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• W2057360928 abstract "Phagocyte NADPH oxidase generates O2·¯ for defense mechanisms and cellular signaling. Myeloid-related proteins MRP8 and MRP14 of the S100 family are EF-hand calcium-binding proteins. MRP8 and MRP14 were co-isolated from neutrophils on an anti-p47phox matrix with oxidase cytosolic factors and identified by mass spectrometry. MRP8 and MRP14 are absent from Epstein-Barr virus-immortalized B lymphocytes, and, coincidentally, these cells display weak oxidase activity compared with neutrophils. MRP8/MRP14 that was purified from neutrophils enhanced oxidase turnover of B cells in vitro, suggesting that MRP8/MRP14 is involved in the activation process. This was confirmed ex vivo by co-transfection of Epstein-Barr virus-transformed B lymphocytes with genes encoding MRP8 and MRP14. In a semi-recombinant cell-free assay, recombinant MRP8/MRP14 increased the affinity of p67phox for cytochrome b 558 synergistically with p47phox. Moreover, MRP8/MRP14 initiated oxidase activation on its own, through a calcium-dependent specific interaction with cytochrome b 558 as shown by atomic force microscopy and a structure-function relationship investigation. The data suggest that the change of conformation in cytochrome b 558, which initiates the electron transfer, can be mediated by effectors other than oxidase cytosolic factors p67phox and p47phox. Moreover, MRP8/MRP14 dimer behaves as a positive mediator of phagocyte NADPH oxidase regulation. Phagocyte NADPH oxidase generates O2·¯ for defense mechanisms and cellular signaling. Myeloid-related proteins MRP8 and MRP14 of the S100 family are EF-hand calcium-binding proteins. MRP8 and MRP14 were co-isolated from neutrophils on an anti-p47phox matrix with oxidase cytosolic factors and identified by mass spectrometry. MRP8 and MRP14 are absent from Epstein-Barr virus-immortalized B lymphocytes, and, coincidentally, these cells display weak oxidase activity compared with neutrophils. MRP8/MRP14 that was purified from neutrophils enhanced oxidase turnover of B cells in vitro, suggesting that MRP8/MRP14 is involved in the activation process. This was confirmed ex vivo by co-transfection of Epstein-Barr virus-transformed B lymphocytes with genes encoding MRP8 and MRP14. In a semi-recombinant cell-free assay, recombinant MRP8/MRP14 increased the affinity of p67phox for cytochrome b 558 synergistically with p47phox. Moreover, MRP8/MRP14 initiated oxidase activation on its own, through a calcium-dependent specific interaction with cytochrome b 558 as shown by atomic force microscopy and a structure-function relationship investigation. The data suggest that the change of conformation in cytochrome b 558, which initiates the electron transfer, can be mediated by effectors other than oxidase cytosolic factors p67phox and p47phox. Moreover, MRP8/MRP14 dimer behaves as a positive mediator of phagocyte NADPH oxidase regulation. The phagocyte respiratory burst depends on the NADPH reduction of molecular oxygen and generation of superoxide anion O2·¯ upon activation of the cell with soluble (chemotactic factors) or particulate (bacteria or fungi) stimuli (1Morel F. Doussière J. Vignais P.V. Eur. J. Biochem. 1991; 201: 523-546Crossref PubMed Scopus (526) Google Scholar). The O2·¯ -generating NADPH oxidase (EC 1.6.99.6) is a heterogeneous complex compartmentalized in resting cells, between plasma membrane, cytosol, and the cytoskeleton, which assembles at the plasma membrane level once activated. Cytochrome b 558 is the membrane redox core of the oxidase complex, which, after activation, transfers electrons from NADPH to molecular oxygen. It is constituted of gp91phox (the β-catalytic subunit) and p22phox (α-subunit), which is necessary in phagocytes to stabilize the 1/1 (α/β) heterodimer (2DeLeo F.R. Burritt J.B. Yu L. Jesaitis A.J. Dinauer M.C. Nauseef M. J. Biol. Chem. 2000; 275: 13986-13993Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Three soluble factors, p67phox, p47phox, and p40phox, participate in the activation process, as does the monomeric G protein Rac1/2. Another monomeric G protein, Rap1A, has been identified in the membrane and proposed as a possible regulation factor of the oxidase complex (3Maly F.-E. Quilliam L.A. Dorseuil O. Der C.J. Bokoch G.M. J. Biol. Chem. 1994; 269: 18743-18746Abstract Full Text PDF PubMed Google Scholar, 4Bokoch G.M. Trends Cell Biol. 1995; 5: 109-113Abstract Full Text PDF PubMed Scopus (109) Google Scholar). In phagocytes, O2·¯ and derivatives are synthesized in large amounts and used as bactericidal tools, but uncontrolled production is also injurious to tissues and triggers inflammatory reactions (5Lambeth J.D. J. Biochem. Mol. Biol. 2000; 33: 427-439Google Scholar, 6Vignais P.V. Cell. Mol. Life Sci. 2002; 59: 1428-1459Crossref PubMed Scopus (634) Google Scholar). Phagocyte NADPH oxidase activity is transitory; it is submitted to a strict up- and down-regulation process. Up to now, oxidase activation has been investigated, but the molecular support of down-regulation has not. In fact, the role of a membrane-associated GTPase-activating protein has been described and related to the deactivation of NADPH oxidase (7Geiszt M. Dagher M.C. Molnar G. Havasi A. Faure J. Paclet M.H. Morel F. Ligeti E. Biochem. J. 2001; 355: 851-858Crossref PubMed Scopus (29) Google Scholar, 8Moskwa P. Dagher M.C. Paclet M.H. Morel F. Ligeti E. Biochemistry. 2002; 41: 10710-10716Crossref PubMed Scopus (20) Google Scholar). Recent observations would support an allosteric regulation of NADPH oxidase activity where cytochrome b 558 is the catalytic subunit. In the oxidase assembly, p67phox is the limiting factor (9Freeman J.L. Lambeth J.D. J. Biol. Chem. 1996; 271: 22578-22582Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 10Koshkin V. Lotan O. Pick E. J. Biol. Chem. 1996; 271: 30326-30329Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 11Paclet M.H. Coleman A.W. Vergnaud S. Morel F. Biochemistry. 2000; 39: 9302-9310Crossref PubMed Scopus (69) Google Scholar). The binding of p67phox to cytochrome b 558 mediates the transition from an inactive to an active conformation of cytochrome b 558 (11Paclet M.H. Coleman A.W. Vergnaud S. Morel F. Biochemistry. 2000; 39: 9302-9310Crossref PubMed Scopus (69) Google Scholar). In the process, p47phox and Rac1/2 are introduced as positive effectors, whereas the p40phox function remains undetermined. In chronic granulomatous disease (CGD) 1The abbreviations used are: CGD, chronic granulomatous disease; AFM, atomic force microscopy; DTT, dithiothreitol; ECL, enhanced chemiluminescence; EBV, Epstein-Barr virus; FACS, fluorescence-activated cell sorting; FAD, flavin adenine dinucleotide; GTPγS, guanosine 5′-3-O-(thio)triphosphate; IPG, immobilized pH gradient; MALDI, matrix-assisted laser desorption/ionization; MRP, myeloid related protein; MRP8/MRP14, mixture (1/1) of MRP8 and MRP14; rMRP8/rMRP14, mixture (1/1) of recombinant MRP8 and recombinant MRP14; MS, mass spectrometry; PBS, phosphate-buffered saline; Phox, phagocyte oxidase; PLA2, phospholipase A2; PMA, phorbol myristate acetate; PMN, polymorphonuclear neutrophil; TOF, time-of-flight., there is no oxidase activity (12Morel F. Boulay F. Doussière J. Vignais P.V. Medecine/Sciences. 1992; 8: 912-920Crossref Scopus (12) Google Scholar). All the components of the phagocyte NADPH oxidase complex are expressed in Epstein-Barr virus-immortalized B lymphocytes. However, in these cells, the activity of stimulated oxidase is 100 times inferior to that of human neutrophils (13Morel F. Cohen Tanugi Cholley L. Brandolin G. Dianoux A.C. Martel C. Champelovier P. Seigneurin J.M. François P. Bost M. Vignais P.V. Biochim. Biophys. Acta. 1993; 1182: 101-109Crossref PubMed Scopus (22) Google Scholar). Both neutrophils and EBV-B cells are qualitatively similar in terms of the nature of their oxidase constituents, but they differ in the concentration and stoichiometry of the cytosolic factors compared with cytochrome b 558 (14Kobayashi S. Imajoh-Ohmi S. Kuribayashi F. Nunoi H. Nakamura M. Kanegasaki S. J. Biochem. (Tokyo). 1995; 117: 758-765Crossref PubMed Scopus (42) Google Scholar). We have recently demonstrated that the weak oxidase activity measured in intact EBV-B lymphocytes was not only a result of the low expression of cytochrome b 558, but also of a possible defect or constraint in the assembly process because of an unfavorable membrane environment (15Paclet M.H. Coleman A.W. Burritt J.B. Morel F. Eur. J. Biochem. 2001; 268: 5197-5208Crossref PubMed Scopus (31) Google Scholar). In fact, once cytochrome b 558 is extracted from the membrane of both cell types, the turnover of reconstituted NADPH oxidase is similar. It is also possible that such a low activity may result from down-regulation because regulatory factors normally present in phagocytes are absent in the B cells. Myeloid-related proteins, MRP8 and MRP14, are two proteins of the S100 family (S100A8 and S100A9, respectively) expressed by myeloid cells and some secretory epithelium (16Itou H. Yao M. Fujita I. Watanabe N. Suzuki M. Nishihira J. Tanaka I. J. Mol. Biol. 2002; 316: 265-276Crossref PubMed Scopus (84) Google Scholar). In myeloid cells, they represent ∼45% of the cytosolic proteins in neutrophils (17Donato R. Int. J. Biochem. Cell Biol. 2001; 33: 637-668Crossref PubMed Scopus (1337) Google Scholar) and 1% in monocytes. MRP8 and MRP14 are not expressed by resident macrophages, although they may be present in macrophages of inflammatory lesions (18Xu K. Geczy C.L. J. Immunol. 2000; 164: 4916-4923Crossref PubMed Scopus (102) Google Scholar). MRPs are known to be markers of inflammation. Their expression by infiltrating neutrophils was assumed to reflect activation stages (19Kerkhoff C. Klempt M. Kaever V. Sorg C. J. Biol. Chem. 1999; 274: 32672-32679Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). In inflammation, high levels of MRP8 and MRP14 were found in the extracellular medium of stimulated neutrophils (20Morel F. Dewald B. Berthier S. Zaoui P. Dianoux A.C. Vignais P.V. Baggiolini M. Biochim. Biophys. Acta. 1994; 1201: 373-380Crossref PubMed Scopus (19) Google Scholar) and these proteins could be markers of inflammatory diseases such as arthritis and ulcerative colitis (17Donato R. Int. J. Biochem. Cell Biol. 2001; 33: 637-668Crossref PubMed Scopus (1337) Google Scholar, 21Kumar R.K. Yang Z. Bilson S. Thliveris S. Cooke B.E. Geczy C.L. J. Leukocyte Biol. 2001; 70: 59-64PubMed Google Scholar). Whether the complex is released by dying cells or is a result of active secretion has not been clarified. The two proteins are deposited onto endothelium venules through a specific MRP14-heparan sulfate proteoglycan interaction (22Robinson M.J. Tessier P. Poulsom R. Hogg N. J. Biol. Chem. 2002; 277: 3658-3665Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). It has been postulated that MRP8 and MRP14 participate in neutrophil migration, phagocytosis, and activation (21Kumar R.K. Yang Z. Bilson S. Thliveris S. Cooke B.E. Geczy C.L. J. Leukocyte Biol. 2001; 70: 59-64PubMed Google Scholar). However, their specific function remains unclear. MRP8 and MRP14 are believed to be present as a 1/1 non-covalent heterodimer, the process of dimer formation being calcium-dependent. Four MRP14 isoforms have been identified, although this is not the case for MRP8 (23Hessian P.A. Fisher L. Eur. J. Biochem. 2001; 268: 353-363Crossref PubMed Scopus (38) Google Scholar). Several studies have reported that both proteins also associate with membrane and cytoskeleton in a calcium-dependent manner (24Roth J. Burwinkel F. van den Bos C. Goebeler M. Vollmer E. Sorg C. Blood. 1993; 82: 1875-1883Crossref PubMed Google Scholar). Recent studies have suggested that MRP8 and MRP14 play a role in potentiating activation of O2·¯ -generating oxidase in bovine neutrophils (25Doussière J. Bouzidi F. Vignais P.V. Biochem. Biophys. Res. Commun. 2001; 285: 1317-1320Crossref PubMed Scopus (25) Google Scholar). Doussière et al. (26Doussière J. Bouzidi F. Vignais P.V. Eur. J. Biochem. 2002; 269: 3246-3255Crossref PubMed Scopus (70) Google Scholar) also reported that MRP8/MRP14 interacts preferentially with the cytosolic factor p67phox, which translocates to plasma membrane upon stimulation. Moreover, various phosphorylated states of MRPs may discriminate subcellular compartmentation (27Guignard F. Mauel J. Markert M. Eur. J. Biochem. 1996; 241: 265-271Crossref PubMed Scopus (60) Google Scholar). Using a proteomics approach, we identified MRP8 and MRP14 associated with oxidase cytosolic factors in a complex isolated from neutrophil cytosol. In contrast, there was neither MRP8 nor MRP14 in the complex isolated from EBV-B cell cytosol. MRP8/MRP14 purified from human neutrophils and recombinant MRP8/MRP14 were shown to complement the cytosol of EBV-B cells and to restore a full oxidase activity, as measured in human neutrophils. These observations were confirmed ex vivo after MRP-encoding gene transfection. We demonstrated further that this effect was mediated through a specific interaction with cytochrome b 558 and a change of conformation that initiated oxidase activation. Materials—Chemical reagents used in this study were obtained from the following sources: Carbolink™ coupling gel (Pierce); l-α-phosphatidylcholine type II-S, PMA, carbenicillin, and Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) (Sigma); ECL Western blotting detection reagents, and FPLC monoQ anion exchange column (Amersham Biosciences, Uppsala, Sweden); immobilized linear pH gradient strips, IPG buffer, pH 3–10 (Bio-Rad); synthetic peptide corresponding to the C-terminal portion of p47phox (ADLILNRSSESTKRKLASAV) (Neosystem, Strasbourg, France), MRP8 (Calgranulin A C19), MRP14 (Calgranulin B C19), goat polyclonal antibody, and donkey anti-goat IgG antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); PIA (Difco); Geneticin 10131– 019 (G418) (Invitrogen Corp., Paisley, United Kingdom); trypsin, sequencing grade (Promega, Charbonnières, France). Rabbit polyclonal antibodies (anti-MRP8/MRP14) were from M. Markert (Central Laboratory of Clinical Chemistry, Lausanne, Switzerland). Mouse monoclonal antibodies 54.1 and 44.1 directed against gp91phox and p22phox, respectively, were prepared by J. B. Burritt and A. Jesaitis (Department of Microbiology, Montana State University, Bozeman, MT). The plasmids pRc-CMV-MRP8, pRc-CMV-MRP14, and pRc-CMV-GFP were kindly provided by H. Perron (BioMerieux, Marcy l'Etoile, France). Human recombinant MRP8 and MRP14 (28Kerkhoff C. Vogl T. Nacken W. Sopalla C. Sorg C. FEBS Lett. 1999; 460: 134-138Crossref PubMed Scopus (60) Google Scholar, 29Nacken W. Sopalla C. Propper C. Sorg C. Kerkhoff C. Eur. J. Biochem. 2000; 267: 560-565Crossref PubMed Scopus (29) Google Scholar) were a generous gift from Dr. C. Kerkhoff (Institute of Experimental Dermatology, University of Münster, Münster, Germany). The cDNA encoding p47phox cloned into the plasmid pGEX-2T was kindly provided by A.W. Segal and F. Wientjes (Department of Medicine, University College London, London, United Kingdom) (30Abo A. Boyhan A. West I. Thrasher A.J. Segal A.W. J. Biol. Chem. 1992; 267: 16767-16770Abstract Full Text PDF PubMed Google Scholar). Lymphoid Cell Lines and Neutrophils—Citrate-sterile venous blood was drawn from healthy patients after obtaining their informed consent. Neutrophils from buffy coats and B lymphocytes were isolated according to previously used methods (31Cohen Tanugi-Cholley 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). Lymphocytes were immortalized with the B95-8 strain of Epstein-Barr virus. The EBV-B lymphocyte cell lines were kept in culture using RPMI 1640 supplemented with 10% (v/v) fetal calf serum, 2 mm l-glutamine at 37 °C in 5% CO2 atmosphere. The cytosolic fractions from the 3 mm diisopropyl fluoro-phosphate-treated and purified neutrophils and EBV-B cells were prepared as described (32Batot G. Martel C. Capdeville N. Wientjes F. Morel F. Eur. J. Biochem. 1995; 234: 208-215Crossref PubMed Scopus (35) Google Scholar, 33Batot G. Paclet M.H. Doussière J. Vergnaud S. Martel C. Vignais P.V. Morel F. Biochim. Biophys. Acta. 1998; 1406: 188-202Crossref PubMed Scopus (39) Google Scholar). EBV-B lymphocytes from a p47phox-deficient CGD patient were kindly provided by M.-A. Pocidalo (INSERM U479, Hôpital Bichat, Paris, France). Recombinant Proteins—Full-length cDNAs encoding p67phox, p47phox, and Rac1 were expressed in Escherichia coli as a glutathione S-transferase fusion protein using pGEX-3X (p67phox) or pGEX-2T (p47phox and Rac1). Protein expression was induced with isopropyl-1-thio-β-d-galactopyranoside (0.2 mm at 20 °C for p67phox and p47phox, 0.1 mm at 37 °C for Rac1) for 3 h. Glutathione S-transferase fusion proteins were affinity-purified from isopropyl-1-thio-β-d-galactopyranoside-induced bacteria on glutathione-Sepharose (11Paclet M.H. Coleman A.W. Vergnaud S. Morel F. Biochemistry. 2000; 39: 9302-9310Crossref PubMed Scopus (69) Google Scholar). After washing in PBS, recombinant proteins were cleaved directly on the matrix using Xa factor (p67phox)or thrombin (p47phox and Rac1) in PBS. Recombinant proteins (p67phox, p47phox, and Rac1) were stored at –20 °C or used in cell-free assay. Isolation of the p47phox, p67phox, p40phox Cytosolic Activation Complex—Anti-p47phox immunoglobulins were immobilized onto a Carbolink™ coupling matrix. Cytosol from either EBV-B lymphocytes or neutrophils was used to affinity-purify the p47phox-, p67phox-, and p40phox-activating factors as a complex (33Batot G. Paclet M.H. Doussière J. Vergnaud S. Martel C. Vignais P.V. Morel F. Biochim. Biophys. Acta. 1998; 1406: 188-202Crossref PubMed Scopus (39) Google Scholar, 34Vergnaud S. Paclet M.H. El Benna J. Pocidalo M.A. Morel F. Eur. J. Biochem. 2000; 267: 1059-1067Crossref PubMed Scopus (59) Google Scholar). The cytosol (50 –100 mg, i.e. 2–5 × 109 cell eq) was preincubated first with an uncoupled Carbolink™ matrix and then with a Carbolink™ matrix coupled with nonspecific immunoglobulins. Unbound proteins were loaded on the anti-p47phox matrix previously equilibrated in PBS, with overnight recycling at 4 °C. After washing in PBS, bound proteins were eluted either with 0.1 m glycine, pH 3, or with 1 m NaCl and then 0.1 m glycine, pH 3, or with 2 mm competitor peptide (used to generate the anti-p47phox antibodies). Glycine eluates were immediately buffered with 1 m Tris-HCl, pH 9.5, dialyzed against PBS containing a mixture of protease inhibitors (10 μm N3α-p-tosyl-l-lysine chloromethyl ketone, 1.8 μm leupeptin, 1.5 μm pepstatin) and stored at –80 °C until further use. To verify the specificity of the recovered proteins, a control experiment was conducted with cytosol from p47phox-deficient EBV-B lymphocytes. MRP8 and MRP14 Purification—MRP8 and MRP14 were purified from the cytosol of unstimulated neutrophils as described (35Van den Bos C. Rammes A. Vogl T. Boynton R. Zaia J. Sorg C. Roth J. Protein Exp. Purif. 1998; 13: 313-318Crossref PubMed Scopus (46) Google Scholar). Briefly, neutrophil cytosol was submitted to 70% (w/v) (NH4)2SO4 precipitation. The 10,000 × g centrifugation supernatant was dialyzed against 50 mm Tris-HCl, pH 8.5, containing 1 mm DTT, 1 mm EDTA, and 1 mm EGTA, and fractionated through FPLC onto a monoQ anion exchange column. The bound MRP8 and MRP14 were eluted with 0.13 m NaCl, as shown on the elution chromatogram (Fig. 1A, left panel), and then dialyzed against PBS. The major elution peak was analyzed by SDS-PAGE (Fig. 1A, right panel, lane 1). This peak presented two peptide bands with an apparent molecular mass of 16 and 11 kDa, identified by Western blotting as MRP14 and MRP8, respectively (Fig. 1A, right panel, lane 2). This fraction was called MRP8/MRP14. The absence of contaminating p47phox, p67phox, and Rac in the purified fraction was checked by Western blot (Fig. 1B, lanes 1) versus a positive control performed on neutrophil cytosol (Fig. 1B, lanes 2). The purified proteins were stored at –80 °C until further use. Oxidase Activity Reconstitution in a Cell-free Assay with Purified Cytochrome b 558 —Cytochrome b 558 was purified from the plasma membranes of 1010 PMA-stimulated neutrophils and from 1010 EBV-B lymphocytes and relipidated with l-α-phosphatidylcholine II-S as reported (11Paclet M.H. Coleman A.W. Vergnaud S. Morel F. Biochemistry. 2000; 39: 9302-9310Crossref PubMed Scopus (69) Google Scholar). It was then quantified by reduced-minus-oxidized difference spectra using an absorption coefficient of 106 mm–1·cm–1 (15Paclet M.H. Coleman A.W. Burritt J.B. Morel F. Eur. J. Biochem. 2001; 268: 5197-5208Crossref PubMed Scopus (31) Google Scholar). Oxidase activity was reconstituted by incubating purified cytochrome b 558 (0.215 pmol) with cytosol isolated from neutrophils or from EBV-B lymphocytes (300 μg) in the presence of 10 μm FAD, 40 μm GTPγS,5mm MgCl2 and an optimal amount of arachidonic acid (80 –100 nmol) in a final volume of 100 μl of PBS. In some experiments the effect of MRP8/MRP14 purified from neutrophil cytosol was investigated. MRP8/MRP14 (0.75–1.3 μg) was preincubated in the presence of 500 nm CaCl2 at room temperature for 20 min. Then, reconstitution was performed by adding first calcium-loaded MRPs, then purified cytochrome b 558, and finally cytosol. In the semi-recombinant system, the activity was reconstituted with purified cytochrome b 558 from neutrophils (0.215 pmol = 2.15 nm) and recombinant proteins: rRac1 (100 nm), rp67phox (0 –300 nm), rp47phox (185 nm), and a mixture (1/1) of rMRP8 and rMRP14 (300 nm) (rMRP8/rMRP14) preincubated for 20 min with 500 nm calcium as previously done. The oxidase activity was estimated by measuring the reduction of ferricytochrome c in the absence or the presence of superoxide dismutase at 550 nm (Σ550 nm = 21.1 mm–1·cm–1) and expressed as turnover, mol of O2·¯ ·s–1·mol of heme b –1 (15Paclet M.H. Coleman A.W. Burritt J.B. Morel F. Eur. J. Biochem. 2001; 268: 5197-5208Crossref PubMed Scopus (31) Google Scholar). High Resolution Two-dimensional Gel Electrophoresis—The neutrophil cytosolic activating factors p47phox, p67phox, and p40phox, isolated as a complex on the Carbolink™ affinity matrix (75 μg), were submitted to high resolution two-dimensional gel electrophoresis as reported (36Garin J. Diez R. Kieffer S. Dermine J.F. Duclos S. Gagnon E. Sadoul R. Rondeau C. Desjardins M. J. Cell Biol. 2001; 152: 165-180Crossref PubMed Scopus (610) Google Scholar). Proteins (75 μg) were precipitated with 7% (v/v) perchloric acid, washed with cold acetone, and solubilized in 2% (v/v) IPG buffer 3–10 containing 8 m urea, 4% (p/v) chaps, 18 mm DTT, and traces of bromophenol blue. The strips were rehydrated with the 100,000 × g ultracentrifugation supernatant (1 h, 20 °C). Proteins were first separated according to their isoelectric point along linear pH gradient strips (pH 3–10). Electrophoresis was performed at 50 μA/strip at 20 °C. Then the strips were equilibrated, first in a solution containing 0.375 m Tris, 6 m urea, 2% (w/v) SDS, 20% (v/v) glycerol, and 130 mm DTT at pH 8, and then in a similar buffer in which DTT was replaced by 135 mm iodoacetamide. After 20 min of incubation at room temperature, the proteins were separated according to their molecular mass using standard SDS-PAGE, and silver-stained as previously described (37Santoni V. Kieffer S. Desclaux D. Masson F. Rabilloud T. Electrophoresis. 2000; 21: 3329-3344Crossref PubMed Scopus (213) Google Scholar), or processed for mass spectrometry (MS) analysis. Trypsin Digestion—Protein bands of interest were excised from a Coomassie Blue-stained 11% SDS-PAGE (38Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and washed with 25 mm NH4HCO3, pH 8.0, for 10 min and then with 50% (v/v) acetonitrile in 25 mm NH4HCO3, pH 8.0, for 10 min at room temperature. This step was repeated three times, and finally the sample was washed in ultrapure water and completely vacuum dried. “In-gel” tryptic digestion was performed for 4 h at 37 °C in 10 –20 μl of 25 mm NH4HCO3, pH 8.0, with 0.3– 0.5 μg of trypsin per sample, depending on the gel volume and protein amount. Peptide Mass Fingerprinting by Matrix-assisted Laser Desorption/Ionization Time-of-flight (MALDI-TOF) Mass Spectrometry—Mass spectra of the tryptic digest were acquired on a Biflex (Bruker Daltonik, Bremen, Germany) MALDI-TOF mass spectrometer equipped with gridless delayed extraction. The tryptic digests (0.5 μl) were deposited onto the target disk on a thin dry layer of matrix (mixture 4:3 (v/v) of a saturated solution of α-cyano-4-hydroxy-trans-cinnamic acid in acetone, and a solution of nitrocellulose (10 mg of nitrocellulose in 1 ml of 50% (v/v) isopropanol and 50% (v/v) acetone). Samples had been washed with 5 μl of 0.1% (v/v) trifluoroacetic acid before drying, as previously described by Garin (36Garin J. Diez R. Kieffer S. Dermine J.F. Duclos S. Gagnon E. Sadoul R. Rondeau C. Desjardins M. J. Cell Biol. 2001; 152: 165-180Crossref PubMed Scopus (610) Google Scholar). The MALDI spectra were analyzed by comparing a list of mass-to-charge ratios (peptide mass fingerprint) acquired for each digested protein to available data bases at prospector.ucsf.edu/ucsfhtml4.0u/msfit.htm. Proteins were identified by MS/MS analysis when no consistent hit was found. Peptide Sequencing by Tandem Mass Spectrometry (MS/MS)—The tryptic digest was extracted with 5% formic acid and then with pure acetonitrile. The digest solution and extracts were vacuum dried, dissolved in 10 μl of 10% formic acid, and desalted with ZipTip™ (Millipore). After elution with 5–10 μl of 50% acetonitrile combined with 0.1% formic acid, the peptide solution was introduced into a glass capillary (MDS Protana) for nanoelectrospray ionization. Tandem mass spectrometry experiments were carried out on a Q-TOF hybrid mass spectrometer (Micromass) to obtain amino acid sequence information. MS/MS sequence information was used for data base searching using the MS-Pattern programs (prospector.ucsf.edu/ucsfhtml4.0u/mspattern.htm) or Peptide Search (www.narrador.embl-heidelberg.de/). Atomic Force Microscopy (AFM) Experimentation—For AFM experiments, the medium contained cytochrome b 558 (0.2 pmol) purified from neutrophils or from EBV-B lymphocytes and incorporated into l-α-phosphatidylcholine liposomes, 40 μm GTPγS, 5 mm MgCl2, and 10 μm FAD (11Paclet M.H. Coleman A.W. Vergnaud S. Morel F. Biochemistry. 2000; 39: 9302-9310Crossref PubMed Scopus (69) Google Scholar). Aliquot fractions (10 μl) were collected from the mixture before and after incubation with 100 nmol of arachidonic acid at room temperature, deposited on a mica surface, and allowed to adhere for 1 min. After three washings with 200 μl of distilled water and overnight desiccation, samples were observed using AFM. In some experiments, the medium also contained purified MRP8/MRP14 (0.75 μg), preloaded or not with Ca2+ (as described for oxidase activity reconstitution) or cytosol (from neutrophils or EBV-B lymphocytes; 300 μg). The AFM measurements were carried out in air at room temperature using a ThermoMicroscopes Explorer AFM in the low amplitude true non-contact mode, as previously described (11Paclet M.H. Coleman A.W. Vergnaud S. Morel F. Biochemistry. 2000; 39: 9302-9310Crossref PubMed Scopus (69) Google Scholar). Liposome height was determined on x images (where x represents the number of liposome images acquired for the same preparation, x > 3) of three different experiments using the ThermoMicroscopes SPMLab software after calculation of the distance between two selected points. Transfection of EBV-B Lymphocytes—Electroporation was used for transfection of pRc-CMV-MRP8, pRc-CMV-MRP14, and pRc-CMV-GFP in EBV-B lymphocytes. Briefly, EBV-B lymphocytes were harvested, washed three times, and suspended at 107 cells/300 μl in RPMI 1640 medium supplemented with 2 mm l-glutamine at room temperature. Cells were then mixed with 50 μg of plasmid DNA and electroporated once in a 4-mm gap electroporation cuvette at 220 V for 10 ms (BTX ECM 830™, Electro Square Porator). Then they were immediately diluted in 6 ml of complete medium containing 10% (v/v) fetal calf serum. FACS analysis of transfected cells (FACScalibur, Becton Dickinson) showed a 14% transfection efficiency 48 h after transfection. Selection of transfected cells began at 48 h after transfection by cultivating cells in the presence of Geneticin (G418) (500 μg/ml). Superoxide production of transfected cells was measured by chemiluminescence. Measurement of NADPH Oxidase Activity in EBV-B Lymphocytes Using Chemiluminescence—Lymphocytes (5 × 105 to 2 × 106) suspended in 50 μl of PBS were added to 200 μl of PBS containing 0.9 mm CaCl2, 0.5 mm MgCl2, 20 mm glucose, 20 μm Luminol, and 10 units/ml horseradish peroxidase. Superoxide production was measured by chemiluminescence after adding 10 μl of a 2 μg/ml PMA solution (39De Mendez I. Garrett M.C. Adams A.G. Leto T.L. J. Biol. Chem. 1994; 269: 16326-16332Abstract Full Text PDF PubMed Google Scholar). Photon emission was recorded at 37 °C for 1 h with a Luminoscan (Labsystem, Pontoise, France). Polyclonal Antibodies against Cytosolic Factors—Polypeptides corresponding to the C-terminal region of p40phox (residues 325–339) and the C-terminal regions of p47phox (residues 371–390) and p67phox (residues 511–526) were synthesized by Neosystem (Strasbourg, France). Anti-sera were used for Ig purification onto a 1-ml protein A-Sepharose CL-4B matrix (34Vergnaud S. Paclet M.H. El Benna J. Pocidalo M.A. Morel F. Eur. J. Biochem. 2000; 267: 1059-1067Crossref PubMed Scopus (59) Google Scholar). SDS-PAGE and Western Blotting—The proteins were fractionated by 10 or 11% SDS-PAGE (" @default.
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• W2057360928 cites W1491943205 @default.
• W2057360928 cites W1501041796 @default.
• W2057360928 cites W1525541654 @default.
• W2057360928 cites W1541051315 @default.
• W2057360928 cites W1556381278 @default.
• W2057360928 cites W1562828870 @default.
• W2057360928 cites W1579785356 @default.
• W2057360928 cites W1587330841 @default.
• W2057360928 cites W1599764734 @default.
• W2057360928 cites W1606357879 @default.
• W2057360928 cites W1749116795 @default.
• W2057360928 cites W1826136818 @default.
• W2057360928 cites W1893323289 @default.
• W2057360928 cites W1964175388 @default.
• W2057360928 cites W1964498325 @default.
• W2057360928 cites W1965669621 @default.
• W2057360928 cites W1975439918 @default.
• W2057360928 cites W1982971424 @default.
• W2057360928 cites W1990595453 @default.
• W2057360928 cites W1997664110 @default.
• W2057360928 cites W1998826717 @default.
• W2057360928 cites W2000588507 @default.
• W2057360928 cites W2000754365 @default.
• W2057360928 cites W2013606705 @default.
• W2057360928 cites W2020324576 @default.
• W2057360928 cites W2020854576 @default.
• W2057360928 cites W2023285881 @default.
• W2057360928 cites W2026346896 @default.
• W2057360928 cites W2026358430 @default.
• W2057360928 cites W2029987169 @default.
• W2057360928 cites W2036811549 @default.
• W2057360928 cites W2043716033 @default.
• W2057360928 cites W2047441819 @default.
• W2057360928 cites W2054159684 @default.
• W2057360928 cites W2067370488 @default.
• W2057360928 cites W2071673910 @default.
• W2057360928 cites W2082355045 @default.
• W2057360928 cites W2088164656 @default.
• W2057360928 cites W2089665911 @default.
• W2057360928 cites W2097544903 @default.
• W2057360928 cites W2100488606 @default.
• W2057360928 cites W2100837269 @default.
• W2057360928 cites W2101108802 @default.
• W2057360928 cites W2114512574 @default.
• W2057360928 cites W2123297305 @default.
• W2057360928 cites W2124922239 @default.
• W2057360928 cites W2135358834 @default.
• W2057360928 cites W2135577920 @default.
• W2057360928 cites W2145108764 @default.
• W2057360928 cites W2161491198 @default.
• W2057360928 cites W2207135385 @default.
• W2057360928 cites W2327302238 @default.
• W2057360928 cites W2370426177 @default.
• W2057360928 cites W2411494675 @default.
• W2057360928 cites W4293247451 @default.
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