From the Department of Microbiology, Montana State
University, Bozeman, Montana 59717, § Inflammation
Program and Departments of Medicine, Veterans Affairs Medical
Center and University of Iowa, Iowa City, Iowa 52242, ¶ Wells
Center for Pediatric Research, Riley Hospital for Children, Indiana
University School of Medicine, Indianapolis, Indiana 46202, and the
Institute of Tropical Medicine, Nagasaki
852-8523, Japan
Received for publication, July 13, 2000, and in revised form, October 6, 2000
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ABSTRACT |
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Despite extensive experimental and clinical
evidence demonstrating the critical role of flavocytochrome
b558 (Cyt b) in the NADPH-dependent oxidase, there is a paucity of direct
structural data defining its topology in the phagocyte membrane. Unlike
other Cyt b-specific monoclonal antibodies, 7D5 binds
exclusively to an extracellular domain, and identification of its
epitope should provide novel insight into the membrane topology of Cyt
b. To that end, we examined biochemical features of 7D5-Cyt
b binding and used the J404 phage display nonapeptide
library to identify the bound epitope. 7D5 precipitated only
heterodimeric gp91-p22phox and not individual or
denatured Cyt b subunits from detergent extracts of human
neutrophils and promyelocytic leukemia cells (gp91-PLB). Moreover, 7D5
precipitated precursor gp65-p22phox complexes from detergent
extracts of the biosynthetically active gp91-PLB cells, demonstrating
that complex carbohydrates were not required for epitope recognition.
Epitope mimetics selected from the J404 phage display library by 7D5
demonstrated that 226RIVRG230 and
160IKNP163 regions of gp91phox were
both bound by 7D5. These studies reveal specific information about Cyt
b membrane topology and structure, namely that
gp91phox residues 226RIVRG230 and
160IKNP163 are closely juxtaposed on
extracytoplasmic domains and that predicted helices containing residues
Gly165-Ile190 and
Ser200-Glu225 are adjacent to each other in
the membrane.
The phagocyte NADPH oxidase is a plasma membrane redox system that
produces superoxide anion (O Determination of structural and functional aspects of epitopes bound by
specific antibodies can provide information about the protein against
which the antibody is directed (11). Antipeptide and antisubunit
polyclonal antibodies against regions of Cyt b have been
used to locate the corresponding epitopes (12, 13) and information
derived from identification of epitope mimetics has led to the
description of anti-Cyt b "antibody imprints" (14). We
continue to elucidate the structure of Cyt b domains
recognized by monoclonal antibodies to better define its transmembrane
topology and gain insight into its functional organization.
Reported epitope mapping data for monoclonal antibodies (mAbs) specific
for Cyt b indicate that they bind cytosolic aspects of the
protein (15-19). However, it has been reported that mAb 7D5 (19) binds
an extracellular Cyt b epitope on intact neutrophils derived
from normal but not CGD patients that lack Cyt b (20). Thus,
7D5 has proven useful in the determination of Cyt b
up-regulation as an indicator of neutrophil activation and granule
exocytosis (21) and for the identification of individuals deficient in Cyt b (20, 22, 23). However, neither the subunit location nor chemical nature of the 7D5 epitope has been elucidated.
In our current studies, we have used phage display and immunological
analyses to identify the 7D5 epitope on Cyt b. Although we
confirmed the inability of 7D5 to recognize Cyt b on
immunoblots, we found that 7D5 immunoprecipitated detergent-solubilized
Cyt b heterodimer containing its fully processed 91kDa
form of gp91 phox and its 65-kDa precursor. Additionally, it
precipitated the deglycosylated gp91phox core
protein,2 suggesting that
neither the mature nor high mannose-containing carbohydrate contributes
significantly to the epitope. Furthermore, our results indicate that
the 226RIVRG230 and
160IKNP163 segments of gp91phox form
the 7D5 epitope and therefore must be exposed on the cell surface.
These sequences of gp91phox were not bound by 7D5 in the
absence of p22phox nor under conditions that disrupted the
heterodimer, suggesting that, although 7D5 binding is confined to
nonlinear but contiguous regions of gp91phox, it depends on
associated p22phox for its conformational integrity. In
combination, these data provide direct evidence for the identity of two
adjacent transmembrane helices in gp91phox.
Chemicals, Reagents, and Materials--
Prestained protein
molecular weight standards were purchased from Life Technologies, Inc.
Reagents for buffers and bacteriological media and Nunc Maxisorb flat
bottom plates for ELISA were purchased from Fisher. Cyanogen
bromide-activated Sepharose CL-4B and GammaBind Sepharose were
purchased from Amersham Pharmacia Biotech. Sequencing data were
obtained using a Sequenase version 2.0 sequencing kit purchased from
U.S. Biochemical Corp. Unless specified, all other reagents were
purchased from Sigma.
Neutrophil Isolation and Flow Cytometry--
Heparinized, venous
blood was obtained from healthy individuals (or from patients with CGD)
in accordance with a protocol approved by the Institutional Review
Board for Human Subjects at the University of Iowa, and neutrophils
were isolated as described previously using Hypaque-Ficoll gradients
after dextran sedimentation (24). Genetic analyses of the individuals
with either X-linked deficiency of gp91phox or autosomal
deficiency of p22phox were performed by Paul G. Heyworth (The
Scripps Research Institute, La Jolla, CA) and John T. Curnutte
(Genentech, Inc.). The individual with autosomal deficiency of
p22phox (A220) had a nucleotide replacement at
position C354 (C354 Cyt b ELISA--
35 µl of relax buffer (10 mM
Hepes, 100 mM KCl, 10 mM NaCl, pH 7.4)
containing 8 pmol of heparin-purified Cyt b (determined by
spectral absorbance at 414 nm, extension coefficient = 21.6 mM Immunoprecipitation of Cyt b--
Following treatment with 4 mM diisopropylfluorophosphate (Sigma) for 15 min on ice,
5 × 106 human PMNs were washed and then resuspended
in 200 µl of lysis buffer (Tris-buffered saline, pH 7.5, containing
0.1 mg/ml pepstatin A, 0.1 mg/ml leupeptin, 1.0% Triton X-100, 0.5%
cetyltrimethylammonium bromide, and 2.0 mM
phenylmethylsulfonyl fluoride). Cells were sonicated at level 10 for
10 s using a probe sonicator (Heat Systems Inc., Farmingdale, NY)
and then incubated on ice for 10 min. Lysates were precleared using
Pansorbin Cells (Calbiochem) as described previously (26) and then
divided into two 100-µl aliquots. For nondenaturing
immunoprecipitations, one of the aliquots was diluted to 1.2 ml with
dilution buffer (50 mM Tris-HCl, pH 7.4, 190 mM NaCl, containing 2.5% Triton X-100). 10-15 µg of monoclonal
antibody, 7D5, or those specific for gp91phox (54.1 ( Antibody and Phage Display Epitope Mapping--
The generation
of monoclonal antibody 7D5 has been described previously (19), and the
production of the J404 nonapeptide phage display library was reported
(28). 7D5 was purified from spent RPMI 1640 media of a hybridoma cell
line using GammaBind Sepharose (Amersham Pharmacia Biotech) according
to the manufacturer's instructions, and purity was assessed by
SDS-PAGE. Mapping of 7D5 with the phage display library and plaque lift
analyses were carried out as described (16), except the
affinity-selected clones were amplified following each round of
selection by replicating as plaques on a lawn of K91 cells (instead of
as K91 colonies on LB agar containing 75 µg/ml kanamycin).
Immunoblots of Phage-displayed Sequences--
5 × 1010 plaque-forming units of phage produced as described
above were disrupted with SDS loading buffer at 100 °C for 5 min and
loaded onto a 5-20% SDS-PAGE gel (29) to separate capsid proteins.
Following transfer to nitrocellulose, the immunoblot was probed with 5 µg/ml 7D5 and detected by goat anti-mouse alkaline phosphatase-conjugated secondary (Bio-Rad) in combination with chromagen reagent (Kirkegaard and Perry Laboratories, Inc.,
Gaithersburg, MD) as described (16).
Specificity of mAb 7D5--
7D5 has been previously reported to
recognize Cyt b expressed at the plasma membrane of
neutrophils but not on cells from X-linked CGD individuals (19, 30).
The absence of either gp91phox or p22phox in X-linked
or autosomal recessive CGD, respectively, results in the absence of
both proteins on the neutrophil membrane (5, 31). Thus, to
rule out the possibility that 7D5 binding is dependent on
transcriptional regulation of only gp91phox and not
p22phox, we compared its binding to cells derived from
homozygous and heterozygous individuals with autosomal and X-linked
inheritance of the disease. Intact neutrophils derived from individuals
deficient in gp91phox (X910) or p22phox
(A220) were probed for surface expression of Cyt
b using 7D5 in flow cytometry (Fig.
1, A and B). In
contrast to neutrophils from normal individuals, 7D5 did not stain the
cell surface of neutrophils deficient in either p22phox or
gp91phox (Fig. 1, A and B). Neutrophils
from the mother and female siblings of an individual with X-linked CGD
displayed bimodal fluorescence, demonstrating that they have both
normal and Cyt b-deficient neutrophils. The identification
of heterogeneous Cyt b expression in their neutrophils
indicates that they possess one copy of a mutant CYBB allele
and therefore are carriers for X-linked CGD (Fig. 1B). Approximately 87% of the neutrophils from the mother and 80% from the
daughter shown in Fig. 1B reduced nitro blue tetrazolium to formazan, indicating that those cells possessed normal
O
To demonstrate further the specificity of immunoreactivity of 7D5 to
Cyt b, human neutrophil Cyt b was purified from
cell membranes as described (38) and bound to 96-well plates. Probing the immobilized Cyt b with 7D5, anti-p22phox mAb
44.1 ( 7D5 Recognizes Native Cyt b Heterodimers--
To gain insight into
the structural nature of the 7D5 epitope, we compared nondenaturing
with denaturing conditions for immunoprecipitation of Cyt b using 7D5.
Using nondenaturing conditions, 7D5,
To determine whether 7D5 bound complex carbohydrates on
gp91phox, we examined the ability of 7D5 to precipitate Cyt
b using biosynthetically active gp91-PLB cells as a source
of both precursor and mature Cyt b (Fig. 3B).
Using nondenaturing conditions, 7D5 precipitated gp91phox,
p22phox, and a small amount of gp65, the gp91phox
precursor that has exclusively high mannose oligosaccharides (Fig.
3B). Since 7D5 precipitated gp65, the complex carbohydrates of gp91phox were not required for 7D5 binding to Cyt
b. These findings were confirmed by the ability of 7D5 to
precipitate the heterodimeric complex consisting of 55-58-kDa core
gp91phox protein-p22phox synthesized in the presence of
tunicamycin2 and also bind heterodimer following digestion
with PNGase F (data not shown). Consistent with the neutrophil
experiments, when gp91-PLB lysates were denatured with 1% SDS and
heat, 7D5 precipitated neither mature subunit nor gp65. In contrast,
Our previous studies on the biosynthesis of Cyt b
demonstrated that pools of uncomplexed gp65 and p22phox
accumulate for a limited time following synthesis in gp91-PLB cells
(26). Therefore, these cells provide a source of native, monomeric Cyt
b subunits from which to test whether 7D5 recognizes individual gp65 or p22phox subunits. Sequential
immunoprecipitations were performed to determine whether 7D5 was
capable of depleting lysates of Cyt b heterodimer and/or
individual subunits (Fig. 4A).
Although 7D5 completely removed gp91-p22phox heterodimer from
lysates after three rounds of immunoprecipitation, monomeric gp65 could
be subsequently precipitated from those lysates using Epitope Mapping Using Phage Display--
To identify whether the
7D5 epitope was located on gp91phox, p22phox, or both
subunits, we selected phage display library clones using a 7D5
immunoaffinity Sepharose bead matrix. Limiting dilutions were performed
on each of three successive eluate samples, to determine the titer and
to provide isolated plaques for plaque lift analysis. A six-log
increase in the number of adherent clones was observed between the
first and third round of selection, suggesting strong enrichment for
peptide sequences by the antibody (data not shown). About 15% of the
plaques from the second round and 95% of the plaques from the third
round elution gave strong signals when probed by 7D5 in a plaque lift
analysis (14), compared with an irrelevant monoclonal (data not shown).
Isolated plaques were then selected for nucleotide sequence analysis as
described (16) (Table I).
Immunoaffinity selection of peptides presented on phage display clones
produced 29 unique amino acid sequences, many of which appeared on
several different phage (Table I). The first phage sequence listed in
Table I showed a five-residue match to the 226RIVRG230 segment of gp91phox, a
region predicted to be extracytoplasmic by hydropathy analysis and by
its proximity to the Asn240 putative glycosylation
site. The recovery of this single RIVRGVGGI peptide
thus provided important evidence supporting the
226RIVRG230 segment of gp91phox as
being part of the epitope (Table I, clone A). In addition to containing
similarity to the 226RIVRG230 segment of
gp91phox, several other phage sequences selected by
7D5, including YKNPWIRGM, LKNPWQRGD,
LPNPWVRGD, and NANPWSRGF, suggested a match to
161KNP163 of gp91phox as
well (Table I, clones B, C, D, and F).
The 161KNP163 region of
gp91phox lies above a predicted transmembrane region 12 residues from Asn149, another possible glycosylation
site, and 63 residues from the 226RIVRG230
segment, just above the fifth predicted transmembrane-spanning domain
(12, 39). Modest matches in several other selected clones (Table I)
also reflected the 161KNP163 segment of
gp91phox. More than 50% of the selected phage peptides
contained an aliphatic or hydrophobic residue, including Leu, Ile, Val,
or Tyr, which when aligned with the
161KNP163 and
226RIVRG230 segments of gp91phox,
corresponded to Ile160 (Table I). Most impressively, these
two regions of gp91phox were represented by four phage peptides
with five-residue identities and additional conservatively substituted
or shifted residues (clones A-D, Table I). In our previous studies,
mapping antibody epitopes or protein-protein interactions, such
extended matches were rare (16, 40). When phage peptides were aligned
with the gp91phox segments 161KNP163
and 226RIVRG230, Trp was represented in
the same position in greater than 91% of the total number of phage
isolates (Table I). Although no residue in the identified
gp91phox sequences fits with this selected residue,
Trp125 immediately outside of the third transmembrane
region of gp91phox is a possible candidate, since all of these
transmembrane regions are likely to be in close proximity.
Trp251 could also be represented by the phage-selected
sequences, yet we were unable to block the binding of 7D5 in flow
cytometry with another antibody that binds this residue (data not
shown). It is also possible that Trp68 of p22phox
contributes the tryptophan in the epitope identified in the phage display mapping and could therefore provide some rationale for the
heterodimer requirement for epitope conformation. Another possibility
is that the Trp selected by the phage clones represented a hydrophobic
"pocket" or "spacer," which bridged the gap between the
extracellular transmembrane loops containing
161KNP163 and
226RIVRG230 sequences.
Several phage sequences also contained an RGD tripeptide motif, which
aligned well within the gp91phox residues
226RIVRGQ231, if flexibility is
provided to allow for a polar residue substitution at
Gln231. The unexpected identification of RGD in several
selected sequences suggested that this surface-accessible region of Cyt
b might interact with integrins in an
RGD-dependent manner (41), yet our attempts to confirm such
an interaction were unsuccessful (data not shown).
Our mapping data indicate that the minimal epitope bound by 7D5
consists of five mapped residues in the
226RIVRG230 segment, and four more matching the
160IKNP163 region. These two regions are likely
to be extracellular based on hydropathy analysis that suggests they are
located immediately adjacent to the extracellular aspect of two
putative membrane-spanning helices (39) (see Fig. 6 for putative
epitope location). The combination of these two regions constitutes a
logical target for binding by 7D5, based on our findings from the
biochemical assays, i.e. the ability of the antibody to
identify an accessible epitope on the plasma membrane of intact
neutrophils combined with its inability to bind denatured protein.
The binding of 7D5 to the selected peptides on the denatured pIII
display protein (42) from 5 × 1010 plaque-forming
units was examined by SDS-PAGE and immunoblotting (Fig.
5, immunoblot). These signal
intensities varied significantly with the displayed sequence, with
YPGWGRNDA and YPGWPRKDL sequences (clones Z and BB, respectively)
producing the strongest signals, the clones containing cysteine pairs
(clones U and W) showing weak signals, and the rest showing
intermediate staining. An irrelevant clone not selected by 7D5 gave no
detectable signal (Fig. 5B). These findings demonstrate that
the selected peptides exhibited varied degrees of binding to 7D5, and
the binding presumably represents the extent to which each denatured
clone was able to represent the Cyt b epitope. The binding
by 7D5 to clones A and B, which have five residues identical to the
putative epitope, were bound less by 7D5 than at least three other
clones with fewer identical residues (Fig. 5, immunoblot;
compare clones A and B with clones Z, BB, and M). This result suggested
that some structure in the displayed epitope mimetic was lost by
denaturation.
Since 7D5 required Cyt b in its native conformation to bind,
the binding of 7D5 to SDS-denatured phage clones may have represented conditions less than optimal to determine which was the best epitope mimetic. Therefore, we analyzed the ability of various intact 7D5-binding phage clones to block the interaction of 7D5 with purified
Cyt b in an ELISA (Fig. 5, bar graph).
Of the eight phage representatives tested, clones M and A (LNTKWLRGD
and RIVRGVGGI, respectively) were most effective at blocking the
interaction of 7D5 with Cyt b in this assay. Both clones
have much greater linear homology to the putative gp91phox
epitope than do clones Z and BB (YPGWGRNDA and YPGWPRKDL,
respectively), which had greater reactivity with 7D5 on the immunoblot
(Fig. 5). Moreover, clone B, which had relatively weak reactivity with 7D5 on the immunoblot despite its high similarity to the putative epitope, showed much greater interaction with 7D5 in the ELISA. The
difference in immunoreactivity between the immunoblot and the ELISA
probably represents the difference in the way each clone is presented
to 7D5; the immunoblot presents denatured protein to 7D5, whereas the
ELISA promotes interactions that reflect those between native proteins.
Our finding that 7D5 required native Cyt b for binding is reflected
most by the ELISA data, which demonstrated that clones of higher
similarity reacted with 7D5 better in native conformation than in
denatured form.
In response to difficulties in predicting the structure of
membrane proteins, epitope mapping of antibodies that bind native protein provides an alternative means of assigning localized structure to individual protein domains, as well as elucidating certain membrane
topology. The apparent complex nature of the 7D5 epitope suggested that
its characterization could provide information about Cyt b
structure beyond localization of regions outside the plasma membrane.
mAb 7D5 has been previously utilized in reports describing molecular
and genetic analysis of CGD and the NADPH oxidase, yet the region(s) of
Cyt b bound by 7D5 have remained undefined. To provide a
better view of the 7D5 epitope and to gain structural information about
Cyt b membrane topology, we investigated the 7D5-Cyt
b interaction to identify which regions of Cyt b
are recognized by the antibody.
Our analysis of 7D5 binding first established its specificity for the
Cyt b protein. 7D5 bound neither to neutrophils from patients with autosomal deficiency of p22phox nor to those from
patients with X-linked CGD. Flow cytometric 7D5 staining of neutrophils
from female carriers of X-linked CGD was bimodal, consistent with the
mosaic expression of Cyt b in such subjects (43). The use of
mAb 7D5 to identify carriers of CGD as well as affected individuals
constitutes an important yet simple screening test for the genetic
types associated with impaired Cyt b expression. Specificity
of 7D5 for Cyt b was further supported by the reactivity of
7D5 to partially purified Cyt b in an ELISA.
7D5 precipitated heterodimeric gp91-p22phox under nondenaturing
conditions but recognized neither individual subunit when Cyt b was
denatured. Anti-p22phox and anti-gp91phox mAbs, Because 7D5 appeared to bind a polypeptide region on Cyt b,
we used phage display epitope mapping to identify residues of Cyt
b involved in 7D5 binding. The phage-displayed sequences
show similarity to both 160IKNP163 and
226RIVRG230 regions of gp91phox, yet no
matches to p22phox sequences could be identified. The diversity
of the 29 selected sequences suggests that the 7D5 epitope involves a
nonlinear or conformational epitope, consistent with the inability of
7D5 to recognize denatured protein on immunoblots. Several phage
clones, A-D from Table I, gave five-residue identities to the
discontinuous region spanning 160IKNP163 and
226RIVRG230 of gp91phox. Such strong
similarity provides credible support to the identification of this
region as the 7D5 epitope, especially because these epitope mimetics
were recovered from a randomly generated peptide library (14, 16, 40).
Moreover, clone A, one of the clones with five residues identical to
the gp91phox sequence, interacted with 7D5 in the native ELISA
better than all clones tested except one (Fig. 5). Tryptophan was also
recovered in nearly every phage clone (Table I), although this residue does not appear in either of the two gp91phox regions
identified. It is possible that this tryptophan represents a residue
from another membrane-spanning helix of gp91phox or a
hydrophobic pocket, potentially bridging
160IKNP163 and
226RIVRG230 of gp91phox. A subset of
four phage clones (Z, AA, BB, and CC, each beginning with YPGW; Table
I) are listed in the reverse orientation (carboxyl to amino, left to
right, respectively) compared with the other clones in Table I. These
clones are unique because 1) they fit the consensus best if written in
reverse order, 2) clones Z and BB were most strongly recognized by 7D5
in immunoblot (Fig. 5), and 3) a synthetic peptide analog comprising
part of clones Z and BB
(NH2-ADNRPWGPYGP-CONH2) was the only
synthetic peptide sequence found to compete with the binding of 7D5 to
immobilized Cyt b in ELISA, albeit only at an
EC50 of 1 mM (data not shown). The ability of
the clones to display differential reactivity in the immunoblot
versus the ELISA is probably due to the complex nature of
the epitope; i.e., the sequences displayed by the phage are
the best linear representatives of an epitope that requires tertiary structure.
This selection of apparent reverse sequences by mAb 7D5 suggests that
this antibody can select epitope mimetics that are not always in the
same orientation as natural epitope. We previously observed the
recovery of some peptides from the J404 phage display library that fit
the consensus if listed in reverse order (40, 46, 47), and the
cross-reactivity of antibodies with retropeptides has been specifically
addressed (48). We also observed that the reverse sequence of a
bioactive peptide shows significantly greater effects than a peptide
bearing a randomly chosen sequence (49). An interaction between the
bacterial ribonuclease barnase and its natural inhibitor barstar, are
also reported to be "strong and relatively insensitive to ideal
geometry" when compared with the interaction between the enzyme and
the nucleotide substrate (50). These findings support the rationale for
our observations that antibodies can interact with peptides by
identifying critical side chain charge distribution and hydrophobicity,
in a way that is not entirely dependent on peptide backbone orientation.
The findings from the immunological analyses and phage display are
summarized in a schematic model of the topology and identity of the 7D5
epitope contained within two separate extracellular regions of
gp91phox (Fig. 6). Previous
attempts to elucidate Cyt b structure, including mapping of
extracellular versus intracellular domains have been based
on hydropathy analyses and enzymatic cleavage experiments (12),
structural homology to known proteins in combination with computer
modeling (39, 51, 52), and identification of the binding regions of
p47phox (40, 53-56) and NADPH and FAD (25, 54, 57, 58)).
Imajoh-Ohmi et al. (12) found that antibodies raised against
gp91phox residues 150-172 stained intact neutrophils, leading
the authors to conclude that that region is exposed on the cell
surface. Our data not only extend those findings but demonstrate that
gp91phox residues 160IKNP163 and
226RIVRG230 are juxtaposed on the
extracytoplasmic face of the plasma membrane (Fig. 6), providing direct
evidence for the identity and positioning of two previously predicted
transmembrane regions (12, 39). Moreover, these results are compatible
with the findings of Wallach et al., which demonstrated that
gp91phox residues Asn132, Asn149, and
Asn240 are glycosylated and, therefore, exposed on the cell
surface (59).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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2), an essential precursor for
other reactive oxygen metabolites critical for
oxygen-dependent microbicidal activity (1-3). A genetic
lesion affecting any one of four of the oxidase components,
gp91phox, p22phox, p47phox, or p67phox,
results in defective oxidase activity and the inability of
phagocytes to kill pathogenic microorganisms, a disorder clinically
recognized as chronic granulomatous disease
(CGD)1 (2, 4-9). Human
neutrophil flavocytochrome b558 (Cyt
b) is a heme-containing, heterodimeric integral membrane
protein composed of subunits gp91phox and p22phox (10).
Cyt b is the electron transferase of the NADPH oxidase, relaying electrons from bound NADPH within the cell to an oxygen acceptor region of Cyt b on the exterior aspect of the cell
membrane where O
2 is formed. In this functional capacity, Cyt
b has been established as an essential component of the
respiratory burst oxidase, although little published experimental data
describe its topology in the membrane.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A), which
resulted in the replacement of Ser118 with Arg. The
individual with deficiency of gp91phox (X910) had a
frameshift in exon 11, resulting in the replacement of nine nucleotides
with eight. Surface-expressed Cyt b was detected using 7D5 by flow
cytometry as described previously (21). Samples were analyzed on a
FACscan flow cytometer (Becton Dickinson, San Jose, CA) at the
University of Iowa Core Flow Cytometry facility. For most experiments,
unfixed neutrophils were used, and a single live gate eliminated debris
and contaminating cells. However, when patient cells were shipped
overnight, it was sometimes necessary to use propidium iodide staining
in combination with an additional gate to further exclude dead cells
from the analysis.
1
cm
1) (25), in 2% octyl glucoside was used to
coat each well of a 96-well Corning Maxisorb ELISA plate overnight at
4 °C. Rinsing and blocking were performed as described (14), except
a different blocking buffer (Hanks' balanced salt buffer with 10 mM HEPES, pH 7.4, 0.5% bovine serum albumin) was used. Cyt
b was probed for 1 h at 25 °C with 80 µl of the indicated mAb
at a concentration of 3 µg/ml. To measure the ability of the intact
phage clones to block the binding of 7D5 to immobilized Cyt
b, the antibody was diluted to 3 µg/ml and exposed to
2.5 × 1012 plaque-forming units for 30 min 25 °C.
Pretreated 7D5 was then exposed to the immobilized Cyt b for
4 h. The reactivities of the mAbs for the Cyt b were determined by
probing the wells with goat anti-mouse secondary antibody conjugated
with horseradish peroxidase as described above.
gp91))
and p22phox (44.1 (
p22)) or an IgG1 control mAb
(Cappel, Organon Teknika Co., Durham, NC) was added to the diluted
lysate and rotated at 4 °C for 4 h. Subsequent steps were
performed as described previously (26). For denatured lysates, the
100-µl aliquot was made 1% SDS, heated to 100 °C for 3-4 min,
and then iced immediately for 5 min. Lysate was then diluted to 1.2 ml
with dilution buffer so that SDS concentration was 0.1%, and
precipitation was performed as described for nondenaturing
immunoprecipitations (above). Alternatively, Cyt b was
precipitated from solubilized gp91-PLB cells, a described previously
human promyelocytic leukemia cell line constitutively expressing
gp91phox (27), using 7D5,
gp91, and
p22. Cyt b
was precipitated from gp91-PLB or a human promyelocytic leukemia cell
line with the CYBB gene deleted (XCGD-PLB) as reported
earlier (26), and denaturing conditions were as described above.
Following 5-20% SDS-PAGE, immune complexes were immunoblotted using a
combination of
gp91 and
p22. Immunoblots were developed using an
enhanced chemiluminescence detection system (SuperSignal Substrate;
Pierce) according to the manufacturer's instructions.
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ABSTRACT
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DISCUSSION
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2-generating capacity. A second daughter also had similar
lyonization with 74% nitro blue tetrazolium-positive neutrophils (not
shown). That three females within the same family would have similar
degrees of lyonization may reflect familial nonrandom X-chromosome
inactivation, which has been identified in other X-linked disorders
(32-37). The percentage of their neutrophils with normal
O
2-producing capability correlated well with the amount of 7D5
binding shown by flow cytometry: e.g. 81.1% of the
neutrophils in the daughter with 80% nitro blue tetrazolium-positive
cells stained with 7D5 (Fig. 1B). These findings indicate
that 7D5-epitope recognition required surface expression of both
gp91phox and p22phox. Moreover, these
results illustrate how 7D5 can be used to identify Cyt
b-deficient individuals or those who are carriers for
X-linked CGD.
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Fig. 1.
Identification of female carriers of
gp91phox deficiency or individuals deficient in
gp91phox or p22phox by flow cytometry.
A, neutrophils (106) from healthy subjects
(father, mother, and normal daughter as indicated) or from an
individual with autosomal deficiency of p22phox
(A220) were probed with 7D5 and analyzed by flow cytometry
as described under "Experimental Procedures." B, a
similar analysis was performed on neutrophils from an individual with
X-linked CGD (X910) or from the mother and female sibling
of an individual with X-linked CGD (mother and daughter carriers as
indicated) and were compared with a healthy individual (normal as
indicated). Dashed histograms represent staining
with IgG1, an isotype control antibody.
p22) (14), anti-gp91phox mAb 54.1 (
gp91) (14), and
an irrelevant mAb suggested that the epitope bound by 7D5 was intact on
the detergent-solubilized protein (Fig.
2). Although the reactivity of 7D5 in the
ELISA was less than that of
p22 or
gp91, all three mAbs
specifically recognized Cyt b. It is possible that the
binding of mAb 7D5 to Cyt b requires a more native or
membrane-resident conformation of the protein than does either
p22
or
gp91, although we were unable to test this directly using our
ELISA.
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Fig. 2.
Analysis of 7D5-Cyt b
interaction by ELISA. One µg of heparin-purified human
neutrophil Cyt b was used to coat wells in an ELISA plate so
that binding by each of three anti-Cyt b mAbs could be determined.
gp91 and
p22 were previously shown to be Cyt
b-specific (14) and were used as positive controls, and the
anti-rhodopsin mAb K42.41 (60) served as a negative control. The
results indicate specific binding of 7D5 to the detergent-solubilized
form of the protein. These data are typical of five separate
analyses.
gp91, and
p22 precipitated
both gp91phox and p22phox from human neutrophil
detergent lysates (Fig. 3A).
When the lysates were denatured with heat and 1% SDS,
gp91 and
p22, binding linear regions of the protein, precipitated
their respective subunits alone. However, 7D5 precipitated neither
subunit after denaturation (Fig. 3A).
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Fig. 3.
Immunoprecipitation (IP) of
Cyt b using 7D5. A, PMNs were
solubilized in lysis buffer, and Cyt b was precipitated with
the indicated antibody using denaturing (DEN) or
nondenaturing (ND) conditions as described under
"Experimental Procedures." Following SDS-PAGE and transfer to
nitrocellulose, subunits of Cyt b, gp91phox and
anti-p22phox, were detected with combined use of gp91 and
p22. Murine IgG1 was used as an isotype control. IgG Hch
and IgG Lch indicate positions of the heavy and light
chains, respectively, of immunoglobulin. Results shown are
representative of three separate experiments. B, gp91-PLB or
X-CGD PLB cells (2 × 106) were solubilized in
radioimmune precipitation buffer, and Cyt b was precipitated
from lysates using mAb 7D5 or
gp91 and
p22 as indicated.
Immunoprecipitations were carried out using nondenaturing conditions or
after lysates were heated to 100 °C in the presence of 1% SDS to
denature all proteins. Following SDS-PAGE, proteins were transferred to
nitrocellulose, and immunoblots were probed with a combination of
gp91 and
p22. Results are representative of three separate
experiments.
gp91 and
p22 precipitated their respective subunits following
denaturation (Fig. 3B). These findings suggest that 7D5
recognized the Cyt b peptide backbone in its native form
only but do not elucidate whether the antibody bound to an epitope
shared by both subunits or if it associated with individual
gp91phox or p22phox subunits in their native conformation.
gp91 (Fig.
4A, arrowheads). Since neither gp91phox
nor p22phox coprecipitated with gp65 using
gp91 following
three rounds of precipitation with 7D5, gp65 was precipitated free of
associated p22phox. The finding that gp65 but neither
gp91phox nor p22phox coprecipitated with
gp91 after
the immunodepletions with 7D5 suggests that heterodimeric
gp91phox-p22phox complexes were completely removed by
7D5 (Fig. 4A). The inability to precipitate and detect by
immunoblotting monomeric p22phox following the three
immunodepletions with 7D5 or those with 7D5 followed by a single
depletion with
gp91 may be due to the rapid degradation of
uncomplexed p22phox or its rapid processing to heterodimeric
form (26) (Fig. 4A). It is likely that the amount of newly
synthesized, monomeric p22phox present in the depleted lysates
may have been below the limits of detection by immunoblotting.
Therefore, we pulse-labeled gp91-PLB cells and subsequently chased for
2 h to allow for partial processing of gp65 to gp91phox
and also to allow the formation of gp91phox-p22phox
complexes (Fig. 4B). As with the unlabeled immunodepletion
experiment, three rounds of precipitation of radiolabeled Cyt
b using 7D5 completely removed
gp91phox-p22phox complexes from lysates (Fig.
4B). In contrast, following depletion with 7D5, subsequent
precipitation with either
gp91 or
p22 revealed that both gp65 and
p22phox remained in depleted lysates free of their
complementary subunit (Fig. 4B). These results suggest that
7D5 precipitated only gp91phox-p22phox or
gp65-p22phox heterodimers.
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Fig. 4.
Immunodepletion of
gp91phox-p22phox and gp65-p22phox using
7D5. A, gp91-PLB cells were solubilized in radioimmune
precipitation buffer, and lysates were immunodepleted using three
successive 7D5 precipitations and subsequently subjected to
precipitation using either gp91 or
p22 as indicated. Following
SDS-PAGE, proteins were transferred to nitrocellulose, and immunoblots
were probed with a combination of anti-gp91phox and
anti-p22phox mAbs. The arrowheads indicate gp65.
B, alternatively, gp91-PLB cells were pulse-labeled with
[35S]methionine for 1 h and then chased 2 h
with unlabeled methionine. Cells were solubilized in radioimmune
precipitation buffer, and lysates were immunodepleted using three
successive 7D5 precipitations followed by either
gp91 or
p22 as
indicated. The arrowhead indicates gp65, and the
line at the left indicates a protein of 60 kDa
unrelated to gp65. We have previously demonstrated that precipitation
of radiolabeled p22phox by
p22 results in uninterpretable
signal due to high background above 50 kDa on autoradiograms (26).
However, this background signal is not due to gp65 or gp91phox.
The panels at the far right in both
A and B illustrate immunoprecipitation
(IP) of gp65/91phox and p22phox by
gp91
and
p22, respectively. Following SDS-PAGE, gels were processed for
autoradiography. Results are representative of two or three separate
experiments.
Peptide sequences selected on a 7D5 immunoaffinity matrix
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Fig. 5.
Immunoreactivity of 7D5 with selected
phage-displayed sequences by ELISA and immunoblotting. Intact
phage display clones showed variable blocking of the binding of 7D5 to
immobilized Cyt b by ELISA. Each well of the ELISA plate was
coated with Cyt b as described for Fig. 2. Following the
pretreatment of 7D5 with the indicated phage clones bearing the unique
sequences listed below as described under "Experimental
Procedures," the binding of 7D5 to the wells was measured. For these
phage clones, immunoblotting (lower section of
the figure) was used to illustrate the immunoreactivity of
7D5 for the denatured form of the phage pIII fusion protein bearing the
selected peptide sequence. Each lane in the
immunoblot represents 5 × 1010 plaque-forming units
of phage clones following separation in the reducing and denaturing gel
conditions. Sequences displayed on the phage clones for both the ELISA
and immunoblot are as follows: clone A (RIVRGVGGI), clone X
(GWIKYRLEG), clone Z (YPGWGRNDA), clone BB (YPGWPRKDL), clone B
(YKNPWIRGM), clone M (LNTKWLRGD), clone U (FRCSWCRGE), clone W
(GECRWCKGD), unselected clone (unsel.), or none. The pIII
capsid protein fused to the unique peptides typically migrates as a
68-kDa protein in these gel conditions. The blot was probed with 3 µg/ml 7D5, and immunoreactive phage peptides were detected using
alkaline-phosphatase conjugated secondary antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
p22
and
gp91, respectively, which bind linear epitopes (16), also
precipitated heterodimeric gp91-p22phox. In contrast to 7D5,
p22 and
gp91 precipitated individual p22phox and
gp91phox subunits after Cyt b had been denatured.
The inability of 7D5 to precipitate either individual subunit following
denaturation of Cyt b is consistent with its failure to recognize
either on an immunoblot. Furthermore, 7D5 recognized neither native
monomers of gp65 nor p22phox, suggesting that individual
subunit conformation alone was not sufficient for binding. These
results suggest two possible explanations for the requirements of 7D5
binding: 1) that both subunits contribute to the epitope or 2) that the
epitope conformation is dependent upon heterodimer assembly, although
the residues constituting the epitope reside on only one of the
subunits. In addition to gp91phox and p22phox, 7D5
precipitated gp65, the ER-resident precursor containing only high
mannose carbohydrate (Figs. 3A and 4, A and
B) (44), which is later modified in the Golgi to include
complex carbohydrates to form gp91phox (45). Moreover, Yamauchi
et al.2 have demonstrated that the
unglycosylated gp91phox core protein can be precipitated by 7D5
from gp91-PLB cells cultured in the presence of tunicamycin, and our
previous studies indicate that the unglycosylated gp91phox core
protein associates with p22phox to form an unglycosylated
heterodimer (26). Together these data indicate that carbohydrate did
not contribute significantly to the 7D5 epitope.
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Fig. 6.
A hypothetical model for the 7D5 epitope and
membrane topology of Cyt b. The epitope was
mapped to two juxtaposed extracytoplasmic regions of gp91phox,
residues 160IKNP163 and
226RIVRG230, represented by
blue and green spheres,
respectively. These residues are predicted by hydropathy analysis of
the protein to exist immediately extracellular to the fourth and fifth
membrane-spanning helices. Residues identified in yellow
lettering were represented by the sequences selected from
the phage display library and thus accessible to 7D5 on the surface of
the cell. The underlined glutamine residue represents polar
residue conservation for the epitope in that position although it was
not represented by selected sequences. Although tryptophan was almost
always found between sequences representing
160INKP163 or
226RIVRGQ231 in individual phage clones, a
tryptophan residue does not exist in this vicinity of the protein.
Therefore, the black W represents the position of
this residue as selected by phage clones.
Our mapping data suggest that the epitope bound by 7D5 was located
entirely on gp91phox, but the presence of p22phox in
the gp91-p22 heterodimer was required for 7D5 binding on human myeloid
cells. It should also be noted that heme insertion is a prerequisite
for heterodimer formation (26) that in turn is essential for
recognition by 7D5. To the extent that heterodimer formation requires
heme coordination by one or both subunits, the 7D5 epitope is at the
least indirectly heme-sensitive. Thus, p22phox, possibly in
combination with heme, appears to impart a structural constraint to
gp91phox that is essential for recognition by 7D5. Further
structural analysis will be necessary to elucidate the influence of
p22phox on the membrane topology and processing of
gp91phox and the possible role of heme in this process.
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ACKNOWLEDGEMENTS |
---|
We also acknowledge the expert technical assistance of Justin K. Fishbaugh and B. E. Hoess at the University of Iowa Core Flow Cytometry Facility.
![]() |
FOOTNOTES |
---|
* This work was performed during the tenure of the following American Heart Association awards: postdoctoral fellowships 9704584S (to J. B. B.) and 9920491Z (to F. R. D.) and Scientist Development Grant 30156N (to J. B. B.). This work was supported in part by Public Health Service Grants RO1 AI 26711 (to A. J. J.), HL 45635 (to M. C. D.), RO1 AI 34879, and HL53592 (to W. M. N) and a Merit Review Award (to W. M. N.) from the Department of Veterans Affairs.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.
These authors contributed equally to this work.
** To whom correspondence should be addressed: 109 Lewis Hall, Dept. of Microbiology, Montana State University, Bozeman, MT 59717. Tel.: 406-994-4811; Fax: 406-994-4926; E-mail: umbaj@gemini.oscs.montana.edu.
Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M006236200
2 A. Yamauchi, L. Yu, A. J. G. Pötgens, F. Kuribayashi, H. Nunoi, S. Kanegasaki, D. Roos, H. L. Malech, M. C. Dinauer, and M. Nakamura, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CGD, chronic granulomatous disease; Cyt b, flavocytochrome b558; mAb, monoclonal antibody; ELISA, enzyme linked immunosorbent assay.
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