Phage Display Epitope Mapping of Human Neutrophil Flavocytochrome b558

IDENTIFICATION OF TWO JUXTAPOSED EXTRACELLULAR DOMAINS*

James B. BurrittDagger, Frank R. DeLeoDagger§, Connie L. McDonald, Justin R. Prigge, Mary C. Dinauer, Michio Nakamura||, William M. Nauseef§, and Algirdas J. Jesaitis**

From the Department of Microbiology, Montana State University, Bozeman, Montana 59717, § Inflammation Program and Departments of Medicine, Veterans Affairs Medical Center and University of Iowa, Iowa City, Iowa 52242,  Wells Center for Pediatric Research, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, Indiana 46202, and the || Institute of Tropical Medicine, Nagasaki 852-8523, Japan

Received for publication, July 13, 2000, and in revised form, October 6, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Despite extensive experimental and clinical evidence demonstrating the critical role of flavocytochrome b558 (Cyt b) in the NADPH-dependent oxidase, there is a paucity of direct structural data defining its topology in the phagocyte membrane. Unlike other Cyt b-specific monoclonal antibodies, 7D5 binds exclusively to an extracellular domain, and identification of its epitope should provide novel insight into the membrane topology of Cyt b. To that end, we examined biochemical features of 7D5-Cyt b binding and used the J404 phage display nonapeptide library to identify the bound epitope. 7D5 precipitated only heterodimeric gp91-p22phox and not individual or denatured Cyt b subunits from detergent extracts of human neutrophils and promyelocytic leukemia cells (gp91-PLB). Moreover, 7D5 precipitated precursor gp65-p22phox complexes from detergent extracts of the biosynthetically active gp91-PLB cells, demonstrating that complex carbohydrates were not required for epitope recognition. Epitope mimetics selected from the J404 phage display library by 7D5 demonstrated that 226RIVRG230 and 160IKNP163 regions of gp91phox were both bound by 7D5. These studies reveal specific information about Cyt b membrane topology and structure, namely that gp91phox residues 226RIVRG230 and 160IKNP163 are closely juxtaposed on extracytoplasmic domains and that predicted helices containing residues Gly165-Ile190 and Ser200-Glu225 are adjacent to each other in the membrane.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The phagocyte NADPH oxidase is a plasma membrane redox system that produces superoxide anion (O&cjs1138;2), an essential precursor for other reactive oxygen metabolites critical for oxygen-dependent microbicidal activity (1-3). A genetic lesion affecting any one of four of the oxidase components, gp91phox, p22phox, p47phox, or p67phox, results in defective oxidase activity and the inability of phagocytes to kill pathogenic microorganisms, a disorder clinically recognized as chronic granulomatous disease (CGD)1 (2, 4-9). Human neutrophil flavocytochrome b558 (Cyt b) is a heme-containing, heterodimeric integral membrane protein composed of subunits gp91phox and p22phox (10). Cyt b is the electron transferase of the NADPH oxidase, relaying electrons from bound NADPH within the cell to an oxygen acceptor region of Cyt b on the exterior aspect of the cell membrane where O&cjs1138;2 is formed. In this functional capacity, Cyt b has been established as an essential component of the respiratory burst oxidase, although little published experimental data describe its topology in the membrane.

Determination of structural and functional aspects of epitopes bound by specific antibodies can provide information about the protein against which the antibody is directed (11). Antipeptide and antisubunit polyclonal antibodies against regions of Cyt b have been used to locate the corresponding epitopes (12, 13) and information derived from identification of epitope mimetics has led to the description of anti-Cyt b "antibody imprints" (14). We continue to elucidate the structure of Cyt b domains recognized by monoclonal antibodies to better define its transmembrane topology and gain insight into its functional organization.

Reported epitope mapping data for monoclonal antibodies (mAbs) specific for Cyt b indicate that they bind cytosolic aspects of the protein (15-19). However, it has been reported that mAb 7D5 (19) binds an extracellular Cyt b epitope on intact neutrophils derived from normal but not CGD patients that lack Cyt b (20). Thus, 7D5 has proven useful in the determination of Cyt b up-regulation as an indicator of neutrophil activation and granule exocytosis (21) and for the identification of individuals deficient in Cyt b (20, 22, 23). However, neither the subunit location nor chemical nature of the 7D5 epitope has been elucidated.

In our current studies, we have used phage display and immunological analyses to identify the 7D5 epitope on Cyt b. Although we confirmed the inability of 7D5 to recognize Cyt b on immunoblots, we found that 7D5 immunoprecipitated detergent-solubilized Cyt b heterodimer containing its fully processed 91kDa form of gp91 phox and its 65-kDa precursor. Additionally, it precipitated the deglycosylated gp91phox core protein,2 suggesting that neither the mature nor high mannose-containing carbohydrate contributes significantly to the epitope. Furthermore, our results indicate that the 226RIVRG230 and 160IKNP163 segments of gp91phox form the 7D5 epitope and therefore must be exposed on the cell surface. These sequences of gp91phox were not bound by 7D5 in the absence of p22phox nor under conditions that disrupted the heterodimer, suggesting that, although 7D5 binding is confined to nonlinear but contiguous regions of gp91phox, it depends on associated p22phox for its conformational integrity. In combination, these data provide direct evidence for the identity of two adjacent transmembrane helices in gp91phox.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals, Reagents, and Materials-- Prestained protein molecular weight standards were purchased from Life Technologies, Inc. Reagents for buffers and bacteriological media and Nunc Maxisorb flat bottom plates for ELISA were purchased from Fisher. Cyanogen bromide-activated Sepharose CL-4B and GammaBind Sepharose were purchased from Amersham Pharmacia Biotech. Sequencing data were obtained using a Sequenase version 2.0 sequencing kit purchased from U.S. Biochemical Corp. Unless specified, all other reagents were purchased from Sigma.

Neutrophil Isolation and Flow Cytometry-- Heparinized, venous blood was obtained from healthy individuals (or from patients with CGD) in accordance with a protocol approved by the Institutional Review Board for Human Subjects at the University of Iowa, and neutrophils were isolated as described previously using Hypaque-Ficoll gradients after dextran sedimentation (24). Genetic analyses of the individuals with either X-linked deficiency of gp91phox or autosomal deficiency of p22phox were performed by Paul G. Heyworth (The Scripps Research Institute, La Jolla, CA) and John T. Curnutte (Genentech, Inc.). The individual with autosomal deficiency of p22phox (A220) had a nucleotide replacement at position C354 (C354 right-arrow A), which resulted in the replacement of Ser118 with Arg. The individual with deficiency of gp91phox (X910) had a frameshift in exon 11, resulting in the replacement of nine nucleotides with eight. Surface-expressed Cyt b was detected using 7D5 by flow cytometry as described previously (21). Samples were analyzed on a FACscan flow cytometer (Becton Dickinson, San Jose, CA) at the University of Iowa Core Flow Cytometry facility. For most experiments, unfixed neutrophils were used, and a single live gate eliminated debris and contaminating cells. However, when patient cells were shipped overnight, it was sometimes necessary to use propidium iodide staining in combination with an additional gate to further exclude dead cells from the analysis.

Cyt b ELISA-- 35 µl of relax buffer (10 mM Hepes, 100 mM KCl, 10 mM NaCl, pH 7.4) containing 8 pmol of heparin-purified Cyt b (determined by spectral absorbance at 414 nm, extension coefficient = 21.6 mM-1 cm-1) (25), in 2% octyl glucoside was used to coat each well of a 96-well Corning Maxisorb ELISA plate overnight at 4 °C. Rinsing and blocking were performed as described (14), except a different blocking buffer (Hanks' balanced salt buffer with 10 mM HEPES, pH 7.4, 0.5% bovine serum albumin) was used. Cyt b was probed for 1 h at 25 °C with 80 µl of the indicated mAb at a concentration of 3 µg/ml. To measure the ability of the intact phage clones to block the binding of 7D5 to immobilized Cyt b, the antibody was diluted to 3 µg/ml and exposed to 2.5 × 1012 plaque-forming units for 30 min 25 °C. Pretreated 7D5 was then exposed to the immobilized Cyt b for 4 h. The reactivities of the mAbs for the Cyt b were determined by probing the wells with goat anti-mouse secondary antibody conjugated with horseradish peroxidase as described above.

Immunoprecipitation of Cyt b-- Following treatment with 4 mM diisopropylfluorophosphate (Sigma) for 15 min on ice, 5 × 106 human PMNs were washed and then resuspended in 200 µl of lysis buffer (Tris-buffered saline, pH 7.5, containing 0.1 mg/ml pepstatin A, 0.1 mg/ml leupeptin, 1.0% Triton X-100, 0.5% cetyltrimethylammonium bromide, and 2.0 mM phenylmethylsulfonyl fluoride). Cells were sonicated at level 10 for 10 s using a probe sonicator (Heat Systems Inc., Farmingdale, NY) and then incubated on ice for 10 min. Lysates were precleared using Pansorbin Cells (Calbiochem) as described previously (26) and then divided into two 100-µl aliquots. For nondenaturing immunoprecipitations, one of the aliquots was diluted to 1.2 ml with dilution buffer (50 mM Tris-HCl, pH 7.4, 190 mM NaCl, containing 2.5% Triton X-100). 10-15 µg of monoclonal antibody, 7D5, or those specific for gp91phox (54.1 (alpha gp91)) and p22phox (44.1 (alpha p22)) or an IgG1 control mAb (Cappel, Organon Teknika Co., Durham, NC) was added to the diluted lysate and rotated at 4 °C for 4 h. Subsequent steps were performed as described previously (26). For denatured lysates, the 100-µl aliquot was made 1% SDS, heated to 100 °C for 3-4 min, and then iced immediately for 5 min. Lysate was then diluted to 1.2 ml with dilution buffer so that SDS concentration was 0.1%, and precipitation was performed as described for nondenaturing immunoprecipitations (above). Alternatively, Cyt b was precipitated from solubilized gp91-PLB cells, a described previously human promyelocytic leukemia cell line constitutively expressing gp91phox (27), using 7D5, alpha gp91, and alpha p22. Cyt b was precipitated from gp91-PLB or a human promyelocytic leukemia cell line with the CYBB gene deleted (XCGD-PLB) as reported earlier (26), and denaturing conditions were as described above. Following 5-20% SDS-PAGE, immune complexes were immunoblotted using a combination of alpha gp91 and alpha p22. Immunoblots were developed using an enhanced chemiluminescence detection system (SuperSignal Substrate; Pierce) according to the manufacturer's instructions.

Antibody and Phage Display Epitope Mapping-- The generation of monoclonal antibody 7D5 has been described previously (19), and the production of the J404 nonapeptide phage display library was reported (28). 7D5 was purified from spent RPMI 1640 media of a hybridoma cell line using GammaBind Sepharose (Amersham Pharmacia Biotech) according to the manufacturer's instructions, and purity was assessed by SDS-PAGE. Mapping of 7D5 with the phage display library and plaque lift analyses were carried out as described (16), except the affinity-selected clones were amplified following each round of selection by replicating as plaques on a lawn of K91 cells (instead of as K91 colonies on LB agar containing 75 µg/ml kanamycin).

Immunoblots of Phage-displayed Sequences-- 5 × 1010 plaque-forming units of phage produced as described above were disrupted with SDS loading buffer at 100 °C for 5 min and loaded onto a 5-20% SDS-PAGE gel (29) to separate capsid proteins. Following transfer to nitrocellulose, the immunoblot was probed with 5 µg/ml 7D5 and detected by goat anti-mouse alkaline phosphatase-conjugated secondary (Bio-Rad) in combination with chromagen reagent (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) as described (16).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Specificity of mAb 7D5-- 7D5 has been previously reported to recognize Cyt b expressed at the plasma membrane of neutrophils but not on cells from X-linked CGD individuals (19, 30). The absence of either gp91phox or p22phox in X-linked or autosomal recessive CGD, respectively, results in the absence of both proteins on the neutrophil membrane (5, 31). Thus, to rule out the possibility that 7D5 binding is dependent on transcriptional regulation of only gp91phox and not p22phox, we compared its binding to cells derived from homozygous and heterozygous individuals with autosomal and X-linked inheritance of the disease. Intact neutrophils derived from individuals deficient in gp91phox (X910) or p22phox (A220) were probed for surface expression of Cyt b using 7D5 in flow cytometry (Fig. 1, A and B). In contrast to neutrophils from normal individuals, 7D5 did not stain the cell surface of neutrophils deficient in either p22phox or gp91phox (Fig. 1, A and B). Neutrophils from the mother and female siblings of an individual with X-linked CGD displayed bimodal fluorescence, demonstrating that they have both normal and Cyt b-deficient neutrophils. The identification of heterogeneous Cyt b expression in their neutrophils indicates that they possess one copy of a mutant CYBB allele and therefore are carriers for X-linked CGD (Fig. 1B). Approximately 87% of the neutrophils from the mother and 80% from the daughter shown in Fig. 1B reduced nitro blue tetrazolium to formazan, indicating that those cells possessed normal O&cjs1138;2-generating capacity. A second daughter also had similar lyonization with 74% nitro blue tetrazolium-positive neutrophils (not shown). That three females within the same family would have similar degrees of lyonization may reflect familial nonrandom X-chromosome inactivation, which has been identified in other X-linked disorders (32-37). The percentage of their neutrophils with normal O&cjs1138;2-producing capability correlated well with the amount of 7D5 binding shown by flow cytometry: e.g. 81.1% of the neutrophils in the daughter with 80% nitro blue tetrazolium-positive cells stained with 7D5 (Fig. 1B). These findings indicate that 7D5-epitope recognition required surface expression of both gp91phox and p22phox. Moreover, these results illustrate how 7D5 can be used to identify Cyt b-deficient individuals or those who are carriers for X-linked CGD.



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Fig. 1.   Identification of female carriers of gp91phox deficiency or individuals deficient in gp91phox or p22phox by flow cytometry. A, neutrophils (106) from healthy subjects (father, mother, and normal daughter as indicated) or from an individual with autosomal deficiency of p22phox (A220) were probed with 7D5 and analyzed by flow cytometry as described under "Experimental Procedures." B, a similar analysis was performed on neutrophils from an individual with X-linked CGD (X910) or from the mother and female sibling of an individual with X-linked CGD (mother and daughter carriers as indicated) and were compared with a healthy individual (normal as indicated). Dashed histograms represent staining with IgG1, an isotype control antibody.

To demonstrate further the specificity of immunoreactivity of 7D5 to Cyt b, human neutrophil Cyt b was purified from cell membranes as described (38) and bound to 96-well plates. Probing the immobilized Cyt b with 7D5, anti-p22phox mAb 44.1 (alpha p22) (14), anti-gp91phox mAb 54.1 (alpha gp91) (14), and an irrelevant mAb suggested that the epitope bound by 7D5 was intact on the detergent-solubilized protein (Fig. 2). Although the reactivity of 7D5 in the ELISA was less than that of alpha p22 or alpha gp91, all three mAbs specifically recognized Cyt b. It is possible that the binding of mAb 7D5 to Cyt b requires a more native or membrane-resident conformation of the protein than does either alpha p22 or alpha gp91, although we were unable to test this directly using our ELISA.



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Fig. 2.   Analysis of 7D5-Cyt b interaction by ELISA. One µg of heparin-purified human neutrophil Cyt b was used to coat wells in an ELISA plate so that binding by each of three anti-Cyt b mAbs could be determined. alpha gp91 and alpha p22 were previously shown to be Cyt b-specific (14) and were used as positive controls, and the anti-rhodopsin mAb K42.41 (60) served as a negative control. The results indicate specific binding of 7D5 to the detergent-solubilized form of the protein. These data are typical of five separate analyses.

7D5 Recognizes Native Cyt b Heterodimers-- To gain insight into the structural nature of the 7D5 epitope, we compared nondenaturing with denaturing conditions for immunoprecipitation of Cyt b using 7D5. Using nondenaturing conditions, 7D5, alpha gp91, and alpha p22 precipitated both gp91phox and p22phox from human neutrophil detergent lysates (Fig. 3A). When the lysates were denatured with heat and 1% SDS, alpha gp91 and alpha p22, binding linear regions of the protein, precipitated their respective subunits alone. However, 7D5 precipitated neither subunit after denaturation (Fig. 3A).



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Fig. 3.   Immunoprecipitation (IP) of Cyt b using 7D5. A, PMNs were solubilized in lysis buffer, and Cyt b was precipitated with the indicated antibody using denaturing (DEN) or nondenaturing (ND) conditions as described under "Experimental Procedures." Following SDS-PAGE and transfer to nitrocellulose, subunits of Cyt b, gp91phox and anti-p22phox, were detected with combined use of alpha gp91 and alpha p22. Murine IgG1 was used as an isotype control. IgG Hch and IgG Lch indicate positions of the heavy and light chains, respectively, of immunoglobulin. Results shown are representative of three separate experiments. B, gp91-PLB or X-CGD PLB cells (2 × 106) were solubilized in radioimmune precipitation buffer, and Cyt b was precipitated from lysates using mAb 7D5 or alpha gp91 and alpha p22 as indicated. Immunoprecipitations were carried out using nondenaturing conditions or after lysates were heated to 100 °C in the presence of 1% SDS to denature all proteins. Following SDS-PAGE, proteins were transferred to nitrocellulose, and immunoblots were probed with a combination of alpha gp91 and alpha p22. Results are representative of three separate experiments.

To determine whether 7D5 bound complex carbohydrates on gp91phox, we examined the ability of 7D5 to precipitate Cyt b using biosynthetically active gp91-PLB cells as a source of both precursor and mature Cyt b (Fig. 3B). Using nondenaturing conditions, 7D5 precipitated gp91phox, p22phox, and a small amount of gp65, the gp91phox precursor that has exclusively high mannose oligosaccharides (Fig. 3B). Since 7D5 precipitated gp65, the complex carbohydrates of gp91phox were not required for 7D5 binding to Cyt b. These findings were confirmed by the ability of 7D5 to precipitate the heterodimeric complex consisting of 55-58-kDa core gp91phox protein-p22phox synthesized in the presence of tunicamycin2 and also bind heterodimer following digestion with PNGase F (data not shown). Consistent with the neutrophil experiments, when gp91-PLB lysates were denatured with 1% SDS and heat, 7D5 precipitated neither mature subunit nor gp65. In contrast, alpha gp91 and alpha p22 precipitated their respective subunits following denaturation (Fig. 3B). These findings suggest that 7D5 recognized the Cyt b peptide backbone in its native form only but do not elucidate whether the antibody bound to an epitope shared by both subunits or if it associated with individual gp91phox or p22phox subunits in their native conformation.

Our previous studies on the biosynthesis of Cyt b demonstrated that pools of uncomplexed gp65 and p22phox accumulate for a limited time following synthesis in gp91-PLB cells (26). Therefore, these cells provide a source of native, monomeric Cyt b subunits from which to test whether 7D5 recognizes individual gp65 or p22phox subunits. Sequential immunoprecipitations were performed to determine whether 7D5 was capable of depleting lysates of Cyt b heterodimer and/or individual subunits (Fig. 4A). Although 7D5 completely removed gp91-p22phox heterodimer from lysates after three rounds of immunoprecipitation, monomeric gp65 could be subsequently precipitated from those lysates using alpha gp91 (Fig. 4A, arrowheads). Since neither gp91phox nor p22phox coprecipitated with gp65 using alpha gp91 following three rounds of precipitation with 7D5, gp65 was precipitated free of associated p22phox. The finding that gp65 but neither gp91phox nor p22phox coprecipitated with alpha gp91 after the immunodepletions with 7D5 suggests that heterodimeric gp91phox-p22phox complexes were completely removed by 7D5 (Fig. 4A). The inability to precipitate and detect by immunoblotting monomeric p22phox following the three immunodepletions with 7D5 or those with 7D5 followed by a single depletion with alpha gp91 may be due to the rapid degradation of uncomplexed p22phox or its rapid processing to heterodimeric form (26) (Fig. 4A). It is likely that the amount of newly synthesized, monomeric p22phox present in the depleted lysates may have been below the limits of detection by immunoblotting. Therefore, we pulse-labeled gp91-PLB cells and subsequently chased for 2 h to allow for partial processing of gp65 to gp91phox and also to allow the formation of gp91phox-p22phox complexes (Fig. 4B). As with the unlabeled immunodepletion experiment, three rounds of precipitation of radiolabeled Cyt b using 7D5 completely removed gp91phox-p22phox complexes from lysates (Fig. 4B). In contrast, following depletion with 7D5, subsequent precipitation with either alpha gp91 or alpha p22 revealed that both gp65 and p22phox remained in depleted lysates free of their complementary subunit (Fig. 4B). These results suggest that 7D5 precipitated only gp91phox-p22phox or gp65-p22phox heterodimers.



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Fig. 4.   Immunodepletion of gp91phox-p22phox and gp65-p22phox using 7D5. A, gp91-PLB cells were solubilized in radioimmune precipitation buffer, and lysates were immunodepleted using three successive 7D5 precipitations and subsequently subjected to precipitation using either alpha gp91 or alpha p22 as indicated. Following SDS-PAGE, proteins were transferred to nitrocellulose, and immunoblots were probed with a combination of anti-gp91phox and anti-p22phox mAbs. The arrowheads indicate gp65. B, alternatively, gp91-PLB cells were pulse-labeled with [35S]methionine for 1 h and then chased 2 h with unlabeled methionine. Cells were solubilized in radioimmune precipitation buffer, and lysates were immunodepleted using three successive 7D5 precipitations followed by either alpha gp91 or alpha p22 as indicated. The arrowhead indicates gp65, and the line at the left indicates a protein of 60 kDa unrelated to gp65. We have previously demonstrated that precipitation of radiolabeled p22phox by alpha p22 results in uninterpretable signal due to high background above 50 kDa on autoradiograms (26). However, this background signal is not due to gp65 or gp91phox. The panels at the far right in both A and B illustrate immunoprecipitation (IP) of gp65/91phox and p22phox by alpha gp91 and alpha p22, respectively. Following SDS-PAGE, gels were processed for autoradiography. Results are representative of two or three separate experiments.

Epitope Mapping Using Phage Display-- To identify whether the 7D5 epitope was located on gp91phox, p22phox, or both subunits, we selected phage display library clones using a 7D5 immunoaffinity Sepharose bead matrix. Limiting dilutions were performed on each of three successive eluate samples, to determine the titer and to provide isolated plaques for plaque lift analysis. A six-log increase in the number of adherent clones was observed between the first and third round of selection, suggesting strong enrichment for peptide sequences by the antibody (data not shown). About 15% of the plaques from the second round and 95% of the plaques from the third round elution gave strong signals when probed by 7D5 in a plaque lift analysis (14), compared with an irrelevant monoclonal (data not shown). Isolated plaques were then selected for nucleotide sequence analysis as described (16) (Table I).


                              
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Table I
Peptide sequences selected on a 7D5 immunoaffinity matrix

Immunoaffinity selection of peptides presented on phage display clones produced 29 unique amino acid sequences, many of which appeared on several different phage (Table I). The first phage sequence listed in Table I showed a five-residue match to the 226RIVRG230 segment of gp91phox, a region predicted to be extracytoplasmic by hydropathy analysis and by its proximity to the Asn240 putative glycosylation site. The recovery of this single RIVRGVGGI peptide thus provided important evidence supporting the 226RIVRG230 segment of gp91phox as being part of the epitope (Table I, clone A). In addition to containing similarity to the 226RIVRG230 segment of gp91phox, several other phage sequences selected by 7D5, including YKNPWIRGM, LKNPWQRGD, LPNPWVRGD, and NANPWSRGF, suggested a match to 161KNP163 of gp91phox as well (Table I, clones B, C, D, and F). The 161KNP163 region of gp91phox lies above a predicted transmembrane region 12 residues from Asn149, another possible glycosylation site, and 63 residues from the 226RIVRG230 segment, just above the fifth predicted transmembrane-spanning domain (12, 39). Modest matches in several other selected clones (Table I) also reflected the 161KNP163 segment of gp91phox. More than 50% of the selected phage peptides contained an aliphatic or hydrophobic residue, including Leu, Ile, Val, or Tyr, which when aligned with the 161KNP163 and 226RIVRG230 segments of gp91phox, corresponded to Ile160 (Table I). Most impressively, these two regions of gp91phox were represented by four phage peptides with five-residue identities and additional conservatively substituted or shifted residues (clones A-D, Table I). In our previous studies, mapping antibody epitopes or protein-protein interactions, such extended matches were rare (16, 40). When phage peptides were aligned with the gp91phox segments 161KNP163 and 226RIVRG230, Trp was represented in the same position in greater than 91% of the total number of phage isolates (Table I). Although no residue in the identified gp91phox sequences fits with this selected residue, Trp125 immediately outside of the third transmembrane region of gp91phox is a possible candidate, since all of these transmembrane regions are likely to be in close proximity. Trp251 could also be represented by the phage-selected sequences, yet we were unable to block the binding of 7D5 in flow cytometry with another antibody that binds this residue (data not shown). It is also possible that Trp68 of p22phox contributes the tryptophan in the epitope identified in the phage display mapping and could therefore provide some rationale for the heterodimer requirement for epitope conformation. Another possibility is that the Trp selected by the phage clones represented a hydrophobic "pocket" or "spacer," which bridged the gap between the extracellular transmembrane loops containing 161KNP163 and 226RIVRG230 sequences.

Several phage sequences also contained an RGD tripeptide motif, which aligned well within the gp91phox residues 226RIVRGQ231, if flexibility is provided to allow for a polar residue substitution at Gln231. The unexpected identification of RGD in several selected sequences suggested that this surface-accessible region of Cyt b might interact with integrins in an RGD-dependent manner (41), yet our attempts to confirm such an interaction were unsuccessful (data not shown).

Our mapping data indicate that the minimal epitope bound by 7D5 consists of five mapped residues in the 226RIVRG230 segment, and four more matching the 160IKNP163 region. These two regions are likely to be extracellular based on hydropathy analysis that suggests they are located immediately adjacent to the extracellular aspect of two putative membrane-spanning helices (39) (see Fig. 6 for putative epitope location). The combination of these two regions constitutes a logical target for binding by 7D5, based on our findings from the biochemical assays, i.e. the ability of the antibody to identify an accessible epitope on the plasma membrane of intact neutrophils combined with its inability to bind denatured protein.

The binding of 7D5 to the selected peptides on the denatured pIII display protein (42) from 5 × 1010 plaque-forming units was examined by SDS-PAGE and immunoblotting (Fig. 5, immunoblot). These signal intensities varied significantly with the displayed sequence, with YPGWGRNDA and YPGWPRKDL sequences (clones Z and BB, respectively) producing the strongest signals, the clones containing cysteine pairs (clones U and W) showing weak signals, and the rest showing intermediate staining. An irrelevant clone not selected by 7D5 gave no detectable signal (Fig. 5B). These findings demonstrate that the selected peptides exhibited varied degrees of binding to 7D5, and the binding presumably represents the extent to which each denatured clone was able to represent the Cyt b epitope. The binding by 7D5 to clones A and B, which have five residues identical to the putative epitope, were bound less by 7D5 than at least three other clones with fewer identical residues (Fig. 5, immunoblot; compare clones A and B with clones Z, BB, and M). This result suggested that some structure in the displayed epitope mimetic was lost by denaturation.



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Fig. 5.   Immunoreactivity of 7D5 with selected phage-displayed sequences by ELISA and immunoblotting. Intact phage display clones showed variable blocking of the binding of 7D5 to immobilized Cyt b by ELISA. Each well of the ELISA plate was coated with Cyt b as described for Fig. 2. Following the pretreatment of 7D5 with the indicated phage clones bearing the unique sequences listed below as described under "Experimental Procedures," the binding of 7D5 to the wells was measured. For these phage clones, immunoblotting (lower section of the figure) was used to illustrate the immunoreactivity of 7D5 for the denatured form of the phage pIII fusion protein bearing the selected peptide sequence. Each lane in the immunoblot represents 5 × 1010 plaque-forming units of phage clones following separation in the reducing and denaturing gel conditions. Sequences displayed on the phage clones for both the ELISA and immunoblot are as follows: clone A (RIVRGVGGI), clone X (GWIKYRLEG), clone Z (YPGWGRNDA), clone BB (YPGWPRKDL), clone B (YKNPWIRGM), clone M (LNTKWLRGD), clone U (FRCSWCRGE), clone W (GECRWCKGD), unselected clone (unsel.), or none. The pIII capsid protein fused to the unique peptides typically migrates as a 68-kDa protein in these gel conditions. The blot was probed with 3 µg/ml 7D5, and immunoreactive phage peptides were detected using alkaline-phosphatase conjugated secondary antibody.

Since 7D5 required Cyt b in its native conformation to bind, the binding of 7D5 to SDS-denatured phage clones may have represented conditions less than optimal to determine which was the best epitope mimetic. Therefore, we analyzed the ability of various intact 7D5-binding phage clones to block the interaction of 7D5 with purified Cyt b in an ELISA (Fig. 5, bar graph). Of the eight phage representatives tested, clones M and A (LNTKWLRGD and RIVRGVGGI, respectively) were most effective at blocking the interaction of 7D5 with Cyt b in this assay. Both clones have much greater linear homology to the putative gp91phox epitope than do clones Z and BB (YPGWGRNDA and YPGWPRKDL, respectively), which had greater reactivity with 7D5 on the immunoblot (Fig. 5). Moreover, clone B, which had relatively weak reactivity with 7D5 on the immunoblot despite its high similarity to the putative epitope, showed much greater interaction with 7D5 in the ELISA. The difference in immunoreactivity between the immunoblot and the ELISA probably represents the difference in the way each clone is presented to 7D5; the immunoblot presents denatured protein to 7D5, whereas the ELISA promotes interactions that reflect those between native proteins. Our finding that 7D5 required native Cyt b for binding is reflected most by the ELISA data, which demonstrated that clones of higher similarity reacted with 7D5 better in native conformation than in denatured form.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In response to difficulties in predicting the structure of membrane proteins, epitope mapping of antibodies that bind native protein provides an alternative means of assigning localized structure to individual protein domains, as well as elucidating certain membrane topology. The apparent complex nature of the 7D5 epitope suggested that its characterization could provide information about Cyt b structure beyond localization of regions outside the plasma membrane. mAb 7D5 has been previously utilized in reports describing molecular and genetic analysis of CGD and the NADPH oxidase, yet the region(s) of Cyt b bound by 7D5 have remained undefined. To provide a better view of the 7D5 epitope and to gain structural information about Cyt b membrane topology, we investigated the 7D5-Cyt b interaction to identify which regions of Cyt b are recognized by the antibody.

Our analysis of 7D5 binding first established its specificity for the Cyt b protein. 7D5 bound neither to neutrophils from patients with autosomal deficiency of p22phox nor to those from patients with X-linked CGD. Flow cytometric 7D5 staining of neutrophils from female carriers of X-linked CGD was bimodal, consistent with the mosaic expression of Cyt b in such subjects (43). The use of mAb 7D5 to identify carriers of CGD as well as affected individuals constitutes an important yet simple screening test for the genetic types associated with impaired Cyt b expression. Specificity of 7D5 for Cyt b was further supported by the reactivity of 7D5 to partially purified Cyt b in an ELISA.

7D5 precipitated heterodimeric gp91-p22phox under nondenaturing conditions but recognized neither individual subunit when Cyt b was denatured. Anti-p22phox and anti-gp91phox mAbs, alpha p22 and alpha gp91, respectively, which bind linear epitopes (16), also precipitated heterodimeric gp91-p22phox. In contrast to 7D5, alpha p22 and alpha gp91 precipitated individual p22phox and gp91phox subunits after Cyt b had been denatured. The inability of 7D5 to precipitate either individual subunit following denaturation of Cyt b is consistent with its failure to recognize either on an immunoblot. Furthermore, 7D5 recognized neither native monomers of gp65 nor p22phox, suggesting that individual subunit conformation alone was not sufficient for binding. These results suggest two possible explanations for the requirements of 7D5 binding: 1) that both subunits contribute to the epitope or 2) that the epitope conformation is dependent upon heterodimer assembly, although the residues constituting the epitope reside on only one of the subunits. In addition to gp91phox and p22phox, 7D5 precipitated gp65, the ER-resident precursor containing only high mannose carbohydrate (Figs. 3A and 4, A and B) (44), which is later modified in the Golgi to include complex carbohydrates to form gp91phox (45). Moreover, Yamauchi et al.2 have demonstrated that the unglycosylated gp91phox core protein can be precipitated by 7D5 from gp91-PLB cells cultured in the presence of tunicamycin, and our previous studies indicate that the unglycosylated gp91phox core protein associates with p22phox to form an unglycosylated heterodimer (26). Together these data indicate that carbohydrate did not contribute significantly to the 7D5 epitope.

Because 7D5 appeared to bind a polypeptide region on Cyt b, we used phage display epitope mapping to identify residues of Cyt b involved in 7D5 binding. The phage-displayed sequences show similarity to both 160IKNP163 and 226RIVRG230 regions of gp91phox, yet no matches to p22phox sequences could be identified. The diversity of the 29 selected sequences suggests that the 7D5 epitope involves a nonlinear or conformational epitope, consistent with the inability of 7D5 to recognize denatured protein on immunoblots. Several phage clones, A-D from Table I, gave five-residue identities to the discontinuous region spanning 160IKNP163 and 226RIVRG230 of gp91phox. Such strong similarity provides credible support to the identification of this region as the 7D5 epitope, especially because these epitope mimetics were recovered from a randomly generated peptide library (14, 16, 40). Moreover, clone A, one of the clones with five residues identical to the gp91phox sequence, interacted with 7D5 in the native ELISA better than all clones tested except one (Fig. 5). Tryptophan was also recovered in nearly every phage clone (Table I), although this residue does not appear in either of the two gp91phox regions identified. It is possible that this tryptophan represents a residue from another membrane-spanning helix of gp91phox or a hydrophobic pocket, potentially bridging 160IKNP163 and 226RIVRG230 of gp91phox. A subset of four phage clones (Z, AA, BB, and CC, each beginning with YPGW; Table I) are listed in the reverse orientation (carboxyl to amino, left to right, respectively) compared with the other clones in Table I. These clones are unique because 1) they fit the consensus best if written in reverse order, 2) clones Z and BB were most strongly recognized by 7D5 in immunoblot (Fig. 5), and 3) a synthetic peptide analog comprising part of clones Z and BB (NH2-ADNRPWGPYGP-CONH2) was the only synthetic peptide sequence found to compete with the binding of 7D5 to immobilized Cyt b in ELISA, albeit only at an EC50 of 1 mM (data not shown). The ability of the clones to display differential reactivity in the immunoblot versus the ELISA is probably due to the complex nature of the epitope; i.e., the sequences displayed by the phage are the best linear representatives of an epitope that requires tertiary structure.

This selection of apparent reverse sequences by mAb 7D5 suggests that this antibody can select epitope mimetics that are not always in the same orientation as natural epitope. We previously observed the recovery of some peptides from the J404 phage display library that fit the consensus if listed in reverse order (40, 46, 47), and the cross-reactivity of antibodies with retropeptides has been specifically addressed (48). We also observed that the reverse sequence of a bioactive peptide shows significantly greater effects than a peptide bearing a randomly chosen sequence (49). An interaction between the bacterial ribonuclease barnase and its natural inhibitor barstar, are also reported to be "strong and relatively insensitive to ideal geometry" when compared with the interaction between the enzyme and the nucleotide substrate (50). These findings support the rationale for our observations that antibodies can interact with peptides by identifying critical side chain charge distribution and hydrophobicity, in a way that is not entirely dependent on peptide backbone orientation.

The findings from the immunological analyses and phage display are summarized in a schematic model of the topology and identity of the 7D5 epitope contained within two separate extracellular regions of gp91phox (Fig. 6). Previous attempts to elucidate Cyt b structure, including mapping of extracellular versus intracellular domains have been based on hydropathy analyses and enzymatic cleavage experiments (12), structural homology to known proteins in combination with computer modeling (39, 51, 52), and identification of the binding regions of p47phox (40, 53-56) and NADPH and FAD (25, 54, 57, 58)). Imajoh-Ohmi et al. (12) found that antibodies raised against gp91phox residues 150-172 stained intact neutrophils, leading the authors to conclude that that region is exposed on the cell surface. Our data not only extend those findings but demonstrate that gp91phox residues 160IKNP163 and 226RIVRG230 are juxtaposed on the extracytoplasmic face of the plasma membrane (Fig. 6), providing direct evidence for the identity and positioning of two previously predicted transmembrane regions (12, 39). Moreover, these results are compatible with the findings of Wallach et al., which demonstrated that gp91phox residues Asn132, Asn149, and Asn240 are glycosylated and, therefore, exposed on the cell surface (59).



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Fig. 6.   A hypothetical model for the 7D5 epitope and membrane topology of Cyt b. The epitope was mapped to two juxtaposed extracytoplasmic regions of gp91phox, residues 160IKNP163 and 226RIVRG230, represented by blue and green spheres, respectively. These residues are predicted by hydropathy analysis of the protein to exist immediately extracellular to the fourth and fifth membrane-spanning helices. Residues identified in yellow lettering were represented by the sequences selected from the phage display library and thus accessible to 7D5 on the surface of the cell. The underlined glutamine residue represents polar residue conservation for the epitope in that position although it was not represented by selected sequences. Although tryptophan was almost always found between sequences representing 160INKP163 or 226RIVRGQ231 in individual phage clones, a tryptophan residue does not exist in this vicinity of the protein. Therefore, the black W represents the position of this residue as selected by phage clones.

Our mapping data suggest that the epitope bound by 7D5 was located entirely on gp91phox, but the presence of p22phox in the gp91-p22 heterodimer was required for 7D5 binding on human myeloid cells. It should also be noted that heme insertion is a prerequisite for heterodimer formation (26) that in turn is essential for recognition by 7D5. To the extent that heterodimer formation requires heme coordination by one or both subunits, the 7D5 epitope is at the least indirectly heme-sensitive. Thus, p22phox, possibly in combination with heme, appears to impart a structural constraint to gp91phox that is essential for recognition by 7D5. Further structural analysis will be necessary to elucidate the influence of p22phox on the membrane topology and processing of gp91phox and the possible role of heme in this process.


    ACKNOWLEDGEMENTS

We also acknowledge the expert technical assistance of Justin K. Fishbaugh and B. E. Hoess at the University of Iowa Core Flow Cytometry Facility.


    FOOTNOTES

* This work was performed during the tenure of the following American Heart Association awards: postdoctoral fellowships 9704584S (to J. B. B.) and 9920491Z (to F. R. D.) and Scientist Development Grant 30156N (to J. B. B.). This work was supported in part by Public Health Service Grants RO1 AI 26711 (to A. J. J.), HL 45635 (to M. C. D.), RO1 AI 34879, and HL53592 (to W. M. N) and a Merit Review Award (to W. M. N.) from the Department of Veterans Affairs.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These authors contributed equally to this work.

** To whom correspondence should be addressed: 109 Lewis Hall, Dept. of Microbiology, Montana State University, Bozeman, MT 59717. Tel.: 406-994-4811; Fax: 406-994-4926; E-mail: umbaj@gemini.oscs.montana.edu.

Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M006236200

2 A. Yamauchi, L. Yu, A. J. G. Pötgens, F. Kuribayashi, H. Nunoi, S. Kanegasaki, D. Roos, H. L. Malech, M. C. Dinauer, and M. Nakamura, unpublished observation.


    ABBREVIATIONS

The abbreviations used are: CGD, chronic granulomatous disease; Cyt b, flavocytochrome b558; mAb, monoclonal antibody; ELISA, enzyme linked immunosorbent assay.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Nauseef, W. M. (1999) Proc. Assoc. Am. Physicians 111, 373-382[Medline] [Order article via Infotrieve]
2. Dinauer, M. C. (1993) Crit. Rev. Clin. Lab. Sci. 30, 329-369[Medline] [Order article via Infotrieve]
3. Clark, R. A. (1999) J. Infect. Dis. 179 Suppl. 2, 309-317
4. Roos, D., De Boer, M., Kuribayashi, F., Meischl, C., Weening, R. S., Segal, A. W., Ahlin, A., Nemet, K., Hossle, J. P., Bernatowska-Matuszkiewicz, E., and Middleton-Price, H. (1996) Blood 87, 1663-1681
5. Segal, A. W. (1987) Nature 326, 88-91[CrossRef][Medline] [Order article via Infotrieve]
6. Royer-Pokora, B., Kunkel, L. M., Monaco, A. P., Goff, S. C., Newburger, R. L., Baehner, R. L., Cole, F. S., Curnutte, J. T., and Orkin, S. H. (1986) Nature 322, 32-38[Medline] [Order article via Infotrieve]
7. Dinauer, M. C., Orkin, S. H., Brown, R., Jesaitis, A. J., and Parkos, C. A. (1987) Nature 327, 717-720[CrossRef][Medline] [Order article via Infotrieve]
8. Volpp, B. D., Nauseef, W. M., and Clark, R. A. (1988) Science 242, 1295-1297
9. Lomax, K. J., Leto, T. L., Nunoi, H., Gallin, J. I., and Malech, H. L. (1989) Science 245, 409-412
10. Jesaitis, A. J. (1995) J. Immunol. 155, 3286-3288
11. MacCallum, R. M., Martin, A. C., and Thornton, J. M. (1996) J. Mol. Biol. 262, 732-745[CrossRef][Medline] [Order article via Infotrieve]
12. Imajoh-Ohmi, S., Tokita, K., Ochiai, H., Nakamura, M., and Kanegasaki, S. (1992) J. Biol. Chem. 267, 180-184[Abstract/Free Full Text]
13. Jesaitis, A. J., Buescher, E. S., Harrison, D., Quinn, M. T., Parkos, C. A., Livesey, S., and Linner, J. (1990) J. Clin. Invest. 85, 821-835
14. Burritt, J. B., Busse, S. C., Gizachew, D., Siemsen, D. W., Quinn, M. T., Bond, C. W., Dratz, E. A., and Jesaitis, A. J. (1998) J. Biol. Chem. 273, 24847-24852[Abstract/Free Full Text]
15. Verhoeven, A. J., Bolscher, B. G. J. M., Meerhof, L. J., van Zwieten, R., Keijer, J., Weening, R. S., and Roos, D. (1989) Blood 73, 1686-1694[Abstract]
16. Burritt, J. B., Quinn, M. T., Jutila, M. A., Bond, C. W., and Jesaitis, A. J. (1995) J. Biol. Chem. 270, 16974-16980[Abstract/Free Full Text]
17. Burritt, J. B., Fritel, G. N., Dahan, I., Pick, E., Roos, D., and Jesaitis, A. J. (2000) Eur. J. Haematol. 65, 407-413[CrossRef][Medline] [Order article via Infotrieve]
18. Batot, G., Martel, C., Capdeville, N., Wientjes, F., and Morel, F. (1995) Eur. J. Biochem. 234, 208-215[Abstract]
19. Nakamura, M., Murakami, M., Koga, T., Tanaka, Y., and Minakami, S. (1987) Blood 69, 1404-1408[Abstract]
20. Emmendorffer, A., Nakamura, M., Rothe, G., Spiekermann, K., Lohmann-Matthes, M. L., and Roesler, J. (1994) Cytometry 18, 147-155[Medline] [Order article via Infotrieve]
21. DeLeo, F. R., Renee, J., McCormick, S., Nakamura, M., Apicella, M., Weiss, J. P., and Nauseef, W. M. (1998) J. Clin. Invest. 101, 455-463[Abstract/Free Full Text]
22. Faizunnessa, N. N., Tsuchiya, T., Kumatori, A., Kurozumi, H., Imajoh-Ohmi, S., Kanegasaki, S., and Nakamura, M. (1997) Hum. Genet. 99, 469-473[CrossRef][Medline] [Order article via Infotrieve]
23. Kuribayashi, F., Kumatori, A., Suzuki, S., Nakamura, M., Matsumoto, T., and Tsuji, Y. (1995) Biochem. Cell Biol. Commun. 209, 146-152
24. Boyum, A. (1968) J. Clin. Invest. 21, 77-89
25. Cross, A. R., Higson, F. K., Jones, O. T., Harper, A. M., and Segal, A. W. (1982) Biochem. J. 204, 479-485[Medline] [Order article via Infotrieve]
26. DeLeo, F. R., Burritt, J. B., Yu, L., Jesaitis, A. J., Dinauer, M. C., and Nauseef, W. M. (2000) J. Biol. Chem. 275, 13986-13993[Abstract/Free Full Text]
27. Zhen, L., King, A. A. J., Xiao, Y., Chanock, S. J., Orkin, S. H., and Dinauer, M. C. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9832-9836[Abstract]
28. Burritt, J. B., Bond, C. W., Doss, K. W., and Jesaitis, A. J. (1996) Anal. Biochem. 238, 1-13[CrossRef][Medline] [Order article via Infotrieve]
29. Laemmli, U. K., and Favre, M. (1973) J. Mol. Biol. 80, 575-599[Medline] [Order article via Infotrieve]
30. Mizuno, Y., Hara, T., Nakamura, M., Ueda, K., Minakami, S., and Take, H. (1988) J. Pediatr. 113, 458-462[Medline] [Order article via Infotrieve]
31. Parkos, C. A., Dinauer, M. C., Jesaitis, A. J., Orkin, S. H., and Curnutte, J. T. (1989) Blood 73, 1416-1420[Abstract]
32. Moser, H., and Emery, A. E. (1974) Clin. Genet. 5, 271-284[Medline] [Order article via Infotrieve]
33. Kaladhar, R. B., Anandavalli, T. E., and Reddi, O. S. (1984) Hum. Genet. 67, 460-462[Medline] [Order article via Infotrieve]
34. Taylor, S. A., Deugau, K. V., and Lillicrap, D. P. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 39-42[Abstract]
35. Ingerslev, J., Schwartz, M., Lamm, L. U., Kruse, T. A., Bukh, A., and Stenbjerg, S. (1989) Clin. Genet. 35, 41-48[Medline] [Order article via Infotrieve]
36. Marcus, S., Steen, A. M., Andersson, B., Lambert, B., Kristoffersson, U., and Francke, U. (1992) Hum. Genet. 89, 395-400[Medline] [Order article via Infotrieve]
37. Ropers, H. H., Wienker, T. F., Grimm, T., Schroetter, K., and Bender, K. (1977) Am. J. Hum. Genet. 29, 361-370[Medline] [Order article via Infotrieve]
38. Parkos, C. A., Allen, R. A., Cochrane, C. G., and Jesaitis, A. J. (1987) J. Clin. Invest. 80, 732-742
39. Parkos, C. A., Quinn, M. T., Sheets, S., and Jesaitis, A. J. (1992) Molecular Basis of Oxidative Damage by Leukocytes , CRC Press, Inc., Boca Raton, FL
40. DeLeo, F. R., Yu, L., Burritt, J. B., Loetterle, L. R., Bond, C. W., Jesaitis, A. J., and Quinn, M. T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7110-7114[Abstract]
41. Hynes, R. O. (1992) Cell 69, 11-25[Medline] [Order article via Infotrieve]
42. Smith, G. P., and Scott, J. K. (1993) Methods Enzymol. 217, 228-257[Medline] [Order article via Infotrieve]
43. Dusi, S., Poli, G., Berton, G., Catalano, P., Fornasa, C. V., and Peserico, A. (1990) Acta Haematol. 84, 49-56[Medline] [Order article via Infotrieve]
44. Yu, L., DeLeo, F. R., Biberstine-Kinkade, K. J., Renee, J., Nauseef, W. M., and Dinauer, M. C. (1999) J. Biol. Chem. 274, 4364-4369[Abstract/Free Full Text]
45. Yu, L., Zhen, L., and Dinauer, M. C. (1997) J. Biol. Chem. 272, 27288-27294[Abstract/Free Full Text]
46. DeLeo, F. R., Nauseef, W. M., Burritt, J. B., Jesaitis, A. J., Clark, R. A., and Quinn, M. T. (1995) J. Biol. Chem. 270, 26246-26251[Abstract/Free Full Text]
47. DeLeo, F. R., Ulman, K. V., Davis, A. R., Jutila, K. L., and Quinn, M. T. (1996) J. Biol. Chem. 271, 17013-17020[Abstract/Free Full Text]
48. Guichard, G., Benkirane, N., Zeder-Lutz, G., van Regenmortel, M. H., Briand, J. P., and Muller, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9765-9769
49. Bommakanti, R. K., Dratz, E. A., Siemsen, D. W., and Jesaitis, A. J. (1995) Biochemistry 34, 6720-6728[Medline] [Order article via Infotrieve]
50. Guillet, V., Lapthorn, A., Hartley, R. W., and Mauguen, Y. (1993) Structure 1, 165-176
51. Taylor, W. R., Jones, D. T., and Segal, A. W. (1993) Protein Sci. 2, 1675-1685[Abstract/Free Full Text]
52. Shatwell, K. P., Dancis, A., Cross, A. R., Klausner, R. D., and Segal, A. W. (1996) J. Biol. Chem. 271, 14240-14244[Abstract/Free Full Text]
53. Sumimoto, H., Kage, Y., Nunoi, H., Sasaki, H., Nose, T., Fukumaki, Y., Ohno, M., Minakami, S., and Takeshige, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5345-5349[Abstract]
54. Segal, A. W., West, I., Wientjes, F., Nugent, J. H. A., Chavan, A. J., Haley, B., Garcia, R. C., Rosen, H., and Scrase, G. (1992) Biochem. J. 284, 781-788[Medline] [Order article via Infotrieve]
55. Leto, T. L., Adams, A. G., and De Mendez, I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10650-10654
56. Leusen, J. H. W., De Boer, M., Bolscher, B. G. J. M., Hilarius, P. M., Weening, R. S., Ochs, H. D., Roos, D., and Verhoeven, A. J. (1994) J. Clin. Invest. 93, 2120-2126
57. Park, M.-Y., Imajoh-Ohmi, S., Nunoi, H., and Kanegasaki, S. (1994) Biochem. Cell Biol. Commun. 204, 924-929
58. Rotrosen, D., Yeung, C. L., Leto, T. L., Malech, H. L., and Kwong, C. H. (1992) Science 256, 1459-1462
59. Wallach, T. M., and Segal, A. W. (1997) Biochem. J. 321, 583-585[Medline] [Order article via Infotrieve]
60. Adamus, G., Zam, Z. S., Arendt, A., Palczewski, K., McDowell, J. H., and Hargrave, P. A. (1991) Vision Res. 31, 17-31[CrossRef][Medline] [Order article via Infotrieve]


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