From the
Groupe de Recherche et d'Etude du
Processus Inflammatoire, Laboratoire d'Enzymologie, Centre Hospitalier
Universitaire de Grenoble, BP 217, 38043 Grenoble,
¶Laboratoire de Chimie des Protéines,
Commissariat à l'Energie Atomique de Grenoble, 38054 Grenoble, and
||Institut de Biologie et Chimie des
Protéines, 69367 Lyon, France
Received for publication, September 23, 2002 , and in revised form, April 15, 2003.
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ABSTRACT |
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INTRODUCTION |
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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 (7, 8). Recent observations would support an allosteric regulation of NADPH oxidase activity where cytochrome b558 is the catalytic subunit. In the oxidase assembly, p67phox is the limiting factor (911). The binding of p67phox to cytochrome b558 mediates the transition from an inactive to an active conformation of cytochrome b558 (11). In the process, p47phox and Rac1/2 are introduced as positive effectors, whereas the p40phox function remains undetermined. In chronic granulomatous disease (CGD)1, there is no oxidase activity (12).
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 (13). 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 b558 (14). 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 b558, but also of a possible defect or constraint in the assembly process because of an unfavorable membrane environment (15). In fact, once cytochrome b558 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 (16). In
myeloid cells, they represent 45% of the cytosolic proteins in
neutrophils (17) and 1% in
monocytes. MRP8 and MRP14 are not expressed by resident macrophages, although
they may be present in macrophages of inflammatory lesions
(18). MRPs are known to be
markers of inflammation. Their expression by infiltrating neutrophils was
assumed to reflect activation stages
(19). In inflammation, high
levels of MRP8 and MRP14 were found in the extracellular medium of stimulated
neutrophils (20) and these
proteins could be markers of inflammatory diseases such as arthritis and
ulcerative colitis (17,
21). 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
(22). It has been postulated
that MRP8 and MRP14 participate in neutrophil migration, phagocytosis, and
activation (21). 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
(23). Several studies have
reported that both proteins also associate with membrane and cytoskeleton in a
calcium-dependent manner
(24).
Recent studies have suggested that MRP8 and MRP14 play a role in
potentiating activation of
-generating
oxidase in bovine neutrophils
(25). Doussière et
al. (26) 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
(27).
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 b558 and a change of conformation that initiated oxidase activation.
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EXPERIMENTAL PROCEDURES |
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Lymphoid Cell Lines and NeutrophilsCitrate-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 (31). 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 (32, 33). EBV-B lymphocytes from a p47phox-deficient CGD patient were kindly provided by M.-A. Pocidalo (INSERM U479, Hôpital Bichat, Paris, France).
Recombinant ProteinsFull-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--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-
-D-galactopyranoside-induced bacteria on
glutathione-Sepharose (11).
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
ComplexAnti-p47phox immunoglobulins were
immobilized onto a CarbolinkTM 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
(33,
34). The cytosol (50
100 mg, i.e. 25 x 109 cell eq) was
preincubated first with an uncoupled CarbolinkTM matrix and then with a
CarbolinkTM 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
N3-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 PurificationMRP8 and MRP14 were purified from the cytosol of unstimulated neutrophils as described (35). Briefly, neutrophil cytosol was submitted to 70% (w/v) (NH4)2SO4 precipitation. The 10,000 x 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.
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Oxidase Activity Reconstitution in a Cell-free Assay with Purified
Cytochrome b558Cytochrome
b558 was purified from the plasma membranes of
1010 PMA-stimulated neutrophils and from 1010 EBV-B
lymphocytes and relipidated with L--phosphatidylcholine II-S
as reported (11). It was then
quantified by reduced-minus-oxidized difference spectra using an absorption
coefficient of 106
mM1·cm1
(15). Oxidase activity was
reconstituted by incubating purified cytochrome b558
(0.215 pmol) with cytosol isolated from neutrophils or from EBV-B lymphocytes
(300 µg) in the presence of 10 µM FAD, 40 µM
GTP
S,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 (0.751.3 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 b558, and finally cytosol. In the
semi-recombinant system, the activity was reconstituted with purified
cytochrome b558 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 (
550 nm = 21.1
mM1·cm1) and expressed
as turnover, mol of
·s1·mol
of heme b1
(15).
High Resolution Two-dimensional Gel ElectrophoresisThe neutrophil cytosolic activating factors p47phox, p67phox, and p40phox, isolated as a complex on the CarbolinkTM affinity matrix (75 µg), were submitted to high resolution two-dimensional gel electrophoresis as reported (36). Proteins (75 µg) were precipitated with 7% (v/v) perchloric acid, washed with cold acetone, and solubilized in 2% (v/v) IPG buffer 310 containing 8 M urea, 4% (p/v) chaps, 18 mM DTT, and traces of bromophenol blue. The strips were rehydrated with the 100,000 x g ultracentrifugation supernatant (1 h, 20 °C). Proteins were first separated according to their isoelectric point along linear pH gradient strips (pH 310). 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 (37), or processed for mass spectrometry (MS) analysis.
Trypsin DigestionProtein bands of interest were excised from a Coomassie Blue-stained 11% SDS-PAGE (38) 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
SpectrometryMass 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
-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 (36). 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 ZipTipTM (Millipore). After elution with 510 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) ExperimentationFor AFM
experiments, the medium contained cytochrome b558 (0.2
pmol) purified from neutrophils or from EBV-B lymphocytes and incorporated
into L--phosphatidylcholine liposomes, 40 µM
GTP
S, 5 mM MgCl2, and 10 µM FAD
(11). 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 (11).
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 LymphocytesElectroporation 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 830TM, 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 ChemiluminescenceLymphocytes (5 x 105 to 2 x 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 (39). Photon emission was recorded at 37 °C for 1 h with a Luminoscan (Labsystem, Pontoise, France).
Polyclonal Antibodies against Cytosolic FactorsPolypeptides corresponding to the C-terminal region of p40phox (residues 325339) and the C-terminal regions of p47phox (residues 371390) and p67phox (residues 511526) were synthesized by Neosystem (Strasbourg, France). Anti-sera were used for Ig purification onto a 1-ml protein A-Sepharose CL-4B matrix (34).
SDS-PAGE and Western BlottingThe proteins were fractionated by 10 or 11% SDS-PAGE (38) and electrotransferred to nitrocellulose as previously described (40). Immunodetection was performed using polyclonal antibodies raised against MRP8 and MRP14, and specific polyclonal antibodies directed against p47phox, p67phox, or p40phox. The two subunits of cytochrome b558 were detected with mouse monoclonal antibodies directed against gp91phox (monoclonal antibody 54.1) and p22phox (monoclonal antibody 44.1). The immune complexes were detected with goat anti-rabbit secondary antibody coupled to alkaline phosphatase or peroxidase. The bound phosphatase activity was measured by staining with nitro blue tetrazolium. The bound peroxidase activity was detected using ECL reagents.
Biochemical AssaysProteins were measured with the Bradford assay using bovine serum albumin as a protein standard (41).
Statistical AnalysisData were given as mean ± S.D. 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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Because there are several isoforms of MRP8 and MRP14, the neutrophil-isolated complex was submitted to two-dimensional gel electrophoresis to determine the specific isoforms co-purifying with Phox proteins (Fig. 3A). Interestingly, two spots with a different isoelectric point were identified as MRP8 by immunoblot (Fig. 3B, left panel), whereas only one spot corresponded to MRP14 (Fig. 3B, right panel).
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MRP8/MRP14 Increases NADPH Oxidase Activity, in Vitro and ex
VivoMRP8 and MRP14 were purified from the cytosol of human
neutrophils in one step on a monoQ anion exchange matrix (FPLC)
(Fig. 1), as described under
"Experimental Procedures." The fraction eluted at 0.13
M NaCl was called purified MRP8/MRP14. The MRP effect on
reconstituted oxidase activity was investigated because they were present in
the complex purified from cytosol of neutrophils, but absent from that of B
cells. The purified MRP8/MRP14 preparation was first reactivated in the
presence of calcium; reconstitution was performed by adding MRP8/MRP14,
optimal amount of cytochrome b558 purified from
neutrophils, and the cytosol of EBV-B lymphocytes
(33). Results are shown in
Fig. 4A. Optimal
oxidase activity was measured in the presence of cytochrome
b558 and neutrophil cytosol. It corresponded to a turnover
of the reconstituted enzyme ranging close to 189 mol of
·s1·mol
of heme b1 (Fig.
4A, lane 5). The turnover decreased to 30
40% of the control when B-cell cytosol was used instead of neutrophil
cytosol (Fig. 4A,
lane 2 versus 5)
(33). The reconstituted
oxidase turnover became similar to the oxidase turnover measured with
neutrophil fractions when Ca2+-loaded MRP8/MRP14 was present
(Fig. 4A, lane 4
versus 5). Purified MRP8/MRP14 was preincubated with increasing
concentrations of calcium (0 1 mM), before being incubated
with cytochrome b558 and cytosol (data not shown), to
verify the calcium dependence of this effect. The results demonstrated that,
in the absence of calcium, MRP8/MRP14 had no impact on the oxidase activity.
The optimal amount of calcium-reactivated MRP8/MRP14 was determined with each
B-cell cytosol preparation used for the cell-free assay
(Fig. 4A,
inset). Furthermore, the results demonstrated that
Ca2+-loaded MRP8/MRP14 was able, on its own, to considerably
increase
production of relipidated and purified cytochrome b558 in
the absence of cytosol (Fig.
4A, lane 3 versus 1), suggesting a direct
MRPs/cytochrome b558 interaction. The absence of
p47phox, p67phox, and Rac from the
fraction containing the purified cytochrome b558
(Fig. 4B, left
panel) was assessed by Western blot and compared with positive control
(neutrophil cytosol) (Fig.
4B, right panel). Because of the sensitivity of
the immunoblotting assay, if potential contaminant cytosolic Phox proteins
were present, their concentration would be more than 108 times
inferior to the one necessary for cytochrome b558
activation in a cell-free assay.
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The specificity of the interaction between MRP8/MRP14 and cytochrome
b558 was investigated by adding purified neutrophil
cytochrome b558 to the reconstitution medium containing
Ca2+-loaded MRP8/MRP14, either after
(Fig. 5A, black
circles) or before (Fig.
5B, black circles) adding EBV-B lymphocyte
cytosol. In the absence of MRP8/MRP14, oxidase activity ranged between 50 and
70 mol of
·s1·mol
of heme b1 (Fig.
5, A and B, open squares),
40% of
control value, as previously shown (Fig.
4A, lane 3). Adding cytochrome
b558 to the reconstitution medium containing
Ca2+-loaded MRP8/MRP14 induced, by itself and in the absence of
EBV-B lymphocyte cytosol, a slight but significant increase in oxidase
activity (Fig. 5C,
black circles). This activity remained stable after 70 min of
incubation on ice. These data showed that, in the presence of MRP8/MRP14, a
different effect was observed depending on how cytochrome
b558 was added to the medium. A 2-fold increase in oxidase
turnover was observed when the cytochrome b558 fraction
was added first to the MRP8/MRP14 fraction, before adding cytosol
(Fig. 5B, black
circles). On the other hand, the activity was similar to that
reconstituted in the absence of MRP8/MRP14 when cytosol was incorporated
before cytochrome b558
(Fig. 5A, black
circle versus open squares). These results suggest a specific and
necessary interaction involving MRP8/MRP14 and relipidated cytochrome
b558 to restore a total oxidase activity in EBV-B cells.
Moreover, these data show that Ca2+-loaded-MRP8/MRP14, on its own,
is sufficient to obtain an active form of cytochrome
b558.
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Effect of Recombinant MRP8 and MRP14 on NADPH Oxidase Activity
Reconstituted in VitroA mixture (1/1) of recombinant MRP8 and
MRP14 (rMRP8/rMRP14), prepared in E. coli
(28,
29) and preloaded with
calcium, was used instead of MRP8/MRP14 purified from neutrophil cytosol, in
the reconstitution experiment that was performed with an optimal amount of
purified cytochrome b558 and cytosol from EBV-B
lymphocytes. Having rMRP8/rMRP14 in the system induced a 50% increase in
reconstituted oxidase activity (88 ± 7 versus 60 ± 5
mol of
·s1·mol
of heme b1)
(Fig. 6A, lane 6
versus 3). However, adding either rMRP8 or rMRP14 alone had no effect
(Fig. 6A, lanes
4 and 5). A significant rise in cytosol-independent activity was
again measured in a medium containing exclusively cytochrome
b558 and rMRP8/rMRP14
(Fig. 6A, lane 2
versus 1).
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A similar MRP effect was observed ex vivo in EBV-B lymphocytes co-transfected with plasmids pRc-CMV-MRP8 and pRc-CMV-MRP14 encoding MRP8 and MRP14, respectively (Fig. 6B). Transfection efficiency was determined by FACS analysis (FACScalibur; Becton Dickinson) and showed that, 48 h after transfection, 14% of the cells expressed the transfected construct. After 19 days of selection for G418 resistance, superoxide production was measured by chemiluminescence upon PMA stimulation. NADPH oxidase activity was optimal 31 days after transfection (Fig. 6B). Expression of either MRP8 or MRP14 alone in EBV-B cells did not significantly increase oxidase activity versus control value. Co-expression of MRP8 and MRP14 is required for enhancing the NADPH oxidase activity of B cells.
Conformation Change of Cytochrome b558
Mediated by MRP8/MRP14 The conformational change in
cytochrome b558 was investigated using AFM
(Fig. 7) to estimate the
potential interaction of Ca2+-loaded MRP8/MRP14 with cytochrome
b558 (Fig.
4, lane 3). AFM experiments were performed by incubating
cytochrome b558 liposomes with purified neutrophil
MRP8/MRP14 and addition of arachidonic acid for activation. The topography of
native cytochrome b558 liposomes was analyzed before and
after "activation." Liposome height was determined as previously
described (11), and reported
in Fig. 7A. First,
adding arachidonic acid to the medium containing cytochrome
b558 liposomes did not modify the liposome height,
whatever the cellular origin of cytochrome b558; liposome
height reached 6 nm. We found a similar distribution of cytochrome
b558 liposome height before and after the stimulus was
added (Fig. 7B,
left panel). The incubation of Ca2+-loaded MRP8/MRP14 with
cytochrome b558 purified from neutrophils or from EBV-B
cells, followed by the addition of arachidonic acid, led to an increase in
liposome height. Fig.
7B (right panel) clearly shows the difference in
the distribution of liposome height values after activation in the system
containing EBV-B cell cytochrome b558 and
Ca2+-loaded MRP8/MRP14. In the absence of calcium, there was no
significant conformation change upon activation (data not shown). A
statistically significant increase in liposome height was observed (8
versus 10 nm) (Fig.
7A) when MRP8/MRP14 was pre-incubated with arachidonic
acid without calcium, and then incubated with cytochrome
b558 liposomes. These results confirm that MRP8/MRP14
interact directly with cytochrome b558 purified from
neutrophils and from EBV-B cells. This interaction depends on the presence of
calcium and arachidonic acid. The effect of MRP8/MRP14 on the conformational
change in cytochrome b558, as confirmed by AFM, is related
to oxidase measurement in a cell-free assay. The results are presented in
Table II. Optimal oxidase
turnover was obtained with the system containing neutrophil fractions as
previously shown. Oxidase turnover was lower when EBV-B-cell cytosol replaced
neutrophil cytosol (56% versus 100%), but unchanged when cytochrome
b558 purified from EBV-B cells was used instead of
neutrophil cytochrome b558
(Table II and Ref.
33). AFM studies showed an
increase in liposome height after activation, indicating an assembly process.
This assembly occurred, whatever the origin of the cytosol used, and could be
fully active (in presence of neutrophil cytosol) or only partially functional
(in presence of EBV-B lymphocyte cytosol). Incubating neutrophil cytochrome
b558 with calcium-loaded MRP8/MRP14, before adding cytosol
from EBV-B lymphocytes, increased oxidase turnover up to 88% of the control
value, suggesting a synergistic effect of MRPs on cytosolic factors in terms
of assembly and conformational change of cytochrome b558
(Fig. 4A, lane
4). Moreover, the interaction of calciumloaded MRP8/MRP14 with cytochrome
b558, confirmed by AFM, was correlated to an increase in
oxidase turnover of purified cytochrome b558 (46
versus 17%, respectively). This effect was observed only when
MRP8/MRP14 was loaded with Ca2+. The preincubation of MRP8/MRP14
with arachidonic acid in the absence of Ca2+ led to an interaction
with cytochrome b558, but not to the conformational change
of the hemoprotein, which initiates oxidase activity.
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MRP8/MRP14 Behaves as a Positive Effector of Oxidase
ActivationThe data reported in the previous section suggest that
MRPs specifically interact with the redox component of NADPH oxidase complex.
We previously proposed an allosteric mechanism for the regulation of the
phagocyte oxidase and demonstrated that the transition from an inactive to an
active oxidase complex initiates the electron transfer from NADPH to oxygen.
In vivo, assembling regulatory factors (p67phox,
p47phox, and Rac) with cytochrome b558
is necessary to obtain an active enzyme. We have demonstrated that, in
vitro, p67phox is the limiting factor; it is involved
in both assembly and activation of oxidase
(11). In the present study, we
wanted to clarify the role of MRP8/MRP14 on oxidase activity at a molecular
level. We therefore assessed the effect of adding rMRP8/rMRP14 to a
semi-recombinant cell-free assay. Oxidase activity was reconstituted in a
medium containing purified cytochrome b558, rRac1, an
increasing concentration of rp67phox, and an optimal
amount of arachidonic acid (Fig.
8). A total reconstitution of oxidase activity was obtained at 200
nM rp67phox
(Fig. 8, open
squares). The rp67phox concentration necessary to
reconstitute an optimal oxidase activity was reduced by a factor of 3 and
5, respectively, when rMRP8/rMRP14 (Fig.
8, black squares) or rp47phox
(Fig. 8, open circles)
was present in the medium. The shift of the bell-shaped curves was maximum
when both rMRP8/rMRP14 and rp47phox were incorporated in
the medium, giving an optimal oxidase activity at 10 nM
rp67phox (Fig.
8, black circles). These data demonstrate that MRP8/MRP14
increased the affinity of p67phox for cytochrome
b558, as did p47phox.
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DISCUSSION |
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MRPs Control Phagocyte Oxidase ActivationThe properties of MRP8/MRP14 were investigated first in bovine neutrophils; they were shown to bind phenylarsine oxide and synergistically to activate NADPH oxidase (25). Furthermore, it was recently suggested that these proteins participate in NADPH oxidase activation, via a preferential association with the cytosolic activating factors of oxidase, mainly p67phox (26). In the present study, MRP8 and MRP14 were co-isolated on an affinity matrix with the soluble factors of neutrophil NADPH oxidase; both components were absent from the cytosol of EBV-B lymphocytes, and coincidentally, these cells displayed a very low oxidase activity compared with neutrophils (13). We demonstrated in vitro and ex vivo, after gene transfection, that complementation of B lymphocytes with MRP8 and MRP14 was sufficient to increase oxidase activity up to control values (Figs. 4 and 6). Moreover, we showed that both MRP8 and MRP14 were necessary in this process, suggesting that at least the dimer MRP8/MRP14 was involved.
There was no oxidase activity even though a normal amount of MRP8 and MRP14 was present (data not shown) in the neutrophils of CGD AR patients (12, 42). A preferential interaction between the MRP8/MRP14 complex and p67phox has been suggested in resting neutrophils (26). This may suggest a co-translocation mechanism. In CGD 47°, the absence of p47phox prevents translocation of p67phox (43), whereas in CGD 67°, p47phox is present and translocates on its own to the plasma membrane, without any MRP8/MRP14.
From these observations, we may conclude that, once translocated to the plasma membrane, the MRP8/MRP14 complex participates in the activation of oxidase as a positive effector of regulation.
MRP8/MRP14 Interaction with Cytochrome b558 Is Mediated by Arachidonic AcidMRPs need calcium to be active. MRPs are EF-hand molecules, which bind calcium selectively and with high affinity (17). The transitory elevation of cytosolic calcium, in response to inflammatory stimuli, initiates reaction cascades beginning in cytosol with phosphorylation of oxidase cytosolic factors, mainly p47phox, but also MRP14. After calcium binding, MRPs associate in a non-covalent oligomeric MRP8/MRP14 structure (heterodimer or heterotetramer) and translocate to the plasma membrane (44). In myelomonocytic cells, phosphorylation of MRP8 and MRP14 has been suggested as the main parameter of subcellular compartmentation (27, 45). The results tend to show that MRP8 and MRP14 must be assembled in an oligomeric structure to have a biological activity. A preferential translocation of phosphorylated MRP14 isoforms was reported (45) in response to a calcium signal.
Arachidonic acid has been suggested to mediate MRP association with membrane structures (46). In fact, a conformational change in the tertiary structure of the MRP8/MRP14 dimer, caused by an interaction with arachidonic acid, may improve its binding on membrane or extracellular surfaces. Arachidonic acid has been shown to enhance oxidase activity in vitro (47) and to induce a significant structural change in cytochrome b558 (48). In vivo, among its multiple functions in neutrophils, arachidonic acid has been reported to have a role in NADPH oxidase activation through phospholipase A2 (PLA2). Cytosolic PLA2 was recently introduced as the major PLA2 activated in stimulated phagocytic-like cell (49, 50). Moreover, transfection of antisense cytosolic PLA2 oligonucleotide completely abolished arachidonic acid release and NADPH oxidase activation in stimulated PLB985 cells, but the translocation of p47phox and p67phox was unaffected (49). These data suggest that arachidonic acid participates in the oxidase complex assembly in vivo. Biological concentrations of arachidonic acid require micromolar levels to elicit most biological activities (51). The binding of MRPs to arachidonic acid may raise its local concentration in the membrane and facilitate MRP interaction with cytochrome b558.
We have seen that both MRP8 and MRP14 were necessary to induce NADPH oxidase activation in our experiments, and that this effect was dependent on calcium and arachidonic acid (Figs. 4 and 6). Most of the S100 proteins formed homodimers, but when both MRP8 and MRP14 were expressed, the heterodimer MRP8/MRP14 was preferred (17, 52). More sensitive immunochemical labeling will be necessary to determine which MRP oligomeric structure is involved, but we can reasonably suggest that the heterodimer MRP8/MRP14 is indeed implicated.
Conformation Change of Cytochrome b558 Initiates Oxidase ActivationOther teams, as well as ours, have demonstrated in vitro that cytochrome b558 is the sole redox component and that p67phox is the limiting cytosolic factor (9, 10) that initiates both assembly of the complex and activation (11). An allosteric regulation mechanism was suggested in which p47phox behaves as a positive effector that increases the affinity of p67phox for cytochrome b558 (53, 54, 11). In vivo, phosphorylated p47phox serves as an adapter protein bringing p67phox into proximity with flavocytochrome b558 (55).
Successfully applying AFM to the structural analysis of oxidase complex assembly opened the way to exploring the structure-function relationships of cytochrome b558 incorporated into liposomes in the presence of MRP8/MRP14. The results of AFM cytochrome b558 topography assessment were correlated to oxidase turnover measured at the molecular level. Purified and relipidated cytochrome b558 displays a low but measurable level of NADPH oxidase activity in vitro in the absence of cytosolic factors: this activity depends on the nature of the phospholipid environment (56, 11). The data presented here show that, in vitro, the sole Ca2+-loaded MRP8/MRP14 modulates the transition from an inactive to an active conformation state of cytochrome b558 in the presence of arachidonic acid. Moreover, we found that adding MRP8/MRP14 to the cytosol of p67phox-deficient EBV-B lymphocytes partly restores oxidase activity, highlighting MRP8/MRP14 capacity to induce the transition between the resting and activated states of oxidase (data not shown).
When the reconstitution assay was performed with physiological effectors such as p67phox and p47phox, MRP8/MRP14 increased the affinity of p67phox for cytochrome b558 and potentiated the effect of p47phox. These findings suggest that MRPs directly interact at the molecular level with cytochrome b558; this association, which depends on calcium and arachidonic acid, induces reorganization in its structural conformation (Figs. 4 and 6, lanes 2) resulting in oxidase activation. These data suggest that MRP8/MRP14 will positively regulate NADPH oxidase activity in cells when cytosolic subunits of oxidase are in limited quantity.
In conclusion, conformation change of cytochrome b558 has been shown to initiate the electron transfer from NADPH to oxygen, and to generate superoxide anions. MRP8/MRP14 is suggested to mediate the transition from an inactive to an active conformation state of cytochrome b558 and to behave physiologically as positive mediators of oxidase regulation. The respective function and modality of binding of both MRPs in the allosteric process should be further investigated.
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FOOTNOTES |
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These authors contributed equally to this work.
** To whom correspondence should be addressed: GREPI EA 2938, Lab. Enzymologie, CHU Grenoble BP 217, 38043 Grenoble, cedex 9, France. Tel.: 33-4-76-76-54-83; Fax: 33-4-76-76-56-08; E-mail: frmorel.enzymo{at}chu-grenoble.fr.
1 The 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; GTPS, 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.
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ACKNOWLEDGMENTS |
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