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The Journal of Immunology, 2003, 170: 6082-6089.
Copyright © 2003 by The American Association of Immunologists

Functional Epitope on Human Neutrophil Flavocytochrome b5581

James B. Burritt2,*, Thomas R. Foubert*, Danas Baniulis*, Connie I. Lord*, Ross M. Taylor*, John S. Mills*, Travis D. Baughan*, Dirk Roos{dagger}, Charles A. Parkos{ddagger} and Algirdas J. Jesaitis*

* Department of Microbiology, Montana State University, Bozeman, MT 59717; {dagger} Sanquin Research and Landsteiner Laboratory of the Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; and {ddagger} Division of Gastrointestinal Pathology, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
mAb NL7 was raised against purified flavocytochrome b558, important in host defense and inflammation. NL7 recognized the gp91phox flavocytochrome b558 subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the 498EKDVITGLK506 region of gp91phox. In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (Km). mAb NL7 did not inhibit translocation of p47phox, p67phox, or Rac to the plasma membrane, and bound its epitope on gp91phox independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91phox on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47phox binding sites. We conclude that the 498EKDVITGLK506 segment resides on the cytosolic surface of gp91phox and represents a region important for oxidase function, but not substrate or cytosolic component binding.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The human phagocyte NADPH oxidase is a multisubunit enzyme complex that produces the superoxide anion (O2-) for the destruction of pathogens (1). This system is activated by a variety of stimuli and exerts toxic effects in most inflammatory processes (2). Individuals deficient in superoxide production due to a genetic lesion in any of four components of this system (p22phox, gp91phox, p47phox, or p67phox) experience severe recurrent infections, often from catalase-positive microbes. This condition is known as chronic granulomatous disease (CGD)3 (3, 4).

In the unstimulated neutrophil, the NADPH oxidase remains in an unassembled inactive state. Activation of the oxidase requires translocation of the cytosolic components p47phox, p40phox, p67phox, and Rac2 from the cytosol to the membrane-resident flavocytochrome b (Cyt b, a heterodimer of gp91phox and p22phox), and occurs within seconds following the appropriate stimulus (5). Once assembled, single electrons from metabolic NADPH are transported across the plasma membrane to a putative extracellular oxygen-binding site to form superoxide. The superoxide reacts with other constituents of the surrounding medium to produce cytotoxic compounds that are then targeted to encountered pathogens, either outside the cell or within the phagocytic vacuole. Cyt b thus appears to play a dual role: serving as a scaffold for the assembly of the cytosolic factors during activation and providing a transmembrane pathway for passage of electrons to extracellular molecular oxygen to form superoxide.

Cyt b -specific mAbs have been applied to identify solvent-accessible regions on both the cytosolic and extracellular aspects of the native protein, as well as regions which are accessible only after denaturation (6, 7). Tertiary structural elements of native Cyt b have further been determined by this method, revealing discontinuous regions of protein that are contained within a single epitope (8, 9). In the current report, we show that both p-iodonitrotetrazolium violet (INT) and cytochrome c reduction are inhibited by mAb NL7, which binds 498EKDVITGLK506, a cytosolic segment of gp91phox. Our data suggest that the inhibitory effect of mAb NL7 is exerted during the initial stages of oxidase activation. NL7 does not interfere directly with NADPH binding, or block the translocation of cytosolic oxidase factors p47phox, p67phox, or Rac to the membrane. Characterization of the oxidase inhibitory effects of mAb NL7 provides information pertinent to both the structural and mechanistic aspects of Cyt b function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals, reagents, and materials

Unless otherwise specified, all reagents were purchased from Sigma-Aldrich (St. Louis, MO). mAb NL7 was produced using standard hybridoma technology. Briefly, three BALB/c mice were immunized three times each with 50 µg of Cyt b, quantitated as heme content by absorbance spectroscopy (10), and solubilized in 40 mM octyl-{beta}-glucopyranoside (OG). Hyperimmune splenocytes were fused with 3PU1 myeloma cells and screened for mAb production by ELISA using OG-solubilized Cyt b. Hybridoma clones producing anti-Cyt b mAbs were cloned twice by limiting dilution and grown in RPMI 1640 medium containing 5% FCS. Ab was collected on Gammabind Sepharose beads (Pharmacia, Piscataway, NJ), and eluted with 100 mM glycine, 150 mM NaCl, pH 2.5, into neutralization buffer and dialyzed against 150 mM NaCl, 10 mM Na2HPO4, pH 7.4.

Immunoblotting

Binding of mAb NL7 to gp91phox was determined using immunoblots of Cyt b as described (9) using protein solubilized in either 0.1% Triton X-100 or 40 mM OG. Human neutrophil Cyt b (30 ng per lane) was partially purified by heparin-agarose affinity chromatography prepared as described (10), or by immunoaffinity purification of Cyt b using mAb 44.1 (8). Following separation of proteins by SDS-PAGE and transfer to nitrocellulose, the blot was probed with 13 nM (2 µg/ml) of the primary Abs NL7, 54.1, or 7D5. After washing four times, each blot was then probed with a 1/1000 dilution of a goat anti-mouse-Ig secondary Ab labeled with alkaline phosphatase (Bio-Rad, Hercules, CA) and developed with a chromagen reagent (Kirkegaard & Perry Laboratories, Gaithersburg, MD).

Phage-display epitope mapping

mAb NL7 (1.2 mg) was subjected to epitope mapping as previously described (9) by selecting peptide sequences that mimic the natural Cyt b epitope from the J404 nonapeptide library (11). The J404 library is a collection of infectious M13-based bacteriophage, in which each clone of the library displays three to five copies of a unique nine-residue peptide displayed at the N terminus of the pIII capsid protein. This library collectively contains at least 5 x 108 unique nine-residue sequences. Use of the peptide library in this way is possible because each phage particle in the library displays a unique peptide on its surface and contains the encoding nucleotide segment within the genome. Briefly, three sequential rounds of selection and amplification of phage-display peptide library clones were conducted using a mAb NL7 immunoaffinity matrix. After three rounds of selection, the sequences of displayed peptides were deduced by nucleotide sequence analysis of the isolated clones as described (12).

Flow cytometry

Human neutrophils were prepared from fresh whole blood collected in citrate anticoagulant from normal human donors and probed by mAbs NL7, 44.1, 7D5, or an irrelevant isotype-matched monoclonal as described (6). Briefly, leukocytes were incubated in 200 nM of each mAb diluted in 10 mM PBS (10 mM phosphate, 150 mM NaCl, pH 7.4) with 4% normal goat serum and 4% nonfat dry milk for 30 min on ice. For intracellular staining, neutrophils were permeabilized by exposure to 0.05% saponin for 30 min on ice before incubation with primary Abs. Saponin (0.05%) was also included during all washes and during incubation with secondary Ab. Final resuspension buffer consisted of PBS containing 0.1 µg/ml propidium iodide to identify permeabilized cells. Data were collected using a BD Biosciences FACScan instrument (Franklin Lakes, CA), and analyses were performed using WinMDI software (version 2.7; The Scripps Research Institute, La Jolla, CA) by gating on neutrophil populations determined from forward and side scatter profiles. Permeabilized cells were identified as having FL2 intensity signals significantly above background.

Cell-free oxidase assay

Plasma membrane-bound Cyt b and cytosolic oxidase factors were prepared as described (13) using LPS-free reagents. A discontinuous 5–20% Percoll gradient was used to fractionate the subcellular constituents. Concentration dependence analysis was used to determine the optimal concentrations of plasma membranes, cytosol, and lithium dodecyl sulfate (LDS) (data not shown). Superoxide assays were performed in Costar 3370 96-well microtiter plates (Cambridge, MA) in assay buffer (47 mM NaH2PO4, 18 mM K2HPO4, 1 mM MgCl2, 1 mM EGTA, and 2 mM NaN3) containing the following additional final concentrations: 100 µM cytochrome c, 125 µM LDS, 10 µM FAD, 200 µM NADPH, 125 µM GTP{gamma}S, 5 x 106 cell equivalents of plasma membranes, and 2 x 106 cell equivalents of cytosol, in a total volume of 200 µl.

Superoxide generation rate was measured as the reduction of cytochrome c over a selected time period, quantitated using {Delta}{epsilon}550 = 21 (cm mM)-1 (14) blanked against wells containing 310 U/ml superoxide dismutase (SOD). In measuring concentration dependence of mAb NL7 on oxidase inhibition, average rates of superoxide generation were reported as the percent of control (irrelevant Ab), where 100% corresponds to ~1.2 mole of O2-/mole Cyt b heme/sec. Aliquots containing different concentrations of each mAb were mixed with an equal volume of membrane fraction (5 µl at 1 x 109 cell equivalents/ml), and allowed to react for 1 h at room temperature before addition of the remaining reagents (causing a 20-fold dilution of the membranes and Ab concentration reported). To test the ability of the mAbs to inhibit superoxide generation after activation, Ab concentration in the cuvette was adjusted to 6.7 µM after production of superoxide was occurring at a maximal linear rate. For INT reduction assays, reaction conditions were identical to cytochrome c reduction except that 100 µM INT was used in place of cytochrome c. Sodium azide is included in these reactions as described (15, 16) to inhibit the activity of heme-containing redox systems such as myeloperoxidase which catalyze the conversion of H2O2 to HOCl and other oxidants. Contribution by these oxidants would otherwise interfere with the measurement of cytochrome c reduction as a function of superoxide production by the NADPH oxidase (17).

To measure possible competitive binding between mAb NL7 and the cytosolic factors by oxidase rate, 7 x 106 cell equivalents of membranes were simultaneously mixed with the indicated amount of cytosol and NL7 in a buffer containing the same final concentrations of FAD, GTP{gamma}S, and LDS as used in the cell-free assay. This mixture was incubated for 1 h at 25°C, diluted 5-fold in an identical buffer containing cytochrome c, and NADPH was added to 200 µM to initiate superoxide generation. In the presence of LDS, the membranes prepared in this way produced superoxide at a high rate (up to 18 mole O2-/mole of Cyt b heme/second), but without a lag phase following the addition of NADPH, suggesting oxidase assembly had occurred. This experiment was conducted at three concentrations of mAb NL7, in varying concentrations of cytosol.

Translocation assay

Association of p47phox and p67phox with neutrophil membranes in the presence of mAb NL7 was examined as described (18). The membrane fraction was prepared as described above using sonication to reduce the amount of p47phox and p67phox trapped in membrane vesicles. Briefly, 4 x 107 cell equivalents (40 µl) of neutrophil membranes were pretreated for 1 h at 25°C in 6.7 µM of either mAb NL7 or an irrelevant mAb. The membranes were then allowed to react with 1.6 x 107 cell equivalents (160 µl) of neutrophil cytosol for 10 min at 25°C in activation buffer (47 mM NaH2PO4, 18 mM K2HPO4, 1 mM MgCl2, 1 mM EGTA, 2 mM NaN3, 125 µM LDS, 10 µM FAD, and 125 µM GTP{gamma}S). Separation of membrane-bound from soluble factors was accomplished by two different methods, both yielding the same results. In one method, the membranes were pelleted by centrifugation at 100,000 x g in a TLA-100.2 rotor for 30 min at 4°C and washed by resuspension in 1.0 ml of activation buffer. This sequence was repeated for two wash steps. Alternatively, the membranes were sedimented through a discontinuous sucrose gradient at 100,000 x g for 30 min at 25°C as described (19). Fractionation of the resulting gradient yielded a clear peak of activity at the 20–50% sucrose interface (data not shown). These fractions were then examined for the presence of cytosolic factors by immunoblot as described (19, 20) using polyclonal rabbit Abs kindly provided by W. M. Nauseef (University of Iowa School of Medicine, Iowa City, IA).

Since the inception of the cell-free oxidase assay (21, 22, 23), several theories have been put forth to explain oxidase activation by anionic amphiphiles (e.g., arachidonate, SDS, or LDS). There is evidence for direct interactions with the cytosolic subunits (24, 25) that induce conformational changes, fostering the notion that certain Src homology 3 binding domains in both p47phox and p67phox remain inaccessible until exposed to anionic amphiphile. Such effects were postulated to mimic in vivo phosphorylation events where Src homology 3 domain interactions are believed to partly control oxidase activity. Other experiments conducted by our lab (26, 27) and others provide evidence that the amphiphiles also interact directly with Cyt b to impart similar conformational effects that may effect partial oxidase control. Collectively, these studies suggest that that the amphiphiles interact with several components of the oxidase to induce full activity.

Binding of mAb NL7 to activated membranes by immunoblot

Experiments were conducted to determine whether mAb NL7 is capable of binding to membranes preactivated with oxidase components including cytosolic factors. For this analysis, a mixture was prepared in the activation buffer (above) containing 10 µM FAD, 125 µM GTP{gamma}S, 1.5 x 108 cell equivalents/ml membranes, 7.7 x 107 cell equivalents/ml cytosol, in either the presence or absence of 125 µM LDS. The mixture was incubated for 15 min at room temperature to activate the membranes, followed by exposure for 45–60 min at 25°C to mAb NL7 at 1.7 µM. Irrelevant mAb was also tested as a control under the same conditions, and purified goat IgG was included at 20 µM in all samples (which did not affect superoxide generation measured by cytochrome c reduction, data not shown) to reduce nonspecific mAb binding. Following washing of the membranes as described for the translocation assay, SDS-PAGE and transfer of proteins to nitrocellulose was conducted so that the IgG subunits associated with the membranes could be determined semiquantitatively using an IgG-specific secondary Ab.

Kinetic analysis

Kinetic analysis of NL7 inhibition was examined with respect to the NADPH concentration as described (28). Serial 0.67-fold dilutions of NADPH were added to the cell-free assay as described above but with membranes that had been kept at 25°C for 1 h in the presence of 3.4 µM mAb NL7 or a PBS control. The rates of superoxide generation were then determined by the cytochrome c reduction assay (above), and plotted as a function of the NADPH concentration. The data were then analyzed by nonlinear regression (GraphPad Prism version 3.00 for Windows; GraphPad Software, San Diego, CA). The mAb NL7-mediated percent inhibition was then plotted from these data by subtracting the percent of activity remaining after NL7 treatment from 100%.

Structure modeling

Most of the C-terminal domain of gp91phox, beginning at Ser291, was modeled using the Web-based program 3D-PSSM (29), available at www.bmm.icnet.uk/~3dpssm (Web site of the Biomolecular Modeling Laboratory of the Imperial Cancer Research Fund, London, U.K.). This program uses a novel Web-based sequence alignment and "threading" program (3D-PSSM) which compares the primary sequence of the protein of interest to those with known three-dimensional structure to determine residues that are structurally equivalent. Identified residues are then used to extend the multiple sequence alignment to new proteins, creating a database of known structures that can be used to model the unknown protein. The program then predicts coordinates for the atoms of a protein of unknown structure, based on a threading algorithm applied to structures selected by 3D-PSSM that are most similar to the unknown structure. These structures were then scored for one-dimensional sequence alignment, matching of predicted secondary structural elements, and solvent accessibility. From this analysis it was determined that the structure with closest fit (lowest E-value) to gp91phox was corn nitrate reductase (30) (Brookhaven Database Coordinate filename, 1CNF). A rasmol file output of the gp91phox fragment was generated by 3D-PSSM which was then converted to pdb file format by a text editor. The file was then examined using the SwissPDB viewer available on the SwissProt Web site (http://www.ebi.ac.uk/swissprot/), or by the Insight II program obtained from Accelerys (San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To produce novel probes of the phagocyte NADPH oxidase, mAbs were generated against partially purified neutrophil Cyt b. One such mAb, mAb NL7, bound a broad band suggestive of gp91phox present in neutrophil membrane extracts prepared by heparin affinity chromatography, shown by immunoblot in Fig. 1A (lane 1, labeled HE for heparin eluate). Specificity of mAb NL7 for the gp91phox subunit was confirmed using Cyt b which had been purified on mAb 44.1 (8) (lane 2, labeled IP for immunopurified) which will immunoprecipitate the Cyt b heterodimer (9). Similar immunoreactivity was also shown using heparin eluate probed by the previously characterized anti-gp91phox mAb 54.1 (lane 3), but not for mAb 7D5 (lane 4) which binds native, but not denatured, gp91phox.



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  		FIGURE 1. Immunoblotting and flow cytometry. A, SDS-PAGE was used to resolve affinity purified Cyt b (30 ng per lane) which had been solubilized in 0.1% Triton X-100. Purification of the protein was first conducted either by heparin affinity chromatography (HE) or by immunoaffinity purification using the Cyt b-specific mAb 44.1 (IP). The electrotransferred protein was probed with 13 nM of the primary Ab indicated, and a goat-anti-mouse Ig secondary Ab was conjugated to alkaline phosphatase for reaction development. B, Flow cytometry of intact human neutrophils probed with anti-Cyt b and control mAbs indicated. Intact neutrophils (1 x 106) were exposed to 200 nM of the mAbs indicated on ice for 30 min before washing and labeling with goat-anti-mouse Ig FITC-conjugated secondary Ab. C, Immunoreactivity of the mAbs indicated is shown to 0.05% saponin-permeabilized cells. The data are representative of at least three separate analyses.

 
The membrane sidedness of the epitope bound by mAb NL7 was examined by flow cytometry of both intact and saponin-permeabilized neutrophils that had been exposed to mAb NL7. This analysis indicated the epitope is not accessible on intact neutrophils (Fig. 1B), compared with the positive control mAb 7D5 which binds an extracellular epitope on human (9) and primate neutrophils (31). However, following permeabilization of neutrophil membranes with 0.05% saponin the staining by mAb NL7 increased significantly, producing a signal that was slightly greater than the positive control mAb 44.1 (Ref.6 ; Fig. 1C). The labeling on permeabilized but not intact, neutrophils by mAb NL7 suggested both a cytosolic and solvent-accessible location of the epitope for NL7 binding on gp91phox of the native protein.

The specificity of mAb NL7 for gp91phox was further confirmed by phage display epitope mapping (9, 11). A six-log increase in adherent phage numbers from the first to third selection was observed and positive plaque lifts (7) confirmed specific selection of immunoreactive sequences had taken place (data not shown). The deduced peptide sequences obtained from third round eluate phage clones showed an obvious similarity to the 498EKDVITGLK506 region of gp91phox (Fig. 2). Each selected peptide shown contains from four to eight residues similar to those of the identified epitope. Residues showing an exact match to residues of the epitope are shown in blue, and those suggesting a conservative substitution are shown in red. The diversity of the J404 phage-display peptide library is estimated to include over 5 x 108 unique nonapeptides (11), and the consensus provided by immunoaffinity selection on mAb NL7 provides a very strong match to the 498EKDVITGLK506 region on gp91phox. Thus, the peptides shown in Fig. 2 collectively provide an unambiguous identification of the epitope on gp91phox bound by mAb NL7.



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  		FIGURE 2. Phage-displayed peptide sequences matching 498EKDVITGLK506 of gp91phox. Three rounds of selection were used to enrich immunoreactive peptide sequences from the J404 phage-display peptide library (11 ), which were then subjected to nucleotide sequence analysis to determine the displayed peptide. These sequences were then compared with the sequence of gp91phox, and found to align with the 497EEKDVITGLKQ507 region of gp91phox as shown. Residues of selected peptides identical to corresponding residues of gp91phox are shown in blue, and those with side chain similarities are shown in red. Only three sequences appearing after the third round of selection did not appear to fit the consensus (not shown), and sequences appearing on more than one occasion are indicated in parentheses.

 
mAb NL7 inhibits oxidase activation

Exposure of mAb NL7 to neutrophil membranes for 1 h at 25°C before oxidase measurement resulted in a concentration-dependent inhibition of NADPH oxidase activity. The half-maximal effect occurred at ~400 nM Ab (Fig. 3). However, addition of NL7 to the system after reaching the maximal superoxide generation rate had no measurable effect when compared with an irrelevant mAb control (Fig. 3, inset), indicating that the inhibitory effects of mAb NL7 were not exerted once the oxidase was assembled and functioning. The ability of mAb NL7 to bind the activated complex was further tested as described below to determine whether the 498EKDVITGLK506 region of gp91phox is accessible to Ab following cytosolic factor translocation.



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  		FIGURE 3. mAb NL7 concentration-dependent inhibition of oxidase activity. Both cytochrome c ({diamondsuit}) and INT reduction ({blacksquare}) were inhibited by mAb NL7 in a cell-free assay using 200 µM NADPH as shown. Serial 4-fold dilutions of each mAb were preincubated for 1 h with neutrophil membranes before analysis, using the electron acceptor indicated. One-hundred percent activity is equivalent to 1.2 mole O2-/mole of Cyt b heme/second. Identical conditions including an isotype-matched irrelevant control mAb did not show significant effects in either cytochrome c ({blacktriangledown}) or INT ({blacktriangleup}) reduction. Cyt b-specific mAbs 44.1 (x) and 54.1 (•) (6 ) also had no effect on oxidase activity when tested at the highest mAb concentration examined. Inset, Monoclonals (NL7 ({diamond}) or irrelevant ({square})) were added to a final concentration of 6.7 µM during the cytochrome c reduction (arrowhead) to test their ability to inhibit superoxide generation once the oxidase had assembled, and are compared with the SOD control (*). Data are representative of five experiments and error bars indicate SEM of four replicates from different experiments.

 
To dissect the effect of mAb NL7 on electron transport in the oxidase, INT was used as an alternative electron acceptor in the cell-free assay. In this application (32), INT was reduced directly by oxidation of the Cyt b flavin moiety. Fig. 3 shows that mAb NL7-mediated inhibition of INT reduction is similar to that of cytochrome c reduction, suggesting that the inhibitory effect of NL7 is due to impaired NADPH binding or electron flow before reduction of FAD or possibly by the impairment of accessibility of INT to FAD. In this system, an isotype-matched irrelevant mAb shows insignificant effect on both cytochrome c and INT reduction, similar to Cyt b-specific mAbs 44.1 and 54.1 (6) when tested at the highest mAb concentration examined.

Binding of mAb NL7 to gp91phox does not perturb translocation of p47phox, p67phox, or Rac during oxidase activation

Previous biochemical analyses of a CGD patient with a Asp500 -> Gly substitution in gp91phox revealed normal Cyt b expression levels, but an impaired translocation of both p47phox and p67phox (33). This mutation is located within the NL7 epitope on gp91phox, suggesting the Ab might inhibit oxidase activation by preventing translocation of p47phox or p67phox. We thus examined the translocation of p47phox, p67phox, and Rac after pretreating 4 x 107 cell equivalents of neutrophil membranes with 6.7 µM mAb NL7 or an isotype-matched irrelevant mAb before addition of the remaining assay constituents including cytosol and LDS. Semiquantitative immunoblotting of these membrane fractions indicated mAb NL7 had no effect on the amount of p67phox or p47phox (Fig. 4A, top panel), or Rac (bottom panel) associated with the membrane by activation with LDS (lane 1) compared with the irrelevant mAb control (lane 2). The level of p47phox, p67phox, and Rac signal from membranes treated without LDS is shown in lane 3, which was subtracted from all densitometry measurements before graphing (Fig. 4C).



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  		FIGURE 4. Cytosolic factor translocation and mAb NL7 binding to activated membranes. A, Top and bottom panels: 4 x 107 cell equivalents of neutrophil plasma membranes were exposed to 6.7 µM of mAb indicated, then reacted with oxidase components including LDS. The membranes were then pelleted and washed to remove unbound cytosolic factors. Translocated p67phox and p47phox (top panel) were detected by immunoblot using polyclonal Abs to each subunit, while Rac was detected with a mAb (bottom panel). B, The similar amount of NL7 Ab subunits bound to cytosol and LDS-activated (lane 1) and unactivated (lane 3) membranes, compared with that of an irrelevant mAb (lane 2). C, Densitometry results for measurements in A and B. For graphing of p47phox ({blacksquare}), p67phox ({square}), and Rac (), the LDS-independent signal (A, lane 3) was subtracted from signal in lanes 1 and 2. For the IgG H subunit signal (), the irrelevant mAb signal (B, lane 2) was subtracted from the signal resulting in lanes 1 and 3. The data are representative of four experiments where they represent SEM, n = 4.

 
Our data indicate that mAb NL7 binding does not inhibit the translocation of p47phox, p67phox, or Rac to the membranes. Our data also showed that while mAb NL7 inhibited oxidase function when exposed to the membranes before activation, it had no measurable effect on activity following activation. However, these data did not preclude the possibility that mAb NL7 may not bind the activated membranes due to epitope sequestration by the cytosolic factors. Therefore, we tested the binding of mAb NL7 to its epitope on gp91phox following the functional binding of the cytosolic factors. Relative immunoblotting signal of the monoclonal subunits (Fig. 4B) was compared for LDS-activated (lane 1) and resting (lane 3) membranes. Nonspecific binding of irrelevant mouse monoclonal was found insignificant in this assay (lane 2). The signal of NL7 IgG H and L chains was similar on membranes irrespective of the activation state of the oxidase, suggesting that translocation of the cytosolic factors to the membrane does not render the mAb NL7 epitope inaccessible.

To further examine whether NL7 affects cytosolic factor translocation, we tested whether an increasing concentration of cytosol could reverse the inhibitory effect of the Ab. After simultaneous exposure of membranes to increasing concentrations of both mAb NL7 and cytosol for 1 h at room temperature in the presence of 125 µM LDS, the activated complex was then diluted in assay medium containing cytochrome c, and the oxidase rate was measured following addition of NADPH (Fig. 5). These activating conditions included higher concentrations of cytosol and a longer preincubation step relative to the conditions depicted in Fig. 3, and therefore produce almost a 5-fold increase in the superoxide generation. At the concentrations of mAb NL7 tested (0, 0.42, and 6.7 µM), the EC50 values for cytosol were 3.79 (3.31–4.35), 2.28 (2.62–3.08), and 2.7 (2.45–2.96 x 106 cell equivalents per well), respectively. Thus, there is no reduction on mAb NL7-mediated oxidase inhibition, due to increasing concentrations of the cytosolic NADPH oxidase factors.



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  		FIGURE 5. Concentration dependence of cytosol and NL7 on oxidase activity. The superoxide generation rate was determined after simultaneously exposing the membranes to NL7 at 0 µM ({blacksquare}), 0.42 µM ({diamondsuit}), and 6.7 µM ({blacktriangleup}) and increasing concentrations of cytosol as indicated as described in Materials and Methods, where the maximum rate (100%) was equal to 18 mol O2-/mol of Cyt b heme/second. Data indicate SEM of four replicates, and are representative of separate analysis.

 
Examination of potential changes in NADPH binding on gp91phox caused by mAb NL7

Homology studies suggested that the 504GLKQKTLYGR513 region of gp91phox may be important for binding of NADPH (34). This region, which slightly overlaps the epitope bound by mAb NL7, could be sequestered by bound Ab, thus obstructing the access of NADPH to gp91phox. To test this hypothesis, we examined the NADPH concentration dependence of the oxidase in the cell-free assay in the presence of 400 nM mAb NL7 compared with the effect of a PBS control (Fig. 6A). A hyperbolic concentration dependence of the superoxide generation rate on NADPH was observed in both the presence and absence of mAb NL7. These data were fit to a curve by nonlinear regression analysis, modeling a one or two NADPH binding site assumption (traced by the thin line or bold line, respectively). The two NADPH binding site model (35) gave a better fit to the data, as shown in Fig. 6A, and as indicated by the corresponding R2 values (Fig. 6B). Nonlinear regression analysis of the two binding site model also indicates that the Michaelis constant (Km) 1 and maximum velocity (Vmax) 1 did not appear affected by mAb NL7 binding, while the only statistically significant effect of mAb NL7 binding was on the low affinity site, in which the Vmax was reduced by 53%.



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  		FIGURE 6. Oxidase inhibition by mAb NL7 corresponding to NADPH concentration. A, The SOD-inhibitable superoxide generation rate was determined after incubation in the absence ({blacktriangleup}) or presence ({blacksquare}) of mAb NL7 at 3.4 µM (a relative rate of 15 corresponds to 0.55 mol O2-/mol of Cyt b heme/second). Nonlinear regression analysis of the oxidase rate was performed for both data sets to examine substrate-binding parameters. The thin line represents the curve fit to the data points modeled to a single NADPH binding site as described in Materials and Methods. The bold line shows the same data fit to a two binding site model. Inset, The percent of NL7-mediated inhibition at the NADPH concentrations tested ({blacktriangleup}). B, The Vmax and Km values corresponding to the binding site models are shown. Comparison of the curve fits with and without mAb NL7 indicate that the two binding site model gives a significantly better fit to the experimental results. Data are representative of four experiments, and error bars represent SEM of four replicates using different experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results indicate that mAb NL7 recognizes the 498EKDVITGLK506 epitope on the cytosolic aspect of native gp91phox, as well as the denatured form of the protein. The binding of mAb NL7 to gp91phox in a variety of conditions enables its use in analysis of the oxidase by several immunologic methods. In addition to binding to a specific segment of gp91phox, mAb NL7 was found to inhibit the function of the NADPH oxidase. These characteristics allowed mAb NL7 to be used as a probe of superoxide production at the level of Cyt b structure and thus provide insight into the possible role of the 498EKDVITGLK506 region of gp91phox in oxidase activation.

The epitope bound by mAb NL7 contains the residue Asp500 of gp91phox, which was previously reported to be the locus of a CGD point mutation that prevented translocation of cytosolic factors p47phox and p67phox (33). This observation led to the hypotheses that Asp500 might be part of a docking site for the cytosolic components or that mutation of this residue perturbed the structure of gp91phox and inhibited translocation indirectly. Our results are consistent with the latter hypothesis and indicate that the inhibitory effects of mAb NL7 binding to the 498EKDVITGLK506 epitope do not appear to directly affect membrane translocation of p47phox, p67phox, or Rac. However, there is evidence that cytosolic oxidase subunits may be translocated en bloc as a complex with p40phox (36), and therefore dock to Cyt b by multiple contact sites. Therefore, obstruction of a single contact by mAb NL7 may be insufficient to prevent membrane translocation, but could inhibit functional interactions between Cyt b and the cytosolic factors.

The observation that mAb NL7 binds 498EKDVITGLK506 but does not inhibit cytosolic factor translocation is consistent with reports that inclusion of peptides mimicking the nearby 484D-I502 region in a cell-free system (37) or by creating a deletion mutant corresponding to 488A-E497 (38), have little effect on NADPH oxidase activity. However, a synthetic peptide corresponding to 491F-G504 of gp91phox was shown to inhibit both cytochrome c reduction (EC50 10 µM) and cytosolic factor translocation (33), supporting the role for this region of gp91phox in some aspect of oxidase function, if not direct binding to cytosolic factors.

Competitive binding between mAb NL7 and the cytosolic factors was further discounted by two separate analyses, thus corroborating our translocation data shown in Fig. 4. First, membranes activated with LDS in the presence of cytosolic factors were found to bind mAb NL7 as strongly as those not exposed to LDS activation and translocation (Fig. 4B, compare lanes 1 and 3). Second, our data relating cytosol and mAb NL7 concentration to oxidase rate indicate that increasing cytosol concentrations did not reverse the degree of mAb NL7 inhibition (Fig. 5). Thus, the mechanism of inhibition by the Ab probably involves independent binding of Ab and cytosolic factors.

The nonlinear regression analysis relating oxidase rate to NADPH concentration indicated the most accurate modeling of the data involved a two NADPH binding site fit (Fig. 6), as previously suggested (35, 39). According to this view, the kinetic parameters influenced by NL7 correspond to the low affinity (~26 µM) NADPH binding site, presumably on gp91phox (Fig. 6B). This Km value is similar to previous reports of ~35 µM for NADPH on human neutrophil membranes (22), between 49 and 94 µM for membranes from stimulated mouse macrophages (28), and below 50 µM in a detergent-solubilized human neutrophil system (40). Because several regions of gp91phox proposed to contribute to the NADPH binding (41) are close in primary sequence to the epitope bound by mAb NL7 (see below), we examined the kinetic data to determine whether the inhibitory effect was the result of competitive binding between mAb NL7 and NADPH. Because the only significant effect mAb NL7 had on oxidase kinetics was to reduce the low affinity Vmax (Fig. 6B), Ab binding is not suggested to compete with NADPH binding.

The two fitted curves in Fig. 6A indicate the degree of NL7-mediated inhibition increased with increasing NADPH concentrations, and this inhibition saturated at ~50% in the presence of 20–30 µM NADPH (summarized in the inset). This result corresponds to the kinetic data fit by a two NADPH binding site model, in which the low Vmax and Km (high affinity) form is not inhibited by the Ab (Fig. 6B). This conclusion is compatible with Fig. 3, which indicates an inability to completely inhibit the oxidase with increasing concentrations of NL7 (inhibition asymptotically approaches ~90% with increasing NL7 concentrations; data not shown).

The conditions in which experiments for Figs. 3 and 6 were done are not identical. Fig. 3 tests mAb NL7 between 7 and 6700 nM as the NADPH concentration is held constant at 200 µM. For Fig. 6, mAb NL7 is held constant at 3400 nM while the NADPH concentration is varied from 2.3 to 89 µM. Therefore, direct comparisons between the two conditions in which component ranges do not overlap may be misleading.

To gain a better understanding of the influence of mAb NL7 binding on oxidase activity, we modeled most of the C-terminal domain of gp91phox (amino acid residues 291S-F565) to the known crystal structure of a related protein, corn nitrate reductase (pdb 1CNF) (30). In the space-filling and ribbon images showing the crystal structure of corn nitrate reductase (Fig. 7, A and C), the blue and purple colors correspond to space-filling representation of the bound and cocrystallized ADP and FAD cofactors, respectively. Cocrystallization with NADPH or its oxidized form was not achieved, so the bound ADP represents the ADP moiety of bound NADPH. The resulting model of gp91phox (superimposed on the coordinates of 1CNF) was viewed in the same orientation as 1CNF, and is shown in Fig. 7, B and D. The amino acid residues of gp91phox predicted to contact the FAD and the ADP moiety of NADPH are shown colored in purple and blue, respectively. The location of 498EKDVITGLK506 of gp91phox, shown in green on this model, suggests the placement of the mAb NL7 epitope. This view predicts the mAb NL7 epitope is close to, but not overlapping, the proposed NADPH or FAD binding site.



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  		FIGURE 7. Structural model comparing the crystal structure of 1CNF (30 ) to the predicted carboxyl portion of gp91phox. A and C, The 1CNF structure is shown in space-filling and ribbon rendition, respectively. The cocrystallized heteroatoms of ADP and FAD in A and C are indicated, respectively, in light blue and purple space-filling representation. The cytosolic domain of gp91phox (291S-F565) was threaded onto the crystal coordinates of 1CNF as described in Materials and Methods and is represented in space-filling and ribbon models shown in B and D, respectively. To allow examination of the regions predicted to coordinate flavin and the ADP moiety of NADPH, the residues that correspond to those in 1CNF shown to form hydrogen bonds with ADP or those that interact directly with flavin are colored in light blue or purple as in the 1CNF structure. The epitope bound by mAb NL7 is identified by green space-filling atoms. The N- and C-terminal residues are colored dark blue and red, respectively.

 
Despite evidence for direct interaction between gp91phox and p67phox (42), the specific residues that mediate this interaction have not been identified, and we were thus unable to assess their relative placement with respect to the NL7 epitope on the modeled surface of gp91phox using structural modeling. However, some residues mediating contact between gp91phox and p47phox have been described, and were modeled to suggest their proximity to the epitope bound by NL7. The model of gp91phox shown in Fig. 7, B and D, was modified so that residues believed to contact p47phox are shown in pink (Fig. 8) (33, 43, 44, 45). This view of gp91phox shows residues 555ESGPRGVHFIF565 in pink (Phe565 is red and represents the C terminus). This region of the molecule has been identified as a potential p47phox contact site based on CGD analysis (46), synthetic peptide inhibition of oxidase activation (43, 45) and translocation (44), and selection by phage display analysis of p47phox affinity matrices (45). Such a placement also suggests that the C-terminal residues of gp91phox, 566NKENF570 (not shown in the model), might lay over the cleft proposed to contain the NADPH binding site of gp91phox and would allow Phe570 to coordinate the isoalloxazine ring of the FAD as originally suggested by Taylor et al. (47) from studies of similarity to the ferridoxin nucleotide reductases. According to this model, 555ESGPRGVHFIF565 is well-removed from the NL7 epitope (shown in green), having at least 15Å separating the two regions at closest approach.



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  		FIGURE 8. Modeled p47phox interactive residues on gp91phox. The modeled structure of gp91phox shown in Fig. 7 was modified to identify residues of the protein believed to contact p47phox. N- and C-terminal residues are shown in dark blue and red, respectively. A, The same view of gp91phox shown in Fig. 7, B and D, and shows possible p47phox interactive domain, 555ESGPRGVHFIF565, in pink. The face of the protein revealed when the molecule is rotated 180° around the y-axis is shown in B, revealing 451FEWFADLL458 also in pink, which may interact with p47phox. According to this modeling, the placement of these two domains suggests that the regions of gp91phox important in the translocation of p47phox are located on opposite faces of the protein.

 
From the model shown in Fig. 8A, the opposite face of gp91phox is obtained when the molecule is rotated 180° around the y-axis and is shown in Fig. 8B. On this face of the structure, the residues of 555ESGPRGVHFIF565 are obstructed from view. However, the other segment of gp91phox previously identified as a potential p47phox binding site, 451FEWFADLL458 (again shown in pink), is positioned in a depression on the surface but has solvent-accessible residues that lie near His354 (purple) which is predicted to interact with the FAD. The model suggests that this region of the molecule is also remote from the NL7 epitope. Interestingly, the two regions of gp91phox reported to interact with p47phox are on opposite sides of the modeled protein. DeLeo et al. (45) also identified the 87STRVRRQL94 region on gp91phox (not included in the model), which resides on the cytoplasmic aspect of the N-terminal half of the molecule as a p47phox interactive site, suggesting that contact between p47phox and Cyt b must be relatively complex.

Current information about the region of gp91phox in the vicinity of the epitope bound by NL7 suggests a more complex role for 498EKDVITGLK506 of gp91phox, beyond that of a docking site for the cytosolic oxidase factors. Our results indicate NL7 binding does not interfere with oxidase substrate or subunit involvement. The results are nevertheless compatible with mAb NL7-mediated oxidase inhibition involving perturbations of electron transport within the complex, or by restricting activation-dependent flexibility of the protein, in which NL7 binding prevents spatial reorganization of a Cyt b domain necessary for electron transport during activation. This latter view is compatible with our evidence (26) and reports of others that suggest Cyt b undergoes structural rearrangement during oxidase activation (48, 49).

Identification of the functional significance of the 498EKDVITGLK506 epitope of gp91phox bound by mAb NL7 provides a link between Cyt b structure and function. Our data indicated that this domain resides on the cytosolic aspect of gp91phox, and that the inhibitory effects of mAb NL7 on oxidase activation are exerted before or during the initial stages of electron transfer. Availability of mAb NL7 as a Cyt b-specific inhibitor of oxidase function may enable a better understanding of oxidase activation and thus control. We conclude that the 498EKDVITGLK506 domain of gp91phox participates in the activation of the oxidase and influences electron transport during superoxide generation. A precise description of the mechanism of oxidase inhibition by mAb NL7 requires additional analysis.


   Footnotes
 
1 This work was supported by American Heart Association Scientist Development Grants 30156 (to J.B.B.), RO1 AI 26711 (to A.J.J.), and RO1 HL54229 (to C.A.P.), a National Arthritis Foundation Biomedical Science grant (to J.S.M.), and the National Arthritis Foundation Postdoctoral Fellowship (to R.M.T.). Back

2 Address correspondence and reprint requests to Dr. James B. Burritt, 109 Lewis Hall, Department of Microbiology, Montana State University, Bozeman, MT 59717. E-mail address: jburritt{at}montana.edu Back

3 Abbreviations used in this paper: CGD, chronic granulomatous disease; Cyt b, flavocytochrome b558; INT, p-iodonitrotetrazolium violet; OG, octyl-{beta}-glucopyranoside; LDS, lithium dodecyl sulfate; FAD, flavin adenine dinucleotide; SOD, superoxide dismutase. Back

Received for publication December 30, 2002. Accepted for publication March 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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