Assembly of the neutrophil respiratory burst oxidase: A direct interaction between p67^PHOX and cytochrome b₅₅₈ II

Abstract

Activation of the phagocyte NADPH oxidase complex requires assembly of the cytosolic factors p47^PHOX, p67^PHOX, p40^PHOX, and Rac with the membrane-bound cytochrome b₅₅₈. We recently established a direct interaction between p67^PHOX and cytochrome b_558. In the present study, we show that removal of the C-terminal domain of p67^PHOX increased its binding to cytochrome b₅₅₈. Whereas phosphorylated p40^PHOX alone did not bind to cytochrome b₅₅₈, phosphorylated p47^PHOX did, and, moreover, it allowed the binding of p40^PHOX to the cytochrome. Furthermore, both increased the binding of p67^PHOX to the cytochrome. Phosphorylated p47^PHOX thus appears to increase the binding of p67^PHOX to cytochrome b₅₅₈ by serving as an adapter, bringing p67^PHOX into proximity with cytochrome b₅₅₈, whereas phosphorylated p40^PHOX may increase the binding by inducing a conformational change that allows p67^PHOX to interact fully with cytochrome b₅₅₈.

The NADPH oxidase of phagocytes plays a pivotal role in host defense against microbial infection. It catalyzes the reduction of oxygen to O at the expense of NADPH. The O is the precursor of potent oxidants used to kill the invading microorganisms (1–4).

The NADPH oxidase is composed of a number of cytosolic and membrane-bound proteins—in the cytosol, p67^PHOX, p47^PHOX, p40^PHOX, and Rac1/Rac2; in the membrane, gp91^PHOX and p22^PHOX—which, together, comprise cytochrome b₅₅₈. When activation takes place, the cytosolic components migrate to the membranes, where they associate with the membrane-bound components to assemble the catalytically active oxidase (5). Cytochrome b₅₅₈, which contains both flavin and heme groups (6), likely is responsible for electron transfer from NADPH to oxygen upon activation of the cells, although it was demonstrated that p67^PHOX also possesses an NADPH-binding site (7). P47^PHOX, which is phosphorylated extensively during the activation of the oxidase (8), probably initiates the assembly of the enzyme, whereas the function of p67^PHOX appears to be to activate catalysis by cytochrome b₅₅₈, although the mechanism of activation is obscure (9). Rac appears to promote the interaction between p67^PHOX and cytochrome b₅₅₈ (10).

During the NADPH oxidase activation process, a number of defined protein–protein binding interactions occur (11). For example, p47^PHOX and p67^PHOX each contain two SH3 domains, the C-terminal SH3 domain of p67^PHOX interacts with p47^PHOX, and the N-terminal SH3 domain of p47^PHOX is able to interact with p22^PHOX (12).

In the present study, we established a direct interaction between p67^PHOX and cytochrome b₅₅₈ (10) and showed that this interaction increased when the C-terminal region of the protein was removed and also when the whole protein was incubated in the presence of phosphorylated p47^PHOX or phosphorylated p40^PHOX.

Experimental Procedures

Expression and Purification of Recombinant Proteins in Escherichia coli.

P67^PHOX and its truncated forms were expressed in E. coli as glutathione S-transferase (GST) fusion proteins and purified with glutathione-Sepharose beads (13). The proteins then were separated from GST while on the beads by cleavage with PreScission protease (Amersham Pharmacia) or eluted from the beads by incubating for 20 min at 4°C with 1 ml of 50 mM Tris⋅HCl, pH 8/5 mM glutathione/150 mM NaCl. P47^PHOX was expressed in E. coli as GST fusion proteins and purified with glutathione-Sepharose beads (Amersham Pharmacia) followed by thrombin cleavage when needed. P40^PHOX cDNA in pET-32a plasmid (a kind gift of A. Fuchs, Centre National de la Recherche Scientifique, Grenoble, France) was transformed in BL21-DE3 (pLysS) E. coli strain and expressed as follows. An overnight culture was diluted 10-fold in fresh Terrific Broth medium containing 100 μg/ml ampicillin and grown for an additional hour at 37°C. The culture then was induced with 0.5 mM isopropyl β-d-thiogalactoside for 4 h at 25°C. Bacteria were harvested by centrifugation and lysed by sonication in 20 mM Hepes, pH 7.9/0.5 M NaCl/10 mM imidazole in the presence of 0.2 mM PMSF/100 μg/ml leupeptin/100 μg/ml pepstatin/0.5 mM diisopropyl fluorophosphate. The homogenate then was centrifuged at 150,000 × g for 15 min, and the supernatant was incubated with Probond resin (Invitrogen) for 1 h at 4°C. The resin then was packed into a gravity column, washed twice in 20 mM Hepes, pH 7.9/0.5 M NaCl/20 mM imidazole, and then again washed twice in 20 mM Hepes, pH 7.9/0.5 M NaCl/30 mM imidazole. The protein then was eluted with 150 mM imidazole and dialyzed against 20 mM Hepes, pH 7.9/0.1 mM DTT/2 mM EGTA. Protein concentrations were determined with the Bio-Rad Assay Kit with BSA as a standard.

Contruction of Truncated p67^phox Forms.

P67^PHOX truncated forms were constructed as described by Dang et al. (13). Briefly, p67^PHOX (1–243), p67^PHOX (1–210), p67^PHOX (1–199), and p67^PHOX (244–526) were obtained by PCR with Pfu polymerase (Stratagene) and wild-type p67^PHOX cDNA cloned into the vector pBK-CMV. The PCR products were ligated into the SmaI site of pGEX-6P1 for p67^PHOX (1–243), BamHI and EcoRI of pGEX-6P1 for p67^PHOX (1–210) and p67^PHOX (1–199), and EcoRI and XhoI sites of pGEX-6P3 for p67^PHOX (244–526) and transformed into DH5α for expression of the proteins. The sequences all were confirmed by DNA sequencing in the Scripps Research Institute molecular biology facility.

Purification of Cytochrome b₅₅₈.

Cytochrome b₅₅₈ was purified from human neutrophil plasma membranes by heparin and hydroxyapatite chromatography and subsequently relipidated and reflavinated as described by Cross et al. (14).

Affinity Precipitation.

For affinity precipitation, 80 pmol of GST, full-length GST-p67^PHOX or truncated GST-p67^PHOX (1–243), GST-p67^PHOX (1–210), GST-p67^PHOX (1–199), or GST-P67^PHOX (244–526) was incubated in the presence of 4.3 pmol of cytochrome b₅₅₈ in 200 μl of 20 mM Hepes, pH 7.5/1% Nonidet P-40/10 mM NaCl/1 mM EGTA for 2 h at room temperature. In some assays, phosphorylated GST-p67^PHOX was used instead. Then, 40 μl of glutathione-Sepharose 4B beads (Amersham Pharmacia) then was added followed by incubation for 2 h at 4°C. After washing five times with the same buffer, the beads were pelleted, resuspended in 40 μl of Laemmli sample buffer, and boiled for 5 min. Proteins were separated by SDS/PAGE on a 12% Tris-glycine gel (Bio-Rad) and transferred onto nitrocellulose. The cytochrome b₅₅₈ on the nitrocellulose then was visualized by using an anti-gp91^PHOX mAb (15). To determine the effect of p47^PHOX and p40^PHOX on p67^PHOX binding to gp91^PHOX, 80 pmol of phosphorylated p47^PHOX or unphosphorylated p47^PHOX, phosphorylated p40^PHOX or unphosphorylated p40^PHOX was added to the assay at the same time as p67^PHOX.

Affinity precipitation of cytochrome b by p47^PHOX was carried out as described above except that GST-p47^PHOX was used instead of GST-p67^PHOX.

For affinity precipitation of cytochrome b by p40^PHOX, 40 pmol of His-tagged p40^PHOX or phosphorylated His-tagged p40^PHOX was incubated in the presence of 2.15 pmol of cytochrome b₅₅₈ in the presence of 100 μl of 20 mM Hepes, pH 7.5/1% Nonidet P-40/10 mM NaCl/1 mM EGTA/40 mM imidazole for 2 h at room temperature and then precipitated with nickel-chelating beads.

Western Blotting.

Nitrocellulose membranes were blocked with 5% nonfat dry milk in borate-buffered saline, pH 8.4 (100 mM boric acid/25 mM borax/75 mM NaCl) for 1 h at room temperature and then incubated with 1:1,000 mouse monoclonal anti-gp91^PHOX antibody overnight. The membranes then were washed extensively and incubated with horseradish peroxidase-conjugated 1:5,000 anti-mouse IgG for 1 h at room temperature. Blots were visualized by using enhanced chemiluminescence Western blotting reagents (Amersham Pharmacia).

Phosphorylation.

Phosphorylation of p67^PHOX by mitogen-activated protein kinase and protein kinase C (Calbiochem) in combination was performed by incubating 80 pmol of p67^PHOX in a reaction mixture containing 40 mM Hepes (pH 7.5), 10 mM MgCl₂, 10 mM DTT, 0.6 mM CaCl₂, 5 μg/ml 1,2-dioleoyl-sn-glycerol (Sigma), 50 μg/ml l-α-phosphatidylserine (Sigma), the indicated kinase, and 50 μM ATP in a total volume of 40 μl for 30 min at 30°C. Phosphorylation of p40^PHOX and p47^PHOX by protein kinase C was performed the same way. The reactions were stopped by adding 5 μM GF109203X, an inhibitor of conventional and novel protein kinase C.

Statistical Analysis.

All of the experiments were repeated a minimum of three times. Results are expressed as means ± SE. Statistical analysis was performed with one-way ANOVA. P < 0.05 was considered to be statistically significant.

Results

P67^PHOX Interacts Directly with Cytochrome b₅₅₈.

The direct binding of p67^PHOX to cytochrome b₅₅₈ was established by affinity-precipitation experiments. Cytochrome b₅₅₈ was incubated in the presence of GST-p67^PHOX or GST. Fig. 1 shows that cytochrome b₅₅₈ was precipitated with GST-p67^PHOX coupled to glutathione-Sepharose beads but not with GST coupled to glutathione-Sepharose beads. The results demonstrate a direct interaction between cytochrome b₅₅₈ and p67^PHOX.

Figure 1.

Affinity precipitation of cytochrome b₅₅₈ with GST-p67^PHOX. Eighty pmol of GST or full-length GST-p67^PHOX was incubated in the presence of 4.3 pmol of cytochrome b₅₅₈ in 200 μl of 20 mM Hepes, pH 7.5/1% Nonidet P-40/10 mM NaCl/1 mM EGTA for 2 h at room temperature. Then, 40 μl of glutathione-Sepharose 4B beads (Pharmacia) was added, followed by incubation for 2 h at 4°C. After washing five times with the same buffer, the beads were pelleted, resuspended in 40 μl of Laemmli sample buffer (19), and boiled for 5 min. Proteins were resolved by SDS/12% PAGE and transferred onto nitrocellulose. The proteins then were detected with a monoclonal anti-gp91^PHOX antibody.

Interaction of Truncated Forms of p67^PHOX with Cytochrome b₅₅₈.

In an attempt to localize the region of p67^PHOX responsible for binding to cytochrome b₅₅₈, a series of truncated GST-p67^PHOX forms were generated and expressed (Fig. 2A). Full-length GST-p67^PHOX or truncated GST-p67^PHOX was used in affinity-precipitation experiments. As shown in Fig. 2 B and C, affinity precipitation of cytochrome b₅₅₈ was increased when the C-terminal GST truncations of p67^PHOX were used. This increase is especially dramatic for GST-p67^PHOX (1–210) and GST-p67^PHOX (1–199). By quantitation, the increase was 153 ± 15% for GST-p67^PHOX (1–243) and 241 ± 27% and 242 ± 24% for GST-p67^PHOX (1–210) and GST-p67^PHOX (1–199), respectively. The C-terminal fragment GST-p67^PHOX (244–526) precipitated cytochrome b₅₅₈ much less effectively. Indeed, scanning of several experiments showed no significant difference compared with control GST. These results suggest that in the N-terminal forms of p67^PHOX, the binding site for cytochrome b₅₅₈ is unmasked, whereas in the full-length p67^PHOX, this site is masked partially by the C-terminal half of the molecule. When this region is removed, the interaction with cytochrome b₅₅₈ is increased.

Figure 2.

Binding of various truncated forms of p67^PHOX to cytochrome b₅₅₈. (A) SDS/PAGE of full-length and truncated GST-p67^PHOX forms expressed and purified as described under Experimental Procedures. The samples were visualized by Coomassie blue staining. (B) Affinity precipitation of cytochrome b₅₅₈ by full-length GST-p67 ^PHOX or truncated GST-p67^PHOX (1–243), GST-p67^PHOX (1–210), GST-p67^PHOX (1–199), or GST-p67^PHOX (244–526). The precipitation was performed as described in Fig. 1. Quantification of binding was determined by scanning densitometry. The data are expressed as percentage of full-length GST-p67^PHOX and are the means ± SE of four experiments.

P67^PHOX (1–210) is active in the cell-free NADPH oxidase system, whereas p67^PHOX (1–199) is inactive (16). It is interesting that the GST counterparts of those two p67^PHOX fragments both are able to bind to cytochrome b₅₅₈. This result suggests that the binding of GST-p67^PHOX (1–199) to cytochrome b₅₅₈ serves to hold the active portion of p67^PHOX to the cytochrome.

Effect of p67^PHOX Phosphorylation, p47^PHOX, and p40^PHOX on the Interaction of p67^PHOX with Cytochrome b₅₅₈.

The above results indicate that a change in p67^PHOX conformation is required for its interaction with cytochrome b₅₅₈. We next aimed to determine whether the phosphorylation of p67^PHOX or the presence of other components of the NADPH oxidase complex could affect the association of cytochrome b₅₅₈ with GST-p67^PHOX. The phosphorylation of p67^PHOX had little effect (Fig. 3B). Even arachidonic acid, which increases the interaction of p47^PHOX with the cytosolic tail of p22^PHOX (17), had no effect (Fig. 3B). Interestingly, when phosphorylated p47^PHOX or phosphorylated p40^PHOX was added to the assay, the coprecipitation of cytochrome b₅₅₈ by GST-p67^PHOX was found to be increased (Fig. 3 A and B). On the other hand, unphosphorylated p47^PHOX and unphosphorylated p40^PHOX had no effect. These data indicate that phosphorylated p47^PHOX and phosphorylated p40^PHOX are able to increase the binding of p67^PHOX to cytochrome b₅₅₈. Phosphorylated p47^PHOX has been shown to interact directly with the cytoplasmic tail of p22^PHOX (17), and we established here its interaction with cytochrome b₅₅₈. Indeed, phosphorylated GST-p47^PHOX was able to affinity-precipitate cytochrome b₅₅₈ in the presence of glutathione-Sepharose beads (Fig. 4 Left), whereas unphosphorylated GST-p47^PHOX was not capable of this interaction. Thus, phosphorylated p47^PHOX might increase the binding of p67^PHOX to cytochrome b by acting as an adapter protein, bringing p67^PHOX into proximity with cytochrome b₅₅₈. In contrast, we were not able to find any interaction of phosphorylated p40^PHOX with cytochrome b₅₅₈. Indeed, phosphorylated or unphosphorylated His-tagged-p40^PHOX was not able to affinity-precipitate cytochrome b in the presence of nickel-chelating beads (Fig. 4 Right). Thus, it is probable that phosphorylated p40^PHOX increases the binding of p67^PHOX with cytochrome b by inducing a conformational change of p67^PHOX, allowing it to fully interact with cytochrome b₅₅₈.

Figure 3.

Phosphorylated p47^PHOX and phosphorylated p40^PHOX increase the binding of p67^PHOX to cytochrome b₅₅₈. (A) Effect of p47^PHOX. (Upper) Affinity precipitation was performed as described in Fig. 1, except that in some assays 80 pmol of phosphorylated p47^PHOX (lane 3) or unphosphorylated p47^PHOX (lane 4) was added. (Lower) Quantification of the binding was determined by scanning densitometry. The data are expressed as the percentage of full-length GST-p67^PHOX and are the means ± SE of three experiments. (B) Effect of phosphorylated p67^PHOX, p40^PHOX. (Upper) Affinity precipitation was performed as described in Fig. 1. Lanes: 3, phosphorylated GST-p67^PHOX was used instead of GST-p67^PHOX; 4, phosphorylated GST-p67^PHOX and 10 μM arachidonic acid were added; 5, phosphorylated p40^PHOX (80 pmol) was added to the assay; 6, unphosphorylated p40^PHOX (80 pmol) was added to the assay. (Lower) Quantification of the binding was determined by scanning densitometry. The data are expressed as percentage of full-length GST-p67^PHOX and are the means ± SE of three experiments.

Figure 4.

Phosphorylated p47^PHOX binds to cytochrome b whereas phosphorylated p40^PHOX does not. (Left) Precipitation of cytochrome b by p47^PHOX. Affinity precipitation was performed as described in Experimental Procedures in the presence of unphosphorylated GST-p47^PHOX (lane 1) or phosphorylated GST-p47^PHOX (lane 2). (Right) Precipitation of cytochrome b by p40^PHOX. Affinity precipitation was performed as described in Experimental Procedures in the presence of unphosphorylated His-tagged p40^PHOX (lane 1) or phosphorylated His-tagged p40^PHOX (lane 2). His-tagged P40^PHOX was precipitated with nickel-chelating beads.

Discussion

In an earlier study, we demonstrated by several methods that p67^PHOX is able to bind directly to cytochrome b₅₅₈ (10). A direct interaction between p67^PHOX and cytochrome b₅₅₈ is in accord with the idea that p67^PHOX regulates the transfer of electrons from NADPH to the flavin (18) because p67^PHOX then would be in proximity to the flavin center, enabling it to perform a regulatory function in this part of the protein. Recently, one specific domain of p67^PHOX, called the activation domain, has been shown to be involved in this regulation (16). The activation domain, p67^PHOX (1–210), binds more strongly to cytochrome b₅₅₈ than does the complete protein, but so does a domain of similar size, p67^PHOX (1–199), which shows no activity in the cell-free NADPH oxidase system. These results suggest that the C truncation that yields p67^PHOX (1–210) and p67^PHOX (1–199) affects the binding of the fragments to cytochrome b₅₅₈ but has nothing to do with oxidase activity.

The effect of p47^PHOX and p40^PHOX on the binding of p67^PHOX to cytochrome b₅₅₈ also was examined. Our results showed that the unphosphorylated proteins had no effect, but that phosphorylated p47^PHOX and phosphorylated p40^PHOX both increased the binding of p67^PHOX to cytochrome b₅₅₈. Phosphorylated p47^PHOX probably serves as an adapter protein, bringing full-length p67^PHOX into proximity with cytochrome b₅₅₈. Phosphorylated p40^PHOX appears to induce a conformational change in p67^PHOX, although the functional significance of this conformational change is unclear. In conclusion, our data establish a direct interaction between p67^PHOX and cytochrome b_558, as demonstrated previously. Furthermore, we have shown that full binding requires a conformation change that, in the intact cells, might be induced by phosphorylated p40^PHOX.

Abbreviation

GST

glutathione S-transferase