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

Abstract

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 181GGPQVNPI18 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.

Cloning and sequencing of the bovine flavocytochrome b subunit proteins, gp91-phox and p22-phox: comparison with other known flavocytochrome b sequences

“…Using a similar analysis, we found that monoclonal mouse anti-human gp91-phox [48] also recognized the bovine gp91-phox protein, resulting in a broad band of approximately 91 kDa. The smearing of gp91-phox in human and bovine samples is consistent with reported heterogeneous glycosylation of human gp91-phox [7], and the size of the bovine gp91-phox smear is approximately the same size as the human protein, suggesting a similar level of posttranslational glycosylation.…”

“…For reference, prestained molecular weight standards (BRL, Bethesda, MD) were included on all gels. Immunoblots were probed with mouse monoclonal anti-human gp91-phox (54.1) [48] or rabbit polyclonal anti-human p22-phox [47], followed by Bio-Rad alkaline phosphatase-conjugated goat anti-mouse IgG or anti-rabbit IgG secondary antibodies, respectively. The blots were developed using a Bio-Rad alkaline phosphatase development kit.…”

Functional Epitope on Human Neutrophil Flavocytochrome b558

“…Human neutrophils were prepared from fresh whole blood collected in citrate anticoagulant from normal human donors and probed by mAbs NL7, 44.1, 7D5, or an irrelevant isotype-matched monoclonal as described (6). Briefly, leukocytes were incubated in 200 nM of each mAb diluted in 10 mM PBS (10 mM phosphate, 150 mM NaCl, pH 7.4) with 4% normal goat serum and 4% nonfat dry milk for 30 min on ice.…”

“…Cyt b -specific mAbs have been applied to identify solventaccessible regions on both the cytosolic and extracellular aspects of the native protein, as well as regions which are accessible only after denaturation (6,7). Tertiary structural elements of native Cyt b have further been determined by this method, revealing discontinuous regions of protein that are contained within a single epitope (8,9).…”

Immunofluorescence and Confocal Microscopy of Neutrophils

“…5 We routinely use the following antibodies for immunofluorescence microscopy of human neutrophils [6][7][8][9][10]. To detect NADPH oxidase components, antibodies specific for p22 phox (mAb 44.1) and gp91 phox (mAb 54.1) [20,21] are now available from Santa Cruz Biotechnology (Santa Cruz, CA); mAb 7D5 detects an epitope of gp91 phox present in mature flavocytochrome b 558 [22] and can be used on intact PMNs to detect protein upregulation at the cell surface [7]; a rabbit mAb specific for human p40 phox (Epitomics, Burlingame, CA); an antibody that specifically detects active Rac (NewEast Biosciences, Malvern, PA); and rabbit antisera from William Nauseef (University of Iowa) specific for p47 phox and p67 phox [6]. Mouse mAbs specific for human CD63, lamp-1, and CD11b are obtained from the University of Iowa Developmental Studies Hybridoma Bank.…”