(Received for publication, July 17, 1995; and in revised form, August 25, 1995)
From the
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 p47
binding 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
) 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
production, but had no effect on
phosphorylation. None of the peptides blocked
O
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
production by
electropermeabilized neutrophils, thereby supporting the interpretation
that this region of p47
interacts with
flavocytochrome b.
Human polymorphonuclear leukocytes (PMNs) play an
important role in host defense against invading microorganisms. PMNs
possess an NADPH-dependent oxidase which is capable of generating
superoxide anion (O
) and other
microbicidal oxygen-derived species (e.g. H
O
, 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
.
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
. 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
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.
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.
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
COOH and COOH
NH
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
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.
Figure 2:
Effect of phage-mapped
p47peptides on NADPH oxidase activity in the
broken cell system. Peptides
QRRRQARPGPQSPG
(
),
SRKRLSQDAYRRNS
(
),
and a control peptide KLSYRPRDSNE (
) were added to the broken
cell NADPH oxidase assay system at the indicated concentrations, and
O
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
generation in the broken cell system (Table 1). Peptide 325-330, a hexapeptide including the
putative phosphorylation site at Ser
, did not inhibit
O
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
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
-generating system, and addition of
peptide to the second step (after the components of the oxidase had
assembled) did not inhibit O
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.
As discussed above,
p47 peptides 315-332, 323-332, and
334-347 all inhibited O
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
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 µM
QRRRQARPGPQSPG
(lane 3), the
complete system with 100 µM
SRKRLSQDAYRRNS
(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.
As shown in Table 2, the peptides that
were inhibitory in the broken cell assay system also inhibited
O 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.
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
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
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 production and translocation in the
broken cell system, the p47
peptides
SRKRLSQDAYRRNS
,
AYRRNSVRFL
, and
QRRRQARPGPQSPG
all inhibited
O
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
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
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 (
5
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.