Abstract
Collagen IV comprises the predominant protein network of basement membranes, a specialized extracellular matrix, which underlie epithelia and endothelia. These networks assemble through oligomerization and covalent crosslinking to endow mechanical strength and shape cell behavior through interactions with cell-surface receptors. A recently discovered sulfilimine (S=N) bond between a methionine sulfur and hydroxylysine nitrogen reinforces the collagen IV network. We demonstrate that peroxidasin, an enzyme found in basement membranes, catalyzes formation of the sulfilimine bond. Drosophila peroxidasin mutants have disorganized collagen IV networks and torn visceral muscle basement membranes, pointing to a critical role for the enzyme in tissue biogenesis. Peroxidasin generates hypohalous acids as reaction intermediates, suggesting a paradoxically anabolic role for these usually destructive oxidants. This work highlights sulfilimine bond formation as what is to our knowledge the first known physiologic function for peroxidasin, a role for hypohalous oxidants in tissue biogenesis, and a possible role for peroxidasin in inflammatory diseases.
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Acknowledgements
This work was supported by the US National Institutes of Health (RO1 DK18381, DK18381-38S1 and 2PO1 DK065123 to B.G.H.), the Mount Desert Island Biological Laboratory Salisbury Cove Research Fund and F.H. Epstein Fellowship (to B.G.H.), the Vanderbilt Division of Nephrology Faculty Development Fund (to R.M.V.) and a Vanderbilt Physician Scientist Development Award (to G.B.). We are grateful to K.L. Rose and W.H. McDonald of the Mass Spectrometry Resource Center at Vanderbilt University for assistance with mass spectrometry, which was supported in part by the Vanderbilt Academic Venture Capital Fund. We acknowledge A. Chisholm and A. Page-McCaw for fruitful discussions during the writing of this manuscript. We thank M. Geizst (Semmelweis University) for the human peroxidasin coding sequence and L. Cooley (Yale University) for the vikingG454 protein trap Drosophila line. P. Todd, N. Danylevych and C. Venkov provided technical assistance.
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G.B. conducted, designed and analyzed data from the PFHR-9 cell culture experiments, purified collagen IV NC1 hexamers from Drosophila and conducted western blotting experiments on Drosophila mutants. C.F.C. conducted mechanistic experiments involving hypohalous acids and peroxidasin. R.M.V. conducted MS and analysis. L.I.F. prepared Drosophila materials, and C.K.-C. performed Drosophila genetics and confocal microscopy. I.A.E.-T. performed overlay experiments involving peroxidasin and other peroxidases. M.R. isolated collagen IV NC1 hexamers and sulfilimine-crosslinked peptides for further analysis. J.-S.K. isolated human peroxidasin expressing HEK293 stable cell lines, and V.P. established the PFHR-9 cell culture system for these studies. L.I.F. generated Drosophila mutant larvae, antibodies and protein reagents. L.I.F., J.H.F. and B.G.H. designed the study and wrote the paper along with G.B. All authors discussed the results and commented on the manuscript.
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Bhave, G., Cummings, C., Vanacore, R. et al. Peroxidasin forms sulfilimine chemical bonds using hypohalous acids in tissue genesis. Nat Chem Biol 8, 784–790 (2012). https://doi.org/10.1038/nchembio.1038
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DOI: https://doi.org/10.1038/nchembio.1038
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