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Originally published online as doi:10.1189/jlb.0905541 on October 23, 2006

Published online before print October 23, 2006
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(Journal of Leukocyte Biology. 2007;81:238-249.)
© 2007 by Society for Leukocyte Biology

Leu505 of Nox2 is crucial for optimal p67phox-dependent activation of the flavocytochrome b558 during phagocytic NADPH oxidase assembly

Xing Jun Li*, Franck Fieschi{dagger}, Marie-Hélène Paclet*, Didier Grunwald{ddagger}, Yannick Campion*, Philippe Gaudin*, Françoise Morel* and Marie-José Stasia*,1

* Groupe de Recherche et d’Etude du Processus Inflammatoire, Université Joseph Fourier, Laboratoire d’Enzymologie, Centre Hospitalier Universitaire, Grenoble, France;
{dagger} Institut de Biologie Structurale, CEA/CNRS/Université Joseph Fourier, Laboratoire des Proteines Membranaires, Grenoble, France; and
{ddagger} Département Réponse et Dynamique Cellulaire/Commissariat à l’Energie Atomique, Grenoble, France

1Correspondence: GREPI, EA UJF Laboratoire d’Enzymologie, Grenoble CHU 38043, Cedex 9, France. E-mail mjstasia{at}chu-grenoble.fr


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ABSTRACT
 
The role of Leu505 of Nox2 on the NADPH oxidase activation process was investigated. An X-CGD PLB-985 cell line expressing the Leu505Arg Nox2 mutant was obtained, exactly mimicking the phenotype of a previously published X91+-CGD case. In a reconstituted cell-free system (CFS), NADPH oxidase and iodonitrotetrazolium (INT) reductase activities were partially maintained concomitantly with a partial cytosolic factors translocation to the plasma membrane. This suggests that assembly and electron transfer from NADPH occurred partially in the Leu505Arg Nox2 mutant. Moreover, in a simplified CFS using purified mutant cytochrome b558 and recombinant p67phox, p47phox, and Rac1proteins, we found that the Km for NADPH and for NADH was about three times higher than those of purified WT cytochrome b558, indicating that the Leu505Arg mutation induces a slight decrease of the affinity for NADPH and NADH. In addition, oxidase activity can be extended by increasing the amount of p67phox in the simplified CFS assay. However, the maximal reconstituted oxidase activity using WT purified cytochrome b558 could not be reached using mutant cytochrome b558. In a three-dimensional model of the C-terminal tail of Nox2, Leu505 appears to have a strategic position just at the entry of the NADPH binding site and at the end of the {alpha}-helical loop (residues 484–504), a potential cytosolic factor binding region. The Leu505Arg mutation seems to affect the oxidase complex activation process through alteration of cytosolic factors binding and more particularly the p67phox interaction with cytochrome b558, thus affecting NADPH access to its binding site.

Key Words: gp91phox • PLB-985 cells • X91+-CGD • {alpha}-helical loop • cytosolic C-terminal tail


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INTRODUCTION
 
Professional phagocytes generate high levels of reactive oxygen species (ROS) by activating a superoxide-generating NADPH oxidase, one of the crucial elements of microbicidal mechanisms. The NADPH oxidase is a multicomponent enzyme composed of a membrane-associated flavocytochrome b558 and cytosolic proteins p67phox, p47phox, p40phox, and Rac [1 , 2 ]. Flavocytochrome b558, the catalytic center of the NADPH oxidase, consists of a heavily glycosylated large ß subunit (gp91phox, Nox2) and a small {alpha} subunit (p22phox). In resting cells, p67phox, p40phox, and p47phox form a cytosolic complex dissociated from cytochrome b558 [3 , 4 ]. In resting neutrophils, this oxidase is inactive in a dissociated state and becomes assembled and activated by exposure to microorganisms or inflammatory mediators. Indeed, upon activation, p47phox is highly phosphorylated and mediates p67phox and/or p40phox translocation to the plasma membrane as an organizer. P67phox, which is essential for oxidase activation, serves as an activator. However, the small GTP-binding protein Rac (Rac1/2) translocates independently of the other cytosolic components [5 ]. P22phox seems to play a docking site role for the cytosolic oxidase components and especially for p47phox [6 , 7 ], whereas Nox2 is the only catalytic element of the NADPH oxidase complex. It contains six {alpha}-helix transmembrane domains in the N-terminus in which two hemes [8 ] and the glycosylation sites are localized [9 ].

The C-terminus of Nox2 is a cytosolic sequence supporting the NADPH and FAD binding sites. On the basis of sequence alignments of gp91phox with flavoproteins and reductases, two regions, 338HPFTLTSA345 and 355IRIVGD360, are proposed as binding sites for FAD. In addition, four cytosolic sequences, namely 405MLVGAGIGVTPF416, 442YWLCRD447, 504GLKQ507, and 535FLCGPE540, are considered to be binding sites for pyrophosphate, ribose, adenine, and the nicotinamide unit of NADPH, respectively [10 11 12 ]. In the predicted three-dimensional structure model of Nox2, another intriguing sequence composed of residues 484–504 and located near the potential adenine of the NADPH binding site has been proposed to form an {alpha}-helical loop covering, in the inactive state of the enzyme, the cleft in which NADPH binds [13 ]. Upon oxidase activation, NADPH access to the binding site could potentially be regulated by the interaction of this loop with oxidase cytosolic factors. This {alpha}-helical loop is specifically found in the Nox-Duox family and yeast membranous ferric reductase (FRE1) but not in other FNR reductases [13 ].

Chronic granulomatous disease (CGD), a rare congenital disorder in which the phagocytic cells fail to generate superoxide (O2–) and characterized by severe recurrent bacterial and fungal infections, results from mutations in one of the four subunits of NADPH oxidase [14 15 16 ]. X91+-CGD cases, characterized by normal expression of a nonfunctional Nox2 protein, are rare and useful CGD variants to study functional domains of Nox2. Indeed, the missense mutations Gly408Glu and Asp500Gly, located in the pyrophosphate binding site for NADPH and in the C-terminal {alpha}-helical loop, respectively, disturb the NADPH oxidase assembly and electron transfer [17 , 18 ]. This suggests that an intimate relation exists between the electron transfer and the oxidase assembly processes in certain Nox2 domains. In addition, in transgenic X-CGD PLB-985 cell models, we demonstrated that point mutations in Asp484 and Asp500, located in the {alpha}-helical loop, were essential to the oxidase assembly and to maintaining the electron transfer from NADPH to FAD [19 ]. Recently, Stasia et al. [20 ] have reported a new X91+-CGD case characterized by a missense mutation Leu505Arg in the potential NADPH binding site of Nox2, close to the predicted cytosolic C-terminal {alpha}-helical loop.

In this study, transfected PLB-985 cells mimicking the previously reported X91+-CGD case were used to elucidate the impact of Leu505Arg mutation on NADPH oxidase activation. Purified Leu505Arg mutant and wild-type (WT) cytochrome b558 purified from transfected PLB-985 cells were used in a simplified cell-free system (CFS) with recombinant proteins p67phox, p47phox, and Rac1 to study the effect of increasing the amount of NADPH and p67phox on NADPH oxidase activity. The three-dimensional model of the C-terminal tail of Nox2 was used to determine the strategic importance of the Leu505 position on the NADPH oxidase activation process.


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MATERIALS AND METHODS
 
Materials
Diisopropylfluorophosphate (DFP), leupeptin, tosyl-L-lysine chloroethyl ketone (TLCK), dimethylformamide (DMF), luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), phorbol 12-myristate 13-acetate (PMA), horse-radish peroxidase (HRPO), cytochrome c (from horse heart, type VI), latex beads (3 µm), heparin-agarose and L-{alpha}-phosphatidylcholine type II-S were obtained from Sigma Chemical (St. Louis, MO). Taq DNA polymerase, ATP, guanosine 5'-3-O- (thio) triphosphate (GTP{gamma}S), NADPH, pepstatin, phenylmethyl sulfonyl fluoride (PMSF) and N-octyl glucoside were from Roche (Meylan, France). Endofree Plasmid Maxi Kit was purchased from Qiagen (Courtaboeuf, France). The Sephaglas kit and molecular weight markers were from Amersham (Buckinghamshire, UK). ECL Western blotting detection reagents, CM sepharose CL-6B, DEAE sepharose CL-6B, octyl sepharose CL-4B, and sephacryl S-300 HR were from Amersham Biosciences, Biotechnology (Uppsala, Sweden). Geneticin (G418) was purchased from Gibco (Cergy Pontoise, France). Monoclonal antibodies mAb449 and mAb48 were kindly provided by Dr. D. Roos (CLB, Amsterdam, the Netherlands). Polyclonal antibody anti-p47phox was purchased from Upstate Biotechnology (New York, NY). Monoclonal antibody anti-gp91phox, 7D5 was purchased from MBL Medical and Biological Laboratories (NaKa-ku Nagoya, Japan). Fetal bovine serum and RPMI 1640 were from Life Technologies (Paisley, Scotland, UK). 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, UK).

Culture and differentiation of PLB-985 cells
WT, X-CGD and transfected PLB-985 cells expressing WT or the mutant Nox2 were grown in RPMI-1640 medium supplemented with 2 mM glutamine, 10% (v/v) fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin in a humidified incubator at 37°C in an atmosphere of 5% CO2 in air. After selection, 0.5 mg/ml G418 was added to maintain the selection pressure. For granulocytic differentiation, WT or derivative PLB-985 cells (5x105 cells/ml) were exposed to 0.5% DMF for 5–7 days [19 , 21 ].

Construction of transfected PLB-985 cell line
Leu505Arg mutation was generated by the replacement of thymine1526 with guanine in 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 and subcloned into the mammalian expression vector pEF-PGKneo [19 , 21 ]. The Leu505Arg Nox2 expression construct was sequenced to confirm the mutation and was electroporated into X-CGD PLB-985 cells. Following electroporation, clones were selected by limiting dilution in 1.5 mg/ml G418.

Cytosol and membrane fraction preparation
After being treated with 3 mM DFP for 15 min on ice, PLB-985 cells or human neutrophils were resuspended at a concentration of 5x108 cells/ml in PBS containing 1 mM PMSF, 2 µM leupeptin, 2 µM pepstatin, and 10 µM TLCK. Cells were disrupted by sonication 3 x 10 s at 40 W using a Branson sonifier. The homogenate was centrifuged at 1,000 g for 15 min at 4°C to remove unbroken cells and nuclei. The postnuclear supernatant was centrifuged at 200,000 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 suspended in the same buffer [22 ].

Purification and relipidation of cytochrome b558 from transfected PLB-985 cells
Cytochrome b558 was purified [23 ]. Briefly, crude membrane pellets from 1.5 x 1010 WT or mutant Nox2-transfected PLB-985 cells were treated with 1M NaCl to eliminate the contamination of myeloperoxidase and solubilized in 2% N-octyl glucoside (wt/vol). After a centrifugation at 200,000 g for 60 min at 4°C, the supernatant was submitted to three successive chromatographic steps [24 , 25 ]. Purified cytochrome b558 was relipidated with L-{alpha}-phosphatidylcholine II-S [25 , 26 ]. Cytochrome b558 purity was assessed by silver-stained SDS-PAGE and Western blot analysis. Cytochrome b558 was quantified by reduced-minus-oxidized difference spectroscopy using Soret band absorption at 426 nm. Liposomes were stored at –80°C.

Purification of recombinant p67phox, p47phox, and Rac1
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-ß-D-galactopyranoside (0.2 mM at 20°C for p67phox and p47phox, 0.1 mM at 37°C for Rac1) for 3 h. Fusion proteins were purified by affinity on glutathione-sepharose and were cleaved directly on the matrix using Xa factor for p67phox or thrombin for p47phox and Rac1 [22 , 26 27 28 ]. Purity and cleavage of recombinant proteins were checked by SDS PAGE and Coomassie blue staining. The efficiency of enzymatic cleavage was more than 90%, and the purity was over 95%. Recombinant proteins were stored at –20°C until used.

FACS analysis, Western blot analysis and spectral assessment of Nox2 expression in PLB-985 cells
Nox2 expression in intact PLB-985 cells was assessed using mAb 7D5 (5 µg/ml) [29 ] or control monoclonal IgG1 (Immunotech, Marseille, France). The expression of cytochrome b558 was examined by Western blot analysis using mAb 48 and mAb 449 directed against Nox2 and p22phox, respectively [19 , 21 ]. The WT or mutant Nox2 expression was further detected indirectly by cytochrome b558 differential spectral analysis. A molecular absorption coefficient of {epsilon}426nm =106 mM–1x cm–1 for the Soret band was used for calculations [30 ]. All experiments were done in triplicate.

NADPH oxidase activity in intact cells
H2O2 production was measured by chemiluminescence [19 , 21 ]. Granulocyte-differentiated PLB-985 cells (5 x 105 cells/well in a 96-well plate) in PBS containing 0.9 mM Ca2+, 0.5 mM Mg2+, 20 mM glucose, 20 µM luminol and 10 U/ml HRPO were stimulated with PMA (80 ng/ml). Relative light units (RLU) were recorded at 37°C over a time course of 60 min in a luminoscan luminometer (Labsystem, Helsinki, Finland) connected to a computer.

NADPH oxidase and iodonitrotetrazolium reductase activities in a CFS assay
In vitro NADPH oxidase activity was measured using plasma membranes (30 µg) obtained from transfected PLB-985 cells and cytosol (300 µg) from human neutrophils in a reaction mixture containing 20 mM glucose, 20 µM GTP{gamma}S, 5 mM MgCl2, and an optimal amount of arachidonic acid in a final volume of 100 µl [31 ]. Iodonitrotetrazolium (INT) reductase activity was performed as described above except that cytochrome c was replaced by INT whose reduction was SOD-insensitive [19 ].

In vitro NADPH oxidase activity was also measured with purified cytochrome b558 (0.2 pmol) and purified recombinant proteins, 300 nM p67phox, 323 nM p47phox, and 100 nM Rac1, or an increasing amount of them. We added 10 µM FAD to the reaction medium when purified cytochrome b558 was used.

Ex vivo p47phox and p67phox translocation in transfected PLB-985 cells
P47phox and p67phox translocation in transfected PLB-985 cells was detected by confocal microscopy analysis. Polyclonal anti-p47phox or anti-p67phox antibodies and monoclonal anti-91phox antibody (7D5) were used as primary antibodies, Alexa Fluor® 488 F(ab’)2 goat anti-rabbit IgG (H+L) and Alexa Fluor 546 F(ab’)2 goat anti-mouse IgG (H+L) (Molecular Probes, Eugene, OR) were added to the system as secondary antibodies. Nuclei were visualized by TO-PRO 3 iodine. Negative controls were done for all cell types using only the secondary antibodies. Cells were examined with a confocal laser-scanning microscope and analyzed with Leica confocal software (Heidelberg, Germany) [19 ].

In addition, ex vivo cytosolic factors translocation was performed using 5 x 107 intact PLB-985 cells incubated with PMA (80 ng/ml) or without PMA for 10 min at 37°C. The cells were then sonicated in ice-cold buffer with protease inhibitors, as described in [17 ]. Unbroken cells were harvested by a centrifugation at 800 g for 10 min at 4°C, and the sonicate was layered on a sucrose gradient consisting of 52% (wt/vol) sucrose, 40% (wt/vol) sucrose, and 15% (wt/vol) sucrose. The interface of the 15/40% sucrose layers as a source of plasma membranes was analyzed by immunoblotting with antipeptide polyclonal antibodies directed against p67phox and p47phox, respectively [17 , 19 , 21 ].

In vitro translocation of cytosolic NADPH oxidase proteins
The translocation of p67phox and p47phox to the plasma membrane was studied using the method described previously by Leusen et al. [17 ] with slight modifications. Briefly, partially purified PLB-985 cell membranes (100 µg) and human neutrophil cytosol (1,000 µg) were stimulated with or without 100 µM SDS and 20 µM GTP{gamma}S. The reisolated membrane fraction from a discontinuous sucrose gradient, as described previously, was analyzed by immunoblot with antipeptide polyclonal antibodies directed against p67phox and p47phox, respectively [19 , 21 ].

Protein determination
Protein content was estimated using the Bradford assay [32 ] or the Pierce® method [33 ].

The three-dimensional model of the cytosolic part of gp91phox
With the program PyMol, a picture of the three-dimensional (3D) model from the C-terminal domain of gp91phox was made and the C-terminal {alpha}-helix (residues 483–507) was highlighted [13 ].


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RESULTS
 
A rare X-linked CGD variant resulting from a missense mutation Leu505Arg has recently been reported [20 ]. This mutant was characterized by a normal level of a nonfunctional cytochrome b558 in the patient’s neutrophils, with a totally abolished NADPH oxidase activity. Homologous sequence analysis showed that Leu505 is highly conserved in the human Nox-Duox family, except in Nox2 of Dictyostelium discoideum and plants in which Leu505 is replaced by Phe and Thr (Fig. 1A ). The conserved characterization of Leu505 suggests that this amino acid residue has a common function. According to the sequence alignment of the ferredoxin-NADP+ reductase family (FNR), Leu505 is located in the potential adenine binding motif of Nox2 NADPH. Interestingly, the Nox-Duox family possesses an additional sequence (residues 484–504) just above this amino acid compared with most FNR members (Fig. 1B) . This region has been predicted to be an {alpha}-helical loop structure in a 3D model of the Nox2 C-terminal tail. It has been proposed that it is involved in binding cytosolic factors during the time course of oxidase activation, promoting the NADPH access to its binding site [13 ]. To investigate the molecular mechanism by which the Leu505Arg mutant inhibits the NADPH oxidase activity, a cellular model of this X+-CGD case was built in the X-CGD PLB-985 cell line previously used to study other X91+-CGD mutations [19 , 21 ].


Figure 1
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  		Figure 1. Amino acid sequence alignment of the potential NADPH binding sites of Nox2 in the Nox/Duox and FNR reductase families. (A) The amino acid residues involved in adenine binding are shown in the light-shaded column. Leucine 505 is shown in the dark-shaded column. (B) The putative adenine binding site in the FNR family from the sequence alignment [12 ]. Helix-{alpha} is shown in the shaded column. YSCFRElA, essential for ferric reductase activity—Saccharomyces cerevisiae; NIA-ASPNI, Nitrate reductase (EC 1.6.6.1)—Aspergillus nidulans; NIA-NEUCR, nitrate reductase (NADPH) (EC 1.6.6.3)—Neurospora crassa; B26616, cytochrome b5 reductase; NIA-SPIOL, nitrate reductase (EC 1.6.6.1) (NR)—Spinacia oleracea (spinach); S15959, nitrate reductase (NAD(P)H) (EC 1.6.6.2)—European white birch; MZENAR, NADH:nitrate reductase (AA at 1) (EC 1.6.6.1)—Zea mays; DMPP-PSEPU, hydroxylase P5 protein (EC 1.14.13.7) phenol 2-mono-oxygenase P5 component—Pseudomonas; S15303, hypothetical protein 7.6—Salmonella typhimurium; MEMC-METCA, methane monooxygenase component C (EC 1.14.13.25) methane hydroxylase—Methylococcus capsula; XYLZ-PSEPU, E 1.2-dioxygenase electron transfer component contains ferredoxin and ferredoxin-NAD(+); PSENAPDOXA, DOXA locus PSENAPDOXA—Pseudomonas purida; FENR-ANASO, ferredoxin-NADP reductase (EC 1.18.1.2) (FNR)—Anabaena spp.; SYONDHF, F N-terminal domain homologous to CpcD proteins of Synechococcus spp.; N-lFNR, ferredoxin:NADP+ oxidoreductase (ferredoxin reductase) (EC 1.18.1.2)—Spinacia olerucea (spinach).

Leu505Arg Nox2-transfected X-CGD PLB-985 cells mimic X91+-CGD neutrophils
As seen in Fig. 2A , FACS analysis showed that the expression level of WT and mutant Nox2 in transfected X-CGD PLB-985 cells was similar to that of the original WT PLB-985 cells [19 ]. A similar amount of cytochrome b558 was observed by immunoblotting (Fig. 2B) . In addition, the reduced-minus-oxidized difference spectrum of transfected PLB-985 cells was equivalent to that of WT PLB-985 cells (Fig. 2C) . No Nox2 or cytochrome b558 was detected in X-CGD PLB-985 cells or empty vector-transfected cells, consistent with the hypothesis that the two subunits of cytochrome b558 can costabilize each other in the granulocytic cell line (Fig. 2) .


Figure 2
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  		Figure 2. Expression of WT and mutated Nox2 in transfected X-CGD PLB-985 cells. (A) Flow cytometry analysis of Nox2 expression. 5x105 differentiated WT or transfected X-CGD PLB-985 cells were incubated with the Nox2 mAb 7D5, as described in Materials and Methods. Mouse IgG1 isotype was used as an irrelevant mAb. (B) Immunoblot analysis of the two subunits of cytochrome b558 (Nox2/p22phox) was performed with 50 µg of 1% Triton X100 soluble extracts from WT or transfected PLB-985 cells, subjected to SDS-PAGE (10% acrylamide gel), blotted onto a nitrocellulose sheet, and detected with mAb 48 and mAb 449. (C) Cytochrome b558 differential spectra were performed with the same extract as described in (B). Results are shown from 1 experiment representative of 3.

The NADPH oxidase activity of the Leu505Arg Nox2-transfected PLB-985 cells was determined in intact cells. After DMF-induced granulocytic differentiation, H2O2 production was measured by luminol-amplified chemiluminescence after activation with 80 ng/ml PMA. WT Nox2-transfected PLB-985 cells had an H2O2 production comparable to the original WT PLB-985 cells, as illustrated in Table 1 . Even though the expression of Leu505Arg Nox2 was normal, this mutant was unable to generate H2O2, as observed in X-CGD PLB-985 cells or empty vector transfected cells (Table 1) . The amount of cytochrome b558 was equivalent in original WT PLB-985 cells and in WT and Leu505Arg Nox2-transfected X-CGD PLB-985 cells, confirming results previously obtained by FACS and Western blot analysis (Table 1) . In conclusion, Leu505Arg Nox2-transfected PLB-985 cells mimic the phenotype of X91+-CGD neutrophils [20 ].


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  		Table 1. Cytochrome b558 Amount and NADPH Activity in WT and Transfected X-CGD PLB-985 Cells

NADPH oxidase and INT reductase activities in a CFS
Surprisingly, roughly 50% of the NADPH oxidase activity reconstituted with membranes from WT PLB-985 cells was obtained with membranes from Leu505Arg Nox2 mutant cells. Similarly, INT reductase activity was half of the activity measured in WT PLB-985 cell membranes (Fig. 3 ). This suggests that in a CFS assay, the oxidase complex of the Leu505Arg Nox2 assembled, and the electron transfer from NADPH to molecular oxygen occurs with half efficiency.


Figure 3
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  		Figure 3. NADPH oxidase and INT reductase activity in a cell-free system assay. Cytochrome c and INT reduction were measured using crude membranes (30 µg) isolated from transfected PLB-985 cells in the presence of neutrophil cytosol (300 µg) and activated with GTP{gamma}S, MgCl2, and arachidonic acid, as described in Materials and Methods. The data represent mean ± SD of 3 separate experiments.

Translocation of cytosolic components of the NADPH oxidase complex
Because Leu505 is located near the C-terminal {alpha}-helical loop, a potential binding site for p47phox and/or p67phox in Nox2, translocation of both proteins was studied in intact transfected PLB-985 cells [19 ]. As shown in Fig. 4A and 4B , red and green fluorescence, representing Nox2 and p47phox or p67phox, respectively, surrounded phagosomal membranes around latex beads in both Leu505Arg- and WT Nox2-transfected cells. No p47phox translocation occurred in the X-CGD PLB-985 cells (data not shown) or empty vector-transfected cells, demonstrating that this translocation is cytochrome b558-dependent. Negative control performed in absence of primary antibodies demonstrated the specificity of the immunodetection (Fig. 4B) . In conclusion, confocal microscopy analysis shows that cytosolic factor translocation to the plasma membrane can take place in the leu505Arg mutant. However, to ascertain these data, in vitro and ex vivo translocation of p47phox or p67phox was assessed using a more quantitative method than the confocal microscopy analysis [17 ]. The in vitro and ex vivo assays reveal that the efficiency of p67phox translocation is diminished in the mutant with respect to the WT Nox2-transfected PLB-985 cells (Fig. 4C 4D) . In addition, in the ex vivo experiment, the p47phox translocation was also was diminished in the mutant. The results shown in Fig. 4 are representative of three separate experiments.


Figure 4
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  		Figure 4. Analysis of the NADPH oxidase assembly in transfected PLB-985 cells. Location of p47phox and Nox2 (A) and location of p67phox and Nox2 (B) during activation of transfected PLB-985 cells. A total of 5 x 105 transfected PLB-985 cells were activated with PMA-treated latex-beads for 15 min on day 6 after 0.5% DMF differentiation. p47phox and p46phox translocation was followed by confocal microscopy analysis, as described in Materials and Methods. Polyclonal anti-p47phox antibody or anti-p67phox and mAb 7D5 were used as primary antibodies, Alexa Fluor 488 F(ab’)2 fragments of goat anti-rabbit IgG (H+L) and Alexa Fluor 546 F(ab’)2 fragments of goat anti-mouse IgG(H+L) were used as second antibodies. WT PLB-985 cells incubated only with the secondary antibodies were used as the negative control. (C) in vitro translocation of p47phox and p67phox to the plasma membrane of transfected PLB-985 cells. NADPH oxidase was activated in vitro in presence (+) or in absence (–) of SDS and GTP{gamma}S, using crude membrane from transfected PLB-985 cells as described in Materials and Methods. (D) Ex vivo translocation of p47phox and p67phox to the plasma membrane of transfected PLB-985 cells previously activated by PMA. P47phox and p67phox was detected in the plasma membranes by Western blot analysis after discontinuous sucrose gradient purification. These data are representative of at least three separate experiments.

Turnover and Km for NADPH of WT and Leu505Arg mutant Cytochrome b558
According to the sequence alignment study in the FNR reductase family, Leu505 is potentially located in the adenine of NADPH binding site of Nox2 (Fig. 1A) [2 , 12 ]. It was of interest to investigate the affinity of the purified mutant flavocytochrome b558 for NADPH. Cytochrome b558 was purified to homogeneity from 1.5 x 1010 intact WT or mutant Nox2-transfected PLB-985 cells after three successive chromatographic steps (Fig. 5 ). Crude membranes from WT or mutant Nox2-transfected PLB-985 cells contain ~70 pmol of cytochrome b558/mg of proteins, 7 times less than what is found in human neutrophils (460 pmol/mg). This result is consistent with the amount of cytochrome b558 found in soluble extract from WT or Leu505Arg-transfected PLB-985 cells of 25 pmol/mg vs. 170 pmol/mg in human neutrophils [25 ]. These results suggest that the level of membrane-bound cytochrome b558 in PLB-985 cells is about 7 times less than in neutrophils. The purified preparation was enriched in cytochrome b558 (2800–3500 pmol/mg protein) with a purification factor of 47–50 and a yield of 4–7% (Fig. 5C) . In the purified proteins, two major bands corresponding to the large and small subunits of cytochrome b558 were observed using immunoblot analysis and silver staining (Fig. 5A) . Reduced minus oxidized difference spectra of purified cytochrome b558 from both mutant- and WT-transfected PLB-985 cells possess the three characteristic peaks at 426 nm, 530 nm, and 558 nm (Fig. 5B) .


Figure 5
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  		Figure 5. Purification of cytochrome b558 from transfected PLB-985 cells (A) Purification table of WT-Nox2 and Leu505Arg Nox2 cytochrome b558 from transfected PLB-985 cells. (B) Differential spectral analysis of cytochrome b558 purified from transfected PLB-985 cells. Reduction was achieved by adding a few grains of sodium dithionite to the sample, and the reduced-minus-oxidized difference spectra were recorded at room temperature with a DU 640 Beckman spectrophotometer. (C) Immunodetection of cytochrome b558 was performed with the membrane fractions isolated at each purification step; 3-µg proteins of different fractions were used for Western blot analysis with mAb 48 and mAb 449 and silver staining (D).

The Km for NADPH and NADH was determined using purified cytochrome b558 in the presence of neutrophil cytosol or recombinant proteins p67phox, p47phox, and Rac1, as detailed in Materials and Methods. The Km of purified WT and Leu505Arg mutant cytochrome b558 for NADPH and NADH was determined using increasing NADPH concentrations with optimal conditions in a CFS assay. The Km for NADPH of WT cytochrome b558 in a CFS assay using neutrophil cytosol and recombinant proteins was 42.2 µM and 46.7 µM, respectively (Table 2 ). Like several FNR reductases, the phagocytic oxidase can utilize NADH as a substrate, but with a lower affinity than for NADPH [34 ]. Indeed, the Km for NADH of purified WT cytochrome b558 was 490.1 µM. Our Km values for NADPH and NADH are in agreement with those obtained by several groups [26 , 35 , 36 ]. However, the Km for NADPH and NADH of the purified mutant cytochrome b558 was approximately threefold higher than those of the purified WT cytochrome b558 (Table 2) . These data suggest that the Leu505Arg mutation disturbed the affinity of the mutant cytochrome b558 for NADPH.


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  		Table 2. Specific NADPH Oxidase Activity and Km for NADPH and NADH of WT and Leu505Arg Cytochrome b558

The turnover of the WT and mutant Leu505Arg purified cytochrome b558 in a CFS assay was measured with optimal conditions, as described previously [27 , 28 ]. The oxidase turnover of the purified mutant Leu505Arg cytochrome b558 was 112 ± 8 and 63 ± 7 mol O2– · s–1 · mol–1 heme b when cytosol or recombinant proteins p67phox, p47phox, and Rac1 were used, respectively (Table 2) . This represents a loss of 35–45% of the oxidase turnover of WT cytochrome b558 in a reconstituted CFS using human neutrophil cytosol or recombinant cytosolic factors. This suggests that 1) part of the NADPH oxidase activity of the purified mutant Leu505Arg cytochrome b558 can be restored using neutrophil cytosol or recombinant proteins p67phox, p47phox, and Rac1; and 2) the Leu505Arg mutation affects cytochrome b558, leading to a decrease in maximal specific oxidase activity. The oxidase turnover was higher when human neutrophil cytosol was used instead of purified recombinant cytosolic factors. Perhaps new partners of oxidase-like MRP8/MRP14 (myeloid-related proteins) present in cytosol improved the NADPH oxidase activity [22 ].

Effect of increasing the amount of recombinant p67phox on purified Leu505Arg cytochrome b558
It has been proposed that p67phox is the essential activator element of the oxidase complex, while p47phox was only an "adaptator" protein [27 , 35 ]. With this in mind, the effect of increasing the amount of recombinant p67phox was evaluated on NADPH oxidase activity in a simplified CFS assay. As shown in Fig. 6 , a concentration of 900 nM p67phox was needed to reach optimal NADPH oxidase activity for the Leu505Arg mutant, while only a concentration of 300 nM was sufficient for the WT flavocytochrome b558. In addition, the maximal turnover of NADPH oxidase was 120 mol O2– · s–1 · mol–1 heme b for the WT cytochrome b558, while this turnover was 90 mol O2–.s–1. mol–1 heme b for the mutant. Using 300 nM p67phox, we verified that an increasing amount of the recombinant proteins Rac1 and p47phox were ineffective in increasing the maximal turnover of the purified Leu505 mutant cytochrome b558 (Table 3 ). However, the turnover of the purified Leu505Arg mutated cytochrome b558 alone was closed to that of the WT cytochrome b558 (38 ± 5 vs. 48 ± 3 mol O2– · s–1 · mol–1 heme b). Finally, it appears that only p67phox protein is the essential factor necessary to obtain the most active conformation of the Leu505Arg mutant flavocytochrome b558. It was concluded that the Leu505Arg mutation affects the binding efficiency of p67phox on the mutant cytochrome b558 during the in vitro NADPH oxidase activation process.


Figure 6
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  		Figure 6. Effect of Leu505Arg mutation on the affinity of Cytochrome b558 for p67phox in the cell-free reconstituted system. Activation of NADPH oxidase in a CFS assay using purified Cytochrome b558 (0.2 pmol) from transfected PLB-985 cells and recombinant cytosolic proteins, p47phox (323 nM), Rac1 (100 nM), and increasing concentrations of p67phox (0–1500 nM) in the presence of 300 µM NADPH and 10 µM FAD. Data are representative of 3 independent experiments.


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  		Table 3. Effect of an increasing amount of p47phox and Rac1 on NADPH oxidase turnover of purified WT and Leu505Arg cytochrome b558


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DISCUSSION
 
The aim of this study was to determine why NADPH oxidase activity was absent in the neutrophils of a patient suffering from X+CGD caused by a missense mutation Leu505Arg in the cytosolic tail of gp91phox [20 ]. Only 18 cases of X+-CGD variants have been reported in the literature. Rarely, missense mutations yield normal gp91phox expression with a total lack of oxidase activity [16 ]. This subtype of CGD has provided interesting information on the NADPH oxidase structure and activation mechanisms [17 , 19 , 21 , 37 38 39 ]. However, the amount of purified neutrophils obtained from patients’ blood is often inadequate to study the molecular mechanisms of the NADPH oxidase defect in such patients in detail. In 1993, an ex vivo cellular model of X-CGD was developed [40 ]. The X chromosome-linked CGD locus was disrupted by homologous recombination in the PLB-985 human myeloid cell line (X-CGD PLB-985 cells). Transfected X-CGD PLB-985 cells had been previously used successfully to study the impact of three Nox2 mutations ({Delta}488–497, His303Gln/Pro304Arg and Asp500Gly), resulting in X91+-CGD on the NADPH oxidase activation [19 , 21 , 38 ]. This confirms that the X-CGD PLB-985 cell line is a useful cellular model to express recombinant Nox2 mutant forms for its structure–function analysis. According to our results, Leu505Arg Nox2-transfected PLB-985 cells exactly mimic the phenotype of the previously reported case of X91+-CGD neutrophils. Mutant Nox2 protein was equally expressed in Leu505Arg Nox2-transfected PLB-985 cells, in WT-Nox2-transfected PLB-985 cells and in WT PLB-985 cells (Table 1 , Fig. 2 ). In addition, the difference spectrum of transfected cells was identical to that of WT PLB-985 cells, suggesting that the mutant cytochrome b558 was correctly processed and targeted to the plasma membrane with heme incorporation.

Although the NADPH oxidase activity was totally abolished in intact Leu505Arg Nox2-transfected PLB-985 cells, oxidase activity was partially restored in an in vitro CFS assay using purified membranes from mutant cells (Fig. 3) . In a previous study, investigating several Nox2 mutants using the same approach, we found that for most of the mutants, results obtained in vitro were correlated with those obtained in intact cells, although residual oxidase activity (20% of the original WT PLB-985 cells) was observed in some mutants that had no oxidase activity in vivo [19 ]. Arachidonic acid, used in the CFS assay, probably leads to a conformational change in cytosolic proteins, promoting more protein–protein interactions with Nox2 than occur in vivo. This can explain that there is a partial electron transfer from NADPH to oxygen through the mutant cytochrome b558, according to the INT and cytochrome c reduction assay results (Fig. 3) .

According to the sequence alignment of cytochrome b558 with members of the FNR family, it was generally admitted that the 504GLKQ507 sequence was a potential site for the adenine binding of NADPH [5 , 12 , 13 , 41 ], suggesting that Leu505 potentially played a role as a binding residue for NADPH. However, Taylor et al. [13 ] emphasized that despite a high similitude of sequences between the FNR family and flavocytochrome b558, the most remarkable difference was the addition of a large insertion of 20 residues, 484–504, forming an {alpha}-helical loop. In their 3D model of the C-terminal tail of Nox2, which was built from the atomic structure of ferredoxin-NADP+ reductase [42 ], the location of the large {alpha}-helical loop insert impaired accessibility to the nucleotide binding site from the cytosol (Fig. 7A and 7B ). It should be noted that Leu505 is at the end of this additional {alpha}-helical loop. This structural element (484–504) contains a highly charged sequence: 494HHDEEKD500. In addition to the Leu505Arg mutation, two other mutations leading to X91+-CGD have been mapped in this region. They correspond to the deletion of the lower part of the helical insertion, {Delta}488–497, and to an amino acid substitution, Asp500Gly, in its middle (Fig. 7C) [17 , 37 ]. Moreover, we have recently reported that other mutations (notably Asp484Thr, His490Thr, and Asp500Gly/Ala/Arg) impair the electron transfer process from NADPH to FAD [19 ] (Fig. 7C) . For all the mutants reported in this inserted sequence (whether or not they are associated with a known case of X91+-CGD), the expression level of membrane cytochrome b558, as well as the heme difference spectra, were comparable to those of WT Nox2 [19 , 37 ]. This indicates that this insertion is not of structural importance for gp91phox integrity, but the defective electron transfer from NADPH to FAD in Asp500Gly/Ala/Arg mutants may result from the disrupted interaction between the cytosolic factors with these mutants. Interestingly, Leusen et al. [17 ] found that Asp500 was involved in binding cytosolic components in human neutrophils. These data provided evidence that these residues are indispensable for NADPH oxidase assembly related to electron transfer. Monoclonal antibody NL7, which binds the 498EKDVITGLK506 region of gp91phox, inhibits the NADPH oxidase activity before or during the initial stages of electron transfer [43 ]. These data suggest that residues 498–506 of gp91phox participate in oxidase activation and influence electron transport for superoxide generation. These cumulative data suggest that Leu505 could have a strategic position controlling the movement of the {alpha}-helical loop as a structural barrier to allow NADPH entry into the cleft rather than a NADPH binding role by itself. To determine the specific function of each of these possibilities, we analyzed the changes in the enzymatic properties of the Leu505Arg-Nox2 flavocytochrome b558.


Figure 7
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  		Figure 7. Surface representation of the 3D model of the cytosolic part of gp91phox. (A) The surface drawn in blue is a top view from the inside of the membrane. FAD and NADPH are represented as sticks using the CPK color code. The surface of the additional {alpha}-helix (residues 483–507), closing access to the NADPH binding site, is sand-colored. (B) The surface structure viewed from the cytoplasm after a 90° rotation in the upper direction of the surface structure presented in (A). (C) Zoom on the {alpha}-helix (residues 483–507) orientated as in (B). Leu505, Asp500, His490, and Asp484 are represented as sticks. The figures were drawn with PyMol.

We found that the Km for NADPH of the purified mutant cytochrome b558 was 3 times higher than those of the purified WT, suggesting that the Leu505Arg mutation disturbed the affinity of the mutant cytochrome b558 for NADPH. However, this effect is not strong enough to suggest involvement of Leu505 in a direct interaction and stabilization of the NADPH. Usually, mutation of residue directly involved in substrate binding has a more dramatic effect on the Km. Indeed, in the spinach FNR enzyme, which is the structural prototype for the Nox2 C-terminal region, the Lys244 and Lys216 residues have been identified, from the crystal structure, as being directly involved in NADP+ binding [42 ]. Mutations of these two residues led to a drastic increase in the NADPH Km, by a factor of 15 to 50 for K244 and K216, respectively [44 ]. Moreover, according to the 3D model, Leu 505 is oriented toward the solvent, which does not favor this type of direct interaction in NADPH binding. Therefore, from the moderate amplitude of the Km modification and the Leu505 location in the 3D model, we assume that Leu505 is not a NADPH-binding residue and that the apparent decrease in NADPH affinity points to an alteration in the efficiency of the activation step.

To analyze cytosolic factors translocation in the Leu505Arg mutant, we first used confocal microscopy analysis as a qualitative method. It showed that in this mutant, the assembly of the NADPH oxidase complex occurs at the phagosomal membrane (Fig. 4A 4B) . However, using more quantitative methods, we demonstrated a less effective translocation of p67phox (Fig. 4C 4D) . In addition, a defect of p47phox translocation to the plasma membranes of the Leu505Arg mutant PLB-985 cells was visible in the ex vivo translocation analysis. Future experiments will help us characterize further the effect of Leu505Arg mutation on the p47phox translocation observed in the ex vivo assay. In conclusion, the Leu505Arg mutation, located at the end of the insertion region, does not have the drastic effect of the Asp500Gly mutation, located in the middle of this {alpha}-helical loop, in NADPH oxidase assembly [17 ]. However, both residues of this region are related by their influence in some aspect of the specific activation event, allowing the transformation of the inactive flavocytochrome b558 into a competent electron transfer enzyme.

The original observation, reported in this study, that this Leu505Arg mutant requires larger amounts of p67phox and not increased amounts of p47phox and Rac1 proteins (Table 3) , to reach its maximal turnover, strengthens a possible link between this Leu505-containing region and a p67phox interaction during activation. This contrasts with the absence of a marked difference in the optimal NADPH oxidase activity turnover between WT and mutated cytochrome b558 measured in a CFS assay independently of the presence of the cytosolic factors. In p67phox, region 199–210 was defined from functional studies of various truncated forms of p67phox as an NADPH oxidase activation domain [45 ]. On the basis of structural studies, this domain has been extended to the amino acids 187–210 [46 ] and was reported to be involved in the regulation of electron transfer [37 ]. P67phox was also suggested to participate directly in electron transfer between NADPH and the oxidase flavin [47 ] and to interact directly with cytochrome b558 [48 ].

In summary, according to the 3D model analysis, Leu505 is at the end of this strategic helix in the C-terminal of gp9phox, which may control the entry of NADPH into the cleft of the NADPH binding site (Fig. 7C) . Leu505 seems not to be directly involved in the binding of the adenine moiety of NADPH but is instead a residue located on the protein surface, which is probably important in the {alpha}-helical loop movements controlled by p67phox interaction during oxidase assembly, leading the NADPH access to its binding site. The probable indirect role of p47phox on this process has to be elucidated. The steric hindrance and/or electrostatic effect caused by the Leu505Arg mutation disturbs an optimal flavocytochrome b558 activity, probably as a consequence of an alteration either of the oxidase complex assembly and/or of the conformational changes occurring during the activation event. The relatively widespread hypothesis of a direct link of gp91phox activation during oxidase assembly, as a consequence of an interaction with p67phox, is illustrated here from the point of view of the flavocytochrome b558. It opens the way to a new perspective in the characterization of the events leading to the initiation of the electron transfer in Nox2.


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ACKNOWLEDGEMENTS
 
The authors are grateful to Prof. Mary C. Dinauer for the generous gift of WT PLB-985 cells and X-CGD PLB-985 cells. The authors are also grateful to Dr. W. Taylor and Prof. A. Segal for the gift of the structural model’s coordinates of the Nox2 C-terminal domain. The antibodies 449 and 48 were generous gifts from Prof. D. Roos. We thank Michelle Guillot and Elisabeth Maquet for technical assistance. Supported by grants from the Université Joseph Fourier, Faculté de Médecine; the Région Rhône-Alpes, Program Emergence; the Ministère de l’Education et de la Recherche; MENRT; and the Direction de la Recherche Régionale Clinique, DRRC. Laboratoire Merck-Sharp and Dohme-Chibret, Program Conjoint de Recherche Tempra/Mira 2001-Région Rhône-Alpes.

Received September 30, 2005; revised August 31, 2006; accepted September 15, 2006.


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