Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St Louis, Missouri, USA.
X-Ray Science Division, Argonne National Laboratory, Lemont, Illinois, USA.
J Biol Chem. 2021 Aug;297(2):100890. doi: 10.1016/j.jbc.2021.100890. Epub 2021 Jun 29.
β-glycoprotein I (βGPI) is an abundant multidomain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as antiphospholipid syndrome (APS). Previous studies have shown that βGPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to βGPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate βGPI's structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of βGPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating βGPI's physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL.
β-糖蛋白 I（βGPI）是一种丰富的多功能血浆蛋白，在凝血和补体级联反应中发挥多种作用。它也是获得性凝血障碍即抗磷脂综合征（APS）中抗磷脂抗体（aPL）的主要靶标。先前的研究表明，βGPI 采用两种可相互转化的生化构象，即氧化和还原，这取决于二硫键的完整性。然而，二硫键对βGPI 结构和功能的确切贡献尚不清楚。在这里，我们用丝氨酸取代半胱氨酸，研究结构域 I（DI）中的 C32-C60 二硫键和结构域 V（DV）中的 C288-C326 二硫键如何调节βGPI 的结构和功能。我们的生物物理和生化研究结果支持这样的假设，即 C32-C60 二硫键起结构作用，而 C288-C326 二硫键是别构的。我们证明，与 C32-C60 键缺失不同，C288-C326 键缺失不会影响蛋白质的热力学稳定性和整体结构，而是使膜结合减弱，使蛋白质在溶液中保持伸长状态。我们还记录到，虽然 C32-C60 键缺失直接损害了致病性抗 DI 抗体对βGPI 的识别，但在阴离子磷脂存在的情况下，C288-C326 二硫键缺失足以破坏复合物的形成。我们得出结论，C288-C326 二硫键作为一种分子开关，能够以氧化还原依赖的方式调节βGPI 的生理功能。我们提出，在具有抗 DI 抗体的 APS 患者中，选择性破坏 C288-C326 二硫键可能是降低 aPL 致病潜力的有效策略。
