Structural analyses of β2-glycoprotein I: is there a circular conformation?

Abstract

BackgroundAntiphospholipid antibodies targeting β2-glycoprotein I (β2GPI) cause thrombosis and pregnancy morbidity in antiphospholipid syndrome (APS) patients. How these antibodies recognize β2GPI remains controversial. ObjectivesThis study aimed to elucidate the structure of β2GPI and evaluate how pathogenic anti–domain I (DI) antibodies recognize it in human plasma. Methodsβ2GPI was made recombinant and purified from human plasma using different protocols. Structural and functional analyses were conducted using orthogonal techniques, namely, electron microscopy, size-exclusion chromatography, single-molecule Förster resonance energy transfer, and microfluidic diffusional sizing. ResultsElectron microscopy and size-exclusion chromatography showed that the structure of β2GPI produced recombinantly and purified from plasma is elongated, even when subjected to conditions previously reported to favor circularization. Single-molecule Förster resonance energy transfer analyses of β2GPI labeled at positions 88 in DII and 278 in DV showed that these residues are located >90 Å apart, consistent with an elongated form. They also documented that the distance between these 2 residues did not change when the protein was reconstituted in human plasma. Microfluidic diffusional sizing documented that β2GPI binds with moderate affinity to a prototypical anti-DI antibody targeting the epitope G40-R43 despite being elongated. ConclusionCirculating β2GPI is elongated and, therefore, fully capable of binding to anti-DI antibodies. Binding of β2GPI to negatively charged phospholipids drives autoantibody recognition by increasing the local concentration of the antigen and not by dramatically changing its conformation. These findings clarify the structural properties of β2GPI, which have important implications for understanding APS pathogenesis and the development of APS diagnostics and therapeutics.