New insights into the membrane topology of the phagocyte NADPH oxidase: Characterization of an anti-gp91-phox conformational monoclonal antibody

Abstract

Cytochrome b 558 is the catalytic core of the phagocyte NADPH oxidase that mediates the production of bactericidal reactive oxygen species. Cytochrome b 558 is formed by two subunits gp91-phox and p22-phox (1/1), non-covalently associated. Its activation depends on the interaction with cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac) leading to an electron transfer from NADPH to molecular oxygen and to the release of superoxide anions. Several studies have suggested that the activation process was linked to a change in cytochrome b 558 conformation. Recently, we confirmed this hypothesis by isolating cytochrome b 558 in a constitutively active form. To characterize active and inactive cytochrome b 558 conformations, we produced four novel monoclonal antibodies (7A2, 13B6, 15B12 and 8G11) raised against a mixture of cytochrome b 558 purified from both resting and stimulated neutrophils. The four antibodies labeled gp91-phox and bound to both native and denatured cytochrome b 558. Interestingly, they were specific of extracellular domains of the protein. Phage display mapping combined to the study of recombinant gp91-phox truncated forms allowed the identification of epitope regions. These antibodies were then employed to investigate the NADPH oxidase activation process. In particular, they were shown to inhibit almost completely the NADPH oxidase activity reconstituted in vitro with membrane and cytosol. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils pointed out the capacity of the monoclonal antibody 13B6 to bind preferentially to the active form of cytochrome b 558. All these data suggested that the four novel antibodies are potentially powerful tools to detect the expression of cytochrome b 558 in intact cells and to analyze its membrane topology. Moreover, the antibody 13B6 may be conformationally sensitive and used as a probe for identifying the active NADPH oxidase complex in vivo.