Peptide-based Antibodies against Glutathione-binding Domains Suppress Superoxide Production Mediated by Mitochondrial Complex I

Title

Peptide-based Antibodies against Glutathione-binding Domains Suppress Superoxide Production Mediated by Mitochondrial Complex I

Creator

Chen J F; Chen C L; Rawale S; Chen C A; Zweier J L; Kaumaya P T P; Chen Y R

Publisher

Journal of Biological Chemistry

Date

2010
2010-01

Description

Complex I (NQR) is a critical site of superoxide (O-2(radical anion)) production and the major host of redox protein thiols in mitochondria. In response to oxidative stress, NQR-derived protein thiols at the 51- and 75-kDa subunits are known to be reversibly S-glutathionylated. Although several glutathionylated domains from NQR 51 and 75 kDa have been identified, their roles in the regulatory functions remain to be explored. To gain further insights into protein S-glutathionylation of complex I, we used two peptides of S-glutathionylated domain ((200)GAGAYI (C) under bar GEETALIESIEGK(219) of 51-kDa protein and (VDSDTL)-V-361 (C) under bar TEEVFPTAGAGTDLR(382) of 75-kDa protein) as chimeric epitopes incorporating a "promiscuous" T-cell epitope to generate two polyclonal antibodies, AbGSCA206 and AbGSCB367. Binding of AbGSCA206 and AbGSCB367 inhibited NQR-mediated O-2(radical anion). generation by 37 and 57%, as measured by EPR spin-trapping. To further provide an appropriate control, two peptides of non-glutathionylated domain ((21)SGDTTAPKKTSFGSLKDFDR(40) of 51-kDa peptide and (100)WNILTNSEKTKKAREGVMEFL(120) of 75-kDa peptide) were synthesized as chimeric epitopes to generate two polyclonal antibodies, Ab51 and Ab75. Binding of A51 did not affect NQR-mediated O-2(radical anion) generation to a significant level. However, binding of Ab75 inhibited NQR- mediated O-2(radical anion) generation by 35%. None of AbGSCA206, AbGSCB367, Ab51, or Ab75 showed an inhibitory effect on the electron transfer activity of NQR, suggesting that antibody binding to the glutathione-binding domain decreased electron leakage from the hydrophilic domain of NQR. When heart tissue homogenates were immunoprecipitated with Ab51 or Ab75 and probed with an antibody against glutathione, protein S-glutathionylation was enhanced in post-ischemic myocardium at the NQR 51-kDa subunit, but not at the 75-kDa subunit, indicating that the 51-kDa subunit of flavin subcomplex is more sensitive to oxidative stress resulting from myocardial infarction.

Subject

oxidative stress; Biochemistry & Molecular Biology; nitric-oxide; proteins; site; generation; s-nitrosylation; postischemic heart; radical formation; bovine heart-mitochondria; nadh-ubiquinone oxidoreductase

Format

Journal Article or Conference Abstract Publication

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Rights

Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).

Pages

3168-3180

Issue

5

Volume

285

Citation

Chen J F; Chen C L; Rawale S; Chen C A; Zweier J L; Kaumaya P T P; Chen Y R, “Peptide-based Antibodies against Glutathione-binding Domains Suppress Superoxide Production Mediated by Mitochondrial Complex I,” NEOMED Bibliography Database, accessed April 22, 2021, https://neomed.omeka.net/items/show/8868.

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