Fused p47phox and p67phox Truncations Efficiently Reconstitute NADPH Oxidase with Higher Activity and Stability Than the Individual Components*

Kentaro EbisuDagger, Teruaki Nagasawa, Kyoji Watanabe, Katsuko Kakinuma§, Kei Miyano, and Minoru Tamura

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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&cjs1138;2 generating activity was remarkably stabilized by using p67N-p47N. The cytosolic proteins fused in the opposite orientation, p47N-p67N, showed similar activity and stability as individual proteins, but with a 4-fold improved efficiency compared with the individual cytosolic factors. In the system efficiency for Rac and affinity for NADPH were also higher than those with the nonfused components. Interestingly, the p67N-p47N showed nearly full activation in the absence of an anionic amphifile in a cell-free system containing cytochrome b558 relipidated with phosphatidylinositol- or phosphatidylserine-enriched phospholipid mixtures. From the results we consider multiple roles of anionic amphifiles in a cell-free activation, which could be substituted by our system. The fact that a fusion produces a more stable complex indicates that interactions among components determine the longevity of the complex. Based on the findings we propose a model for the topology among p47N, p67N, and cytochrome b558 in the active complex.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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-alpha -Phosphatidylcholine (PC) (soybean), L-alpha -phosphatidylethanolamine (PE) (bovine brain), L-alpha -phosphatidylinositol (PI) (bovine liver), phosphatidylserine (PS) (bovine brain), cholesterol, omega -aminooctyl-agarose, heparin-agarose, cytochrome c (horse heart), thrombin (bovine plasma), and GTPgamma S were purchased from Sigma Aldrich. Diisopropyl fluorophosphate and n-heptyl-beta -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).

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-beta -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, omega -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-beta -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.

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&cjs1138;2 Generation-- The standard mixture contained p67N and p47N (2 µM each) or a fusion between p67N and p47N (2 µM), PM (5 µg), and Rac (7 µM), which had been treated with 100 µM GTP, in a total volume of 50 µl of buffer D (4 mM MgCl2, 20 mM potassium phosphate buffer, pH 7.0) containing 10 µM GTP. The mixture was supplemented with 200 µM SDS and incubated at 25 °C for 5 min. Four 10-µl aliquots of each reaction mixture were transferred into microplate wells, which contained 200 µM NADPH and 80 µM cytochrome c in 240 µl of buffer D. Superoxide generation was measured by monitoring the absorbance increase at 550 nm with a microplate-reader Spectraclassic (Tecan, Grödig, Austria). NADPH oxidase activity was expressed as micromole of O&cjs1138;2 formed/min per mg of PM protein. The assays were sometimes repeated in the presence of superoxide dismutase (80 µg/ml) to verify actual O&cjs1138;2 generation (7).

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-beta -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&cjs1138;2 formed/min per nmol of cyt. b558.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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.


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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.

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.


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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&cjs1138;2 generation was assayed as described under "Experimental Procedures." The data are expressed as mean ± S.D. from three experiments. B, purified and lipidated cyt. b558 was used instead of PM. Other experimental conditions are as described under "Experimental Procedures."

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.


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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.

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.


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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.

                              
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Table I
Effect of fusion on the affinity for Rac and Km for NADPH of the reconstituted oxidase
The reconstitution system contained fused (p47N-p67N and p67N-p47N) or nonfused p47N and p67N with GTP-loaded rac and the plasma membrane. EC50, Km, and Vmax values were determined from the data in Figs. 4A and 5 by nonlinear least squares fits to the Michaelis-Menten equation.

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.


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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.

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.


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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.

                              
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Table II
Half-lives of NADPH oxidase activity reconstituted with fused or nonfused p47N and p67N
The half-lives of the oxidase with nonfused components or p67N-p47N were determined by a first-order plot of the data in Fig. 6, A and B. A time course experiment with p47N-p67N was performed in the same way and the half-life was determined as described above. The values in parentheses are the half-lives with GTPgamma 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).

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 GTPgamma 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 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&cjs1138;2 generating activity in the absence of SDS. When PM was used as a source of cyt. b558, the activity was 0.7 µmol/min/mg, which is 22% of that with SDS. When p47N-p67N or p67N-p47N was used the activity increased, but the ratio to the activity with SDS was not much changed. On the other hand, when purified cyt. b558 was used, a quite different result was obtained. The activity in the absence of SDS was greatly increased using p67N-p47N to 5.6 µmol/min/nmol cyt. b558, which corresponds to 75% of the activity in the presence of SDS. This activity was very stable (t1/2 = 360 min), even more stable than the activity in the presence of SDS (Table II). These results clearly show that fusion eliminates a requirement of anionic amphifiles for activation. In other words, cyt. b558 becomes active just by mixing with p67N-p47N and GTP-Rac.

                              
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Table III
SDS-independent activation of NADPH oxidase by p67N-p47N with purified cyt. b558
The reconstitution of the oxidase was carried out with p47N-p67N, p67N-p47N, or nonfused p47N and p67N (nonfused) using PM or purified cyt. b558. The mixture was incubated for 5 min at 25 °C in the presence or absence of SDS and assayed for superoxide generation as described under "Experimental Procedures."

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.


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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.

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&cjs1138;2/min/nmol of cyt. b558, which was 47% of the activity with our optimal lipid composition. Addition of PI or PS to the relipidation mixture increased the O&cjs1138;2 generating activity to almost the maximal level. This result indicates that p67N-p47N produces a substantial activity with neutrophil membrane composition and that acidic PLs enhances the activation.

                              
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Table IV
SDS-independent activation of the oxidase by p67N-p47N with cyt. b558 relipidated with neutrophil-type lipid or PI (PS) enriched lipid
Purified cyt. b558 was relipidated with optimal lipid composition as described under "Experimental Procedures" or neutrophil-type composition, which contained PC (30), PE (28.4), PI (2.6), PS (10.3), SM (12.9), and cholesterol (15.5) % weight of the total lipid. In some experiments, the ratio of PI or PS was increased; Namely, PC (24.7), PE (23.2), PI (29), PS (0), SM (10.5), and cholesterol (12.6) mg/ml for "PI-enriched" or PC (24.7), PE (23.2), PI (0), PS (29), SM (10.5), and cholesterol (12.6) % weight for "PS-enriched." The total lipid concentration was kept at 1 mg/ml cyt. b558 preparation. Other experimental conditions are as described under "Experimental Procedures."


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 GTPgamma S (Table II) indicating that the deactivation observed here is not due to GTP hydrolysis.

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.


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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 alpha  and beta , respectively. PRR of p22 (alpha ) 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&cjs1138;2 generating activity of the oxidase in PM from phorbol ester-activated neutrophils (30).

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.

    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.

    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.

Dagger 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.

    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-alpha -phosphatidylcholine; PE, L-alpha -phosphatidylethanolamine; PI, L-alpha -phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; PL, phospholipid; SH3, Src homology 3; PRR, proline-rich region; TPR, tetratricopeptide repeats; TLCK, tosyl-L-lysyl chloromethylketone; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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