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

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

doi:10.1074/jbc.m109.056846

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 ((200)GAGAYICGEETALIESIEGK(219) of 51-kDa protein and (361)VDSDTLCTEEVFPTAGAGTDLR(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 O2-* 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 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.

Highlights

In mitochondria, the generation of O2. and the oxidants derived from it can act as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence
It has been documented that Complex I is the major component of the electron transport chain to host protein thiols, which comprise structural thiols involved in the ligands of iron-sulfur clusters and the reactive/regulatory thiols that are thought to function in antioxidant defense and redox signaling [4, 5]
We have reported previously that in oxidative damage to NQR, the C206 moiety of the 51-kDa subunit plays a unique role as a reactive thiol, based on the evidence of immunospin trapping with 5,5-dimethyl pyrroline N-oxide and mass spectrometry [9]

Summary

Introduction

The generation of O2. and the oxidants derived from it can act as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence. The Complex I-derived regulatory thiols have been implicated in the regulation of respiration, nitric oxide utilization [6, 7], and redox status of mitochondria [1,2,3]. It has been well documented that the 51- and 75-kDa subunits of the NQR hydrophilic domain are two of the major polypeptides that host regulatory thiols in mitochondria [4, 5, 12]. Based on an EPR spin-trapping study, GSSG-induced glutathionylation of NQR at the 51- and 75-kDa subunits affects the O2. High dosage of GSSG or diamide-induced glutathionylation tends to decrease the catalytic function of NQR and increase enzyme-mediated O2. Knowledge of the function of each GS-binding domain is imperative, if we are to better understand the redox regulation mediated by NQR

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Conclusion