From the Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan and § Biophotonics Information Laboratories/MMBS, Faculty of Science, The University of Tokyo, Misaki, Kanagawa 238-0225, Japan
Received for publication, February 6, 2001, and in revised form, April 3, 2001
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ABSTRACT |
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Activation of the neutrophil NADPH oxidase occurs
via assembly of the cytosolic regulatory proteins p47phox,
p67phox, and Rac with the membrane-associated
flavocytochrome b558. Following cell-free
activation, enzymatic activity is highly labile (Tamura, M., Takeshita,
M., Curnutte, J. T., Uhlinger, D. J., and Lambeth, J. D. (1992) J. Biol. Chem. 267, 7529-7538). To try to
stabilize the activity and investigate the nature of the complex,
fusion proteins between p47N-(1-286) and p67N-(1-210) were
constructed. In a cell-free system, a fusion protein, p67N-p47N, had an
8-fold higher efficiency and produced a higher activity than the
individual proteins, and also resulted in an 8-fold improved efficiency
for Rac and a lowered Km for NADPH.
O The superoxide-generating phagocyte NADPH oxidase (Phox/Nox-2)
functions in host defense against microbial infection (1, 2). The
enzyme is dormant in resting cells and becomes active upon cell
stimulation. The activation is thought to occur via assembly of
cytosolic components, p47phox
(p47),1 p67phox
(p67), and Rac with the membrane-associated flavocytochrome
b558 (cyt. b558), which
consists of p22phox (p22) and gp91phox (gp91) although
the structure of the complex has remained unclear (3-6). Other two
factors p40phox and rap1A are also assumed to be involved in
the enzyme regulation although they are not essential for the activation.
The activated NADPH oxidase is highly labile, complicating
investigations of the subunit structure and preventing isolation of the
active enzyme complex (7). In a previous study (8) using a cell-free
system consisting of cytosol and plasma membrane (PM) we showed that
the stability is dramatically improved by chemical cross-linking, and
suggested that the deactivation is caused by dissociation of proteins
from the complex. Cross-linking was useful in stabilizing the oxidase,
but it was difficult to isolate the active complex because the
cross-linked complex resisted solubilization (8). We have also
attempted cross-linking in a system comprised of recombinant cytosolic
factors and purified cyt. b558, but the effect
of cross-linking was not as dramatic as in the crude
system.2
Fusion proteins between cytochrome P450s and their reductase,
NADPH-cytochrome P450 reductase have been genetically engineered (9,
10). Subsequent studies revealed that fusion facilitates electron
transfer from NADPH to several cytochrome P450s via flavins (FMN and
FAD) (11, 12). This suggested to us that fusion might be useful for the
stabilization of the phagocyte oxidase activity. In addition, the use
of fusion proteins can provide basic information on the structure of
the complex, e.g. topology and interactions between
components. Interactive domain structures such as Src homology 3 (SH3)
domain and proline-rich region (PRR) were identified in p47 and p67
(13). The latter structure was also found in p22 (a subunit of cyt.
b558) (14). In addition, PX domain and tetratricopeptide repeats (TPR) were identified in p47 and p67, respectively (15). More recently, Han et al. (16) identified an activation domain in the middle of p67, and De Leo et al.
(17) found a cationic region in C-terminal of p47. The interactions among these domains, especially between SH3 and PRR, have been extensively studied (4, 18-22). However, neither the actual interactions nor the topology of the components in the active complex are unknown (5).
We have herein genetically engineered two fusion proteins between
truncated forms of p47 and p67 and have examined their properties. The
truncated forms have previously been shown to be capable of reconstituting enzymatic activity (16, 23). We find that both expressed
fusion constructs can efficiently activate the oxidase in a cell-free
reconstitution system. In addition, one of them produced an oxidase
complex with higher activity and remarkably higher stability than the
individual components. In this system, the activity requirement for
anionic amphifiles was eliminated. Based on these results, we propose a
model for the topology of these cytosolic factors in the active complex.
Materials--
pGEX-6P, Escherichia coli BL21,
glutathione-Sepharose, DEAE-Sepharose, CM-Sepharose, and PreScission
Protease were purchased from Amersham Pharmacia Biotech (Little
Chalfont, United Kingdom). The oligonucleotide primers for mutations
were synthesized by the same manufacturer. BamHI and
EcoRI were obtained from Toyobo Co. (Tokyo, Japan).
L- Preparation of PM from Human Neutrophils--
Human blood was
obtained from healthy volunteers with informed consent. The separation
of neutrophils and the fractionation of PM were performed as previously
described (24).
Purification of Cytochrome b558--
Neutrophils
were obtained from porcine blood basically according to the method of
Fujii and Kakinuma (25) with several
modifications. The cells suspended in buffer A (60 mM KCl,
18 mM NaCl, 2.3 mM MgCl2, 1 mM PMSF, 3 µM TLCK, 1 mM ATP, and
6% (w/v) sucrose, 6 mM PIPES, pH 7.4) were subjected to
nitrogen cavitation at 500 p.s.i. for 20 min at 4 °C. The
mixture was centrifuged at 800 × g for 5 min, and the
supernatant was centrifuged at 140,000 × g for 1 h in a sucrose gradient (50 and 30% (w/v) in buffer A). The turbid
yellow band between two phases was taken up, diluted twice with buffer
B (140 mM NaCl, 1 mM PMSF, 1 mM
EGTA, 50 mM PIPES, pH 7.4), centrifuged at 250,000 × g for 1.5 h in a compact ultracentrifuge (Hitachi
CS100GX), and resuspended in buffer B. The fraction was referred to as
"plasma membrane (PM)." The purification of cyt.
b558 from PM was essentially followed by Abo
et al. (26). NaCl solution (5 M) was added to
the fraction and allowed the final concentration to 1 M.
After centrifugation at 140,000 × g for 1 h, the
pellet was suspended in 50 mM PIPES (pH 7.4) containing 50 mM NaCl, 1 mM PMSF, 1 mM EGTA, 0.5 mM dithiothreitol, and 16% (v/v) glycerol. The suspension
was solubilized by 50 mM
n-heptyl- Expression of Recombinant p47 (Full-length), p67 (Full-length),
p67N, and Rac--
Full-length p67 was overexpressed in Sf9
cells and purified with Q-Sepharose as described (27). The cDNAs
for p47 (in pVL1393), p67N (residues 1-210; in pGEX-2T), and Rac
(Rac1[C189S] in pGEX-2T) were generous gifts from Drs.
Chang-Hoon Han and Dave Lambeth (Department of Biochemistry,
Emory University School of Medicine). After amplification with
polymerase chain reaction using specific 5' and 3' primers, both
attached with EcoRI restriction linker, p47 cDNA was
subcloned into pGEX-6P (EcoRI fragment) and transfected into
E. coli BL21 strain by the TSS method (28). Recombinant p47,
p67N, and Rac were expressed in E. coli as glutathione
S-transferase fusion proteins, purified with
glutathione-Sepharose beads, released from glutathione
S-transferase domain by thrombin (for p67N and Rac) or
PreScission Protease (for p47), and dialyzed against 20 mM
potassium phosphate buffer (pH 7.0). By using this system, p47
(full-length) was obtained in a relatively good yield (2-3 mg/1 liter
of medium) without any proteolytic
fragmentation,3
which has often been observed and precluded the production of full-length p47 in E. coli system. All the proteins were
purified to >95% homogeneity.
Construction of Recombinant Plasmids for Fusions--
DNA
fragment encoding p47N-(1-286) was amplified from pVL1393-p47 with
polymerase chain reaction using specific 5' and 3' primers, both
attached with EcoRI restriction linker. For p67N-p47N fusion, the p67N cDNA in pGEX-2T was mutated to remove the stop codon by converting TGA (stop codon) to TCA (Ser) using primers 5'-GGCATCTGTGGTGGATTCAGAATTCATCGTGAC-3' and
5'-GTCACGATGAATTCTGAATCCACCACAGATGCC-3' by a site-directed
mutagenesis Quick Change kit (Stratagene, La Jolla, CA). Then, p47N
cDNA was ligated to pGEX-2T-p67N (EcoRI fragment). All
the truncated and mutated sequences were verified by DNA sequencing by
primer extension. For p47N-p67N fusion, p47N cDNA was subcloned
into pGEX-6P (EcoRI fragment) and engineered to change the
EcoRI site into the BamHI site and to eliminate the stop codon by converting TGA to TCA (Ser codon). p67N cDNA was
engineered to change the BamHI site into EcoRI
site and ligated to pGEX-6P-p47N (EcoRI fragment).
Expression and Purification of Fusion Proteins--
The proteins
were expressed in E. coli BL21 strain as fusion proteins
with glutathione S-transferase and purified with
glutathione-Sepharose beads, and cleaved from glutathione
S-transferase with thrombin (for p67N-p47N) or PreScission
Protease (for p47N-p67N). The resulting fusion proteins have
Ser-Glu-Phe as a linker peptide between two Phox proteins.
Reconstitution of NADPH Oxidase and Assay for O
For the reconstitution with purified cyt. b558,
the reaction mixture contained p67N and p47N (125 nM each)
or a fused protein between p67N and p47N (125 nM) with
GTP-treated Rac (430 nM) and cyt.
b5582 (6.3 nM) (10 µl
of the suspension) in buffer D (800 µl total) containing 1 mM FAD. By this process the
n-heptyl- Fusion Proteins Used in This Study--
We designed fusion
proteins using C-terminal-truncated forms of both p47 and p67. These
truncations were previously shown to support a full activation in a
reconstitution system (16, 23). Fig.
1A illustrates the domain
structures of p47 and p67 truncations and their fusion proteins.
C-terminal-truncated p47 (p47N) (residues 1-286) contains the PX
domain and two SH3 domains, whereas the C-terminal-truncated p67 (p67N)
(residues 1-210) contains TPR and the activation domain. We produced
two kinds of the fusion proteins in different order, designated
p47N-p67N and p67N-p47N. In the former, PX domain is located at the
N-terminal end and the activation domain is at the C terminus. In the
latter, the TPR domain is located at the N terminus and the SH3 regions
are located at the C terminus. Fig. 1B shows
SDS-polyacrylamide gel electrophoresis of the proteins expressed in
E. coli and purified with glutathione-Sepharose beads. Each
protein showed a single major band at the expected size.
The Reconstituted Activity of NADPH Oxidase--
Fig.
2A shows the superoxide
generating activity of the oxidase reconstituted with truncated and
fusion proteins with PM. The activity with p47N was 50% higher than
that with full-length p47. p47N-p67N showed 40% higher activity than
nonfused p67N and p47N, whereas p67N-p47N showed almost twice the
activity of nonfused components. Fig. 2B shows the oxidase
activity with purified cyt. b558. The activity
with nonfused p47N and p67N was similar to that with full-length p47
and p67N, or full-length of both p47 and p67 (data not shown). The
activity with a fusion p67N-p47N was higher than that with the nonfused
components. On the other hand, the activity with p47N-p67N was similar
to that with nonfused p47N and p67N. This result indicates that fusion
(especially p67N-p47N) greatly improves the ability of the components
to induce the oxidase activity.
Effect of Fusion on the Efficiency of the Components--
The
efficiencies (i.e. EC50 values) of the fusion
proteins in stimulating NADPH oxidase activity were compared with the
nonfused components (Fig. 3). The
EC50 values estimated for nonfused components, p47N-p67N,
and p67N-p47N were 0.81, 0.22, and 0.10 µM, respectively, indicating that fusion (particularly the p67N-p47N construct) greatly
improves the affinity of the components within the active complex. The
Vmax with p67N-p47N was 6.2 µmol/min/nmol of
cyt. b558, which was twice the activity seen
using the nonfused components, while the Vmax
with p47N-p67N was similar to that with nonfused components. This
result indicates that p67N-p47N is a very efficient activator of the
oxidase.
Concentration Dependence for Rac and Cytochrome
b558--
Fig. 4A
shows the concentration dependence for Rac in the cell-free system
using fused or nonfused p47N and p67N. The EC50 value for
Rac was 0.5 µM with individual cytosolic proteins, and this was dramatically lowered by using p67N-p47N (0.06 µM, Table I). Meanwhile,
the EC50 value with p47N-p67N decreased to one-half of that
of the nonfused components. The Vmax value was
in the following rank order: p67N-p47N > p47N-p67N > nonfused components. The result shows that fusion improves the affinity
of Rac for other components in the complex. Whereas Fig. 4B
shows the concentration dependence for cyt.
b558. The concentration dependence for cyt. b558 with nonfused components was not changed by
fusions, p67N-p47N or p47N-p67N.
Concentration Dependence for NADPH--
The Km
value for NADPH was determined in the assay of the oxidase
reconstituted with the fused or nonfused components (Fig.
5). The Km with the
nonfused components was 50 µM, which was decreased to 17 and 23 µM by p67N-p47N and p47N-p67N, respectively (Table
I). The Vmax value was increased by 51% with p67N-p47N and slightly with p47N-p67N. This result shows that fusion
between p47N and p67N influences the NADPH-binding site, which is
thought to be in cyt. b558.
Stability of NADPH Oxidase Reconstituted--
Fig.
6A shows the stability of the
oxidase activity reconstituted with PM at 25 °C. After 25 min
incubation, the oxidase with p67N-p47N showed the same activity as the
initial one while that with nonfused components had only 20% activity
of the initial one. The half-life (t1/2) was 16 min
with nonfused p47N and p67N and was prolonged to 240 min by the fusion
of p67N-p47N. While the half-life with p47N-p67N was 28 min (Table
II). When purified cyt.
b558 was used in the reconstitution, the
half-life with nonfused p47N and p67N was 38 min, which was expanded to
290 min (Fig. 6B and Table II). The half-life with p67N-p47N
was 22 min, similar to that with nonfused components. This result
indicates that fused p47N and p67N (especially p67N-p47N) produces much
more stable complex than the nonfused components. When full-length p47
and p67 (nonfused) were used, the stability was the same as that with
truncated p47 and p67. Thus C-terminal regions of these components do
not appear to be involved in the longevity of the complex.
Rac, a small GTPase, is thought to regulate the lifetime of the oxidase
by converting its state from active to inactive upon hydrolysis of
loaded GTP (3, 29). Therefore, we examined the stability of Rac loaded
with GTP SDS-independent Activation with Fusion Proteins--
Table
III shows the activity reconstituted in
the presence and absence of SDS. As reported by Hata et al.
(23), the truncated p47 and p67 produced some O Effect of Acidic PLs in Relipidation of Cytochrome
b558--
Fig. 7 shows the
effect of acidic PLs in relipidation of cyt.
b5582 on activity in the absence of
SDS. When the content of PI or PS was increased, the reconstituted
oxidase activity with p67N-p47N increased in a
dose-dependent manner. However, the activity with nonfused
components was not changed much by PS or PI. In contrast, in the
presence of SDS, the activity induced by fused or nonfused components
was constant regardless of the content of acidic PLs (data not shown).
When PI was directly added to the assay mixture, the oxidase activity
was not influenced suggesting that PI is not effective as a free form
but effective when incorporated into the vesicles.
In the above experiments, we used an optimized mixture of lipids. Here
we tested the lipid composition of neutrophil PM (30, 31) in the
relipidation and examined the reconstitution with p67N-p47N in the
absence of SDS. As shown in Table IV,
with natural PL content of the activity was 3.0 µmol
of O The initial aim of this study was to investigate how the stability
of the oxidase is related to complex formation using molecular fusions
between p47 and p67. Two novel fusion proteins between truncated forms
of p47 and p67 were made, and both were found to be effective in
activating the oxidase in a cell-free system. In particular, one of the
fusions, p67N-p47N, produced much higher activity than the nonfused
components. In addition, the stability of the enzyme was remarkably
improved by using p67N-p47N while the reverse fusion p47N-p67N did not
improve the stability.
Kinetic studies showed that p67N-p47N is more effective in activating
the oxidase, showing a much lower EC50 than the
corresponding nonfused components. The p67N-p47N fusion markedly
lowered the EC50 value for Rac, suggesting that fusion
strengthened the affinity of Rac for p67N, cyt.
b558 or both. Interestingly, p67N-p47N produced a significantly lower Km for NADPH. This suggests
that the fusion modifies the tertiary structure of the NADPH-binding site in cyt. b558 by interacting either directly
or indirectly. It is also possible that p67 itself could provide part
of the NADPH-binding site as proposed by Dang et al. (32).
However, Nisimoto et al. (33) showed that mutations in the
p67 activation domain does not affect the Kd of the
oxidase for NADPH, and the effect of p67 on activity is consistent with
that on the rate of the hydride transfer from NADPH to FAD of gp91,
suggesting that p67 itself may not directly contribute binding energy
for NADPH.
As for the stability the role of a small GTPase Rac has been suggested
(29). Conversion of active form to inactive form by GTP hydrolysis may
dominate the lifetime of the oxidase. The present study, however, shows
that the deactivation is not influenced by changing Rac activator from
GTP to GTP In this study p47N-p67N fusion had a slightly higher activity and
similar stability compared with nonfused components. The EC50 value for Rac, the Km for NADPH,
and the efficiency were all intermediate between those of nonfused
components and p67N-p47N. These facts apparently show that p47N-p67N
and p67N-p47N can both reconstitute the oxidase. This seems strange
because the reverse connection must cause a drastic change in the
topology of the subunits in the complex, which must be strict in the
active complex of NADPH oxidase. To solve this dilemma we tried to find a model, in which the findings described here are satisfied.
Fig. 8 is the schematic illustration of a
possible model for the arrangement of components p47, p67, and cyt.
b558 in the active complex. In the figure, the
complex is illustrated as a bottom view, i.e. seen from the
cytoplasmic side. Experimental data with fusions indicates that the
activation domain C terminus should be located close to the PX domain N
terminus, and the TPR domain N terminus should be close to the SH3
domain C terminus. In the model the PRR of p22 (represented by P)
interacts with two SH3 regions simultaneously as suggested by two
groups (21, 34). Also, the domains of p47 and p67 are arranged
circularly with the activation domain of p67 fixed on cyt.
b558. In our fusion experiment, the stability
was dramatically improved by p67N-p47N in which the activation domain
is directly fixed by linking with the PX domain. On the other hand, the
stability was not much improved by p47N-p67N, in which the domain is
indirectly set by a link between SH3 and TPR domains. That is why the
former produces a very stable complex and the latter does not, but
nevertheless it shows a similar activity as the nonfused proteins. We
speculate that orientation of the activation domain at the correct
position is centrally important for activation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-Phosphatidylcholine (PC) (soybean),
L-
-phosphatidylethanolamine (PE) (bovine brain),
L-
-phosphatidylinositol (PI) (bovine liver), phosphatidylserine (PS) (bovine brain), cholesterol,
-aminooctyl-agarose, heparin-agarose, cytochrome c (horse
heart), thrombin (bovine plasma), and GTP
S were purchased from Sigma
Aldrich. Diisopropyl fluorophosphate and
n-heptyl-
-D-thioglucoside were
obtained from Wako Pure Chemicals (Osaka, Japan). GTP, FAD,
phenylmethanesulfonyl fluoride (PMSF), PIPES, and sphingomyelin (SM)
(bovine brain) were obtained from Nacalai Tesque (Kyoto, Japan). All
the phospholipids used were of the highest grade of the manufacturers
(98-99% purity).
-D-thioglucoside and the mixture
was immediately centrifuged at 100,000 × g for 1 h. The supernatant was subjected to a three-bed column
(DEAE-Sepharose,
-aminooctyl-agarose, and CM-Sepharose) equilibrated
with buffer C (50 mM NaCl, 1 mM PMSF, 0.2 mM dithiothreitol, 3 µM TLCK, 17 mM n-heptyl-
-D-thioglucoside, 50 mM PIPES, pH 6.5) containing 16% (v/v) glycerol. The
flow-through fractions were applied to a heparin-agarose column
equilibrated with buffer C containing 8% glycerol and 1 mM
EGTA, and the column was eluted with NaCl gradient (0.05-1.5
M) in the same buffer. After concentration with an
Ultrafree Biomax 30k (Millipore Corp., Bedford, MA), the fraction
containing cyt. b558 was immediately mixed with
PL suspension (PC (31), PE (3), PI (29), SM (33), cholesterol (12) weight % of total lipid in buffer C) to give a final concentration of
1 mg/ml cyt. b558 preparation. We happened to
find that this PL composition effective for the oxidase activation and
designated it as "optimal composition." In some experiments, PL
composition was varied, but total lipid content in the suspension was
always maintained at 1 mg/ml. The specific content of heme in purified cyt. b558 preparation was 2.1-2.5 nmol of
heme/mg of protein.
-D-thioglucoside concentration was
diluted to 0.2 mM (CMC: 30 mM). The mixture was
supplemented with 100 µM SDS and incubated for 5 min at
25 °C. The mixture was transferred to the cuvette containing 80 µM cytochrome c and the reaction was started
with addition of 200 µM NADPH. Absorbance change at 550 nm was measured with a spectrophotometer UV160A (Shimadzu, Kyoto).
Superoxide generation was expressed as micromole of O
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ABSTRACT
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Fig. 1.
Fusion proteins used in this work.
A, truncations p47N-(1-286), p67N-(1-210), and their
fusion proteins are schematically shown featuring domain structures.
p47N possesses a PX domain (including PRR) and two SH3 domains whereas
p67N has a TPR domain. The amino acid residues at the hinges of both
fusions are Ser-Glu-Phe. Dotted lines and symbols
are the regions and domains truncated. PRR is represented by
P and the cationic region by +++. B,
SDS-polyacrylamide gel electrophoresis of the truncations and fusions.
The proteins (1 µg each) were loaded on a 15% (w/v) gel. After
electrophoresis the proteins were stained with Coomassie Blue. Expected
molecular mass of p47N, p67N, and their fusions were 33, 24, and 57 kDa, respectively.
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Fig. 2.
NADPH oxidase activity reconstituted with
fused or nonfused p47 and p67. A, the cell-free
activation mixture contained a fused (2 µM) or nonfused
p47N and p67N (2 µM each), GTP-loaded Rac (7 µM), PM (5 µg), GTP (10 µM), and SDS (200 µM) in 50 µl volume. In some experiments full-length
p47 was used instead of p47N (p47FL). The reaction was initiated by the
addition of assay mixture including NADPH (200 µM) and
cytochrome c (80 µM). O
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Fig. 3.
Concentration dependence for fused or
nonfused p47N and p67N in the reconstitution of the oxidase. The
reconstitution system included different concentrations of fused
(p67N-p47N or p47N-p67N) or nonfused p47N and
p67N (Nonfused), the concentrations of nonfused components
being synchronized. The PM was used as the cyt.
b558 source. Other experimental conditions are
as described in the legend to Fig. 2A. The data are
expressed as mean ± S.D. from three experiments.
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Fig. 4.
Concentration dependence for Rac and cyt.
b558 in the activation using fusion
proteins. A, the reconstitution system contained
various concentrations of GTP-loaded Rac with fused
(p67N-p47N and p47N-p67N) or nonfused p47N and
p67N (Nonfused) with PM. Other experimental conditions are
as described in the legend to Fig. 2A. The data are
expressed as mean ± S.D. from three experiments. B,
the reconstitution system contained different concentrations of cyt.
b558 with p47N-p67N (closed
triangle), p67N-p47N (closed square), or nonfused p47N
and p67 (open square) as well as GTP-loaded Rac. Other
experimental conditions are as described in the legend to Fig.
2B.
Effect of fusion on the affinity for Rac and Km for NADPH of
the reconstituted oxidase
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Fig. 5.
Effect of fusion on
Km for NADPH of the oxidase
reconstituted. The reconstitution system contained fused
(p47N-p67N and p67N-p47N) or nonfused p47N
and p67N (Nonfused) with GTP-loaded Rac and PM. The assay
mixture contained different concentrations of NADPH. Other experimental
conditions are as described in the legend to Fig. 2A. The
data are expressed as mean ± S.D. from three experiments.
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Fig. 6.
Stability of NADPH oxidase activity
reconstituted with p67N-p47N or nonfused components. A,
the reconstitution system contained p67N-p47N (2 µM) or
nonfused p67N and p47N (2 µM each) with GTP-loaded Rac (7 µM) and the PM (5 µg). After activation with SDS for 10 min, the mixture was kept at 25 °C for a given time. Then the
mixture was supplemented with NADPH and superoxide generation was
measured as described in the legend to Fig. 2A . Activities
are expressed as the percentages of initial activities. The initial
activities with p67N-p47N and nonfused components were 3154 ± 44 and 2730 ± 32 nmol/min/mg of membrane protein, respectively.
B, purified cyt. b558 was used in the
reconstitution system. In some experiments full-length p47 and p67 were
used (open triangle). Other experimental conditions are as
described in A. The initial activities with p67N-p47N and
nonfused p47N/p67N and full-length p47/p67 were 5558 ± 2.5, 4638 ± 45, and 5565 ± 55 nmol/min/mg of PM protein,
respectively.
Half-lives of NADPH oxidase activity reconstituted with fused or
nonfused p47N and p67N
S-loaded Rac (upper row). In some experiments full-length p47 and
p67 were used as components (a) or the activation was
performed in the absence of SDS (b) (lower row).
S, a non-hydrolyzable GTP analog, however, the stability of
the oxidase was not changed (Table II, in parentheses). Thus GTP
hydrolysis is not involved in the deactivation of the oxidase at least
for initial 30 min in the reconstitution system.
SDS-independent activation of NADPH oxidase by p67N-p47N with purified
cyt. b558
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Fig. 7.
Effect of PI or PS on the activation with
p67N-p47N or nonfused components in the absence of SDS. The
purified cyt. b558 was relipidated with a PL
mixture containing PC (0.62), PE (0.06), PI or PS (0-0.58), SM (0.51),
cholesterol (0.24) (mg) to give the final PL concentration of 1 mg/ml
cyt. b558 preparation. The activation was
performed with p67N-p47N (squares) or the nonfused
components (triangles). Other experimental conditions are as
described in the legend to Fig. 2B.
SDS-independent activation of the oxidase by p67N-p47N with cyt.
b558 relipidated with neutrophil-type lipid or PI (PS) enriched
lipid
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES
S (Table II) indicating that the deactivation observed
here is not due to GTP hydrolysis.
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Fig. 8.
A model for the topology among p47N, p67N,
and cyt. b558 in the active complex.
The figure is a view from the bottom (cytoplasmic side) of the complex.
The small and large subunits of cyt. b558 (p22
and gp91) are designated as and
, respectively. PRR of p22 (
)
is represented by "P." N-terminal and C-terminal of
p47/p67 are shown by "N" and "C,"
respectively. "R" represents Rac protein, the location
of which is tentative. Background spots represent polar heads of
phospholipids in lipid bilayer. Other domain abbreviations are as
defined in the legend to Fig. 1.
As the truncated p47 and p67 showed maximal activity, tail-tail interaction between p47 (PR domain) and p67 (SH3 domain) (4, 5, 21) may not be essential in the final stage of activation. However, the idea is well accepted that p47 mediates the binding of p67 to the cytochrome. Then a question arises, how do the truncated forms of p67 and p47 interact with each other in the activated complex? The answer is not clear at present but a possibility is an interaction between the N-terminal regions of p47 and p67. In relation to this, we find that a point mutation in the PX domain of p47 elevates EC50 of p67N in the reconstitution,4 suggesting that the PX domain interacts with the activation domain or with the TPR region of p67. Further study will be required to clarify this point.
One of the most interesting findings in this study is that p67N-p47N does not require SDS for activation. This indicates that the enzyme reconstituted with the fusion protein is spontaneously active when mixed with cyt. b558 and GTP-loaded Rac. It is apparent that fusion of p47 and p67 substitutes in some manner for SDS. One role of activating anionic amphifiles is thought to release the self-blocking of p47, permitting the complex formation with cyt. b558. There have been reports suggesting two intramolecular interactions within the p47 molecule. One is between SH3 domains and the cationic region (23, 35), and the other is between PX and SH3 domains (34). In the truncated p47, however, the first interaction is already eliminated while the second interaction is still valid. We consider that the N terminally fused p67N interferes with the second self-blocking and keeps p47N fully open. This is supported by the fact that either nonfused components or the reverse fusion p47N-p67N cannot fully activate the oxidase without SDS. Nevertheless, the possibility cannot be ruled out that fusion also influences the conformation of p67, which has also been assumed as a target of anionic amphifiles (23, 36).
Anionic amphifile-independent activation has been reported by other groups. Hata et al. (23) reported that truncated p47 and p67 produced a partial activation (44%) with Rac and PM without anionic amphifile. We confirmed that a partial activation (22%) was produced with truncated p47 and p67 in the absence of SDS. In contrast to this, p67N-p47N produced a complete activation without SDS, which was even higher than that with nonfused components in the presence of SDS. Very recently, Gorzalczany et al. (37) reported that prenylated Rac with p67 can activate cyt. b558 in the absence of anionic amphifile and p47. Our system cannot be compared with theirs because we used non-prenylated Rac in the reconstitution.
We found an effect of acidic phospholipids on the activation,
i.e. PI and PS in relipidation of cyt.
b558 enhances the activation. As this effect was
evident in SDS-independent activation, acidic PL in the vesicle must
replace one effect of SDS. Cyt. b558 is highly
positive-charged (pI values calculated for p22 and gp91 are 10.0 and
9.7, respectively) as are p47 PX domain (pI 9.1) and p67N (pI 9.2).
Negative charges incorporated into the vesicles might therefore
facilitate the access of p47 and p67N to cyt. b558 by eliminating the charge repulsion.
Negative charges in the vesicles may also facilitate the binding of
cationic C-terminal of Rac1. Kreck et al. (38) reported that
PI in the membrane facilitates Rac1 insertion to the membrane. Another
possible mechanism is that acidic PL in vesicles directly interacts
with cyt. b558 and induces an activating
conformational change in cyt. b558. Although the
exact mechanism is not clear at present, this finding can be
related to our previous work which showed that acidic PLs enhance
O
The mechanism for SDS activation has not been fully elucidated. However, the present results enable us to dissect the multiple roles of SDS in a cell-free activation as follows: 1) to release the self-blocking between SH3 and cationic regions; 2) to release the self-blocking between PX and SH3 domains; and 3) to change the membrane charges. As above, role 1 is substituted by truncation (deletion of C-terminal region), and role 2 is presumably substituted by fusion with p67N at the N-terminal end, and finally role 3 may be executed by acidic PLs incorporated into the vesicle membrane. In cells, role 1 is probably accomplished by phosphorylation of p47 by a protein kinase (39), and roles 2 and 3 might be performed by cellular anionic amphifiles such as arachidonate (34) or phosphatidate (40-42) formed on cell activation. We found that acidic PLs do not increase on cell activation by phorbol ester (30).
In summary, we genetically engineered fusion proteins between p47 and
p67 and could stabilize the enzyme complex successfully. Based on
kinetic studies of the enzyme reconstituted with fused or nonfused
components we propose a model for the physical arrangement of the
cytosolic components in the active complex. Finally, we emphasize that
protein fusion may be useful as a general technique to stabilize
multicomponent enzymes or functional assemblies of proteins although
the approach may not always be valid owing to the steric hindrance or
topological limitations of the complexes.
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ACKNOWLEDGEMENT |
---|
We are indebted to Yasuaki Komeima, Shingo Masuda, and Tetsutaro Kai (this department) for technical assistance, and Drs. Yaeta Endo and Kei-ichi Kato (this department) for discussions and instruments. We are grateful to Drs. Komei Shirabe (Department of Biochemistry, Oita Medical University), Chang-Hoon Han and Dave Lambeth (Department of Biochemistry, Emory University), and Takuo Shiraishi (Yamagata Advanced Technology Research Center) for helpful discussions and samples. We are also thankful to Dr. Shuichi Saheki and Kazue Murakami (Ehime University Health Care Center) for obtaining human blood.
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FOOTNOTES |
---|
* 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.
Present address: Chemo-Sero-Therapeutic Research Institute,
Ohkubo, Kumamoto 860-8568, Japan.
¶ To whom correspondence should be addressed. Tel.: 81-89-927-9938; Fax: 81-89-927-8546; E-mail: miketamu@en3.ehime-u.ac.jp.
Published, JBC Papers in Press, May 1, 2001, DOI 10.1074/jbc.M101122200
2 M. Tamura, unpublished data.
3 K. Ebisu and M. Tamura, unpublished results.
4 K. Ebisu and M. Tamura, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
p47, p47phox;
p67, p67phox;
p22, p22phox;
gp91, gp91phox;
p47N, C terminus-truncated p47phox (residues
1-286);
p67N, C terminus-truncated p67phox (residues 1-210);
cyt. b558, flavocytochrome
b558;
PMSF, phenylmethanesulfonyl fluoride;
PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid);
PM, plasma membrane;
PC, L--phosphatidylcholine;
PE, L-
-phosphatidylethanolamine;
PI, L-
-phosphatidylinositol;
PS, phosphatidylserine;
SM, sphingomyelin;
PL, phospholipid;
SH3, Src homology 3;
PRR, proline-rich
region;
TPR, tetratricopeptide repeats;
TLCK, tosyl-L-lysyl
chloromethylketone;
GTP
S, guanosine
5'-3-O-(thio)triphosphate.
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