Goodpasture's (GP) disease is an autoimmune disorder which affects the glomerular basement membrane (GBM) and pulmonary vasculature resulting in death for 50% of cases. In GP disease, antibodies specifically target the non-collagenous 1 (NC1) domain of the ?3 and 5 chains of the collagen IV network. The NC1 domain occurs as hexamers that are covalently crosslinked by a sulfilimine chemical bond (-S=N-), which renders the NC1 hexamer inert to binding of GP autoantibodies. This bond is formed by the recently identified enzyme peroxidasin (PXDN) which putatively utilizes hydrogen peroxide as an initial oxidant to form hypobromous acid (HOBr) as the terminal oxidant for S=N bond formation. The highly reactive nature of HOBr suggests that peroxidasin complexes with the NC1 domain in vivo to maximize the potential efficiency of S=N bond formation and decrease collateral oxidative stress on the surrounding tissue. The pathogenesis of Goodpasture's disease may involve the disruption of PXDN function and normal cross-linking which would lead to proteolysis and self-immunization with auto-antigenic peptide fragments. The objective of this particular proposal is to determine peroxidasin's role in the pathogenesis of Goodpasture's disease. My pilot data has revealed novel anti-PXDN antibodies in GP patients which inhibit S=N bond formation. Additionally, I have found that PXDN can form the S=N bond using nucleotide monophosphates for oxidation rather than hydrogen peroxide (H2O2). These findings lead to the central hypothesis that anti-peroxidasin antibodies play a key role in the pathogenesis of GP disease by preventing the formation of the sulfilimine bond. This hypothesis will be interrogated with three specific aims:
Aim 1 : To characterize anti-peroxidasin antibodies in Goodpasture's patients. I have identified Goodpasture's patients who have anti-peroxidasin antibodies. We hypothesize that anti-peroxidasin antibodies target the peroxidase domain and inactivate S=N bond forming activity.
Aim 2 : To determine the time course of appearance and binding characteristics of anti-peroxidasin antibodies in Goodpasture's disease patients. Serial serum samples from 30 patients before the appearance of Goodpasture's disease will give temporal resolution to anti-PXDN antibodies. We hypothesize that anti-peroxidasin antibodies play a causal role in Goodpasture's disease.
Aim 3 : To identify the initial oxidant source used to form the S=N bond by peroxidasin. Hydrogen peroxide is the putative oxidant for peroxidasin but preliminary data shows that PXDN is capable of using nucleotide monophosphates as the initial oxidant for S=N bond formation. We hypothesize peroxidasin uses O2 and extracellular nucleotides as initial oxidants to form HOBr for the biosynthesis of the S=N bond. The achievement of these aims will provide insight into a novel mechanism of autoimmune disease pathogenesis and potentially enable early diagnosis of GP disease through understanding the inhibition of an unprecedented enzymatic mechanism in tissue biosynthesis.
The proposed research is relevant to public health through better understanding of autoimmunity, particularly Goodpasture's disease, and a new autoantibody which could be a harbinger of disease and lead to early diagnosis treatment opportunities. The insight gained from this study also pertains to the NIH's mission of better understanding human disease through a more detailed grasp of basement membrane autoimmune complexes and fundamental aspects of normal tissue genesis through the actions of peroxidasin.
|McCall, A Scott; Cummings, Christopher F; Bhave, Gautam et al. (2014) Bromine is an essential trace element for assembly of collagen IV scaffolds in tissue development and architecture. Cell 157:1380-92|