Phage Display Epitope Mapping of Human Neutrophil Flavocytochromeb 558

Abstract

. 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 gp91 phox core protein, 2 suggesting that neither the mature nor high man-* This work was performed during the tenure of the following American Heart Association awards: postdoctoral fellowships 9704584S

Functional Epitope on Human Neutrophil Flavocytochrome b558

“…The specificity of mAb NL7 for gp91 phox was further confirmed by phage display epitope mapping (9,11). A six-log increase in adherent phage numbers from the first to third selection was observed and positive plaque lifts (7) confirmed specific selection of immunoreactive sequences had taken place (data not shown).…”

“…1A (lane 1, labeled HE for heparin eluate). Specificity of mAb NL7 for the gp91 phox subunit was confirmed using Cyt b which had been purified on mAb 44.1 (8) (lane 2, labeled IP for immunopurified) which will immunoprecipitate the Cyt b heterodimer (9). Similar immunoreactivity was also shown using heparin eluate probed by the previously characterized anti-gp91 phox mAb 54.1 (lane 3), but not for mAb 7D5 (lane 4) which binds native, but not denatured, gp91 phox .…”

“…Tertiary structural elements of native Cyt b have further been determined by this method, revealing discontinuous regions of protein that are contained within a single epitope (8,9). In the current report, we show that both p-iodonitrotetrazolium violet (INT) and cytochrome c reduction are inhibited by mAb NL7, which binds 498 EKDVITGLK 506 , a cytosolic segment of gp91 phox .…”

Direct Interaction of the Novel Nox Proteins with p22phox Is Required for the Formation of a Functionally Active NADPH Oxidase

“…The results of the present study suggest that the Nox homologues studied are similar in this regard. Interestingly, almost nothing is known regarding the precise mechanism of p22phox and Nox2 association, and only epitope mapping provides some information regarding the amino acid sequences coming into proximity with each other (31,32). Altogether, the function of p22phox is incompletely understood (2).…”

Rapid Detection of Intracellular p47phox and p67phox by Flow Cytometry; Useful Screening Tests for Chronic Granulomatous Disease

“…However, further identification of CGD subgroups is necessary before genetic analysis, which is often labor-intensive and time-consuming. The membrane-bound subunits, gp91phox and p22phox, can be analyzed by flow cytometry using monoclonal antibodies (mAbs), such as 7D5 that identifies the extracellular domain of flavocytochrome b558 [7,8]. In contrast, the cytosolic components, p47phox and p67phox, are usually investigated by immunoblot analysis that requires a large sample of blood and takes longer to perform.…”