Phage display epitope mapping of human neutrophil flavocytochrome b(558) -Identification of two juxtaposed extracellular domains

Despite extensive experimental and clinical evidence demonstrating the crit ical role of flavocytochrome b(558) (Cyt b) in the NADPH-dependent oxidase, there is paucity of direct structural data defining its topology in the ph agocyte membrane. Unlike other Cyt b-specific monoclonal antibodies, 7D5 bi nds exclusively to an extracellular domain, and identification of its epito pe should provide novel insight into the membrane topology of Cyt b. To tha t end, we examined biochemical features of 7D5-Cyt b binding and used the J 404 phage display nonapeptide library to identify the bound epitope. 7D5 pr ecipitated only heterodimeric gp91-p22(phox) and not individual or denature d Cyt b subunits from detergent extracts of human neutrophils and promyeloc ytic leukemia cells (gp91-PLB). Moreover, 7D5 precipitated precursor gp65-p 22(phox) complexes from detergent extracts of the biosynthetically active g p91-PLB cells, demonstrating that complex carbohydrates were not required f or epitope recognition. Epitope mimetics selected from the J404 phage displ ay library by 7D5 demonstrated that (226)RIVRG(230) and (IKNP163)-I-160 reg ions of gp91(phox) were both bound by 7D5, These studies reveal specific in formation about Cyt b membrane topology and structure, namely that gp91(pho x) residues (226)RIVRG(230) and (IKNP163)-I-160 closely juxtaposed on extra cytoplasmic domains and that predicted helices containing residues Gly(165) -Ile(190) and Ser(200)-Glu(226) are adjacent to each other in the membrane.