558 B Flavocytochrome Functional Epitope on Human Neutrophil

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Functional Epitope on Human Neutrophil Flavocytochrome b558 James B. Burritt, Thomas R. Foubert, Danas Baniulis, Connie I. Lord, Ross M. Taylor, John S. Mills, Travis D. This information is current as Baughan, Dirk Roos, Charles A. Parkos and Algirdas J. of September 28, 2021. Jesaitis J Immunol 2003; 170:6082-6089; ; doi: 10.4049/jimmunol.170.12.6082

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

1 Functional Epitope on Human Neutrophil Flavocytochrome b558

James B. Burritt,2* Thomas R. Foubert,* Danas Baniulis,* Connie I. Lord,* Ross M. Taylor,* John S. Mills,* Travis D. Baughan,* Dirk Roos,† Charles A. Parkos,‡ and Algirdas J. Jesaitis* mAb NL7 was raised against purified flavocytochrome b558, important in host defense and inflammation. NL7 recognized the phox gp91 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 phox phox the oxidase substrate Michaelis constant (Km). mAb NL7 did not inhibit translocation of p47 , p67 , 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 phox to the proposed NADPH binding site, but significant separation from the proposed p47 binding sites. We conclude that the Downloaded from 498EKDVITGLK506 segment resides on the cytosolic surface of gp91phox and represents a region important for oxidase function, but not substrate or cytosolic component binding. The Journal of Immunology, 2003, 170: 6082–6089.

he human phagocyte NADPH oxidase is a multisubunit b thus appears to play a dual role: serving as a scaffold for the enzyme complex that produces the superoxide anion assembly of the cytosolic factors during activation and providing a (O Ϫ) for the destruction of pathogens (1). This system is transmembrane pathway for passage of electrons to extracellular T 2 http://www.jimmunol.org/ activated by a variety of stimuli and exerts toxic effects in most molecular oxygen to form superoxide. inflammatory processes (2). Individuals deficient in superoxide Cyt b -specific mAbs have been applied to identify solvent- production due to a genetic lesion in any of four components of this accessible regions on both the cytosolic and extracellular aspects system (p22phox, gp91phox, p47phox,orp67phox) experience severe re- of the native protein, as well as regions which are accessible only current infections, often from catalase-positive microbes. This condi- after denaturation (6, 7). Tertiary structural elements of native Cyt tion is known as chronic granulomatous disease (CGD)3 (3, 4). b have further been determined by this method, revealing discon- In the unstimulated neutrophil, the NADPH oxidase remains in tinuous regions of protein that are contained within a single an unassembled inactive state. Activation of the oxidase requires epitope (8, 9). In the current report, we show that both p-iodo- phox phox translocation of the cytosolic components p47 , p40 , nitrotetrazolium violet (INT) and cytochrome c reduction are in- by guest on September 28, 2021 p67phox, and Rac2 from the cytosol to the membrane-resident fla- hibited by mAb NL7, which binds 498EKDVITGLK506, a cytosolic vocytochrome b (Cyt b, a heterodimer of gp91phox and p22phox), segment of gp91phox. Our data suggest that the inhibitory effect of and occurs within seconds following the appropriate stimulus (5). mAb NL7 is exerted during the initial stages of oxidase activation. Once assembled, single electrons from metabolic NADPH are NL7 does not interfere directly with NADPH binding, or block the transported across the plasma membrane to a putative extracellular translocation of cytosolic oxidase factors p47phox, p67phox,orRac oxygen-binding site to form superoxide. The superoxide reacts to the membrane. Characterization of the oxidase inhibitory effects with other constituents of the surrounding medium to produce cy- of mAb NL7 provides information pertinent to both the structural totoxic compounds that are then targeted to encountered patho- and mechanistic aspects of Cyt b function. gens, either outside the cell or within the phagocytic vacuole. Cyt Materials and Methods Chemicals, reagents, and materials *Department of Microbiology, Montana State University, Bozeman, MT 59717; †Sanquin Research and Landsteiner Laboratory of the Academic Medical Center, Unless otherwise specified, all reagents were purchased from Sigma-Al- University of Amsterdam, Amsterdam, The Netherlands; and ‡Division of Gastroin- drich (St. Louis, MO). mAb NL7 was produced using standard hybridoma testinal Pathology, Department of Pathology and Laboratory Medicine, Emory Uni- technology. Briefly, three BALB/c mice were immunized three times each versity, Atlanta, GA 30322 with 50 ␮gofCytb, quantitated as heme content by absorbance spectros- ␤ Received for publication December 30, 2002. Accepted for publication March copy (10), and solubilized in 40 mM octyl- -glucopyranoside (OG). Hy- 21, 2003. perimmune splenocytes were fused with 3PU1 myeloma cells and screened for mAb production by ELISA using OG-solubilized Cyt b. Hybridoma The costs of publication of this article were defrayed in part by the payment of page clones producing anti-Cyt b mAbs were cloned twice by limiting dilution charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and grown in RPMI 1640 medium containing 5% FCS. Ab was collected on Gammabind Sepharose beads (Pharmacia, Piscataway, NJ), and eluted 1 This work was supported by American Heart Association Scientist Development with 100 mM glycine, 150 mM NaCl, pH 2.5, into neutralization buffer and Grants 30156 (to J.B.B.), RO1 AI 26711 (to A.J.J.), and RO1 HL54229 (to C.A.P.), dialyzed against 150 mM NaCl, 10 mM Na HPO , pH 7.4. a National Arthritis Foundation Biomedical Science grant (to J.S.M.), and the Na- 2 4 tional Arthritis Foundation Postdoctoral Fellowship (to R.M.T.). Immunoblotting 2 Address correspondence and reprint requests to Dr. James B. Burritt, 109 Lewis phox Hall, Department of Microbiology, Montana State University, Bozeman, MT 59717. Binding of mAb NL7 to gp91 was determined using immunoblots of E-mail address: [email protected] Cyt b as described (9) using protein solubilized in either 0.1% Triton X-100 3 Abbreviations used in this paper: CGD, chronic granulomatous disease; Cyt b, fla- or 40 mM OG. Human neutrophil Cyt b (30 ng per lane) was partially ␤ purified by heparin-agarose affinity chromatography prepared as described vocytochrome b558; INT, p-iodonitrotetrazolium violet; OG, octyl- -glucopyranoside; LDS, lithium dodecyl sulfate; FAD, flavin adenine dinucleotide; SOD, super- (10), or by immunoaffinity purification of Cyt b using mAb 44.1 (8). Fol- oxide dismutase. lowing separation of proteins by SDS-PAGE and transfer to nitrocellulose,

the blot was probed with 13 nM (2 ␮g/ml) of the primary Abs NL7, 54.1, in a buffer containing the same final concentrations of FAD, GTP␥S, and or 7D5. After washing four times, each blot was then probed with a 1/1000 LDS as used in the cell-free assay. This mixture was incubated for 1 h at dilution of a goat anti-mouse-Ig secondary Ab labeled with alkaline phos- 25¡C, diluted 5-fold in an identical buffer containing cytochrome c, and phatase (Bio-Rad, Hercules, CA) and developed with a chromagen reagent NADPH was added to 200 ␮M to initiate superoxide generation. In the (Kirkegaard & Perry Laboratories, Gaithersburg, MD). presence of LDS, the membranes prepared in this way produced superoxide Ϫ at a high rate (up to 18 mole O2 /mole of Cyt b heme/second), but without Phage-display epitope mapping a lag phase following the addition of NADPH, suggesting oxidase assem- mAb NL7 (1.2 mg) was subjected to epitope mapping as previously de- bly had occurred. This experiment was conducted at three concentrations of scribed (9) by selecting peptide sequences that mimic the natural Cyt b mAb NL7, in varying concentrations of cytosol. epitope from the J404 nonapeptide library (11). The J404 library is a col- Translocation assay lection of infectious M13-based bacteriophage, in which each clone of the library displays three to five copies of a unique nine-residue peptide dis- Association of p47phox and p67phox with neutrophil membranes in the pres- played at the N terminus of the pIII capsid protein. This library collectively ence of mAb NL7 was examined as described (18). The membrane fraction contains at least 5 ϫ 108 unique nine-residue sequences. Use of the peptide was prepared as described above using sonication to reduce the amount of library in this way is possible because each phage particle in the library p47phox and p67phox trapped in membrane vesicles. Briefly, 4 ϫ 107 cell displays a unique peptide on its surface and contains the encoding nucle- equivalents (40 ␮l) of neutrophil membranes were pretreated for1hat otide segment within the genome. Briefly, three sequential rounds of se- 25¡Cin6.7␮M of either mAb NL7 or an irrelevant mAb. The membranes lection and amplification of phage-display peptide library clones were con- were then allowed to react with 1.6 ϫ 107 cell equivalents (160 ␮l) of ducted using a mAb NL7 immunoaffinity matrix. After three rounds of neutrophil cytosol for 10 min at 25¡C in activation buffer (47 mM

selection, the sequences of displayed peptides were deduced by nucleotide NaH2PO4,18mMK2HPO4, 1 mM MgCl2, 1 mM EGTA, 2 mM NaN3, 125 sequence analysis of the isolated clones as described (12). ␮M LDS, 10 ␮M FAD, and 125 ␮M GTP␥S). Separation of membrane- bound from soluble factors was accomplished by two different methods, Flow cytometry both yielding the same results. In one method, the membranes were pel- Downloaded from ϫ Human neutrophils were prepared from fresh whole blood collected in leted by centrifugation at 100,000 g in a TLA-100.2 rotor for 30 min at citrate anticoagulant from normal human donors and probed by mAbs NL7, 4¡C and washed by resuspension in 1.0 ml of activation buffer. This se- 44.1, 7D5, or an irrelevant isotype-matched monoclonal as described (6). quence was repeated for two wash steps. Alternatively, the membranes ϫ Briefly, leukocytes were incubated in 200 nM of each mAb diluted in 10 were sedimented through a discontinuous sucrose gradient at 100,000 g mM PBS (10 mM phosphate, 150 mM NaCl, pH 7.4) with 4% normal goat for 30 min at 25¡C as described (19). Fractionation of the resulting gradient serum and 4% nonfat dry milk for 30 min on ice. For intracellular staining, yielded a clear peak of activity at the 20Ð50% sucrose interface (data not neutrophils were permeabilized by exposure to 0.05% saponin for 30 min shown). These fractions were then examined for the presence of cytosolic http://www.jimmunol.org/ on ice before incubation with primary Abs. Saponin (0.05%) was also factors by immunoblot as described (19, 20) using polyclonal rabbit Abs included during all washes and during incubation with secondary Ab. Final kindly provided by W. M. Nauseef (University of Iowa School of Medi- resuspension buffer consisted of PBS containing 0.1 ␮g/ml propidium io- cine, Iowa City, IA). dide to identify permeabilized cells. Data were collected using a BD Bio- Since the inception of the cell-free oxidase assay (21Ð23), several the- sciences FACScan instrument (Franklin Lakes, CA), and analyses were ories have been put forth to explain oxidase activation by anionic amphi- performed using WinMDI software (version 2.7; The Scripps Research philes (e.g., arachidonate, SDS, or LDS). There is evidence for direct in- Institute, La Jolla, CA) by gating on neutrophil populations determined teractions with the cytosolic subunits (24, 25) that induce conformational changes, fostering the notion that certain Src homology 3 binding domains from forward and side scatter profiles. Permeabilized cells were identified phox phox as having FL2 intensity signals significantly above background. in both p47 and p67 remain inaccessible until exposed to anionic amphiphile. Such effects were postulated to mimic in vivo phosphorylation Cell-free oxidase assay events where Src homology 3 domain interactions are believed to partly by guest on September 28, 2021 control oxidase activity. Other experiments conducted by our lab (26, 27) Plasma membrane-bound Cyt b and cytosolic oxidase factors were pre- and others provide evidence that the amphiphiles also interact directly with pared as described (13) using LPS-free reagents. A discontinuous 5Ð20% Cyt b to impart similar conformational effects that may effect partial oxi- Percoll gradient was used to fractionate the subcellular constituents. Con- dase control. Collectively, these studies suggest that that the amphiphiles centration dependence analysis was used to determine the optimal concen- interact with several components of the oxidase to induce full activity. trations of plasma membranes, cytosol, and lithium dodecyl sulfate (LDS) (data not shown). Superoxide assays were performed in Costar 3370 96- Binding of mAb NL7 to activated membranes by immunoblot well microtiter plates (Cambridge, MA) in assay buffer (47 mM NaH PO , 2 4 Experiments were conducted to determine whether mAb NL7 is capable of 18 mM K2HPO4, 1 mM MgCl2, 1 mM EGTA, and 2 mM NaN3) containing the following additional final concentrations: 100 ␮M cytochrome c, 125 binding to membranes preactivated with oxidase components including ␮M LDS, 10 ␮M FAD, 200 ␮M NADPH, 125 ␮M GTP␥S, 5 ϫ 106 cell cytosolic factors. For this analysis, a mixture was prepared in the activation ␮ ␮ ␥ ϫ 8 equivalents of plasma membranes, and 2 ϫ 106 cell equivalents of cytosol, buffer (above) containing 10 M FAD, 125 M GTP S, 1.5 10 cell ϫ 7 in a total volume of 200 ␮l. equivalents/ml membranes, 7.7 10 cell equivalents/ml cytosol, in either ␮ Superoxide generation rate was measured as the reduction of cyto- the presence or absence of 125 M LDS. The mixture was incubated for ⌬⑀ ϭ 15 min at room temperature to activate the membranes, followed by ex- chrome c over a selected time period, quantitated using 550 21 (cm Ϫ ␮ mM) 1 (14) blanked against wells containing 310 U/ml superoxide dis- posure for 45Ð60 min at 25¡C to mAb NL7 at 1.7 M. Irrelevant mAb was mutase (SOD). In measuring concentration dependence of mAb NL7 on also tested as a control under the same conditions, and purified goat IgG ␮ oxidase inhibition, average rates of superoxide generation were reported as was included at 20 M in all samples (which did not affect superoxide the percent of control (irrelevant Ab), where 100% corresponds to ϳ1.2 generation measured by cytochrome c reduction, data not shown) to reduce Ϫ nonspecific mAb binding. Following washing of the membranes as de- mole of O2 /mole Cyt b heme/sec. Aliquots containing different concentrations of each mAb were mixed with an equal volume of membrane scribed for the translocation assay, SDS-PAGE and transfer of proteins to fraction (5 ␮lat1ϫ 109 cell equivalents/ml), and allowed to react for 1 h nitrocellulose was conducted so that the IgG subunits associated with the at room temperature before addition of the remaining reagents (causing a membranes could be determined semiquantitatively using an IgG-specific 20-fold dilution of the membranes and Ab concentration reported). To test secondary Ab. the ability of the mAbs to inhibit superoxide generation after activation, Ab Kinetic analysis concentration in the cuvette was adjusted to 6.7 ␮M after production of superoxide was occurring at a maximal linear rate. For INT reduction as- Kinetic analysis of NL7 inhibition was examined with respect to the says, reaction conditions were identical to cytochrome c reduction except NADPH concentration as described (28). Serial 0.67-fold dilutions of that 100 ␮M INT was used in place of cytochrome c. Sodium azide is NADPH were added to the cell-free assay as described above but with included in these reactions as described (15, 16) to inhibit the activity of membranes that had been kept at 25¡Cfor1hinthepresence of 3.4 ␮M heme-containing redox systems such as myeloperoxidase which catalyze mAb NL7 or a PBS control. The rates of superoxide generation were then

the conversion of H2O2 to HOCl and other oxidants. Contribution by these determined by the cytochrome c reduction assay (above), and plotted as a oxidants would otherwise interfere with the measurement of cytochrome c function of the NADPH concentration. The data were then analyzed by reduction as a function of superoxide production by the NADPH oxidase (17). nonlinear regression (GraphPad Prism version 3.00 for Windows; Graph- To measure possible competitive binding between mAb NL7 and the Pad Software, San Diego, CA). The mAb NL7-mediated percent inhibition cytosolic factors by oxidase rate, 7 ϫ 106 cell equivalents of membranes was then plotted from these data by subtracting the percent of activity were simultaneously mixed with the indicated amount of cytosol and NL7 remaining after NL7 treatment from 100%. 6084 OXIDASE INHIBITION BY AN ANTI-gp91phox Ab

Structure modeling munoreactivity was also shown using heparin eluate probed by the phox phox 291 previously characterized anti-gp91 mAb 54.1 (lane 3), but not for Most of the C-terminal domain of gp91 , beginning at Ser , was mod- phox eled using the Web-based program 3D-PSSM (29), available at www. mAb 7D5 (lane 4) which binds native, but not denatured, gp91 . bmm.icnet.uk/ϳ3dpssm (Web site of the Biomolecular Modeling Labora- The membrane sidedness of the epitope bound by mAb NL7 tory of the Imperial Cancer Research Fund, London, U.K.). This program was examined by flow cytometry of both intact and saponin-per- uses a novel Web-based sequence alignment and “threading” program (3D- meabilized neutrophils that had been exposed to mAb NL7. This PSSM) which compares the primary sequence of the protein of interest to those with known three-dimensional structure to determine residues that analysis indicated the epitope is not accessible on intact neutro- are structurally equivalent. Identified residues are then used to extend the phils (Fig. 1B), compared with the positive control mAb 7D5 multiple sequence alignment to new proteins, creating a database of known which binds an extracellular epitope on human (9) and primate structures that can be used to model the unknown protein. The program neutrophils (31). However, following permeabilization of neutro- then predicts coordinates for the atoms of a protein of unknown structure, based on a threading algorithm applied to structures selected by 3D-PSSM phil membranes with 0.05% saponin the staining by mAb NL7 that are most similar to the unknown structure. These structures were then increased significantly, producing a signal that was slightly greater scored for one-dimensional sequence alignment, matching of predicted sec- than the positive control mAb 44.1 (Ref. 6; Fig. 1C). The labeling ondary structural elements, and solvent accessibility. From this analysis it on permeabilized but not intact, neutrophils by mAb NL7 sug- was determined that the structure with closest fit (lowest E-value) to phox gested both a cytosolic and solvent-accessible location of the gp91 was corn nitrate reductase (30) (Brookhaven Database Coordinate phox filename, 1CNF). A rasmol file output of the gp91phox fragment was gen- epitope for NL7 binding on gp91 of the native protein. phox erated by 3D-PSSM which was then converted to pdb file format by a text The specificity of mAb NL7 for gp91 was further confirmed editor. The file was then examined using the SwissPDB viewer available on by phage display epitope mapping (9, 11). A six-log increase in the SwissProt Web site (http://www.ebi.ac.uk/swissprot/), or by the Insight adherent phage numbers from the first to third selection was ob- Downloaded from II program obtained from Accelerys (San Diego, CA). served and positive plaque lifts (7) confirmed specific selection of Results immunoreactive sequences had taken place (data not shown). The deduced peptide sequences obtained from third round eluate phage To produce novel probes of the phagocyte NADPH oxidase, mAbs clones showed an obvious similarity to the 498EKDVITGLK506 were generated against partially purified neutrophil Cyt b. One region of gp91phox (Fig. 2). Each selected peptide shown contains such mAb, mAb NL7, bound a broad band suggestive of gp91phox

from four to eight residues similar to those of the identified http://www.jimmunol.org/ present in neutrophil membrane extracts prepared by heparin af- epitope. Residues showing an exact match to residues of the finity chromatography, shown by immunoblot in Fig. 1A (lane 1, epitope are shown in blue, and those suggesting a conservative labeled HE for heparin eluate). Specificity of mAb NL7 for the substitution are shown in red. The diversity of the J404 phage- gp91phox subunit was confirmed using Cyt b which had been pu- display peptide library is estimated to include over 5 ϫ 108 unique rified on mAb 44.1 (8) (lane 2, labeled IP for immunopurified) nonapeptides (11), and the consensus provided by immunoa- which will immunoprecipitate the Cyt b heterodimer (9). Similar im- ffinity selection on mAb NL7 provides a very strong match to the by guest on September 28, 2021

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 con- FIGURE 2. Phage-displayed peptide sequences matching 498EKDVIT- ducted either by heparin affinity chromatography (HE) or by immunoaf- GLK506 of gp91phox. Three rounds of selection were used to enrich immu- finity purification using the Cyt b-specific mAb 44.1 (IP). The electrotrans- noreactive peptide sequences from the J404 phage-display peptide library ferred protein was probed with 13 nM of the primary Ab indicated, and a (11), which were then subjected to nucleotide sequence analysis to deter- goat-anti-mouse Ig secondary Ab was conjugated to alkaline phosphatase mine the displayed peptide. These sequences were then compared with the for reaction development. B, Flow cytometry of intact human neutrophils sequence of gp91phox, and found to align with the 497EEKDVITGLKQ507 probed with anti-Cyt b and control mAbs indicated. Intact neutrophils (1 ϫ region of gp91phox as shown. Residues of selected peptides identical to corre- 106) were exposed to 200 nM of the mAbs indicated on ice for 30 min before sponding residues of gp91phox are shown in blue, and those with side chain washing and labeling with goat-anti-mouse Ig FITC-conjugated secondary Ab. similarities are shown in red. Only three sequences appearing after the third C, Immunoreactivity of the mAbs indicated is shown to 0.05% saponin-per- round of selection did not appear to fit the consensus (not shown), and se- meabilized cells. The data are representative of at least three separate analyses. quences appearing on more than one occasion are indicated in parentheses. The Journal of Immunology 6085

498EKDVITGLK506 region on gp91phox. Thus, the peptides shown Binding of mAb NL7 to gp91phox does not perturb translocation in Fig. 2 collectively provide an unambiguous identification of the of p47phox, p67phox, or Rac during oxidase activation phox epitope on gp91 bound by mAb NL7. Previous biochemical analyses of a CGD patient with a Asp500 3 Gly substitution in gp91phox revealed normal Cyt b expression lev- els, but an impaired translocation of both p47phox and p67phox (33). mAb NL7 inhibits oxidase activation This mutation is located within the NL7 epitope on gp91phox, sug- Exposure of mAb NL7 to neutrophil membranes for1hat25¡C gesting the Ab might inhibit oxidase activation by preventing before oxidase measurement resulted in a concentration-dependent translocation of p47phox or p67phox. We thus examined the trans- inhibition of NADPH oxidase activity. The half-maximal effect location of p47phox, p67phox, and Rac after pretreating 4 ϫ 107 cell occurred at ϳ400 nM Ab (Fig. 3). However, addition of NL7 to the equivalents of neutrophil membranes with 6.7 ␮M mAb NL7 or an system after reaching the maximal superoxide generation rate had isotype-matched irrelevant mAb before addition of the remaining no measurable effect when compared with an irrelevant mAb assay constituents including cytosol and LDS. Semiquantitative control (Fig. 3, inset), indicating that the inhibitory effects of mAb immunoblotting of these membrane fractions indicated mAb NL7 NL7 were not exerted once the oxidase was assembled and func- had no effect on the amount of p67phox or p47phox (Fig. 4A, top panel), tioning. The ability of mAb NL7 to bind the activated complex or Rac (bottom panel) associated with the membrane by activation was further tested as described below to determine whether the with LDS (lane 1) compared with the irrelevant mAb control (lane 2). 498EKDVITGLK506 region of gp91phox is accessible to Ab follow- The level of p47phox, p67phox, and Rac signal from membranes treated ing cytosolic factor translocation. without LDS is shown in lane 3, which was subtracted from all den- Downloaded from To dissect the effect of mAb NL7 on electron transport in the sitometry measurements before graphing (Fig. 4C). oxidase, INT was used as an alternative electron acceptor in the Our data indicate that mAb NL7 binding does not inhibit the phox phox cell-free assay. In this application (32), INT was reduced directly translocation of p47 , p67 , or Rac to the membranes. Our by oxidation of the Cyt b flavin moiety. Fig. 3 shows that mAb data also showed that while mAb NL7 inhibited oxidase function NL7-mediated inhibition of INT reduction is similar to that of when exposed to the membranes before activation, it had no mea- cytochrome c reduction, suggesting that the inhibitory effect of surable effect on activity following activation. However, these data http://www.jimmunol.org/ 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. by guest on September 28, 2021

FIGURE 3. mAb NL7 concentration-dependent inhibition of oxidase activity. Both cytochrome c (ࡗ) and INT reduction (f) were inhibited by FIGURE 4. Cytosolic factor translocation and mAb NL7 binding to ac- ␮ mAb NL7 in a cell-free assay using 200 M NADPH as shown. Serial tivated membranes. A, Top and bottom panels:4ϫ 107 cell equivalents of 4-fold dilutions of each mAb were preincubated for 1 h with neutrophil neutrophil plasma membranes were exposed to 6.7 ␮M of mAb indicated, membranes before analysis, using the electron acceptor indicated. One- Ϫ then reacted with oxidase components including LDS. The membranes hundred percent activity is equivalent to 1.2 mole O2 /mole of Cyt b heme/ were then pelleted and washed to remove unbound cytosolic factors. Trans- second. Identical conditions including an isotype-matched irrelevant con- located p67phox and p47phox (top panel) were detected by immunoblot using trol mAb did not show significant effects in either cytochrome c ( )orINT polyclonal Abs to each subunit, while Rac was detected with a mAb (bot- (Œ) reduction. Cyt b-specific mAbs 44.1 (ϫ) and 54.1 (F) (6) also had no tom panel). B, The similar amount of NL7 Ab subunits bound to cytosol effect on oxidase activity when tested at the highest mAb concentration and LDS-activated (lane 1) and unactivated (lane 3) membranes, compared examined. Inset, Monoclonals (NL7 (छ) or irrelevant (Ⅺ)) were added to with that of an irrelevant mAb (lane 2). C, Densitometry results for measure- a final concentration of 6.7 ␮M during the cytochrome c reduction (arrow- ments in A and B. For graphing of p47phox (f), p67phox (Ⅺ), and Rac (o), the head) to test their ability to inhibit superoxide generation once the oxidase LDS-independent signal (A, lane 3) was subtracted from signal in lanes 1 and Data are rep- 2. For the IgG H subunit signal (u), the irrelevant mAb signal (B, lane 2) was .(ء) had assembled, and are compared with the SOD control resentative of five experiments and error bars indicate SEM of four repli- subtracted from the signal resulting in lanes 1 and 3. The data are represen- cates from different experiments. tative of four experiments where they represent SEM, n ϭ 4. 6086 OXIDASE INHIBITION BY AN ANTI-gp91phox Ab 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 ␮M LDS, the activated complex was then diluted in assay medium containing cytochrome c, and the oxidase rate was measured fol- Downloaded from lowing addition of NADPH (Fig. 5). These activating conditions included higher concentrations of cytosol and a longer preincuba- tion 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 ␮M), the

EC50 values for cytosol were 3.79 (3.31Ð4.35), 2.28 (2.62Ð3.08), http://www.jimmunol.org/ and 2.7 (2.45Ð2.96 ϫ 106 cell equivalents per well), respectively. Thus, there is no reduction on mAb NL7-mediated oxidase inhibition, due to increasing concentrations of the cytosolic FIGURE 6. Oxidase inhibition by mAb NL7 corresponding to NADPH concentration. A, The SOD-inhibitable superoxide generation rate was de- NADPH oxidase factors. termined after incubation in the absence (Œ) or presence (f) of mAb NL7 at 3.4 ␮M (a relative rate of 15 corresponds to 0.55 mol O Ϫ/mol of Cyt Examination of potential changes in NADPH binding on 2 b heme/second). Nonlinear regression analysis of the oxidase rate was per- gp91phox caused by mAb NL7 formed for both data sets to examine substrate-binding parameters. The Homology studies suggested that the 504GLKQKTLYGR513 re- thin line represents the curve fit to the data points modeled to a single gion of gp91phox may be important for binding of NADPH (34). NADPH binding site as described in Materials and Methods. The bold line by guest on September 28, 2021 This region, which slightly overlaps the epitope bound by mAb shows the same data fit to a two binding site model. Inset, The percent of Œ NL7, could be sequestered by bound Ab, thus obstructing the ac- NL7-mediated inhibition at the NADPH concentrations tested ( ). B, The V and K values corresponding to the binding site models are shown. cess of NADPH to gp91phox. To test this hypothesis, we examined max m Comparison of the curve fits with and without mAb NL7 indicate that the the NADPH concentration dependence of the oxidase in the cell- two binding site model gives a significantly better fit to the experimental free assay in the presence of 400 nM mAb NL7 compared with the results. Data are representative of four experiments, and error bars repre- effect of a PBS control (Fig. 6A). A hyperbolic concentration de- sent SEM of four replicates using different experiments. pendence of the superoxide generation rate on NADPH was ob- served 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%.

Discussion Our results indicate that mAb NL7 recognizes the 498EKDVIT- GLK506 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 FIGURE 5. Concentration dependence of cytosol and NL7 on oxidase oxidase by several immunologic methods. In addition to binding to activity. The superoxide generation rate was determined after simulta- phox neously exposing the membranes to NL7 at 0 ␮M(f), 0.42 ␮M(ࡗ), and a speciﬁc segment of gp91 , mAb NL7 was found to inhibit the 6.7 ␮M(Œ) and increasing concentrations of cytosol as indicated as de- function of the NADPH oxidase. These characteristics allowed mAb scribed in Materials and Methods, where the maximum rate (100%) was NL7 to be used as a probe of superoxide production at the level of Cyt Ϫ equal to 18 mol O2 /mol of Cyt b heme/second. Data indicate SEM of four b structure and thus provide insight into the possible role of the replicates, and are representative of separate analysis. 498EKDVITGLK506 region of gp91phox in oxidase activation. The Journal of Immunology 6087

The epitope bound by mAb NL7 contains the residue Asp500 of The conditions in which experiments for Figs. 3 and 6 were gp91phox, which was previously reported to be the locus of a CGD done are not identical. Fig. 3 tests mAb NL7 between 7 and 6700 point mutation that prevented translocation of cytosolic factors nM as the NADPH concentration is held constant at 200 ␮M. For p47phox and p67phox (33). This observation led to the hypotheses Fig. 6, mAb NL7 is held constant at 3400 nM while the NADPH that Asp500 might be part of a docking site for the cytosolic com- concentration is varied from 2.3 to 89 ␮M. Therefore, direct components or that mutation of this residue perturbed the structure of parisons between the two conditions in which component ranges gp91phox and inhibited translocation indirectly. Our results are do not overlap may be misleading. consistent with the latter hypothesis and indicate that the inhibitory To gain a better understanding of the influence of mAb NL7 effects of mAb NL7 binding to the 498EKDVITGLK506 epitope do binding on oxidase activity, we modeled most of the C-terminal not appear to directly affect membrane translocation of p47phox, domain of gp91phox (amino acid residues 291S-F565) to the known p67phox, or Rac. However, there is evidence that cytosolic oxidase crystal structure of a related protein, corn nitrate reductase (pdb subunits may be translocated en bloc as a complex with p40phox 1CNF) (30). In the space-filling and ribbon images showing the (36), and therefore dock to Cyt b by multiple contact sites. There- crystal structure of corn nitrate reductase (Fig. 7, A and C), the blue fore, obstruction of a single contact by mAb NL7 may be insuf- and purple colors correspond to space-filling representation of the ficient to prevent membrane translocation, but could inhibit func- bound and cocrystallized ADP and FAD cofactors, respectively. tional interactions between Cyt b and the cytosolic factors. Cocrystallization with NADPH or its oxidized form was not The observation that mAb NL7 binds 498EKDVITGLK506 but achieved, so the bound ADP represents the ADP moiety of bound does not inhibit cytosolic factor translocation is consistent with NADPH. The resulting model of gp91phox (superimposed on the

reports that inclusion of peptides mimicking the nearby 484D-I502 coordinates of 1CNF) was viewed in the same orientation as Downloaded from region in a cell-free system (37) or by creating a deletion mutant 1CNF, and is shown in Fig. 7, B and D. The amino acid residues corresponding to 488A-E497 (38), have little effect on NADPH ox- of gp91phox predicted to contact the FAD and the ADP moiety of idase activity. However, a synthetic peptide corresponding to 491F- NADPH are shown colored in purple and blue, respectively. The G504 of gp91phox was shown to inhibit both cytochrome c reduc- location of 498EKDVITGLK506 of gp91phox, shown in green on ␮ tion (EC50 10 M) and cytosolic factor translocation (33), this model, suggests the placement of the mAb NL7 epitope. This phox supporting the role for this region of gp91 in some aspect of view predicts the mAb NL7 epitope is close to, but not overlap- http://www.jimmunol.org/ oxidase function, if not direct binding to cytosolic factors. ping, the proposed NADPH or FAD binding site. Competitive binding between mAb NL7 and the cytosolic factors Despite evidence for direct interaction between gp91phox and was further discounted by two separate analyses, thus corroborating p67phox (42), the specific residues that mediate this interaction our translocation data shown in Fig. 4. First, membranes activated have not been identified, and we were thus unable to assess their with LDS in the presence of cytosolic factors were found to bind mAb relative placement with respect to the NL7 epitope on the modeled NL7 as strongly as those not exposed to LDS activation and translo- surface of gp91phox using structural modeling. However, some res- cation (Fig. 4B, compare lanes 1 and 3). Second, our data relating idues mediating contact between gp91phox and p47phox have been cytosol and mAb NL7 concentration to oxidase rate indicate that in- described, and were modeled to suggest their proximity to the creasing cytosol concentrations did not reverse the degree of mAb epitope bound by NL7. The model of gp91phox shown in Fig. 7, B by guest on September 28, 2021 NL7 inhibition (Fig. 5). Thus, the mechanism of inhibition by the Ab and D, was modified so that residues believed to contact p47phox probably involves independent binding of Ab and cytosolic factors. are shown in pink (Fig. 8) (33, 43Ð45). This view of gp91phox The nonlinear regression analysis relating oxidase rate to shows residues 555ESGPRGVHFIF565 in pink (Phe565 is red and NADPH concentration indicated the most accurate modeling of the represents the C terminus). This region of the molecule has been data involved a two NADPH binding site fit (Fig. 6), as previously identified as a potential p47phox contact site based on CGD analysis suggested (35, 39). According to this view, the kinetic parameters (46), synthetic peptide inhibition of oxidase activation (43, 45) and influenced by NL7 correspond to the low affinity (ϳ26 ␮M) translocation (44), and selection by phage display analysis of phox phox NADPH binding site, presumably on gp91 (Fig. 6B). This Km p47 affinity matrices (45). Such a placement also suggests that value is similar to previous reports of ϳ35 ␮M for NADPH on the C-terminal residues of gp91phox, 566NKENF570 (not shown in human neutrophil membranes (22), between 49 and 94 ␮M for the model), might lay over the cleft proposed to contain the membranes from stimulated mouse macrophages (28), and below NADPH binding site of gp91phox and would allow Phe570 to co- 50 ␮M in a detergent-solubilized human neutrophil system (40). ordinate the isoalloxazine ring of the FAD as originally suggested Because several regions of gp91phox proposed to contribute to the by Taylor et al. (47) from studies of similarity to the ferridoxin NADPH binding (41) are close in primary sequence to the epitope nucleotide reductases. According to this model, 555ESGPRGVH- bound by mAb NL7 (see below), we examined the kinetic data to FIF565 is well-removed from the NL7 epitope (shown in green), determine whether the inhibitory effect was the result of compet- having at least 15Å separating the two regions at closest approach. itive binding between mAb NL7 and NADPH. Because the only From the model shown in Fig. 8A, the opposite face of significant effect mAb NL7 had on oxidase kinetics was to reduce gp91phox is obtained when the molecule is rotated 180o around

the low affinity Vmax (Fig. 6B), Ab binding is not suggested to the y-axis and is shown in Fig. 8B. On this face of the structure, compete with NADPH binding. the residues of 555ESGPRGVHFIF565 are obstructed from view. The two fitted curves in Fig. 6A indicate the degree of NL7- However, the other segment of gp91phox previously identified as mediated inhibition increased with increasing NADPH concentra- a potential p47phox binding site, 451FEWFADLL458 (again tions, and this inhibition saturated at ϳ50% in the presence of shown in pink), is positioned in a depression on the surface but 20Ð30 ␮M NADPH (summarized in the inset). This result corre- has solvent-accessible residues that lie near His354 (purple) sponds to the kinetic data fit by a two NADPH binding site model, which is predicted to interact with the FAD. The model suggests

in which the low Vmax and Km (high afﬁnity) form is not inhibited that this region of the molecule is also remote from the NL7 by the Ab (Fig. 6B). This conclusion is compatible with Fig. 3, which epitope. Interestingly, the two regions of gp91phox reported to indicates an inability to completely inhibit the oxidase with increasing interact with p47phox are on opposite sides of the modeled pro- concentrations of NL7 (inhibition asymptotically approaches ϳ90% tein. DeLeo et al. (45) also identiﬁed the 87STRVRRQL94 re- with increasing NL7 concentrations; data not shown). gion on gp91phox (not included in the model), which resides on 6088 OXIDASE INHIBITION BY AN ANTI-gp91phox Ab

FIGURE 7. Structural model compar- ing 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 rendi- tion, respectively. The cocrystallized het- eroatoms 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 Materi- als 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 Downloaded from 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 http://www.jimmunol.org/ and red, respectively.

the cytoplasmic aspect of the N-terminal half of the molecule as for electron transport during activation. This latter view is com- a p47phox interactive site, suggesting that contact between patible with our evidence (26) and reports of others that suggest p47phox and Cyt b must be relatively complex. Cyt b undergoes structural rearrangement during oxidase acti- Current information about the region of gp91phox in the vi- vation (48, 49). by guest on September 28, 2021 cinity of the epitope bound by NL7 suggests a more complex Identification of the functional significance of the 498EKD- role for 498EKDVITGLK506 of gp91phox, beyond that of a dock- VITGLK506 epitope of gp91phox bound by mAb NL7 provides a ing site for the cytosolic oxidase factors. Our results indicate link between Cyt b structure and function. Our data indicated NL7 binding does not interfere with oxidase substrate or sub- that this domain resides on the cytosolic aspect of gp91phox, and unit involvement. The results are nevertheless compatible with that the inhibitory effects of mAb NL7 on oxidase activation are mAb NL7-mediated oxidase inhibition involving perturbations exerted before or during the initial stages of electron transfer. of electron transport within the complex, or by restricting ac- Availability of mAb NL7 as a Cyt b-specific inhibitor of oxidase tivation-dependent flexibility of the protein, in which NL7 bind- function may enable a better understanding of oxidase activation and ing prevents spatial reorganization of a Cyt b domain necessary thus control. We conclude that the 498EKDVITGLK506 domain of

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, 555ESG- PRGVHFIF565, 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. The Journal of Immunology 6089 gp91phox participates in the activation of the oxidase and influences 27. Palicz, A., T. R. Foubert, A. J. Jesaitis, L. Marodi, and L. C. McPhail. 2001. electron transport during superoxide generation. A precise description Phosphatidic acid and diacylglycerol directly activate NADPH oxidase by inter- acting with enzyme components. J. Biol. Chem. 276:3090. of the mechanism of oxidase inhibition by mAb NL7 requires addi- 28. Sasada, M., M. J. Pabst, and R. B. Johnston Jr. 1983. Activation of mouse peri- tional analysis. toneal macrophages by lipopolysaccharide alters the kinetic parameters of the superoxide-producing NADPH oxidase. J. Biol. Chem. 258:9631. 29. Kelley, L. A., R. M. MacCallum, and M. J. Sternberg. 2000. Enhanced genome References annotation using structural profiles in the program 3D-PSSM. J. Mol. Biol. 299:499. 1. Nauseef, W. M. 1999. The NADPH-dependent oxidase of phagocytes. Proc. 30. Lu, G., W. H. Campbell, G. Schneider, and Y. Lindqvist. 1994. Crystal structure Assoc. Am. Physicians 111:373. of the FAD-containing fragment of corn nitrate reductase at 2.5 A resolution: 2. Segal, B. H., T. L. Leto, J. I. Gallin, H. L. Malech, and S. M. Holland. 2000. relationship to other flavoprotein reductases. Structure 2:809. Genetic, biochemical, and clinical features of chronic granulomatous disease. 31. Yamauchi, A., L. X. Yu, A. J. G. Po¬tgens, F. Kuribayashi, H. Nunoi, Medicine 79:170. S. Kanegasaki, D. Roos, H. L. Malech, M. C. Dinauer, and M. Nakamura. 2001. 3. Berendes, H., R. A. Bridges, and R. A. Good. 1957. Fatal granulomatosus of Location of the epitope for 7D5, a monoclonal antibody raised against human childhood: clinical study of a new syndrome. Minn. Med. 40:309. phox flavocytochrome b558, to the extracellular peptide portion of primate gp91 . 4. Landing, B. H., and H. S. Shirkey. 1957. A syndrome of recurrent infection and Microbiol. Immunol. 45:249. infiltration of viscera by pigmented lipid histiocytes. Pediatrics 20:431. 32. Cross, A. R., J. L. Yarchover, and J. T. Curnutte. 1994. The superoxide-gener- 5. Clark, R. A. 1999. Activation of the neutrophil respiratory burst oxidase. J. Infect. ating system of human neutrophils possesses a novel diaphorase activity: evi- Dis. 179(Suppl. 2):S309. dence for distinct regulation of electron flow within NADPH oxidase by p67phox 6. Burritt, J. B., M. T. Quinn, M. A. Jutila, C. W. Bond, and A. J. Jesaitis. 1995. and p47phox. J. Biol. Chem. 269:21448. Topological mapping of neutrophil cytochrome b epitopes with phage-display 33. Leusen, J. H. W., M. De Boer, B. G. J. M. Bolscher, P. M. Hilarius, libraries. J. Biol. Chem. 270:16974. R. S. Weening, H. D. Ochs, D. Roos, and A. J. Verhoeven. 1994. A point mu- 7. Burritt, J. B., G. N. Fritel, I. Dahan, E. Pick, D. Roos, and A. J. Jesaitis. 2000. tation in gp91phox of cytochrome b of the human NADPH oxidase leading to Epitope identification for human neutrophil flavocytochrome b monoclonals 48 558 defective translocation of the cytosolic proteins p47phox and p67phox. J. Clin. and 449. Eur. J. Haematol. 65:407.

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