title: The extracellular A-loop of dual oxidases affects the specificity of reactive oxygen species release.
abstract: NADPH oxidase (Nox) family proteins produce superoxide (O2 (⨪)) directly by transferring an electron to molecular oxygen. Dual oxidases (Duoxes) also produce an O2 (⨪) intermediate, although the final species secreted by mature Duoxes is H2O2, suggesting that intramolecular O2 (⨪) dismutation or other mechanisms contribute to H2O2 release. We explored the structural determinants affecting reactive oxygen species formation by Duox enzymes. Duox2 showed O2 (⨪) leakage when mismatched with Duox activator 1 (DuoxA1). Duox2 released O2 (⨪) even in correctly matched combinations, including Duox2 + DuoxA2 and Duox2 + N-terminally tagged DuoxA2 regardless of the type or number of tags. Conversely, Duox1 did not release O2 (⨪) in any combination. Chimeric Duox2 possessing the A-loop of Duox1 showed no O2 (⨪) leakage; chimeric Duox1 possessing the A-loop of Duox2 released O2 (⨪). Moreover, Duox2 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA2 showed enhanced O2 (⨪) release, and Duox1 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA1 acquired O2 (⨪) leakage. Although we identified Duox1 A-loop residues (His(1071), His(1072), and Gly(1074)) important for reducing O2 (⨪) release, mutations of these residues to those of Duox2 failed to convert Duox1 to an O2 (⨪)-releasing enzyme. Using immunoprecipitation and endoglycosidase H sensitivity assays, we found that the A-loop of Duoxes binds to DuoxA N termini, creating more stable, mature Duox-DuoxA complexes. In conclusion, the A-loops of both Duoxes support H2O2 production through interaction with corresponding activators, but complex formation between the Duox1 A-loop and DuoxA1 results in tighter control of H2O2 release by the enzyme complex.
authors.affiliation: From the Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan, tueyama@kobe-u.ac.jp.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Ueyama
authors.affiliation: From the Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Sakuma
authors.affiliation: From the Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Ninoyu
authors.affiliation: From the Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hamada
authors.affiliation: CNRS UMR8200 Laboratoire Stabilité Génétique et Oncogenèse, Université Paris-Sud, Institut Gustave Roussy, Villejuif 94805, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Dupuy
authors.affiliation: Department of Physiology, Faculty of Medicine, Semmelweis University, H-1444 Budapest, Hungary, "Lendület" Peroxidase Enzyme Research Group of the Semmelweis University and the Hungarian Academy of Sciences, H-1444 Budapest, Hungary, and.
authors.forename: Miklós
authors.initials: M
authors.lastname: Geiszt
authors.affiliation: Molecular Defenses Section, Laboratory of Host Defenses, NIAID, National Institutes of Health, Rockville, Maryland 20852.
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Leto
authors.affiliation: From the Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan, naosaito@kobe-u.ac.jp.
authors.forename: Naoaki
authors.initials: N
authors.lastname: Saito
mesh_terms: D002462:Cell Membrane / Q000201:enzymology; D000074623:Dual Oxidases; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000378:metabolism*; D013481:Superoxides / Q000737:chemistry / Q000378:metabolism; D013963:Thyroid Hormones / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D013963:Thyroid Hormones; D013481:Superoxides; D006861:Hydrogen Peroxide; D000074623:Dual Oxidases; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C402799:NOX1 protein, human; C431513:DUOX1 protein, human; C431515:DUOX2 protein, human; D010100:Oxygen
keywords: Glycosylation; Hydrogen Peroxide; NADPH Oxidase; Superoxide Ion; Thyroid Hormone