3 - Peroxidasin-Catalysed Oxidative Modifications of Proteins in the Extracellular Matrix

Abstract

Peroxidasin has been recently identified as a mammalian heme peroxidase that catalyses the formation of an essential crosslink in collagen IV in the extracellular matrix (ECM). This crosslink was identified as sulfilimine bond between methionine and hydroxylysine resuidues in the non-collagenous domain. Heme peroxidases catalyse the oxidation of a variety of substrates by hydrogen peroxide (H2O2). These include bromide (Br–) to give hypobromous acid (HOBr), which is proposed to be responsible for the collagen IV crosslink. However, the specificity of peroxidasin and the exact mechanism of sulfilimine formation are not completely understood. We investigated peroxidasin activity and the cross-linking of collagen IV in isolated ECM from cultured epithelial cells. We found that formation of the sulfilimine crosslink requires both H2O2 and Br–. It did not form in presence of other halides such as chloride, iodide or the pseudohalide thiocyanate, with both of the latter inhibiting the reaction. Acetaminophen (paracetamol), thioxanthine, and 4-aminobenzoic acid hydrazide, which all inhibit myeloperoxidase, had minimal effect on the peroxidasin-dependent crosslink. The specificity and inhibitory profile of peroxidasin is therefore different from myeloperoxidase. HOBr has been shown to form sulfilimine bonds in peptides containing Met and Lys, but it also undergoes other reactions with proteins, including the formation of a specific product bromotyrosine (Br-Tyr)(1). Using LC-MS, we detected Br-Tyr in the noncollagenous domain of collagen IV isolated from cultured cells, and to a lesser extent in the rest of matrix and the cell extract. Br-Tyr levels increased in cells grown in the presence of Br–, and decreased in presence of phloroglucinol, a potent inhibitor of cross-linking. Cross-linking was already maximal without added Br–, suggesting that the reaction is selective only at low peroxidasin activity. These results indicate that cross-linking of collagen IV requires Br–, and is susceptible to modulation by endogenous and exogenous substances. Furthermore, peroxidasin-mediated oxidative modification of proteins in the ECM might not be limited to formation of the sulfilimine crosslink. 1. Ronsein GE, Winterbourn CC, Di Mascio P, Kettle AJ. Cross-linking methionine and amine residues with reactive halogen species. Free radical biology & medicine. 2014;70:278-87.