The phagocyte respiratory burst oxidase that generates the superoxide radical plays a central role in host defense and the inflammatory response. Genetic defects in oxidase subunits result in chronic granulomatous disease (CGD), a syndrome characterized by an absent respiratory burst and recurrent infections. A low-potential cytochrome in the plasma membrane is the focal point for oxidase assembly, and contains both flavin and heme redox centers for transfer of electrons from NADPH to O2. This phagocyte-specific cytochrome is comprised of gp91phox, a 91 kD glycoprotein encoded by an X-linked gene that is the site of mutations in X-linked CGD, and p22phox, a non-glycosylated peptide derived from an autosomal CGD locus. Upon oxidase activation, the cytosolic oxidase subunits, p47phox and p67phox, and a small GTPase, Rac, associate with the plasma membrane, and appear to interact directly with the cytochrome to initiate electron transfer. A ras-related small GTPase, Rap1a, co-purifies with the cytochrome b, and may also participate in regulation of oxidase activity. The structural and functional relationships between various oxidase subunits remain incompletely understood. The overall goal of this project is to develop a clearer understanding of how the phagocyte cytochrome b functions in the regulated production of superoxide. The project has four specific objectives, many of which take advantage of a gp91phox-deficient phagocyte cell line recently developed by gene targeting. First, the topologic organization of the cytochrome complex with respect to the plasma membrane will be investigated using site-directed mutagenesis of potential glycosylation sites and by developing monoclonal and antipeptide antibodies using a new mouse model of X-linked CGD. Second, specific cytochrome residues that participate in the transfer of electrons through flavin and heme centers will be identified using site-directed mutagenesis. Third, domains in the gp91phox cytochrome subunit that function in the assembly and regulation of oxidase will be identified, using a strategy that emphasizes site-directed mutagenesis of candidate gp91phox domains followed by expression and analysis of mutant cytochrome function in intact phagocytes. Fourth, a Rap1a-deficient cell line will be generated by gene targeting to further investigate the interaction of the Rap1a GTPase with cytochrome b and its function in the respiratory burst oxidase. Alternative systems using expression of Rap1a transgenes in cultured cells will also be explored. These studies will provide further insight into the superoxide-generating system of the phagocyte, which may lead to new approaches in modulating superoxide formation in the inflammatory response.
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