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The Journal of Immunology, 2004, 173: 7349-7357.
Copyright © 2004 by The American Association of Immunologists

Site-Specific Inhibitors of NADPH Oxidase Activity and Structural Probes of Flavocytochrome b: Characterization of Six Monoclonal Antibodies to the p22phox Subunit1

Ross M. Taylor*, James B. Burritt*, Danas Baniulis*, Thomas R. Foubert*, Connie I. Lord*, Mary C. Dinauer{dagger}, Charles A. Parkos{ddagger} and Algirdas J. Jesaitis2,*

* Department of Microbiology, Montana State University, Bozeman, Montana 59717; {dagger} Departments of Pediatrics and Medical and Molecular Genetics, Indiana University Medical Center, Indianapolis, IN 46202; and {ddagger} Department of Pathology and Laboratory Medicine, Division of Gastrointestinal Pathology, Emory University, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the human phagocyte NADPH oxidase, an enzyme complex that initiates a cascade of reactive oxygen species important in the elimination of infectious agents. This study reports the generation and characterization of six mAbs (NS1, NS2, NS5, CS6, CS8, and CS9) that recognize the p22phox subunit of the Cyt b heterodimer. Each of the mAbs specifically detected p22phox by Western blot analysis but did not react with intact neutrophils in FACS studies. Phage display mapping identified core epitope regions recognized by mAbs NS2, NS5, CS6, CS8, and CS9. Fluorescence resonance energy transfer experiments indicated that mAbs CS6 and CS8 efficiently compete with Cascade Blue-labeled mAb 44.1 (a previously characterized, p22phox-specific mAb) for binding to Cyt b, supporting phage display results suggesting that all three Abs recognize a common region of p22phox. Energy transfer experiments also suggested the spatial proximity of the mAb CS9 and mAb NS1 binding sites to the mAb 44.1 epitope, while indicating a more distant proximity between the mAb NS5 and mAb 44.1 epitopes. Cell-free oxidase assays demonstrated the ability of mAb CS9 to markedly inhibit superoxide production in a concentration-dependent manner, with more moderate levels of inhibition observed for mAbs NS1, NS5, CS6, and CS8. A combination of computational predictions, available experimental data, and results obtained with the mAbs reported in this study was used to generate a novel topology model of p22phox.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neutrophils and other phagocytes serve a critical role in innate immunity by migrating to sites of infection and tissue injury to carry out effector functions critical for a productive inflammatory response. Following cell stimulation, important neutrophil functions include the production and release of cytokines, the internalization of infectious agents, and the generation of a host of microbicidal agents including reactive oxygen species (1, 2, 3). The phagocyte NADPH oxidase is a multisubunit enzyme complex that initiates a cascade of reactive oxygen species by extracting electrons from NADPH for reduction of molecular oxygen (4, 5). Because the inappropriate generation of reactive oxygen species can result in unwanted damage to host tissue (2, 6), the oxidase remains in an inactive state in unstimulated cells with components of the enzyme complex residing in both the membrane (flavocytochrome b (Cyt b)3) and cytoplasmic (p40phox, p47phox, p67phox, Rac) compartments. Following cell stimulation, the cytoplasmic oxidase components translocate to the membrane environment and assemble with Cyt b to form the catalytically active NADPH oxidase. The superoxide anion produced by this enzyme complex serves as a precursor for a variety of toxic oxygen metabolites (including hydrogen peroxide, hypochlorous acid, peroxynitrite, and ozone) that contribute to the resolution of infection (7).

Cyt b is a heterodimeric integral membrane protein composed of a heavily glycosylated 570-residue large subunit (gp91phox; Nox2) and a 195-residue small subunit (p22phox) that serves as the central, catalytic core of the NADPH oxidase complex (1, 5, 8). Primary structure analysis suggests that the N-terminal half of gp91phox contains six potential transmembrane (TM) {alpha} helices with conserved histidine residues that coordinate the two heme prosthetic groups (5, 9, 10, 11). The C-terminal, cytosolic domain of gp91phox shows sequence homology with the FNR enzyme family (12) and has been shown to coordinate the flavin adenine dinucleotide (13) and NADPH (14) cofactors that serve as redox centers for the generation of superoxide anion. Recent work has demonstrated gp91phox to represent the prototype of a large family of redox enzymes (Nox/Duox) that have a broad tissue distribution and generate reactive oxygen species with a growing range of proposed biological functions (7, 15, 16).

The p22phox subunit of the Cyt b heterodimer has been suggested to contain three (17) or four (18) potential TM helices and has a primary sequence that is highly conserved between species (17). Coexpression of gp91phox and p22phox is required for efficient assembly of the mature, catalytically active Cyt b heterodimer (1, 19), and naturally occurring mutations in p22phox have been identified that result in an inactive NADPH oxidase complex (1, 20, 21). The cytoplasmic domain of p22phox contains a proline-rich region involved in well-characterized interactions with the cytoplasmic oxidase component p47phox (22, 23, 24, 25). In support of this notion, a recent peptide walking study has characterized this proline-rich region and additional regions of p22phox that mayparticipate in protein-protein interactions with both p47phox and p67phox (18). Phage display epitope mapping of the p22phox-specific mAb 44.1 has identified a tertiary structure element on the cytoplasmic aspect of p22phox composed of residues 29TAGRF33 and 183PQVNPI188, where 32RF lies within 5 Å of 185VN as determined by TrNOESY nuclear magnetic resonance methods (26). Current evidence suggests that p22phox serves primarily as a scaffolding protein to juxtapose gp91phox and the cytosolic oxidase components in the activated complex, although additional roles in the regulation of electron transfer remain possible. Interestingly, cotransfection of p22phox into a heterologous expression system with the gp91phox homologue Nox1 appears to facilitate the production of reactive oxygen species (27), and immunofluorescence microscopy studies have suggested the colocalization of p22phox with Nox1 and Nox4 in vascular smooth muscle cells (28). Coupled with the wide tissue distribution of p22phox mRNA (15), such observations support the possibility that p22phox plays a wider role in the biology of reactive oxygen species by serving as a common subunit for multiple members of the Nox/Duox protein family. Overall, a genuine understanding of the role of p22phox in the production of reactive oxygen species has been hindered by a lack of direct biochemical evidence to support topology models and identify structural features of this component of the Cyt b heterodimer.

In this study, we report the generation and characterization of six novel mAbs that recognize various regions of p22phox. The p22phox-reactive mAbs were epitope mapped by phage display analysis, used to probe the surface structure and topology of the Cyt b heterodimer, and were examined for effects on catalytic activity of the NADPH oxidase complex. A novel topology model of p22phox has been generated based on consideration of experimental data and computational analysis of the p22phox primary sequence. The mAbs reported in this study provide valuable reagents for structure/function analysis of Cyt b and, potentially, additional members of the Nox enzyme family.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Dodecylmaltoside was purchased from Fluka (Buchs, Switzerland) and octylglucoside, PMSF, and DTT from Calbiochem (La Jolla, CA). Cascade Blue (CCB) acetyl azide was purchased from Molecular Probes (Eugene, OR). Cyanogen bromide-activated Sepharose 4B, protein A-Sepharose, and GammaBind Plus-Sepharose beads were obtained from Pharmacia (Peapeck, NJ); Centricon concentrators from Millipore (Bedford, MA) and the goat anti-mouse (H + L) alkaline phosphatase secondary Ab and Econo-Pac 10 DG desalting columns (30 x 10 ml) from Bio-Rad (Hercules, CA). ProSieve color molecular mass markers were purchased from BMA (Rockland, ME); isopropyl-{beta}-D-thiogalactopyranoside from Promega (Madison, WI); Western blot developing reagents (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD) and nitrocellulose (0.45-µm pore size) was from Schleicher & Schuell (Keene, NH). The elution peptide AC-PQVRPI-CONH2 (AC, acetylation at the N terminus; CONH2, amidation at the C terminus) was obtained from Macromolecular Resources (Fort Collins, CO) and powder for Luria-Bertani and Luria-Bertani agar was from VWR (West Chester, PA). SDS, 0.2-µm polyethersulfone syringe filters (Whatman), acrylamide, bis-acrylamide, NaCl, NaH2PO4, KCl, EDTA, EGTA, NaN3, chloroform, and HPLC grade methanol were purchased from Fisher Scientific (Pittsburgh, PA). The bicinchoninic acid protein determination kit was obtained from Pierce (Rockford, IL); the Escherichia coli strain BL21-CodonPlus (DE3)-RIL was purchased from Stratagene (La Jolla, CA) and Maxisorb 96-well ELISA plates were obtained from Corning (Corning, NY). The anti-rhodopsin mAb K16 was produced in-house by standard hybridoma culture technology. The X-linked chronic granulomatous disease (CGD) mouse strain C57B1/BJxE129 was generated by Genentech (South San Francisco, CA). Neutrophil plasma membranes for Cyt b immunoaffinity purification were prepared as previously described (29) and stored at –70°C before cytochrome purification. All other reagents were obtained from Sigma-Aldrich (St. Louis, MO). The instrumentation used for absorption spectroscopy, fluorescence spectroscopy, and high-speed centrifugation of small-volume samples was as described previously (30).

Ab production

All mAbs reported in this study were produced and purified using standard hybridoma technology as previously described (31). The mAbs NS1, NS2, and NS5 were generated in normal mice, while mAbs CS6, CS8, and CS9 were generated in an X-linked CGD mouse strain.

ELISA, Western blotting, and FACS analysis

Screening of hybridoma clones and characterization of Cyt b-reactive Abs by ELISA was conducted as previously described (32). Color development in ELISA wells was also judged both in the absence of primary Ab and in the presence of an irrelevant primary Ab (the anti-rhodopsin mAb K16) as negative controls.

Hybridoma clones that gave strong positive ELISA signals were further screened for reactivity toward subunits of the Cyt b heterodimer by Western blot analysis. Following separation of neutrophil membrane extracts by SDS-PAGE and transfer to nitrocellulose (~300 ng Cyt b/lane), membranes were probed with primary Ab (2 µg/ml) in diluting buffer (PBS (pH 7.3), 3% goat serum (v/v), 1% BSA, 0.2% Tween 20 (v/v), and 0.1% thimerosal) for 3 h at room temperature. After washing four times in 10 mM HEPES (pH 7.3), 250 mM NaCl, and 0.2% Tween 20, each blot was then probed with a 1/1000 dilution of a goat anti-mouse secondary Ab labeled with alkaline phosphatase and developed with a nitroblue tetrazolium/5-bromo-4-chloro-indolyl phosphate chromagen reagent.

FACS analysis evaluating the binding of mAbs to intact human neutrophils was conducted as previously described (31).

Phage display epitope mapping

Phage display epitope mapping for each of the p22phox-specific mAbs was conducted using previously described methods (26, 33, 34).

Computational analysis of p22phox topology

The prediction of putative p22phox TM {alpha} helices was conducted using six available web-based computational methods (DAS, HMMTOP, PHDHTM, TMHMM, TMPRED, and SOSUI). The human p22phox amino acid sequence was examined using the default setting for each method. The final topology model of p22phox was generated manually by evaluation both computational results and existing experimental data.

Immunoprecipitation studies

To assess the ability of the various p22phox-specific mAbs to bind detergent-solubilized Cyt b, immunoprecipitation experiments were conducted using neutrophil membrane extracts. For immunoprecipitation experiments, 3 ml of neutrophil membranes (1.5 x 109 cell equivalents) was washed with 1 M NaCl and centrifuged at 114,000 x g for 30 min at 4°C. Membrane pellets were resuspended by brief sonication in 3 ml of 10 mM HEPES (pH 7.3), 100 mM KCl, 10 mM NaCl, 2 mM PMSF, and 1 µl/ml P8340 (a mammalian protease inhibitor mixture) and then brought to 0.8% dodecylmaltoside (DDM) by addition from 20% stock solution. Membrane extracts were rotated for 1 h at room temperature and then subjected to centrifugation at 114,000 x g for 30 min at 14°C. The resulting supernatant fractions were collected and analyzed for Cyt b content by absorption spectroscopy using {epsilon}414 = 131,000 M–1cm–1 (35).

For immunoprecipitation, 10 µl of PBS or each respective mAb (2 mg/ml stocks; 620 nM final in immunoprecipitation reactions) was added to 200 µl of neutrophil membrane extract (~220 nM Cyt b) and samples were incubated at room temperature for 20 min. Following this incubation, 200 µl of the above mixture was added to 30 µl of packed GammaBind Plus-Sepharose beads (that had been previously equilibrated in PBS) and the resulting mixtures were rotated for 15 min at room temperature. Following brief centrifugation, the Cyt b content of resulting supernatant fractions was determined by absorption spectroscopy. It serves to note that beads alone consistently precipitated ~15% of the total heme spectrum from detergent-solubilized neutrophil membrane fractions under these experimental conditions. Immunoprecipitation experiments were conducted on three separate membrane extracts using different batches of neutrophil membrane fractions.

CCB labeling of mAbs

The various mAbs were labeled with CCB acetyl azide (using the protocol for labeling at room temperature) and subsequently characterized as previously described (30). This particular procedure resulted in a similar labeling stoichiometry for all mAbs (molar ratio of ~5–6:1 CCB:mAb).

Purification of Cyt b

Construction of the mAb 44.1 affinity matrix and immunoaffinity purification of Cyt b from neutrophil plasma membranes were conducted as previously described (11, 30).

Fluorescence resonance energy transfer studies

Solutions containing Cyt b, CCB-labeled mAbs, and unlabeled mAbs were kept on ice before use. Instrument settings for fluorescence measurements were as described elsewhere (30). For energy transfer measurements, CCB-labeled mAbs were diluted to 10 nM in PBS/0.03% DDM and filtered before use. After background fluorescence had been established with a filtered solution of PBS/0.03% DDM (in a separate cuvette), fluorescence of the labeled mAb was monitored for a short time before addition of other agents. Cyt b (typically 30 nM final) was then added directly to the stirred microcuvette and fluorescence emission was monitored until a steady state was achieved. For competition experiments, 20 µl of unlabeled mAb (13 µM stock solution; ~500 nM final) was next added to the fluorescence cuvette and fluorescence emission monitored. In some competition experiments, 10-µl aliquots of unlabeled mAb (13 µM stock solutions) were added to an equal volume of purified Cyt b (950 nM stock concentration) and the mixture was preincubated on ice for 10 min to allow complex formation before addition to the fluorescence cuvette containing the gp91phox-specific CCB-mAb 54.1 (10 nM).

Cell-free oxidase assay

Neutrophil membranes and cytosol for cell-free assays were prepared as described previously (36) using LPS-free reagents, with a discontinuous 5–20% Percoll gradient used to fractionate the subcellular constituents. The Cyt b content of resulting membrane fractions was determined from reduced minus oxidized difference spectra. Briefly, isolated membrane fractions were brought to 1% octylglucoside, 2 mM PMSF, and 1 µl/ml P8340 and allowed to solubilize for 5 min at room temperature before obtaining the absorption spectrum. Reduced spectra were then obtained following addition of 1 µl of 1 M sodium dithionite and heme content was determined from {Delta}{epsilon}559 = 29,300 M–1 cm–1 (35).

Superoxide assays were performed to examine the effects of the various p22phox-specific mAbs on catalytic activity of the NADPH oxidase complex. In these studies, 35 µl of mAb (13 µM stock solution) was added to 5 µl of neutrophil membrane fraction (~700 nM Cyt b in starting neutrophil membrane fractions) and the mixtures were incubated for 30 min on ice before addition of 17 µl of the mixture to duplicate wells of a 96-well microtiter plate. Total activity of membranes was judged by a similar preincubation of membrane fractions with cell-free assay buffer in the absence of mAbs. A multichannel pipetter was next used to simultaneously add 163 µl of assay buffer (47 mM NaH2PO4, 18 mM K2HPO4, 1 mM MgCl2, 1 mM EGTA, and 2 mM NaN3) containing 100 µM cytochrome c, 125 µM lithium dodecyl sulfate, 10 µM flavin adenine dinucleotide, and 3–4 µl of neutrophil cytosol (1 x 109 cell equivalents/ml) to an entire row of the microtiter plate (each row contained a total activity sample and all Abs examined in this study). After a 5-min incubation at room temperature, superoxide production was initiated by the simultaneous addition of 20 µl of a 20 mM NADPH stock solution to an entire row of the microtiter plate. Superoxide generation was then measured as the rate of cytochrome c reduction, quantitated using {Delta}{epsilon}550 = 21 (cm mM)–1 (37) with individual wells blanked against themselves at time zero. To ensure that the observed cytochrome c reduction resulted from superoxide production, reaction wells containing 310 U/ml superoxide dismutase were also evaluated. Studies examining the ability of mAbs to inhibit superoxide production stimulated by the recombinant fusion protein chimera 3 (38) were conducted in a similar fashion with 900 nM chimera 3 in each well used in place of neutrophil cytosol.

In similar studies evaluating the concentration dependence of the mAbs NS1 and CS9 on oxidase activity, 5-µl aliquots of stock solutions containing various concentrations of each mAb (6.5–6700 nM mAb final in the preincubation mixture) were mixed with an equal volume of membrane fraction (~350 nM Cyt b final in the resulting preincubation mixture) and allowed to react for 1 h at room temperature. Membranes treated in this fashion were then analyzed for superoxide production (supported by neutrophil cytosol) as described above following addition of 170 µl of assay buffer containing the listed components of the cell-free oxidase assay.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The generation of six mAbs recognizing p22phox

Following immunization of normal and X-linked CGD mice, established hybridoma cell lines producing IgG species were screened by ELISA for reactivity to neutrophil membrane extracts, with positive clones further screened by Western blot analysis for reactivity to either the large or small subunit of the Cyt b heterodimer. The Western blot shown in Fig. 1 demonstrates that mAbs NS1, NS2, NS5, CS6, CS8, and CS9 all specifically recognize a band in neutrophil membrane extracts (~22 kDa) of the anticipated molecular mass of the p22phox subunit of Cyt b. A band of the same relative molecular mass is also specifically detected by mAb 44.1 (Fig. 1, lane 7), an epitope-mapped mAb previously shown to recognize a discontinuous epitope on p22phox (26). Similar reactivities were observed using purified Cyt b in the place of neutrophil membrane extracts, confirming specificity of the mAbs for p22phox (data not shown).



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  		FIGURE 1. Western blot analysis with six novel p22phox-specific mAbs. Following separation of neutrophil membrane extracts by SDS-PAGE (~300 ng Cyt b/lane) and transfer to nitrocellulose, membranes were probed with the various mAbs (2 µg/ml final concentration). The previously characterized p22phox-specific mAb 44.1 was also examined for comparative purposes. Lane 1, mAb NS1; lane 2, mAb NS2; lane 3, mAb NS5; lane 4, mAb CS6; lane 5, mAb CS8; lane 6, mAb CS9; lane 7, mAb 44.1 (molecular mass standards are also shown in lane 7). Each of the Abs examined strongly and specifically recognized p22phox in neutrophil membrane extracts. A minor species at ~40 kDa was detected with each of the Abs tested and represents a p22phox dimer commonly observed following SDS-PAGE.

 
Phage display epitope mapping and FACS analysis

To gain a more comprehensive understanding of the determinants recognized by the p22phox-specific mAbs, each of the mAbs was epitope mapped by phage display analysis (33, 34). Following selection of phage and confirmation of Ab cross-reactivity, the primary structures of displayed peptides that conferred Ab binding were deduced from nucleotide sequence analysis. In each instance, alignment of phage sequences resulted in a consensus peptide sequence that was compared with the primary structure of p22phox. Phage display consensus peptide sequences and the resulting p22phox core epitope identification based on these results are shown in Fig. 2. Concerning mAb NS5, preliminary analysis of >100 selected phage clones with the program FINDMAP (39, 40) suggests that in addition to p22phox 78KLFGPF83, residues 51LLEYPRG57 might also contribute to a complex epitope (A. Jesaitis, unpublished data).



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  		FIGURE 2. Phage display epitope mapping for six p22phox-specific mAbs. Following selection and amplification, nucleotide sequence analysis was conducted on isolated phage clones to investigate primary structure determinants of p22phox recognized by each respective mAb. A, Shows only a phage consensus sequence for mAb NS1 since this consensus peptide could not be readily identified in the p22phox primary structure. Identified phage consensus peptides were aligned to corresponding regions of p22phox for the remaining mAbs as follows: mAb NS2 (B), mAb NS5 (C), mAb CS6 (D), mAb CS8 (E), and mAb CS9 (F). Amino acid identities and highly conservative substitutions are shown in bold.

 
Since mAbs NS1, NS2, and NS5 appeared to recognize regions that were distinct from the mAb 44.1 epitope (mAb 44.1 was previously shown to recognize a discontinuous epitope comprised of 29TAGRF33 and 183PQVNPI188 on the cytoplasmic portion of p22phox (26)), FACS analysis was conducted to characterize the ability of the mAbs to bind the neutrophil cell surface. Similar to results obtained with mAb 44.1 (33), mAbs NS1, NS2, and NS5 did not bind intact neutrophils by FACS analysis, indicating that their respective epitopes were not accessible on the cell surface (data not shown). FACS analysis also demonstrated that mAbs CS6, CS8, and CS9 did not recognize the surface of intact neutrophils (data not shown), as anticipated in light of the proximity of these epitopes to the cytoplasmic mAb 44.1 epitope.

Resonance energy transfer studies investigating the assignment of overlapping epitopes

The assignment of at least partially overlapping epitopes for mAbs 44.1, CS6, and CS8 suggested these Abs to represent a good test case for confirmation of phage display results by alternative biochemical methods. Our group has recently shown CCB-labeled mAb 44.1 to represent a valuable probe for analysis of Ab binding to purified Cyt b by fluorescence resonance energy transfer methods (30). Using this method, quenching of donor fluorescence is observed when the Cyt b heme prosthetic groups (the acceptors) are brought within close proximity of the CCB molecules (the donors) and such quenching provides a sensitive and convenient measure of the formation of a mAb:Cyt b complex in solution.

In the present study, the ability of various mAbs to disrupt a preformed CCB-mAb 44.1:Cyt b complex was evaluated by monitoring fluorescence emission following addition of unlabeled mAbs 1) whose epitopes were assigned to the same p22phox region as mAb 44.1 (mAbs CS6 and CS8); 2) that recognize the cytoplasmic domain of the Cyt b large subunit gp91phox (mAbs 54.1 and NL7); and 3) that recognize the integral membrane protein rhodopsin (irrelevant mAb K16). The structure model presented for Cyt b (see Fig. 7; see below for discussion of model) depicts core epitope regions mapped by phage display analysis for the Cyt b-specific mAbs used in this study.



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  		FIGURE 7. Structure model of Cyt b highlighting gp91phox and p22phox-specific mAb epitopes. The topology model for gp91phox will be discussed elsewhere (D. Baniulis and A. J. Jesaitis, manuscript in preparation) and highlights mapped epitope regions for mAbs 54.1, NL7, and 7D5. Two coordination states are proposed for the heme prosthetic group near the cytoplasmic surface of Cyt b (gp91phox His101/gp91phox His209 and alternatively gp91phox His101/p22phox His94) to account for available experimental data (9 42 43 ). The novel topology model for p22phox was generated by hydropathy analysis with six different computational methods, with the resulting model manually refined to account for available experimental data. Results obtained by phage display epitope mapping of the p22phox-specific mAbs are depicted on this model, with the exception of mAb NS1 whose phage consensus peptide did not readily align to the p22phox primary sequence. EC, extracellular; IC, intracellular.

 
As shown in Fig. 3, quenching of CCB-mAb 44.1 (10 nM) donor fluorescence is observed following addition of immunoaffinity-purified Cyt b (30 nM final) to the fluorescence cuvette, indicating the formation of a CCB-mAb 44.1:Cyt b complex. A substantial relaxation of fluorescence quenching (84 ± 8% relaxation, n = 3) was observed following addition of a 50-fold molar excess of unlabeled mAb 44.1 (Fig. 3, curve a), suggesting a direct competition between labeled and unlabeled mAb 44.1 for binding sites on the Cyt b heterodimer (resulting in the physical separation of the donor/acceptor pair). Analogous competition experiments conducted with mAbs CS6 (data not shown) and CS8 (Fig. 3, curve b) demonstrated a similar relaxation of fluorescence quenching following addition of a 50-fold molar excess of unlabeled Ab to the preformed CCB-mAb 44.1:Cyt b complex (77 ± 10% relaxation for mAb CS6, n = 3 and 81 ± 6% relaxation for mAb CS8, n = 3). These results directly support phage display data, suggesting that mAbs 44.1, CS6 and CS8 bind to a similar structural determinant of the Cyt b heterodimer. In contrast, no relaxation of fluorescence quenching was observed when similar experiments were conducted with the gp91phox-specific mAb 54.1 (Fig. 3, curve c), the gp91phox-specific mAb NL7 (data not shown), or an irrelevant, anti-rhodopsin mAb K16 (data not shown). Similar to mAb 44.1, mAbs 54.1 and NL7 recognize the cytoplasmic surface of Cyt b (31, 33) and have been shown to efficiently bind detergent-solubilized Cyt b by fluorescence resonance energy transfer under these experimental conditions (R. M. Taylor and A. J. Jesaitis, unpublished data). Additional control experiments demonstrated that preincubation of DDM-solubilized Cyt b with unlabeled mAb 44.1 does not interfere with the binding of CCB-mAb 54.1 (data not shown). These data further demonstrate that although mAbs 44.1 and 54.1 both bind the cytoplasmic surface of Cyt b under these conditions, they do not compete with each other for binding due to the physical separation of their respective epitopes.



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  		FIGURE 3. Confirmation of overlapping epitopes for mAbs 44.1, CS6, and CS8 by fluorescence resonance energy transfer. Following formation of a CCB-mAb 44.1:Cyt b complex, various mAbs were added to the fluorescence cuvette to assess their ability to physically disrupt the donor-acceptor pair. The relaxation of fluorescence quenching observed when unlabeled mAb 44.1 (curve a), CS8 (curve b), or 54.1 (curve c) was added to the CCB-mAb 44.1:Cyt b complex. Arrows indicate the addition of both Cyt b (30 nM final) and unlabeled mAb (500 nM final) to the fluorescence cuvette containing CCB-mAb 44.1 (10 nM).

 
Analysis of epitope proximity for nonoverlapping epitopes by fluorescence resonance energy transfer and characterization of Ab binding to detergent-solubilized Cyt b

The results obtained with mAbs CS6 and CS8 suggested that similar studies might prove valuable for estimating the spatial proximity of the epitopes bound by CCB-mAb 44.1 and other p22phox-specific mAbs whose phage display maps 1) suggested an epitope nearby in the primary sequence (mAb CS9); 2) suggested epitopes more distant in the primary sequence (mAbs NS2 and NS5); or 3) provided a consensus sequence not readily identified in the p22phox primary structure (mAb NS1). Fig. 4 (curve b) demonstrates that addition of a 50-fold molar excess of mAb CS9 to the preformed CCB-mAb 44.1:Cyt b complex caused a relaxation of fluorescence quenching (44 ± 4% relaxation, n = 3), suggesting the ability to compete with CCB-mAb 44.1 for binding to a common surface region of p22phox. Similar studies conducted with mAb NS1 (data not shown) also demonstrated a substantial relaxation in fluorescence quenching (55 ± 18% relaxation, n = 3). Experiments conducted with mAb NS2 (data not shown) and mAb NS5 (Fig. 4, curve c) showed no observable relaxation in fluorescence quenching, suggesting an inability of these Abs to displace CCB-mAb 44.1 from the preformed CCB-mAb 44.1:Cyt b complex.



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  		FIGURE 4. Analysis of epitope proximity for p22phox-specific mAbs with nonoverlapping epitopes by fluorescence resonance energy transfer. Following formation of a CCB-mAb 44.1:Cyt b complex, various mAbs were added to the fluorescence cuvette to assess their ability to physically disrupt the donor-acceptor pair. The relaxation of fluorescence quenching observed when unlabeled mAb 44.1 (curve a), CS9 (curve b), or NS5 (curve c) was added to the CCB-mAb 44.1:Cyt b complex. Arrows indicate the addition of both Cyt b (30 nM final) and unlabeled mAb (500 nM final) to the fluorescence cuvette containing CCB-mAb 44.1 (10 nM).

 
Since weaker binding affinities might provide an explanation for the significantly diminished (mAbs CS9 and NS1) or lack (mAbs NS2 and NS5) of competition with CCB-mAb 44.1 relative to results obtained with mAbs CS6 and CS8, the ability of the various mAbs to immunoprecipitate Cyt b from neutrophil membrane extracts was evaluated. Fig. 5 shows representative curves demonstrating that mAb CS9 immunoprecipitates Cyt b (65 ± 5% of the total heme spectrum depleted, n = 3) with a somewhat greater efficiency than mAb 44.1 (53 ± 4% of the total heme spectrum depleted, n = 3) under these experimental conditions (~220 nM Cyt b and 620 nM mAb in the assay). In these studies, mAbs NS1 and NS5 also immunoprecipitated Cyt b (42 ± 3% and 51 ± 7% of the total heme spectrum depleted, respectively, n = 3), while mAb NS2 showed no ability to immunoprecipitate Cyt b (Fig. 5). Resonance energy transfer experiments also demonstrated that while CCB-labeled mAbs NS5 and CS9 efficiently bound to detergent-solubilized Cyt b (10 nM CCB-mAb and 30 nM Cyt b final in the fluorescence cuvette), CCB-mAb NS2 showed no observable binding under these conditions (data not shown). Taken together, the above results suggest that mAbs NS1 and CS9 bind a region of p22phox that partially overlaps the mAb 44.1 epitope, whereas mAb NS5 binds a region with no measurable overlap with the mAb 44.1 epitope.



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  		FIGURE 5. Immunoprecipitation studies examining the binding of mAbs 44.1, NS2, NS5, and CS9 to detergent-solubilized Cyt b. Following solubilization of neutrophil membrane fractions with the nonionic detergent DDM, immunoprecipitation experiments were conducted to monitor the ability of the indicated mAbs to bind detergent-solubilized Cyt b. The Cyt b Soret absorption band was monitored as a measure of Cyt b content of each resulting supernatant fraction. Curve a, Incubation of membrane extract and GammaBind beads in the absence of mAb (total Cyt b); curve b, immunoprecipitation by mAb NS2; curve c, immunoprecipitation by mAb 44.1; curve d, immunoprecipitation by mAb NS5; and curve e, immunoprecipitation by mAb CS9.

 
Effect of p22phox-specific mAbs on catalytic activity of the NADPH oxidase complex

A recent study by our group reported the inhibition of NADPH oxidase activity by the Cyt b, large subunit-specific Ab mAb NL7 (31). In the interest of characterizing additional inhibitors of NADPH oxidase function, the p22phox-specific mAbs were preincubated with isolated neutrophil membrane fractions (the source of Cyt b) and then evaluated for effects on superoxide generation in an in vitro cell-free assay system. Fig. 6A summarizes results obtained in these experiments for each of the six p22phox-specific mAbs (967 nM mAb final in the cell-free assays). Although mAbs NS2 and K16 (an irrelevant anti-rhodopsin monoclonal IgG) caused only a slight reduction of superoxide production (7 ± 5% and 6 ± 5% inhibition, respectively), moderate reductions were observed in the presence of mAbs NS1 (42 ± 4% inhibition), NS5 (17 ± 6% inhibition), CS6 (30 ± 5% inhibition), and CS8 (24 ± 5% inhibition). Preincubation of neutrophil membranes with mAb CS9 resulted in the most pronounced reduction in superoxide production (75 ± 3% inhibition) under these assay conditions. Overall, similar results were obtained when inhibition studies were performed with a previously described p67phox(1–212)-Rac1(1–192) GST fusion protein (chimera 3 (38)) in the place of neutrophil cytosol for activation of superoxide production (data not shown).



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  		FIGURE 6. Analysis of the effects of p22phox-specific mAbs on superoxide generation by the NADPH oxidase complex. Following preincubation of neutrophil membrane fractions with p22phox-specific mAbs, cell-free superoxide production assays were conducted to monitor catalytic activity of the NADPH oxidase complex with neutrophil cytosol used as the source of soluble oxidase components. A, Summary of results obtained when each of the mAbs reported in this study was examined for effects on superoxide production rates (967 nM mAb final in the cell-free assays). This analysis represents results obtained from six data points for each mAb (from three independent experiments), with the anti-rhodopsin mAb K16 provided as an irrelevant Ab control. B, Concentration-dependent effects on NADPH oxidase activity observed when neutrophil membrane fractions (~350 nM Cyt b in the preincubation reaction) were preincubated with increasing levels of the indicated mAbs NS1, CS9, or K16 (6.5–6700 nM mAb in the preincubation reaction). Addition of the preincubation mixtures to the cell-free assay resulted in a 20-fold dilution of the above listed concentrations. Neutrophil membranes were preincubated in the absence of Ab to determine total NADPH oxidase activity and the anti-rhodopsin mAb K16 was evaluated as an irrelevant mAb control.

 
A more detailed characterization of the inhibition of NADPH oxidase activity by mAbs NS1 and CS9 was derived from mAb concentration-dependence studies conducted with neutrophil cytosol used as the source of cytosolic oxidase components. Fig. 6B demonstrates the well-behaved inhibition of oxidase function when mAbs NS1 and CS9 were preincubated with neutrophil membranes. In these studies, inhibition of superoxide production was observed when the Ab concentrations included in the cell-free assays (~5–21 nM final for CS9; ~5–84 nM final for NS1) were in modest excess of the Cyt b content of neutrophil membrane fractions (~17.5 nM Cyt b final).

Generation of a novel topology model for p22phox

The structural model of Cyt b shown in Fig. 7 proposes a novel topology for p22phox that accounts for both computational predictions (Table I) and available experimental data. Core epitope regions identified by phage display analysis have been shaded and regions of Cyt b believed to comprise complex epitopes are shown for mAbs 7D5, 44.1, and NS5. Although sequence analysis by computational methods suggests that p22phox contains two to four hydrophobic regions that may comprise TM {alpha} helices (Table I), we exclude the assignment of TM1 and TM2 based on 1) the cytoplasmic localization of p22phox residues (29TAGRF33) involved in the discontinuous mAb 44.1 epitope (26) that was assigned to TM1 by computational methods and 2) results obtained by analysis of phage display data with the program FINDMAP suggesting p22phox residues involved in a discontinuous mAb NS5 epitope (51LLEYPRG57); A. J. Jesaitis, unpublished data) that were assigned to TM2 by computational methods. The topology model in Fig. 7 is also consistent with FACS data demonstrating the inability of p22phox-specific mAbs to bind the neutrophil surface (33) and experimental data mapping regions of p22phox important for interactions with soluble oxidase components (18, 22, 24) by proposing a cytoplasmic location for these regions. The proposed topology model also assigns membrane-spanning segments to the predominantly hydrophobic region of p22phox, comprised of residues 91–126, that was roughly identified by each of the computational methods shown in Table I. Although the structure model in Fig. 7 assumes interactions between the TM regions of Cyt b, it is important to note that absolutely no biochemical data has been obtained describing the protein-protein interface between gp91phox and p22phox.


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  		Table I. Computational analysis of p22phox topology

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The generation and characterization of six mAbs recognizing p22phox

Although the p22phox subunit of the Cyt b heterodimer serves as an obligate component in the functional NADPH oxidase complex, major questions remain concerning the subunit’s structure, TM topology, and role in the regulation of superoxide production. The present study introduces six novel mAbs that have been characterized with respect to recognition of the Cyt b heterodimer and effects on catalytic activity of the NADPH oxidase complex. Each of the mAbs specifically detected p22phox in Western blot analysis of neutrophil membrane extracts, indicating the utility of these reagents in studies examining protein expression profiles p22phox. In addition, each of the mAbs (with the exception of mAb NS2), immunoprecipitated the Cyt b heterodimer from neutrophil membrane extracts to roughly a similar degree as mAb 44.1, a p22phox-specific mAb that has found broad utility for immunoaffinity purification of Cyt b (11), analysis of p22phox structure (26), and the detection of anionic lipid-induced conformational changes in Cyt b (30). The ability to bind and immunoprecipitate p22phox from detergent extracts will provide a valuable means for directly examining the possibility that p22phox forms functional heterodimers with homologues of gp91phox (Nox family members) in a wide variety of tissues (15, 27, 28). Additionally, although we have used only human Cyt b for binding experiments, amino acid sequence alignment reveals that human p22phox shares 87% identity with the murine and bovine forms of the protein (see also Ref.17), suggesting that the mAbs reported in this study represent valuable probes for analysis of Cyt b from a variety of different species.

Phage display epitope mapping provided strong evidence that mAbs NS2, NS5, and CS9 recognize distinct regions of the p22phox primary sequence, with mAbs CS6 and CS8 binding a region of p22phox that has been previously characterized as the core region of the discontinuous mAb 44.1 epitope (26, 33). Current efforts are directed at sequencing large numbers of selected phage clones for each of the p22phox-specific mAbs for attempts to identify residues involved in complex epitopes by the recently described computational method FINDMAP (39, 40).

The inability of the p22phox-specific mAbs to bind intact neutrophils in FACS analysis provides support for topology models localizing these respective regions to the cytoplasmic portion of Cyt b. It is important to note that the inability to bind intact neutrophils cannot rule out the possibility of extracellular regions of p22phox that are simply masked on the cell surface. The Cyt b structural model in Fig. 7 proposes a novel topology for p22phox that accounts for results obtained in this study and consideration of available experimental data. This model differs from previous predictions (17, 18) by assigning only two membrane spanning domains and a completely cytoplasmic localization for the first 90-aa residues of p22phox. Although the various models suggest potential p22phox topologies, direct biochemical evidence will be required for a genuine understanding of the structure of this subunit of the Cyt b heterodimer.

Analysis of epitope proximity by fluorescence resonance energy transfer

Recent efforts by our group with CCB-labeled mAb 44.1 have shown this Ab conjugate to represent a convenient probe for monitoring complex formation with Cyt b by fluorescence resonance energy transfer methods (30). In the current study, each of the p22phox-specific mAbs was tested for the ability to disrupt a preformed CCB-mAb 44.1:Cyt b complex (competition experiments) in the hopes of obtaining a gross understanding of epitope proximity on surface of the Cyt b heterodimer.

Resonance energy transfer studies demonstrated the ability of mAbs CS6 and CS8 to disrupt the CCB-mAb 44.1:Cyt b complex to a similar degree as mAb 44.1 itself, supporting phage display results suggesting that these mAbs to recognize a common region of p22phox. These results demonstrates the utility of resonance energy transfer methods as a solution-based assay for conducting competition experiments to investigate protein-protein interactions in cases where appropriate donor/acceptor pairs are inherent or can be engineered into the proteins of interest.

Energy transfer studies conducted in a similar fashion with mAbs NS1 and CS9 also indicated a measurable epitope proximity with the mAb 44.1 epitope. It is of interest to highlight the smaller relaxation of fluorescence quenching observed with mAb CS9 (suggesting less efficient competition with CCB-mAb 44.1) relative to similar experiments conducted with mAbs recognizing common primary structure determinants (mAbs 44.1, CS6, and CS8). Since mAbs 44.1 and CS9 were shown to bind Cyt b with comparable efficiency, these results suggest the diminished competition by mAb CS9 to result from the ~13-residue spacing between the mAb 44.1 and CS9 core epitope regions. This observation suggests that resonance energy transfer-based mAb competition experiments may be surprisingly sensitive to epitope distance and/or orientation. The ability of mAb NS1 to displace CCB-mAb 44.1 in competition experiments was of interest since the strong peptide consensus obtained for mAb NS1 by phage display analysis was not readily identified in the p22phox primary sequence. These data support a working hypothesis contending that mAb NS1 recognizes a strongly discontinuous epitope that shows some degree of spatial proximity to the mAb 44.1 epitope on the cytoplasmic surface of the Cyt b heterodimer.

In contrast to results obtained with mAbs CS6, CS8, CS9, and NS1, mAbs NS2 and NS5 showed a complete inability to displace CCB-mAb 44.1 in resonance energy transfer experiments. Phage display analysis indicated that the NS5 core epitope resides ~100 residues to the N-terminal side of the core mAb 44.1 epitope, demonstrating a significant separation in the primary sequence. Since mAb NS5 immunoprecipitated Cyt b with a similar efficiency as mAb 44.1 and a CCB-NS5 conjugate efficiently bound low concentrations of Cyt b in energy transfer studies, these results suggest the mAb 44.1 and NS5 epitopes reside in spatially distinct regions on the native Cyt b surface. In support of this notion, it serves to highlight control experiments conducted in this study with mAb 44.1 and the gp91phox-specific mAb 54.1 indicating that the cytoplasmic surface of Cyt b is sufficiently large to simultaneously bind two distinct, intact mAbs. The inability of mAb NS2 to displace CCB-mAb 44.1 in similar experiments was anticipated in light of results demonstrating the inability of this mAb to bind detergent-solubilized Cyt b.

Although immunoprecipitation and energy transfer experiments provided a qualitative understanding of the ability of the various mAbs to recognize detergent-solubilized Cyt b, it will be of interest to determine the actual association/dissociation constants for the various mAbs to more rigorously evaluate competition experiments. In addition, the generation and use of Fab will result in more highly defined, monovalent probes for such studies. Although the lack of atomic structure information for p22phox renders discussions of epitope proximity vague, evaluation of high-resolution lysozyme:Fab x-ray structures indicates that each respective mAb epitope comprises only a relatively minor portion of the overall molecular surface (~20%) and provides some insight into the nature of nonoverlapping and partially overlapping epitopes on this relatively small, model protein Ag (41).

Effect of p22phox mAbs on catalytic activity of the NADPH oxidase complex

Previous studies demonstrating the inhibitory effects of the gp91phox-specific mAb NL7 on superoxide production by the NADPH oxidase complex (31) led us to examine effects of the p22phox-specific mAbs on catalytic function of the oxidase. In the present study, preincubation of neutrophil membrane fractions with mAb CS9 (~130-fold molar excess mAb:Cyt b) resulted in markedly reduced rates of superoxide production, with more modest inhibition observed for mAbs NS1, NS5, CS6, and CS8. The complete lack of inhibition with mAb NS2 was consistent with immunoprecipitation and energy transfer experiments demonstrating the inability of this Ab to efficiently recognize the native Cyt b surface structure. In these studies, the overall degrees of Ab inhibition were strikingly similar when either neutrophil cytosol or a recombinant p67phox-Rac1 chimera was used to stimulate superoxide production.

The direct correlation with binding to the Cyt b heterodimer and some degree of inhibition of oxidase activity for Abs recognizing p22phox is in contrast to results showing that although both of the gp91phox-specific mAbs 54.1 and NL7 bind detergent-solubilized Cyt b, only mAb NL7 binding results in marked inhibition of oxidase function (31). In previous studies characterizing the effect of mAb NL7 on superoxide production, oxidase inhibition did not appear to result from either reduced translocation of the cytosolic oxidase components or impaired binding of the NADPH cofactor (31). To provide a detailed mechanistic characterization of the inhibitory p22phox-specific mAbs, it will be of interest to evaluate effects of these mAbs on cofactor binding, translocation of cytosolic oxidase components, and anionic lipid-induced conformational changes in Cyt b.

Concerning the structural basis of oxidase assembly, it is of interest to highlight phage display results indicating the core mAb CS9 epitope (assigned to p22phox 165KKPSE169) to reside near a proline-rich region of p22phox (residues 151–160) previously shown to be important for binding the cytosolic oxidase factors p47phox and p67phox (18, 25). Phage display mapping results also indicate that the core mAb NS5 epitope (assigned to p22phox 77VKLFGP82) resides near a region of p22phox that may serve to bind the cytosolic oxidase component p67phox (18). Since multiple sites on Cyt b have been characterized as potentially important for binding cytosolic oxidase components to form the catalytically active NADPH oxidase complex, the site-specific inhibitors of superoxide production reported in this study represent a potentially valuable set of probes for biochemical investigation of these proposed protein-protein interactions. Additionally, the ability of the p22phox-specific mAbs to inhibit superoxide production supported by both neutrophil cytosol and a recombinant p67phox-Rac1 fusion protein may allow for detailed characterization of the individual roles of cytosolic oxidase components in assembly and activation of the NADPH oxidase complex.


   Acknowledgments
 
We gratefully acknowledge Jeannie Gripentrog for the preparation of neutrophil plasma membrane fractions. We also thank Dr. Edgar Pick for providing the chimera 3 construct and for helpful discussions concerning expression and purification.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by an Arthritis Foundation Postdoctoral Fellowship (to R.M.T.), American Heart Association Scientist Development Grant 30156 (to J.B.B.), and Grants RO1 HL45635 (to M.C.D.), RO1 HL54229 (to C.A.P.), and RO1 AI 26711 (to A.J.J.). Back

2 Address correspondence and reprint requests to Dr. Algirdas J. Jesaitis, Department of Microbiology, Montana State University, 109 Lewis Hall, Bozeman, MT 59717. E-mail address: umbaj{at}gemini.oscs.montana.edu Back

3 Abbreviations used in this paper: Cyt b, flavocytochrome b; CCB, Cascade Blue; CGD, chronic granulomatous disease; DDM, dodecylmaltoside. Back

Received for publication June 4, 2004. Accepted for publication September 30, 2004.


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