Abstract

Beta-2-Glycoprotein I (β2GPI) is the main autoantigenic target of antiphospholipid syndrome (APS) with antibodies leading to clinical manifestations. There are two known structural isomers of β2GPI, a J shape and a circular shaped one. The transition between these structures is incompletely understood, with the functional implications unknown. β2GPI is a substrate of the protease plasmin, which cleaves within the fifth domain of β2GPI leading to altered cellular binding. Very little is currently known regarding the structure and function of this protein variant. We present the first comprehensive structural characterisation plasmin-clipped β2GPI and the associated implications for pathogenic antibody binding to this protein.

Methods β2GPI was purified using a novel acid-free process from healthy control plasma and cleaved with plasmin. Cleavage was confirmed by SDS-PAGE. Structural characterisation was undertaken using dynamic light scattering (DLS), small angle X-ray scattering (SAXS), ion mobility mass spectrometry (IMMS) and molecular dynamics simulation (MD). Activity was tested using inhibition of β2GPI ELISAs with patient samples and cleaved β2GPI in the fluid phase and cellular binding by flow cytometry using HUVEC cells.

Results DLS revealed a significantly smaller hydrodynamic radius for plasmin-clipped β2GPI (p=0.0043). SAXS and MD analysis indicated a novel S-like structure of β2GPI only present in the plasmin-clipped sample whilst IMMS showed a different structure distributions in plasmin clipped compared to non-clipped B2GPI. The increased binding of autoantibodies was shown for plasmin-clipped β2GPI (p=0.056), implying a greater exposure of pathogenic epitopes following cleavage.

Conclusions Cleavage of β2GPI by plasmin results in the production of a unique S-shaped structural conformation and higher patient antibody binding. This novel structure may explain the loss of binding to phospholipids and increase in anti-angiogenic potential described previously for plasmin-clipped β2GPI.

Competing Interest Statement

The authors have declared no competing interest.

Funding Statement

We thank the Medical Research Foundation for a Fellowship to T. C. R. M. (MRF-057-0004-RG-MCDO-C0800) and a Versus Arthritis Senior Fellowship (ShS/SRF/22977). H.F.B. was supported by an UCB BIOPHARMA SPRL/BBSRC PhD Studentship (BB/P504725/1). C.J.L. was supported by the EPSRC Centre for Doctoral Training in Emergent Macromolecular Therapies (000033549) and IPSEN Bioinnovation (EP/L015218/1). S.J.P. was supported by the CCP-SAS project, a joint EPSRC (EP/K039121/1) and NSF (CHE-1265821) grant. P.D is supported by an EPSRC Grant (EP/P006485/1). Wellcome Collaborative Award in Science (209250/Z/17/Z) to K.T.

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This study was carried out in accordance with the recommendations of London Hampstead Research Ethics Committee Ref No 12/LO/0373 with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the London Hampstead Research Ethics Committee Ref No 12/LO/0373.

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Data Availability Statement

All data are contained within this manuscript and data are available on request.

Abbreviationsβ2GPIβ2 glycoprotein IHChealthy controlMDmolecular dynamicsRGradius of gyrationSAXSsmall angle X-ray scatteringIEXIon Exchange ChromatographyELISAEnzyme Linked Immunosorbent Assay