Characterising the activity of peroxidasin in the extracellular matrix

Abstract

Peroxidasin is a rising star in the field of redox biology. It is a peculiar heme peroxidase that uniquely contains non-catalytic domains in addition to the peroxidase domain. Peroxidasin catalyses the formation of an unusual sulfilimine bond between methionine and hydroxylysine in the non-collagenous domain of collagen IV in the extracellular matrix (ECM). The mechanism is proposed to involve the generation of hypobromous acid (HOBr) in a rare example of an anabolic function of this powerful oxidant and destructive bactericide. Peroxidasin is implicated in many pathologies, including cardiovascular diseases, fibrosis and cancer. A lot is unknown about the exact mechanism of collagen IV cross-linking, the substrate specificity and selectivity of peroxidasin, and regulation of its activity. One of the main questions that surrounded peroxidasin since its discovery is whether it causes collateral oxidative modifications of biomolecules. In this thesis, I investigated the selectivity and modulation of the activity of peroxidasin in the ECM. I optimised an in situ model system and confirmed the activity of peroxidasin embedded in the ECM. The results demonstrate that peroxidasin is highly efficient in cross-linking collagen IV using low concentrations of bromide and hydrogen peroxide. Peroxidasin catalyses the oxidation of exogenous NADH to bromohydrin, providing direct evidence that it generates free HOBr in the decellularised ECM. I investigated the modulation of collagen IV cross-linking by peroxidase substrates and scavengers of HOBr. Physiological levels of thiocyanate and urate modulate collagen IV cross-linking. Overall, peroxidasin shows preference for bromide over other physiological substrates, although the basis of this specificity is not yet known. Scavengers of HOBr, like methionine and glutathione are inefficient in inhibiting the sulfilimine bond in collagen IV. These observations demonstrate selectivity of the reactions of HOBr produced by peroxidasin for collagen IV cross-linking. I investigated whether HOBr produced by peroxidasin in the ECM undergoes reactions beyond cross-linking collagen IV. I developed mass spectrometry methods to measure markers of oxidative modifications of proteins. The results show that peroxidasin indeed catalyses the formation of 3-bromotyrosine, a specific marker of the reaction of HOBr with tyrosine. The levels of 3-bromotyrosine on proteins are significantly lower when peroxidasin is inhibited or knocked out. Formation of 3-bromotyrosine is more susceptible to inhibition by thiocyanate, urate and nitrite than the cross-linking of collagen IV. These results suggest selectivity of HOBr reactions towards the deliberate sulfilimine bond formation over the presumably collateral reactions with other amino acid residues on proteins. Taken together, the evidence I present here demonstrates that the reactions of peroxidasin in the ECM are highly specific for, but not limited to, the cross-linking of collagen IV. It strengthens the hypothesis that HOBr generated by peroxidasin is targeted towards collagen IV, though likely to diffuse a short distance in the ECM. The novel finding that peroxidasin contributes to the formation of 3-bromoytrosine on proteins brings it to the fore as an enzymatic source of oxidative modifications of proteins. It also raises more questions about the extent and effect of peroxidasin-catalysed oxidative modifications of proteins in physiology and pathology.