Graziano Colombo1, Marco Clerici2, Daniela Giustarini3, Nicola Portinaro4, Salvatore Badalamenti5, Ranieri Rossi3, Aldo Milzani1, Isabella Dalle-Donne6. 1. Department of Biosciences, Università degli Studi di Milano, Milan, Italy. 2. Department of Biosciences, Università degli Studi di Milano, Milan, Italy; Department of Biomedical Sciences for Health, Università degli Studi di Milano, Milan, Italy. 3. Department of Evolutionary Biology, University of Siena, Siena, Italy. 4. Clinica ortopedica e traumatologica, Humanitas Clinical and Research Center, Rozzano, Milan, Italy. 5. Humanitas Clinical and Research Center - Nephrology Unit, Rozzano, Milan, Italy. 6. Department of Biosciences, Università degli Studi di Milano, Milan, Italy. Electronic address: quack@unimi.it.
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. GENERAL SIGNIFICANCE: These results highlight that oxidized fibrinogen, instead of HSA, is the key protein for intermolecular dityrosine formation in human plasma.
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. GENERAL SIGNIFICANCE: These results highlight that oxidized fibrinogen, instead of HSA, is the key protein for intermolecular dityrosine formation in human plasma.
Authors: A V Bychkova; A D Vasilyeva; A E Bugrova; M I Indeykina; A S Kononikhin; E N Nikolaev; M L Konstantinova; M A Rosenfeld Journal: Dokl Biochem Biophys Date: 2017-07-20 Impact factor: 0.788
Authors: Alexandra Vasilyeva; Lyubov Yurina; Alexander Shchegolikhin; Maria Indeykina; Anna Bugrova; Alexey Kononikhin; Eugene Nikolaev; Mark Rosenfeld Journal: Biomolecules Date: 2020-06-17