title: A central role for intermolecular dityrosine cross-linking of fibrinogen in high molecular weight advanced oxidation protein product (AOPP) formation.
abstract: BACKGROUND
  Advanced oxidation protein products (AOPPs) are dityrosine cross-linked and carbonyl-containing protein products formed by the reaction of plasma proteins with chlorinated oxidants, such as hypochlorous acid (HOCl). Most studies consider human serum albumin (HSA) as the main protein responsible for AOPP formation, although the molecular composition of AOPPs has not yet been elucidated. Here, we investigated the relative contribution of HSA and fibrinogen to generation of AOPPs.
  
  
  METHODS
  AOPP formation was explored by SDS-PAGE, under both reducing and non-reducing conditions, as well as by analytical gel filtration HPLC coupled to fluorescence detection to determine dityrosine and pentosidine formation.
  
  
  RESULTS
  Following exposure to different concentrations of HOCl, HSA resulted to be carbonylated but did not form dityrosine cross-linked high molecular weight aggregates. Differently, incubation of fibrinogen or HSA/fibrinogen mixtures with HOCl at concentrations higher than 150 μM induced the formation of pentosidine and high molecular weight (HMW)-AOPPs (>200 k Da), resulting from intermolecular dityrosine cross-linking. Dityrosine fluorescence increased in parallel with increasing HMW-AOPP formation and increasing fibrinogen concentration in HSA/fibrinogen mixtures exposed to HOCl. This conclusion is corroborated by experiments where dityrosine fluorescence was measured in HOCl-treated human plasma samples containing physiological or supra-physiological fibrinogen concentrations or selectively depleted of fibrinogen, which highlighted that fibrinogen is responsible for the highest fluorescence from dityrosine.
  
  
  CONCLUSIONS
  A central role for intermolecular dityrosine cross-linking of fibrinogen in HMW-AOPP formation is shown.
  
  
  CONCLUSIONS
  These results highlight that oxidized fibrinogen, instead of HSA, is the key protein for intermolecular dityrosine formation in human plasma.
authors.affiliation: Department of Biosciences, Università degli Studi di Milano, Milan, Italy.
authors.forename: Graziano
authors.initials: G
authors.lastname: Colombo
authors.affiliation: Department of Biosciences, Università degli Studi di Milano, Milan, Italy; Department of Biomedical Sciences for Health, Università degli Studi di Milano, Milan, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Clerici
authors.affiliation: Department of Evolutionary Biology, University of Siena, Siena, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Giustarini
authors.affiliation: Clinica ortopedica e traumatologica, Humanitas Clinical and Research Center, Rozzano, Milan, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Portinaro
authors.affiliation: Humanitas Clinical and Research Center - Nephrology Unit, Rozzano, Milan, Italy.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Badalamenti
authors.affiliation: Department of Evolutionary Biology, University of Siena, Siena, Italy.
authors.forename: Ranieri
authors.initials: R
authors.lastname: Rossi
authors.affiliation: Department of Biosciences, Università degli Studi di Milano, Milan, Italy.
authors.forename: Aldo
authors.initials: A
authors.lastname: Milzani
authors.affiliation: Department of Biosciences, Università degli Studi di Milano, Milan, Italy. Electronic address: quack@unimi.it.
authors.forename: Isabella
authors.initials: I
authors.lastname: Dalle-Donne
mesh_terms: D062107:Advanced Oxidation Protein Products / Q000097:blood / Q000378:metabolism*; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D015153:Blotting, Western; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D006997:Hypochlorous Acid / Q000494:pharmacology; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D008970:Molecular Weight; D010084:Oxidation-Reduction / Q000187:drug effects; D050050:Protein Carbonylation / Q000187:drug effects; D012709:Serum Albumin / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D062107:Advanced Oxidation Protein Products; D003432:Cross-Linking Reagents; D012709:Serum Albumin; D014443:Tyrosine; D006997:Hypochlorous Acid; D005340:Fibrinogen; D001120:Arginine; C062187:pentosidine; C007543:dityrosine; D008239:Lysine
keywords: Advanced oxidation protein products; Albumin; Dityrosine; Fibrinogen; Human plasma; Hypochlorous acid