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* GREPI EA 2938, Laboratory Enzymologie/DBPC, CHU Grenoble, Grenoble, France; and
Laboratory Chimie des protéines, ERIT-M-CEA Grenoble, Grenoble, France
1Correspondence: GREPI EA 2938, Laboratory Enzymologie/DBPC, CHU Grenoble BP 217, 38043, Grenoble Cedex 9, France. E-mail: mhpaclet{at}chu-grenoble.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50 mol O2– ·s–1·mol heme b–1). Analysis of phox complex components by Western blot and mass spectrometry showed the presence of cytosolic factors (especially p67-phox) and structural proteins (moesin, ezrin). To investigate the difference in activity of phox complexes, we evaluated the effect of MRP8 and MRP14, specifically expressed in neutrophils, on the activity of the B lymphocyte complex. MRPs induce the switch between the partially and the fully "open" cytochrome b558 conformation. Moreover, their effect was independent of p67-phox. Data point out two potential cytochrome b558 activation states.—Paclet, M-H., Berthier, S., Kuhn, L., Garin, J., Morel, F. Regulation of phagocyte NADPH oxidase activity: identification of two cytochrome b558 activation states.
Key Words: NADPH oxidase complex • MRP • p67-phox
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5
6)
. Upon cell stimulation, the complex assembles through specific protein/protein interactions. p67-phox mediates by itself cytochrome b558 activation via a specific interface with gp91-phox, leading to electron transfer from NADPH in cytosol to hemes via flavin adenine dinucleotide (FAD) (7
8
9)
. Finally, oxygen is reduced to superoxide anion O2·–. A defect in NADPH oxidase activity is illustrated in chronic granulomatous disease (CGD), a rare genetic disorder characterized by severe and recurrent infections due to the incapacity of phagocytes to kill invading microbes. CGD is caused by mutations in any one of the four genes encoding the major oxidase subunits: gp91-phox, p22-phox, p47-phox, and p67-phox (10)
.
The NADPH oxidase is also present in nonphagocytic cells such as B lymphocytes, but in these cells the catalytic activity of the enzyme is reduced due to a low expression of cytochrome b558 (11
, 12)
and also to a possible defect or constraint in the assembly process because of an unfavorable membrane environment (13)
. Moreover, we recently discovered that two calcium binding proteins, MRP8 (S100A8) and MRP14 (S100A9), widely expressed in neutrophils but absent in B lymphocytes, were able to increase NADPH oxidase activity of Epstein-Barr virus (EBV) immortalized B cells (14)
. To clarify the enzyme regulation process, we developed a method to isolate the reconstituted assembled NADPH oxidase complex through the purification of cytochrome b558.
In the 1980s, several teams tried to purify the NADPH oxidase complex from PMA-stimulated neutrophil membranes (15
16
17)
. The isolated fractions were able to produce superoxide anions but the NADPH oxidase activity was low, probably due to the high lability of the activated enzyme and the loss of regulatory proteins during detergent extraction. Development of cell-free NADPH oxidase assays allowed reconstitution of the fully activated complex in vitro with native or recombinant proteins in the presence of an amphiphile reagent (18
, 19)
. In this condition, oxidase activity was inducible and transitory, with maximum turnover in the range of 100 mol O2– ·s–1·mol heme b–1. A change in cytochrome b558 conformation upon cytosolic factor assembly was suggested by atomic force microscopy (9)
. Recent studies have pointed out that membrane cytochrome b558 could be activated in vitro by chimeric proteins containing specific moieties of p67-phox and Rac (20
, 21)
. According to the nature of the chimera, NADPH oxidase activation could occur in the absence of an amphiphile agent (21)
and/or the activity could be stabilized (20)
. However, the critical point was the inability to use such methods to analyze the physiological regulation process.
Here we used a combination of both ex vivo and in vitro approaches to study some regulation aspects of the phagocyte NADPH oxidase activation. We isolated the whole NADPH oxidase complex through the purification of cytochrome b558 on an affinity matrix in order to identify its constituents and investigate its capacity to generate O2·–. Data clearly showed that the recovered assembled complex was active in the absence of an amphiphile agent. Moreover, the capacity of electron transfer was dependent on the nature of cytosolic regulatory proteins present in the cells. Results focused on the role of MRP8/MRP14 and pointed out at least two independent cytochrome b558 activation states.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Lymphoid cell lines and neutrophils
Neutrophils from buffy coats and B lymphocytes were isolated according to previous methods (22)
. 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 and in 5% CO2 atmosphere. Cytosolic fractions from the 3 mM DFP-treated and purified neutrophils and EBV-B cells were prepared as described (23)
. EBV-B lymphocytes from a p47-phox-deficient CGD patient were kindly provided by M. A. Pocidalo (INSERM U479, Hôpital Bichat, Paris, France).
MRP8 and MRP14 purification
MRP8 and MRP14 were purified from the cytosol of unstimulated neutrophils as described (14)
. 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 fast protein liquid chromatography on a monoQ anion exchange column. Bound MRP8 and MRP14 were eluted with 0.13 M NaCl, then dialyzed against PBS. The major elution peak was analyzed by SDS-PAGE and presented only two peptide bands identified as MRP8 and MRP14 by Western blot. The absence of contaminating p47-phox, p67-phox, and Rac in the purified fraction was checked by Western blot compared with a positive control performed on neutrophil cytosol (14)
. The purified MRP complex was called MRP8/MRP14 and was stored at –80°C until further use.
Recombinant proteins
Full-length cDNAs encoding p67-phox, Rac1, MRP8, and MRP14 were expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein using pGEX-3X (p67-phox), pGEX-2T (Rac1), or pGEX-5X-2 (MRP8 and MRP14). Protein expression was induced with isopropyl thiogalactoside (IPTG) (0.2 mM at 20°C for p67-phox, MRP8, and MRP14, and 0.1 mM at 37°C for Rac1) for 3 h. GST fusion proteins were affinity purified from IPTG-induced bacteria on glutathione-Sepharose (9
, 14)
. After washing in PBS, recombinant proteins were cleaved directly on the matrix using Xa factor (rp67-phox, rMRP8, and rMRP14) or thrombin (rRac1) in PBS. Recombinant proteins were stored at –20°C until further use.
Isolation of oxidase complex
Purified neutrophils were suspended at 108 cells/ml in PBS supplemented with 0.9 mM CaCl2 and 0.5 mM MgCl2. Neutrophils were then activated at 37°C in the presence of 5 µM cytochalasin B for 15 min and 810 nM PMA for 10 min. This step is required to obtain an activated cytochrome b558 conformation able to bind cytosolic regulatory proteins and remove granule proteases. In parallel, an experiment was performed with neutrophils not stimulated by PMA. Crude plasma membranes and cytosol from 4 x 109 resting or phorbol ester-stimulated neutrophils were obtained as reported previously (23)
. Membrane proteins were solubilized using 68 mM n-octyl glucoside for 20 min at 4°C. After centrifugation at 200,000 g, the solubilized extract was submitted to ion exchange chromatography formed by a mixture (1V/1V/1V) of CM-Sepharose, DEAE Sepharose, and N-amino octyl-Sepharose, combined with heparin agarose affinity chromatography, as described for neutrophil cytochrome b558 purification (23)
. Once cytochrome b558 was bound to heparin agarose, the purification buffer [containing 100 mM HEPES, pH 7.2, 100 mM KCl, 10 mM NaCl, 1 mM EDTA, 0.1 mM DTT, 20% (v/v) glycerol, 0.1% (w/v) Triton X-100, and a mixture of protease inhibitors: 10 µM N
-p-tosyl-L-lysine chloromethyl ketone, 1.8 µM leupeptin, and 1.5 µM pepstatin] was replaced by the same buffer containing 40 mM n-octyl-glucoside instead of Triton X-100. Then cytosol either from resting ("unstimulated" control) or stimulated neutrophils or from EBV-B lymphocytes (25 mg) was deposited on the heparin agarose matrix. A control experiment was performed without loading cytosol onto heparin-bound cytochrome b558. In some experiments, a mixture of rMRP8 and rMRP14 (120 nmol) preloaded with 500 nM calcium was used instead of cytosol. The column was extensively washed with the purification buffer containing 40 mM n-octyl-glucoside. Once the absorbance at 280 nm was null, cytochrome b558 and the associated proteins were eluted using a NaCl gradient (0–0.5 M). Elution of cytochrome b558 was followed by measuring the "reduced minus oxidized" differential spectrum. Oxidase activity of the fractions containing cytochrome b558 was determined. Fractions with a high oxidase activity were pooled and applied to a Sephacryl S-300 column equilibrated in the purification buffer. After filtration, the heme-containing fractions were analyzed by Western blot and their oxidase activity was measured.
NADPH oxidase activity of the purified complex
Cytochrome b558 was then quantified by "reduced minus oxidized " difference spectra and by measuring the difference of absorbance between the Soret band at 426 nm and the valley at 410–411 nm. The absorption coefficient used was 106 mM–1 · cm–1 at 426 nm (9)
. NADPH oxidase activity was measured after mixing purified oxidase complex (0.2 pmol cytochrome b558/assay) with 10 µM FAD, 40 µM GTP
S, and 5 mM MgCl2 in a final volume of 100 µl PBS and adding 150 µM NADPH (final concentrations). NADPH oxidase activity was estimated by measuring the superoxide dismutase (SOD) -sensitive cytochrome c reduction at 550 nm (
550 nm=21.1 mM–1·cm–1) and expressed as turnover, mol O2– · s–1 · mol heme b–1 (9)
. In some experiments, recombinant proteins (rRac1 and rp67-phox) or purified MRP8/MRP14 were incubated with the complex in the presence of arachidonic acid (1 mM). In the case of MRP, MRP8/MRP14 (0.78 µg) was preincubated with 500 nM CaCl2 at 25°C for 20 min before contact with the phox complex.
Protein digestion
Protein bands were manually excised from Coomassie blue-stained 1D gels. Excised gel bands were washed several times with destaining solutions (25 mM NH4HCO3 for 15 min, then with 50% (v/v) acetonitrile containing 25 mM NH4HCO3 for 15 min). Gel pieces were then dehydrated with 100% acetonitrile and submitted to drying. Gel pieces were then incubated with a reducing solution (25 mM NH4HCO3 containing 10 mM dithiothreitol) for 1 h at 56°C, then with an alkylating solution (25 mM NH4HCO3 containing 55 mM iodoacetamide) for 45 min at 37°C. After reduction and alkylation, gels were washed several times with the destaining solutions, and finally with pure water for 15 min, before being treated again with 100% acetonitrile. Depending on protein amount, 2 to 3 µl of 0.1 µg/µl modified trypsin (Promega, sequencing grade) in 25 mM NH4HCO3 was added over the gel spots. After 30 min of incubation, 7 to 10 µl of 25 mM NH4HCO3 was added to cover the gel spots before incubation overnight at 37°C.
Matrix-assisted laser desorption ionization: time of flight mass spectrometry (MALDI-TOF-MS) analyses and identification of proteins
For MALDI-TOF-MS analyses, a 0.5 µl aliquot of peptide mixture was mixed with 0.5 µl matrix solution [-cyano-4-hydroxycinnamic acid at half saturation in 60% acetonitrile/0.1% trifluoroacetic acid (TFA) (v/v)]. The resulting solution was automatically spotted on a MALDI-TOF target plate, dried, and rinsed with 2 µl of 0.1% TFA. Peptides mixtures were then analyzed with a MALDI-TOF mass spectrometer (Autoflex, Bruker Daltonik, Bremen, Germany) in reflector/delayed extraction mode over a mass range of 0–4200 Da. For each sample, spectrum acquisition was obtained with an average of 200 laser shots after an external calibration using a mixture of four synthetic peptides (angiotensin II, m/z 1046.54 Da; substance P, m/z 1347.74 Da; bombesin, m/z 1619.82 Da, and ACTH clip18–39, m/z 2465.20 Da). Spectra were then annotated (XMass software, Bruker Daltonik) and peptide mass fingerprints obtained were submitted to database searches against the Swissprot Trembl database with an intranet 1.9 version of MASCOT software. MASCOT search parameters used with MS data are database = Swiss Prot Trembl, enzyme = trypsin/P, 1 miscleavage allowed, variable modifications = acetyl (N-ter)/oxidation (M)/carbamidomethyl (C)/FMA + 1, peptide tolerance = 100 ppm, monoisotopic and [M+H]+ (protonated molecular ions). When a protein is characterized by a MASCOT score higher than 70, at least six peptides, and a coverage higher than 20%, the protein is automatically validated. When one of these parameters is not respected, a manual inspection of the data is needed based on the error between the experimental and theorical mass values from each peptide of the peptide mass fingerprint, the number of miscleavages, and the annotation. If no or unclear identification is obtained after a MALDI-TOF analysis, the sample is submitted to a LC-MS/MS analysis.
Nano-liquid chromatography: mass spectrometry/mass spectrometry (nano-LC-MS/MS) analysis of proteins
For nano-LC-MS/MS analysis, peptides were extracted from the gel pieces by diffusion: once in 50% acetonitrile for 15 min, then once in 5% formic acid, and finally once in 100% acetonitrile. The pooled supernatants were then transferred to microcentrifuge tubes and dried under vaccum on a centrifugal evaporator. The dried extracted peptides were solubilized in water containing 2% acetonitrile and 7% trifluoroacetic acid before being transferred in vials compatible with nano-LC-MS/MS analysis (CapLC and Q-TOF Ultima, Waters, Milford, MA, USA). The method consisted of a 35 min run at a flow rate of 200 nl/min using a gradient from two solvents: A (2% acetonitrile: 97.9% water: 0.1% formic acid) and B (80% acetonitrile: 19.92% water: 0.08% formic acid). The system includes a 300 µm x 5 mm PepMap C18 precolumn in order to preconcentrate peptides and a 75 µm x 150 mm C18 column (LC Parkings Dionex) used for peptides elution. Spectra were calibrated thanks to fragmentation of the green fluorescent protein in MS/MS mode. MS and MS/MS data were acquired and processed automatically using MassLynx 3.5 software (Waters). MASCOT search parameters used with MS/MS data are: database = Swiss Prot Trembl, enzyme = trypsin/P, 1 miscleavage allowed, variable modifications = acetyl (N-ter)/oxidation (M)/carbamidomethyl (C)/FMA+1, protein tolerance = 0.4 Da, peptide tolerance = 0.4 Da, peptide charge = 2+/3+. A proteinthat has at least two peptides, each with a Mascot MOWSE score higher than 40, is automatically validated. When this parameter is not respected, the fragmentation spectrum from each peptide of the protein is manually interpreted using conventional fragmentation rules (24)
.
Polyclonal antibodies
Rabbit polyclonal antibodies were raised against polypeptides corresponding to the C-terminal region of p47-phox (residues 371–390), p67-phox (residues 511–526), p40-phox (residues 325–339), gp91-phox (residues 562–569), p22-phox (residues 184–195), and to the internal region shared by Rac1 and Rac2 (residues 123–145) (23
, 25)
. Immunoglobulins were purified from rabbit antisera on 1 ml protein A-Sepharose.
SDS-PAGE and Western blot
The proteins were fractionated by 10% or 11% SDS-PAGE (26)
and electrotransferred to nitrocellulose, as described previously (27)
. Immunodetection was performed using rabbit polyclonal antibodies raised against p47-phox, p67-phox, p40-phox, gp91-phox, or p22-phox (dilution 1:1000) or goat polyclonal antibodies directed againt MRP8 or MRP14 (dilution 1:1000). The immune complexes were detected with goat anti-rabbit secondary antibody combined with peroxidase. Bound peroxidase activity was detected using ECL reagents.
Statistical analysis
Data were expressed as the mean ± SD. Statistical analysis was performed using the unpaired t test. The results are reported when significantly different (P<0.05) from the controls.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Once the absorbance at 280 nm was null, the heparin column was washed with purification buffer containing 40 mM octyl-glucoside instead of Triton X-100. The detergent exchange step was an essential step to obtain a functional NADPH oxidase complex.
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Then neutrophil cytosol from stimulated neutrophils was deposited onto the heparin-bound cytochrome b558 to allow interactions between cytosolic proteins and cytochrome b558. We expected that cytosolic factors present in stimulated cytosol were in a suitable conformation for association with cytochrome b558. The complex was eluted with a NaCl gradient (0–0.5 M). The elution profile is illustrated in Fig. 1
. Cytochrome b558 elution was followed by measurement of the reduced minus oxidized absorption spectrum of the cytochrome b558 and by Western blot (Fig. 2
A, B). NADPH oxidase activity of eluted fractions presenting a cytochrome b558 spectrum was measured by the SOD-sensitive cytochrome c reduction assay in the presence of NADPH and FAD, but without addition of anionic amphiphile as activator. As shown in Fig. 2A
, all the cytochrome b558-containing fractions were able to "constitutively" produce superoxide anions in the presence of NADPH with a turnover ranging from 40 to 100 mol O2– ·s–1 · mol heme b–1 depending on the fraction. The maximum turnover in the absence of stimulus (100 mol O2– ·s–1·mol heme b–1) is comparable to the turnover of NADPH oxidase reconstituted with neutrophil fractions in a cell-free system activated by arachidonic acid. A control experiment was performed by subjecting to fractionation membranes and cytosol from unstimulated neutrophils (Fig. 2
, – stimulation). In this case, after heparin-agarose, NADPH oxidase activity of cytochrome b558-containing fractions in the absence of activator was strongly decreased compared with that obtained with fractions derived from PMA-stimulated neutrophils. The maximum activity was in the range of 15 to 20 mol O2– · s–1 · mol heme b–1, confirming that the PMA treatment was required to favor cytochrome b558/cytosolic factor assembly. As the eluted fractions were able to produce a large amount of superoxide anions, we suspected that cytochrome b558 was in an active conformation and that regulatory NADPH oxidase factors responsible for cytochrome b558 activation were associated with it. The presence of cytosolic factors p67-phox, p47-phox, and p40-phox in these fractions was examined by Western blot (Fig. 2C
). Cytochrome b558 was eluted with a NaCl concentration between 0.3 and 0.5 M (Fig. 2A
, fractions 15–23). We noticed that a large part of cytosolic factors (p67-phox, p47-phox, and p40-phox) was eluted independently from cytochrome b558, with a low NaCl concentration (0.2 M) (Fig. 2C
, fractions 8–16). It has been described that p47-phox is able to bind to heparin agarose (28)
, and we have shown that p47-phox/p67-phox/p40-phox were partly associated in neutrophil cytosol (14)
. This suggests that the first peak eluted from heparin corresponded to the cytosolic complex. In the complex derived from stimulated cells, some cytosolic factors were also recovered along with cytochrome b558 (Fig. 2C
, + stimulation, fractions 18–21), especially p67-phox. In these latter fractions (Fig. 2C
, + stimulation, fractions 18–21), the ratio p67-phox/p47-phox was clearly increased compared with the ratio in fractions containing only cytosolic factors (Fig. 2C
, fractions 8–16). This indicates a strong association of p67-phox with cytochrome b558 (Fig. 2B
). We controlled that cytosolic phox factors recovered in the cytochrome b558 fractions came exclusively from cytosol, as shown by their absence in the membrane-soluble extract (Fig. 2C
, lane C vs. E). In contrast, in the complex derived from unstimulated cell fractions, the amount of cytosolic phox factors, especially p67-phox, was very low, suggesting a weak association with cytochrome b558 and confirming that PMA stimulation was required to obtain the right cytochrome b558 conformation able to bind cytosolic regulators (Fig. 2C
, – stimulation, fractions 18–21).
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At this stage we pooled active fractions eluted from heparin and containing both cytochrome b558 and cytosolic factors (Fig. 2
, H fractions). The heparin pool (Fig. 2
, H fractions) was submitted to a gel filtration chromatography to eliminate proteins not specifically associated with cytochrome b558. Elution was performed overnight with the purification buffer containing 40 mM octylglucoside. Cytochrome b558 elution was followed up by measuring the "reduced minus oxidized" absorption spectrum of cytochrome b558 (Fig. 3
A). In the fractions derived from stimulated cells, the presence of cytosolic factors was shown by Western blot in cytochrome b558-containing fractions (Fig. 3B
). p67-phox clearly eluted in two separated pools (Fig. 3A
). The first one eluted concomitantly with cytochrome b558 (Fig. 3A
, fractions 38–42); in these fractions, both p47-phox and p40-phox were slightly immunodetected (Fig. 3B
). The second pool of p67-phox (Fig. 3A
, fractions 44–48) eluted independently of cytochrome b558 and represented the cytosolic factor complex elution. In contrast, cytosolic phox factors could not be detected in the cytochrome b558-containing fractions derived from the purification performed with unstimulated neutrophil fractions (data not shown).
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Purified NADPH oxidase complex was able to constitutively produce superoxide anions in the absence of stimulation
After gel filtration, cytochrome b558-containing fractions derived from stimulated cells were analyzed for their capacity to produce constitutively superoxide anions in the presence of the substrates, NADPH and oxygen. As shown in Fig. 3C
, some fractions (fractions 37–40, black squares) had a SOD-sensitive NADPH-dependent cytochrome c reductase activity in the absence of activator. Oxidase turnover was in the range of 50 to 125 mol O2– · s–1·mol heme b–1, depending on the fraction (Fig. 3C
, black squares), and was significantly different from that measured with Sephacryl fractions derived from resting cells (Fig. 3C
, open squares). The fraction with the maximum turnover was called neutrophil complex (Fig. 3C
, PMN Cx). To confirm that NADPH oxidase measured in the absence of arachidonic acid was specific and not due to an experiment artifact, a control purification was performed under the same conditions but without the addition of neutrophil cytosol. After the gel filtration, SOD-sensitive cytochrome c reduction was measured in the fractions containing cytochrome b558 in the absence of stimulus. In this case, NADPH oxidase measured was 5 mol O2– · s–1 · mol heme b–1 (Fig. 3C
, black rhombs). This activity was significantly different from that of the fraction called neutrophil phox complex (Fig. 3C
, PMN Cx), confirming that neutrophil cytosol was able to activate heparin-bound cytochrome b558. Along with cytochrome b558 and cytosolic factors, several proteins were present in the neutrophil phox complex, as shown after protein separation on SDS-PAGE (Fig. 3C
, right panel). MALDI-TOF mass spectrometry analysis, followed by sequencing peptides, with tandem mass spectrometry (MS-MS) of major bands present in the neutrophil phox complex pointed out the presence of moesin, a member of the ezrin-radixin-moesin (ERM) family of proteins, and coronin, an actin binding protein (Table 1
). Coronin has been shown to interact with the C terminus domain of p40-phox (29)
and moesin with p47-phox (30)
. Two proteins involved in glucose metabolism were also identified: the 6-phosphofructo-2-kinase involved in glycolysis and 6-phosphogluconate dehydrogenase, an enzyme involved in hexose monophosphate shunt, the major pathway that produced NADPH by reducing NADP+ (Table 1)
. Their association with the phox complex is probably due to a close proximity of these proteins with cytosolic factors in the neutrophil compartments.
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NADPH oxidase complex reconstituted with neutrophil cytochrome b558 and B lymphocyte cytosol
To evaluate the specificity of protein/protein interactions in the recovered complex, neutrophil cytosol was replaced by EBV-B lymphocyte cytosol prepared either from control cells or from cells deficient in p67-phox or p47-phox. The purification process was similar to that described for the neutrophil phox complex. The heparin-agarose and Sephacryl S300 elution profiles were comparable to those shown for neutrophil phox complex (data not shown). NADPH oxidase activity of cytochrome b558-containing fractions after gel filtration was measured in the absence of activator. Some fractions derived from the purification performed with control EBV-B lymphocyte cytosol were able to constitutively produce superoxide anions in the absence of activator (Fig. 4
A). The maximum NADPH oxidase turnover was 50 mol O2– ·s–1·mol heme b–1. This activity was comparable to that reconstituted in vitro with neutrophil cytochrome b558 and B lymphocyte cytosol after stimulation with an optimum concentration of arachidonic acid (9)
. The fraction presenting the highest turnover was called B lymphocyte phox complex (Fig. 4A
, BL Cx). Along with gp91-phox and p22-phox, several proteins were visualized by SDS-PAGE (Fig. 4B
, right panel), but p67-phox was the sole phox cytosolic factor clearly immunodetected (Fig. 4B
, left panel). The level of p67-phox in these fractions was at least 4-fold inferior to the level in the neutrophil phox complex (Fig. 4B
vs. Fig. 3B
).
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Rac was not immunodetected in either neutrophil or B lymphocyte phox complexes (after gel filtration) (Fig. 5
A). We evaluated the Rac detection threshold with polyclonal Rac antibodies. These antibodies detect both recombinant and native Rac. The detection threshold was 10 pmol recombinant Rac (Fig. 5A
). A weak band was observed in the heparin fraction containing 7 pmol cytochrome b558 (Fig. 5A
, Hep). In phox complexes eluted from gel filtration, cytochrome b558 concentration was decreased by at least 3-fold (Fig. 5A
, Seph). Even if Rac was present in an equimolar quantity, the amount would be too low to be detected. To determine Rac involvement in the purified phox complexes, we measured constitutive activity in the absence of GTPS. Results clearly showed an activity decrease of 40% in the absence of GTPS (Fig. 5B
), confirming involvement of the monomeric G-protein Rac in constitutive NADPH oxidase activity.
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We have shown that the phox complex prepared with control B lymphocyte cytosol was able to constitutively transfer electrons from NADPH to O2 in the absence of stimulus, but the rate represented 40% of that of neutrophil complex (Fig. 6
A). This activity was due specifically to the NADPH oxidase, as complexes prepared with p47-phox-deficient cytosol (Fig. 6A
) or p67-phox-deficient cytosol (data not shown) displayed no significant catalytic activity (Fig. 6A
). This result suggests that cytochrome b558 in the B lymphocyte complex was not in a completely active conformation to transfer the maximum rate of electrons. We tested several components that were potentially able to activate NADPH oxidase. First, the B lymphocyte complex was incubated with arachidonic acid for 10 min before measuring superoxide anion production. Results shown on Fig. 6B
clearly demonstrated that arachidonic acid alone had no effect on the constitutive NADPH oxidase activity of the B lymphocyte complex (control or deficient in p47-phox). Then we evaluated the effect of adding MRP8 and MRP14 to the B lymphocyte complex. In a previous study we demonstrated that a combination of MRP8 and MRP14, two calcium binding proteins expressed in neutrophils but not in B lymphocytes, were able to increase NADPH oxidase of B lymphocytes in vivo and in vitro by a direct effect on cytochrome b558. A mixture of MRP8 and MRP14, purified from neutrophil cytosol and preloaded with calcium, was incubated for 10 min with the B lymphocyte complex in the presence of arachidonic acid. NADPH oxidase activity of the control B lymphocyte phox complex was almost doubled after MRP8/MRP14 addition, reaching a maximum NADPH oxidase turnover (125 mol O2– · s–1 · mol heme b–1). No significant increase was obtained with the p47-phox-deficient complex (Fig. 6B
). This result indicates that the constitutive NADPH oxidase activity of the control B lymphocyte complex could still be up-regulated by MRP8/MRP14 in the presence of calcium and arachidonic acid. All the data suggest that the cytochrome b558 activation process occurs in at least two steps: one depending only on cytosolic phox factors (B lymphocyte complex) and a second depending on "helper" regulatory proteins like MRP8 and MRP14. Both effects seem to be cumulative.
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In an earlier work we showed that MRP8 and MRP14 were associated with the cytosolic regulatory complex in neutrophil cytosol (14)
; recently it has been reported that MRP8/MRP14 interacted with p67-phox and Rac (31)
. To elucidate whether the effect of MRP observed on the B lymphocyte phox complex was due to a direct interaction of MRP with cytochrome b558 or cytosolic factors, another experiment was conducted. We evaluated the direct effect of MRP8 and MRP14 on heparin-bound cytochrome b558 in the absence of cytosolic factors. First, MRP8 and MRP14 were prepared as recombinant proteins in E. coli. Proteins were expressed as GST fusion proteins, purified on glutathione-Sepharose, and both MRP8 and MRP14 were obtained after a direct cleavage of GST on Sepharose. The presence of rMRP8 and rMRP14 in the purified fractions was analyzed by Western blot (Fig. 7
A, left panel) and the purity of the proteins was controlled by SDS-PAGE (Fig. 7A
, right panel). Purification of the complex formed by cytochrome b558 and MRP8/MRP14 was achieved as described in Materials and Methods for the neutrophil phox complex, except that instead of loading neutrophil cytosol on cytochrome b558-bound heparin, a mixture of rMRP8 and rMRP14 preincubated with calcium was deposited. Elution of cytochrome b558 was followed as described previously. Fractions containing the highest cytochrome b558 concentration were pooled and further purified on gel filtration (Fig. 7B, H
fractions). After elution from Sephacryl-S300, cytochrome b558-containing fractions were analyzed for their constitutive SOD-sensitive cytochrome c reductase activity. As observed with previous purifications, one fraction was able to produce constitutively superoxide anions in the range of 25 mol O2– · s–1 · mol heme b–1 (Fig. 7C
, Cyt.b558/MRP Cx). This activity measured in the absence of stimulus was significantly higher than that measured with the fraction issuing from the control purification (Fig. 3C
, black rhombs) performed with neutrophil cytochrome b558 without addition of cytosol or MRP (Fig. 7C
, 25 mol O2– ·s–1·mol heme b–1 vs. 5 mol O2– ·s–1·mol heme b–1). Western blot analysis of the "Cyt.b558/MRP Cx" showed the presence of gp91-phox and p22-phox. MRP8 and MRP14 were never immunodetected in the Cyt.b558/MRP Cx, suggesting either that their association with cytochrome b558 was not stable or that the quantity was under the detection threshold of the ECL method used. That the Cyt.b558/MRP Cx was able to constitutively produce a low rate of superoxide anions indicates that cytochrome b558 was partially activated by MRP8/MRP14. We further investigated the effect of p67-phox and Rac on this fraction. Incubation of p67-phox, Rac with the Cyt.b558/MRP Cx in the presence of arachidonic acid led to a significant increase of superoxide anion production to 50 mol O2– ·s–1·mol heme b–1 (Fig. 8
). This raise was directly correlated with the presence of p67-phox and Rac, as arachidonic acid alone did not affect the Cyt.b558/MRP Cx activity (Fig. 8)
.
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Stabilization of the active cytochrome b558 conformation by MRP8 and MRP14
Next, we examined how long cytochrome b558 in isolated complexes remained active and able to constitutively produce superoxide anions. NADPH oxidase activity of the Cyt.b558/MRP Cx was measured until 30 days after purification. The constitutive activity of the Cyt.b558/MRP Cx was unchanged after storage at –80°C (Fig. 8)
. In contrast, the control cytochrome b558 prepared in the same experimental conditions (without relipidation) but in the absence of cytosol and MRP8/MRP14 (Fig. 3C
, black rhombs) was no longer able to produce superoxide anions after 30 days storage at –80°C (data not shown). This result suggested that in the isolated complex, MRP8/MRP14 stabilized the partially active form of cytochrome b558. Moreover, after 30 days Cyt.b558/MRP Cx activity could be enhanced a second time by p67-phox, Rac, and arachidonic acid (Fig. 8)
.
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DISCUSSION |
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, 32)
. In this system, however, phagocyte membrane lipids were required for proper assembly in the absence of activator. In our model, octyl glucoside-solubilized cytochrome b558 was activated by cytosolic proteins in the absence of lipids or amphiphile agent, suggesting that heparin-bound cytochrome b558 was in an appropriate conformation to allow assembly with cytosolic factors. Once assembled, the complex was able to transfer electrons even if phospholipids were absent. It is well known that phospholipids are essential in vitro to reconstitute a high level of NADPH oxidase activity with purified cytochrome b558 (33)
. In vivo, lipids are implicated in the translocation of cytosolic phox factors (34)
and in dissociating Rac from Rho-GDI (35)
. In addition, we suspected that phopholipids play an important role in maintaining cytochrome b558 conformation in the membrane; this conformation is probably conserved after heparin binding, allowing interaction with cytosolic regulators. Once assembled, the complex no longer requires a lipid environment to produce superoxide anions.
In the isolated phox complex, constitutive NADPH oxidase turnover was dependent on the source of the cytosol used: it was maximum with neutrophil cytosol (120 mol O2– ·s–1·mol heme b–1) and intermediate with B lymphocyte cytosol (50 mol O2– ·s–1·mol heme b–1). The amount of p67-phox, the main effector of activation, was clearly reduced in the B lymphocyte phox complex compared with that detected in the neutrophil phox complex. It is well known that expression of p47-phox and p67-phox was lower in B lymphocytes than in neutrophils (50% and 30%, respectively) (12)
. The reduced expression could not fully explain the decrease in phox complex activity because, in the isolation protocol, cytosolic proteins were in excess compared with heparin-bound cytochrome b558. We hypothesized that other regulators were present in neutrophil cytosol and improved the binding of p67-phox onto cytochrome b558.
We have demonstrated that two calcium binding proteins, MRP8 and MRP14, abundant in neutrophils and absent from EBV-B lymphocytes, were able to activate NADPH oxidase by a direct effect on cytochrome b558 (14)
. Here, addition of calcium-loaded MRP to the phox complex prepared with B lymphocyte cytosol increases the constitutive capacity of cytochrome b558 to produce superoxide anions until maximum NADPH oxidase turnover occurs. In a recent study it has been reported that MRP8/MRP14 associate with cytosolic factors, especially p67-phox and Rac (31)
. Here we observed that heparin-bound cytochrome b558 was partially activated by recombinant MRP8/MRP14 in the absence of cytosolic factors and arachidonic acid, confirming a direct interaction of MRP8/MRP14 with cytochrome b558. The resulting cytochrome b558/MRP complex was able to transfer electrons at a rate significantly higher than that of cytochrome b558 alone.
The constitutive activity of isolated phox complexes can be explained by different cytochrome b558 activation states mediated by p67-phox and MRP8/MRP14. Like MRP8/MRP14, p67-phox can induce in the absence of any amphiphile agent a switch from an inactive to a partially "open" cytochrome b558 conformation that is able to transfer electrons. The fully open conformation is obtained in vitro as in vivo only in the presence of both effectors, p67-phox and MRP8/MRP14 (neutrophil cytosol). Use of p47-phox-deficient cytosol leads to the formation of an inactive phox complex, unable to produce superoxide anions, demonstrating that in our system, like in vivo, p47-phox is required for an efficient interaction between p67-phox and cytochrome b558 and that in a physiological environment, p47-phox is an organizer for an optimal NADPH oxidase assembly (20)
.
In conclusion, we demonstrated by isolating "phox complex" that the positive regulators p67-phox and MRP8/MRP14 can induce independently a partial conformation change in heparin-bound cytochrome b558 in the absence of arachidonic acid (Table 2
). In our experimental conditions, membrane phospholipids are not required for this interaction. The effect of both proteins (p67-phox and MRP8/MRP14) is cumulative, suggesting that they bind to cytochrome b558 at different sites. Moreover, combining both proteins is necessary to obtain a fully open cytochrome b558 conformation able to transfer electrons at a maximum rate. Data point out at least two cytochrome b558 activation states related to the level of NADPH oxidase turnover.
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ACKNOWLEDGMENTS |
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Received for publication July 21, 2006. Accepted for publication November 9, 2006.
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). Oxidase turnover of purified fractions derived from PMA-stimulated (+ stimulation, 
) or resting (– stimulation, 
) neutrophils was determined in the absence of amphiphile agent by incubating cytochrome b558-containing fraction (0.2 pmol heme/assay) with 150 µM NADPH in the presence of 10 µM FAD, 40 µM GTP
). Results are presented as the average of at least three experiments ± SD. *Activity statistically different (P<0.05) from the constitutive activity obtained with the (– cytosol) in the absence of arachidonic acid.
