title: Structure-function analysis of peroxidasin provides insight into the mechanism of collagen IV crosslinking.
abstract: Basement membranes provide structural support and convey regulatory signals to cells in diverse tissues. Assembly of collagen IV into a sheet-like network is a fundamental mechanism during the formation of basement membranes. Peroxidasin (PXDN) was recently described to catalyze crosslinking of collagen IV through the formation of sulfilimine bonds. Despite the significance of this pathway in tissue genesis, our understanding of PXDN function is far from complete. In this work we demonstrate that collagen IV crosslinking is a physiological function of mammalian PXDN. Moreover, we carried out structure-function analysis of PXDN to gain a better insight into its role in collagen IV synthesis. We identify conserved cysteines in PXDN that mediate the oligomerization of the protein into a trimeric complex. We also demonstrate that oligomerization is not an absolute requirement for enzymatic activity, but optimal collagen IV coupling is only catalyzed by the PXDN trimers. Localization experiments of different PXDN mutants in two different cell models revealed that PXDN oligomers, but not monomers, adhere on the cell surface in "hot spots," which represent previously unknown locations of collagen IV crosslinking.
authors.affiliation: Department of Physiology, Semmelweis University, Budapest, Hungary; "Momentum" Peroxidase Enzyme Research Group of the Semmelweis University and the Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Enikő
authors.initials: E
authors.lastname: Lázár
authors.affiliation: Department of Physiology, Semmelweis University, Budapest, Hungary; "Momentum" Peroxidase Enzyme Research Group of the Semmelweis University and the Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Zalán
authors.initials: Z
authors.lastname: Péterfi
authors.affiliation: Department of Physiology, Semmelweis University, Budapest, Hungary; "Momentum" Peroxidase Enzyme Research Group of the Semmelweis University and the Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Gábor
authors.initials: G
authors.lastname: Sirokmány
authors.affiliation: Department of Physiology, Semmelweis University, Budapest, Hungary; "Momentum" Peroxidase Enzyme Research Group of the Semmelweis University and the Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Hajnal A
authors.initials: HA
authors.lastname: Kovács
authors.affiliation: Laboratory of Proteomics Research, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Eva
authors.initials: E
authors.lastname: Klement
authors.affiliation: Laboratory of Proteomics Research, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary; Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA.
authors.forename: Katalin F
authors.initials: KF
authors.lastname: Medzihradszky
authors.affiliation: Department of Physiology, Semmelweis University, Budapest, Hungary; "Momentum" Peroxidase Enzyme Research Group of the Semmelweis University and the Hungarian Academy of Sciences, Szeged, Hungary. Electronic address: geiszt@eok.sote.hu.
authors.forename: Miklós
authors.initials: M
authors.lastname: Geiszt
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017209:Apoptosis; D001485:Basement Membrane / Q000378:metabolism*; D015153:Blotting, Western; D002384:Catalysis; D049109:Cell Proliferation; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000502:physiology*; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D051379:Mice; D018345:Mice, Knockout; D009195:Peroxidase / Q000502:physiology*; D010544:Peroxidases; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D017472:Receptors, Interleukin-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013329:Structure-Activity Relationship; D000097423:Peroxidasin
chemical_list: D000951:Antigens, Neoplasm; D024141:Collagen Type IV; D003432:Cross-Linking Reagents; D016326:Extracellular Matrix Proteins; D009195:Peroxidase; D010544:Peroxidases; D012333:RNA, Messenger; D017472:Receptors, Interleukin-1; D000097423:Peroxidasin; C535215:PXDN protein, human
keywords: Collagen IV; Crosslink; Peroxidase; Peroxidasin; Reactive oxygen species