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.
Dupuy D'Angeac
Arnaud
A
Laboratoire de Chimie Biomoléculaire, UM II, 8 rue de l'Ecole Normale, Montpellier 34216, Cedex 5, France. dangeac@univ-montp2.fr
Stefas
Ilias
I
Graafland
Hubert
H
De Lamotte
Frédéric
F
Rucheton
Marcel
M
Palais
Caroline
C
Eriksson
Anna-Karin
AK
Bosc
Priscille
P
Rosé
Caroline
C
Chicheportiche
Robert
R
D000818:Animals; D017153:Antibodies, Anticardiolipin / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D019921:Biotinylation; D002308:Cardiolipins; D019281:Dimerization; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D053482:beta 2-Glycoprotein I
D017153:Antibodies, Anticardiolipin; D000911:Antibodies, Monoclonal; D002308:Cardiolipins; D006023:Glycoproteins; D011134:Polysaccharides; D053482:beta 2-Glycoprotein I; C053311:biotin hydrazide; D001710:Biotin