From the
Cytochrome b of human neutrophils is the central
component of the microbicidal NADPH-oxidase system. However, the
folding topology of this integral membrane protein remains
undetermined. Two random-sequence bacteriophage peptide libraries were
used to map structural features of cytochrome b by determining
the epitopes of monoclonal antibodies (mAbs) 44.1 and 54.1, specific
for the p22
The NADPH-oxidase system of neutrophils is a host-defensive
plasma membrane redox system that produces superoxide anion
(O)(1, 2) , which subsequently is converted to a variety
of other toxic oxygen species that kill invading microbes and cause
damage to tissue(3) . Humans lacking this enzyme system are
unable to produce neutrophil-generated superoxide and suffer recurrent
bacterial infections, granulomatous lesions of multiple organs, and
early death (4). This condition was first reported in
1957(5, 6) , and is known as chronic granulomatous
disease(3, 4, 7) .
Cytochrome b (also known as flavocytochrome b, cytochrome b
A number of studies have provided
information about cytochrome b native structure. Electron
microscopy and immunochemical analysis were used to localize cytochrome b in the neutrophil and eosinophil(16, 17) . In
one of these studies, we found that the epitopes of cytochrome b containing the amino acid residues
The protein
sequence of the carboxyl-terminal half of gp91
These ambiguities in structure indicate that
additional approaches for determining the topology of this protein are
required. In this study, we have identified the epitopes bound by two
monoclonal antibodies that recognize a specific subunit of cytochrome b using random peptide phage-display libraries. In addition,
we present data relating the accessibility of these epitopes on native
cytochrome b to their respective antibodies.
Following electrophoresis, protein samples were transferred to
nitrocellulose as described previously(8) . Monoclonal
antibodies 44.1 and 54.1 were diluted to 2 µg/ml in diluting buffer
(3% (v/v) goat serum, 1% (w/v) BSA, 0.2% (v/v) Tween 20, 0.1% (w/v)
thimerosal in PBS) and incubated with separate regions of the blot for
1 h at room temperature with continuous rocking. The blot was developed
using alkaline phosphatase-conjugated goat anti-mouse IgG and a
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium phosphatase
substrate system.
To determine the structure of immunogenic surface regions of
cytochrome b and acquire additional information about its
membrane topology, monoclonal antibodies were produced against the
Triton X-100-solubilized wheat germ agglutinin and
heparin-Ultrogel-purified cytochrome b protein(8) .
Hybridoma supernatants were screened for neutrophil-specific
IgG-producing clones that recognized intact or saponin-permeabilized
neutrophils and either subunit of heparin-purified cytochrome b. Numerous clones were identified, and two were chosen that
recognized either the light or heavy chain of cytochrome b.
Epitope mapping of cytochrome b was carried out using these
cytochrome b-specific antibodies and phage random
peptide-display library
technology(25, 27, 33, 34) . FACS and
immunosedimentation analysis were then used to confirm accessibility of
the epitope on native cytochrome b to antibody, and placement
of the epitope relative to the plasma membrane. Finally, confirmation
of the specificity of the mAb for both the cytochrome b subunit and the phage peptide-bearing pIII protein selected by the
mAb was demonstrated by Western blot analysis.
Binding of mAb 44.1 and 54.1 to both the specific
cytochrome b subunit and phage pIII protein confirmed the
presence of a similar epitope on each. In addition, mAbs did not bind
the pIII protein on phage displaying irrelevant peptides in the random
region. Therefore, the sequence of the peptide alone, expressed in the
variable region simulates the natural epitope recognized by the mAb.
Although the variable peptide of the selected phage may not represent
the complete and exact epitope, it clearly contains the residues
sufficient for recognition by the mAb.
In order to determine the
accessibility of the identified epitopes to mAb on native cytochrome b in the cell, FACS analysis was performed on purified
leukocytes. While saponin-permeabilized cells stained strongly with mAb
44.1 at 50 µg/ml (Fig. 5, traceB), intact
cells show background staining when incubated with either mAb 44.1 (Fig. 5, traceA) or an irrelevant mAb (data
not shown). A similar background level of staining intensity was noted
when cells were permeabilized and stained with FITC-conjugated
secondary antibody only (Fig. 5, traceC).
Histogram A of Fig. 5shows a sample of cells that were incubated
with mAb 44.1 but not previously saponin-permeabilized. The majority of
the cells of this histogram show background fluorescence, but a small
population stains as strongly as the saponin-permeabilized cells shown
in histogram B. This small population of highly fluorescent cells in
histogram A probably represents cells with membranes inadvertently
damaged and permeabilized during preparation, as this small group was
also found to stain strongly with propidium iodide (data not shown).
Propidium iodide stains cellular DNA and was used in the final wash of
all cells to indicate cells with permeabilized membranes.
Because prospects for x-ray crystal or NMR solution
structures of cytochrome b are currently limited, modeling of
protein structures must be carried out within constraints imposed by
identification of surface domains and functional and structural
features. Epitope mapping using random phage-display libraries offers
another tool to aid in determining surface features of proteins. We
have used two such libraries to identify the epitopes recognized by
each of two cytochrome b-specific mAbs and characterized the
accessibility of these epitopes to mAb binding on the native protein.
The unique peptides expressed on phage obtained following the third
round of selection were compared to the cytochrome b amino
acid sequences, and regions of remarkable similarity were identified.
Each mAb was then characterized according to its ability to bind either
denatured, spectrally active but detergent-solubilized cytochrome b, or native cytochrome b in intact and
saponin-permeabilized neutrophils. This information was then used to
help predict if a particular region of cytochrome b is exposed
to antibody on the cytosolic or external surface of the neutrophil. Our
epitope mapping data suggest the epitope recognized by mAb 44.1
includes the amino acids 181-188 of p22
Bacteriophage epitope mapping exploits the
specificity of the monoclonal antibody and the unique sequence of the
peptide expressed on selected phage. When mAbs that recognize linear
epitopes are used, this technique allows identification of protein
epitopes with little ambiguity. Clearly, not all mAbs recognize linear
epitopes; thus, the approach may not be universally applicable.
However, it is conceivable the information might be obtained in those
cases that support epitopes corresponding to different regions in the
same molecule, split by a fold or invagination of the peptide
backbone(43, 44) .
Phage selected by mAb 44.1 suggest
this mAb may be able to recognize some sequences without regard to the
orientation of the peptide backbone. Phage 2 of Fig. 2, selected
by mAb 44.1, expresses the sequence PRVQIL, which contains five of the
residues found in PQVRPI, four of which are in reverse order. The
ability of this mAb to recognize an epitope may involve recognition of
exposed amino acid side chains and charge placement (45) rather
than the stereospecific alignment of side chains on the peptide
backbone. This result supports our recent finding that the reverse
sequence of certain synthetic peptides mimicking the structure of the N-formyl peptide chemoattractant receptor (FPR) retain a
diminished, but clearly measurable inhibitory activity in
reconstitution of FPR-G-protein complexes in detergent
solution(46) .
To gain information about the accessibility of
the mAb to the native epitope, FACS analysis was used with
saponin-permeabilized and intact neutrophils. This analysis strongly
suggests the
Our results repeatedly showed that mAb 54.1
failed to bind cytochrome b on intact or saponin-permeabilized
cells. Hydropathy predictions suggest the
The epitope recognized by mAb 54.1 appears
to be less accessible than the epitope recognized by mAb 44.1, as
determined by immunoprecipitation (data not shown). This information
supports our immunosedimentation data (Fig. 8), further
suggesting that the
Rap1A was
found to be present in immunoprecipitates of both mAbs 44.1 and 54.1.
This result suggests the epitopes recognized by the respective mAbs are
probably not blocked by the interaction with Rap1A. The
We are grateful to Dr. George P. Smith (University of
Missouri, Columbia, MO) for helpful discussions, hexapeptide phage
library material, and host E. coli strains; and Steven Cwirla
(Affymax Research Institute, Palo Alto, CA) for helpful information
regarding ligation of degenerate oligonucleotides and additional host E. coli strains. We thank Craig Johnson (Montana State
University) for making synthetic peptides, Cindy Bozic (Macromolecular
Resources, Fort Collins, CO), and Vladimir Kanazin and Patrice Mascolo
(Montana State University) for synthetic oligonucleotides. We
appreciate the technical assistance of Robert Rath (Montana State
University) in the preparation of selected figures.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and gp91
cytochrome b chains, respectively. The unique
peptides of phage selected by mAb affinity purification were deduced
from the phage DNA sequences. Phage selected by mAb 44.1 displayed the
consensus peptide sequence GGPQVXPI, which is nearly identical
to
GGPQVNPI
of p22
.
Phage selected by mAb 54.1 displayed the consensus sequence
PKXAVDGP, which resembles
PKIAVDGP
of gp91
. Western blotting demonstrated
specific binding of each mAb to the respective cytochrome b subunit and selected phage peptides. In flow cytometric analysis,
mAb 44.1 bound only permeabilized neutrophils, while 54.1 did not bind
intact or permeabilized cells. However, mAb 54.1 immunosedimented
detergent-solubilized cytochrome b in sucrose gradients. These
results suggest the
GGPQVNPI
segment of
p22
is accessible on its intracellular surface,
but the
PKIAVDGP
region on
gp91
is not accessible to antibody, and
probably not on the protein surface.
, cytochrome b
, and
cytochrome b
) is the central redox
component of the phagocyte NADPH-oxidase system of human neutrophils.
This component is a heterodimeric integral membrane protein composed of
91-kDa (gp91
)(
)
and
22-kDa (p22
) subunits(8, 9) . At
least two heme groups are coordinated by these subunits(10) ,
and FAD and NADPH binding activities have been
demonstrated(11, 12, 13) . The primary structure
of gp91
includes two asparagine-linked
glycosylation sites (9) and contains five possible transmembrane
regions suggested by hydropathy analysis(14, 15) . p22
contains three possible
transmembrane regions, one of which includes a His-94 residue,
conserved between species, that probably coordinates one of the
cytochrome b heme irons.
KQSISNSESGPRG
of gp91
and
EARKKPSEEEAAA
of
p22
are surface-accessible epitopes of native
cytochrome b(16) . Rotrosen et al. (18) found that synthetic peptides corresponding to the carboxyl
terminus of gp91
inhibited NADPH-oxidase
activation in electrically permeabilized cells, and antipeptide
antibodies directed against this region prevented superoxide formation
in a cell-free system. In addition, p22
contains a proline-rich region in the carboxyl-terminal
tail, which may provide Src homology domain binding sites, for
p47
or
p47
/p67
complexes(19) . These data suggest functional roles
for the carboxyl termini of both subunits, which are presumed to occupy
cytosolic locations. Initial analysis of the folding topology of
cytochrome b has been reported by Imajoh-Ohmi et
al.(20) , who determined accessibility of the subunits to
anti-peptide antibodies and proteolytic enzymes. Two regions of
gp91
were exposed to proteolytic enzymes on the
outer surface of the cell, while p22
was not
found to be sensitive to such external proteolysis(10) . The
carboxyl termini of both subunits were accessible to antibody on the
internal surface of the plasma membrane(20) .
shows some similarity to other NAD(P)H-oxidoreductases (12, 21) such as ferredoxin NADP reductase, a
flavoprotein for which the crystal structure is known. Studies by Pick
and co-workers (22) have shown that cytochrome b binds
FAD and can function as a superoxide generating NAD(P)H-oxidase, even
without added cytosolic constituents normally required for superoxide
production in other cell-free systems (23, 24). These results have
prompted speculation that cytochrome b is the only electron
transporting component of the NADPH oxidase and that its nucleotide
binding domains may resemble ferredoxin reductase (12, 13) or other redox proteins. However, the
structural studies by Imajoh-Ohmi et al. suggest that major
portions of the putative nucleotide binding domains are extracellular.
This contention is also supported by the evidence of Umei et al. (47-49) suggesting that the putative NAD(P)H binding
component of the oxidase is present in neutrophils of normal and
chronic granulomatous disease patients and is thus not part of
cytochrome b.
Reagents
Diisopropyl fluorophosphate, Tween 20,
SDS, acrylamide, bisacrylamide, ammonium persulfate, TEMED,
Hank's solution, FITC-labeled goat anti-mouse IgG, bovine serum
albumin (BSA), and Histopaque were purchased from Sigma. Prestained
protein molecular weight standards were purchased from Life
Technologies, Inc. Western blots were developed with anti-mouse or
anti-rabbit immunoglobulin purchased from Bio-Rad, and
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium chromogen
was purchased from Kirkegaard and Perry Laboratories, Inc.
(Gaithersburg, MD). Cyanogen bromide-activated Sepharose CL-4B was
purchased from Pharmacia Biotech Inc. Sequencing data were obtained
using a Sequenase version 2.0 sequencing kit purchased from U. S.
Biochemical Corp.
Epitope Library and Bacterial Strains
Two random
phage-display libraries were used in this study. A hexapeptide
phage-display library and Escherichia coli strains K91 and
MC1061 were kindly provided by Dr. George P. Smith (25) (University of Missouri, Columbia, MO), and a nonapeptide
phage-display library was produced in our
laboratory.()
Affinity Purification
Affinity purification
of phage bearing epitopes bound by mAbs was performed as follows; 5
10
phage (5 µl) from the hexapeptide phage
peptide-display library (25) or 1
10
phage
(75 µl) from the nonapeptide library were combined with 1.0 ml of
Sepharose beads conjugated with 4 mg of either mAb 44.1 or 54.1. The
beads were mixed with the phage at 4 °C for 16 h by gentle
inversion. The mixture was then loaded into a 5-ml plastic column
barrel (Evergreen), and unbound phage were removed by washing with 50
ml of phage buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.5% Tween 20 (v/v), 1 mg/ml BSA). Bound phage were eluted
from the column with 2.0 ml of eluting buffer (0.1 M glycine,
pH 2.2), and the pH of the eluate was neutralized immediately with four
drops of 2 M Trizma base(27) . The titer of phage
(nonapeptide library) was determined for each column eluate by plaque
assay according to standard procedures(28) . The column matrices
were preserved for reuse in second and third round affinity
purifications by washing with 10 ml of PBS, pH 7.0, followed by 3.0 ml
of PBS containing 0.02% sodium azide. The column was stored at 4 °C
until the next affinity purification and was prepared for reuse by
rinsing with 20 ml of phage buffer prior to mixing with amplified
phage. As a control for antibody-specific selection, one column was
prepared without antibody bound to the beads; all steps of affinity
purification of phage were carried out on this control column, and a
sample of the resulting phage was sequenced.
Phage Amplification
Eluate phage were amplified in
host K91 ``starved'' E. coli cells, which were
prepared as described previously (27, 29) to maximize
phage attachment and infection. The entire volume of the first eluate,
minus a small amount used for titering, was added to 200 µl of
starved K91 cells and incubated 15 min at room temperature without
shaking. Two ml of LB broth (30) with tetracycline at 0.2
µg/ml or kanamycin at 0.75 µg/ml (depending on the library
used) was added to the cells, which were then incubated with aeration
at 37 °C for 45 min. The infected cells were spread with a sterile
glass rod on the surface of LB agar containing tetracycline (40
µg/ml) or kanamycin (75 µg/ml) in a sterile 9 24-inch
glass baking dish. After 24-48 h of incubation at 37 °C, the
antibiotic-resistant colonies were suspended in 25 ml of TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) by gentle scraping with a
bent glass rod. Phage were harvested from the culture as
described(27) , and diluted in 1.0 ml of phage buffer and
allowed to mix with the column matrix, which was also resuspended in 1
ml of phage buffer. These steps were repeated so that a total of three
purifications and two amplifications were used to select and amplify
adherent phage from the library.
Sequencing Phage
One hundred µl of phage from
the final column eluate were used to infect starved K91 cells as
described above. Serial 100-fold dilutions of infected cells were used
to inoculate LB agar plates containing the appropriate dilution, which
were then incubated overnight at 37 °C. Isolated colonies were used
to inoculate 2 ml of 2 YT (30) containing the
appropriate antibiotic, and the minipreps were incubated overnight at
37 °C in a shaking water bath. DNA from the isolated phage was
prepared and sequenced according to the directions of the Sequenase
version 2.0 kit (U. S. Biochemical Corp.). An oligonucleotide primer
with the sequence 5`-GTT TTG TCG TCT TTC CAG ACG-3` was used to
determine the nucleotide sequence of the unique region of the phage,
and autoradiographs were viewed using a Molecular Dynamics model 400E
PhosphorImager.
Large Scale Purification of Selected Phage
Some
phage bearing hexapeptides of interest (Fig. 2, sequences 3 and
27-29) were propagated by infecting 750 µl of mid-log phase
K91 cells with 20 µl of phage supernatant saved from sequencing
minipreps. The phage were produced in a method similar to
amplifications described above, except the infected cells were grown in
100 ml of 2 YT with 40 µg/ml tetracycline, and the purified
phage were resuspended in 400 µl of TBS.
Figure 2:
Hexapeptide and nonapeptide sequences
selected on mAb affinity matrices. Plaques of phage isolated from mAb
44.1 Sepharose (A) and mAb 54.1 Sepharose (B) were
used to inoculate individual cultures and phage DNA samples prepared
from the cultures were sequenced as described under ``Materials
and Methods.'' Both hexapeptide and nonapeptide sequences are
shown, and the region of cytochrome b matching the consensus
phage peptide sequence is indicated. Phage amino acid residues
demonstrating exact matches to the cytochrome b amino acid
sequence are indicated by boldlettering. Some phage
peptide sequences (3, 7, 24, 27, 29, and 44) were multiply recovered.
These phage sequences represent results of two separate
experiments.
Neutrophil Preparation and FACS Analysis
Human
neutrophils were purified from citrated blood using Histopaque
gradients as described by Boyum(31) . Purified cells were
incubated on ice for 15 min with 2 mM diisopropyl
fluorophosphate to inhibit serine proteases. 5 10
cells were used for each sample to determine antibody binding by
FACS analysis. Some cell samples were permeabilized on ice for 10 min
by adding 500 µl of saponin solution (0.01% saponin, 0.1% gelatin
in Dulbecco's PBS) and pelleted by centrifugation as before.
Permeabilized cells were incubated on ice for 30 min with 80 µl of
the primary antibody (usually 50 µg/ml in saponin solution), then
washed once with 3.0 ml of saponin solution, centrifuged to collect,
and resuspended in 80 µl of the FITC-conjugated goat anti-mouse
antibody diluted 1:150 in saponin solution, and incubated on ice for 30
min. Permeabilized cells were washed once with 3.0 ml of saponin
solution containing propidium iodide at 10 µg/ml, pelleted as
before, and resuspended in 500 µl of FACS buffer (Dulbecco's
PBS containing 10% rabbit serum). In separate experiments, 100 µl
of mAb 44.1 at 10 µg/ml was incubated at 37 °C for 30 min with
2
10
transducing units (27) phage expressing
phage sequence 3 or 27 (Fig. 2) prior to incubation with the
saponin-permeabilized cells to determine if the phage-expressed peptide
could compete with the natural epitope for binding by the mAb. Control
samples in all experiments consisted of cells not incubated with either
primary or secondary mAb, cells not incubated with primary mAb, and
cells incubated with both an isotype-matched primary mAb and the
labeled secondary mAb. Staining of some samples of intact cells was
performed as above without treatment with saponin solution.
Fluorescence intensity of the FITC-labeled cells was determined on a
Becton Dickinson FACScan model FACS analyzer with a
15-milliwatt argon-ion laser using CONSORT 30 and LYSYS software
according to the manufacturer's directions.
Western Blotting
Immunoaffinity-purified phage
bearing peptides resembling a region of cytochrome b were
isolated and grown as indicated above. Approximately 5
10
purified phage in 20 µl of TBS were heated in a
boiling water bath for 5 min with an equal volume of SDS sample buffer
(3.3% (w/v) SDS, 167 mM Tris-Cl, pH 6.8, 33% (v/v) glycerol,
0.03% (w/v) bromphenol blue, 0.035% (v/v) 2-mercaptoethanol).
Heparin-Ultrogel-purified cytochrome b, prepared as
described(8) , was combined with an equal volume of SDS sample
buffer without heating. Protein samples were separated by SDS-PAGE at
room temperature on 12% (w/v) polyacrylamide gels as
described(8, 10) , and electrophoretic mobility of
sample proteins were compared to prestained protein standards.
Cytochrome b Immunosedimentation
Purified
cytochrome b (10 µg) was diluted in 100 µl of relax
buffer (8) containing 50 µg of mAb (44.1, 54.1, irrelevant,
or none) and then incubated overnight at 4 °C. The pretreated
cytochrome b was then loaded onto a 1.36-ml continuous
5-20% sucrose gradient in relax buffer and centrifuged at 53,000
RPM in a Beckman TLS-55 rotor for seven hours at 4 °C. The gradient
was manually fractionated into 12 120-µl samples from the top. A
20-µl sample of each fraction was separated by SDS-PAGE and
transferred to nitrocellulose membranes as described above. Cytochrome b was detected by Western blot using a rabbit
anti-p22 polyclonal primary antibody. Western
blots were digitized and quantitated using an image analysis system as
described(32) .
Identification of Monoclonal Antibody Epitopes
To
identify the amino acid sequence of the epitopes of cytochrome b recognized by the reactive mAbs, a nonapeptide phage-display
library capable of binding to the mAbs was created. Using this library,
mAb-binding epitopes were selected from a collection of 5
10
unique nine-residue sequences of all 20 amino acids. The
epitopes thus mimic the original immunogenic cytochrome b epitope. By sequencing the relevant region of the phage genome,
the original cytochrome b epitope was deduced(35) .
Confirmation of the epitope selection was achieved using a second
epitope library kindly provided by George P. Smith at the University of
Missouri, Columbia(27) . Three cycles of immunoaffinity
purification and amplification were used to select phage expressing
peptides bound by either mAb 44.1 or 54.1. Fig. 1shows selection
and amplification of phage bound by mAb columns. An increase of about 5
logs was observed for the population of binding phage for each mAb, and
about 3 logs for phage that interacted with the control column without
mAb.
Figure 1:
Selection
of phage from a phage-display library by affinity chromatography. M13
phage expressing random nonapeptides were incubated with Sepharose
beads conjugated with mAb 44.1 (), 54.1 (
), or a sham matrix
containing no antibody (
). Adherent phage numbers (plaque-forming
units) were determined in samples of each column eluate by plating
appropriate dilutions on a lawn of late log phase K91 cells and
determining phage number as described under ``Materials and
Methods.''
The random insert region was sequenced in 34 phage selected by
mAb 44.1 from the nonapeptide library and 27 from the hexapeptide
library as described under ``Materials and Methods.'' Fig. 2shows 33 of the 37 sequences (89%) from the nonapeptide
library, which exhibited an obvious consensus pattern matching a region
of cytochrome b shown in Fig. 3. This consensus peptide
sequence, GGPQVXPI, closely resembles GGPQVNPI
of p22
(Fig. 3A). The remaining four sequences did
not resemble any region of cytochrome b or each other (data
not shown). Nineteen of the 27 sequences (70%) from the hexapeptide
library showed similarity to the same cytochrome b epitope,
and the same nucleotide sequence coding for the PQVRPI peptide was
recovered in 17 of the 19 cases. The remaining eight hexapeptide
sequences showed no consensus (data not shown). Phage expressing the
hexapeptide sequences PQVRPI, FKRGVD, LRRGID, and PKGAYD (Fig. 2;
sequences 3 and 27-29, respectively) were isolated and propagated
for further study by Western blot and FACS analysis.
Figure 3:
Location of similarity between
antibody-selected phage sequences and primary structure of cytochrome b. The deduced amino acid sequences of the cytochrome b subunits, p22 and gp91 are shown with the identified epitopes boxed and putative transmembrane regions underlined.
Phage selected
by mAb 54.1 expressed the consensus amino acid sequence
PKXAVDGP (the GP is adjacent in the constant pIII region in
all phage except phage sequence 34), which is similar to PKIAVDGP
of gp91
(Fig. 3B). All phage sequenced that were
selected from each library by mAb 54.1 suggested a match to this
putative gp91
epitope. Peptides of phage
selected on the column without antibody suggested no match to
cytochrome b, or to each other (data not shown).
Immunological Analysis
Western blotting analysis
was used to show specificity of the mAb for both the cytochrome b subunit and the unique hexapeptide expressed on the phage. As
shown in Fig. 4, mAb 54.1 specifically recognized
gp91 (laneA), which migrates
between the 68- and 97-kDa molecular size markers, and a 20-kDa
proteolytic fragment. The appearance of this 20-kDa immunoreactive
fragment can be enhanced in cytochrome b samples treated with
V8 protease (10). This mAb also bound to the phage pIII protein
expressing the FKRGVD peptide (Fig. 2, sequence 27), which
migrates at about 64 kDa (laneC). mAb 54.1 also
recognized pIII proteins of phage expressing the LRRGID and PKGAYD
peptides (Fig. 2, sequences 28 and 29, respectively) by Western
blot with equal intensity (data not shown). Phage expressing the PQVRPI
peptide (Fig. 2, sequence 3) was not recognized by mAb 54.1 (laneB), confirming the specificity of mAb 54.1 for
the former sequences.
Figure 4:
Specificity of mAb for cytochrome b and phage pIII proteins. Intact phage expressing known amino acid
sequences (lanesB, C, E, and F) and partially purified cytochrome b (lanesA and D) were separated on SDS-PAGE
polyacrylamide gels and transferred to nitrocellulose as described
under ``Materials and Methods.'' Samples were Western blotted
with mAb 54.1 (lanes A-C) or mAb 44.1 (lanesD-F). LanesB and F contain proteins of phage sequence 3 (PQVRPI), and lanesC and E contain proteins of phage sequence 27
(FKRGVD). Results were confirmed by four separate
experiments.
As seen in Fig. 4, mAb 44.1 bound to a
band migrating at 22 kDa in laneD (p22) and a less intense band at 44 kDa
(subunit dimer). The pIII protein of phage containing the PQVRPI
peptide (Fig. 2, sequence 3) was also recognized by mAb 44.1 (laneF), but not the pIII protein of phage
expressing the FKRGVD peptide (Fig. 2, sequence 27) in laneE. The pIII protein migrates at an apparent molecular
mass of 64 kDa. This mobility appears slow, considering the size of the
protein is 406 amino acids(36) ; however, our migration rate
compares favorably with other reported PAGE mobilities for this protein
(37-40).
Figure 5:
FACS analysis of neutrophils stained with
mAb 44.1. Intact (A) or saponin-permeabilized human
neutrophils (B and C) were incubated with either 50
µg/ml primary antibody 44.1 and a secondary FITC-conjugated
antibody (A and B) or the secondary antibody only (C). Cells were then analyzed for fluorescence intensity using
a Becton Dickinson FACScan model FACS analyzer as described
under ``Materials and Methods.'' The results were
reproducible in four separate experiments using neutrophils from
different donors.
To
demonstrate that this binding was competed by a phage-expressed
epitope, intact phage expressing the PQVRPI peptide (Fig. 2,
sequence 3) were used to pretreat mAb 44.1 and block binding of the mAb
to the GGPQVNPI
epitope of
p22
. Cells incubated with 10 µg/ml mAb 44.1
pretreated with phage (Fig. 6, traceA) showed
a 10-fold reduction in staining intensity compared to cells incubated
with the same concentration of untreated mAb (Fig. 6, traceB). No inhibition occurred if mAb 44.1 was pretreated
with the same concentration of phage expressing the irrelevant peptide,
FKRGVD (Fig. 2, sequence 27) (data not shown). In contrast,
intact and permeabilized cells displayed background staining when
incubated with mAb 54.1 (Fig. 7, traces A and B, respectively), as did permeabilized cells stained with the
secondary FITC-labeled antibody only (Fig. 7, traceC). Although a small shift in staining intensity was seen
if the cells stained by mAb 54.1 were first permeabilized (Fig. 7, traceB), this shift was also seen in
permeabilized cells incubated with an irrelevant primary mAb (data not
shown) or with labeled secondary mAb only ( Fig. 5and 7, traceC). This population of cells staining at a low
intensity probably represents residual fluorescent label present within
the cells following washing.
Figure 6:
Specific phage block mAb 44.1 binding to
permeabilized neutrophils. Neutrophils were incubated with mAb 44.1
following pretreatment of the antibody with 2
10
phage expressing the peptide PQVRPI (A) or with mAb 44.1
not pretreated with phage (B). Cells were then exposed to
FITC-conjugated secondary antibody as described under ``Materials
and Methods,'' and the effect of the phage-displayed PQVRPI
peptide on mAb 44.1 staining was determined as a function of reduction
in cell fluorescence intensity. Results were confirmed in two separate
experiments.
Figure 7:
FACS analysis of neutrophils stained with
mAb 54.1. Intact (A) or saponin-permeabilized human
neutrophils (B and C) were incubated with either 50
µg/ml primary antibody 54.1 and a secondary FITC-conjugated
antibody (A and B) or the secondary antibody only (C). Cells were then analyzed for fluorescence intensity using
a Becton Dickinson FACScan model FACS analyzer as described
under ``Materials and Methods.'' The results were
reproducible in four separate experiments using neutrophils from
different normal human donors.
Because a mAb generally recognizes the
cognate epitope on native antigen, the inability to detect staining
with mAb 54.1 by FACS analysis was surprising. However, since the
original immunogen was detergent-solubilized and partially purified
cytochrome b, the mAb may have been generated in response to
an epitope accessible only in this form of the protein. To determine
whether mAb 54.1 binds to heparin-purified, spectrally active
cytochrome b, a rate-zonal immunosedimentation analysis in
detergent-containing sucrose gradients (41) was performed.
Purified cytochrome b (10 µg) was pretreated overnight at
4 °C with mAb 54.1, 44.1, an irrelevant mAb, or no mAb, as
described under ``Materials and Methods.'' Following
centrifugation on a 5-20% Triton X-100-containing sucrose
gradient and fractionation, Western blots were performed on samples
from each fraction using a rabbit polyclonal antibody specific for
p22(16) . Densitometry was used to
measure relative amounts of cytochrome b in each fraction,
which was then plotted as a function of fraction number (Fig. 8).
Untreated cytochrome b and a sample exposed to an irrelevant
mAb sedimented to a position in the gradient compatible with its 5.6 S
sedimentation coefficient(41) . When treated with mAb 44.1, the
entire sedimentation profile was shifted from fractions 4 and 5 to
fraction 8. Cytochrome b treated with mAb 54.1 displayed a
bimodal distribution with a new peak with a sedimentation coefficient
of 10 S or greater, suggesting that a majority (>50%) of detergent
soluble cytochrome b is recognized by the mAb. Since
approximately 27% of the cytochrome b remains unaffected when
pretreated with mAb 54.1 over a range of 50-500 µg/ml (data
not shown), the result suggests that part of the population of
cytochrome b has a sequestered epitope, inaccessible to mAb
54.1. Immunoprecipitation of cytochrome b using mAbs 44.1 and
54.1 confirmed the above immunosedimentation data, as
immunoprecipitation of cytochrome b from 2% octyl glucoside
extracts of neutrophil membranes by mAb 54.1 was only half as effective
as immunoprecipitation by mAb 44.1 (data not shown). In addition, Rap1A
was found to be associated with the cytochrome b complexes (42) immunoprecipitated by both mAbs 44.1 and 54.1 (data not
shown).
Figure 8:
Immunosedimentation of mAbs
Detergent-solubilized cytochrome b. Heparin-Ultrogel-purified
human cytochrome b was incubated with mAb 44.1 (), 54.1
(
), irrelevant (
), or no mAb (
) overnight at 4 °C
and sedimented in a 5-20% sucrose gradient as described under
``Materials and Methods.'' The relative amount of cytochrome b in each fraction of the gradient was detected with a rabbit
anti-p22 polyclonal antibody by Western blot analysis. Signal
intensities were measured as described previously (32) and plotted for
each fraction number. Results were confirmed by three separate
experiments.
(
GGPQVNPI
) and mAb 54.1 binds amino
acids 382-389 of gp91
(
PKIAVDGP
). The data include a
number of unique phage sequences from each library to support the
identification of the epitope for each mAb. Moreover, data obtained
from the two libraries are complementary. An excellent example that
shows agreement between the libraries is the similarity of hexapeptide
3 to nonapeptides 4 and 5 (Fig. 2). The three phage-displayed
peptides were selected by mAb 44.1 from two different libraries and
express the sequence PQVRPI. The chance recovery of two identical
six-residue peptides is 1/32
, or about 1 in 1
billion(25) .
GGPQVNPI
epitope of
p22
is accessible on the cytosolic but not
external aspect of the plasma membrane in neutrophils. The possibility
that the epitope is resident, but masked on the external surface of the
cell and subsequently made accessible during permeabilization, is
unlikely. Neighboring regions have been shown to be accessible to mAb
in ``slam-frozen'' molecular distillation dried cells (16) and freeze-thaw permeabilized cells(20) . These
regions overlap a p22
domain clearly shown to
be involved in interaction with cytosolic p47
of the NADPH-oxidase system. Together, these data suggest
that the epitope bound by mAb 44.1 is made accessible because of
membrane permeabilization, and not because of disruption and subsequent
exposure of the cytochrome b epitope by the action of saponin
on the molecule itself. In addition, they strongly support the
conclusion that this region of cytochrome b must be on the
surface of the molecule.
PKIAVDGP
region of gp91
may exist on an external loop between the fourth and fifth
putative transmembrane regions, assuming a five membrane-spanning
domain model of cytochrome b(9) . The work of
Imajoh-Ohmi (20) supports this notion and identified an
extracellular papain cleavage site in the region of the epitope. Their
results, however, are incompatible with the proposed structure of the
gp91
NADPH and flavin-binding
regions(11, 12, 13, 47, 48, 49) .
Because mAb 54.1 binds denatured gp91
on
Western blots, but does not bind native cytochrome b in either
intact or permeabilized neutrophils, the intracellular or extracellular
location of
PKIAVDGP
still remains to be
confirmed. The epitope, however, must retain a conformation that is
either not recognized by the mAb or is in a sterically unfavorable
environment. This region has a charge and lies in the middle of a
relatively hydrophilic sequence and is thus not likely to be integrated
in the plasma membrane. Since solubilized, partially purified
cytochrome b is immunosedimented by mAb 54.1, it is a greater
possibility that accessibility to this area of the protein is blocked
by unknown inter- or intramolecular associations. Possible candidates
are other NADPH-oxidase components, including Rap1A since it is known
to dissociate from cytochrome b in sucrose gradient
separations(50) .
PKIAVDGP
region of
gp91
may be sensitive to the association with
another cytosolic component. According to this view of the assembled
complex, another NADPH-oxidase subunit or accessory group could
sterically block or disrupt the conformation of the epitope on native
cytochrome b in permeabilized and intact neutrophils. Varying
dissociation of the component from cytochrome b during
purification would explain accessibility of the epitope on some, but
not all of the detergent-solubilized or heparin-Ultrogel-purified
cytochrome b molecules (Fig. 8). Complete separation of
the component from cytochrome b under denaturing conditions
could account for strong staining of gp91
by
mAb 54.1 on Western blot, which is similar to the level of staining of
p22
by mAb 44.1 (Fig. 4).
PKIAVDGP
epitope of mAb 54.1 might,
however, be affected by the presence of accessory nucleotides. This
epitope region lies between, and close to (<45 amino acids) proposed
binding sites for both FAD and NADPH, as suggested by sequence
similarities with other nucleotide-binding
proteins(12, 13) . Further studies by this method of
epitope mapping, with activated cytochrome b or additional
mAbs specific for other regions of cytochrome b may provide
sufficient structural evidence to design a more accurate model for
neutrophil cytochrome b. Additionally, screening phage
libraries with protein components of the NADPH-oxidase system may
identify protein regions involved in forming the active NADPH-oxidase (26) and suggest a molecular architecture for the phagocyte
oxidase system.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.