The autoantigens 60-kd Ro/SSA (Ro 60) and β2-glycoprotein I (β2GPI) are both displayed on the surface membrane of apoptotic cells. Epitope-spreading experiments have suggested that these autoantigens may be present as a complex on the apoptotic cell surface. This study was undertaken to investigate whether β2GPI interacts with Ro 60 on apoptotic cells and alters the binding of anti–Ro 60 IgG.
The interaction between soluble recombinant Ro 60 fragments and β2GPI was investigated in vitro by direct and saturation binding assays using native human β2GPI and recombinant domain deletion mutants. Binding of β2GPI to early and late apoptotic cells was assessed by multiparameter flow cytometry, and specificity of binding was determined by competitive inhibition with soluble recombinant Ro 60 and anti–Ro 60 IgG.
The Ro 60 fragment expressing a surface-exposed epitope (apotope) bound with high affinity (Kd = ∼15 nM) to domain V of β2GPI in vitro. Beta2-glycoprotein I bound to the surface of apoptotic cells in a dose-dependent manner and was blocked by the Ro 60 apotope fragment. In reciprocal competitive inhibition studies, β2GPI blocked the binding of anti–Ro 60 autoantibodies to apoptotic cells in a dose-dependent manner, and anti–Ro 60 IgG inhibited the binding of β2GPI. Moreover, β2GPI showed a 2-fold increase in binding to apoptotic cells that overexpress Ro 60 on the surface.
These results demonstrate that Ro 60 functions as a novel receptor for β2GPI on the surface of apoptotic cells. The formation of Ro 60–β2GPI complexes may protect against anti–Ro 60 autoantibody–mediated tissue injury.