Topological Mapping of Neutrophil Cytochrome b Epitopes with Phage-display Libraries

Cytochrome b of human neutrophils is the central component of the microbicidal NADPH-oxidase system. However, the folding topology of this integral membrane protein remains undetermined. Two random-sequence bacteriophage peptide libraries were used to map structural features of cytochrome b by determining the epitopes of monoclonal antibodies (mAbs) 44.1 and 54.1, specific for the p22phox and gp91phox cytochrome b chains, respectively. The unique peptides of phage selected by mAb affinity purification were deduced from the phage DNA sequences. Phage selected by mAb 44.1 displayed the consensus peptide sequence GGPQVXPI, which is nearly identical to 181GGPQVNPI188 of p22phox. Phage selected by mAb 54.1 displayed the consensus sequence PKXAVDGP, which resembles 382PKIAVDGP389 of gp91phox. Western blotting demonstrated specific binding of each mAb to the respective cytochrome b subunit and selected phage peptides. In flow cytometric analysis, mAb 44.1 bound only permeabilized neutrophils, while 54.1 did not bind intact or permeabilized cells. However, mAb 54.1 immunosedimented detergent-solubilized cytochrome b in sucrose gradients. These results suggest the 181GGPQVNPI188 segment of p22phox is accessible on its intracellular surface, but the 382PKIAVDGP389 region on gp91phox is not accessible to antibody, and probably not on the protein surface. Cytochrome b of human neutrophils is the central component of the microbicidal NADPH-oxidase system. However, the folding topology of this integral membrane protein remains undetermined. Two random-sequence bacteriophage peptide libraries were used to map structural features of cytochrome b by determining the epitopes of monoclonal antibodies (mAbs) 44.1 and 54.1, specific for the p22phox and gp91phox cytochrome b chains, respectively. The unique peptides of phage selected by mAb affinity purification were deduced from the phage DNA sequences. Phage selected by mAb 44.1 displayed the consensus peptide sequence GGPQVXPI, which is nearly identical to 181GGPQVNPI188 of p22phox. Phage selected by mAb 54.1 displayed the consensus sequence PKXAVDGP, which resembles 382PKIAVDGP389 of gp91phox. Western blotting demonstrated specific binding of each mAb to the respective cytochrome b subunit and selected phage peptides. In flow cytometric analysis, mAb 44.1 bound only permeabilized neutrophils, while 54.1 did not bind intact or permeabilized cells. However, mAb 54.1 immunosedimented detergent-solubilized cytochrome b in sucrose gradients. These results suggest the 181GGPQVNPI188 segment of p22phox is accessible on its intracellular surface, but the 382PKIAVDGP389 region on gp91phox is not accessible to antibody, and probably not on the protein surface. The NADPH-oxidase system of neutrophils is a host-defensive plasma membrane redox system that produces superoxide anion (O)(1Badwey J.A. Karnovsky M.L. Annu. Rev. Biochem. 1980; 49: 695-726Crossref PubMed Scopus (839) Google Scholar, 2Morel F. Doussiere J. Vagnais P.V. Eur. J. Biochem. 1991; 201: 523-546Crossref PubMed Scopus (523) Google Scholar), which subsequently is converted to a variety of other toxic oxygen species that kill invading microbes and cause damage to tissue(3Smith R.M. Curnutte J.T. Blood. 1991; 77: 673-686Crossref PubMed Google Scholar). Humans lacking this enzyme system are unable to produce neutrophil-generated superoxide and suffer recurrent bacterial infections, granulomatous lesions of multiple organs, and early death (4). This condition was first reported in 1957(5Landing B.H. Shirkey H.S. Pediatrics. 1957; 20: 431-438PubMed Google Scholar, 6Berendes H. Bridges R.A. Good R.A. Minn. Med. 1957; 40: 309-312PubMed Google Scholar), and is known as chronic granulomatous disease(3Smith R.M. Curnutte J.T. Blood. 1991; 77: 673-686Crossref PubMed Google Scholar, 4Curnutte J.T. Clin. Immunol. Immunopathol. 1993; 67: S2-S15Crossref PubMed Scopus (153) Google Scholar, 7Dinauer M.C. Orkin S.H. Brown R. Jesaitis A.J. Parkos C.A. Nature. 1987; 327: 717-720Crossref PubMed Scopus (260) Google Scholar). Cytochrome b (also known as flavocytochrome b, cytochrome b588, cytochrome b559, and cytochrome b−245) is the central redox component of the phagocyte NADPH-oxidase system of human neutrophils. This component is a heterodimeric integral membrane protein composed of 91-kDa (gp91phox) 1The abbreviations used are: phoxphagocyte oxidasemAbmonoclonal antibodyFACSfluorescence-activated cell scannerFITCfluorescein isothiocyanateBSAbovine serum albuminPBSphosphate-buffered salineTBSTris-buffered salinePAGEpolyacrylamide gel electrophoresis. 1The abbreviations used are: phoxphagocyte oxidasemAbmonoclonal antibodyFACSfluorescence-activated cell scannerFITCfluorescein isothiocyanateBSAbovine serum albuminPBSphosphate-buffered salineTBSTris-buffered salinePAGEpolyacrylamide gel electrophoresis.1 and 22-kDa (p22phox) subunits(8Parkos C.A. Allen R.A. Cochrane C.G. Jesaitis A.J. J. Clin. Invest. 1987; 80: 732-742Crossref PubMed Scopus (311) Google Scholar, 9Parkos C.A. Quinn M.T. Sheets S. Jesaitis A.J. Molecular Basis of Oxidative Damage by Leukocytes. CRC Press Inc., Boca Raton, FL1992Google Scholar). At least two heme groups are coordinated by these subunits(10Quinn M.T. Mullen M.L. Jesaitis A.J. J. Biol. Chem. 1992; 267: 7303-7309Abstract Full Text PDF PubMed Google Scholar), and FAD and NADPH binding activities have been demonstrated(11Koshkin V. Pick E. FEBS Lett. 1994; 338: 285-289Crossref PubMed Google Scholar, 12Rotrosen D. Yeung C.L. Leto T.L. Malech H.L. Kwong C.H. Science. 1992; 256: 1459-1462Crossref PubMed Scopus (313) Google Scholar, 13Segal A.W. West I. Wientjes F. Nugent J.H.A. Chavan A.J. Haley B. Garcia R.C. Rosen H. Scrase G. Biochem. J. 1992; 284: 781-788Crossref PubMed Scopus (289) Google Scholar). The primary structure of gp91phox includes two asparagine-linked glycosylation sites (9Parkos C.A. Quinn M.T. Sheets S. Jesaitis A.J. Molecular Basis of Oxidative Damage by Leukocytes. CRC Press Inc., Boca Raton, FL1992Google Scholar) and contains five possible transmembrane regions suggested by hydropathy analysis(14Parkos C.A. Dinauer M.C. Walker L.E. Allen R.A. Jesaitis A.J. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3319-3323Crossref PubMed Scopus (242) Google Scholar, 15Royer-Pokora B. Kunkel L.M. Monaco A.P. Goff S.C. Newburger R.L. Baehner R.L. Cole F.S. Curnutte J.T. Orkin S.H. Nature. 1986; 322: 32-38Crossref PubMed Scopus (586) Google Scholar). p22phox contains three possible transmembrane regions, one of which includes a His-94 residue, conserved between species, that probably coordinates one of the cytochrome b heme irons. phagocyte oxidase monoclonal antibody fluorescence-activated cell scanner fluorescein isothiocyanate bovine serum albumin phosphate-buffered saline Tris-buffered saline polyacrylamide gel electrophoresis. phagocyte oxidase monoclonal antibody fluorescence-activated cell scanner fluorescein isothiocyanate bovine serum albumin phosphate-buffered saline Tris-buffered saline polyacrylamide gel electrophoresis. A number of studies have provided information about cytochrome b native structure. Electron microscopy and immunochemical analysis were used to localize cytochrome b in the neutrophil and eosinophil(16Jesaitis A.J. Buescher E.S. Harrison D. Quinn M.T. Parkos C.A. Livesey S. Linner J. J. Clin. Invest. 1990; 85: 821-835Crossref PubMed Scopus (104) Google Scholar, 17Ginsel L.A. Onderwater J.J.M. Fransen J.A.M. Verhoeven A.J. Roos D. Blood. 1990; 76: 2105-2116Crossref PubMed Google Scholar). In one of these studies, we found that the epitopes of cytochrome b containing the amino acid residues 547KQSISNSESGPRG559 of gp91phox and 162EARKKPSEEEAAA174 of p22phox are surface-accessible epitopes of native cytochrome b(16Jesaitis A.J. Buescher E.S. Harrison D. Quinn M.T. Parkos C.A. Livesey S. Linner J. J. Clin. Invest. 1990; 85: 821-835Crossref PubMed Scopus (104) Google Scholar). Rotrosen et al. (18Rotrosen D. Kleinberg M.E. Nunoi H. Leto T. Gallin J.I. Malech H.L. J. Biol. Chem. 1990; 265: 8745-8750Abstract Full Text PDF PubMed Google Scholar) found that synthetic peptides corresponding to the carboxyl terminus of gp91phoxinhibited NADPH-oxidase activation in electrically permeabilized cells, and antipeptide antibodies directed against this region prevented superoxide formation in a cell-free system. In addition, p22phox contains a proline-rich region in the carboxyl-terminal tail, which may provide Src homology domain binding sites, for p47phox or p47phox/p67phox complexes(19De Mendez I. Garrett M.C. Adams A.G. Leto T.L. J. Biol. Chem. 1994; 269: 16326-16332Abstract Full Text PDF PubMed Google Scholar). These data suggest functional roles for the carboxyl termini of both subunits, which are presumed to occupy cytosolic locations. Initial analysis of the folding topology of cytochrome b has been reported by Imajoh-Ohmi et al.(20Imajoh-Ohmi S. Tokita K. Ochiai H. Nakamura M. Kanegasaki S. J. Biol. Chem. 1992; 267: 180-184Abstract Full Text PDF PubMed Google Scholar), who determined accessibility of the subunits to anti-peptide antibodies and proteolytic enzymes. Two regions of gp91phox were exposed to proteolytic enzymes on the outer surface of the cell, while p22phox was not found to be sensitive to such external proteolysis(10Quinn M.T. Mullen M.L. Jesaitis A.J. J. Biol. Chem. 1992; 267: 7303-7309Abstract Full Text PDF PubMed Google Scholar). The carboxyl termini of both subunits were accessible to antibody on the internal surface of the plasma membrane(20Imajoh-Ohmi S. Tokita K. Ochiai H. Nakamura M. Kanegasaki S. J. Biol. Chem. 1992; 267: 180-184Abstract Full Text PDF PubMed Google Scholar). The protein sequence of the carboxyl-terminal half of gp91phoxshows some similarity to other NAD(P)H-oxidoreductases (12Rotrosen D. Yeung C.L. Leto T.L. Malech H.L. Kwong C.H. Science. 1992; 256: 1459-1462Crossref PubMed Scopus (313) Google Scholar, 21Taylor W.R. Jones D.T. Segal A.W. Protein Sci. 1993; 2: 1675-1685Crossref PubMed Scopus (108) Google Scholar) such as ferredoxin NADP reductase, a flavoprotein for which the crystal structure is known. Studies by Pick and co-workers (22Koshkin V. Pick E. FEBS Lett. 1994; 338: 285-289Crossref PubMed Scopus (65) Google Scholar) have shown that cytochrome b binds FAD and can function as a superoxide generating NAD(P)H-oxidase, even without added cytosolic for superoxide in other cell-free These results have that cytochrome b is the only component of the NADPH oxidase and that its binding may ferredoxin (12Rotrosen D. Yeung C.L. Leto T.L. Malech H.L. Kwong C.H. Science. 1992; 256: 1459-1462Crossref PubMed Scopus (313) Google Scholar, 13Segal A.W. West I. Wientjes F. Nugent J.H.A. Chavan A.J. Haley B. Garcia R.C. Rosen H. Scrase G. Biochem. J. 1992; 284: 781-788Crossref PubMed Scopus (289) Google Scholar) or other redox However, the structural studies by Imajoh-Ohmi et al. suggest that of the binding are This is by the of et al. that the binding component of the oxidase is in neutrophils of and chronic granulomatous and is not of cytochrome These in structure that for determining the topology of this protein are In this we have the epitopes bound by two monoclonal antibodies that a specific subunit of cytochrome b peptide In addition, we data the accessibility of these epitopes on native cytochrome b to respective bovine serum albumin and were from protein were from Western were or from and was from and was from data were a from U. S. Two libraries were used in this A and and were provided by J. G. Science. 1990; PubMed Scopus Google Scholar) of and a was in B. F. R. K. M. T. and A. J. purification of phage epitopes bound by was as phage from the phage J. G. 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PubMed Scopus Google Scholar) to phage and The of the first a used for was added to of and without Two of in Molecular Scholar) or on the was added to the cells, which were for The were a on the surface of containing or in a of the were in of by a Phage were from the as 1993; PubMed Scopus Google Scholar), and in of phage and to the which was in of phage These were that a of three and two were used to and phage from the of phage from the were used to as of were used to containing the which were were used to of in Molecular Scholar) containing the and the were in a DNA from the phage was and to the of the S. the sequence was used to the sequence of the unique region of the and were a Molecular phage of and were by of of phage from The phage were in a to the were in of and the phage were in of neutrophils were from as by A. J. Clin. Invest. Scholar). were on for to were used for each to antibody binding by cell were permeabilized on for by of in and by as were on for of the primary antibody in of to and in of the antibody in and on for were of containing as and in of containing In of mAb 44.1 was for phage phage sequence or to the to the peptide the for binding by the in of not primary or not primary and both primary mAb and the of some of intact was as without of the was determined on a a and to the phage peptides a region of cytochrome b were and as phage in of were in a for of cytochrome b, as C.A. Allen R.A. Cochrane C.G. Jesaitis A.J. J. Clin. Invest. 1987; 80: 732-742Crossref PubMed Scopus (311) Google Scholar), was of without Protein were by on polyacrylamide as C.A. Allen R.A. Cochrane C.G. Jesaitis A.J. J. Clin. Invest. 1987; 80: 732-742Crossref PubMed Scopus (311) Google Scholar, M.T. Mullen M.L. Jesaitis A.J. J. Biol. 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Biol. 1994; Scholar). of the was a provided by the of 1993; PubMed Scopus Google Scholar). of purification and were used to phage peptides bound by mAb 44.1 or and of phage bound by mAb of about was for the of binding phage for each and about for phage that the without The region was in phage selected by mAb 44.1 from the and from the as and of the from the which consensus a region of cytochrome b shown in This consensus peptide GGPQVXPI, resembles 181GGPQVNPI188 of p22phox The did not region of cytochrome b or each other not of the from the similarity to the cytochrome b and the sequence for the peptide was in of the The consensus not Phage the and and were and for by Western and Phage selected by mAb 54.1 the consensus amino acid sequence is in the region in phage phage sequence which is to 382PKIAVDGP389 of gp91phox phage that were selected from each by mAb 54.1 suggested a to this of phage selected on the without antibody suggested to cytochrome b, or to each other not Western blotting analysis was used to of the mAb for both the cytochrome b subunit and the unique on the shown in mAb 54.1 gp91phox which between the and and a proteolytic The of this can be in cytochrome b This mAb bound to the phage protein the peptide sequence which about mAb 54.1 of phage the and peptides and by Western not Phage the peptide sequence was not by mAb 54.1 the of mAb 54.1 for the sequences. in mAb 44.1 bound to a in (p22phox) and a The protein of phage containing the peptide sequence was by mAb 44.1 but not the protein of phage the peptide sequence in E. The protein of This the of the protein is amino R. J. 1992; PubMed Scopus Google other reported for this protein of mAb 44.1 and 54.1 to both the specific cytochrome b subunit and phage protein the of a on In addition, did not bind the protein on phage peptides in the the sequence of the peptide in the region the by the the peptide of the selected phage may not the and contains the residues for by the In to the accessibility of the epitopes to mAb on native cytochrome b in the cell, analysis was on mAb 44.1 intact mAb 44.1 or mAb not A of was were permeabilized and antibody only A of a of that were mAb 44.1 but not The of the of this but a as as the shown in B. This of in A probably and permeabilized as this was found to not DNA and was used in the of to permeabilized that this binding was by a intact phage the peptide sequence were used to mAb 44.1 and binding of the mAb to the 181GGPQVNPI188 of p22phox. mAb 44.1 phage a in to the of mAb mAb 44.1 was the of phage the sequence not In intact and permeabilized displayed mAb 54.1 A and as did permeabilized the antibody only a in was the by mAb 54.1 were first permeabilized this was in permeabilized primary mAb not or mAb only and This of a probably the analysis of neutrophils mAb or human neutrophils and were primary antibody 54.1 and a antibody and or the antibody only were for a as and The results were in neutrophils from human a mAb the on native the to mAb 54.1 by analysis was However, the was detergent-solubilized and cytochrome b, the mAb may have been in to accessible only in this of the mAb 54.1 binds to cytochrome b, a analysis in sucrose C.A. Allen R.A. Cochrane C.G. Jesaitis A.J. 1988; PubMed Scopus Google Scholar) was cytochrome b was mAb 54.1, or as and on a sucrose and Western were on from each a antibody specific for A.J. Buescher E.S. Harrison D. Quinn M.T. Parkos C.A. Livesey S. Linner J. J. Clin. Invest. 1990; 85: 821-835Crossref PubMed Scopus (104) Google Scholar). was used to of cytochrome b in each which was as a function of number cytochrome b and a exposed to mAb to a in the its C.A. Allen R.A. Cochrane C.G. Jesaitis A.J. 1988; PubMed Scopus Google Scholar). mAb the was from and to Cytochrome b mAb 54.1 displayed a a a of or that a of cytochrome b is by the of the cytochrome b remains mAb 54.1 a of not the that of the of cytochrome b has a to mAb of cytochrome b 44.1 and 54.1 the as of cytochrome b from of neutrophil by mAb 54.1 was only half as as by mAb 44.1 not In addition, was found to be the cytochrome b M.T. Parkos C.A. Walker Orkin S.H. Dinauer M.C. Jesaitis A.J. Nature. PubMed Scopus Google Scholar) by both 44.1 and 54.1 not for crystal or of cytochrome b are of protein be by of surface and functional and structural libraries to in determining surface features of have used two such libraries to the epitopes by each of two cytochrome and the accessibility of these epitopes to mAb binding on the native The unique peptides on phage the of were to the cytochrome b amino acid and regions of similarity were mAb was to its to bind but detergent-solubilized cytochrome b, or native cytochrome b in intact and neutrophils. This information was used to a region of cytochrome b is exposed to antibody on the cytosolic or external surface of the data suggest the by mAb 44.1 includes the amino of and mAb 54.1 binds amino of gp91phox The data a number of unique phage from each to the of the for each data from the two libraries are that between the libraries is the similarity of to and The three peptides were selected by mAb 44.1 from two libraries and the sequence The of two identical peptides is or about in J. G. Science. 1990; PubMed Scopus Google Scholar). the of the monoclonal antibody and the unique sequence of the peptide on selected that epitopes are this of protein epitopes not the may not be However, is the information be in that epitopes corresponding to regions in the by a or of the peptide F. A. A. R. 1993; PubMed Scopus Google Scholar, R.M. M.E. J. Immunol. 1994; PubMed Scopus Google Scholar). Phage selected by mAb 44.1 suggest this mAb may be to some without to the of the peptide Phage of selected by mAb the sequence which contains five of the residues found in of which are in The of this mAb to may of exposed amino acid and V. A. 1993; Full Text PDF PubMed Scopus Google Scholar) the of on the peptide This that the sequence of synthetic peptides the structure of the peptide a but in of in Jesaitis A.J. PubMed Scopus Google Scholar). information about the accessibility of the mAb to the native analysis was used and intact neutrophils. This analysis the 181GGPQVNPI188 of p22phox is accessible on the cytosolic but not external of the plasma membrane in neutrophils. The that the is but on the external surface of the cell and subsequently accessible is regions have been shown to be accessible to mAb in A.J. Buescher E.S. Harrison D. Quinn M.T. Parkos C.A. Livesey S. Linner J. J. Clin. Invest. 1990; 85: 821-835Crossref PubMed Scopus (104) Google Scholar) and permeabilized S. Tokita K. Ochiai H. Nakamura M. Kanegasaki S. J. Biol. Chem. 1992; 267: 180-184Abstract Full Text PDF PubMed Google Scholar). These regions a p22phox domain shown to be in cytosolic p47phox of the NADPH-oxidase system. these data suggest that the bound by mAb 44.1 is accessible of membrane and not of and of the cytochrome b by the of on the In addition, the that this region of cytochrome b be on the surface of the results that mAb 54.1 to bind cytochrome b on intact or cells. suggest the 382PKIAVDGP389 region of gp91phox may on external between the and transmembrane regions, a five domain of cytochrome C.A. Quinn M.T. Sheets S. Jesaitis A.J. Molecular Basis of Oxidative Damage by Leukocytes. CRC Press Inc., Boca Raton, FL1992Google Scholar). The of Imajoh-Ohmi S. Tokita K. Ochiai H. Nakamura M. Kanegasaki S. J. Biol. Chem. 1992; 267: 180-184Abstract Full Text PDF PubMed Google Scholar) this and in the region of the are the structure of the gp91phox NADPH and V. Pick E. 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Chem. 1986; Full Text PDF PubMed Google Scholar). mAb 54.1 binds gp91phox on Western but not bind native cytochrome b in intact or permeabilized neutrophils, the intracellular or of 382PKIAVDGP389 remains to be The a that is not by the mAb or is in a This region has a and in the of a sequence and is not to be in the plasma cytochrome b is immunosedimented by mAb 54.1, is a that accessibility to this of the protein is by or are other NADPH-oxidase is known to from cytochrome b in sucrose L.A. Jesaitis A.J. Quinn M.T. Science. 1991; PubMed Scopus Google Scholar). The by mAb 54.1 to be accessible the by mAb as determined by not This information data that the 382PKIAVDGP389 region of gp91phox may be sensitive to the cytosolic to this of the NADPH-oxidase subunit or or the of the on native cytochrome b in permeabilized and intact neutrophils. of the component from cytochrome b purification accessibility of the on but not of the detergent-solubilized or cytochrome b of the component from cytochrome b for of gp91phox by mAb 54.1 on Western which is to the of of p22phox by mAb 44.1 was found to be in of both 44.1 and This the epitopes by the respective are probably not by the The 382PKIAVDGP389 of mAb 54.1 be by the of This region and to amino binding sites for both FAD and as suggested by sequence other D. Yeung C.L. Leto T.L. Malech H.L. Kwong C.H. Science. 1992; 256: 1459-1462Crossref PubMed Scopus (313) Google Scholar, 13Segal A.W. West I. Wientjes F. Nugent J.H.A. Chavan A.J. Haley B. Garcia R.C. Rosen H. Scrase G. Biochem. J. 1992; 284: 781-788Crossref PubMed Scopus (289) Google Scholar). studies by this of cytochrome b or specific for other regions of cytochrome b may provide structural to a for neutrophil cytochrome phage libraries protein of the NADPH-oxidase system may protein regions in the NADPH-oxidase Jesaitis A.J. Quinn M.T. Biol. 1994; Scholar) and suggest a for the phagocyte oxidase system. are to of for phage and E. and for information of and E. for synthetic and and for synthetic the of in the of selected