Small Angle X-Ray Scattering Reveals the Conformation of β₂glycoprotein I and the Conformational Binding Epitopes of Autoantibodies

Despite the fact that autoantibodies to a variety of antigens have been described in patients with the antiphospholipid syndrome (APS), it is now generally accepted that β₂glycoprotein I (β₂GPI) is a primary target of pathogenic autoantibodies in APS patients. Unraveling the immunodominant epitope of β₂GPI at the molecular/conformational level may help to understand the disease and develop specific therapies. β₂GPI is an abundant plasma protein that, upon interaction with negative phospholipids, has been shown to undergo a conformational change resulting in the exposure of a pathogenic epitope on domain I. Although the conformation of β₂GPI has been previously reported by small angle X-ray scattering (SAXS), we are the first to show the interaction with autoimmune anti-β₂GPI antibodies. We previously showed that antibodies towards the cryptic domain I epitope are highly associated with both thrombosis and pregnancy morbidity. Previous studies indicated that amino acids R39-R43 and the interlinking region between domains I and II are part of this cryptic epitope. However, these studies used point-mutated variants of β₂GPI, and the results could be influenced by structural changes compared to native β₂GPI.

In this study, we visualized the conformation of β₂GPI in solution by SAXS and its interaction with two anti-domain I antibodies, obtained via B cell isolation from two APS patients. Antibody P2-6 previously showed affinity for domain I regardless of the conformation of β₂GPI, whereas antibody P1-117 only recognized domain I of β₂GPI upon binding to anionic phospholipids. Via point-mutated variants of β₂GPI we previously demonstrated that P1-117 is reactive against the cryptic domain I epitope including epitope G40-R43. In a first set of SAXS experiments, we visualized plasma purified β₂GPI in its native conformation. We found that, due to the flexibility of domain I-III of β₂GPI, β₂GPI is able to adapt into several different conformations ranging from a sort of circular shape into a S shape, confirming results of previous studies. Upon increasing the pH, the conformation of β₂GPI changed into a more erect fish-hook conformation resembling the crystal structure of β₂GPI. The three-dimensional structures of both antibodies were determined by SAXS and allowed to react with β₂GPI in the open conformation (Figure 1). Antibody P2-6 interacted mainly with the amino terminal end of domain I of β₂GPI which is available in both the native and open conformations of β₂GPI. In contrast, antibody P1-117 interacted with a broader part of the protein, identifying a discontinuous and conformational epitope covering domain I, the interlinking region and a small part of domain II. Importantly, (part of) the epitope is not available in the native closed conformation of β₂GPI due to its interaction with domain V (circular conformation) or due to a carbohydrate chain positioned on the interface of domain I-II (S shaped conformation). These data confirm the importance of the conformation of β₂GPI coated on the solid phase of diagnostic anti-β₂GPI assays to avoid false negative classification of APS patients.

In conclusion, our findings contribute to elucidating the conformation of β₂GPI in solution and clearly indicate that pathogenic autoantibodies bind a discontinuous conformational epitope. Identification of this conformational epitope will allow optimization of peptide-based therapies and also epitope-specific diagnostic assays that will enable the stratification of patients according to their risk on thrombosis/pregnancy morbidity.

View largeDownload slide

View largeDownload slide

Close modal