©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Topological Mapping of Neutrophil Cytochrome b Epitopes with Phage-display Libraries (*)

James B. Burritt , Mark T. Quinn (1), Mark A. Jutila (1), Clifford W. Bond , Algirdas J. Jesaitis (§)

From the (1)Departments of Microbiology and Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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, 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.

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 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 gp91inhibited 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) .

The protein sequence of the carboxyl-terminal half of gp91shows 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.

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.


MATERIALS AND METHODS

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.

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.

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) .


RESULTS

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.

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 gp91epitope. 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).

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.


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.




DISCUSSION

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(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) .

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 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.

Our results repeatedly showed that mAb 54.1 failed to bind cytochrome b on intact or saponin-permeabilized cells. Hydropathy predictions suggest the 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) .

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 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).

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 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.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant 5RO1 AI 26711, National Science Foundation EPSCOR Grant RII-891879, Council For Tobacco Research Grant 2918R2 (to A. J. J.), an Arthritis Foundation Biomedical Science Grant and National Institutes of Health FIRST Award AR 20929 (to M. T. Q.), and Public Health Service Grant DMB 900058P to the Pittsburgh Supercomputing Facility. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 406-994-2901; Fax: 406-994-4926.

The abbreviations used are: phox, phagocyte oxidase; mAb, monoclonal antibody; FACS, fluorescence-activated cell scanner; FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis.

J. B. Burritt, F. R. DeLeo, K. W. Doss, M. T. Quinn, C. W. Bond, and A. J. Jesaitis, unpublished data.


ACKNOWLEDGEMENTS

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.


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