Physicochemical and immunological studies on mitochondrial DNA modified by peroxynitrite: implications of neo-epitopes of mitochondrial DNA in the etiopathogenesis of systemic lupus erythematosus

Abstract

Background and objective

Recent evidence has demonstrated that mitochondria possess their own nitric oxide synthase (mtNOS) and can produce endogenous reactive-nitrogen-species (RNS) including peroxynitrite (ONOO^–). This study was undertaken to investigate the role of mitochondrial DNA (mtDNA) damage by ONOO^– in systemic lupus erythematosus (SLE) autoimmunity.

Methods

MtDNA was isolated from fresh goat liver and modified by ONOO^–, generated by synergistic action of nitric oxide (NO) and superoxide (O ^– ₂ ) donors. Modifications occurring in mtDNA were characterized by physicochemical techniques. SLE patients (n = 50) with varying disease activity according to the SLE Disease Activity Index (SLEDAI) and healthy controls (n = 34) were evaluated for antibodies to native and ONOO^–-modified mtDNA by immunoassays. Gel retardation assays were performed to cross-examine the immunoassay results using affinity-purified SLE immunoglobulin G (IgG). Nitrosative stress in SLE patients was studied by measuring nitrotyrosine and inducible NO synthase (iNOS).

Results

The ONOO^– caused extensive damage to mtDNA as evident by ultraviolet (UV) hyperchromicity and loss of florescence intensity. Thermal melting studies, agarose gel electrophoresis and nuclease S1 digestibility clearly indicate structural perturbation in mtDNA by ONOO^–. Quenching studies with specific NO or O ^– ₂ quenchers confirmed that the damaging agent was ONOO^–. SLE autoantibodies exhibited enhanced binding with ONOO^–-mtDNA as compared to their native analog. Interestingly, not only was there an increased number of subjects positive for ONOO ^– -mtDNA, but also the levels of anti-ONOO ^– -mtDNA antibodies were statistically significantly higher among SLE patients whose SLEDAI scores were ≥ 20 as compared with SLE patients with lower SLEDAI scores (SLEDAI < 20). Normal healthy controls showed negligible binding with either antigen. Furthermore, SLE patients had higher levels of nitrotyrosine and iNOS compared with their respective healthy controls.

Conclusions

Our novel results provide an important insight into the immunological basis of anti-DNA autoantibody generation in SLE. Our data conclude that modification of mtDNA by ONOO ^– causes structural perturbations, resulting in the generation of neo-epitopes, and making it a potential immunogen in SLE. The mtDNA modified by ONOO ^– may be useful in evaluating the progression of SLE and in elucidating the mechanisms of disease pathogenesis.