©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Domain of p47 That Interacts with Human Neutrophil Flavocytochrome b(*)

(Received for publication, July 17, 1995; and in revised form, August 25, 1995)

Frank R. DeLeo William M. Nauseef (2) Algirdas J. Jesaitis (1) James B. Burritt (1) Robert A. Clark (3) Mark T. Quinn (§)

From the  (1)Departments of Veterinary Molecular Biology and Microbiology, Montana State University, Bozeman, Montana 59717, the (2)Department of Medicine, Department of Veteran Affairs Medical Center and University of Iowa, Iowa City, Iowa 52242, and the (3)Department of Medicine, University of Texas Health Science Center and Audie L. Murphy Memorial Veteran Affairs Medical Center, San Antonio, Texas 78284

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The NADPH-dependent oxidase of human neutrophils is a multicomponent system including cytosolic and membrane proteins. Activation requires translocation of cytosolic proteins p47, p67, and Rac2 to the plasma membrane and association with the membrane flavocytochrome b to assemble a functioning oxidase. We report the location of a region in p47 that mediates its interaction with flavocytochrome b. From a random peptide phage display library, we used biopanning with purified flavocytochrome b to select phage peptides that mimicked potential p47binding residues. Using this approach, we identified a region of p47 from residue 323 to 342 as a site of interaction with flavocytochrome b. Synthetic peptides SRKRLSQDAYRRNS, AYRRNSVRFL, and QRRRQARPGPQSPG inhibited superoxide (O(2)) production in the broken cell system with IC of 18, 57, and 15 µM, respectively. AYRRNSVRFL and its derivative peptides inhibited phosphorylation of p47. However, the functional importance of this peptide was independent of its effects on phosphorylation, since AYRRNAVRFL inhibited O(2) production, but had no effect on phosphorylation. None of the peptides blocked O(2) production when added after enzyme activation, suggesting that they inhibited the assembly, rather than the activity, of the oxidase. Furthermore these peptides inhibited membrane association of p47 in the broken cell translocation assay and O(2) production by electropermeabilized neutrophils, thereby supporting the interpretation that this region of p47 interacts with flavocytochrome b.


INTRODUCTION

Human polymorphonuclear leukocytes (PMNs)^1 play an important role in host defense against invading microorganisms. PMNs possess an NADPH-dependent oxidase which is capable of generating superoxide anion (O(2)) and other microbicidal oxygen-derived species (e.g. H(2)O(2), HOCl) when activated by various particulate and soluble stimuli(1, 2) . The NADPH oxidase is a multicomponent enzyme system which is unassembled in resting PMNs but assembles on the plasma membrane in activated PMNs(3, 4) . The critical importance of the PMN NADPH oxidase in normal host defense is most dramatically illustrated by the frequent and severe infections seen in patients with chronic granulomatous disease(5, 6) . The PMNs from such patients lack a functionally competent oxidase and, when stimulated, fail to generate O(2).

Essential components of the NADPH oxidase include plasma membrane and cytosolic proteins. The key plasma membrane component is a heterodimeric flavocytochrome b which is composed of a 91-kDa glycoprotein (gp91) and a 22-kDa protein (p22)(7, 8, 9, 10) . Flavocytochrome b serves to transfer electrons from NADPH to molecular oxygen, resulting in the generation of O(2). In PMN membranes, a low molecular weight GTP-binding protein, Rap1A, is associated with flavocytochrome b and plays an important role in NADPH oxidase regulation in vivo(11, 12, 13) . Cytosolic proteins p47, p67, and a second low molecular weight GTP-binding protein, Rac2, are absolutely required for NADPH oxidase activity(14, 15, 16, 17, 18) , and these three proteins associate with flavocytochrome b to form the functional NADPH oxidase(19, 20, 21) . Additionally, a cytosolic protein, p40, has recently been identified, but its role in oxidase function is not completely defined(22) .

According to the current model of NADPH oxidase assembly, p47 and p67 translocate en bloc to associate with flavocytochrome b during PMN activation(23, 24) . Rac2 translocates simultaneously but independently of the other two cytosolic components to associate with the membrane-bound NADPH oxidase(25, 26) . Studies of oxidase assembly in PMNs of patients with various forms of chronic granulomatous disease suggest that p47 binds directly to flavocytochrome b(20) , and at least six regions of flavocytochrome b have been identified as potential sites for interaction with p47, including four sites on gp91 and two sites on p22(27, 28, 29, 30, 31, 32, 33, 34) . In contrast, the complementary sites of interaction presented on p47 have not been fully characterized(29, 33, 34) . In previous studies, peptides mimicking p47 residues AYRRNSVRFL inhibited phosphorylation of p47, O(2) production, and translocation of cytosolic components in the broken cell system (35) , suggesting that this might be a possible site of interaction between p47 and flavocytochrome b.

In the present work, we used an approach combining the screening of a random peptide phage display library with the functional analysis of synthetic peptides to define residues in p47 which interact with flavocytochrome b. Our data indicate that the region encompassing amino acids 323-342 comprises a functionally important domain in the association of p47 with flavocytochrome b.


EXPERIMENTAL PROCEDURES

Materials

NADPH, ferricytochrome c (horse heart, type VI), superoxide dismutase, and FAD were obtained from Sigma. GTPS (5`-triphosphate) was obtained from Boehringer Mannheim and arachidonic acid (sodium salt) from NuChek Prep (Elysian, MN). NHS-LC-biotin was obtained from Pierce, and streptavidin was from Fisher. Dextran, Ficoll, and Percoll were obtained from Pharmacia Biotech Inc., and Hypaque was obtained from Winthrop Laboratories (New York).

Preparation and Fractionation of PMNs

Human PMNs were isolated from heparinized venous blood using sequential dextran sedimentation, differential density sedimentation in Hypaque-Ficoll gradients, and hypotonic lysis of erythrocytes as described previously (35, 36) . Two different methods were employed for the isolation of PMN membranes and cytosol with no difference in the experimental results. Purified PMNs were treated with 2 mM diisopropyl fluorophosphate for 20 min at 4 °C, washed, resuspended in relaxation buffer (KCl, 100 mM; NaCl, 3 mM; MgCl(2), 3.5 mM; EGTA, 1.25 mM; Pipes, 10 mM, pH 7.3) and disrupted by N(2) cavitation(35) . Nuclei and unbroken cells were pelleted (200 times g, 6 min, 5 °C) and the supernatant loaded on top of an isotonic discontinuous gradient of Percoll as described previously(37) . Cytosol was collected from the top of the gradient and clarified before use. The plasma membrane fraction was collected, Percoll removed by centrifugation, and the membrane washed prior to use. Alternatively, purified PMNs (5 times 10^8/ml in 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 15 µg/ml leupeptin, 10 µg/ml chymostatin, 1 mM EGTA, and 10 mM Hepes, pH 7.0) were disrupted by N(2) cavitation as described previously. Membrane and cytosolic fractions were prepared from the cavitate by sequential centrifugation as described by Fujita et al.(36) .

Preparation and Purification of Flavocytochrome b

Human neutrophil flavocytochrome b was purified from 10 neutrophils following the methods of Parkos et al.(7, 38) . The purified flavocytochrome b was >95% pure, as determined by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining(7, 38) , and immunoblotting with anti-cytochrome b antibodies confirmed the presence of both gp91 and p22 (not shown).

Random Sequence Peptide Bacteriophage Display Library Analysis

Fifty µg of human neutrophil flavocytochrome b was biotinylated to a final biotin/protein ratio of 20:1 (39) . Three rounds of biopanning were performed as described previously (39) using 10 µg/pan of biotinylated flavocytochrome b in 0.2% Triton X-100 and 75 µl (round 1 only) of a nonapeptide phage display library (J404-3)(40) . The bacteriophage eluates of the first two pans were amplified in K91 Escherichia coli cells on solid Luria-Bertani (LB) agar dishes containing 100 µg/ml kanamycin to approximately 10 plaque-forming units. The phage were then extracted(41) , and one-third of the eluate was used in the next round. The third round eluate was plated in K91 cells on LB plates, and plaques were then randomly picked and sequenced using a gene III-specific primer(40) . Dried sequencing gels were analyzed on a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Peptide Synthesis

Peptides SRKRLSQDAYRRNS, QRRRQARPGPQSPG, RQARPGPQ, KLSYRPRDSNE, and AVEGGMKPVKLLVGC were synthesized by the Montana State University Peptide Synthesis Facility. Peptide sequences and purity (>95%) were confirmed by mass spectrometry. The p47 peptides PPRRSSIRNA, HQRSRKRLSQD, AYRRNSVRFL, and gp91 peptide RGVHFIF were synthesized at the Core Peptide Facility at the University of Iowa and displayed >95% purity via high performance liquid chromatography analysis. The p47 (323-332)-related peptides ( Table 1and Table 2) were obtained from Macromolecular Resources (Colorado State University, Fort Collins, CO), and sequence and purity were confirmed by mass spectrometry.





Broken Cell NADPH Oxidase Reconstitution and Translocation Assay

NADPH oxidase activity was measured using two different versions of the broken cell system, and results with the two systems were similar. In some studies, the broken cell system used arachidonic acid as the activating agent, and activity was determined as described by Nauseef et al.(35) . In other experiments, activity was measured in an SDS-activated system as described by DeLeo et al.(32) . To analyze peptide inhibition, peptides dissolved in broken cell assay buffer were added to the reaction mixture at the indicated concentrations before, and after the 2-min incubation with agonist and maximum rate of O(2) production was determined as described(32) .

Two broken cell translocation assays were used with similar results. Studies of AYRRNSVRFL and related peptides were performed using 91 µM arachidonic acid as the activating agent, and translocation was determined as described by Park and Babior(42) . In other experiments, the broken cell translocation assay of Verhoeven et al.(43) was used.

Analysis of Phosphorylated Proteins

Conditions used to assess the effect of various peptides on the phosphorylation of p47 mirrored closely those for quantification of O(2) generation and have been described in detail previously(35) . Relative quantitation of p47 phosphorylation was obtained by densitometric analysis of resultant autoradiographs.

Electrophoresis and Immunoblotting

Proteins were separated by SDS-polyacrylamide gel electrophoresis and electroblotted as described previously(21, 35) . Immunoblots were probed with primary antibody, rabbit anti-p47(21, 35, 44) , and followed by either iodinated protein A or alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (Bio-Rad). Relative amounts of translocated proteins were quantitated by densitometric analysis of the immunoreactive p47 detected by autoradiography or colorimetric assay.

Preparation and Electropermeabilization of Human Neutrophils

Histopaque-purified neutrophils were washed once with ice-cold permeabilization buffer (140 mM KCl, 10 mM Hepes, 10 mMD-glucose, 1 mM MgCl(2), 0.193 mM CaCl(2), and 1 mM EGTA, pH 7.2) and then resuspended in the same buffer supplemented with 100 µM GTPS, 1 mM ATP, and 2 mM NADPH, to a final concentration of 10^7 cells/ml and stored on ice. Aliquots of 800 µl were placed in 0.4-cm electroporation cuvettes and permeabilized with a Bio-Rad gene pulser using two consecutive pulses (with brief stirring between pulses) of 3.75 kV/cm with a 25-microfarad capacitor ( = 0.3-0.5 ms) at 4 °C. The cells were then incubated on ice for 5 and 30 min with or without 1 mM of the appropriate peptide. Subsequently, the cells were diluted into supplemented permeabilization buffer containing 100 µM cytochrome c ± 1 mM peptide (final concentration of 5 times 10^6 cells/ml) and then stimulated with 1 µg/ml phorbol 12-myristate 13-acetate (reference cuvette contained 25 µg/ml superoxide dismutase). The maximal rate of superoxide dismutase-inhibitable reduction of cytochrome c was determined on a Cary dual-beam spectrophotometer (Varian, Melbourne, Australia) at 550 nm and 25 °C for 10 min. Electropermeabilized neutrophils retained 92.3 ± 1.9 and 49.8 ± 6.3% of their ability to produce O(2) when incubated for 5 and 30 min after pulsing, respectively, compared with control, nonpermeabilized cells and were found to be 98% permeable to trypan blue 5 min after permeabilization.


RESULTS

Biopanning with Purified Flavocytochrome b

To identify the region or regions in p47 that associate with flavocytochrome b, a random sequence nonapeptide bacteriophage display library was screened with purified flavocytochrome b. The predicted amino acid sequences from 94 of these affinity selected bacteriophage were analyzed and three dominant consensus motifs were identified (Fig. 1). When compared with the amino acid sequence of p47, these motifs mapped to residues 323-342 in p47, a region which contains multiple sites for serine/threonine kinase phosphorylation and includes a previously identified area of potential interaction with flavocytochrome b at residues 323-332(35, 45) . The strongest homology among phage peptides was evident in those representative of the p47 region QARPG, and eight phage isolates contained three to four residues identical to this region. Additionally, seven phage isolates contained peptides that were homologous to p47 residues LQQRRRQ (six of these contained three-residue matches), which immediately precede the QARPG region. In combination, 21 phage isolates were representative of the p47 region LQQRRRQARPG (Fig. 1). Conservative substitutions and one residue shifts give many of these phage peptides even more similarity to the indicated region. This is especially evident for residues 335-337, where arginine appears to be substituted by a positively charged histidine or lysine.


Figure 1: Flavocytochrome b-binding phage displaying sequences of homology with p47. The random regions of bacteriophage recovered from flavocytochrome b biopanning were sequenced and the putative motifs aligned. Residues identical to the corresponding p47 sequence are underlined, conservative substitutions are in bold, and residues shifted in position are represented by italics. Bacteriophage peptide sequences representative of p47 regions indicated are present in both NH(2) COOH and COOH NH(2) forms, and the X2/X3 designations indicate the number of clones recovered with that specific sequence.



A third consensus sequence mapped to p47YRRNS and was represented by 21 phage peptides, and conservative substitutions or one-residue shifts give many phage representative of this area an even greater similarity. Nauseef et al.(35) previously found that p47 residues AYRRNSVRFL represented a functionally important domain involved in NADPH oxidase assembly. Certain residues within this region appear to be critically important for structural constraints, as the exclusion of these residues renders the peptide ineffective at inhibiting O(2) production in the broken cell assay(35) . Thus, the mapping of this site using a random sequence library provides direct evidence confirming the biological relevance of this site.

Peptide Inhibition in the Broken Cell Assay

To determine if the p47 regions selected from the phage display library approach were relevant for the association of p47 with flavocytochrome b and, thus, NADPH oxidase activity, peptides representative of these regions were synthesized and analyzed in a broken cell NADPH oxidase assay. In a previous study, p47 peptides PPRRSSIRNA and HQRSRSRKRLSQD did not affect O(2) production significantly in the broken cell assay at concentrations up to 500 µM(35) . These peptides encompass potential phosphorylation sites, are also positively charged, and precede the 323-342 region of p47 mapped by the phage display library. In contrast, the peptides mimicking portions of p47 residues 315-347, SRKRLSQDAYRRNS, AYRRNSVRFL, and QRRRQARPGPQSPG, all inhibited O(2) production in the broken cell assay in a dose-dependent manner with IC values of 18, 57, and 15 µM, respectively (see Fig. 2and Table 1). Surprisingly, a shorter peptide derived from QRRRQARPGPQSPG, RQARPGPQ, was much less inhibitory (IC 750 µM) (see Fig. 2). Apparently, the loss of key flanking residues and/or the potential phosphorylation site at Ser, or most likely, loss of conformational constraint rendered RQARPGPQ ineffective at inhibiting O(2) production in the broken cell system. Charge alone does not seem to be the major determinant of inhibition in the broken cell assay. A control, unrelated peptide (KLSRPRDSNE) and two p47 peptides (PPRRSSIRNA and HQRSRSRKRLSQD) all contain a similar percentage of positively charged residues (27.3, 30.0, and 46.2%, respectively) as do QRRRQARPGPQSPG and SRKRLSQDAYRRNS (28.6 and 35.7%, respectively), but had no inhibitory activity in the broken cell system at concentrations up to 500 µM.


Figure 2: Effect of phage-mapped p47peptides on NADPH oxidase activity in the broken cell system. Peptides QRRRQARPGPQSPG (bullet), SRKRLSQDAYRRNS (box), and a control peptide KLSYRPRDSNE () were added to the broken cell NADPH oxidase assay system at the indicated concentrations, and O(2) generation was measured as described under ``Experimental Procedures.'' The results are expressed as a percent of control activity and represent the mean ± S.D. of three separate experiments.



Previous studies indicated that p47 peptide 323-332 is important in the assembly of a functioning NADPH oxidase(35) . In order to define more precisely the residues in this region which are critical for oxidase assembly, we compared the effects of various peptides derived from p47 323-332 on O(2) generation in the broken cell system (Table 1). Peptide 325-330, a hexapeptide including the putative phosphorylation site at Ser, did not inhibit O(2) production in the broken cell system. Similarly the addition of Tyr or Phe to RRNSVR did not make the peptide inhibitory. However, the addition of both aromatic residues to RRNSVR resulted in inhibitory activity, albeit still less than that of the parent peptide (Table 1). Substitution of tryptophan either for Phe or for Tyr in p47 peptide 323-332 did not alter the inhibitory effects of the parent peptide on O(2) production. The substitution S328A, wherein the phosphorylation site is replaced by an alanine, did not compromise the inhibitory activity of the peptide.

In previous studies, we found that p47 323-332 inhibited the first step (activation) of a two-stage broken cell O(2)-generating system, and addition of peptide to the second step (after the components of the oxidase had assembled) did not inhibit O(2) generation(35) . To determine, in a similar fashion, whether peptides SRKRLSQDAYRRNS and QRRRQARPGPQSPG inhibited assembly of the oxidase, they were added to the broken cell system after activation. Added in this manner, the peptides, at 100 µM, exhibited only minimal inhibition of the oxidase (78.9% ± 3.2 and 78.7% ± 4.1 of control activity, respectively, versus 16.7 ± 1.2% and 15.5 ± 0.5% of control when added before activation; see Fig. 2) This observation is consistent with studies of Kleinberg et al.(27) and our previous studies(32) . These data indicate that prior to full activation of the NADPH oxidase, peptides representative of regions on oxidase components that participate in protein-protein interactions block assembly.

Inhibition of the Phosphorylation and Translocation of p47

Previously, we reported that the inhibitory effects of p47 323-332 on O(2) generation are paralleled in assays of p47 phosphorylation and translocation in the broken cell system(35) . Similar studies of this family of p47 323-332-related peptides were performed (Table 1). In general, the effect of a given peptide on O(2) generation was paralleled by its effect on translocation, supporting the idea that p47 323-332 exerts its inhibitory effect on O(2) production by blocking translocation of p47 and assembly of a functioning oxidase. In contrast, the effects of these peptides on phosphorylation of p47 did not parallel effects on O(2) production. RRNSVR, YRRNSVR, and RRNSVRF reduced phosphorylation of p47 but did not block O(2) production. The S328A mutation did not inhibit phosphorylation but did block O(2) production and translocation. Taken together, these data suggest that in the broken cell system the interactive site(s) in p47 323-332 which associate with flavocytochrome b do not depend on their being phosphorylated.

As discussed above, p47 peptides 315-332, 323-332, and 334-347 all inhibited O(2) production in the broken cell system when added prior to activation but were ineffective when added after assembly, suggesting that these peptides blocked assembly by interfering with the association of p47 with flavocytochrome b. To confirm that p47 peptides SRKRLSQDAYRRNS and QRRRQARPGPQSPG were blocking assembly of the NADPH oxidase rather than activity, these peptides were tested in the broken cell translocation assay at 100 µM. Both peptides inhibited the translocation of p47 to the membrane compared to a control sample (Fig. 3). Peptide QRRRQARPGPQSPG inhibited translocation to 4% of control (Fig. 3), whereas peptides SRKRLSQDAYRRNS and AYRRNSVRFL inhibited p47 translocation to the membrane to 35 and 16% ( Fig. 3and Table 1) of control, respectively. In contrast, peptides representative of p47 residues preceding and partially encompassing 315-342 (PPRRSSIRNA and HQRSRSRKRLSQD) did not inhibit O(2) production or translocation of p47 to the membrane in broken cell assay systems(35) .


Figure 3: Effect of p47 phage-mapped peptides on the translocation of p47 to the membrane in a cell-free system. The effect of p47 peptides on translocation of p47 to the membrane in a broken cell translocation assay was evaluated as described under ``Experimental Procedures.'' The presence of p47 (arrowhead) was assessed in reisolated membranes from the complete system (i.e. SDS + membrane + cytosol; lane 1), the system without SDS (lane 2), the complete system with 100 µMQRRRQARPGPQSPG (lane 3), the complete system with 100 µMSRKRLSQDAYRRNS (lane 4), and the system without cytosol (i.e. membrane only + SDS; lane 5). The relative amount of p47 in the membrane fraction was quantitated by densitometry of the blots and is shown below each lane. The results, expressed as percent of control, are representative of two separate experiments.



Inhibition of Superoxide Production in Electropermeabilized Neutrophils

The broken cell assay for O(2) production does not mimic entirely, the NADPH oxidase assembly in the intact PMN(45) . For that reason, the ability of active p47 peptides to inhibit in electropermeabilized neutrophils was evaluated. Peptides SRKRLSQDAYRRNS, AYRRNSVRFL, and QRRRQARPGPQSPG were incubated with the permeabilized neutrophils for 5 min and phorbol 12-myristate 13-acetate-stimulated O(2) production by these cells was determined. We chose a 5-min treatment time in these experiments for two reasons: 1) the cells were determined to be fully permeable by this time (>98% permeable), and 2) at longer incubation times the permeabilized cells failed to retain a level of O(2) generating capacity that reasonably reflected that in the intact cell (>92% of control activity was maintained after 5 min of incubation, whereas <50% activity was present after 30 min).

As shown in Table 2, the peptides that were inhibitory in the broken cell assay system also inhibited O(2) production in permeabilized neutrophils (40-50% inhibition compared with controls). Similarly, peptides that were inactive in the broken cell assay (RQARPGPQ and RRNSVR) also had no effect on permeabilized cells and served as negative controls (Table 2). In addition, we also tested the gp91 carboxyl-terminal peptide RGVHFIF as a positive control, since it was shown previously by Kleinberg et al.(27) to inhibit oxidase activity in electropermeabilized cells. Our data (Table 2) were consistent with their results. Two irrelevant peptides (AVEGGMKPVKLLVGC and KLSYRPRDSNE) were used to determine nonspecific peptide effects in permeabilized cells, and their molecular weights (1500.3 and 1364.5, respectively) were similar to that of AYRRNSVRFL (1281.6), SRKRLSQDAYRRNS (1719.1), and QRRRQARPGPQSPG (1590.8). Thus, in support of our findings in the broken cell assays, our data indicate that the active p47 peptides identified represent sites which participate in the assembly of the active NADPH oxidase in vivo.


DISCUSSION

The cytosolic NADPH oxidase protein p47 has been shown to associate with flavocytochrome b, and several sites have been identified in carboxyl-terminal domains of both gp91 and p22 that are important for this interaction(27, 28, 29, 30, 31) . Additionally, our recent studies suggest that p47 also binds to a region close to the amino terminus of gp91(32) . Recent studies have examined which regions in p47 participate in the binding to flavocytochrome b. Two reports demonstrated that Src homology 3 domains in p47 may interact with p22(29, 33, 34) . In addition, Nauseef et al.(35) screened p47 peptides containing phosphorylation sites and found that one of these peptides (323-332) represents a functionally important domain in p47. This peptide, AYRRNSVRFL, inhibits O(2) production, phosphorylation, and translocation of p47 in the broken cell system(35) . Thus, the data suggested that this p47 domain might play a role in oxidase assembly, although no direct evidence supported this possibility.

In the present studies using random peptide phage display library analysis, we provide direct evidence that p47 residues 323-332, as well as the adjacent region extending to residue 342, are important in the binding of p47 to flavocytochrome b. Thus, the entire binding domain encompasses residues 323-342 of p47, and synthetic peptides representing these adjacent sites and overlapping with the 323-332 region were potent inhibitors of NADPH oxidase activity.

The phage display library analysis of potential flavocytochrome b-binding domains in p47 indicated that among the 94 phage sequenced the strongest homologies to p47 were YRRNS, LQQRRRQ, and QARPG. Moreover, p47 peptides encompassing these residues (SRKRLSQDAYRRNS, AYRRNSVRFL, and QRRRQARPGPQSPG) all inhibited superoxide production and translocation of p47 in the cell-free system. The inhibition of O(2) production was shown to be dose-dependent and targeted assembly of the oxidase rather than activity, as demonstrated by the inhibition of translocation by these peptides and their ineffectiveness when added after assembly of the oxidase.

In addition to inhibiting O(2) production and translocation in the broken cell system, the p47 peptides SRKRLSQDAYRRNS, AYRRNSVRFL, and QRRRQARPGPQSPG all inhibited O(2) production significantly in intact, permeabilized neutrophils, suggesting that they do indeed represent biologically relevant sites involved in NADPH oxidase assembly. Electropermeabilized neutrophils, which retained nearly all of their ability to generate O(2) when compared with nonpermeabilized cells, may provide a more accurate representation of the in vivo situation. The inhibitory effect of the p47 peptides on O(2) production in permeabilized neutrophils supports the data from the phage library analysis and broken cell system and confirms that p47 residues 323-342 are important for oxidase activation.

Phosphorylation of p47 is required for activation of the NADPH oxidase in neutrophils(46, 47, 48) ; however, this requirement for phosphorylation is not observed in the broken cell system(35, 45) . Previously, it has been suggested that phosphorylation of p47 may function, in part, to neutralize positively charged regions of the protein, thus allowing it to interact with the membrane or target protein. In the broken cell system, the addition of anionic detergent (SDS) or arachidonic acid appears to bypass the need for p47 phosphorylation by imparting negative charge to the protein. Consistent with this hypothesis, the p47 region identified here as an important domain for association with flavocytochrome b and NADPH oxidase assembly (323-342), is within a larger region (314-347) that contains 11 positively charged residues, one protein kinase C phosphorylation site (Ser), and is surrounded by several other potential sites for phosphorylation by protein kinase C and tyrosine kinase(49) . Previously, Joseph et al.(50) reported that any polybasic peptide (geq5 basic residues) could nonspecifically inhibit NADPH oxidase activity; however, they also found that an 11 residue peptide containing six lysines had no effect on oxidase activity. We have analyzed a number of polybasic peptides, including some peptides with the same number of basic residues as in our active peptides, that had no inhibitory effect on NADPH oxidase activity in a broken cell assay system ( (35) and Table 1) and in electropermeabilized neutrophils (Table 2). Thus, the inhibition of NADPH oxidase activity by the active p47 peptides is specific to their sequences and/or the charge distribution represented by their sequences, and the apparent ``nonspecific'' inhibition of oxidase activity by polybasic peptides unrelated to the NADPH oxidase, as reported by Joseph et al.(50) , is due to the blocking of specific binding interactions (in this case between p47 and gp91) that involve basic amino acid-enriched domains on one or both of the interacting proteins.

In conclusion, the data presented, based on the complementary approaches of random peptide phage display library analysis and peptide inhibition in both broken cell and permeabilized cell systems, demonstrate that p47 residues 323-342 interact with flavocytochrome b and are required for the association of these two NADPH oxidase components. The association is sequence-specific and appears to require certain conformational constraints. The elucidation of the sites of interaction between human neutrophil NADPH oxidase component proteins will lead to a further understanding of the regulation of this system.


FOOTNOTES

*
This work was supported in part by an Arthritis Foundation Biomedical Science Grant (to M. T. Q.), National Science Foundation EPSCoR Grant R II-891878 (to M. T. Q.), and National Institutes of Health Grants AR40929 (to M. T. Q.), AI28412 (to R. A. C. and W. M. N.), AI20866 (R. A. C. and W. M. N.), and HL34327 (W. M. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717. Tel.: 406-994-5721; Fax: 406-994-4303.

(^1)
The abbreviations used are: PMN, polymorphonuclear leukocyte; GTPS, guanosine 5`-3-O-(thio)triphosphate; Pipes, 1,4-piperazinediethanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Jan Renee, Kevin Leidal, Sally McCormick, and Linda R. Loetterle for expert technical assistance.


REFERENCES

  1. Badwey, J. A., and Karnovsky, M. L. (1980) Annu. Rev. Biochem. 49,695 [CrossRef][Medline] [Order article via Infotrieve]
  2. Baggiolini, M., Boulay, F., Badwey, J. A., and Curnutte, J. T. (1993) FASEB J. 7, 1004-1010
  3. Clark, R. A. (1990) J. Infect. Dis. 161, 1140-1147 [Medline] [Order article via Infotrieve]
  4. Segal, A. W., and Abo, A. (1993) Trends. Biochem. Sci. 18, 43-47 [CrossRef][Medline] [Order article via Infotrieve]
  5. Smith, R. M., and Curnutte, J. T. (1991) Blood 77, 673-686 [Medline] [Order article via Infotrieve]
  6. Dinauer, M. C. (1993) Crit. Rev. Clin. Lab. Sci. 30, 329-369 [Medline] [Order article via Infotrieve]
  7. Parkos, C. A., Allen, R. A., Cochrane, C. G., and Jesaitis, A. J. (1987) J. Clin. Invest. 80, 732-742 [Medline] [Order article via Infotrieve]
  8. Segal, A. W. (1989) J. Clin. Invest. 83, 1785-1793 [Medline] [Order article via Infotrieve]
  9. Rotrosen, D., Yeung, C. L., Leto, T. L., Malech, H. L., and Kwong, C. H. (1992) Science 256, 1459-1462 [Medline] [Order article via Infotrieve]
  10. Segal, A. W., West, I., Wientjes, F., Nugent, J. H. A., Chavan, A. J., Haley, B., Garcia, R. C., Rosen, H., and Scarce, G. (1992) Biochem. J. 284, 781-788 [Medline] [Order article via Infotrieve]
  11. Quinn, M. T., Parkos, C. A., Walker, L., Orkin, S. H., Dinauer, M. C., and Jesaitis, A. J. (1989) Nature 342, 198-200 [CrossRef][Medline] [Order article via Infotrieve]
  12. Maly, F.-E., Quilliam, L. A., Dorseuil, O., Der, C. J., and Bokoch, G. M. (1994) J. Biol. Chem. 269, 18743-18746 [Abstract/Free Full Text]
  13. Gabig, T. G., Crean, C. D., Mantel, P. L., and Rosli, R. (1995) Blood 85, 804-811 [Abstract/Free Full Text]
  14. Volpp, B. D., Nauseef, W. M., and Clark, R. A. (1988) Science 242, 1295-1297 [Medline] [Order article via Infotrieve]
  15. Lomax, K. J., Leto, T. L., Nunoi, H., Gallin, J. I., and Malech, H. L. (1989) Science 245, 409-412 [Medline] [Order article via Infotrieve]
  16. Leto, T. L., Lomax, K. J., Volpp, B. D., Nunoi, H., Sechler, J. M. G., Nauseef, W. M., Clark, R. A., Gallin, J. I., and Malech, H. L. (1990) Science 248, 727-730 [Medline] [Order article via Infotrieve]
  17. Knaus, U. G., Heyworth, P. G., Evans, T., Curnutte, J. T., and Bokoch, G. M. (1991) Science 254, 1512-1515 [Medline] [Order article via Infotrieve]
  18. Abo, A., and Pick, E. (1991) J. Biol. Chem. 266, 23577-23585 [Abstract/Free Full Text]
  19. Clark, R. A., Volpp, B. D., Leidal, K. G., and Nauseef, W. M. (1990) J. Clin. Invest. 85, 714-721 [Medline] [Order article via Infotrieve]
  20. Heyworth, P. G., Curnutte, J. T., Nauseef, W. M., Volpp, B. D., Pearson, D. W., Rosen, H., and Clark, R. A. (1991) J. Clin. Invest. 87, 352-356 [Medline] [Order article via Infotrieve]
  21. Quinn, M. T., Evans, T., Loetterle, L. R., Jesaitis, A. J., and Bokoch, G. M. (1993) J. Biol. Chem. 268, 20983-20987 [Abstract/Free Full Text]
  22. Wientjes, F. B., Hsuan, J. J., Totty, N. F., and Segal, A. W. (1993) Biochem. J. 296, 557-561 [Medline] [Order article via Infotrieve]
  23. Park, J. W., Ma, M., Ruedi, J. M., Smith, R. M., and Babior, B. M. (1992) J. Biol. Chem. 267, 17327-17332 [Abstract/Free Full Text]
  24. Iyer, S. S., Pearson, D. W., Nauseef, W. M., and Clark, R. A. (1994) J. Biol. Chem. 269, 22405-22411 [Abstract/Free Full Text]
  25. Heyworth, P. G., Bohl, B. P., Bokoch, G. M., and Curnutte, J. T. (1994) J. Biol. Chem. 269, 30749-30752 [Abstract/Free Full Text]
  26. Dorseuil, O., Quinn, M. T., and Bokoch, G. M. (1995) J. Leukocyte Biol. 58, 108-113 [Abstract]
  27. Kleinberg, M. E., Malech, H. L., and Rotrosen, D. (1990) J. Biol. Chem. 265, 15577-15583 [Abstract/Free Full Text]
  28. Leusen, J. H. W., Bolscher, B. G. J. M., Hilarius, P. M., Weening, R. S., Kaulfersch, W., Seger, R. A., Roos, D., and Verhoeven, A. J. (1994) J. Exp. Med. 180, 2329-2334 [Abstract]
  29. Leto, T. L., Adams, A. G., and de Mendez, I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10650-10654 [Abstract/Free Full Text]
  30. Leusen, J. H. W., De Boer, M., Bolscher, B. G. J. M., Hilarius, P. M., Weening, R. S., Ochs, H. D., Roos, D., and Verhoeven, A. J. (1994) J. Clin. Invest. 93, 2120-2126 [Medline] [Order article via Infotrieve]
  31. Nakanishi, A., Imajo-Hohmi, S., Fujinawa, T., Kikuchi, H., and Kanegasaki, S. (1992) J. Biol. Chem. 267, 19072-19074 [Abstract/Free Full Text]
  32. DeLeo, F. R., Yu, L., Burritt, J. B., Loetterle, L. R., Bond, C. W., Jesaitis, A. J., and Quinn, M. T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7110-7114 [Abstract]
  33. Sumimoto, H., Kage, Y., Nunoi, H., Sasaki, H., Nose, T., Fukumaki, Y., Ohno, M., Minakami, S., and Takeshige, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5345-5349 [Abstract]
  34. Finan, P., Shimizu, Y., Gout, I., Hsuan, J., Truong, O., Butcher, C., Bennett, P., Waterfield, M. D., and Kellie, S. (1994) J. Biol. Chem. 269, 13752-13755 [Abstract/Free Full Text]
  35. Nauseef, W. M., McCormick, S., Renee, J., Leidal, K. G., and Clark, R. A. (1993) J. Biol. Chem. 268, 23646-23651 [Abstract/Free Full Text]
  36. Fujita, I., Takeshige, K., and Minakami, S. (1987) Biochim. Biophys. Acta 931, 41-48 [Medline] [Order article via Infotrieve]
  37. Borregaard, N., Heiple, J. M., Simons, E. R., and Clark, R. A. (1983) J. Cell Biol. 97, 52-61 [Abstract]
  38. Quinn, M. T., Parkos, C. A., and Jesaitis, A. J. (1995) Methods Enzymol. 255, 476-487 [Medline] [Order article via Infotrieve]
  39. Smith, G. P., and Scott, J. K. (1993) Methods Enzymol. 217, 228-257 [Medline] [Order article via Infotrieve]
  40. Burritt, J. B., Quinn, M. T., Jutila, M. A., Bond, C. W., and Jesaitis, A. J. (1995) J. Biol. Chem. 270, 16974-16980 [Abstract/Free Full Text]
  41. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , pp. 4.21-4.38, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  42. Park, J. W., and Babior, B. M. (1992) J. Biol. Chem. 267, 19901-19906 [Abstract/Free Full Text]
  43. Verhoeven, A. J., Leusen, J. H. W., Kessels, G. C. R., Hilarius, P. M., De Bont, D. B. A., and Liskamp, R. M. J. (1993) J. Biol. Chem. 268, 18593-18598 [Abstract/Free Full Text]
  44. Volpp, B. D., Nauseef, W. M., Donelson, J. E., Moser, D. R., and Clark, R. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7195-7199 [Abstract]
  45. Nauseef, W. M. (1993) Eur. J. Haematol. 51, 301-308 [Medline] [Order article via Infotrieve]
  46. Hayakawa, T., Suzuki, K., Suzuki, S., Andrews, P. C., and Babior, B. M. (1986) J. Biol. Chem. 261, 9109-9115 [Abstract/Free Full Text]
  47. Rotrosen, D., and Leto, T. L. (1990) J. Biol. Chem. 265, 19910-19915 [Abstract/Free Full Text]
  48. Heyworth, P. G., and Badwey, J. A. (1990) Biochim. Biophys. Acta 1052, 299-305 [CrossRef][Medline] [Order article via Infotrieve]
  49. El Benna, J., Faust, L. P., and Babior, B. M. (1994) J. Biol. Chem. 269, 23431-23436 [Abstract/Free Full Text]
  50. Joseph, G., Gorzalczany, Y., Koshkin, V., and Pick, E. (1994) J. Biol. Chem. 269, 29024-29031 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.