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• W2101692904 abstract "Duox2 (and probably Duox1) is a glycoflavoprotein involved in thyroid hormone biosynthesis, as the thyroid H2O2 generator functionally associated with Tpo (thyroperoxidase). So far, because of the impairment of maturation and of the targeting process, transfecting DUOX into nonthyroid cell lines has not led to the expression of a functional H2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-generating activity in the particulate fractions from DUOX2- and DUOX1-transfected HEK293 and Chinese hamster ovary cells. The particulate fractions of these cells stably or transiently transfected with human or porcine DUOX cDNA demonstrate a functional NADPH/Ca2+-dependent H2O2-generating activity. The immature Duox proteins had less activity than pig thyrocyte particulate fractions, and their activity depended on their primary structures. Human Duox2 seemed to be more active than human Duox1 but only half as active as its porcine counterpart. TPO co-transfection produced a slight increase in the enzymatic activity, whereas p22phox, the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O2·¯ but H2O2. Using a spin-trapping technique combined with electron paramagnetic resonance spectroscopy, we confirmed this result but also demonstrated that the partially glycosylated form of Duox2, located in the endoplasmic reticulum, generates superoxide in a calcium-dependent manner. These results suggest that post-translational modifications during the maturation process of Duox2 could be implicated in the mechanism of H2O2 formation by favoring intramolecular superoxide dismutation. Duox2 (and probably Duox1) is a glycoflavoprotein involved in thyroid hormone biosynthesis, as the thyroid H2O2 generator functionally associated with Tpo (thyroperoxidase). So far, because of the impairment of maturation and of the targeting process, transfecting DUOX into nonthyroid cell lines has not led to the expression of a functional H2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-generating activity in the particulate fractions from DUOX2- and DUOX1-transfected HEK293 and Chinese hamster ovary cells. The particulate fractions of these cells stably or transiently transfected with human or porcine DUOX cDNA demonstrate a functional NADPH/Ca2+-dependent H2O2-generating activity. The immature Duox proteins had less activity than pig thyrocyte particulate fractions, and their activity depended on their primary structures. Human Duox2 seemed to be more active than human Duox1 but only half as active as its porcine counterpart. TPO co-transfection produced a slight increase in the enzymatic activity, whereas p22phox, the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O2·¯ but H2O2. Using a spin-trapping technique combined with electron paramagnetic resonance spectroscopy, we confirmed this result but also demonstrated that the partially glycosylated form of Duox2, located in the endoplasmic reticulum, generates superoxide in a calcium-dependent manner. These results suggest that post-translational modifications during the maturation process of Duox2 could be implicated in the mechanism of H2O2 formation by favoring intramolecular superoxide dismutation. Reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; BMPO, 5-tert-butyloxycarbonyl 5-methyl-1-pyrroline N-oxide; EPR, electron paramagnetic resonance; SOD, superoxide dismutase, GFP, green fluorescent protein; CHO, Chinese hamster ovary. have emerged as important molecules involved in regulating essential cell functions, such as growth and differentiation (1Burdon R.H. Free Radic. Biol. Med. 1995; 18: 775-794Crossref PubMed Scopus (1069) Google Scholar). NAD(P)H oxidases are a major source of ROS. Phagocyte oxidase is the oxidase that has been investigated most thoroughly (2Babior B.M. Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar). It catalyzes the production of superoxide by the one-electron reduction of oxygen, using NADPH as the electron donor. The catalytic moiety of the phagocyte NADPH oxidase is gp91phox, a plasma membrane-associated flavohemoprotein. Recently, it was discovered that gp91phox belongs to a family consisting of several very similar oxidases. Seven NOX (NADPH oxidase) and DUOX/ThOX (dual oxidase/thyroid oxidase) genes have been identified that encode different NADPH oxidases with differing mRNA tissue expression. The Nox family comprises gp91phox, now known as Nox2; Nox1, which is predominantly expressed in the colon (3Suh Y.A. Arnold R.S. Lassegue B. Shi J. Xu X. Sorescu D. Chung A.B. Griendling K.K. Lambeth J.D. Nature. 1999; 401: 79-82Crossref PubMed Scopus (1284) Google Scholar); Nox3, cloned from fetal kidney (4Kikuchi H. Hikage M. Miyashita H. Fukumoto M. Gene (Amst.). 2000; 254: 237-243Crossref PubMed Scopus (125) Google Scholar); Nox4 found in the kidney cortex (5Geiszt M. Kopp J.B. Varnai P. Leto T.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8010-8014Crossref PubMed Scopus (715) Google Scholar, 6Shiose A. Kuroda J. Tsuruya K. Hirai M. Hirakata H. Naito S. Hattori M. Sakaki Y. Sumimoto H. J. Biol. Chem. 2001; 276: 1417-1423Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar); and Nox5, expressed in the testis, spleen, and lymph nodes (7Banfi B. Molnar G. Maturana A. Steger K. Hegedus B. Demaurex N. Krause K.H. J. Biol. Chem. 2001; 276: 37594-37601Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). In addition to the basic structure of gp91phox, Nox5 has a long intracellular N-terminal domain with four calcium binding sites implicated in its Ca2+-dependent activation (8Banfi B. Tirone F. Durussel I. Knisz J. Moskwa P. Molnar G.Z. Krause K.H. Cox J.A. J. Biol. Chem. 2004; 279: 18583-18591Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). The biological functions of these Nox proteins are now under investigation. They are involved in signal transduction related to cell growth and cancer (9Brar S.S. Corbin Z. Kennedy T.P. Hemendinger R. Thornton L. Bommarius B. Arnold R.S. Whorton A.R. Sturrock A.B. Huecksteadt T.P. Quinn M.T. Krenitsky K. Ardie K.G. Lambeth J.D. Hoidal J.R. Am. J. Physiol. 2003; 285: C353-C369Crossref PubMed Scopus (226) Google Scholar, 10Chamulitrat W. Schmidt R. Tomakidi P. Stremmel W. Chunglok W. Kawahara T. Rokutan K. Oncogene. 2003; 22: 6045-6053Crossref PubMed Scopus (91) Google Scholar, 11Mitsushita J. Lambeth J.D. Kamata T. Cancer Res. 2004; 64: 3580-3585Crossref PubMed Scopus (267) Google Scholar) and to angiogenesis (12Arbiser J.L. Petros J. Klafter R. Govindajaran B. McLaughlin E.R. Brown L.F. Cohen C. Moses M. Kilroy S. Arnold R.S. Lambeth J.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 715-720Crossref PubMed Scopus (404) Google Scholar). Duox1 and Duox2 are large homologues of Nox2 with an N-terminal extension comprising two EF-hand motifs, an additional transmembrane helix, and a peroxidase homology ectodomain (see Fig. 4A) (13Dupuy C. Ohayon R. Valent A. Noel-Hudson M.S. Deme D. Virion A. J. Biol. Chem. 1999; 274: 37265-37269Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 14De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). DUOX genes have been identified in the thyroid gland, where they are strongly expressed (13Dupuy C. Ohayon R. Valent A. Noel-Hudson M.S. Deme D. Virion A. J. Biol. Chem. 1999; 274: 37265-37269Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 14De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). However, the DUOX are also expressed on the mucosal surfaces of the trachea and the bronchi (15Geiszt M. Witta J. Baffi J. Lekstrom K. Leto T.L. FASEB J. 2003; 17: 1502-1504Crossref PubMed Scopus (419) Google Scholar) and in the airway epithelial cells (16Schwarzer C. Machen T.E. Illek B. Fischer H. J. Biol. Chem. 2004; 279: 36454-36461Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 17Forteza R. Salathe M. Miot F. Forteza R. Conner G.E. Am. J. Respir. Cell Mol. Biol. 2005; 32: 462-469Crossref PubMed Scopus (200) Google Scholar), where it has been suggested that Duox1 is the isoform responsible for acid production and secretion in airways (16Schwarzer C. Machen T.E. Illek B. Fischer H. J. Biol. Chem. 2004; 279: 36454-36461Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) and plays a critical role in mucin expression (18Shao M.X. Nadel J.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 767-772Crossref PubMed Scopus (229) Google Scholar). DUOX2 was also expressed throughout the digestive tract, where it was found to be functional (19Dupuy C. Pomerance M. Ohayon R. Noel-Hudson M.S. Deme D. Chaaraoui M. Francon J. Virion A. Biochem. Biophys. Res. Commun. 2000; 277: 287-292Crossref PubMed Scopus (59) Google Scholar, 20El Hassani R.A. Benfares N. Caillou B. Talbot M. Sabourin J.C. Belotte V. Morand S. Gnidehou S. Agnandji D. Ohayon R. Kaniewski J. Noel-Hudson M.S. Bidart J.M. Schlumberger M. Virion A. Dupuy C. Am. J. Physiol. 2005; 288: G933-G942Crossref PubMed Scopus (192) Google Scholar), in addition to the salivary gland and rectum (15Geiszt M. Witta J. Baffi J. Lekstrom K. Leto T.L. FASEB J. 2003; 17: 1502-1504Crossref PubMed Scopus (419) Google Scholar). It has been suggested that Duox2, which was identified by purifying thyroid NADPH oxidase, may constitute the catalytic core of this enzyme and generate the H2O2 used by Tpo to catalyze the biosynthesis of thyroid hormones at the apical surface of the thyrocytes (13Dupuy C. Ohayon R. Valent A. Noel-Hudson M.S. Deme D. Virion A. J. Biol. Chem. 1999; 274: 37265-37269Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). Although no functional Duox-based H2O2-generating system has yet been reconstituted (21De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (157) Google Scholar), this proposal is corroborated by a recent report of permanent and severe congenital hypothyroidism in a patient with a biallelic inactivating mutation in the DUOX2 gene (22Moreno J.C. Bikker H. Kempers M.J. van Trotsenburg A.S. Baas F. de Vijlder J.J. Vulsma T. Ris-Stalpers C. N. Engl. J. Med. 2002; 347: 95-102Crossref PubMed Scopus (398) Google Scholar). Because Duox2 is also co-expressed with lactoperoxidase in the salivary gland and rectum, it has been suggested that it may be the source of H2O2 for lactoperoxidase-catalyzed reactions implicated in a host defense mechanism (15Geiszt M. Witta J. Baffi J. Lekstrom K. Leto T.L. FASEB J. 2003; 17: 1502-1504Crossref PubMed Scopus (419) Google Scholar). A Duox-based enzyme is also involved in stabilizing the cuticle of Caenorhabditis elegans by the peroxidase-catalyzed formation of tyrosine-tyrosine bonds (23Edens W.A. Sharling L. Cheng G. Shapira R. Kinkade J.M. Lee T Edens H.A. Tang X. Sullards C. Flaherty D.B. Benian G.M. Lambeth J.D. J. Cell Biol. 2001; 154: 879-891Crossref PubMed Scopus (319) Google Scholar). This means that Duox2 must generate H2O2 for peroxidase reactions catalyzed either by distinct peroxidases, such as Tpo and lactoperoxidase, or by the peroxidase-like ectodomain of Duox2 itself (23Edens W.A. Sharling L. Cheng G. Shapira R. Kinkade J.M. Lee T Edens H.A. Tang X. Sullards C. Flaherty D.B. Benian G.M. Lambeth J.D. J. Cell Biol. 2001; 154: 879-891Crossref PubMed Scopus (319) Google Scholar). On the basis of their homology with gp91phox/Nox2, the Duox proteins are believed to generate superoxide anions that could dismute into H2O2 (24Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2492) Google Scholar). Here, using particulate fractions from DUOX2-transfected nonthyroid cells, we provide data showing for the first time that the partially N-glycosylated form of the Duox2 protein generates H2O2 via dismutation of the superoxide anion ( O2·¯) in a NADPH/Ca2+-dependent manner. Interestingly, we found that the level of H2O2 formation activity depended on the primary structure of the Duox proteins, since human Duox1 appeared to be less active than human Duox2, and porcine Duox2 was twice as active as its human counterpart. Tpo, found recently to be associated with Duoxs in human thyrocyte (25Wang D. De Deken X. Milenkovic M. Song Y. Pirson I. Dumont J.E. Miot F. J. Biol. Chem. 2005; 280: 3096-3103Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), had little effect on the H2O2-generating activity. On the other hand, p22phox, the 22-kDa subunit associated with the glycosylated 91-kDa subunit (gp91phox/Nox2) of the leukocyte NADPH oxidase and recently found interacting with Nox1 (26Takeya R. Ueno N. Kami K. Taura M. Kohjima M. Izaki T. Nunoi H. Sumimoto H. J. Biol. Chem. 2003; 278: 25234-25246Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 27Ambasta R.K. Kumar P. Griendling K.K. Schmidt H.H. Busse R. Brandes R.P. J. Biol. Chem. 2004; 279: 45935-45941Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar) and Nox4 (27Ambasta R.K. Kumar P. Griendling K.K. Schmidt H.H. Busse R. Brandes R.P. J. Biol. Chem. 2004; 279: 45935-45941Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar), had no effect. These results highlight the key impact of the maturation of Duox on H2O2-generating activity. Isolation of Pig Thyroid Follicles—Pig thyroid follicles were prepared by the method previously described (28Morand S. Chaaraoui M. Kaniewski J. Deme D. Ohayon R. Noel-Hudson M.S. Virion A. Dupuy C. Endocrinology. 2003; 144: 1241-1248Crossref PubMed Scopus (67) Google Scholar), except that 20 μm hemin was added to the culture medium. Cell Culture—CHO and HEK293 cells (Flp-In-293), purchased from Invitrogen, were cultured in Ham's F-12 medium (Invitrogen) and in Dulbecco's modified Eagle's medium (high glucose) (Invitrogen) respectively, supplemented with 10% fetal calf serum, 20 μm hemin (28Morand S. Chaaraoui M. Kaniewski J. Deme D. Ohayon R. Noel-Hudson M.S. Virion A. Dupuy C. Endocrinology. 2003; 144: 1241-1248Crossref PubMed Scopus (67) Google Scholar), 1% penicillin, 1% streptomycin, 1% Fungizone, and 100 μg/ml Zeocin (Invitrogen). The human NOX5b-transfected and control-transfected HEK293 cells (kind gift of K. H. Krause, Biology of Aging Laboratories, University of Geneva) (7Banfi B. Molnar G. Maturana A. Steger K. Hegedus B. Demaurex N. Krause K.H. J. Biol. Chem. 2001; 276: 37594-37601Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar) were cultured in the presence or absence of 400 μg/ml G418 instead of Zeocin. Stable and Transient Cell Transfection—The stable human DUOX1- and DUOX2-expressing cell lines were established using the Flp-In system (Invitrogen). The protocol accompanying the kit was used without modification. Human DUOX1 and DUOX2 cDNAs were subcloned in pcDNA5/FRT vector (Invitrogen) designed for use with the Flp-In system. When cotransfected with the pOG44 Flp recombinase expression plasmid into an Flp-In-293 cell line, the pcDNA5/FRT vector containing DUOX1 or DUOX2 cDNA was integrated in an Flp recombinase-dependent manner into the genome. Stable cell lines were established by exposure to 100 μg/ml hygromycin. In transient cell transfection experiments, HEK293 cells reaching 50–60% confluence were transfected using the calcium phosphate precipitation procedure and the Invitrogen protocol. HEK293 cells were incubated overnight with calcium phosphate in the presence of 50 μgof pcDNA3.1-human DUOX2 or pcDNA3.1-porcine DUOX2. 24 h after transfection, 20 μm hemin was added to the medium. After 48 h, the cells were harvested. CHO cells were transiently transfected, using the FuGENE (Roche Applied Science) transfection reagent instead of the calcium phosphate procedure. The pcDNA3.1-GFP vector was purchased from Invitrogen. Full-length 3060-kilobase pair human TPO cDNA (kindly provided by B. Rapoport, Cedars-Sinai Research Institute, Los Angeles, CA) was cloned into the vector pcDNA3.1 as previously described (29Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google Scholar). The human p22phox cDNA (kindly provided by M. Dinauer, Cancer Research Institute R4, Indiana University School of Medicine, Indianapolis, IN) was cloned into the EcoRI sites of the vector pcDNA3.1 (Invitrogen). The Flp-In-293 cells stably expressing the human Duox2 were incubated overnight with calcium phosphate in the presence of 50 μg of pcDNA3.1-h TPO or pcDNA3.1-hp22phox or both. 24 h after transfection, 20 μm hemin was added to the medium. After 48 h, the cells were harvested. Preparation of the Cellular Particulate Fraction—Cells were washed with PBS and scraped into the same solution supplemented with a mixture of protease inhibitors (5 μg/ml aprotinin, 5 μg/ml leupeptin, 1 μg/ml pepstatin, 157 μg/ml benzamidine). After centrifuging at 200 × g for 10 min at 4 °C, the cell pellet was homogenized using a motor-driven Teflon pestle homogenizer in 2 ml of 50 mm sodium phosphate buffer containing 0.25 m sucrose, 0.1 mm dithiothreitol, 1 mm EGTA (pH 7.2), and the mixture of protease inhibitors. After centrifuging at 200,000 × g for 30 min, the pellet was resuspended in 0.5 ml of 50 mm sodium phosphate buffer (pH 7.2) containing 0.25 m sucrose, 1 mm MgCl2, and the mixture of protease inhibitors. The same protocol was used to obtain follicular particulate fractions, except that follicles from two Petri dishes (about 2 × 107 cells) were collected by centrifuging at 200 × g for 7 min before being resuspended in 2 ml of 50 mm sodium phosphate buffer containing 0.25 m sucrose, 0.1 mm dithiothreitol, 1 mm EGTA (pH 7.2), and the mixture of protease inhibitors. Preparation of Thyroid Plasma Membranes—The preparation of plasma membranes was as previously described (30Dupuy C. Kaniewski J. Deme D. Pommier J. Virion A. Eur. J. Biochem. 1989; 185: 597-603Crossref PubMed Scopus (49) Google Scholar). Fresh porcine thyroid glands (150 g) were obtained immediately after slaughter and transferred to the laboratory on ice. Fat and connective tissues were removed. The glands were cut into small pieces and homogenized, first with a Sorvall Omni mixer (30 s, 11,000 rpm) in 150 ml of 50 mm sodium phosphate, pH 7.2, containing 1 mm EGTA, 2 mm MgCl2, 1 mm dithiothreitol, and 16 mg/ml phenylmethylsulfonyl fluoride (buffer A) and then with an Ultra-Turrax (45 s, 60% Vmax/150 ml) after dilution to 600 ml with buffer A. The homogenate was filtered through six layers of cheesecloth and centrifuged at 4200 × g for 15 min. The pellet was resuspended in 50 mm sodium phosphate, pH 7.2, containing 2 mm MgCl2 and 0.25 m sucrose (buffer B) and washed twice by centrifugation at 4200 × g for 15 min. The final pellet was resuspended in buffer B and mixed with 47 ml of a stock Percoll solution (1 volume of 2.5 m sucrose plus 9 volumes of Percoll); the volume was adjusted to 222 ml with buffer B. After centrifugation in a Sorvall SS34 rotor at 13,000 rpm (20,000 × g) for 30 min, the turbid layer sedimenting at about 1 cm from the top of the gradient was collected and washed with 0.1 m KCl in buffer B by centrifugation at 5000 × g for 15 min. The final pellet was resuspended in 15 ml of buffer B (membrane preparation) quick-frozen in liquid nitrogen, and stored at –80 °C. Western Blot Analysis—SDS-PAGE and immunoblot analyses were performed as described previously (31Caillou B. Dupuy C. Lacroix L. Nocera M. Talbot M. Ohayon R. Deme D. Bidart J.M. Schlumberger M. Virion A. J. Clin. Endocrinol. Metab. 2001; 86: 3351-3358PubMed Google Scholar). Depending on the experiments, an anti-Duox antibody raised against a 14-amino acid peptide encompassing the Leu410–Thr423 portion of human Duox2, which is exactly conserved in porcine Duox2, or raised against the first intracellular domain Glu636–Arg1039 of human Duox2, were used to probe the immunoblots. Immune complexes were detected with an alkaline phosphatase-coupled, anti-rabbit IgG antibody (Promega). Human Duox1 was immunodetected using anti-Duox1 antibody kindly given by F. Miot, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles, Brussels) (14De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). Human thyroperoxidase was immunodetected with a polyclonal rabbit anti-porcine Tpo antibody (dilution 1:2000) obtained as previously described (32Virion A. Courtin F. Deme D. Michot J.L. Kaniewski J. Pommier J. Arch. Biochem. Biophys. 1985; 242: 41-47Crossref PubMed Scopus (28) Google Scholar). The anti-GFP antibody was purchased from Invitrogen. The anti-p22phox antibody was purchased from Santa Cruz (Tebu-France). Determination of the Protein Content—The protein content was determined using the Bradford method (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Measurement of the H2O2 Production of the Particulate Fractions— The NADPH-dependent H2O2-generating activity of the particles was determined as previously described (34Leseney A.M. Deme D. Legue O. Ohayon R. Chanson P. Sales J.P. Pires de Carvalho D. Dupuy C. Virion A. Biochimie (Paris). 1999; 81: 373-380Crossref PubMed Scopus (56) Google Scholar). Samples of cellular particulate fraction were incubated at 30 °C in 0.5 ml of 200 mm sodium phosphate, pH 7.4, containing 1 mm NaN3, 1 mm EGTA, 1.5 mm CaCl2, and 0.1 μm FAD. The reaction was started by adding 100 μm NADPH. 100-μl samples were collected at times up to 30 min and mixed with 10 μl of 3 n HCl to stop the reaction by destroying the remaining NAD(P)H. The Ca2+ dependence of the H2O2 formation was determined by assaying parallel samples without Ca2+ in the presence of 1 mm EGTA. The amount of H2O2 in each sample was measured in 200 mm, pH 7.8, phosphate buffer by monitoring the decrease in 0.25 μm scopoletin fluorescence in the presence of horseradish peroxidase in a Hitachi F-2000 spectrofluorimeter. In some experiments, the fluorescence (excitation 320 nm; emission, 420 nm) was measured in a Fluostar spectrofluorimeter (BMG LabTech) after adding to each aliquot 90 μlof200 mm sodium phosphate buffer, pH 7.4, containing 500 μm homovanilic acid and 3 μg/ml horseradish peroxidase. 10 μlof3 n NaOH was added to neutralize the medium after incubating for 15 min with HCl. The activity was measured using a standard curve obtained by incubating increasing amounts of H2O2 (0–10 μm) in the same solution. Both methods measured the H2O2 produced either directly or through O2·¯ dismutation and gave the same results. All superoxide anions that could have been generated during incubation spontaneously disproportionated to form H2O2 before horseradish peroxidase and its substrate (scopoletin or homovanilic acid) were added. Measurement of H2O2 Release from Open Follicles and Cells—The H2O2 released by porcine cells and HEK293 transiently transfected cells in the medium was determined by measuring the oxidation of homovanilic acid into its fluorescent derivative in the presence of horseradish peroxidase (28Morand S. Chaaraoui M. Kaniewski J. Deme D. Ohayon R. Noel-Hudson M.S. Virion A. Dupuy C. Endocrinology. 2003; 144: 1241-1248Crossref PubMed Scopus (67) Google Scholar). Cells and follicles from six wells were washed twice with Hepes-buffered Earle's solution and incubated for 45 min at 37 °C in 0.8 ml of Hepes-buffered Earle's solution containing 0.44 mm homovanilic acid, 10 μg/ml (2 units/ml) horseradish peroxidase, and 1 mm sodium azide, with or without 5 μm ionomycin. At the end of the incubation, 200 μl of medium from each well was collected, and the fluorescence level was measured (320- and 420-nm excitation and emission, respectively). The H2O2 release was determined using a standard curve obtained by incubating increasing amounts of H2O2 (0–25 μm) in the same solution. Generation of Duox2 Constructs—Eight cysteine residues are present in the N-terminal domain of the pig Duox2, and their roles have been analyzed in a previous study (35Morand S. Agnandji D. Noel-Hudson M.S. Nicolas V. Buisson S. Macon-Lemaitre L. Gnidehou S. Kaniewski J. Ohayon R. Virion A. Dupuy C. J. Biol. Chem. 2004; 279: 30244-30251Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). These cysteine residues were replaced by glycines by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit from Stratagene. Deletion of the extracellular domain of Duox2 was also created using the same kit with the pig DUOX2-pcDNA3.1 vector as a template. The Δ151–555 deletion was created using 5′-GACGTGGTGCTGCCCTTCCAGAGGTCCAGTGCCCTGCAGCCCAATGTC-3′ and 5′-GACATTGGGCTGCAGGGCACTGGACCTCTGGAAGGGCAGCACCACGTC-3′ as mutagenic oligonucleotides. The constructs were confirmed by sequencing. Measurement of Superoxide Formation by Spin Trapping and Electron Paramagnetic Resonance Spectroscopy (EPR)—The spin trap 5-tert-butyloxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO) was synthesized using the procedure described by Zhao et al. (36Zhao H. Joseph J. Zhang H. Karoui H. Kalyanaraman B. Free Radic. Biol. Med. 2001; 31: 599-606Crossref PubMed Scopus (331) Google Scholar). A typical incubation mixture contained 0.1 mm NADPH, 1 mm NaN3, 1 mm EDTA, 1.5 mm CaCl2, 0.1 μm FAD, and 50 mm BMPO in 200 mm sodium phosphate, pH 7.4. Proteins from particulate fractions of HEK293 cells stably transfected with human NOX5b (25 μg) or from particulate fractions of HEK293 cells transiently transfected with porcine Duox2 (370 μg) were mixed with the previous mixture and incubated for 30 min at 37 °C (total incubation volume 700 μl). The reaction mixture was quenched by freezing in liquid nitrogen and thawed and centrifuged for 5 min at 10,000 rpm at 4 °C just before EPR analysis. The supernatant was rapidly transferred into the Aqua-X sample cell fitted in an shq001 cavity (Bruker, Wissembourg, France), and data accumulation was started immediately. To analyze superoxide production in the thyroid cell, pig thyroid follicles were used for the EPR experiment. Open follicles (∼2 × 106 cells) were incubated for 30 min at 37 °C in 0.8 ml of Hepes-buffered Earle's solution (pH 7.2) containing 1 mm sodium azide, 1 mm CaCl2, 0.1 mm EDTA, 1 μm ionomycin, and 50 mm BMPO. The reaction mixture was analyzed as indicated above. All measurements were carried out at 20 °C in a Bruker EPR Elexsys 500 spectrometer operating at X-band frequency (9.82 GHz). The following instrument settings were used: field modulation frequency, 100 kHz; field modulation amplitude, 0.2 milliteslas; time constant, 0.081 s; microwave power, 5.1 milliwatts; scan time, 41.9 s; averaged scans, 9. BMPO-OOH and BMPO-OH spectra were identified by comparison with incubations performed in the presence of xanthine/xanthine oxidase (for BMPO-OOH) as previously described (36Zhao H. Joseph J. Zhang H. Karoui H. Kalyanaraman B. Free Radic. Biol. Med. 2001; 31: 599-606Crossref PubMed Scopus (331) Google Scholar). As expected, the BMPO-OOH spin adduct spontaneously decomposed to the BMPO-OH spin adduct with a half-life of about 15 min (36Zhao H. Joseph J. Zhang H. Karoui H. Kalyanaraman B. Free Radic. Biol. Med. 2001; 31: 599-606Crossref PubMed Scopus (331) Google Scholar), and its formation was completely abolished in the presence of SOD. The relative amounts of BMPO-OOH and BMPO-OH adducts observed in the incubation mixtures were estimated by Xsophe software simulations (Bruker). The second peaks of the BMPO-OOH and BMPO-OH adducts are superimposed (maximum, 348.8 milliteslas; minimum, 349.2 milliteslas), and the amplitude of this peak was used to quantify the amounts of superoxide generated in our incubations. The term “BMPO-OOH spin adduct” will be used instead of “BMPO-OOH and BMPO-OH spin adducts” throughout this work for brevity's sake. NADPH/Ca2+-dependent H2O2 Generation Catalyzed by Duox2 and Duox1—Using the Flp-In system, two stable, isogenic HEK293 cell lines were generated expressing human Duox1 and Duox2, respectively. This system has the advantage of making it possible to integrate DUOX1 and DUOX2 into the cells at the same specific genomic location, thus generating two cell lines with comparable expression of DUOX1 and DUOX2. Two antibodies directed against the median domain of each of these proteins were used to evaluate their respective levels of expression by Western blot analysis (Fig. 1A). The antibodies revealed Duox1/2 as two bands with apparent molecular masses of 150 and 165 kDa, corresponding to the nonglycosylated and partially glycosylated form, respectively (21De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (157) Google Scholar, 28Morand S. Chaaraoui M. Kaniewski J. Deme D. Ohayon R. Noel-Hudson M.S. Virion A. Dupuy C. Endocrinology. 2003; 144: 1241-1248Crossref PubMed Scopus (67) Google Scholar). The highly glycosylated 175-kDa form of Duox, thought to be the mature protein involved in the active NADPH oxidase at the plasma membrane (21De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (157) Google Scholar, 28Morand S. Chaaraoui M. Kaniewski J. Deme D. Ohayon R. Noel-Hudson M.S. Virion A. Dupuy C. Endocrinology. 2003; 144: 1241-1248Crossref PubMed Scopus (67) Google Scholar)," @default.
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