ii) binding of β(2)GPI-bh to phosphatidylserine-coated microtitre plates generated high molecular mass polymers in a time-dependent manner. Native β(2)GPI did not polymerize in these conditions. These polymers did not bind more strongly to AnPLs than the monomer β(2)GPI. However, in solution at 1 μM β(2)GPI-bh essentially appeared as a dimer as revealed by light-scattering analysis. SPR (surface plasmon resonance) analysis showed that the increased affinity of β(2)GPI-bh for AnPL monolayers was due to a lower dissociation rate constant compared with native β(2)GPI. Finally, the monoclonal human aCL (auto-immune anti-cardiolipin antibody) EY2C9 bound to β(2)GPI-bh but did not bind to monomeric native and oxidized β(2)GPI. It is likely that the dimeric quaternary structure of β(2)GPI-bh is in fact responsible for the appearance of the epitopes targeted by the EY2C9 antibody