Peptide-based antibodies against glutathione-binding domains suppress superoxide production mediated by mitochondrial complex

Jingfeng Chen, Chwen Lih Chen, Sharad Rawale, Chun An Chen, Jay L. Zweier, Pravin T P Kaumaya, Yeong Renn Chen

Research output: Contribution to journalArticle

Abstract

Complex I (NQR) is a critical site of superoxide (O2 -.) 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 (200GAGAYICGEETALIESIEGK219 of 51-kDa protein and 361VDSDTLCTEEVFPTAGAGTDLR382 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 O2-. generation by 37 and 57%, as measured by EPR spin-trapping. To further provide an appropriate control, two peptides of non-glutathionylated domain ( 21SGDTTAPKKTSFGSLKDFDR40 of 51-kDa peptide and 100WNILTNSEKTKKAREGVMEFL120 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 O2-. generation to a significant level. However, binding of Ab75 inhibited NQR-mediated O2-. 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.

Original languageEnglish (US)
Pages (from-to)3168-3180
Number of pages13
JournalJournal of Biological Chemistry
Volume285
Issue number5
DOIs
StatePublished - Jan 29 2010
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

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