From the Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
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ABSTRACT |
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The superoxide generating NADPH oxidase of phagocytes consists, in resting cells, of a membrane-associated electron transporting flavocytochrome (cytochrome b559) and four cytosolic proteins as follows: p47phox, p67phox, p40phox, and the small GTPase, Rac(1 or 2). Activation of the oxidase is consequent to the assembly of a membrane-localized multimolecular complex consisting of cytochrome b559 and the cytosolic components. We used "peptide walking" (Joseph, G., and Pick, E. (1995) J. Biol. Chem. 270, 29079-29082) for mapping domains in the amino acid sequence of p47phox participating in the molecular events leading to the activation of NADPH oxidase. Ninety-five overlapping pentadecapeptides, with a four-residue offset between neighboring peptides, spanning the complete p47phox sequence, were tested for the ability to inhibit NADPH oxidase activation in a cell-free system. This consisted of solubilized macrophage membranes, recombinant p47phox, p67phox, and Rac1, and lithium dodecyl sulfate, as the activator. Eight functional domains were identified and labeled a-h. These were (N- and C-terminal residue numbers are given for each domain) as follows: a (21-35); b (105-119); c (149-159); d (193-207); e (253-267); f (305-319); g (325-339), and h (373-387). Four of these domains (c, d, e, and g) correspond to or form parts of regions shown before to participate in NADPH oxidase assembly. Thus, domain c corresponds to a region on the N-terminal boundary of the first src homology 3 (SH3) domain, whereas domains d and e represent more precisely defined sites within the full-length first and second SH3 domains, respectively. Domain g overlaps an extensively investigated arginine-rich region. Domains a and b, in the N-terminal half of p47phox, and domains f and h, in the C-terminal half, represent newly identified entities, for which there is no earlier experimental evidence of involvement in NADPH oxidase activation. "Peptide walking" was also applied to the identification of domains in p47phox mediating binding to p67phox. This was done by quantifying, by enzyme-linked immunosorbent assay, the binding of p67phox, in solution, to a series of 95 overlapping biotinylated p47phox peptides, attached to streptavidin-coated 96-well plates. A single proline-rich domain (residues 357-371) was found to bind p67phox in the absence and presence of lithium dodecyl sulfate.
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INTRODUCTION |
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Phagocytic cells produce, in response to appropriate stimuli, a
variety of oxygen-derived toxic radicals, all of which are derived from
superoxide (O2).1
O
2 is generated by the NADPH-derived one-electron reduction of
molecular oxygen, catalyzed by a membrane-associated heterodimeric flavocytochrome (cytochrome b559), composed of a
91-kDa glycoprotein (gp91phox) and a 22-kDa protein
(p22phox), and incorporating two redox centers, FAD and two
hemes (reviewed in Refs. 1-3). The conversion of cytochrome
b559 from the resting to the activated state is,
most likely, the result of a conformational change leading to a high
turnover electron flow from NADPH to oxygen, through the redox centers.
This change in conformation is brought about by protein-protein
interactions involving the two cytochrome b559
subunits and four cytosolic regulatory proteins, p47phox,
p67phox, p40phox, and the small GTPase Rac(1 or 2)
(reviewed in Refs. 4 and 5). It is commonly assumed that activation
leads to the formation of a membrane-localized multimolecular
structure, known as the NADPH oxidase complex. The physical expression
of this, in the intact cell, is the translocation of parts of
p47phox and p67phox to the plasma membrane (6)
associated with the phosphorylation of p47phox at multiple
sites (7). Whether the activation of NADPH oxidase involves
translocation of p40phox and Rac to the membrane is as yet an
unsettled question.
The conversion of cytochrome b559 from the inactive to the active conformation can be achieved in vitro in a cell-free system, consisting, in its most elementary form, of membranes and cytosol from resting cells exposed to a critical concentration of certain fatty acids (8-11) or anionic amphiphiles (12). An advanced version of the cell-free system, consisting of solubilized membrane or purified and lipid-reconstituted cytochrome b559 combined with purified recombinant cytosolic components (13, 14), served as a valuable tool for the analysis of intermolecular interactions in the assembly of NADPH oxidase. p47phox plays a pivotal role in the activation of NADPH oxidase, so far it is the only cytosolic component for which unequivocal evidence is available for physical binding to cytochrome b559 (reviewed in Ref. 4).
A number of methodologies have been utilized in the past for the detection of protein-protein interactions between p47phox and other NADPH oxidase components and for the identification of domains in the amino acid sequence of p47phox essential for its participation in the assembly of the complex (reviewed in Ref. 5). We recently described a new approach to mapping functionally significant domains in proteins, based on the use of overlapping peptides spanning the complete sequence of the protein under scrutiny. This methodology, termed "peptide walking," was applied to the analysis of Rac1 domains involved in the activation of NADPH oxidase (15). In the present paper we applied peptide walking to the analysis of p47phox domains involved in the activation of NADPH oxidase. This research had two goals as follows: 1) evaluate the reliability of the method by its ability to detect domains identified before by other approaches, and 2) assess the capacity of the method to identify previously unknown domains. Finally, peptide walking was used as a method to identify domains in p47phox participating in interaction with p67phox.
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EXPERIMENTAL PROCEDURES |
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Chemicals and Reagents
Synthetic Peptides--
Ninety-five overlapping
pentadecapeptides spanning the complete p47phox amino acid
sequence, from the N to the C terminus, were synthesized by the
multipin synthesis method (16) by Chiron Mimotopes, Clayton, Australia.
Peptides overlapped by 11 residues, with the exception of peptides 94 and 95 which shared 12 residues. Peptides were biotinylated at the N
terminus and were amidated at the C terminus. The biotin group was
attached to the N terminus by the intermediary of a four-residue spacer
consisting of SGSG. The purity of the peptides ranged from 60 to 70%.
The freeze-dried peptides were dissolved in a mixture of 75 parts
dimethyl sulfoxide and 25 parts water (v/v), to a concentration of 1.5 mM, divided into aliquots of 30 µl and frozen at
75 °C.
Chemicals--
The following chemicals were obtained from Sigma:
ferricytochrome c (from horse heart, 95%), NADPH
(tetrasodium salt, 95%), bovine serum albumin (crystallized, >97%;
BSA), 3,3',5.5'-tetramethylbenzidine, free base, polyoxyethylene
sorbitan monolaurate (Tween 20), streptavidin (from Streptomyces
avidinii, affinity purified), and ImmunoProbe biotinylation kit
(product number BK-101). Guanosine 5'-3-O-(thio)triphosphate (GTPS) was obtained from Boehringer Mannheim.
n-Octyl-
-D-glucopyronoside (octyl glucoside)
was a product of Pfanstiehl. Lithium dodecyl sulfate (LiDS) and common
laboratory chemicals (at the highest purity available) were obtained
from Merck.
Antibodies-- A polyclonal antibody against purified baculovirus-derived recombinant p67phox was prepared in goats (17) and kindly provided, as whole serum, by Dr. Thomas L. Leto (National Institutes of Health, Bethesda, MD). Rabbit polyclonal antibody to BSA (whole serum) was obtained from Sigma. Peroxidase-conjugated affinity purified rabbit anti-goat IgG and goat anti-rabbit IgG were purchased from Jackson ImmunoResearch.
Preparation of Recombinant Cytosolic NADPH Oxidase
Components--
Recombinant p47phox and p67phox were
prepared from baculovirus-infected Sf9 cells, as described by
Leto et al. (17), and purified by ion exchange
chromatography, as recently described (18). Recombinant Rac1 was
isolated from E. coli transformed with Rac1 cDNA
subcloned into the bacterial expression vector pGEX2T, as described by
Kwong et al. (19). The glutathione
S-transferase-Rac1 fusion protein was affinity purified on
glutathione-agarose, cleaved by thrombin, and subjected to nucleotide
exchange to GTPS, as described before (20). Baculoviruses carrying
cDNA for p47phox and p67phox and Rac1 cDNA
subcloned in the pGEX2T vector were kind gifts of Dr. Thomas L. Leto
(National Institutes of Health, Bethesda, MD).
Biotinylation of p47phox
Purified recombinant p47phox (at a protein concentration of 0.5 mg/ml) was biotinylated with biotinamidocaproate N-hydroxysulfosuccinimide ester, using the ImmunoProbe kit (Sigma) and following the instructions provided with the kit. The biotin/protein ratio varied from 3 to 3.5 mol of biotin per mol of p47phox.
Preparation of Solubilized Macrophage Membranes
Macrophages were obtained from the peritoneal cavity of guinea pigs injected with mineral oil 5-6 days earlier. Membranes were prepared from cells disrupted by sonication, as reported before (21), but using a modified homogenization buffer, which consisted of 130 mM NaCl, 8 mM Na+, potassium phosphate buffer, pH 7, 340 mM sucrose, 1 mM MgCl2, 1 mM EGTA, 1 mM dithioerythritol, 2 mM NaN3, 10 µM GDP, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, and 21 µM leupeptin. The membranes were solubilized by 40 mM octyl glucoside, as described previously (22), and freed of octyl glucoside by dialysis for 18 h at 4 °C against solubilization buffer (22), lacking detergent. The concentration of cytochrome b559 in the solubilized membrane preparation was measured as described before (23).
Determination of Protein Concentration
This was measured by the method of Bradford (24), modified for use with 96-well plates, as described in technical bulletin 1177EC (Bio-Rad). BSA was used as standard.
Cell-free NADPH Oxidase Assay
LiDS-elicited cell-free O2 production assays were
performed in 96-well, flat bottomed, polystyrene plates (Greiner) at
25 °C. All components were diluted in assay buffer, consisting of 65 mM Na+, potassium phosphate buffer, pH 7, 0.2 mM cytochrome c, 1 mM EGTA, 1 mM MgCl2, 10 µM FAD, and 2 mM NaN3. Reaction mixtures (100 µl total
volume) consisted of (in order of addition) the following: 25 µl of
assay buffer (containing or not p47phox peptide); 25 µl of a
mixture of solubilized membrane (40 nM cytochrome b559), p67phox, and Rac1-GTP
S (0.8 µM, each); 25 µl of p47phox (0.24 µM); and 25 µl of assay buffer containing 0.52 mM LiDS. The final concentrations of NADPH oxidase
components in the assay were 10 nM cytochrome
b559, 200 nM p67phox and
Rac1, and 60 nM p47phox. The final concentration of
LiDS was 130 µM. The plates were shaken for 10 s,
once after the addition of the mixture of membrane, p67phox,
and Rac1, again following the addition of p47phox, and for 1.5 min upon the addition of LiDS. O
2 production was initiated by
the addition of 10 µl of 5 mM NADPH (resulting in a final
concentration of 454 µM) and quantified by following the rate of cytochrome c reduction, at 550 nm, in a kinetic
assay performed in a Spectramax 340 microplate reader (Molecular
Devices), using Softmax Pro software. Results were expressed as
nanomoles of O
2/min (calculated from the linear portion of the
curve) produced by the amount of membrane present in one microplate
well (corresponding to 1 pmol of cytochrome
b559). Each measurement was performed in
triplicate and expressed as the mean value.
Inhibition of NADPH Oxidase Activation by p47phox Peptides
The effect of overlapping p47phox pentadecapeptides on
NADPH oxidase activation was tested in the cell-free system described
in the preceding section. Peptides (stock solutions of 1.5 mM in 75% dimethyl sulfoxide, 25% water) were diluted in
assay buffer to reach a concentration of 120 µM, and
amounts of 25 µl were added per well as the first component of the
100-µl reaction mixtures (resulting in a final concentration of
peptide of 30 µM). Control mixtures contained 25 µl of
assay buffer supplemented with 1.5% (v/v) dimethyl sulfoxide, a
concentration identical to that found in peptide-containing wells. In
this experimental set up, the addition of peptide preceded that of
p47phox and the induction of activation by LiDS. In a parallel
series of experiments, we studied the effect of adding p47phox
peptides to the cell-free assay after the completion of activation. In
these experiments, the components of the assay mixture were added in
the following order: a mixture of solubilized membrane, p47phox, p67phox and Rac1-GTPS in a volume of 50 µl (resulting in the final concentrations of components indicated
before); 25 µl of assay buffer containing 0.39 mM LiDS
(resulting in a final concentration of 130 µM); and following incubation with mixing for 1.5 min, 25 µl of assay buffer containing 120 µM peptide and 130 µM LiDS
(resulting in a final concentration of peptide of 30 µM).
The assay mixtures were incubated, with mixing, for 30 s and
O
2 production initiated by the addition of 10 µl of 5 mM NADPH (resulting in a final concentration of 454 µM).
An Enzyme-linked Immunosorbent Assay for the Detection of Protein-Peptide and Protein-Protein Interactions among NADPH Oxidase Components Applied to the Binding of p67phox to p47phox-derived Peptides
All experiments were performed in 96-well microplates, with flat bottoms and rounded corners (MaxiSorp C96, product 430341, Nunc). 100 µl/well of a 7 µg/ml solution of streptavidin in water were added (= 11.66 pmol of streptavidin/well), and the plates were exposed to circulating air at 37 °C for 18-24 h, to allow the solution to evaporate completely. The streptavidin-coated plates could be kept at 4 °C, under vacuum, in the presence of silica gel dessicant, for up to 4 weeks. Assuming maximal coating with streptavidin, each well should be capable of binding close to 50 pmol of biotin. The plates were washed four times with phosphate-buffered saline (PBS), consisting of 10 mM sodium phosphate buffer, pH 7.2, and 140 mM NaCl, supplemented with 0.1% (v/v) Tween 20 (PBS-T). All washing procedures were performed using an automatic microplate washer (Wellwash type 4 Mk2, Denley Instruments). After removal of washing buffer, nonspecific absorption was blocked by adding to each well 200 µl of 10% (v/v) liquid milk (1% fat) in PBS and shaking the plates for 1 h at room temperature. The plates were washed four times with PBS-T, and to the wells were added 100-µl volumes of biotinylated p47phox peptides or biotinylated p47phox protein, dissolved in 10% milk in PBS, to result in 50 pmol of peptide or protein per well. Binding of biotinylated compounds to the streptavidin-coated plates was allowed to proceed for 30 min at room temperature, with shaking. The plates were again washed four times with PBS-T and, following the removal of washing buffer, 100-µl volumes p67phox, dissolved in 10% milk in PBS, were added, to result in 50 pmol of protein per well. The plates were shaken for 30 min at room temperature to allow the interaction between the surface-attached p47phox peptides or p47phox protein and p67phox, in solution, to proceed. In control experiments, p67phox was replaced by 50 pmol/well of BSA. Unbound protein was removed by washing the plates four times with PBS-T, and the specifically bound p67phox and nonspecifically bound BSA were detected by the addition of 100 µl/well of goat anti-p67phox and rabbit anti-BSA antisera, diluted 1/1000 in 10% milk in PBS, respectively. The plates were incubated with the antibodies for 30 min at room temperature, with shaking, and unbound antibody was removed by four washes with PBS-T. Bound immunoglobulin was detected by the addition of a 1/10,000 dilution, in 10% milk in PBS, of peroxidase-conjugated anti-goat and anti-rabbit IgG antibodies, respectively, and incubation for 30 min at room temperature, with shaking. The plates were washed four times with PBS-T and once with distilled water, and bound peroxidase was detected by the addition of 100 µl/well of 0.1 mg/ml 3,3'5,5'-tetramethylbenzidine and 0.006% H2O2 in 0.1 M sodium acetate buffer, pH 5.4. After incubation for 30 min at room temperature, the reaction was stopped by the addition of 50 µl/well of 2 N H2SO4. Absorbance at 450 nm was read against blank wells, containing only the peroxidase assay reagent, in a Spectramax 340 microplate reader (Molecular Devices).
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RESULTS |
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Clusters of Overlapping p47phox Peptides Inhibit NADPH
Oxidase Activation in a Cell-free Assay--
Ninety five
pentadecapeptides spanning sequentially the complete amino acid
sequence of p47phox, starting at the first 15 residues at the N
terminus and overlapping by 11 residues, were tested for an effect on
the activation of NADPH oxidase in a cell-free system, consisting of
solubilized macrophage membrane and recombinant p47phox,
p67phox, and Rac1-GTPS. Fig. 1
lists the peptides utilized in these experiments and the location of
the corresponding sequences within the p47phox protein.
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p67phox Binds to Surface-immobilized p47phox and to a Single Cluster of p47phox Peptides-- The first purpose of the experiments described in this section of the study was the design of a simple, quantitative, and generally applicable methodology for assessing protein to protein binding among components of the NADPH oxidase complex. It was next intended to extend this technique to protein to peptide binding, as an approach to mapping domains on one component involved in interaction with a second component. In the past, protein-protein interactions between members of the NADPH oxidase complex were investigated by a variety of techniques as follows: binding of one component (or fragment of it) in the fluid phase to a second component (or fragment of it) immobilized on agarose beads (31) or nitrocellulose membrane (32), the yeast two-hybrid system (33), and surface plasmon resonance (biosensor) (34). In addition, information on specific domains involved in the interaction between two components was gained by assessing the inhibitory effect of selected synthetic peptides, corresponding to these domains, on the binding of one protein to another (31, 35). Recently, random sequence peptide phage display library analysis was introduced as a powerful technique for identifying domains on one component responsible for interaction with another component (36).
Although protein-protein interaction assays based on binding of one protein to the surface of 96-well plates, with the second protein being present in solution, have been described before (37-39), this approach has not been previously applied to the study of the interaction between components of the NADPH oxidase complex. We here describe its use in the study of the interaction between p47phox and p67phox. This model was chosen based on evidence, originating from several groups of investigators, that p47phox and p67phox bind to each other constitutively to form a complex of a Mr of 240-300,000 (40-43) and on our recent results showing the formation of a stable complex between highly purified recombinant p47phox and p67phox, upon gel filtration of a mixture of the two components.2 With the purpose of identifying domains in p47phox involved in binding of p67phox, we made use of a technique originally developed for identifying linear epitopes in protein sequences recognized by antibodies. This consists of attaching multiple overlapping biotinylated peptides, spanning the sequence of the protein antigen, to streptavidin-coated 96-well plates, followed by reacting these with the antibody under study in the fluid phase (44). Therefore, we attached the 95 overlapping biotinylated p47phox pentadecapeptides (used before in the inhibition of NADPH oxidase activation experiments) and intact biotinylated p47phox protein to 96-well plates, previously coated with streptavidin, as described under "Experimental Procedures." Addition to the plates of p67phox, in the fluid phase, resulted in its binding to intact p47phox and to 2 out of 95 peptides (peptides 357-371 and 361-375) (Fig. 6A). In control experiments, it was found that BSA did not bind to either surface-attached intact p47phox or to any of 95 surface-attached p47phox peptides (Fig. 6B). As apparent from the list of peptides in Fig. 1, the two peptides binding p67phox shared a domain extending from residues 357 to 371, which was labeled pr (proline-rich) to indicate the presence of five proline residues. The location of domain pr in the p47phox sequence is indicated in Fig. 3. It is of interest that peptides 357-371 and 361-375 were found not to inhibit NADPH oxidase activation (Fig. 2).
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DISCUSSION |
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In the present report we describe the application of peptide walking to the identification of domains, in the linear amino acid sequence of p47phox, which participate in the assembly of an enzymatically active NADPH oxidase complex in vitro. We used this methodology before for the definition of oxidase activation-related domains in Rac1 (15). Although overlapping peptides were tested exclusively as inhibitors of NADPH oxidase activation in this earlier report, we have now extended their use to binding studies involving p47phox peptides and intact p67phox.
The assumptions underlying the use of peptide walking for the identification of domains in p47phox involved in the activation of NADPH oxidase were the following. 1) Inhibition of activity by peptides containing part of or a complete domain is the result of competition between peptide and p47phox protein for a common target, and proof for this is provided by results of Michaelis-Menten analysis, demonstrating a competitive inhibition pattern with respect to p47phox. 2) The use of overlapping peptides results in a high likelihood for a domain or part of it to be present in several neighboring peptides, assuring more sensitive and reliable detection than that offered by the use of selected individual peptides. 3) Peptide walking is, by definition, detecting domains involved in NADPH oxidase activation in the cell-free system, and their role in the elicitation of an oxidative burst in whole cells has to be evaluated by additional methods.
The main goal of this research was to assess the capacity of peptide walking to serve as a screening method for the identification of functional domains in p47phox. The success of this methodology was demonstrated by its ability to both detect domains identified before by other techniques and to reveal novel domains. Thus, we identified eight domains (11-15 residues) in the linear sequence of p47phox participating in NADPH oxidase activation in vitro. Four of these correspond to regions identified previously by other methods (domains c-e and g), and four represent newly identified domains (a, b, f, and h) for which no experimental evidence was available for involvement in activation. The reality of two additional domains (a1 and a2), located in the vicinity of domain a, based solely on the inhibitory action of single peptides will have to be confirmed by other methods.
Domain a spans residues 21-35 and is rich in hydrophobic amino acids.
It does not share residues with domains a1 and
a2, but the possibility cannot be excluded that we are
dealing with a larger region composed of all three nearly contiguous
modules. Recently, it was found that the N-terminal regions of
p47phox and p40phox contain a previously unidentified
domain, termed PX, also shown to be present in the Cpk class of
phosphatidylinositol 3-kinase and in several yeast proteins (Bem1p and
Scd2) (45). In p47phox, domain PX was proposed to span residues
20-125, and it is possible that the domain might also include a region
N-terminal to residue 20 (45). It is of interest that domains a and
a2 are located within the limits of the PX region and
contain no less than six PX consensus residues. Should the PX domain
extend further toward the N terminus, it might possibly also include
domain a1. Potential ligands for PX domains are SH3 domains
and, putatively, Rho-type GTPases (46). The PX region also incorporates
domain b (residues 105-119), which contains two PX consensus residues
(phenylalanines 117 and 118). It should be noted that the PX domain
was proposed as a new entity exclusively on the basis of a sequence
similarity search, and no experimental proof existed for its
participation in NADPH oxidase activation. Therefore, it appears that
the N-terminal region of p47phox, extending from residue 5 to
119, is required for O2 production in a semi-recombinant
cell-free system. The only evidence in the literature in support of
this is the finding that O
2 production by K562 cells
transfected with a vector containing cDNA coding for
p47phox, lacking the 150 N-terminal residues, was reduced by
65% in comparison to cells transfected with full-length
p47phox cDNA (47).
Domain c spans residues 149-159 and is adjacent to the N-terminal
limit of the first SH3 domain. A region extending from residue 151 to
155 was found to be required for O2 production in transfected K562 cells and for binding of the N-terminal SH3 domain of
p47phox to p22phox in vitro and in whole
cells (47, 48). Some authors (5) consider it as merely part of the
N-terminal SH3 domain, and the fact that it was highlighted by peptide
walking supports the contention that it represents an independent
domain.
Domain d spans residues 193-207 and is located in the C-terminal half
of the N-terminal SH3 domain of p47phox. p47phox
contains two SH3 domains (residues 161-212 and 231-281) (49, 50), and
these were found to play a key role in the assembly of the NADPH
oxidase complex (33, 35, 47, 48). It has been reported that the
N-terminal SH3 domain is alone responsible for binding to the
p22phox subunit of cytochrome b559 and
participates in an intramolecular interaction with the C-terminal
proline-rich domain of p47phox (residues 360-370) (48, 51).
Both claims were recently contradicted on the basis of their
reexamination by three binding assays, applied in parallel (52).
Peptide walking revealed a subdomain, within the 60-amino acid long
N-terminal SH3 domain of p47phox, which appears to be of
special importance in NADPH oxidase assembly. SH3 domains are unique
protein-protein signaling motifs, the target of which are proline-rich
sequences, adopting a polyproline type II helix conformation (reviewed
in Ref. 53). SH3 domains make contact with these ligands via three
hydrophobic sites; the first two sites (S1, S2) contain highly
conserved residues interacting with a universal PXXP motif,
whereas the third site (S3) contains nonconserved residues and
determines target specificity. Domain d contains Trp-193 and Pro-206,
two conserved residues found in the S2 site, and Trp-204, a
nonconserved residue in the S3 site. Trp-193 is one of the most
strictly conserved residues in SH3 domains (51); a Trp-193 Arg
mutation resulted in the loss of the ability of the mutant protein to
support O
2 production in the transfected K562 cell (48) and
cell-free (51) models. Also, Pro-206
Leu and Trp-204
Ala
mutations resulted in the total abolishment of O
2 production
in transfected K562 cells. It is significant that peptide 193-207,
which exerted the most potent inhibition of oxidase activation among
peptides of cluster d (94.4 ± 1.8%), also contained all three
essential residues.
Domain e spans residues 253-267 and is located close to the center of
the C-terminal SH3 domain. It was defined on the basis of the moderate
oxidase activation inhibitory activity of a single peptide (253-267).
The domain contains Trp-263, a highly conserved residue in all SH3
domains (54) which forms part of the S2 site. Indeed, K562 cells
transfected with a Trp-263 Arg p47phox exhibit a partial
reduction in O
2 production (48).
It is remarkable that the two SH3 subdomains emphasized by peptide walking contain five out of the seven tryptophans present in p47phox, including three functionally essential tryptophans (Trp-193, Trp-204, and Trp-263). In addition, a sixth tryptophan is located in domain a, discussed above. Recently Quinn and colleagues (55) have reported that treatment of p47phox with oxidase-activating concentrations of SDS or arachidonate, as well as protein kinase C-mediated phosphorylation of p47phox, cause quenching of intrinsic tryptophan fluorescence, indicative of a conformational change. It is tempting to speculate that at least some of the tryptophans situated in the domains revealed by peptide walking are involved in protein-protein interactions related to NADPH oxidase activation.
Domains f (residues 305-319) and g (residues 329-339) were defined by
the oxidase inhibitory activity of two clusters of peptides rich in
basic amino acids and of low hydrophobicity. The boundary separating
the two domains is arbitrary, and it is possible that we are dealing
with a single large domain spanning residues 301-339, containing a
high proportion (38.46%) of basic residues, principally arginine. A
functionally important site within p47phox, encompassing
residues 323-332 (56) or 324-331 (57), was described on the basis of
inhibition, by selected peptides, of O2 generation and
translocation of p47phox to the membrane in a cell-free system.
A region extending from residues 323 to 342 was also identified from a
random phage display library, by biopanning with cytochrome
b559 (58) and p67phox (59), suggesting
that it serves as a binding site in p47phox for both cytochrome
b559 and p67phox, probably at successive
steps in the process of oxidase assembly. Domain f, as such, was not
reported before as an independent, functionally important region, and
whether it represents a distinct domain will have to be established by
additional methods. A p47phox deletion mutant lacking residues
236-350, which includes domains e, f, and g, showed a markedly reduced
capacity to support O
2 production in a cell-free system (60).
The positively charged region extending from residue 303 to 379 also
contains all the serines phosphorylated in the course of phagocyte
activation leading to an oxidative burst (61). The cationic nature of
the peptides inhibiting NADPH oxidase activation, on which definition
of domains f and g was based, raises the issue of a possible
nonspecific, charge-mediated effect. Indeed, we have reported
sequence-unrelated inhibition of NADPH oxidase activation by basic
homopolymers (29). The positive charge of the C terminus of
p47phox is considered to be of functional significance in the
interaction of p47phox with other oxidase components and may
serve as the target for neutralization by phosphorylation in
vivo or anionic amphiphile action in vitro (58, 61).
However, inhibition of NADPH oxidase activation by cluster f and g
peptides appears to be sequence-specific, as indicated by
Michaelis-Menten analysis demonstrating competitive inhibition with
respect to p47phox. Furthermore, earlier work involving the
comparison of the oxidase inhibitory effects of selected peptides,
broadly corresponding to domain g, with peptides containing a similar
proportion of basic residues, showed convincingly that charge was not a
major determinant of inhibition (56-58).
Domain h spans residues 373-387, close to the C terminus of the protein. There is no previous description of this domain in p47phox as being essential for NADPH oxidase activation; thus, p47phox truncated at residue 367 exhibited normal oxidase-activating ability in the cell-free system (60). It has been reported that preincubation of p47phox with an antibody to residues 379-390, or deletion of residues 378-390, leads to the loss of the ability of native p47phox to bind to p67phox (60). It is of potential significance that the domain contains Ser-379, the phosphorylation of which appears to be of key importance for oxidase assembly (62). Also, a peptide partly overlapping domain h (residues 378-390) could serve as a substrate for phosphorylation by protein kinase C and by a kinase present in neutrophil cytosol (63). Therefore, it is possible that the stretch of amino acids surrounding Ser-379 might be an additional target for an activation related conformational change, with LiDS mimicking in vitro the phosphorylation occurring in the intact phagocyte.
The second part of our work describes the use of peptide walking for identifying sites of interaction on p47phox with the cytosolic protein, p67phox. For this purpose, biotinylated p47phox peptides were attached to streptavidin-coated 96-well plates, and p67phox was added in solution. By applying this method, a single p67phox-binding domain was identified, corresponding to residues 357-371 (domain pr). Binding to p47phox peptides was specific for p67phox, amphiphile-independent, and was competed for by intact p47phox protein. No additional p67phox-binding domains were revealed by performing the assay in the presence of the oxidase activating amphiphile, LiDS. The principal candidate, among NADPH oxidase components, for the role of amphiphile target was thought to be p47phox (32, 35, 55), but recent evidence indicates that both p47phox and p67phox are subject to unfolding by anionic amphiphiles (64). This is in agreement with earlier indirect evidence for an effect of amphiphiles on p67phox (32, 65, 66). The design of our binding assay did not allow the investigation of the effect of LiDS on p47phox since p47phox was replaced by a series of peptides which, by definition, represent the "unfolded" state of the molecule. Therefore, the likely target of LiDS in the binding assay would have been the native component, p67phox. Our results, however, show that binding of p67phox to domain pr on p47phox is not dependent on or influenced by amphiphile, indicating that unfolding of p67phox is not a prerequisite for the establishment of this particular bond. Although the effect of amphiphile on p47phox could not be explored in our experiments, results derived from the two-hybrid system indicate that unfolding of p47phox is also not required for exposure of domain pr and binding of p67phox to it (64).
Domain pr, revealed by the binding assay, corresponds closely to the
C-terminal proline-rich motif, shown to interact with the C-terminal
SH3 domain in p67phox (31, 35, 47, 67, 68). This tail-to-tail
interaction is not involved in NADPH oxidase activation in the
cell-free system. Thus, it was shown that deletion mutants of
p47phox, lacking part of the proline-rich domain, were unable
to bind p67phox but were, nevertheless, capable of supporting
O2 production in the cell-free system (60). Reciprocally,
p67phox deletion mutants, lacking the C-terminal SH3 domain and
therefore incapable of binding to the proline-rich domain of
p47phox, were capable of supporting O
2 production in
the cell-free system (64, 68, 69). This explains the fact that domain
pr was not detected by the inhibition of NADPH oxidase activity assay. Our conclusions concerning the lack of involvement of domain pr in
cell-free oxidase activation might not apply to events in the intact
cell. Work in the transfected cell model, indeed, suggests that
tail-to-tail interactions between p47phox and p67phox
might contribute to oxidase activation by optimizing p67phox
recruitment to the membrane (47, 70).
The binding assay, whether performed in the absence or presence of LiDS, did not reveal the binding of p67phox, via its N-terminal proline-rich domain, to the C-terminal SH3 domain of p47phox. This interaction was reported to occur under NADPH oxidase activating conditions (32, 47, 48). A likely explanation for this failure is that binding of the relatively small proline-rich motif, in p67phox, to the large C-terminal SH3 domain in p47phox involves several ligand binding sites, separated by distances exceeding 15 residues and requiring the conservation of secondary structure. The assay also failed to demonstrate binding of p67phox to domain g, identified by inhibition of oxidase activation, and was recently reported as a p67phox-binding region on the basis of random peptide phage-display analysis of p67phox (59). The reason for this failure could not be established.
In conclusion, overlapping biotinylated peptides, spanning the complete p47phox sequence, proved to be effective tools for the identification of domains participating in NADPH oxidase activation. This is shown by the ability of the technique to (a) confirm the existence of domains identified earlier by other methodologies, (b) narrow down the borders of domains described previously, and (c) identify novel domains. The use of peptide walking for the identification of domains in p47phox involved in binding p67phox was only partially successful. Thus, the method failed to detect two domains, identified by other methodological approaches.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. L. Leto (National Institutes of Health, Bethesda) for providing baculoviruses carrying cDNAs for p47phox and p67phox and Rac1 plasmid in E. coli and S. Hanft for word processing.
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FOOTNOTES |
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* This work was supported by the Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, the Israel Science Foundation Grant 10/94, and the David and Natalie Roberts Chair in Immunopharmacology.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.
To whom correspondence should be addressed: Dept. of Human
Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv
69978, Israel. Tel.: 972-3-640-7872; Fax: 972-3-642-9119; E-mail:
epick{at}ccsg.tau.ac.il.
1
The abbreviations used are: O2,
superoxide; Tween 20, polyoxyethylenesorbitan monolaurate; LiDS,
lithium dodecyl sulfate; GTP
S, guanosine
5'-3-O-(thio)triphosphate; PBS, phosphate-buffered saline;
SH3, src homology 3; BSA, bovine serum albumin.
2 Y. Gorzalczany, V. Diatchuk, O. Lotan, A. Toporik, N. Pugach, G. Joseph, and E. Pick, manuscript in preparation.
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REFERENCES |
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