From the Herman B Wells Center for Pediatric Research, Departments
of Pediatrics (Hematology/Oncology) and Medical and
Molecular Genetics, James Whitcomb Riley Hospital for Children, Indiana
University School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
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Site-directed mutagenesis was used to generate a
series of mutants harboring point or multiple substitutions within the
hydrophilic, polybasic domain of gp91phox encompassed by
residues 86-102, which was previously identified as a site of
interaction with p47phox during phagocyte NADPH oxidase
assembly. Recombinant wild-type or mutant gp91phox was
expressed in a human myeloid leukemia cell line in which the endogenous
gp91phox gene was disrupted by gene targeting. NADPH oxidase
activity was measured in a cytochrome c reduction assay
following granulocytic differentiation of cells that expressed
recombinant gp91phox. Expression of a gp91phox mutant
in which amino acids 89-97 were replaced with nine alternate amino
acids abolished NADPH oxidase activity. Expression of gp91phox
mutants R89T, D95A, D95R, R96A, R96E, or K102T did not significantly affect NADPH oxidase activity. However, mutations of individual or
paired arginine residues at positions 91 and 92 had substantial effects
on superoxide generation. The R91E/R92E mutation completely abolished
both NADPH oxidase activity and membrane-translocation of the cytosolic
oxidase proteins p47phox, p67phox, Rac1, and Rac2. The
phorbol 12-myristate 13-acetate-induced rate of superoxide production
was reduced by ~75% in cells expressing R91T/R92A, R91E, or R92E
gp91phox along with an increased lag time to the maximal rates
of superoxide production relative to cells expressing wild-type
gp91phox. Taken together, these results demonstrate that
Arg91 and Arg92 of gp91phox are
essential for flavocytochrome b558 function in
granulocytes and suggest that these residues participate in the
interaction of gp91phox with the cytosolic oxidase proteins.
The phagocyte NADPH oxidase (respiratory burst oxidase) is a
multicomponent enzyme that catalyzes the transfer of electrons from
NADPH to molecular oxygen to form superoxide, a precursor of toxic
oxidants that are important for host defense against microorganisms.
The redox site of the oxidase is flavocytochrome b558, which is a membrane-associated heterodimer
composed of gp91phox and p22phox. NADPH oxidase
activity also requires the cytosolic proteins p47phox,
p67phox, and either of the small G-proteins Rac1 or Rac2
(reviewed in Refs. 1 and 2). The p47phox and p67phox
subunits are present as a complex in the cytosol of resting
neutrophils, along with p40phox, the latter of which is not
required for superoxide formation in cell-free NADPH oxidase assays but
may stabilize p67phox in intact cells (3). While the precise
function of each of the oxidase subunits is incompletely defined, it is
well documented that gp91phox, p22phox,
p47phox, and p67phox are absolutely required for
superoxide production in intact cells, since a deficiency in any one of
these proteins results in chronic granulomatous disease
(CGD),1 a rare genetic
disorder characterized by severe recurrent bacterial and fungal
infections (see Ref. 4 for review).
The oxidase is dormant in resting phagocytes until cellular activation
by inflammatory stimuli signals the assembly of flavocytochrome b558, p47phox, p67phox, and Rac
into the active respiratory burst enzyme. The mechanism of activation
and assembly of the oxidase is currently under investigation. According
to the current paradigm (reviewed in Ref. 2), flavocytochrome b558 serves as a docking site for
p47phox and p67phox, which translocate to the plasma
membrane as a unit after one or both of these cytosolic proteins become
phosphorylated. The p47phox subunit appears to play an
important role in mediating the initial phases of oxidase assembly. In
cell-free assays of superoxide production, participation of
p47phox was shown to precede that of p67phox in
formation of the active NADPH oxidase (5). In addition, p67phox
translocation does not occur in p47phox-deficient CGD, whereas,
in the genetic absence of p67phox, translocation of
p47phox is not impaired (6). Current evidence suggests that Rac
translocates to the membrane concurrently (7) with, but as a separate
unit from, p47phox and p67phox (8); however, it is not
clear if translocation of p67phox is a prerequisite for
translocation of Rac. While Rac has been shown to interact directly
with p67phox (9-12), Rac2 translocates to flavocytochrome
b558 even in the absence of p47phox and
p67phox as shown in the analysis of intact CGD neutrophils
deficient in either p47phox or p67phox (13, 14) or in
cell-free reactions using p47phox- or p67phox-deficient
cytosol (13).
Multiple sites within both gp91phox and p22phox have
been proposed as binding sites for p47phox including a
hydrophilic, polybasic domain within gp91phox between residues
86 and 102. A portion of this domain was identified as a site of
interaction with p47phox by DeLeo and colleagues using random
sequence peptide phage display analysis (15). This domain is likely to
reside at the intracytoplasmic face of the membrane based on current
models of the topologic organization of gp91phox (16). Peptides
derived from this sequence are potent inhibitors of superoxide
formation (15, 17) and translocation of the cytosolic subunits
p47phox and p67phox (17) in cell-free assays.
In the present study, we used site-directed mutagenesis of
gp91phox amino acids 86-102 to identify key residues within
this hydrophilic domain that are involved in NADPH oxidase assembly
and function. Mutant derivatives of gp91phox were expressed in
a myelomonocytic cell line that lacks endogenous gp91phox
expression due to gene targeting. We found that replacement of a pair
of arginine residues at positions 91 and 92 with two glutamic acid
residues abolished superoxide production and inhibited the stable
translocation of the cytosolic oxidase proteins p47phox,
p67phox, Rac1, and Rac2 to the membranes of activated
granulocytes. Replacement of both arginine residues with neutral rather
than acidic amino acids did not abolish NADPH oxidase activity but
resulted in an increased lag time to maximal rates of enzyme activity
and an overall decrease in superoxide formation. We conclude that
Arg91 and Arg92 of gp91phox participate
in a critical step in assembly of the active oxidase complex.
Materials--
The following reagents were purchased from Sigma:
N,N-dimethylformamide, diisopropyl fluorophosphate,
Me2SO, phorbol 12-myristate 13-acetate (PMA), zymosan,
cytochrome c, superoxide dismutase, FAD, and NADPH.
Complete, EDTA-free, protease inhibitor tablets were purchased from
Boehringer Mannheim.
Cell Culture and Differentiation--
Wild-type PLB-985 cells, a
human myeloid leukemia cell line, and X-CGD PLB-985 cells, a derivative
line in which the X-linked gp91phox gene has been disrupted by
gene targeting, were maintained as described (18). For granulocytic
differentiation, cells at a starting density of 1 × 105 cells/ml were exposed to 0.5%
N,N-dimethylformamide for 6 days (18).
Site-directed Mutagenesis and Expression Plasmids--
Mutations
of individual or multiple amino acids in the hydrophilic, polybasic
domain of gp91phox between residues 86 and 102 were introduced
into a full-length wild-type gp91phox cDNA cloned into the
NotI site of the multiple cloning site of pBluescript II
KS(+) (Stratagene) using the Quik-Change mutagenesis kit (Stratagene)
or the Sculptor in vitro mutagenesis system (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Wild-type or mutant gp91phox cDNAs were verified by
dideoxynucleotide sequencing and were subcloned into the pEF-PGKpac
mammalian expression vector (19). The mutant gp91phox
expression constructs were resequenced to confirm the mutations, and
all constructs were linearized with KpnI prior to
electroporation into X-CGD PLB-985 cells. All preparations and
manipulations of plasmids were performed using standard protocols (20).
Following electroporation, clones were selected by limiting dilution in 2 µg/ml puromycin and screened for gp91phox expression by
immunoblot. To minimize any clone-to-clone variation in recombinant
gp91phox expression or NADPH oxidase activity, 4-6 independent
clones determined to express transgenic gp91phox were pooled
and used for subsequent analysis.
Protein Extraction and Immunoblot Analysis--
Cultured
granulocyte-differentiated PLB-985 and derivative cell lines were
harvested by centrifugation at 500 × g for 10 min at
4 °C and washed once with cold phosphate-buffered saline. After diisopropyl fluorophosphate treatment for 10 min on ice, whole cell
extracts were made as described previously (21).
Expression of recombinant wild-type and mutant gp91phox and
endogenous p22phox proteins were analyzed by immunoblot using
monoclonal antibodies 49 and 449, respectively (22) (kindly provided by
A. Verhoeven and D. Roos, Central Laboratory of the Netherlands Blood
Transfusion Service) as described previously (18). Scanning
densitometry was employed to measure the relative intensity of the
gp91phox signal using a Silver Scan II scanner and Image 1.60 software (W. Rasband, National Institutes of Health). The relative
levels of gp91phox expression were confirmed using two
polyclonal gp91phox antibodies (23, 24).
Measurement of NADPH Oxidase Activity--
A continuous
cytochrome c reduction assay was used to quantitate the
superoxide dismutase-inhibitable superoxide formation by
granulocyte-differentiated PLB-985 cells and derivative cell lines
(18). The assay was performed at 37 °C using a Thermomax microplate
reader (Molecular Devices) and associated SOFTMAX version 2.02 software
on whole cells after stimulation with PMA (100 ng/ml) or opsonized
zymosan (6 mg/ml) freshly prepared as described (25). Superoxide
production was quantitated using an extinction coefficient of 21.1 mM
NADPH oxidase activity in selected PLB-985 derived cell lines was also
analyzed in a cell-free assay. For fractionation of membrane-associated
and cytosolic proteins, cells were harvested and diisopropyl
fluorophosphate-treated as described above, resuspended to 1 × 108 cells/ml in relaxation buffer (19) and sonicated at
20% power (Sonics and Materials, Inc.) for 3 × 6 s at
4 °C. The membrane and cytosolic fractions were prepared by
sequential centrifugation (26) and were added to a standard reaction
mixture containing 100 µM cytochrome c, 10 µM FAD, 100 µM SDS in relaxation buffer with or without superoxide dismutase. After a 3-min incubation at
25 °C, the reaction was initiated with 200 µM NADPH
and was monitored at 550 nm using a Thermomax microplate reader.
Measurement of Translocation of Cytosolic NADPH Oxidase
Components--
PLB-985 and derivative cell lines were harvested by
centrifugation at 500 × g for 10 min at 4 °C,
washed in phosphate-buffered saline, and resuspended at 1 × 108 cells/ml in relaxation buffer. The NADPH oxidase was
activated by stimulating the cells with 1 µg/ml PMA in
Me2SO or with Me2SO alone for 10 min at
37 °C. Cells were then placed on ice and, following the addition of
12 ml of ice-cold phosphate-buffered saline, were centrifuged at
500 × g for 10 min at 4 °C. Cells were adjusted to
1 × 108 cells/ml in relaxation buffer and disrupted
by sonication for 3 × 6 s at 20% power (Sonics and
Materials) at 4 °C. Disrupted cells were centrifuged at 500 × g for 10 min at 4 °C, and the supernatants were collected
and centrifuged at 2,000 × g for 10 min at 4 °C.
The supernatants in a volume of 1 ml were then layered on a
discontinuous sucrose gradient (1.5 ml of 20% over 1 ml of 38%) and
centrifuged at 204,000 × g for 30 min. After
centrifugation, the top 600 µl was collected as cytosol, and a
distinct band at the gradient interface was collected as the membrane
fraction. The membrane fractions were mixed with 3.5 ml of cold
relaxation buffer and centrifuged at 368,000 × g for
30 min, and the pellets were resuspended in 100 µl of relaxation
buffer with protease inhibitors. The cytosol and membrane fractions
were stored at A hydrophilic domain of gp91phox encompassed by amino
acids 86-102 has been implicated as a contact point for
p47phox in the assembly of the active NADPH oxidase complex
(15, 17). The purpose of this study was to examine the requirements for an intact 86-102 domain in superoxide production and to identify specific residues within this domain that were essential for normal gp91phox function. We used site-directed mutagenesis to
generate a set of gp91phox mutants in which multiple or
individual amino acid residues were replaced with alternate residues
(Fig. 1). We postulated that charged
amino acids within the 86-102 sequence, which contains five basic
residues and one acidic residue, might mediate electrostatic interactions with p47phox and/or other cytosolic oxidase
subunits. Hence, charged amino acid residues were replaced with neutral
or oppositely charged residues, thereby altering the local
electrostatic charge within this hydrophilic domain. WT or mutant
cDNAs were cloned into a mammalian expression vector and
transfected into X-CGD PLB-985 cells, a derivative of the myelocytic
cell line PLB-985 in which the coding sequence of the gp91phox
gene was disrupted by gene targeting (18) and which, therefore, lack
endogenous gp91phox expression and NADPH oxidase activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 cm
1 for cytochrome
c. Data were analyzed using SOFTMAX to determine Vmax over a 3-min interval and the time to
Vmax, measured as the elapsed time from the
start of the assay until the maximum reaction rate was obtained.
Statistical analysis was performed using InStat 2.0 software.
80 °C until SDS-PAGE analysis. For analysis of
translocation of cytosolic proteins, the membrane fractions were
separated by SDS-PAGE, and the proteins were transferred to
nitrocellulose and immunoblotted sequentially with polyclonal
anti-p47phox, polyclonal anti-p67phox (27) (both kindly
provided by David Lambeth, Emory University), monoclonal anti-Rac1
(Upstate Biotechnology, Inc.), polyclonal anti-Rac2 (7) (kindly
provided by Gary Bokoch, The Scripps Research Institute), and
polyclonal anti-Rap1a (28) (kindly provided by Mark Quinn, Montana
State University) and developed with the ECL detection system (Amersham
Pharmacia Biotech) as described previously (18). Integrated
densitometry was employed to measure the relative intensity of the
protein signal using an Eagle Eye II Still Video System and associated
software (Stratagene). Statistical analysis was performed using InStat
2.0 software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Mutagenesis of an arginine- and lysine-rich
domain of gp91phox. The wild-type amino acid sequence from
residues 86-102 is shown in the top line, and
mutant sequences are shown below. Periods
indicate residues that are identical to the wild-type sequence. Point
substitutions are indicated by the one-letter amino acid abbreviation
of the mutated residue.
We assessed the expression of recombinant wild-type or mutant
gp91phox proteins by immunoblotting whole cell extracts from
PLB-985-derived granulocytes with a monoclonal antibody specific for an
undefined epitope of gp91phox (Fig.
2). The level of expression of
recombinant mutant gp91phox proteins was calculated relative to
that seen for recombinant WT gp91phox in X-CGD PLB-985 cells
(Fig. 2). Similar relative levels of expression were seen when blots
were probed with two different polyclonal anti-gp91phox
antibodies (data not shown). The gp91phox mutant in which amino
acids 89-97 were replaced with nine alternate residues was expressed
at only one-twentieth the level of recombinant WT gp91phox
(Fig. 2). All of the mutant gp91phox polypeptides harboring
single or double point mutations were expressed between 10 and 95% of
recombinant WT gp91phox levels (Fig. 2). The expression of
p22phox in the transfected X-CGD cell lines was rescued in
proportion to the level of expression of the recombinant mutant
gp91phox (data not shown).
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We next examined the effect of these gp91phox mutations on
PMA-stimulated NADPH oxidase activity in intact PLB-985-derived
granulocytes. Superoxide production was completely absent in cells
expressing mutant gp91phox in which residues 89-97 had been
replaced with nine alternate residues (Fig.
3A). While this mutant was
only poorly expressed, previous studies have shown that expression of
even small amounts of recombinant wild-type gp91phox can
reconstitute considerable oxidase activity in X-CGD neutrophils (18,
29-31).
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Single or double point mutations in the gp91phox 86-102 domain
also resulted in altered NADPH oxidase activity, with the most dramatic
effects observed with mutations at Arg91 and
Arg92. PMA-stimulated superoxide production was completely
abolished in cells expressing recombinant gp91phox in which
both Arg91 and Arg92 were replaced with acidic
residues (R91E/R92E). When these same arginine residues were both
substituted with neutral amino acid residues (R91T/R92A), oxidase
activity was markedly decreased, although not abolished (Figs.
3A and 4A). In
addition, cells expressing the gp91phox mutant R91T/R92A
displayed a more than 3-fold increase in the lag period between PMA
stimulation and the onset of maximal rates of superoxide production
(Figs. 3B and 4A). In earlier studies, we
generated transgenic X-CGD PLB-985 cells lines expressing low levels of
recombinant wild-type gp91phox that were associated with
decreased rates of superoxide production but without an increased lag
time (29),2 suggesting that
delayed onset of Vmax is independent of low
oxidase activity. Replacement of individual arginine residues at
position 91 or 92 with glutamic acid (R91E or R92E) also resulted in
decreased and delayed PMA-stimulated oxidase activity. However, alanine substitution of either Arg91 or Arg92 with
alanine residues (R91A or R92A) resulted in increased oxidase activity,
although only that of the R91A mutant reached statistical significance
(Fig. 3, A and B). Superoxide production was not significantly affected by neutral substitutions at basic residues 89, 96, or 102 (R89T, R96A, or K102T) or at aspartic acid 95 (D95A); a
charge reversal at position Asp95 by substitution with
arginine (D95R) or at position Arg96 by substitution with
glutamic acid (R96E) also had no statistically significant effect on
oxidase activity (Fig. 3A).
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Superoxide production in selected cell lines was also measured
following stimulation with opsonized zymosan. The overall rate of
superoxide production was significantly reduced in cells expressing the
gp91phox mutants R91E, R92E, or R91T/R92A (Figs. 4B
and 5A). However, unlike the
PMA-induced respiratory burst, the lag time to maximum rates of
superoxide production after opsonized zymosan activation was not
significantly increased in these mutants (Figs. 4B and 5B). The NADPH oxidase was still completely nonfunctional in
cells expressing R91E/R92E gp91phox upon stimulation with
opsonized zymosan (Figs. 4B and 5A).
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We also examined whether R91T/R92A gp91phox or R91E/R92E gp91phox could support superoxide production in a cell-free assay. No superoxide dismutase-inhibitable superoxide production was detected utilizing membranes from cells expressing R91E/R92E gp91phox (Fig. 4C). However, NADPH oxidase activity using membranes from cells expressing R91T/R92A gp91phox was similar to that seen with membranes containing WT gp91phox (Fig. 4C), without any delay in superoxide production.
Finally, we investigated whether the R91E/R92E mutation in
gp91phox, which abolished NADPH oxidase activity, affected the
PMA-induced translocation of the cytosolic oxidase proteins
p47phox, p67phox, and Rac to the cell membrane.
Membranes from X-CGD PLB-985 granulocytes expressing recombinant WT
gp91phox or R91E/R92E gp91phox as well as the parental
X-CGD PLB-985 cell line were extracted following PMA stimulation;
separated by SDS-PAGE; and analyzed by sequential immunoblotting with
anti-p47phox, anti-p67phox, anti-Rac1, and anti-Rac2,
(Fig. 6A). To adjust for any
unequal loading of lanes, the amounts of p47phox,
p67phox, Rac1, and Rac2 were normalized against the level of
the membrane-associated small G protein Rap1a. In agreement with
previous studies using normal and X-CGD neutrophils (13), PMA
activation of PLB-985 granulocytes expressing recombinant WT
gp91phox resulted in membrane translocation of p47phox,
p67phox, and Rac2, whereas translocation failed to occur or was
significantly reduced in X-CGD PLB-985 granulocytes (Fig. 6,
A and B). PLB-985 granulocytes had readily
detectable levels of Rac1, which upon PMA-induced activation, was also
detected in the membrane fraction of cells expressing wild-type
gp91phox (Fig. 6). Similar to Rac2, Rac1 translocation was
markedly decreased in X-CGD PLB-985 cells (Fig. 6). PLB-985
granulocytes expressing the mutant R91E/R92E gp91phox resembled
X-CGD cells in that PMA-induced translocation of p47phox,
p67phox, and both Rac1 and Rac2 was absent (Figs. 6,
A and B).
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DISCUSSION |
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The phagocyte NADPH oxidase is a multicomponent enzyme assembled from membrane-associated and cytosolic components upon cellular activation by inflammatory stimuli. The objective of this study was to examine the role of a hydrophilic domain encompassing residues 86-102 of gp91phox in oxidase assembly and function. This region was previously identified by random sequence peptide phage display library analysis as a potential site of interaction with p47phox (15). Consistent with this observation, synthetic peptides derived from residues 86-102 have been shown to inhibit superoxide production in the cell-free superoxide assay (15, 17) and in electropermeabilized neutrophils (32), and can block the translocation of p47phox and p67phox to flavocytochrome b558 in a cell-free system (17). In this study, we used site-directed mutagenesis to probe the role of specific charged amino acid residues within this region of gp91phox, hypothesizing that electrostatic interactions between charged residues in gp91phox and p47phox may be important for assembly of the active oxidase complex. We focused our initial analysis on the functional effect of these mutations in intact granulocytic cells since the protein-protein interactions required for oxidase assembly in intact cells appear to differ from those utilized in the cell-free oxidase assay. For example, while two carboxyl-terminal SH3 domains of the p67phox subunit are required for superoxide production in Epstein-Barr virus-transformed B cells (33), the truncation of these domains does not interfere with oxidase activity in the cell-free assay (33). Also, phosphorylation of the p47phox subunit is required for NADPH activity in intact cells (34) but not in the cell-free system (35). In fact, p47phox is not required for superoxide production in the cell-free system if large amounts of p67phox are supplied (36, 37).
We found that replacement of amino acids 89-97 of gp91phox with nine alternate residues completely abolished oxidase activity. This gp91phox mutant was expressed at only 5% of the level of recombinant WT gp91phox, suggesting that the mutant protein had decreased stability. Hence, we cannot rule out the possibility that the absence of enzymatic activity was due to a perturbation of overall gp91phox structure rather than specific to the replacement of residues 89-97. Single or double substitutions within the 86-102 domain generally had much less of an effect on the level of gp91phox expression. Mutation of individual amino acid residues at Arg89, Asp95, Arg96, or Lys102, did not significantly affect superoxide production, even when the mutation resulted in a reversal of electrostatic charge. However, as discussed below, striking effects on oxidase activity were observed with mutations of arginine residues at positions 91 and 92, particularly when Arg91 and Arg92 were mutated simultaneously. It is relevant that superoxide production was not completely abolished by mutation of any single amino acid, suggesting that at least two amino acids within this gp91phox domain must be altered to produce a nonfunctional flavocytochrome b558. This is consistent with the lack of reported cases of X-CGD due to point missense mutations or deletions in this region of gp91phox (38).
A gp91phox mutant in which a pair of arginine residues at positions 91 and 92 was substituted with two glutamic acid residues was nonfunctional in both intact cells and in the cell-free oxidase assay. In addition, we found that the cytosolic oxidase subunits p47phox, p67phox, Rac1, and Rac2 failed to translocate to membranes of cells expressing the R91E/R92E gp91phox mutant, suggesting that these arginine residues play an essential role in assembly of the active oxidase complex. Taken together with previous peptide studies that identified residues 85-93 within gp91phox as a binding site for p47phox (15, 17), these data provide strong support of an important functional role for this polybasic region of gp91phox in the translocation of p47phox. Most reports agree that translocation of p47phox is a prerequisite for p67phox translocation (5, 6, 13); therefore, the failure of p67phox to translocate to the membrane of gp91phox R91E/R92E is probably secondary to the inhibition of p47phox translocation. The sequences within p47phox that are involved in the proposed interaction with this polybasic gp91phox domain are unknown. Binding of p47phox to peptides corresponding to gp91phox residues 85-93 was detected in the absence of p47phox phosphorylation, which normally occurs with activation of oxidase assembly in intact phagocytes (15). A domain encompassing p47phox residues 323-332 appears to bind gp91phox (35, 39, 40) in a phosphorylation-independent fashion (35). However, this p47phox sequence contains multiple basic residues, so an interaction with the polybasic 86-102 domain of gp91phox would seem unlikely.
Hence, it appears that the interaction of flavocytochrome b558 with p47phox is quite complex and involves multiple sites of interaction. Other putative p47phox binding sites within gp91phox include residues 27-46 (17) and 434-457 (15, 17), a domain surrounding Asp500 in gp91phox (41), and a seven-amino acid sequence in the extreme carboxyl-terminal tail of gp91phox (15, 42-44). A proline-rich motif in the carboxyl terminus of p22phox between residues 149 and 162 has also been shown to be essential for assembly of the oxidase complex and appears to function as a binding site for the p47phox SH3 domains located between residues 151 and 214 and residues 225 and 284 (44-46). Less is known about potential interactions between flavocytochrome b558 and p67phox. An activation domain within p67phox has recently been identified that may function as a point of contact with either gp91phox or p22phox (47).
In the current study, we found that the membrane association of both Rac1 and Rac2 was markedly decreased in X-CGD PLB-985 cells and in cells expressing the mutant R91E/R92E gp91phox. Deficient Rac2 translocation in the absence of flavocytochrome b558 confirms previous results in X-CGD neutrophils (13). To our knowledge, ours is the first report that PMA-induced translocation of Rac1 is also dependent upon expression of flavocytochrome b558. While either Rac1 or Rac2 is required for maximal oxidase activity in the cell-free assay (48-50), the relative roles of Rac1 versus Rac2 in neutrophil superoxide production are uncertain. In human neutrophils, Rac2 accounts for >95% of the Rac species present (49, 51). However, Rac1 was identified as the GTP-binding protein necessary for oxidase activity in guinea pig phagocytes (48) and appears to be as abundant as Rac2 in murine neutrophils3 and in human PLB-985 granulocytes (Fig. 6). Furthermore, NADPH oxidase activity is not abolished in neutrophils of Rac2-negative mice generated after targeted disruption of the rac2 gene, indicating that Rac2 does not play an irreplaceable role in the function of this enzyme (52).
From our experiments, we cannot distinguish whether decreased Rac1/2 translocation in X-CGD PLB-985 granulocytes and in R91E/R92E gp91phox-expressing granulocytes is due to a direct effect on the interaction of Rac with flavocytochrome b558 or an indirect effect resulting from deficient translocation of another oxidase component. While specific associations between p67phox and either Rac1 or Rac2 have been identified (9, 10, 12), it is less clear whether Rac1 or Rac2 interacts directly with flavocytochrome b558. In p47phox- or p67phox-deficient neutrophils from CGD patients, PMA-induced translocation of Rac2 was normal (13, 14, 53), suggesting that the membrane association of Rac2 is independent of either of these two oxidase proteins. Therefore, the dependence of Rac2 translocation on the presence of flavocytochrome b558 (13) implies a direct interaction between Rac2 and the flavocytochrome. In contrast to Rac2, Dusi et al. (14, 53) found that Rac1 translocation did not occur in either p47phox- or p67phox-deficient CGD neutrophils. Taken together, these data indicate that while stable membrane association of both Rac1 and Rac2 is dependent on expression of flavocytochrome b558, these two GTPases associate with phagocyte membranes by different mechanisms. Our own data are consistent with a model of oxidase assembly whereby Arg91 and Arg92 in gp91phox are required for translocation of p47phox, which associates directly with flavocytochrome b558, and, indirectly, for translocation of p67phox (which binds to p47phox) and Rac1 (which binds to p67phox). Decreased Rac2 translocation may reflect an indirect effect of the gp91phox R91E/R92E mutation on a putative Rac2-flavocytochrome b558 interaction.
Other mutations in gp91phox Arg91 and/or Arg92 that eliminated a net positive charge at these two positions (rather than the double charge reversal resulting from the R91E/R92E substitution) had more subtle effects on oxidase activity. Replacement of both arginines at positions 91 and 92 with neutral residues or replacement of either Arg91 or Arg92 with an acidic residue resulted in a substantial decrease in superoxide production following stimulation with PMA or opsonized zymosan and an increased lag time for maximal PMA-induced enzyme activity. However, alanine substitution of either Arg91 or Arg92 resulted in an enhanced oxidase activity, although this was statistically significant only for the R91A gp91phox mutant. Hence, it appears that alterations in the local electrostatic charge at residues 91 and 92 of gp91phox can modulate oxidase function. Since the 86-102 domain of gp91phox has been implicated as a binding site for p47phox, it is noteworthy that Kleinberg and co-workers found that the lag time between oxidase activation and the onset of superoxide production in the cell-free system was decreased if the neutrophil membranes were preincubated with p47phox-containing cytosol (5) or pure recombinant p47phox (54) but not with purified Rac (54). This result suggested that p47phox forms an early activation intermediate with the membrane. The lag in the onset of the maximal rates of superoxide formation for the R91E, R92E, and R91T/R92A gp91phox mutants following stimulation with PMA could thus reflect a decreased affinity between p47phox and the mutant gp91phox.
That no alteration in the kinetics of superoxide formation by the
R91T/R92A gp91phox mutant cells was seen in the cell-free
system is not surprising given the discrepancies between the cell-free
and whole cell oxidase assays highlighted above. However, it is less
clear why there was no delay in the onset of
Vmax following stimulation of gp91phox
R91T/R92A phagocytes with opsonized zymosan. We speculate that this
observation reflects differences in the kinetics of activation of
p47phox translocation induced by phorbol ester
versus opsonized zymosan. While PMA directly activates
protein kinase C, phagocytosis of opsonized zymosan activates tyrosine
kinases, leading to activation of phosphatidylinositol-specific
phospholipase C and the formation of diacylglycerol and inositol
1,4,5-trisphosphate (55), which, respectively, activate protein kinase
C and increase intracellular free Ca2+. However, inhibition
of protein kinase C with staurosporine or 1,5-isoquinolinesulfonyl-2-methylpiperazine inhibits PMA-stimulated NADPH oxidase activity and p47phox phosphorylation, whereas
these inhibitors do not affect oxidase activity and cause only a
partial inhibition of p47phox phosphorylation in response to
opsonized zymosan (56). This observation suggests that phagocytosis of
opsonized zymosan phosphorylates p47phox and activates the
oxidase by a protein kinase C-independent pathway. Differences in PMA-
and opsonized zymosan-induced activation were also observed in recent
studies of Downey and colleagues, who found that PD098059, a potent and
selective inhibitor of mitogen-activated protein kinase, significantly
inhibited the respiratory burst induced by opsonized zymosan but not of
that induced by PMA (57). Taken together, these data indicate that the
signaling pathways utilized by PMA differ from those involved in
opsonized zymosan-mediated neutrophil activation. It is plausible that
these differences result in alterations in the kinetics of
p47phox phosphorylation and/or other factors that regulate
translocation. These, in turn, could either directly or indirectly
affect the affinity of p47phox for the
Arg91/Arg92-containing gp91phox domain
and the rate of superoxide production.
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ACKNOWLEDGEMENTS |
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We thank Youyan Zhang for assistance with densitometry and Donna Fischer and Jeanne Wallen for assistance with manuscript preparation.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Herman B Wells Center for Pediatric Research, Cancer Research Institute, Indiana University School of Medicine, 1044 W. Walnut, Indianapolis, IN 46202. Tel.: 317-274-8645; Fax: 317-274-8679; E-mail: mdinauer{at}iupui.edu.
2 K. J. Biberstine-Kinkade, L. Yu, and M. C. Dinauer, unpublished data.
3 C. Kim and M. Dinauer, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: CGD, chronic granulomatous disease; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; WT, wild type.
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REFERENCES |
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