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

Antiphospholipid antibodies targeting β₂-glycoprotein I (β₂GPI) cause thrombosis and pregnancy morbidity in antiphospholipid syndrome (APS) patients. How these antibodies recognize β₂GPI remains controversial.

This study aimed to elucidate the structure of β₂GPI and evaluate how pathogenic anti-domain I (DI) antibodies recognize it in human plasma.

β₂GPI 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.

Electron 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.

Circulating 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.