Peptide-based antibodies against glutathione-binding domains suppress superoxide production mediated by mitochondrial complex I.
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.
Chen
Jingfeng
J
Division of Cardiovascular Medicine, Department of Internal Medicine, Davis Heart & Lung Research Institute, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA.
Chen
Chwen-Lih
CL
Rawale
Sharad
S
Chen
Chun-An
CA
Zweier
Jay L
JL
Kaumaya
Pravin T P
PT
Chen
Yeong-Renn
YR
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry*; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D017202:Myocardial Ischemia / Q000473:pathology; D018384:Oxidative Stress; D010455:Peptides / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides
D000906:Antibodies; D000939:Epitopes; D010455:Peptides; D013481:Superoxides; D042967:Electron Transport Complex I; D005978:Glutathione