Beta 2 glycoprotein I is a substrate of thiol oxidoreductases

Abstract

M. Failure of the Ipsogen MutaScreen kit to detect the JAK2 617VϾF mutation in samples with additional rare exon 14 mutations: implications for clinical testing and report of a novel 618CϾF mutation in addition to 617VϾF [letter]. Blood. 2010;115(15) To the editor: Beta 2 glycoprotein I is a substrate of thiol oxidoreductasesBeta 2 glycoprotein I (␤2GPI) is an abundant plasma protein recognized as the major autoantigen in the antiphospholipid syndrome. Although the crystal structure of ␤2GPI has been resolved, 1,2 its normal function remains unknown. We have been intrigued by the presence of a C-terminal cysteine (Cys326), which forms a loop-back disulfide link in the fifth domain of ␤2GPI. In the current study we examined ␤2GPI's potential to participate in thiol exchange reactions with the thiol oxidoreductases thioredoxin-1 (TRX-1) and protein disulfide isomerase (PDI). The incorporation of free thiols into ␤2GPI after reaction with TRX-1 or PDI was shown by labeling the products of this reaction with the selective sulfhydryl probe N a -(3-maleimidylpropionyl) biocytin (MPB). The biotinylated proteins were visualized by Western blotting with streptavidin-horseradish peroxidase. Because ␤2GPI does not contain unpaired cysteines, no labeling was observed after incubation with MPB ( Figure 1A). Free thiols could not be introduced into ␤2GPI by incubation with the reducing agent dithiothreitol (DTT) alone. However, free thiols could be introduced into ␤2GPI after incubation with the reduced forms of the thiol oxidoreductases TRX-1 and PDI, identifying ␤2GPI as a thiol oxidoreductase substrate ( Figure 1A-C). An interesting effect caused by the reduction of ␤2GPI by TRX-1 was a marked decrease in the affinity of anti-␤2GPI monoclonal and polyclonal antibodies as noted on the immunoblots.To determine the cysteine residue(s) in the ␤2GPI molecule involved in thiol exchange reactions, ␤2GPI treated with the TRX-1/TRX-1 reductase/NADPH system and labeled with MPB was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Figure 1B). Gel bands were excised, digested, and analyzed by liquid chromatography-tandem mass spectrometry. Mass spectral data were searched using Mascot (Version 2.2; Matrix Science) or converted to MzXML file format using ReAdW (Version 4.0.2) 3 and submitted to the database search program X!Tandem (Release 2008.12.01). 4 The analysis revealed Cys326 to be predominantly labeled with biotin (F.H.P., S.R., M.Q., M.J.R., J.W.H.W., K.T., Y.I., J.Y.Z., R.G., J.C.Q., B.G., W.E.H., P.J.H., S.A.K., manuscript submitted). The structural features of the disulfide bond containing Cys326 and all disulfide bonds of the 2 structures of ␤2GPI (PDB 1C1Z and 1QUB) were determined using the disulfide bond analysis tool available at www.cancerresearch.unsw.edu.au/CRCWeb. nsf/page/DisulfideϩBondϩAnalysis. 5 The analysis showed that the Cys288-Cys326 disulfide is a Ϫ/ϩ right-handed hook (Ϫ/ϩRHHook) configuration in both crystal structures of the protein. 1,2 Although there is no other structu...

Lateral self-association of VWF involves the Cys2431-Cys2453 disulfide/dithiol in the C2 domain

“…Two candidates are thioredoxin and protein disulfide isomerase (PDI). Thioredoxin has been shown to cleave the Cys288-Cys326 disulfide bond in ␤ 2 -glycoprotein I in plasma, [32][33][34] as well as in 2 other disulfide bonds with the same ϪRHStaple configuration as the VWF Cys2451-Cys2468 bond. 28,29 Conversely, PDI [35][36][37] is on the surface of platelets 38 and platelet microparticles 39 and so will be in the vicinity of VWF during thrombus formation.…”

Naturally occurring free thiols within β2-glycoprotein I in vivo: nitrosylation, redox modification by endothelial cells, and regulation of oxidative stress–induced cell injury

“…However, ␤2GPI could not be labeled with the free thiol-binding reagent MPB, indicating no free thiols in the purified protein. 29 To underline the relevance of these and future studies pertaining to the redox status of ␤2GPI, evidence for the natural occurrence of free thiols within ␤2GPI in vivo needed to be shown. To further this aim, a novel ELISA method was developed whereby serum is incubated with the biotinylated thiol binding reagent MPB.…”

“…Direct quantification of ␤2GPI protein loading with the use of anti-␤2GPI could not be used for this purpose because pretreatment of ␤2GPI with activated TRX-1 altered its immunoreactivity to both polyclonal and monoclonal anti-␤2GPI antibodies. 29 For pull-down experiments, r␤2GPI/TRX-1/DTT mixture after incubation with EAhy926 cells was removed, labeled with MPB as described earlier, and subjected to nickel chromatography (described in detail in the supplemental data). The eluted material was transferred to PVDF membranes and probed with streptavidin-horseradish peroxidase.…”

Antigen and substrate withdrawal in the management of autoimmune thrombotic disorders

“…52 ␤ 2 GPI can also be oxidized directly. 53 Approximately 80% of circulating plasma ␤ 2 GPI is maintained in the reduced form by platelet and endothelial cell oxidoreductases, 54,55 which may protect cells from hydrogen peroxide-induced apoptosis. 56 Free cysteines in reduced ␤ 2 GPI, particularly Cys 288 and Cys 326 , are susceptible to oxidation and nitrosylation as is the Cys 32 -Cys 60 disulfide bond in domain 1, which spans the primary antigenic region.…”