Abstract 4917: 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 reactive/regulatory protein thiols in mitochondria. In response to oxidative stress, NQR-derived protein thiols at the 51 kDa and 75 kDa subunits are known to be reversibly S-glutathiolated. Although several glutathiolated domains from NQR 51 kDa and 75 kDa have been identified, their role in the catalytic and regulatory function remain to be determined. To gain further insights into protein S-glutathiolation of complex I in myocardial ischemia and reperfusion, we used two S-glutathiolated peptides (200GAGAYIC206GEETALIESIEGK219 of 51-kDa protein and 361VDSDTLC367TEEVFPTAGAGTDLR382 of 75-kDa protein) as chimeric epitopes incorporating a “promiscuous” T cell epitope to generate two polyclonal antibodies, AbGSC206 and AbGSC367. Binding of AbGSC206 and AbGSC367 inhibited NQR-mediated O2•− generation by 37% and 57%, as measured by EPR spin-trapping with DEPMPO. To further provide an appropriate control, two non-glutathiolated peptides (21SGDTTAPKKTSFGSLKDFDR40 of 51-kDa and 100WNILTNSEKTKKAREGVMEFL120 of 75-kDa) were synthesized as chimeric epitopes to generate two high titer polyclonal antibodies, Ab51 and Ab75. Binding of A51 did not affect NQR-mediated O2•− generation. However, binding of A75 inhibited NQR-mediated O2•− generation by 35%. None of AbGSC206, AbGSC367, Ab51, or Ab75 showed an inhibitory effect on the electron transfer activity of NQR, suggesting that antibody binding to the GS-binding domain decreased electron leakage for superoxide generation mediated by complex I. Purified Ab51 and Ab75 showed high immunological specificity to the NQR of rat myocardial tissue and the mouse cardiac myocyte HL-1 cell line. Rats were subjected to 30 min of coronary ligation followed by 24 hours of reperfusion. When tissue homogenates were immunoprecipitated with Ab51 or Ab75 and probed with an antibody against glutathione, protein S-glutathiolation was enhanced by 60.8±5.7% (p<0.05, n = 6) in the post-ischemic myocardium at the NQR 51 kDa subunit, but not significantly changed at the NQR 75 kDa subunit, suggesting that the NQR 51 kDa subunit is more sensitive to oxidative stress caused by myocardial infarction.