Abstract
Beta-2-glycoprotein I (β2GPI) is a blood protein and the major antigen in the autoimmune disorder antiphospholipid syndrome (APS). β2GPI exists mainly in closed or open conformations and comprises of 11 disulfides distributed across five domains. The terminal Cys288/Cys326 disulfide bond at domain V has been associated with different cysteine redox states. The role of this disulfide bond in conformational dynamics of this protein has not been investigated so far. Here, we report on the enzymatic driven reduction by thioredoxin-1 (recycled by Tris(2-carboxyethyl)phosphine; TCEP) of β2GPI. Specific reduction was demonstrated by Western blot and mass spectrometry analyses confirming majority targeting to the fifth domain of β2GPI. Atomic force microscopy images suggested that reduced β2GPI shows a slightly higher proportion of open conformation and is more flexible compared to the untreated protein as confirmed by modelling studies. We have determined a strong increase in the binding of pathogenic APS autoantibodies to reduced β2GPI as demonstrated by ELISA. Our study is relevant for understanding the effect of β2GPI reduction on the protein structure and its implications for antibody binding in APS patients.
Highlights
Beta-2-glycoprotein I (β2GPI) is a blood protein and the major antigen in the autoimmune disorder antiphospholipid syndrome (APS). β2GPI exists mainly in closed or open conformations and comprises of 11 disulfides distributed across five domains
There is a dramatic increase in binding of anti-Domain I (DI) antibodies from APS patients to reduced β2GPI, the structural alteration upon reduction may be more subtle than first hypothesized
This may be explained by an increased flexibility of the molecule, rather than a traditional shift of conformation from closed to open
Summary
Beta-2-glycoprotein I (β2GPI) is a blood protein and the major antigen in the autoimmune disorder antiphospholipid syndrome (APS). β2GPI exists mainly in closed or open conformations and comprises of 11 disulfides distributed across five domains. Beta-2-glycoprotein I (β2GPI) is a blood protein and the major antigen in the autoimmune disorder antiphospholipid syndrome (APS). Β2GPI exists mainly in closed or open conformations and comprises of 11 disulfides distributed across five domains. The terminal Cys288/Cys[326] disulfide bond at domain V has been associated with different cysteine redox states The role of this disulfide bond in conformational dynamics of this protein has not been investigated so far. Our study is relevant for understanding the effect of β2GPI reduction on the protein structure and its implications for antibody binding in APS patients. The beta-sheet rich domains are separated by flexible random coil regions that allow the protein to exhibit two major conformational states; the closed (circular) conformation and open (linear) conformation. The role of post-translational modifications in altering the structural conformation of β2GPI has been investigated[26,27], the potential relationship between alternative structures of β2GPI and the different functions of the molecule is not understood fully
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