The Role of Peroxidasin in Basement Membrane Physiology and Human Disease

Abstract

Basement membranes are a distinct form of extracellular matrix responsible for signal transduction and mechanical integrity throughout development, in mature tissues, and during wound healing. The collagen IV scaffold of basement membranes relies on a sulfilimine crosslink (S=N) between methionine and lysine for its essential function of maintaining basement membrane architecture. The sulfilimine crosslink is formed by the heme peroxidase, peroxidasin. The precise mechanism by which peroxidasin forms sulfilimine crosslinks, or if this crosslinking process is involved in disease, remains largely unknown. Biochemical investigation of peroxidasin-catalyzed formation of the crosslink found that bromide (Br^-) appeared to be the preferred enzymatic cofactor through its conversion to hypobromous acid (HOBr). Through my development of Br-free salts, purified proteins, and in vitro cell culture models of basement membranes, Br^- was shown to be essential to the formation physiologically observed levels of sulfilimine crosslink. I further investigated the underlying mechanism of sulfilimine formation with chemical crosslinking, mass spectrometry analysis, and modeling. These approaches cumulatively supported the presence of a S-Br⁺ (bromosulfonium-ion) intermediate by the crosslinked methionine of the NC1 domain of collagen IV as the key reaction intermediate and energetic basis for bromines role in sulfilimine crosslinking. The essentiality of Br was therefore tested in vivo in Drosophila by developing novel Br-free culture techniques. I found that dietary Br-deficiency is lethal in Drosophila while Br-replenishment restores viability, demonstrating a physiologic Br^- requirement. Importantly, through electron and fluorescence microscopy, I was also able to show that Br-deficient flies phenocopy the developmental and basement membrane defects observed in peroxidasin mutants which indicates a functional connection between Br^-, collagen IV, and peroxidasin. These data collectively established that Br^- is required for sulfilimine crosslinking of collagen IV, an event critical for basement membrane assembly and tissue development. Thus, bromine is an essential trace element for animals through the enzymatic activity of peroxidasin, and Br-deficiency may be relevant to basement membrane alterations observed in patients undergoing dialysis, receiving total parenteral nutrition, and in some smoking related disease. I also sought to test the hypothesis that anti-peroxidasin autoantibodies occur in a specific rapidly progressive glomerulonephritis known as Goodpasture’s disease (GP). Goodpasture’s Disease is characterized by anti-collagen IV NC1 antibodies and the sulfilimine crosslink is thought to modulate immunogenicity of the collagen IV epitopes. Many GP patients have concurrent autoantibodies which recognize myeloperoxidase (MPO), a structurally related heme peroxidase to peroxidasin. Through testing multiple independent patient cohorts by immunoassay, I found anti-peroxidasin autoantibodies in GP patient sera, both before and at the time of clinical presentation. Unexpectedly, the anti-peroxidasin specific antibodies cross-react with coated, but not native MPO, accounting for a subset of the historically characterized dual-positive (anti- collagen IV and anti-MPO) patients. I also found that anti-peroxidasin antibodies inhibited HOBr production, suggesting a possible contribution of these antibodies in GP pathogenesis within this subset of anti-peroxidasin positive patients. These studies demonstrate chemical, biochemical, and tissue level evidence for the role of peroxidasin and Br^- in the assembly of sulfilimine-crosslinked collagen IV scaffolds in basement membranes and peroxidasin’s potential role in disease, including as a novel autoantigen in a subset Goodpasture’s disease patients.