Crucial Role of Two Potential Cytosolic Regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH Oxidase Activation
2005; Elsevier BV; Volume: 280; Issue: 15 Linguagem: Inglês
10.1074/jbc.m500226200
ISSN1083-351X
AutoresXing Jun Li, Didier Grünwald, Jacques Mathieu, Françoise Morel, Marie José Stasia,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoAssembly of cytosolic factors p67phox and p47phox with cytochrome b558 is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop 191TSSTKTIRRS200 and the C-terminal 484DESQANHFAVHHDEEKD500 of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47phox translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loopNox4-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the α-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47phox membrane translocation. This suggests that the α-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD. Assembly of cytosolic factors p67phox and p47phox with cytochrome b558 is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop 191TSSTKTIRRS200 and the C-terminal 484DESQANHFAVHHDEEKD500 of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47phox translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loopNox4-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the α-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47phox membrane translocation. This suggests that the α-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD. Baldridge and Gerard (1Baldridge C.W. Gerard R.W. Am. J. Physiol. 1933; 103: 235-236Crossref Google Scholar, 2Cross A.R. Segal A.W. Biochim. Biophys. Acta. 2004; 1657: 1-22Crossref PubMed Scopus (367) Google Scholar) discovered the respiratory burst in which dramatic oxygen consumption occurred in neutrophils during bacteria phagocytosis. Subsequent research showed that the NADPH oxidase complex of phagocytic cells is responsible for the innate host defense by producing superoxide anions (O2˙−) and reactive oxygen species to kill invading bacterials and fungi (3Sbarra A.J. Karnovsky M.L. J. Biol. Chem. 1959; 234: 1355-1362Abstract Full Text PDF PubMed Google Scholar, 4Mandell G.L. Infect. Immun. 1974; 9: 337-341Crossref PubMed Google Scholar, 5Babior B.M. Curnutte J.T. Kipnes R.S. J. Lab. Clin. Med. 1975; 85: 235-244PubMed Google Scholar, 6Babior B.M. Kipnes R.S. Curnutte J.T. J. Clin. Invest. 1973; 52: 741-744Crossref PubMed Scopus (2133) Google Scholar). The importance of this system is demonstrated by chronic granulomatous disease (CGD), 1The abbreviations used are: CGD, chronic granulomatous disease; PMA, phorbol 12-myristate 13-acetate; fMLP, formylmethionylleucylphenylalanine; DMF, dimethylformamide; GTPγS, 5′-3-O-(thio)triphosphate; BCS, broken cell system; WT, wild type; FACS, fluorescence-activated cell sorting; INT, iodonitrotetrazolium. characterized by severe and recurrent life-threatening infections, resulting in defective NADPH oxidase activity (7Dinauer M.C. Crit. Rev. Clin. Lab. Soc. 1993; 30: 329-369Crossref PubMed Scopus (111) Google Scholar, 8Babior B.M. Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar, 9Goldblatt D. Thrasher A.J. Clin. Exp. Immunol. 2000; 122: 1-9Crossref PubMed Scopus (105) Google Scholar, 10Heyworth P.G. Cross A.R. Curnutte J.T. Curr. Opin. Immunol. 2003; 15: 578-584Crossref PubMed Scopus (337) Google Scholar). However, recent evidence indicates that the killing activity of neutrophils is mediated through activation of proteases by K+ flux, and the large-conductance Ca2+-activated K+ channel is essential for innate immunity (2Cross A.R. Segal A.W. Biochim. Biophys. Acta. 2004; 1657: 1-22Crossref PubMed Scopus (367) Google Scholar, 11Reeves E.P. Lu H. Jacobs H.L. Messina C.G. Bolsover S. Gabella G. Potma E.O. Warley A. Roes J. Segal A.W. Nature. 2002; 416: 291-297Crossref PubMed Scopus (902) Google Scholar). NADPH oxidase is a multicomponent complex, composed of a heterodimeric transmembrane protein known as flavocytochrome b558, cytosolic proteins p47phox, p67phox, and p40phox, and two small GTPase, Rac2 and Rap1A. Cytochrome b558, the redox core of the respiratory burst oxidase, consists of a large glycoprotein gp91phox or Nox2 and a small protein p22phox (12Vignais P.V. Cell. Mol. Life. Sci. 2002; 59: 1428-1459Crossref PubMed Scopus (631) Google Scholar). Nox2 is the catalytic center that transfers the electrons from intracellular NADPH to extracellular O2. It contains two nonidentical hemes and consensus-binding sequences for FAD and NADPH (13Segal A.W. West I. Wientjes F. Nugent J.H. Chavan A.J. Haley B. Garcia R.C. Rosen H. Scrace G. Biochem. J. 1992; 284: 781-788Crossref PubMed Scopus (291) Google Scholar, 14Rotrosen D. Yeung C.L. Leto T.L. Malech H.L. Kwong C.H. Science. 1992; 256: 1459-1462Crossref PubMed Scopus (315) Google Scholar, 15Cross A.R. Rae J. Curnutte J.T. J. Biol. Chem. 1995; 270: 17075-17077Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 16Yu L. Quinn M.T. Cross A.R. Dinauer M.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7993-7998Crossref PubMed Scopus (178) Google Scholar). NADPH oxidase becomes activated and generates O2˙− after cytosolic factor assembly with cytochrome b558. In the resting state, p67phox connects p40phox and p47phox by its PB1 and C-terminal SH3 domains with the PC domain of p40phox and the proline-rich region of p47phox, respectively (17Lapouge K. Smith S.J. Groemping Y. Rittinger K. J. Biol. Chem. 2002; 277: 10121-10128Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Upon activation, a series of protein-protein interactions occurs. The phosphorylation of p47phox causes a conformational change of the protein, which leads to binding the membrane phosphoinositides and the proline-rich region of p22phox with its PX domains and SH3, respectively (18Leto T.L. Adams A.G. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (246) Google Scholar, 19DeLeo F.R. Ulman K.V. Davis A.R. Jutila K.L. Quinn M.T. J. Biol. Chem. 1996; 271: 17013-17020Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 20Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 21DeLeo F.R. Quinn M.T. J. Leukoc. Biol. 1996; 60: 677-691Crossref PubMed Scopus (457) Google Scholar, 22Ago T. Kuribayashi F. Hiroaki H. Takeya R. Ito T. Kohda D. Sumimoto H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4474-4479Crossref PubMed Scopus (181) Google Scholar). p22phox serves as a docking site for p47phox and an accessory for p67phox. p67phox was shown to be involved in both assembly and activation of the oxidase complex, whereas p47phox proceeded as a positive effector and increased the affinity of p67phox with cytochrome b558 (23Paclet M.H. Coleman A.W. Vergnaud S. Morel F. Biochemistry. 2000; 39: 9302-9310Crossref PubMed Scopus (69) Google Scholar, 24Vergnaud S. Paclet M.H. El Benna J. Pocidalo M.A. Morel F. Eur. J. Biochem. 2000; 267: 1059-1067Crossref PubMed Scopus (59) Google Scholar). Current evidence suggests that there is a direct interaction between p67phox and cytochrome b558, promoted by Rac1-p67phox binding (25Dang P.M. Cross A.R. Babior B.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3001-3005Crossref PubMed Scopus (96) Google Scholar, 26Dang P.M. Cross A.R. Quinn M.T. Babior B.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4262-4265Crossref PubMed Scopus (79) Google Scholar). However, the precise sequence of the p67phox and cytochrome b558 interaction has not been fully elucidated. In PMA-stimulated phagocytes from X-CGD patients, no translocation of p47phox, p67phox, and p40phox to the membranes occurs, indicating that gp91phox is absolutely required for the binding of cytosolic components to cytochrome b558 (27Dusi S. Nadalini K.A. Donini M. Zentilin L. Wientjes F.B. Roos D. Giacca M. Rossi F. J. Immunol. 1998; 161: 4968-4974PubMed Google Scholar). Sequence alignment of Nox2 with members of the ferredox-in-reductase family demonstrates the presence of six transmembrane α-helices in the N-terminal hydrophobic region, four cytosolic regions, the 1MGNWVAVNEGL11sequence, the B-loop 70PVCRNLLSFLRGSSACCSTRIRRQLDRNLTFHK102, the D-loop 191TSSTKTIRRS200, and a C-terminal region containing binding sites for FAD, NADPH, and cytosolic components of NADPH oxidase (12Vignais P.V. Cell. Mol. Life. Sci. 2002; 59: 1428-1459Crossref PubMed Scopus (631) Google Scholar). A peptide inhibitory study, random-sequence peptide phage analysis, and the mutagenesis approach suggested that complementary binding sites of gp91phox encompassing residues 451FEWFADLL458 and 559RGVHFIF565 presumably interact with cytosolic factors upon stimulation (19DeLeo F.R. Ulman K.V. Davis A.R. Jutila K.L. Quinn M.T. J. Biol. Chem. 1996; 271: 17013-17020Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 21DeLeo F.R. Quinn M.T. J. Leukoc. Biol. 1996; 60: 677-691Crossref PubMed Scopus (457) Google Scholar, 28Taylor W.R. Jones D.T. Segal A.W. Protein Sci. 1993; 2: 1675-1685Crossref PubMed Scopus (108) Google Scholar, 29Nakanishi A. Imajoh-Ohmi S. Fujinawa T. Kikuchi H. Kanegasaki S. J. Biol. Chem. 1992; 267: 19072-19074Abstract Full Text PDF PubMed Google Scholar, 30Leusen J.H. de Boer M. Bolscher B.G. Hilarius P.M. Weening R.S. Ochs H.D. Roos D. Verhoeven A.J. J. Clin. Invest. 1994; 93: 2120-2126Crossref PubMed Google Scholar, 31Thrasher A.J. Keep N.H. Wientjes F. Segal A.W. Biochim. Biophys. Acta. 1994; 1227: 1-24Crossref PubMed Scopus (212) Google Scholar, 32DeLeo F.R. Yu L. Burritt J.B. Loetterle L.R. Bond C.W. Jesaitis A.J. Quinn M.T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7110-7114Crossref PubMed Scopus (133) Google Scholar, 33Park M.Y. Imajoh-Ohmi S. Nunoi H. Kanegasaki S. Biochem. Biophys. Res. Commun. 1997; 234: 531-536Crossref PubMed Scopus (19) Google Scholar, 34Zhen L. Yu L. Dinauer M.C. J. Biol. Chem. 1998; 273: 6575-6581Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 35Biberstine-Kinkade K.J. Yu L. Dinauer M.C. J. Biol. Chem. 1999; 274: 10451-10457Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Furthermore, mutations in Nox2 found in X+CGD cases (C369R, G408E, E568K, and H303N/P304R) have been reported to abolish the oxidase assembly (36Leusen J.H. Meischl C. Eppink M.H. Hilarius P.M. de Boer M. Weening R.S. Ahlin A. Sanders L. Goldblatt D. Skopczynska H. Bernatowska E. Palmblad J. Verhoeven A.J. van Berkel W.J. Roos D. Blood. 2000; 95: 666-673PubMed Google Scholar, 37Stasia M.J. Lardy B. Maturana A. Rousseau P. Martel C. Bordigoni P. Demaurex N. Morel F. Biochim. Biophys. Acta. 2002; 1586: 316-330Crossref PubMed Scopus (31) Google Scholar, 38Bionda C. Li X.J. Bruggen R.V. Eppink M. Roos D. Morel F. Stasia M.J. Hum. Genet. 2004; 115: 418-427Crossref PubMed Scopus (26) Google Scholar, 39Nauseef W.M. Histochem. Cell Biol. 2004; 122: 277-291Crossref PubMed Scopus (322) Google Scholar, 40Yu L. Cross A.R. Zhen L. Dinauer M.C. Blood. 1999; 94: 2497-2504Crossref PubMed Google Scholar). In addition, Taylor et al. (28Taylor W.R. Jones D.T. Segal A.W. Protein Sci. 1993; 2: 1675-1685Crossref PubMed Scopus (108) Google Scholar) had predicted a three-dimensional structure of Nox2 using ferredoxin-NADP+ reductase as template and proposed that an α-helical loop 491FAVHHDEEKDVITG504 covers the cleft in which NADPH binds. During oxidase activation, access of NADPH into the binding site could potentially be regulated by interaction of this loop with cytosolic oxidase factors. However, there were conflicting results for the role of this potential α-helical loop. Leusen and co-workers found that cytosolic factors p47phox and p67phox did not translocate to the plasma membrane of phagocytes from an X+CGD case because of the D500G missense mutation located in this sequence of gp91phox. In addition, a peptide mimicking 491–504 residues of Nox2 inhibited the translocation of p47phox and p67phox to the membrane (30Leusen J.H. de Boer M. Bolscher B.G. Hilarius P.M. Weening R.S. Ochs H.D. Roos D. Verhoeven A.J. J. Clin. Invest. 1994; 93: 2120-2126Crossref PubMed Google Scholar). In contrast, in another X+CGD case consisting of a Δ488–497 gp91phox deletion in the α-helical loop, translocation of p47phox and p67phox to the plasma membrane occurred normally (40Yu L. Cross A.R. Zhen L. Dinauer M.C. Blood. 1999; 94: 2497-2504Crossref PubMed Google Scholar). Recently Dinauer and co-workers have studied the role of a second cytosolic region of Nox2, the B loop, on oxidase activity (35Biberstine-Kinkade K.J. Yu L. Dinauer M.C. J. Biol. Chem. 1999; 274: 10451-10457Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). They found that two Arg at positions 91 and 92 were essential for NADPH oxidase activity and assembly. However, the role of the second intracytosolic polybasic loop of Nox2, the D-loop 191TSSTKTIRRS200, has never been elucidated. Recently, large homologs of gp91phox, called Nox and Duox, have been identified. They are present in various tissues of nonphagocytic cells and can generate low amounts of O2˙−, suggested to be involved in cell signaling, host defense, hypoxia response, and also as proton transport (41Lambeth J.D. Curr. Opin. Hematol. 2002; 9: 11-17Crossref PubMed Scopus (232) Google Scholar). The family contains seven members in humans. Nox1, Nox3, and Nox4 resemble Nox2 in that they consist of six α-helix transmembrane regions in the N-terminal, with two intracytosolic loops (B and D-loops) and a cytosolic C-terminal tail containing the putative binding sites for FAD and NADPH (42Bokoch G.M. Knaus U.G. Trends Biochem. Sci. 2003; 28: 502-508Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). A recent study indicates that p51 and p41 (homolog of p67phox and p47phox, respectively) regulate Nox1 to generate O2˙− (43Banfi B. Clark R.A. Steger K. Krause K.H. J. Biol. Chem. 2003; 278: 3510-3513Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 44Geiszt M. Lekstrom K. Witta J. Leto T.L. J. Biol. Chem. 2003; 278: 20006-20012Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 45Takeya 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 (325) Google Scholar, 46Cheng G. Lambeth J.D. J. Biol. Chem. 2004; 279: 4737-4742Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 47Tucker K.A. Lilly M.B. Heck Jr., L. Rado T.A. Blood. 1987; 70: 372-378Crossref PubMed Google Scholar). This suggests that Nox proteins need some cytosolic components to be activated or regulated. Meanwhile, the molecular mechanisms of the regulation of Nox2 homologs has not been fully elucidated. The aim of this work was to investigate the role of two regions of Nox2, the intracytosolic D-loop 191TSSTKTIRRS200 and the C-terminal α-helical loop 484DESQANHFAVHHDEEKD500, on oxidase activity and assembly by means of the mutagenesis approach in the X-CGD PLB-985 cell model (37Stasia M.J. Lardy B. Maturana A. Rousseau P. Martel C. Bordigoni P. Demaurex N. Morel F. Biochim. Biophys. Acta. 2002; 1586: 316-330Crossref PubMed Scopus (31) Google Scholar). Results suggest that these two regions are critical for NADPH oxidase activity, especially the charged amino acids Arg195, Arg198, Arg199, Asp484, and Asp500. The D-loop of Nox2 is probably involved in electron transfer from FAD to oxygen independently of cytosolic factor translocation, whereas the C-terminal α-helical loop is crucial for the assembly of oxidase and electron transfer from NADPH to FAD. Chimeric Nox2 proteins containing the D-loop of Nox1/3/4 support NADPH oxidase activity, suggesting that this region should play a similar role on Nox analog activation. Finally, two “super-mutants” of the D-loop of Nox2, with a significant increase in oxidase activity, were obtained, suggesting that the R199Q and D-loopNox4 mutations optimize the NADPH oxidase activation process. Materials—Phorbol 12-myristate 13-acetate (PMA), dimethylformamide (DMF), diisopropylfluorophosphate, and horseradish peroxidase, cytochrome c (horse heart, type VI), and latex beads were obtained from Sigma. TaqDNA polymerase, ATP, GTPγS, and NADPH were from Roche Applied Science. Endofree Plasmid Maxi Kit was purchased from Qiagen. The Sephaglas kit and molecular weight markers for SDS-PAGE were from Amersham Biosciences. Nitrocellulose sheets for Western blotting were purchased from Bio-Rad. G418 was purchased from Invitrogen. Monoclonal antibodies 449 and 48 were kindly provided by Dr. D. Roos (Sanquin Research at CLB, Amsterdam, the Netherlands). Polyclonal antibody anti-p47phox (rabbit antiserum) was purchased from Upstate Biotechnology, Inc. (New York, NY). Monoclonal antibody specific for gp91phox, 7D5 was kindly provided by Dr. M. Nakamura (Nagasaki University, Nagasaki, Japan). Fetal bovine serum and RPMI 1640 were from Invitrogen. Site-directed Mutagenesis and Expression of Recombinant gp91phox in X-CGD PLB-985 Cell Line—Mutations were introduced into the wild-type (WT) Nox2 cDNA in pBluescript II KS(+) vector using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The sequence of WT and the mutated gp91phox cDNA were verified by dideoxynucleotide sequencing (Genome Express, Grenoble, France). The WT or mutant Nox2 cDNA were subcloned into the mammalian expression vector pEF-PGKneo as previously described (37Stasia M.J. Lardy B. Maturana A. Rousseau P. Martel C. Bordigoni P. Demaurex N. Morel F. Biochim. Biophys. Acta. 2002; 1586: 316-330Crossref PubMed Scopus (31) Google Scholar). The pEF-PGKneo constructs were electroporated into X-CGD PLB-985 cells in which the gp91phox gene was disrupted by gene targeting, resulting in the absence of Nox2 expression and NADPH oxidase activity as previously described (37Stasia M.J. Lardy B. Maturana A. Rousseau P. Martel C. Bordigoni P. Demaurex N. Morel F. Biochim. Biophys. Acta. 2002; 1586: 316-330Crossref PubMed Scopus (31) Google Scholar, 38Bionda C. Li X.J. Bruggen R.V. Eppink M. Roos D. Morel F. Stasia M.J. Hum. Genet. 2004; 115: 418-427Crossref PubMed Scopus (26) Google Scholar). Clones were selected by limiting dilution in 1.5 mg/ml G418. Cell Culture and Granulocyte Differentiation—WT, X-CGD, and transfected PLB-985 cells expressing WT or the mutant were maintained in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine at 37 °C in a 5% CO2 atmosphere. After selection, 0.5 mg/ml G418 was added to maintain the selection pressure. PLB-985 cells (5 × 105 cells/ml) were exposed to 0.5% DMF for 5–7 days, providing granulocytic differentiation as described previously (38Bionda C. Li X.J. Bruggen R.V. Eppink M. Roos D. Morel F. Stasia M.J. Hum. Genet. 2004; 115: 418-427Crossref PubMed Scopus (26) Google Scholar). Analysis of Nox2 Protein Expression—PLB-985 cells (5 × 105) were incubated with 10 μg/ml of mAb 7D5 directed against Nox2 or control monoclonal IgG1 (Immunotech, Marseille, France). Then the cells were incubated with phycoerythrin-labeled goat-F(ab′)2 fragment anti-mouse-Ig (Beckman Coulter, Marseille, France). Finally, flow cytometry analysis (FACS) was performed (FACSCalibur, BD Biosciences) (48Stasia M.J. Brion J.P. Boutonnat J. Morel F. J. Infect Dis. 2003; 188: 1593-1604Crossref PubMed Scopus (19) Google Scholar). Sorting of 7D5-positive PLB-985 cells was done on a FACSVantage Diva (BD Biosciences) instrument at a sheath pressure of 12 p.s.i. with a 70-μm nozzle. Cells were collected into phosphate-buffered saline supplemented with 0.5% bovine serum albumin and routinely contained >97% of 7D5-positive cells. Viability after sorting was >95%. After centrifugation of the cell suspension, the cell pellet was resuspended in culture medium. The expression of recombinant Nox2 was also examined by Western blot using monoclonal antibody 48 developed with the ECL detection system (Amersham Pharmacia Biotech) as described previously (49Verhoeven A.J. Bolscher G.J.M. Meerhof L. van Zwieten R. Keijer J. Weening R.S. Roos D. Blood. 1989; 73: 1686-1694Crossref PubMed Google Scholar). The WT or mutant Nox2 expression was further detected indirectly by cytochrome b558 differential spectral analysis. A molecular extinction coefficient of ∑426 nm = 106 mm–1 × cm–1 for the Soret band was used for calculations (50Batot G. Paclet M.H. Doussière J. Vergnaud S. Martel C. Vignais P.V. Morel F. Biochim. Biophys. Acta. 1998; 406: 188-202Crossref Scopus (39) Google Scholar). All experiments were done in triplicate. Cytosol and Membrane Fraction Preparation from Transfected PLB-985 Cell Lines—108 PLB-985 cells were treated with 3 mm diisopropylfluorophosphate for 15 min on ice and resuspended in 1 ml of phosphate-buffered saline containing 1 mm phenylmethylsulfonyl fluoride, 2 μm leupeptin, 2 μm pepstatin, and 10 μm 1-chloro-3-tosylamido-7-amino-2-heptanone. Cells were disrupted by sonication, and the homogenate was centrifuged at 1,000 × g for 15 min at 4 °C. The supernatant was withdrawn and centrifuged at 152,000 × g for 1 h at 4 °C. This high speed supernatant was referred to as the cytosol, and the pellet consisting of crude membranes was resuspended in the same buffer, as described before (51Berthier S. Paclet M.H. Lerouge S. Roux F. Vergnaud S. Coleman A.W. Morel F. J. Biol. Chem. 2003; 278: 25499-25508Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Measurement of NADPH Oxidase Activity in Intact Cells—H2O2 production was measured as described previously (38Bionda C. Li X.J. Bruggen R.V. Eppink M. Roos D. Morel F. Stasia M.J. Hum. Genet. 2004; 115: 418-427Crossref PubMed Scopus (26) Google Scholar). For each well in a 96-well plate, 5 × 105 granulocytic differentiated PLB-985 cells in phosphate-buffered saline with 0.9 mm Ca2+ and 0.5 mm Mg2+ was mixed with 20 mm glucose, 20 μm luminol, and 10 units/ml horseradish peroxidase. We added 80 ng/ml PMA to initiate the reaction. Luminescence was recorded every 30 s for a total of 90 min at 37 °C using a Luminoscan® luminometer (Labsystems, Helsinki, Finland). In some experiments, PMA was replaced by 4 × 10–7m fMLP. Detection of NADPH Oxidase Activity in a Broken Cell System— NADPH oxidase activity in vitro was measured in a homologous BCS using previously described protocols (52Cohen-Tanugi L. Morel F. Pilloud-Dagher M.C. Seigneurin J.M. François P. Bost M. Vignais P.V. Eur. J. Biochem. 1991; 202: 649-655Crossref PubMed Scopus (51) Google Scholar). Briefly, plasma membranes obtained from transgenic PLB-985 cells and cytosol of control human neutrophils were added to a reaction mixture containing 20 mm glucose, 20 μm GTPγS, 5 mm MgCl2, and arachidonic acid in a final volume of 100 μl. After incubation for 10 min at 25 °C, the oxidase activation was initiated in the presence of 100 μm cytochrome c and 150 μm NADPH. The specificity of the O2˙− production was checked by adding 50 μg/ml superoxide dismutase to stop the kinetic reduction. Iodonitrotetrazolium Reductase Activity—Diaphorase activity was measured under the same BCS assay conditions, except that the 100 μm cytochrome c was replaced by 50 μm INT (53Cross A.R. Yarchover J.L. Curnutte J.T. J. Biol. Chem. 1994; 269: 21448-21454Abstract Full Text PDF PubMed Google Scholar). Analysis of in Vivo p47phox Translocation—In vivo p47phox translocation was detected by confocal microscopy analysis according to (38Bionda C. Li X.J. Bruggen R.V. Eppink M. Roos D. Morel F. Stasia M.J. Hum. Genet. 2004; 115: 418-427Crossref PubMed Scopus (26) Google Scholar) with little modified. The 5 × 105 differentiated PLB-985 cells deposited on coverslips were activated with PMA-treated latex beads (3 μm in diameter) at 37 °C for 15 min. The cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100. After extensive washing, cells were incubated for 1 h at room temperature with the diluted polyclonal anti-p47phox (Upstate Biotechnology Inc.). After washing, cells were incubated in 5% bovine serum albumin/phosphate-buffered saline buffer containing a 2-μg/ml Alexa Fluor® 488 F(ab′)2 fragment of goat anti-rabbit IgG(H+L) (Molecular Probes, Eugene, OR) for 1 h. Finally, PLB-985 cells were stained with 3 μm TO-PRO 3 iodide for 10 min to visualize the nuclei. Cells were examined with a confocal laser scanning microscope and analyzed with Leica confocal software (Heidelberg, Germany). In some experiments, a 10 μg/ml mAb 7D5 and a 4-μg/ml Alexa Fluor® 546 F(ab′)2 fragment of goat anti-mouse IgG(H+L) (Molecular Probes) were added to the system as primary and secondary antibodies, respectively, to detect Nox2. Analysis of Translocation of p47phox in Vitro—In vitro p47phox translocation to the plasma membrane was measured using a classic protocol (37Stasia M.J. Lardy B. Maturana A. Rousseau P. Martel C. Bordigoni P. Demaurex N. Morel F. Biochim. Biophys. Acta. 2002; 1586: 316-330Crossref PubMed Scopus (31) Google Scholar). Briefly, membranes purified from PLB-985 cells (100 μg) were preincubated with human neutrophil cytosol (1,000 μg) for 2 min at 30 °C and activated with (+) or without 100 μm SDS and 20 μm GTPγS for 15 min at 30 °C. Membranes were collected between the 45 and 20% (w/v) sucrose layers after centrifugation (30,000 rpm × 1 h in an SW41 rotor (Beckman) at 18 °C) and analyzed using immunoblotting with anti-peptide polyclonal antibody directed against p47phox (24Vergnaud S. Paclet M.H. El Benna J. Pocidalo M.A. Morel F. Eur. J. Biochem. 2000; 267: 1059-1067Crossref PubMed Scopus (59) Google Scholar). Protein Determination—Protein content was estimated using the Bradford assay (54Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216412) Google Scholar) or the Pierce® method (55Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18645) Google Scholar). To further understand the role of the D-loop (191TSSTKTIRRS200) and the C-terminal (484DESQANHFAVHHDEEKD500) in Nox2 upon oxidase activation, homologous sequence analysis in the ferredoxin-NADP+ reductase family was done (Fig. 1). The D-loop is a polybasic region, containing Lys195, Arg198, and Arg199, which are conserved in the ferredoxin-NADP+ reductase family. In the C-terminal, the charged amino acids encompassing residues 484–500 are also highly conserved in this family, especially Asp484, His490, Asp 496, and Asp500. We postulated that charged amino acids within these two domains might be important for the active conformation of Nox2 and/or for further binding with cytosolic factors. Hence charged amino acid residues were replaced with neutral or reverse-charged residues by site-directed mutagenesis to alter the local electrostatic charge within these two regions (Fig. 2). To further elucidate the function of the D-loop in the Nox family, the D-loop of Nox2 was replaced with the same region of their homologs Nox1, Nox3, and Nox4. Twelve mutants of the D-loop (Fig. 2A) and eight of the α-helical loop in the C-terminal of Nox2 (Fig. 2B) were generated by directed mutagenesis and stably transfected in X-CGD PLB-985 cells, which lack endogenous Nox2 expression because of gene targeting (56Zhen L. King A.A. Xiao Y. Chanock S.J. Orkin S.H. Dinauer M.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9832-9836Crossref PubMed Scopus (183) Google Scholar). We also produced two mutants known to disturb cytosolic factor translocation to the plasma membrane, the D500G mutant, which had reproduced a human X+CGD case (30Leusen J.H. de Boer M. Bolscher B.G. Hilarius P.M. Weening R.S. Ochs H.D. Roos D. Verhoeven A.J. J. Clin. Invest. 1994; 93: 2120-2126Crossref PubMed Google Scholar), and the RR9192EE mutant of the B loop of Nox2 previously studied by Biberstine-Kindade et al. (35Biberstine-Kinkade K.J. Yu L. D
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