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

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

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

Methods: βGPI 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.

Results: Electron microscopy and size-exclusion chromatography showed that the structure of βGPI 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 βGPI 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 βGPI binds with moderate affinity to a prototypical anti-DI antibody targeting the epitope G40-R43 despite being elongated.

Conclusion: Circulating βGPI is elongated and, therefore, fully capable of binding to anti-DI antibodies. Binding of βGPI 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 βGPI, which have important implications for understanding APS pathogenesis and the development of APS diagnostics and therapeutics.