title: Myeloperoxidase-mediated protein lysine oxidation generates 2-aminoadipic acid and lysine nitrile in vivo.
abstract: Recent studies reveal 2-aminoadipic acid (2-AAA) is both elevated in subjects at risk for diabetes and mechanistically linked to glucose homeostasis. Prior studies also suggest enrichment of protein-bound 2-AAA as an oxidative post-translational modification of lysyl residues in tissues associated with degenerative diseases of aging. While in vitro studies suggest redox active transition metals or myeloperoxidase (MPO) generated hypochlorous acid (HOCl) may produce protein-bound 2-AAA, the mechanism(s) responsible for generation of 2-AAA during inflammatory diseases are unknown. In initial studies we observed that traditional acid- or base-catalyzed protein hydrolysis methods previously employed to measure tissue 2-AAA can artificially generate protein-bound 2-AAA from an alternative potential lysine oxidative product, lysine nitrile (LysCN). Using a validated protease-based digestion method coupled with stable isotope dilution LC/MS/MS, we now report protein bound 2-AAA and LysCN are both formed by hypochlorous acid (HOCl) and the MPO/H2O2/Cl-system of leukocytes. At low molar ratio of oxidant to target protein Nε-lysine moiety, 2-AAA is formed via an initial Nε-monochloramine intermediate, which ultimately produces the more stable 2-AAA end-product via sequential generation of transient imine and semialdehyde intermediates. At higher oxidant to target protein Nε-lysine amine ratios, protein-bound LysCN is formed via initial generation of a lysine Nε-dichloramine intermediate. In studies employing MPO knockout mice and an acute inflammation model, we show that both free and protein-bound 2-AAA, and in lower yield, protein-bound LysCN, are formed by MPO in vivo during inflammation. Finally, both 2-AAA and to lesser extent LysCN are shown to be enriched in human aortic atherosclerotic plaque, a tissue known to harbor multiple MPO-catalyzed protein oxidation products. Collectively, these results show that MPO-mediated oxidation of protein lysyl residues serves as a mechanism for producing 2-AAA and LysCN in vivo. These studies further support involvement of MPO-catalyzed oxidative processes in both the development of atherosclerosis and diabetes risk.
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, OH 44195, United States; Department of Chemistry, Cleveland State University, Cleveland, OH 44115, United States.
authors.forename: Hongqiao
authors.initials: H
authors.lastname: Lin
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, OH 44195, United States.
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Levison
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, OH 44195, United States; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH 44195, United States.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Buffa
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, OH 44195, United States; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH 44195, United States.
authors.forename: Ying
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, OH 44195, United States; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH 44195, United States.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Fu
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, OH 44195, United States; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH 44195, United States.
authors.forename: Zeneng
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, OH 44195, United States; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH 44195, United States; Department of Chemistry, Cleveland State University, Cleveland, OH 44115, United States.
authors.forename: Valentin
authors.initials: V
authors.lastname: Gogonea
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, OH 44195, United States; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH 44195, United States.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: DiDonato
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, OH 44195, United States; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH 44195, United States; Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, OH 44195, United States. Electronic address: Hazens@ccf.org.
authors.forename: Stanley L
authors.initials: SL
authors.lastname: Hazen
mesh_terms: D015074:2-Aminoadipic Acid / Q000378:metabolism*; D000818:Animals; D050197:Atherosclerosis / Q000378:metabolism / Q000473:pathology; D003920:Diabetes Mellitus / Q000378:metabolism / Q000473:pathology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006997:Hypochlorous Acid / Q000378:metabolism; D007249:Inflammation / Q000378:metabolism* / Q000473:pathology; D007962:Leukocytes / Q000378:metabolism / Q000473:pathology; D008239:Lysine / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009570:Nitriles / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000235:genetics*; D009195:Peroxidase / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011506:Proteins / Q000378:metabolism*; D012307:Risk Factors; D053719:Tandem Mass Spectrometry
chemical_list: D009570:Nitriles; D011506:Proteins; D015074:2-Aminoadipic Acid; D006997:Hypochlorous Acid; D006861:Hydrogen Peroxide; D009195:Peroxidase; D008239:Lysine
keywords: 2-aminoadipic acid; Atherosclerosis; Chloramine; Diabetes; Hypochlorous acid; Inflammation; Lysine; Lysine nitrile; Myeloperoxidase; Neutrophil