From the Wells Center for Pediatric Research, Riley
Hospital for Children, Indiana University School of Medicine,
Indianapolis, Indiana 46202 and the ¶ Inflammation Program and
Department of Medicine, Veterans Administration Medical Center and
University of Iowa, Iowa City, Iowa 52242
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The redox center of the phagocyte NADPH oxidase
is flavocytochrome b558, a transmembrane
protein with two subunits, gp91phox and p22phox. In
this study we investigated the identity, subcellular localization, and
maturation of a putative 65-kDa gp91phox precursor (p65).
Expressing the gp91phox cDNA in an in vitro
transcription and translation system, we found that synthesis of p65
required endoplasmic reticulum (ER) microsomes. Sucrose density
gradient centrifugation of postnuclear supernatants obtained from a
PLB-985 derived cell line with a constitutively expressed
gp91phox transgene demonstrated that p65 co-sedimented with the
ER marker protein calreticulin and myeloperoxidase precursors.
Unexpectedly, the majority of p22phox was found in subcellular
compartments containing the mature 91-kDa form of gp91phox and
not with p65, suggesting that heterodimer formation may occur in a
post-ER compartment. The heme synthesis inhibitor, succinyl acetone,
reduced the abundance of mature gp91phox and p22phox
but had little or no impact on p65. These studies demonstrate (a) gp91phox is synthesized as a glycosylated
65-kDa precursor in the ER, (b) heterodimer formation is
not a co-translational process, and (c) heme insertion is a
determinant in the formation of a stable heterodimer but does not
appear to affect the stability of p65.
Activated phagocytes release granule contents and generate
reactive oxygen species to kill ingested microorganisms. Potent reactive oxygen species such as hydrogen peroxide and hypochlorous acid
generated by phagocytes originate from a superoxide anion (O Flavocytochrome b558, the redox center of the
electron transport complex (14, 15), is a heterodimer composed of a
glycosylated 91-kDa subunit, gp91phox, and a nonglycosylated
22-kDa subunit, p22phox (2, 4, 16, 17). The C terminus of
gp91phox contains homology with known nucleotide (FAD and
NADPH) binding domains of other flavoproteins (18-20). In addition, we
have recently shown that both heme prosthetic groups contained within
flavocytochrome b558 are localized within
gp91phox, further supporting the concept that gp91phox
is the subunit mediating electron transport (21). However, neither
gp91phox itself nor the combination of individual
gp91phox and p22phox subunits were able to replace the
intact gp91/p22 heterodimer in supporting O Clarification of the biosynthetic pathway of flavocytochrome
b558 is a logical step toward understanding the
determinants for the association of gp91phox and
p22phox, which are important for both stability and function of
the cytochrome. The mature form of gp91phox migrates as a broad
band in SDS-PAGE with an average size of ~91 kDa because of variable
carbohydrate processing (25). In previous studies, we and others have
described a 65-kDa immunochemically related intermediate of
gp91phox with high mannose carbohydrate side chains (p65) (24,
26, 27). This species was detected in SA-treated PLB-985 cells in which
both p22phox and mature gp91phox are absent (24) and
also in B-cell lines from p22phox-deficient CGD patients (26).
The mature form of gp91phox has post-translationally modified
N-linked carbohydrate, which resists digestion with
endoglycosidase H, indicating that side chains undergo additional
processing by mannosidases in the Golgi complex (16, 24, 26). However,
the actual sequence of events in the post-translational processing of
gp91phox and the subcellular localization of the putative
65-kDa precursor have not yet been characterized.
We have previously established a cultured cell model of X-linked CGD by
targeted disruption of the gp91phox gene in PLB-985 cells (28).
After the stable transfection of wild-type gp91phox cDNA
into X-CGD PLB-985 cells, expression of the putative 65-kDa precursor
and mature gp91phox was constitutive, making it possible to
monitor the relationship of their co-expression in biosynthetically
active cells. These studies show that gp91phox is generated as
a glycosylated 65-kDa precursor in the ER and further suggest that its
association with p22phox is not co-translational but is
augmented by heme insertion.
Materials--
[35S]Methionine/cysteine (7.18 mCi/0.5 ml) was obtained from Amersham Pharmacia Biotech. Peptide
N-glycosidase F (PNGase F), endoglycosidase H (Endo H), and
fluorescein isothiocyanate-conjugated goat anti-mouse IgG were obtained
from Boehringer Mannheim. A rabbit reticulocyte lysate kit for
transcription and translation reactions was purchased from Promega,
Inc. (Madison, WI). Mouse anti-CD11b-fluorescein isothiocyanate and
mouse IgG2b-fluorescein isothiocyanate were from Immunotech, Inc.
(Westbrook, ME). All other reagents were purchased from Sigma.
Antibodies specific for gp91phox and p22phox, monoconal
antibodies 48 and 449, respectively, were kindly provided by D. Roos
and A. Verhoeven, and monoclonal antibodies 54.1 and 44.1, also
specific for gp91phox and p22phox, respectively, were
provided by A. J. Jesaitis, M. T. Quinn, and J. B. Burritt. Rabbit
polyclonal antibody specific for p67phox was generously
provided by P. Heyworth. An antibody specific for Cell Differentiation and Acquisition of NADPH Oxidase
Activity--
The human promyelocytic cell line PLB-985 (obtained from
P. Newburger, University of Massachusetts), PLB-985 X-CGD, and gp91 PLB
cells (28, 29) were maintained in RPMI 1640 supplemented with 10%
fetal bovine serum as described previously (24). For granulocytic
differentiation, cells were induced with 0.5%
N,N-dimethylformamide for 5 days in
CO2-independent medium (Life Technologies, Inc.) in the
absence of fetal bovine serum but supplied with Nutridoma-SP (Boehringer Mannheim) and 2 mM L-glutamine.
During differentiation, the acquisition of O In Vitro Transcription and Translation--
The cDNA for
gp91phox was transcribed and translated in vitro
using a rabbit reticulocyte lysate assay according to the
manufacturer's instructions. 5-10 µl of each reaction was resolved
by 5-20% SDS-PAGE, and gels were fixed, soaked in 1.2 M
sodium salicylate, dried, and then exposed to Kodak X-Omat film for
2-6 h.
Subcellular Fractionation of Transfected PLB-985
Cells--
Subcellular fractionations were performed as described
previously (30). Briefly, gp91 PLB cells were seeded at 2 × 105 cells/ml in culture medium and cultured for 60 h.
1.5-2.0 × 107 cells were washed once in 30 ml of
phosphate-buffered saline and N2 cavitated in 0.5 ml of
relaxation buffer as described by Borregaard et al. (31).
Postnuclear supernatants were loaded on a 10-60% continuous sucrose
gradient and centrifuged at 35,000 rpm (151,000 × gav) in an SW41 rotor (Beckman Instruments) for 15 h at 4 °C, and 0.5-ml fractions were collected from the
bottom of each tube. The linearity of these gradients was determined using a refractometer and was similar to that previously reported (r2 = 0.993) (30). The distribution of protein
in the gradient was measured with the BCA protein assay (Pierce). Two
separate protein peaks were identified in fractions 8 and 20, containing 110 and 340 µg/ml protein, respectively. To identify
gp91phox, p65, p22phox, p47phox, calreticulin
(CRT), and Deglycosylation Analysis--
20 µl of each sucrose gradient
fraction was denatured by heating in the presence of 0.2% SDS and then
diluted 2-fold with the appropriate enzyme buffer. Digestions with
PNGase F or Endo H were performed using 0.5 milliunit of PNGase F and
10 milliunits of Endo H, respectively, for 1 h at 37 °C as per
the manufacturer's instructions. 6× SDS sample buffer was added to
digestions, and samples were resolved by SDS-PAGE.
Analysis of mRNA by Northern Blotting--
Total cellular
RNA was isolated, separated on a 1.0% formaldehyde agarose gel system
(24), and then transferred to Magnacharge nylon membrane (Micron
Separations Inc., Westboro, MA). Blots were hybridized with
32P-labeled cDNA probes to human gp91phox,
p22phox, and Flow Cytometric Analysis of Flavocytochrome b558 and
CD11b Surface Expression--
Cells treated with SA or
phosphate-buffered saline (control) for 5 days were stained with 7D5 (a
generous gift of Dr. Michio Nakamura, Nagasaki University, Japan), a
gp91phox monoclonal antibody that reacts with an extracellular
epitope of the cytochrome (32, 33). Surface expression of CD11b
(Immunotech) was assessed in a similar fashion, and IgG1 and IgG2b were
used as antibody isotype controls for 7D5 and anti-CD11b, respectively. Following staining, samples were analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
SDS-PAGE and Immunoblotting--
Intact cells (5 × 107/ml) were solubilized in a buffer containing 1% Triton
X-100, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1 mM EDTA (24), and proteins were resolved by 12% SDS-PAGE
(34). Alternatively, 20 µl of each sucrose gradient fraction was
resolved by 10% SDS-PAGE. Proteins were transferred to nitrocellulose
(35), and blots were probed with antibodies specific for
gp91phox, p22phox, p47phox, p67phox,
myeloperoxidase (MPO), CRT, and The gp91phox Precursor, p65, Is Synthesized and
Co-translationally Modified in Endoplasmic Reticulum--
We and
others have postulated that gp91phox is synthesized as a 65-kDa
precursor (p65) in the ER and that subsequent carbohydrate processing
in the Golgi complex yields mature gp91phox (24, 26). To
identify the initial precursor of gp91phox, we used cDNA
encoding gp91phox to prime biosynthesis in an in
vitro transcription and translation system. We have previously
demonstrated that in this system the nonglycosylated core polypeptide
precursor of MPO is generated using MPO cDNA (30). When purified ER
microsomes were then added to the system, both unglycosylated and
high-mannose type N-linked glycosylated forms of precursor
MPO were produced (30). Using gp91phox cDNA in the absence
of ER microsomes, gp91phox was translated as a 58-kDa
polypeptide (Fig. 1), a finding
consistent with the size of the gp91phox core protein
previously described by Parkos et al. (16) and others
following removal of carbohydrate with PNGase F (24, 25, 36). In the
presence of added ER microsomes, both the 58-kDa core polypeptide and
p65 were generated (Fig. 1). Because generation of p65 was dependent on
the presence of ER microsomes containing the necessary machinery for
N-linked glycosylation, these data clearly indicate that p65
is a precursor of the 91-kDa form of gp91phox and is
synthesized in the ER. Using a similar assay system to delineate sites
of N-linked glycosylation, Wallach and Segal (37) observed
that gp91phox cDNA was translated into polypeptides of
~50 kDa in the absence of ER microsomes and variably glycosylated
species of 53-60 kDa when ER microsomes were present. Thus, the
identity of the primary translation product was not established. By
contrast, our data demonstrate that these species are 58 kDa when
translated without microsomes and 65 kDa with added microsomes. The
reasons for these differences are uncertain, but may be related to
technical variations in the in vitro translation and/or gel
electrophoresis system.
We have previously established a cultured cell model of X-linked CGD by
targeted disruption of the gp91phox gene in PLB-985
myelomonoblastic cells (28). These cells are unable to produce
O
Although both gp91phox and p22phox were associated with
organelles that co-sedimented with those containing a 59-kDa heavy
subunit of mature MPO (lysosomes and/or azurophilic granules), we found that the intracellular pool of gp91phox and p22phox was
not lysosomal, because plasma membrane expression of the cytochrome was
not up-regulated when these cells were differentiated and then treated
with dihydrocytochalasin B and 1 mM
formylmethionylleucylphenylalanine (data not shown). However,
Heme Insertion Augments gp91/p22 Heterodimer Formation at the
Post-translational Level--
Synthesis of heme is essential for
complete processing of hemoproteins such as MPO and for enzymatic
activity (38-40). To determine whether inhibition of heme synthesis
affected flavocytochrome b558 biosynthesis in
gp91 PLB cells, we treated these cells with SA and examined the
expression of gp91phox, p22phox, and p65 after 5 days
of granulocytic differentiation with
N,N-dimethylformamide. As shown in Fig.
3A, differentiated gp91 PLB
cells developed significant O
Unaltered expression of p65 in SA-treated cells suggested that
transcription and/or translation of gp91phox mRNA was not
blocked by inhibition of heme synthesis. Northern blot analysis showed
similar levels of transgenic gp91phox mRNAs in both
undifferentiated and differentiated gp91 PLB cells in the absence or
presence of SA (Fig. 4). The abundance of
endogenous p22phox mRNA increased following differentiation
and was unaffected by SA treatment (Fig. 4). Thus, the decreased
expression of the 91-kDa form of gp91phox and of
p22phox by SA was not because of reduced levels of
gp91phox or p22phox transcripts. These data along with
the presence of p65 in SA-treated cells suggest that heme plays a role
in the post-translational processing of flavocytochrome
b558 rather than gene expression.
We have previously shown that the transgenic expression of
gp91phox also resulted in its plasma membrane association,
indicating it is fully processed in these cells (29). Because a small
portion of mature gp91phox was found in the plasma
membrane-enriched fractions of gp91 PLB cells (Fig. 2A)
(fractions 15-18), reduced expression of mature gp91phox in
SA-treated gp91 PLB cells should correlate with its reduced cell
surface expression. To confirm this hypothesis, we examined plasma
membrane expression of gp91phox in SA-treated or untreated
differentiated gp91 PLB cells by flow cytometry using the monoclonal
antibody, 7D5 (32, 33). 7D5 recognizes an extracellular epitope of
gp91phox (21).2 As
shown in Fig. 5A, cells not
treated with SA contain surface-expressed gp91phox. In
contrast, treatment with SA reduced gp91phox surface expression
to a level comparable with that of the IgG1 control antibody, a finding
consistent with data obtained by immunoblotting (compare Figs.
3B and 5A). Decreased expression of
gp91phox by SA treatment was specific for heme-containing
proteins, as the cell surface expression of the
Flavocytochrome b558 is primarily expressed
in phagocytic cells. In neutrophils, it has been well established that
~85% of the cytochrome is found in the membranes of specific
granules and gelatinase-containing granules with the remainder residing in the secretory vesicles and plasma membrane (31, 41). Stability of
flavocytochrome b558 is dependent on
co-expression of both gp91phox and p22phox (16, 22).
Recently, a putative 65-kDa precursor (p65) of gp91phox was
described in Epstein-Barr virus-transformed B-cell lines derived from
individuals with X-CGD and p22phox-deficient CGD (26, 27) and
also in the human promyelocytic leukemia cell line, PLB-985 (24).
Although antibodies specific for the peptide backbone of
gp91phox also recognize p65, p65 is undetectable in mature
neutrophils, perhaps because of their low biosynthetic activity and the
highly proteolytic environment.
Our current studies investigated directly the processing of
gp91phox using gp91phox-transfected PLB-985 X-CGD cells
in which mature gp91phox as well as p65 were constitutively
expressed. No 58-kDa gp91phox core polypeptide was detected in
the transfected cells, suggesting that the addition of asparagine
N-linked glycosylation was a co-translational process during
biosynthesis. Our data from in vitro transcription and
translation assays using gp91phox cDNA strongly support
that gp91phox is synthesized and co-translationally modified as
p65. These results are supported by the earlier work of Wallach and
Segal (37) who found that the initial translational product of
gp91phox cDNA is glycosylated in the presence of ER
microsomes. Previously, we found that p65 contained high mannose-type
carbohydrates as determined by its sensitivity to Endo H digestion
(24); however, mature gp91phox was Endo H-resistant, consistent
with its acquisition of complex carbohydrate side chains after
further processing in the Golgi complex (16, 24).
The distribution of p22phox in sucrose gradients paralleled
that of the 91-kDa form of gp91phox, which suggested that
p22phox did not associate with p65 in the ER but that
heterodimer formation occurs later in flavocytochrome
b558 biosynthesis. Our co-sedimentation data are
consistent with the observations in SA-treated gp91 PLB cells in which
p65 was persistently expressed in contrast to the decreased expression
of p22phox and the mature 91-kDa form of gp91phox and
also supported by the observations of Porter et al. (26) who
were able to detect p65 in p22phox-deficient CGD individuals
who lacked the 91-kDa form of gp91phox secondarily. We have
previously demonstrated that both complete processing of
N-linked carbohydrates and correct targeting of gp91phox to plasma membranes can be achieved in the absence of
p22phox in non-myeloid COS7 cells (24, 21), indicating that an
association of p65 with p22phox is not a prerequisite for these
events. These earlier findings are consistent with our current results,
because co-sedimentation of p22phox with gp91phox in
sucrose gradients is not observed until gp91phox is modified to
its 91-kDa form (Fig. 2A) (fractions 9-14). The compartment
in which heterodimer formation occurs between p22phox and
gp91phox or an intermediate species as it is processed from its
p65 to mature 91-kDa form is likely to be post-ER or Golgi. Heterodimer formation appears to be important for increased stability of both subunits against degradation in the proteolytic environment in phagocytes and may also facilitate the subsequent targeting of the
gp91/p22 heterodimer to the plasma membrane or, in neutrophils, specific granule membranes.
In SA-treated cells, p65 was more stable than either p22phox or
mature gp91phox, suggesting that heme incorporation augments
heterodimer formation in an as of yet undefined way. Previous studies
using p22phox-deficient lymphoblasts also revealed that the
abundance of a 65-kDa protein detected with antibody specific for
gp91phox was unaffected in the absence of p22phox (26);
however, formation of the mature form of gp91phox could be
rescued by expression of transgenic p22phox (26). Because
p22phox neither binds heme nor is glycosylated, its stability
appears to be regulated by the abundance of heme-containing
gp91phox. Heme insertion has no impact on p65 stability either
because it occurs during or after carbohydrate modification to produce the 91-kDa form of gp91phox or because heme-associated
gp91phox is required for subsequent p22phox binding.
Because we have recently shown that both heme prosthetic groups are
contained solely within gp91phox, neither heme is directly
involved in dimerization but may impart a required conformation for
heterodimer formation (21). We recently reported that impaired MPO
biosynthesis because of a missense mutation (R569W) resulted in the
inability of MPO to acquire heme and subsequent peroxidase activity in
transfected K562 cells (40). In those cells, MPO containing the R569W
mutation was not processed to mature heavy and light subunits,
suggesting that heme insertion was necessary for protein maturation
(40). In a similar fashion, impaired heme insertion resulting from SA
treatment blocked heterodimer formation in flavocytochrome
b558. Because p65 expression is unaffected by SA
treatment and heterodimers are not formed, the fate of p65 synthesized
in the absence of heme synthesis is unknown.
Further studies are necessary to elucidate how the association between
gp91phox and p22phox promotes their increased
expression in comparison with that of the unassembled subunits. The
sites of interaction between the two subunits have not yet been
described, and further studies will also be needed to address the
timing of heme insertion and its role in flavocytochrome
b558 stability. It is likely that observations
in this system will have application to the biosynthesis and targeting
of other heme-containing multisubunit proteins.
INTRODUCTION
Top
Abstract
Introduction
References
2)
produced by the NADPH-dependent oxidase (1). The NADPH oxidase is a multicomponent enzyme complex that includes an integral membrane protein, flavocytochrome b558 (2-4),
and four cytosolic protein components, p47phox (5-7),
p67phox (5, 8), p40phox (9), and a small GTP-binding
protein (Rac) (10, 11). In unstimulated phagocytes the oxidase is
unassembled and inactive. However, during phagocyte activation the
cytosolic components translocate to the plasma and/or phagosomal
membrane to form the functional enzyme complex (12), which generates
O
2 by transferring electrons from cytosolic NADPH to molecular
oxygen (13). A genetic defect affecting flavocytochrome
b558, p47phox, or p67phox
results in a rare inherited disorder known as chronic granulomatous disease (CGD)1 (1).
Individuals afflicted with CGD develop recurrent and often
life-threatening bacterial and fungal infections because of defective
microbial killing (1).
2 production in a
cell-free NADPH oxidase reconstitution assay, indicating that assembly
of the fully functional enzyme complex requires specific interactions
between subunits (21). In addition, stable expression of
gp91phox in phagocytes closely correlates with expression of
p22phox as has been observed in CGD patients with mutations in
either flavocytochrome b558 subunit (22, 23) and
has also been shown by in vitro studies using the heme
synthesis inhibitor, succinyl acetone (SA) (24).
EXPERIMENTAL PROCEDURES
-COP, clone M3A5,
was obtained from Sigma.
2-generating
capacity was determined by either nitro blue tetrazolium reduction or
by the reduction of cytochrome c following stimulation with
0.10 µg/ml 4
-phorbol 12-myristate 13-acetate (24). In the studies
using SA, cells were differentiated with
N,N-dimethylformamide in the presence of 10.0 µg/ml SA or in the presence of an equal volume of sterile
phosphate-buffered saline for 5 days prior to experimentation. Cell
growth, viability, and morphological changes were unaffected by SA
(data not shown).
-COP distribution, 20 µl of each fraction was mixed 1:1
with 2× SDS sample buffer and resolved by 10% SDS-PAGE, and
immunoblots were probed with the indicated antibodies (see above).
-actin, and the intensity of the signals was
quantitated by densitometry.
-COP (see above). Immunoblots were
developed using an enhanced chemiluminescence detection system (SuperSignal Substrate, Pierce) according to the manufacturer's instructions.
RESULTS
View larger version (24K):
[in a new window]
Fig. 1.
In vitro biosynthesis of the
gp91phox precursor, p65. The cDNA for gp91phox
was transcribed/translated in vitro using a rabbit
reticulocyte lysate assay. In the absence ( ) of ER microsomes,
gp91phox was translated as a 58-kDa polypeptide. 65-kDa
glycosylated forms of the initial transcripts were generated when
translation was initiated in the presence (+) of ER
microsomes as indicated.
2 after granulocytic differentiation because of the absence
of endogenous gp91phox (29). Stable transfection of X-CGD
PLB-985 cells with the wild-type gp91phox cDNA under
control of the constitutively active EF1
promoter resulted in
continuous expression of gp91phox even in undifferentiated
cells and restored O
2-generating capacity upon granulocytic
differentiation (28, 29). To further address the subcellular location
of p65 in gp91phox-transfected cells (gp91 PLB), we separated
subcellular organelles by sucrose density gradient centrifugation as
described previously (30). Following centrifugation, 0.5-ml fractions
were collected, and organelles were identified by the presence of
specific marker proteins as described previously (30). ER (CRT and
90-kDa precursor MPO), lysosomes (59-kDa heavy subunit of mature MPO),
Golgi (
-COP), plasma membrane (cell surface biotinylation), and
cytosol (p47phox) were identified and compared with the
sedimentation of gp91phox, p22phox, and p65 (Fig.
2A). Fractions containing peak
levels of gp91phox (fractions 10-13) corresponded to those
containing peak levels of p22phox (fractions 10-13) (Fig.
2A). gp91phox in fraction 12 was resistant to
digestion with Endo H, indicating that its subcellular localization is
likely that of a post-ER and/or post-Golgi vesicle, because it has
already undergone carbohydrate modification (Fig. 2B).
Fractions 7-9 contained peak levels of p65 that co-sedimented with ER
markers CRT and precursor MPO (90 kDa) (Fig. 2A). In
contrast to gp91phox, p65 was found to be susceptible to
digestion with both Endo H and PNGase F consistent with previous
reports (24, 26) and with its localization in the ER (Fig.
2B). Though peak levels of p65 were found in fractions 7-9,
its decreasing distribution toward lesser density in the gradient was
countered by the increasing distribution of gp91phox in those
same fractions (fractions 7-13) (Fig. 2A). The overall distributions of p65 and gp91phox revealed an apparent
ER-to-Golgi continuum separated in the sucrose density gradient as
fraction 7 contained mainly p65, whereas fraction 13 contained only
gp91phox (Fig. 2A, top panel). Only the
mature form of gp91phox was found in fractions corresponding to
the
-COP-associated organelles, implying that p65 had been
completely processed to mature gp91phox prior to reaching the
region of the Golgi associated with
-COP (Fig. 2A). It is
notable that the distribution of
-COP may reflect only a subset of
Golgi vesicles and/or part of the Golgi complex (Fig. 2A).
We additionally observed that the majority of gp91phox and
p22phox was intracellular with only a small fraction associated
with biotinylated plasma membrane proteins (fractions 16-18) (Fig. 2A). This observation has been confirmed by
immunofluorescence microscopy as well (29). Finally,
p22phox distribution appeared unassociated with ER-associated
p65, suggesting that heterodimer formation was not a co-translational
event and likely occurred in a post-ER compartment (Fig.
2A).
View larger version (39K):
[in a new window]
Fig. 2.
Subcellular distribution of gp91phox,
p22phox, and p65. Postnuclear supernatants of
undifferentiated gp91 PLB cells expressing gp91phox were
separated by centrifugation on 10-60% sucrose gradients as described
under "Experimental Procedures." A, gradient fractions
were resolved by 10% SDS-PAGE and then probed with antibodies to
gp91phox, p22phox, CRT, MPO, p47phox, and
-COP, as indicated. A gradient from PLB-985 X-CGD cells was included
(gp91phox KO) to show nonspecific
immunoreactivity just below 65 kDa. The bottom panel
indicates distribution of surface-biotinylated proteins derived from
gp91 PLB cells. Results are representative of at least three to four
separate experiments. B, 20-40 µl of the indicated gp91
PLB gradient fractions were digested with Endo H or PNGase F and
separated by SDS-PAGE, and the immunoblots were probed with antibody to
gp91phox. Results are representative of at least three to four
separate experiments.
-glucuronidase, a lysosomal marker, was released following treatment
with dihydrocytochalasin B and formylmethionylleucylphenylalanine,
indicating that degranulation had occurred (data not shown). Therefore,
the intracellular pool of gp91phox and p22phox was not
contained within lysosomal or azurophilic granule membranes.
2-generating capacity in
comparison with that in undifferentiated cells that do not express the
cytosolic oxidase components p47phox and p67phox (Fig.
3C). The O
2-generating capacity in differentiated
gp91 PLB cells was reduced significantly upon SA treatment (Fig.
3A). Immunoblot analysis of whole cell extracts showed that
decreased activity was associated with reduced expression of both
p22phox and gp91phox (Fig. 3B). The induced
expression of other oxidase components p47phox and
p67phox, non-heme-containing proteins, was unaffected by SA
treatment (Fig. 3C). The SA-dependent reduction
of transgenic gp91phox expression is consistent with our
previous findings on endogenously expressed gp91phox in
differentiated PLB-985 cells (24), providing further evidence that heme
incorporation is important for stable expression of the gp91/p22
heterodimer. By contrast, expression of p65 was unaffected by SA
treatment, which is a finding similar to what has been described in
SA-treated PLB-985 cells (Fig. 3B) (24). Furthermore, these data suggest that p22phox does not interact with p65 in the
absence of heme incorporation, because stable expression of
p22phox is not supported by the continued presence of p65 in
SA-treated cells.
View larger version (32K):
[in a new window]
Fig. 3.
Effect of SA on O 2-generating
capacity and expression of NADPH oxidase components in differentiated
gp91 PLB cells. Undifferentiated (D0) gp91 PLB cells or
those differentiated (D5) for 5 days in the absence
(
SA) or presence (+SA) of SA were analyzed for
O
2-generating activity (A), expression of
gp91phox and p22phox (B), or expression of
p47phox and p67phox (C) by immunoblotting as
described under "Experimental Procedures." The 65-kDa precursor of
gp91phox is indicated by an arrow. Results are
expressed as the mean ± S.D. of at least three experiments.
View larger version (68K):
[in a new window]
Fig. 4.
Northern blot analysis of gp91phox
and p22phox expression in gp91 PLB cells. Total cellular
RNA was isolated from undifferentiated gp91 PLB cells (D0)
and those differentiated (D5) for 5 days in the absence
( SA) or presence (+SA) of SA and analyzed by
Northern blot analysis. Each lane was loaded with 10 µg of
RNA, and mRNA for actin was probed to show equivalent loading.
Results shown are from one representative experiment performed three
times.
2-integrin (CD11b/CD18), a non-heme-containing
heterodimer, was unaffected, demonstrating that normal protein
processing was unaffected by SA treatment (Fig. 5B).
View larger version (30K):
[in a new window]
Fig. 5.
SA-dependent reduction of plasma
membrane-associated flavocytochrome
b558. Undifferentiated gp91 PLB cells
not treated ( SA) or those treated (+SA) with SA
for 5 days were stained with 7D5, which recognizes an extracellular
epitope of gp91phox and was analyzed by flow cytometry
(A). Mouse IgG1 was used as an isotype control in
A. Alternatively, these cells were analyzed for surface
expression of CD11b to determine the effect of SA on normal protein
processing (B). IgG2b was used as an isotype control in
B. Results shown are from one representative experiment
performed three times.
DISCUSSION
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Algirdas J. Jesaitis, James B. Burritt, and Mark T. Quinn at Montana State University for their generous contribution of anti-gp91phox and p22phox monoclonal antibodies (54.1 and 44.1) and Dr. Michio Nakamura (Nagasaki University, Japan) for graciously providing 7D5, the anti-flavocytochrome b558 monoclonal antibody used for flow cytometry. In addition, Drs. Dirk Roos and Arthur J. Verhoeven (Central Laboratory of the Netherlands Blood Transfusion Service) provided anti-gp91phox and p22phox monoclonal antibodies 48 and 449, respectively.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Public Health Service Grants 5-T32-AI07343-09 (to F. R. D.), RO1 HL45635 and PO1 HL 353586 (to M. C. D.), AI-34879 (to W. M. N.) and a Veterans Administration Merit Review (to W. M. N.).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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Wells Center for
Pediatric Research, Cancer Research Bldg., Rm. 466, 1044 W. Walnut St.,
Indianapolis, IN 46202. Tel.: 317-274-8645; Fax: 317-274-8679; E-mail:
mdinauer{at}iupui.edu.
The abbreviations used are:
CGD, chronic
granulomatous disease; phox, phagocyte oxidase; SA, succinyl
acetone; PAGE, polyacrylamide gel electrophoresis; PNGase F, peptide
N-glycosidase F; Endo H, endoglycosidase H; CRT, calreticulin; MPO, myeloperoxidase; ER, endoplasmic reticulum; -COP, a 110-kDa subunit of the coat proteins.
2 A. Yamauchi, L. Yu, A. Postgens, F. DeLeo, F. Kuribayashi, H. Nunoi, S. Kanegasaki, D. Roos, W. Nauseef, H. Malech, M. Dinauer, and M. Nakamura, personal communication.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|