An important host defense system of phagocytes is a plasma membrane associated NADPH-oxidase that reduces molecular oxygen to superoxide. The components of the oxidase and their correspondence with specific genetic loci have been incompletely defined. Recently, the gene and cDNA for the product lacking in the classic X-linked form of chronic granulomatous disease (CGD) (a spectrum of inherited disorders in which the oxidase is incapacitated) was cloned by """"""""reverse genetics"""""""" and shown to encode a 90 kD membrane glycoprotein subunit of the phagocyte b-cytochrome heterodimer. In the proposed research, the structure, assembly and function of the cytochrome will be pursued using a combination of molecular and cell biology techniques, which will provide insight into the functional basis of CGD and extend knowledge about the Phagocyte oxidase. cDNA has been cloned for a 22 kd polypeptide that is intimately associated with the 90 kd product of the X-CGD gene to form the b-cytochrome heterodimer. Cases of autosomal recessive, cytochrome negative CGD will be analyzed for a defect in the gene encoding the 22 kD subunit or abnormal regulation of 90 kD mRNA expression. Selected cases of X-CGD will studied to identify mutations in coding sequence that impair b-cytochrome function. Antibodies to specific domains of each cytochrome subunit will be prepared to examine b-cytochrome biosynthesis, membrane topology, and function. The characterization of critical functional and structural domains will also be approached by developing non- phagocytic and b-cytochrome-deficient phagocytic cell systems for introduction of cDNAs (or modified derivatives prepared by in vitro mutagenesis) for the 90 kd and 22 kD subunits.
|Kume, A; Dinauer, M C (1994) Retrovirus-mediated reconstitution of respiratory burst activity in X-linked chronic granulomatous disease cells. Blood 84:3311-6|
|Maly, F E; Schuerer-Maly, C C; Quilliam, L et al. (1993) Restitution of superoxide generation in autosomal cytochrome-negative chronic granulomatous disease (A22(0) CGD)-derived B lymphocyte cell lines by transfection with p22phax cDNA. J Exp Med 178:2047-53|
|Dinauer, M C; Orkin, S H (1992) Chronic granulomatous disease. Annu Rev Med 43:117-24|
|Dinauer, M C; Pierce, E A; Erickson, R W et al. (1991) Point mutation in the cytoplasmic domain of the neutrophil p22-phox cytochrome b subunit is associated with a nonfunctional NADPH oxidase and chronic granulomatous disease. Proc Natl Acad Sci U S A 88:11231-5|
|Schapiro, B L; Newburger, P E; Klempner, M S et al. (1991) Chronic granulomatous disease presenting in a 69-year-old man. N Engl J Med 325:1786-90|
|Dinauer, M C; Pierce, E A; Bruns, G A et al. (1990) Human neutrophil cytochrome b light chain (p22-phox). Gene structure, chromosomal location, and mutations in cytochrome-negative autosomal recessive chronic granulomatous disease. J Clin Invest 86:1729-37|
|Dinauer, M C; Curnutte, J T; Rosen, H et al. (1989) A missense mutation in the neutrophil cytochrome b heavy chain in cytochrome-positive X-linked chronic granulomatous disease. J Clin Invest 84:2012-6|