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.
