Structural analyses of ₂ -glycoprotein I: is there a circular conformation?

Background: Antiphospholipid antibodies targeting (beta(2)-glycoprotein I ((32GPI) cause thrombosis and pregnancy morbidity in antiphospholipid syndrome (APS) patients. How these antibodies recognize (beta(2)GPI remains controversial.Objectives: This study aimed to elucidate the structure of (32GPI and evaluate how pathogenic anti-domain I (DI) antibodies recognize it in human plasma.Methods: (beta(2)GPI was made recombinant and purified from human plasma using different protocols. Structural and functional analyses were conducted using orthog-onal techniques, namely, electron microscopy, size-exclusion chromatography, single-molecule Forster resonance energy transfer, and microfluidic diffusional sizing.Results: Electron microscopy and size-exclusion chromatography showed that the structure of (beta(2)GPI produced recombinantly and purified from plasma is elongated, even when subjected to conditions previously reported to favor circularization. Single-molecule Forster resonance energy transfer analyses of (beta(2)GPI labeled at positions 88 in DII and 278 in DV showed that these residues are located >90 angstrom 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 (beta(2)GPI binds with moderate affinity to a prototypical anti-DI antibody targeting the epitope G40-R43 despite being elongated.Conclusion: Circulating (beta(2)GPI is elongated and, therefore, fully capable of binding to anti-DI antibodies. Binding of (beta(2)GPI to negatively charged phospholipids drives auto-antibody recognition by increasing the local concentration of the antigen and not by dramatically changing its conformation. These findings clarify the structural properties of (beta(2)GPI, which have important implications for understanding APS pathogenesis and the development of APS diagnostics and therapeutics.