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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2497-2504
Functional Analysis of NADPH Oxidase in Granulocytic Cells
Expressing a  488-497 gp91phox Deletion
Mutant
By
Lixin Yu,
Andrew R. Cross,
Ling Zhen, and
Mary C. Dinauer
From the Department of Pediatrics (Hematology-Oncology) and Medical
and Molecular Genetics, Herman B Wells Center for Pediatric Research,
Riley Hospital for Children, Indiana University School of Medicine,
Indianapolis, IN; and the Department of Molecular and Experiment
Medicine, The Scripps Research Institute, La Jolla, CA.
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ABSTRACT |
Chronic granulomatous disease (CGD) is a group of inherited
disorders in which phagocytes are unable to generate superoxide (O2 ) due to genetic defects in any 1 of 4 essential NADPH oxidase components. Mutations in the X-linked gene for
gp91phox, the large subunit of the flavocytochrome
b558 heterodimer, account for the majority of CGD.
An X-CGD patient in which a splice junction mutation results in an
in-frame deletion of 30 nucleotides encoding amino acids 488 to 497 of
gp91phox ( 488-497 gp91phox)
has previously been reported. In this study, we generated myeloid PLB-985 cells expressing the mutant 488-497
gp91phox to further characterize its functional
properties. These cells mimicked the phenotype of the patient's
neutrophils with normal expression of a nonfunctional 488-497
gp91phox flavocytochrome. Translocation of
p47phox and p67phox to
488-497 gp91phox PLB-985 plasma membranes was
not affected, as determined both in activated intact cells and in the
cell-free system. Furthermore, a synthetic peptide corresponding to
residues 488-497 of gp91phox was relatively
ineffective in inhibiting O2 production in
the cell-free oxidase assay (IC50, ~500 µmol/L), suggesting that
residues 488-497 of gp91phox are not directly
involved in oxidase assembly. Mutant 488-497 gp91phox flavocytochrome failed to support
iodonitrotetrazolium (INT) reduction, showing a disruption of electron
transfer from NADPH to the FAD center of gp91phox.
However, the FAD binding capacity of the mutant flavocytochrome was
normal, as measured by equilibrium dialysis. Taken together, these
results suggest that the 488-497 deletion in
gp91phox disrupts electron transfer to FAD, either
due to a defect in NADPH binding or to impaired delivery of electrons
from NADPH.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PHAGOCYTES PLAY a critical role in host
defense by producing reactive oxygen species against invading
microorganisms. One of the most important enzymes in producing
microbicidal oxidants is the superoxide
(O2)-generating NADPH
oxidase.1 The NADPH oxidase is a multicomponent enzyme
complex whose redox center is a membrane-associated flavocytochrome b558 heterodimer composed of
gp91phox and p22phox. In
addition, three cytosolic oxidase subunits,
p47phox, p67phox, and a low
molecular weight GTP binding protein Rac, are required for high level
production of O2. In resting phagocytes,
the dormant oxidase is unassembled. However, upon phagocyte activation,
the active oxidase complex is rapidly formed by translocation of the
cytosolic oxidase components to the plasma membrane via interactions
with the cytochrome.2 Subsequently, electrons are
transferred from cytosolic NADPH to molecular oxygen (O2)
at the external face of the membrane to generate
O2.3
Genetic deficiency of NADPH oxidase activity results in chronic
granulomatous disease (CGD), a rare inherited disorder of host defense.
Patients with CGD develop recurrent, often life-threatening bacterial
and fungal infections due to impaired microbicidal oxidant generation
by the patient's phagocytes. CGD is caused by genetic defects in any 1 of the 4 oxidase components, p47phox,
p67phox, gp91phox, and
p22phox. Mutations in the X-linked gene for
gp91phox account for approximately two thirds of
CGD, with the remaining cases due to autosomal recessive mutations in
the genes encoding p22phox,
p47phox, or
p67phox.1
The NADPH oxidase catalyzes the transfer of electrons from the
substrate NADPH to O2, via intermediate flavin (FAD) and
heme prosthetic groups, to produce
O2.3,4 The
gp91phox polypeptide appears to be the oxidase
subunit responsible for mediating electron transfer. We recently have
shown that the 2 heme groups incorporated into the cytochrome
heterodimer are located within
gp91phox.5 The carboxyl terminus of
gp91phox contains homologies to consensus FAD and
NADPH binding domains of members of ferredoxin-NADP+
reductase (FNR) family,6-8 although
p67phox has also been reported recently to contain
a functional NADPH-binding site and may also participate in the NADPH
binding.9,10 Coexpression of both
gp91phox and p22phox subunits
are required to assemble a functional flavocytochrome capable of
supporting O2 production.5
In addition, expression of gp91phox in phagocytes
is stabilized by association with its partner
p22phox.11,12
The majority of missense mutations or in-frame deletions identified in
X-CGD result in apparent instability of the
gp91phox polypeptide, with either absent or
markedly reduced expression of the mutant flavocytochrome
b558. Rare mutations in which expression of
flavocytochrome b558 is preserved have been
informative in identifying important structural-function relationships
of the cytochrome.13-15 An X-CGD patient in which a splice
junction mutation results in an in-frame deletion of 30 nucleotides
encoding amino acids 488 to 497 of gp91phox
( 488-497 gp91phox) has previously been
reported.16 A detailed functional analysis of the mutant
cytochrome could not be performed due to the death of the patient. In
this study, we stably transfected the 488-497 gp91phox cDNA into X-CGD PLB-985 cells, which lack
endogenous gp91phox expression due to gene
targeting,17 to create a cell line expressing the mutant
488-497 gp91phox flavocytochrome. This approach
allowed us to perform functional studies on 488-497
gp91phox PLB-985 cells to further characterize the
defect resulting in failure of O2
production. We found that deletion of gp91phox
residues 488-497 did not affect translocation of the cytosolic subunits
p47phox and p67phox to plasma
membranes of activated 488-497 gp91phox PLB-985
cells. However, mutant 488-497 gp91phox
flavocytochrome failed to support iodonitrotetrazolium (INT) reduction,
showing a defect of the proximal electron transfer pathway from NADPH
to the FAD center of gp91phox. Partially purified
488-497 gp91phox had a normal capacity for FAD
binding as determined by equilibrium dialysis against FAD. These
results suggest that the 488-497 deletion disrupted electron
transfer from NADPH to FAD, either due to a defect in NADPH binding or
to impaired electron delivery from NADPH.
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MATERIALS AND METHODS |
In vitro mutagenesis and expression of recombinant
gp91phox in promyelocytic PLB-985 cells.
The 488-497 gp91phox cDNA, which has an in-frame
deletion of 30 nucleotides,16 was generated by
oligonucleotide-directed mutagenesis by using the Sculptor in vitro
mutagenesis kit (Amersham, Arlington Heights, IL). The
mutant cDNA was verified by dideoxynucleotide sequencing and then
subcloned into the Not I site of the mammalian expression
vector, pEF-PGKpac. The pEF-PGKpac vector contains a mammalian EF-1
promoter to drive constitutive gp91phox expression
and a linked expression cassette for puromycin-N
acetyltransferase.18 In parallel, the vector containing the
full-length human wild-type (WT) gp91phox cDNA was
also constructed. The WT or the 488-497
gp91phox-containing vectors were transfected by
electroporation into X-CGD PLB-985 cells.17 Clones were
selected by limiting dilution in the presence of puromycin (1 µg/mL).
To minimize any potential clone-to-clone variation in recombinant
gp91phox expression or NADPH oxidase activity, 3 independent clones determined to express relatively higher levels of
recombinant gp91phox were pooled and used for
subsequent analysis.
Cell culture and granulocytic differentiation.
X-CGD PLB-985 cells (X-CGD), transfected PLB-985 cells expressing WT,
or the deletion mutant (488-497) gp91phox were
maintained in RPMI 1640 medium containing 10% fetal calf serum and 2 mmol/L L-glutamine. To induce expression of endogenous NADPH oxidase
subunits, cells were differentiated for 5 days by exposure to 0.5%
dimethylformamide (DMF). Under these conditions, more than 80% of the
cells had undergone granulocytic differentiation as determined by
observation of morphological changes and nitroblue tetrazolium (NBT)
test.12
Immunoblot and confocal microscopy analysis of recombinant
gp91phox expression.
To evaluate cell surface expression of flavocytochrome
b558, immunostaining with the 7D5 monoclonal
antibody was performed as described.5,18 After staining,
3,000 to 5,000 cells were deposited on glass slides by centrifugation
at 450 rpm for 5 minutes and were observed by confocal microscopy.
Expression of recombinant gp91phox and
p22phox was also determined by immunoblotting as
described previously.12
Translocation analysis of cytosolic oxidase components.
Granulocyte-differentiated cells (1 × 108) were
collected, washed, and then resuspended in 1 mL of relaxation buffer
consisting of 10 mmol/L PIPES, pH 7.3, 100 mmol/L KCl, 3.5 mmol/L
MgCl2, 3 mmol/L NaCl, and 1 mmol/L EGTA. To activate the
NADPH oxidase assembly, cells were treated with either phorbol
12-myristate 13-acetate (PMA) or dimethyl sulfoxide (DMSO)
vehicle control at a final concentration of 500 ng/mL for 10 minutes at
37°C. After the addition of 12 mL of cold phosphate-buffered saline (PBS) to stop the activation, cells were pelleted,
resuspended in 1.5 mL of the relaxation buffer, and disrupted by
sonication for 3 times at 6 seconds each at 20% power at
4°C. Subsequently, the sonicates were spun for 8 minutes at
500g, followed by 10 minutes at 2,000g. A total
of 0.75 mL of the resulting supernatant was loaded on a
discontinuous sucrose gradient (1.5 mL of 20% over 1.5 mL of 38%) and
centrifuged at 41,000 rpm (204,275g) in an SW55 rotor (Beckman
Instruments, Fullerton, CA) for 40 minutes at 4°C.
After centrifugation, the 0.6 mL fraction of the top of the gradient
was collected as cytosol, and a distinct band located in the interface
of the 20% and 38% sucrose gradient was collected as the plasma
membranes (~0.6 mL). To remove sucrose, the membranes were mixed with
3.5 mL of cold PBS and centrifuged at 55,000 rpm (368,000g) for
30 minutes, and the resulting pellets were resuspended in 100 µL of
relaxation buffer. Translocation of cytosolic oxidase components
p47phox and p67phox to plasma
membrane was detected by immunoblotting as described previously.12 Translocation assay was also performed in a
cell-free system. Briefly, 100 µg of cellular membranes was mixed
with 300 µg of cytosol isolated from granulocyte-differentiated cells
indicated in the figure legends in 0.5 mL of relaxation buffer
containing 10 µmol/L GTP s. After the addition of 100 µmol/L
sodium dodecyl sulfate (SDS), the mixture was incubated for 5 minutes
at 37°C and then centrifuged for 30 minutes at 55,000 rpm
(368,000g) at 4°C. The resulting pellets were collected and
used for immunoblotting.
Measurement of NADPH oxidase activity.
O2 production by
granulocyte-differentiated cells was measured both in whole cells and
in the cell-free oxidase assay by monitoring the reduction of
cytochrome c at 550 nm using a Thermomax microplate reader.12 In the assay using intact cells, PMA at a final
concentration of 0.1 µg/mL was used to activate the NADPH oxidase of
granulocyte-differentiated cells (a total of 2.5 × 105 cells in 200 µL of volume in a well). The cell-free
assays using SDS as activator were performed as described
previously,19,20 using flavocytochrome
b558 partially purified from membranes of the
PLB-985 cell lines and cytosol from neutrophil or
granulocyte-differentiated PLB-985 cells. Membrane and cytosolic
fractions were prepared by continuous centrifugation followed by cell
disruption by sonication.21 Michaelis-Menton kinetics were
analyzed using GraphPad Prism (San Diego, CA). For peptide inhibition
assays, peptides were dissolved in assay buffer and added to the
reaction mixture before the addition of SDS (100 µmol/L). Protein
concentration was determined by BCA assay (Pierce, Rockford,
IL). INT reductase activity was measured as described
previously.20
Purification, relipidation, and reflavination of flavocytochrome
b558.
Flavocytochrome b558 was partially purified from 3 × 109 cell equivalents of salt-washed PLB-985 cell
membranes using the method described previously for flavocytochrome
b558 purification from neutrophil
membranes.22 This method uses mixed-bed (carboxylmethyl [CM], diethyl aminoethyl [DEAE] Sepharose CL-6B,
amin-octyl agarose) and heparin chromatography. Relipidation and
reflavination of the partially purified flavocytochrome
b558 was also performed as described previously
using phosphatidylcholine (type IIS; Sigma, St Louis,
MO). For the determination of FAD binding constants, portions of the partially purified cytochrome were relipidated in the
absence of FAD.
Spectroscopy.
Reduced minus oxidized difference spectra of detergent-solubilized
membranes and partially purified flavocytochrome
b558 were recorded as described previously using a
Perkin-Elmer Lamda 18 spectrophotometer (Perkin-Elmer, Norwalk,
CT).13
Determination of the affinity of flavocytochrome
b558 for FAD.
The dissociation constant for FAD binding by the flavocytochrome
b558 preparations was determined by equilibrium
dialysis using Sialomed equilibrium dialyzers (AmiKa Corp, Columbus,
MD). One-hundred-microliter aliquots (130 to 150 nmol/L in
concentration) of partially purified flavocytochrome
b558 samples that had been relipidated (but not
reflavinated) were placed in one side of the dialysis chamber and
dialyzed against 100 µL of the same buffer containing 100 nmol/L FAD.
FAD standards were made by serial dilution of a freshly prepared stock
solution of FAD. The concentration of the stock solution was determined
from the absorbance at 450 nm using an extinction coefficient of 11.3 mmol/L1. All FAD-containing solutions were protected
from light. The concentration of flavocytochrome
b558 was determined spectrophotometrically using an
extinction coefficient of 21.6 mmol/L1 at 559 nm for
the reduced-minus oxidized heme (10.8 mmol/L1 per
mole flavocytochrome b558).22 Samples
were dialyzed on ice for 4 hours, and the contents of each chamber were
removed for FAD analysis. All samples and FAD standards were heated in a 100°C water bath for 3 minutes to release enzyme-bound FAD and centrifuged to remove denatured protein.
FAD was estimated using a modification of the method of Hinkkanen and
Decker.23 The assay mixture consisted of 80 to 100 µL of
sample (or FAD standard), 20 mmol/L 3, 5-dichlorobenzene sulfonic acid,
200 µmol/L 4-amino antipyrene, 4 U/mL horseradish peroxidase, 0.2 U/mL apo-D-amino acid oxidase, and 35 mmol/L D-proline in a total
volume of 250 µL 100 mmol/L Tris, pH 8.6. The assay was performed at
37°C and the rate of formation of
N-(4-antipyryl)-3-chloro-5-sulfonate-p-benzoquinone monoimine
was observed for 60 minutes by measuring the increase in absorbance at
512 nm. The FAD concentration of the samples was calculated from a
standard curve of 0 to 200 nmol/L FAD plotted against maximum rate of
A512. The Kd (FAD) for each sample was calculated from the final concentrations of FAD in the sample and
buffer compartments, and the concentration of flavocytochrome b558 was added.
| |
RESULTS AND DISCUSSION |
488-497 gp91phox PLB-985 cells
mimic the phenotype of X-CGD 488-497
gp91phox neutrophils.
Stable expression of recombinant 488-497
gp91phox in X-CGD PLB-985 cells mimicked the
phenotype originally reported for neutrophils isolated from an X-CGD
patient with the same 488-497 deletion in
gp91phox.16 Expression of 488-497
gp91phox in transfected PLB-985 cells was examined
by immunoblotting (Fig 1B). The level of
recombinant 488-497 gp91phox was similar to that
of recombinant WT gp91phox expressed in PLB-985
cells. A marked increase in expression of p22phox
was seen in both transgenic 488-497 gp91phox and
WT gp91phox PLB-985 cells (Fig 1B). This is
consistent with previous observations indicating that coexpression of
both gp91phox and p22phox and
subsequent heterodimer formation is important for stable expression of
each flavocytochrome b558
subunit.11,12,24 We have previously shown that the
transgenically expressed recombinant WT gp91phox is
processed and targeted normally into the plasma membrane in promyelocytic PLB-985 cells.18 To determine whether
488-497 gp91phox was expressed in the plasma
membranes, transfected cells were stained with 7D5, a monoclonal
antibody that interacts with an extracellular epitope of
gp91phox, and examined by confocal microscopy. As
shown in Fig 1A, membrane surface staining was present in both
488-497 gp91phox as well as WT
gp91phox PLB-985 cells. No positive signal above
the background was obtained in X-CGD PLB-985 cells, consistent with the
absence of gp91phox in the cells (Fig 1B).
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| Fig 1.
Expression of recombinant WT gp91phox
and 488-497 gp91phox flavocytochrome
b558 in transgenic PLB-985 cells. X-CGD PLB cells
were transfected with either WT or 488-497
gp91phox cDNAs, and the expression of recombinant
gp91phox/p22phox heterodimer
was examined by immunoblotting (B). Five micrograms of cellular
membranes was loaded. (A) Confocal microscopy observation of membrane
surface expression of recombinant gp91phox. The
indicated cells were stained with the gp91phox
monoclonal antibody, 7D5, as described previously,18 and
mouse IgG1 was used as an isotype control. Imaging amplifications:
×360 for 7D5 staining and ×148 for IgG1 staining.
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We next performed reduced minus oxidized difference spectroscopy on
transgenically expressed flavocytochrome b558
partially purified from membranes isolated from 488-497
gp91phox, WT gp91phox, and
X-CGD PLB-985 cells. Virtually identical spectra characteristic of
flavocytochrome b558 were seen for both the
488-497 gp91phox and WT
gp91phox flavocytochrome preparations,
demonstrating the normal incorporation of heme groups in the
gp91phox deletion mutant
(Fig 2). As expected, X-CGD samples lacked
specific absorption at 558 nm (Fig 2).
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| Fig 2.
Reduced minus oxidized difference spectrum of
flavocytochrome b558 samples partially purified
from membranes of X-CGD, WT gp91phox, and
488-497 gp91phox PLB-985 cells. Flavocytochrome
b558 samples were partially purified from cellular
membranes of the indicated cells and dithionite-reduced minus oxidized
difference spectrum of the samples were analyzed as described in the
Materials and Methods. Results shown are from one representative of
triplicate analyses.
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NADPH oxidase activity in 488-497 gp91phox
PLB-985 cells was determined in both intact cells and in the cell-free
oxidase assay. After granulocytic differentiation for 5 days to induce
the expression of the endogenous p47phox and
p67phox oxidase subunits, WT
gp91phox PLB-985 cells produced
O2 after stimulation with PMA, as
expected (Table 1). In contrast, 488-497
gp91phox PLB-985 cells were unable to generate
O2 (Table 1). To confirm that the
cellular defect in the NADPH oxidase in 488-497
gp91phox PLB-985 cells was related to the mutation
in gp91phox, cell-free oxidase assays were
performed using combinations of cytosol and membranes prepared from WT
gp91phox and 488-497
gp91phox PLB-985 cells. As shown in
Table 2, membranes isolated from 488-497
gp91phox PLB-985 cells failed to support
O2 generation in combination with
cytosol from either WT gp91phox or 488-497
gp91phox PLB-985 cells, demonstrating that absence
of NADPH oxidase activity resulted from a defect in the cellular
membranes containing the mutant flavocytochrome
b558.
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Table 2.
Superoxide Production in the Cell-Free Oxidase Assay
Using Cellular Membranes and Cytosol Isolated From Transgenic
PLB-985 Cells
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Assembly of the NADPH oxidase by translocation of
p47phox and p67phox to the
plasma membrane is not affected by the 488-497 deletion
in gp91phox.
Upon phagocyte activation, cytosolic oxidase components translocate to
the plasma membrane to assemble the functional NADPH oxidase. Multiple
contact points between p47phox and the
gp91phox and p22phox subunits
of flavocytochrome b558 have been described
previously.14,25-29 Among these, a missense mutation
predicting an Asp-Gly substitution at residue 500 of
gp91phox has been reported to lead to defective
translocation of p47phox and
p67phox.14 The proximity of the
488-497 deletion in gp91phox to Asp500 prompted
us to test whether translocation of p47phox and
p67phox to the plasma membrane during oxidase
assembly was affected by this deletion. As shown in
Fig 3A, PMA-stimulated translocation of
p47phox and p67phox to the
plasma membrane in intact 488-497 gp91phox
PLB-985 cells was similar to that seen for WT
gp91phox PLB-985 cells. A similar result was
obtained in a cell-free oxidase reconstitution system using SDS for
activation (Fig 3B). The band seen below the 68 kD marker protein is
the high mannose 65-kD precursor of
gp91phox.24 We have previously shown
that this species is localized in the ER, as determined by cell
fractionation using a 10% to 60% continuous sucrose
gradient.24 The membranes isolated by discontinuous sucrose
gradient (20% and 38%) in the current experiment may be contaminated
with intracellular membranes including ER, which may account for the
presence of the precursor in the preparation (Figs 1B and 3B). A small
amount of p47phox but not
p67phox was seen in WT and 488-497
gp91phox-transfected PLB-985 granulocytes even in
the absence of PMA, as well as in X-CGD PLB-985 cells (Fig 3A and B).
This likely reflects nonspecific binding of p47phox
to membranes, which has also been observed by others.30
Alternatively, an unexpected priming of PLB-985 granulocytes during
culture at 37°C may cause the translocation of
p47phox to the membrane, which is
flavocytochrome-independent.
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| Fig 3.
Translocation of p47phox and
p67phox to plasma membrane in intact cells
activated with PMA and in the cell-free oxidase reconstitution assay
stimulated with SDS. (A) The indicated cells undergone granulocytic
differentiation for 5 days were stimulated with (+) or without ( )
PMA (500 ng/mL) for 10 minutes at 37°C, and the plasma membranes
were prepared on discontinuous sucrose gradients and analyzed for
translocation of p47phox and
p67phox by immunoblot analysis using
p47phox and p67phox antibodies
(left panel). After stripping, the blots were reprobed with
gp91phox and p22phox monoclonal
antibodies to show an equal loading (right panel). Each lane was loaded
with 5 µg of proteins. (B) Membranes separated from the indicated
cells were mixed with 3-fold of neutrophil cytosol in the cell-free
oxidase reconstitution assay. After 10 minutes of incubation at
25°C in the presence (+) or absence () of 100 µmol/L SDS,
the membranes were reisolated by ultracentrifugation and detected for
the translocation of p47phox and
p67phox (left panel) by immunoblot analysis as
described in (A). Each lane was loaded with 5 µg of proteins.
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To provide additional evidence that gp91phox
residues 488-497 are not essential for oxidase assembly, we synthesized
a peptide corresponding to residues 488-497 and tested its ability to
inhibit O2 production in the cell-free
oxidase assay using membrane and cytosol isolated from normal
neutrophils. As shown in Fig 4, peptide 488-497 inhibited oxidase activity only at high concentrations (IC50,
~500 µmol/L), whereas a peptide derived from residues 86-102 of
gp91phox (containing a probable
p47phox binding motif28,29) had an IC50
of 2 µmol/L (Fig 4). The 488-497 peptide was also much less potent at
inhibiting O2 production compared with
other peptides derived from gp91phox domains
proposed as binding sites for cytosolic oxidase components, including
peptide 491-504 containing Asp500 (IC50, 10 µmol/L),14 peptide 559-570 (IC50, 28 µmol/L),31 and peptide 550-569 (IC50, 4 µmol/L).32 Consistent with our results,
Kanegasaki's group has reported that IC50 of peptide 484-502 was
greater than 300 µmol/L.32
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| Fig 4.
Effect of peptide 488-497 on NADPH oxidase activity in
the cell-free oxidase assay. Plasma membrane (8 µg) and cytosolic
fractions (20 µg) separated from normal neutrophils were used in the
cell-free assay. Peptide 488-497 of gp91phox ( )
as well as a control peptide corresponding to residues 86-102 of
gp91phox containing a putative
p47phox binding site 86-93 ( ) were added to the
assay before the addition of SDS, and O2
generation was measured. The activity of superoxide production in the
absence of peptides was 216 ± 22 nmol/min/mg of membrane proteins.
The data represent the mean ± SD of 3 separate experiments.
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Electron transport from NADPH to FAD is disrupted in
488-497 gp91phox.
We have previously shown that electrons are transferred to INT
primarily from the flavin center of
gp91phox.4 We have used this method to
show that the proximal electron transfer pathway (from NADPH to flavin)
is normal in a mutant form of gp91phox in which
there is an amino acid substitution affecting one of the heme redox
potentials.13 It was therefore of interest to see if the
proximal electron transport pathway of the 488-497 mutant was
functional. Partially purified flavocytochrome b558 from membranes of WT gp91phox PLB-985 cells was
able to support INT reductase activity as efficiently as
flavocytochrome b558 purified from neutrophils (not
shown). In contrast, flavocytochrome b558 from
488-497 gp91phox PLB-985 cells was incapable of
INT reductase activity, suggesting that either flavin or NADPH binding
is affected in this mutant. As expected, the equivalent purification
fraction from X-CGD PLB-985 cells also had no activity
(Fig 5).
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| Fig 5.
INT reductase activity in flavocytochrome
b558 purified from membranes of X-CGD, 488-497
gp91phox, and WT gp91phox
PLB-985 cells. INT reductase activity was determined in the 96-well
microtiter plate assay as described in the Materials and Methods. Each
well contained the equivalent of 0.5 pmol flavocytochrome
b558 and 2.5 × 106 cell equivalents
of neutrophil cytosol. The results are expressed as the mean ± SEM.
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The 488-497 mutant flavocytochrome
b558 has a normal affinity for FAD.
To evaluate the capacity of the 488-497 gp91phox
mutant to bind FAD, we measured the affinity of the partially purified
flavocytochrome b558 preparations for FAD by
equilibrium dialysis as described in the Materials and Methods. Both WT
gp91phox and 488-497
gp91phox flavocytochrome preparations had virtually
identical affinities for FAD of approximately 66 nmol/L
(Table 3). These values are consistent with
the literature values of 20 to 85 nmol/L.33-35 An
equivalent volume of the X-CGD PLB fraction eluted from the heparin
column that corresponded to the peak fractions of the 488-497
gp91phox and WT gp91phox
flavocytochrome b558 showed no ability to bind FAD.
In all cases, the recovery of FAD from each equilibrium
dialysis experiment (sample + buffer) was 100% ± 4%.
Analysis of NADPH binding in the 488-497
gp91phox flavocytochrome b558.
Residues 488-497 have been postulated to lie near the NADPH binding
domain of gp91phox.36 To address
whether the 488-497 mutation alters the affinity of flavocytochrome
b558 for NADPH, we measured the Km for
NADPH on mutant flavocytochrome partially purified from membranes of 488-497 gp91phox PLB-985 cells. The NADPH
oxidase has the ability to use NADPH or NADH as substrate, although the
Km for NADPH is approximately 10-fold lower (~40
µmol/L).37-39 The Km for NADPH of the enzyme in the cell-free system using neutrophil membranes was found to be 57.1 ± 1.8 µmol/L (n = 22), and the Km of purified
neutrophil flavocytochrome b558 was 27.5 ± 1.3 µmol/L (n = 17; A. Cross, unpublished data). The
Km of the flavocytochrome purified from membranes of WT
gp91phox PLB-985 cells was found to be 30.4 ± 1.5 µmol/L (not shown). No activity was evident in the cytochrome
purified from the 488-497 gp91phox PLB-985
cells. Increasing the substrate concentration to as high as 4.9 mmol/L
NADPH did not induce any O2 formation
from the 488-497 mutant and neither did the addition of 4.9 mmol/L
NADH (Fig 6). This suggests that either the
488-497 gp91phox cannot bind the substrate NADPH
or it cannot support electron transfer from NADPH to FAD.
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| Fig 6.
O2 generating activity of
flavocytochrome b558 purified from membranes of
X-CGD, 488-497 gp91phox, and WT
gp91phox PLB-985 cells. Cell-free
O2 assays were performed as described in the
Materials and Methods using the equivalent of 0.5 pmol flavocytochrome
b558 per well and 2.5 × 106 cell
equivalents of neutrophil cytosol. ( ) The maximum rate of
O2 production with 4.9 mmol/L NADPH as
substrate (mean ± SEM); ( ) the maximum rate of
O2 production with 4.8 mmol/L NADH as
substrate.
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|
Over the past 10 years, several groups have tried to identify the NADPH
binding component of the oxidase complex by labeling with
32P- or 3H-labeled NADPH analogues. However,
different results have been reported by different groups, including a
66-kD cytosolic protein,40 an approximately 32-kD cytosolic
protein,41 a 52-kD membrane-associated protein,42 and p67phox,9
therefore leaving the issue still uncertain. By comparison with known
NADPH binding regions of members of Ferredoxin-NADP+
Reductase family, Segal's group,6 Rotrosen et
al,8 and Sumimoto et al7 have proposed that the
apparent NADPH binding pocket resides in the carboxyl terminal portion
of gp91phox. This postulation has subsequently been
strongly supported by the experiments showing that reflavinated and
relipidated membrane fractions isolated from normal neutrophils are
capable of supporting O2 generation in
the absence of any cytosolic proteins.43,44
We attempted to analyze the ability of the 488-497 mutant
flavocytochrome b558 to bind NADPH by affinity
labeling using the photoaffinity label
[4-N-(4-azido-2-nitrophenyl) aminobutyryl] NAD[32P].45 Despite using a number of
different experimental conditions, it was not possible to convincingly,
or reproducibly, label WT gp91phox from either
neutrophils or WT gp91phox PLB-985 cell membranes.
Therefore, we were not able to unambiguously determine if the
488-497 mutant can bind NADPH.
Taylor et al36 have predicted a 3-dimensional structure of
gp91phox using Ferredoxin-NADP+
Reductase as a template and proposed that an -helical loop composed of residues 484-503 lies over the NADPH binding cleft. During oxidase
activation, access of NADPH into the binding site could potentially be
regulated by interactions of this loop with cytosolic oxidase
components.36 Although the impaired translocation of cytosolic oxidase subunits reported for a gp91phox
mutant with Asp500Gly substitution indirectly supports this
hypothesis,14 we found no evidence that
gp91phox residues 488-497 are required for
interactions with cytosolic oxidase components during oxidase assembly.
Deletion of these residues did not affect translocation of
p47phox and p67phox, and the
peptide corresponding to residues 488-497 was a weak inhibitor of
superoxide production in the cell-free oxidase assay. It therefore is
likely that the 488-497 deletion in gp91phox either
leads to a failure of NADPH to bind or a defect in the subsequent
transfer of electrons to FAD. Either would be consistent with the
observed absence of INT reductase activity for the 488-497 gp91phox flavocytochrome b558.
The data presented here further imply that simply removing the loop is
insufficient to expose a functional NADPH binding site.
The majority of patients with CGD present in early childhood with
severe, recurrent bacterial and fungal infections. However, the CGD
patient with the 488-497 gp91phox mutation was
in good health until 69 years of age, when he developed an infection
due to Burkholderia cepacia.16 Patient neutrophils were reported to have trace amounts of
O2 production, which may have accounted
for this milder phenotype, although we could not confirm the presence
of residual O2-generating activity in
488-497 gp91phox PLB-985 cells. Also, note that
the grandson of this patient died at 5 years of age due to
Burkholderia cepacia pneumonia,16 suggesting that
other factors influencing host defense may have accounted for the late
age of presentation in the index patient.
| |
ACKNOWLEDGMENT |
The authors thank Drs Dirk Roos and Arthur J. Verhoeven (Central
Laboratory of The Netherlands Blood Transfusion Service, Amsterdam, The
Netherlands) for kindly providing
anti-gp91phox and anti-p22phox
monoclonal antibodies 48 and 449, respectively. In addition, Dr David
Lambeth (Emory University, Atlanta, GA) provided
polyclonal anti-p47phox and Dr Paul Heyworth (The
Scripps Research Institute, La Jolla, CA) provided
polyclonal anti-p67phox antibodies. The
anti-flavocytochrome b558 monoclonal antibody 7D5
was a generous gift from Dr Michio Nakamura (Nagasaki University, Nagasaki, Japan).
| |
FOOTNOTES |
Submitted March 18, 1999; accepted May 28, 1999.
Supported in part by Grants No. RO1 HL45635 and PO1 HL 353586 to M.C.D.
and AI-24838 to A.R.C.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Mary C. Dinauer, MD, PhD, Wells Center for
Pediatric Research, Cancer Research Bldg, Room 466, 1044 W Walnut St,
Indianapolis, IN 46202; e-mail: mdinauer{at}iupui.edu.
| |
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ABSTRACT
INTRODUCTION