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 Medical Center,
Indianapolis, Indiana 46702
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ABSTRACT |
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Site-directed mutagenesis was used to generate a
series of substitutions and deletions in the carboxyl-terminal 11 residues of gp91phox, the 91-kDa subunit of the phagocyte NADPH
oxidase flavocytochrome b558. This region
encompasses 559RGVHFIF565, implicated as a
contact point for the cytosolic oxidase subunit p47phox during
oxidase activation, and a carboxyl-terminal phenylalanine (Phe570), which corresponds in position to a highly
conserved aromatic residue that interacts with the flavin group in the
ferredoxin-NADP+ reductase flavoenzyme family, of which
gp91phox is a member. Mutant proteins were expressed in human
myeloid leukemia cells which lack expression of endogenous
gp91phox due to targeted disruption of the X-linked
gp91phox gene. Although specific residues within
559RGVHFIF565 had previously been identified by
alanine scanning as essential for peptide inhibition of oxidase
activity in a cell-free assay, comparable substitutions in the
gp91phox polypeptide had either no or only a modest effect on
oxidase activity in whole cells. Replacement of nonpolar with polar or charged residues had greater effects on oxidase activity, but were also
associated with decreased gp91phox expression, suggesting that
overall protein structure was perturbed. No stable gp91phox
protein was detected upon deletion of the terminal 11 amino acids. Alanine substitution or deletion of the carboxyl-terminal
Phe570 in gp91phox resulted in a 2-fold reduction
in superoxide production. This contrasts with a 300-800-fold
reduction reported for comparable mutations in pea
ferredoxin-NADP+ reductase, which suggests that structural
or functional differences exist between the carboxyl terminus of
gp91phox and other ferredoxin-NADP+ reductases.
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INTRODUCTION |
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Neutrophils and other phagocytic leukocytes possess an NADPH oxidase (respiratory burst oxidase) that generates large quantities of superoxide during the respiratory burst (1). Upon phagocyte activation by opsonized bacteria or other inflammatory stimuli, the active NADPH oxidase complex is rapidly assembled at the plasma membrane from cytosolic and membrane components to catalyze the transfer of electrons from NADPH to molecular oxygen. Oxidase subunits include two polypeptides, gp91phox and p22phox, which form a membrane-bound phagocyte flavocytochrome b558 heterodimer, and three cytosolic proteins, p47phox, p67phox, and a low molecular weight GTP-binding protein, Rac (1-3). A fourth cytosolic protein, p40phox, is present as a complex with p67phox in resting neutrophil cytosol (4, 5), but does not appear to play a direct role in superoxide production. Superoxide and its derivatives are essential for normal microbicidal activity, and genetic defects in the NADPH oxidase result in chronic granulomatous disease (CGD),1 a syndrome characterized by life-threatening fungal and bacterial infections (1). Mutations in the X-linked gene for gp91phox account for the majority of cases of CGD, and the p22phox subunit is defective in an uncommon autosomal recessive subgroup of CGD (1). The remaining cases of autosomal recessive CGD result from mutations in the genes encoding p47phox or p67phox (1).
Current evidence suggests that flavocytochrome b558 functions as the redox center of the NADPH oxidase, and is regulated by the cytosolic oxidase subunits p47phox, p67phox, and Rac, which translocate to the cell membrane upon neutrophil activation (6-13). The p47phox subunit appears to mediate the first steps of interaction with the flavocytochrome in assembling the active oxidase complex (6, 7, 14), although it is not required in vitro if high levels of p67phox and Rac are supplied (15, 16). Sites within flavocytochrome b558 that function as contact points with p47phox have been identified in both the gp91phox and p22phox subunits (2, 3). The cytochrome has an NADPH-binding site and bears a flavin group that acts as the initial acceptor of a pair of electrons from NADPH (17-23), although recently p67phox has also been reported to contain a functional NADPH-binding site (24). The subsequent one-electron transfer to molecular oxygen is mediated by a pair of heme groups in the flavocytochrome heterodimer that are embedded within the membrane (25-28).
The gp91phox subunit of flavocytochrome
b558 is a 570-amino acid membrane glycoprotein
with multiple hydrophobic domains in the amino-terminal half followed
by a hydrophilic carboxyl terminus (1). The extreme carboxyl terminus
of gp91phox, which resides at the cytoplasmic face of the
membrane (29, 30), has been implicated as both a docking site for the
cytosolic oxidase subunit p47phox and as a participant in NADPH
binding. A variety of approaches have suggested that gp91phox
residues 559RGVHFIF565 interact with
p47phox at an early step of oxidase assembly (14, 29, 31-34).
Residues 400-570 of gp91phox also contain regions with
sequence homologies to the NADP+-binding site of
ferredoxin-NADP+ reductase flavoenzymes (17-20, 22). An
aromatic amino acid at or near the carboxyl terminus is highly
conserved among members of the ferredoxin-NADP+ reductase
flavoprotein family, including gp91phox which has a
carboxyl-terminal phenylalanine (Phe570). Crystallographic
analysis of the spinach ferredoxin-NADP+ reductase has
localized the carboxyl-terminal aromatic side chain to the
NADP+-binding pocket, where it may interact with the
isoalloxazine ring of FAD in the absence of NADP+ (35).
Altering the aromatic character or deleting this conserved residue in
the pea ferredoxin-NADP+ reductase leads to major
impairments in catalytic efficiency of the enzyme (36).
Although the majority of missense mutations in gp91phox identified in patients with X-linked CGD result in apparent structural instability of the protein, in rare cases, gp91phox expression is preserved but the mutant polypeptide forms a non-functional flavocytochrome b558 (37, 38). These latter mutations have been informative in identifying residues critical for gp91phox function. The expression and analysis of mutant gp91phox polypeptides generated by site-directed mutagenesis has not been previously reported, at least in part due to the lack of systems in which adequate levels of functional recombinant gp91phox can be readily expressed. To undertake a more systematic analysis of structure-function relationships in gp91phox using this approach, we developed a human myeloid leukemia cell line that lacks endogenous gp91phox after targeted disruption of the X-linked gp91phox gene (39). This "X-CGD" cell line has been a valuable tool for expression of recombinant gp91phox, which, in the case of wild-type recombinant gp91phox, assembles with p22phox to form a functional flavocytochrome b558 heterodimer (39-41).
In the present study, we have used site-directed mutagenesis to probe the role of the carboxyl terminus of gp91phox in NADPH oxidase activity. We examined both the requirements for an intact 559RGVHFIF565 sequence and for a carboxyl-terminal aromatic residue in supporting superoxide production by intact granulocytic cells. Mutant gp91phox cDNAs encoding polypeptides with amino acid substitutions or deletions in the carboxyl terminus were transfected into the X-CGD myeloid cell line for analysis of expression and function. The results suggest that the distal carboxyl terminus is an important determinant for gp91phox stability, but that neither an intact 559RGVHFIF565 sequence nor a carboxyl-terminal aromatic residue are absolutely essential for NADPH oxidase activity in intact granulocytic cells. Alanine substitutions had either no or only a modest effect on NADPH oxidase activity. The greatest effects on superoxide production were seen with substitution of polar or charged residues for hydrophobic amino acids in 559RGVHFIF565, but these were also associated with reduced expression of gp91phox, suggesting that the overall protein structure was perturbed.
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EXPERIMENTAL PROCEDURES |
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Plasmids-- A full-length wild-type gp91phox cDNA, extending from 12 nucleotides upstream of the initiator ATG to a SacI site in the 3'-untranslated region (39), was cloned into the NotI site in the multiple cloning site of pBluescript II KS(+) (Stratagene). This construct was used as a phagemid to produce single-stranded DNA for oligonucleotide-directed mutagenesis of specific codons. Mutations were introduced into the carboxyl-terminal region of the gp91phox cDNA using the Sculptor in vitro mutagenesis system (Amersham), and verified by dideoxynucleotide sequencing. Deletion mutants were made by replacement of nucleotides with a premature stop codon. Mutant gp91phox cDNAs were subcloned into the NotI site of the pEF-PGKneo mammalian expression vector (39), or a related vector, pEF-PGKpac, which contains a linked expression cassette for puromycin-N-acetyltransferase instead of neomycin phosphotransferase. The mutated gp91phox cDNA expression constructs were resequenced to confirm the mutations and were linearized with KpnI prior to electroporation into a human myeloid leukemia cell line (see below). Preparation and other manipulations of plasmid DNAs were performed by standard protocols (42). Restriction enzymes and other reagents for molecular biology were obtained from Promega, Boehringer Mannheim, New England Biolabs, and U.S. Biochemical Corp.
Cell Lines-- Wild-type PLB-985 cells (43), a human myeloid leukemia cell line, and a derivative line in which the X-linked gp91phox gene has been disrupted by gene targeting (X-CGD PLB-985 cells) were maintained as described (39). X-CGD PLB-985 cells do not express gp91phox protein and lack NADPH oxidase activity. After electroporation of expression constructs into X-CGD PLB-985 cells, clones were selected by limiting dilution in either 1.5 mg/ml G418 or 3 µg/ml puromycin (39). Since up to one-third of clones selected for resistance to the linked antibiotic marker will not have a full-length transgene and therefore fail to express any transgenic gp91phox mRNA,2 clones were analyzed for recombinant gp91phox expression by either Northern blot and/or immunoblot with gp91phox-specific probes. For subsequent analysis, three to five independent clones determined to express transgenic gp91phox were pooled to minimize potential clone-to-clone variation in recombinant gp91phox expression and NADPH oxidase activity. PLB-985 and derivative cell lines were differentiated into granulocytes with dimethylformamide as described (39).
Analysis of gp91phox Expression--
Northern blot and
immunoblot analyses were performed as described previously (39). A
gp91phox monoclonal antibody (mAb 48) (44) was kindly provided
by A. Verhoeven and D. Roos (Central Laboratory of the Netherlands
Blood Transfusion Service). Polyclonal gp91phox antibodies
raised against a gp91phox peptide (residues 86-102), a
-galactosidase-gp91phox fusion protein, and a
p22phox peptide have previously been described (45-47).
Scanning densitometry was employed to measure the relative intensity of
the gp91phox signals on immunoblots developed with the ECL
detection system (Amersham) and performed using a Silver Scan II
scanner and image 1.45 software (W. Rasband, National Institutes of
Health) (40). Different exposures as well as serial dilutions of cell
extracts were scanned to ensure that measurements were taken within a
linear response range.
Assay of Superoxide Formation-- A continuous cytochrome c reduction assay was used for quantitative measurement of superoxide dismutase-inhibitable superoxide formation by granulocyte-differentiated PLB-985 cell lines (39). The assay was performed at 37 °C using a Thermomax microplate reader and associated SOFTMAX Version 2.02 software (Molecular Devices) on whole cells after stimulation with either phorbol myristate acetate (100 ng/ml) or formyl-methionyl-leucyl-phenylalanine (FMLP) (1 µM).
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RESULTS |
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To examine the role of the carboxyl terminus of the
gp91phox subunit of flavocytochrome b558
in NADPH oxidase activity, site-directed mutations were introduced into
the gp91phox cDNA (Fig. 1).
Mutant forms of gp91phox that were generated included a series
of alanine replacements within the
559RGVHFIF565 domain at positions
(Arg559, Val561, Phe563,
Ile564, and Phe565) previously identified as
crucial for oxidase inhibition by alanine-substituted peptides derived
from this region (33). Substitutions with a more polar or charged amino
acid were prepared at positions 561, 563, and 565. Two mutants with
double substitutions (R559A/V561A and I564T/F565V) were also generated.
A mutant gp91phox with an alanine-substituted
His562 residue was also prepared, which would be predicted
to have no impact on oxidase activity based on peptide inhibition
studies (33). To investigate the requirement for a carboxyl-terminal aromatic residue for gp91phox function, the phenylalanine at
position 570 was substituted with an alanine (F570A) or deleted
(F570) by insertion of a premature stop codon at this position.
Finally, the effect of entirely deleting the last 10 residues
(
560-570) by introduction of a premature stop codon at position 560 was examined.
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The mutated gp91phox cDNAs were subcloned into mammalian
expression vectors under control of the EF1- promoter, and the
expression of mutant polypeptides evaluated by immunoblot analysis
(Fig. 2) after stable transfection of a
derivative of human myeloid leukemia PLB-985 cells which lack
endogenous gp91phox due to targeted disruption of the
gp91phox gene (X-CGD PLB-985 cells) (39). Note that the
wild-type gp91phox protein migrates as a diffuse band centered
at
91 kDa (Fig. 2), and the heterogeity in size of immunoreactive
species most likely reflects variations in glycosylation. Two related
vectors were used in these studies, pEF-PGKneo and pEF-PGKpac, that
differ only in the selectable marker gene upstream of the EF1-
promoter/gp91phox cDNA cassette. Using the pEF-PGKneo
vector, which has a linked neomycin phosphotransferase marker gene,
expression of recombinant wild-type gp91phox cDNA is
10-20% of the endogenous gp91phox level in
granulocyte-induced wild-type PLB-985 cells (39). During the course of
these studies, we observed that higher levels of expression of
recombinant wild-type gp91phox (25-50% of wild-type PLB-985)
were obtainable using pEF-PGKpac,2 for reasons which remain
unclear. The majority of gp91phox mutants reported here were
expressed using the pEF-PGKneo vector, and their expression compared
with X-CGD PLB-985 cell lines transfected with a pEF-PGKneo vector
containing the wild-type gp91phox cDNA (Fig. 2, upper
two panels). Several mutants prepared later during the course of
this study utilized pEF-PGKpac, and expression was compared with
wild-type p91phox expressed using pEF-PGKpac (Fig. 2,
lower panel). One mutant, F570
, was expressed in both
vector backgrounds, but most mutants were not re-cloned into pEF-PGKpac
as it was felt that the conclusions would not be substantially
altered.
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A summary of the relative levels of expression of recombinant
gp91phox derivatives in granulocyte-induced PLB-985 cells is
shown in Table I. Several of the
gp91phox mutants appeared to be unstable, with deletion of the
carboxyl-terminal 11 amino acids having the greatest impact on protein
expression. No gp91phox was detected in immunoblots of cell
extracts prepared from either undifferentiated (not shown) or
granulocyte-induced cells transfected with the 560-570 construct,
using either the monoclonal antibody MoAb48 (Fig. 2A),
directed against an uncharacterized epitope on gp91phox, or two
different gp91phox polyclonal antisera (not shown). In cells
expressing F563T, F565D, or FI564/F565V gp91phox mutants, very
small but detectable amounts of the gp91phox polypeptide were
present. More modest reductions in the relative level of
gp91phox expression were seen for several other mutants (V561A,
V561T, V561E, F563A, F565A, and F570
). The relative levels of the
remaining mutants were not significantly different from wild-type
recombinant gp91phox as expressed with the corresponding
pEF-PGKneo or pEF-PGKpac vector. The amount of recombinant protein
detected was unrelated to the abundance of the transgene-derived
gp91phox mRNAs, which were all similar (not shown). As was
previously observed with expression of wild-type recombinant
gp91phox (39), expression of mutant derivatives of
gp91phox rescued expression of the cytochrome p22phox
subunit in X-CGD PLB-985 cells, in proportion to the relative abundance
of the mutant gp91phox subunit (not shown).
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To provide evidence that the wild-type and mutant gp91phox are
successfully delivered to the plasma membrane when transgenically expressed in X-CGD PLB-985 cells, we stained the transfected and parental X-CGD cells with a monoclonal antibody, 7D5, and examined cell
surface expression of flavocytochrome b558 by
flow cytometry and confocal microscopy. The 7D5 antibody reacts with an
extracellular epitope on flavocytochrome b558
(49). Since the mutant gp91phox proteins were expressed at
variable levels, we chose F570 as an example of a mutant that was
expressed at relatively high levels, comparable to the wild-type
recombinant gp91phox, and F563T as an example of poorly
expressed gp91phox mutant. As seen in Fig.
3, the 7D5 antibody produced a complete shift in fluorescence intensity in flow cytometry of cells transfected with wild-type gp91phox, and confocal microscopy showed
immunofluorescent staining on the cell surface. Similar results were
obtained for wild-type PLB-985 granulocytes (not shown), whereas no
staining was seen for X-CGD PLB-985 cells (Fig. 3).
Immunofluorescent staining of cells expressing recombinant F570
gp91phox was similar to that seen for cells expressing
recombinant wild-type gp91phox. However, only weak staining of
F563T gp91phox-expressing cells was seen, consistent with the
results seen by immunoblotting (Fig. 2). Taken together, these
observations demonstrate that the transgenic expression of wild-type
and mutant gp91phox in X-CGD PLB-985 cells results in the
plasma membrane expression of flavocytochrome
b558, at levels that correlate with the amount of gp91phox seen in immunoblots of cell extracts.
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We next examined the impact of carboxyl-terminal mutations in
gp91phox on NADPH oxidase activity in intact cells after
granulocyte differentiation (Table I). A continuous cytochrome
c reduction assay was used to quantitate superoxide
formation after NADPH oxidase activation by phorbol myristate acetate.
X-CGD PLB-985 cells expressing wild-type recombinant gp91phox
at only 21-37% of that seen in wild-type PLB-985 granulocytes exhibited NADPH oxidase activity that was not significantly different from wild-type PLB-985 granulocytes (Table I). This is consistent with
our previous studies which have shown that expression of wild-type
recombinant gp91phox at 10% of wild-type levels fully
reconstitutes NADPH oxidase activity, and that cells expressing as
little as
5% of wild-type gp91phox levels exhibit 40-60%
of wild-type activity (39-41, 50). These observations have suggested
that gp91phox and flavocytochrome b558
are normally present in excess and not rate-limiting for superoxide
formation in wild-type neutrophils or PLB-985 granulocytes.
All mutants within the 559RGVHFIF565 with
detectable gp91phox expression were capable of supporting at
least a small amount of superoxide generation (Table I). The lag time
between stimulation of cells with phorbol myristate acetate and the
onset of superoxide generation (2 min) was the same for cells
expressing mutant gp91phox derivatives as for wild-type
gp91phox (not shown). Alanine substitutions at
Arg559, His562, or F563A did not affect NADPH
oxidase activity significantly, as was also seen for the double
R559A/V561A substitution. Point substitutions at Val561,
Ile564, and Phe565 resulted in a 2-5-fold
reduction in the rate of superoxide formation relative to wild-type
PLB-985 cells; the greatest reduction seen in the V561E mutant, which
was only
40% as active as the V561T mutant despite similar levels
of gp91phox expression. Superoxide formation observed with the
F565D gp91phox derivative, which was expressed at very low
levels, was 20-fold less than wild-type. In cells transfected with the
F563T or I564T/F565V mutants, in which expression was also very low,
NADPH oxidase activity appeared to be even further reduced but was
difficult to measure reliably using the cytochrome c assay.
Note that the above comparisons do not take into account variations in
recombinant gp91phox expression levels, and therefore the
relative function of gp91phox mutants expressed in reduced
amounts may be an underestimate. All of the above gp91phox
mutants supported the reduction of nitro blue tetrazolium dye, with the
intensity of resultant formazan staining proportional to enzyme
activity measured by the cytochrome c reduction assay. However, NADPH oxidase activity was undetectable by either the cytochrome c or nitro blue tetrazolium assay in cells
transfected with the
560-570 mutant, which did not express
detectable
91 kDa gp91phox protein. Superoxide generation in
response to fMLP was studied for several mutants (R559A, V561A, V561E,
V561T, and F565A) (not shown). The kinetics of superoxide formation
were unaffected, and the rank order of the relative magnitude of the
response corresponded to that seen for phorbol myristate acetate.
Mutant derivatives of gp91phox in which the final
carboxyl-terminal residue, Phe570, was substituted with an
alanine or deleted entirely (F570) were studied to test the
hypothesis that an aromatic amino acid at this position is critical for
NADPH oxidase enzymatic activity, as observed for pea
ferredoxin-NADP+ reductase (36), another member of the
ferredoxin-NADP+ reductase family. Although the F570A
mutant was expressed at levels comparable to wild-type recombinant
gp91phox, the rate of superoxide formation was reduced by
2-fold (Table I). Deletion of the carboxyl-terminal residue
(F570
) resulted in a
4-fold reduction in expression of
recombinant gp91phox and an almost 10-fold reduction in NADPH
oxidase activity compared with wild-type cells (Table I). When the
relative level of recombinant F570
protein was increased by using
pEF-PGKpac for expression, an almost 2-fold reduction in NADPH-oxidase
activity relative to wild-type cells was still observed (Table I).
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DISCUSSION |
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The phagocyte flavocytochrome b558 is a plasma membrane-associated heterodimer that is the electron transfer center of the superoxide-generating NADPH oxidase. The objective of this study was to use site-directed mutagenesis to probe the role of the distal carboxyl terminus of the gp91phox subunit, which contains domains implicated in the binding of NADPH and in a critical interaction with p47phox during assembly of the active oxidase complex. This is the first reported study using site-directed mutagenesis to investigate structure-function relationships in gp91phox, and utilized an X-CGD myeloid cell line for expression of mutant gp91phox polypeptides. For functional analysis, we specifically focused on analysis of NADPH oxidase activity in intact granulocytic cells, since the protein-protein interactions required for oxidase assembly and function in the cell-free assay do not always reflect those relevant in whole cells. For example, phosphorylation of p47phox is required for NADPH oxidase activity in neutrophils (12, 51, 52), but not in the cell-free system (53). As another example, the p67phox subunit contains two SRC homology 3-binding domains that are essential for oxidase function in whole cells, but are dispensable in the cell-free system (54).
Previous studies on X-CGD patients with deletions involving the carboxyl terminus of gp91phox have suggested that this domain is required for the stability of this protein. Based on computer modeling analysis, the extreme carboxyl terminus of gp91phox has been proposed to be buried within the interior of the protein, where it interacts with the flavin group (22). A complete lack of gp91phox and flavocytochrome b558 has been reported in X-CGD when the last 40-50 amino acids of gp91phox are absent (45, 55), and a 5-fold reduction in gp91phox expression was observed with deletion of the last six residues of gp91phox (56). In this study, we found that many of the gp91phox carboxyl-terminal mutants were expressed at significantly reduced amounts in the X-CGD PLB-985 cells compared with wild-type recombinant gp91phox. Although expression of alanine-substituted derivatives were generally comparable to wild-type recombinant gp91phox, decreased amounts of gp91phox were observed when apolar residues within the 559RGVHFIF565 sequence were replaced with polar or charged residues. This was particularly marked with polar substitutions at positions Phe563 and Phe565. A mutant gp91phox with the double replacement I564T/F565V was also expressed at very low levels. Removal of the last residue of gp91phox resulted in a modest decrease in gp91phox expression, and no recombinant gp91phox was detected at all when the last 11 residues were deleted. These observations support the concept that the carboxyl terminus of gp91phox is involved in maintaining a stable conformation of gp91phox and/or in heterodimer formation with p22phox, which plays an important role in the intracellular stability of each cytochrome subunit (1).
There has been great interest in characterizing the specific contact sites in NADPH oxidase subunits that mediate assembly of the active oxidase complex and regulate electron transfer through flavocytochrome b558 (2, 3). A number of studies have suggested that interactions between p47phox and the 559RGVHFIF565 domain encompassed within the distal carboxyl terminus of gp91phox play a key role in this process. An antibody directed against the carboxyl-terminal 13 amino acids of gp91phox inhibits superoxide formation in the cell-free assay (29). Synthetic peptides derived from this region inhibit NADPH oxidase activity in permeabilized neutrophils (29, 34) and in the cell-free oxidase assay (29, 31, 57). A peptide corresponding to gp91phox residues 559RGVHFIF565 is the minimum sequence capable of oxidase inhibition (IC50 = 38 µM) (33), which can be abolished by preincubation of neutrophil membranes with p47phox-containing neutrophil cytosol (14). When incubated with neutrophil cytosol, a similar carboxyl-terminal gp91phox peptide can be cross-linked to p47phox (31). Finally, a strategy using random sequence peptide bacteriophage display libraries of identified peptides with homologies to RGVHFIF as ligands of recombinant p47phox (32).
One goal of the current study was to further examine the role of the 559RGVHFIF565 domain of gp91phox with regards to NADPH oxidase activity in intact granulocytic cells. Specific residues within this sequence (Arg559, Val561, Phe563, Ile564, and Phe565) have been identified by alanine scanning as important for the function of this domain, based on the relative ability of alanine-substituted peptides to inhibit oxidase activity in a cell-free assay (33). However, we found that comparable alanine substitutions in the gp91phox polypeptide resulted in either no or only a modest reduction in NADPH oxidase activity when the mutant gp91phox was expressed in whole cells. The elimination of the positive charge at position 559 (Arg559), which had been proposed to mediate a critical ionic interaction between gp91phox and another oxidase subunit (33), had no significant impact on gp91phox expression or NADPH oxidase activity. Replacement of nonpolar residues with polar or charged residues in 559RGVHFIF565 resulted in relatively greater reductions in superoxide generation, but these mutations also were associated with apparent decreased stability of the gp91phox polypeptide, suggesting that overall protein structure was perturbed. Hence, it is not possible to rule out long-range effects on other domains within the gp91phox as the primary cause of decreased NADPH oxidase activity seen with alterations in the nonpolar character of these residues. It would also be of considerable interest to examine p47phox translocation in gp91phox mutants in cases where superoxide formation appeared to be affected, which would provide information as to whether enzyme assembly or catalytic activity was impaired as a result of the gp91phox mutation. However, as noted above, mutations in gp91phox in which NADPH oxidase activity was affected also resulted in an apparent reduction in the stability of gp91phox. Because of the low level of expression of these gp91phox mutants, quantitation of p47phox translocation could not be determined reliably.
There are a number of possible explanations for the observed discrepancy between the 559RGVHFIF565 peptide inhibition data and the results obtained in this study. A kinetic analysis of oxidase inhibition by 559RGVHFIF565 has suggested that this peptide acts as a non-competitive inhibitor (57), which might explain why many of the mutations in the gp91phox polypeptide did not have a profound effect. Alternatively, additional sites of interaction between p47phox and gp91phox may obviate the requirement for an intact 559RGVHFIF565 domain for oxidase assembly in intact cells. A proline-rich region in the cytoplasmic carboxyl terminus of p22phox is a target for SRC homology 3 domains within p47phox (47, 58-62). A P156H point mutation in this domain, identified in a CGD patient homozygous for a mutant p22phox gene, abolishes translocation of p47phox and NADPH oxidase activity (47, 58, 59, 61). This interaction between p47phox and the cytochrome thus appears to be critical for oxidase assembly in intact granulocytes. A D500G mutation in gp91phox, identified in a patient with X-linked CGD, is also associated with virtually absent oxidase activity and deficient translocation of p47phox (63). Residues 77-93 in gp91phox is another domain that appears to interact with p47phox, as peptides homologous to this sequence bind to recombinant p47phox and inhibit oxidase function in the cell-free assay (32). Finally, it is also possible that single or multiple substitutions other than those studied within the 559RGVHFIF565 sequence are required to fully inhibit oxidase assembly in whole cells. However, we found that the ability to analyze the impact of more drastic alterations was limited by a lack of mutant protein stability.
The presence of an aromatic amino acid as the final or penultimate
residue is highly conserved among ferredoxin-NADP+
reductase family members, including gp91phox, which contains a
carboxyl-terminal phenylalanine (Phe570). The aromatic ring
has been proposed to maintain enzyme structure in the absence of
NADP+ by acting as a pseudosubstrate (18, 22, 35). X-ray
diffraction analysis of spinach ferredoxin-NADP+ reductase
indicates that the carboxyl-terminal 19 residues, which lie just distal
to the NADP+-binding domain, form an -helix/
-strand
region that extends into the FAD site, so that the terminal tyrosine
residue interacts closely with the flavin ring (35). Altering the
aromatic character of the corresponding residue in gp91phox,
Phe570, or deleting it entirely resulted in a
2-fold
reduction in the rate of superoxide generation relative to cells
expressing similar levels of wild-type recombinant gp91phox.
Alanine substitution of the other aromatic residues (Phe563
and Phe565) in the carboxyl terminus of gp91phox
also resulted in, at most, a 3-fold reduction in NADPH activity. These
data contrast to the
300-850-fold reduction in catalytic activity
observed when non-aromatic replacements of the carboxyl-terminal tyrosine residue or its deletion were created in spinach
ferredoxin-NADP+ reductase (36). Our results suggest that
there are structural and/or functional differences between the extreme
carboxyl-terminal domain of gp91phox and the corresponding
aromatic residues in other members of the ferredoxin-NADP+
reductase family.
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ACKNOWLEDGEMENTS |
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We thank Ling Lin Li and Shelley Bennett for help with maintaining cell lines, Brian Freie for assistance with densitometry, Susan Grigsby for flow cytometry, Ruben Sandoval (R.E.B.E.L. Imaging Facility, Department of Nephrology) for assistance with confocal microscopy, David Skalnik and Karla Kinkade for critical review of this manuscript, and Donna Fischer for assistance with manuscript preparation.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant RO1HL45635 and a Biomedical Research Grant from the Arthritis Foundation.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, James Whitcomb Riley Hospital for Children, Rm. 2600, 702 Barnhill Drive, Indianapolis, IN 46702. Tel.:
317-274-8645; Fax: 317-724-8679.
1 The abbreviations used are: CGD, chronic granulomatous disease; fMLP, formyl-methionyl-leucyl-phenylalanine; PIPES, 1,4-piperazinediethanesulfonic acid.
2 L. Zhen and M. Dinauer, unpublished observations.
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