The phagocyte respiratory burst oxidase that generates the superoxide radical plays a central role in host defense and the inflammatory response. Assembly of the active oxidase complex requires the participation of both membrane and cytosolic proteins, and is regulated by small GTPases. A phagocyte-specific b-type flavocytochrome heterodimer, located in the plasma and, in neutrophils, specific granule membranes, is the focal point for oxidase assembly, and contains both the flavin and heme redox centers for transfer of electrons from NADPH to molecular oxygen. Genetic defects in oxidase proteins, including the two flavocytochrome subunits, result in chronic granulomatous disease (CGD), a syndrome characterized by an absent respiratory burst, recurrent infections, and chronic granulomas. The structural and functional relationships between the various oxidase subunits and the assembly of the active NADPH oxidase complex remain incompletely understood. This low-potential flavocytochrome is a heterodimer comprised of gp91 phox, a 91-kDa glycoprotein encoded by an X-linked gene that is the site of mutations in X-linked CGD, and p221-phOx, a non-glycosylated peptide derived from an autosomal CGD locus. The proposed studies take a genetic approach to investigating the structure and function of the oxidase flavocytochrome b and its role as a focal point for assembly of the active NADPH oxidase complex. The project has 3 specific objectives, which take advantage of a gp91 phox deficient phagocyte cell line developed by gene targeting as well as heterologous cell systems we have developed for expression of functional recombinant oxidase subunits. First, the relative roles of gp91 phox and p22phox in flavocytochrome b biosynthesis and function will be examined using heterologous cells for expression of unassembled subunits, which are otherwise unstable in phagocytes. Second, identification of domains in gp91 phox and p22 phox that function in the assembly and regulation of oxidase activity will pursued, using a strategy that emphasizes site-directed mutagenesis of candidate hydrophilic regions followed by expression and analysis of function in intact cells. Third, functional domains important for flavocytochrome b trafficking and NADPH oxidase assembly during phagocytosis will be investigated using similar approaches in heterologous and phagocytic cell lines. 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 and host defense.
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