The polymorphonuclear leukocyte (PMN) carries out its critical microbicidal functions in large part through a stimulus-induced burst of oxidative metabolism which generates superoxide anion and other toxic products of oxygen. An activatable NADPH oxidase system responsible for oxygen reduction does not function in the cells of patients with chronic granulomatous disease (CGD). Studies in a reconstituted cell-free oxidase system have demonstrated requirements for components in the membrane (e.g. cytochrome b558) and the cytosol. Recent studies have identified two discrete cytosolic oxidase components,, proteins of 47 and 67 kDa, and demonstrated selective absence of one or the other of these protein from the PMN of patients with two different types of autosomal CGD. Based on the recent cloning of both p47 and p67 as well as sequencing and functional expression of the p47 cDNAs, detailed studies of structure/function relationships and the genetic determinants of autosomal CGD are proposed. Synthetic peptides and recombinant proteins including deletion and site- directed mutants will be examined in defined cell-free systems in order to map the structural determinants of function to specific domains within the two proteins. Specific considerations for p5=47 will include sites and mechanisms of stimulus-dependent phosphorylation, N-myristoylation of the N-terminal glycine, potential regulatory role of a region homologous to the src oncogene family, and determinants of GTP binding. The sequencing of the p67 clones will be completed and, based on predicted amino acid sequence, structure/function relationships will be assessed including the basis for GTP binding and the potential presence of a substrate (NADPH) binding site. Patients with autosomal CGD attributable to p47 or p67 deficiency will be studied at the levels of DNA, mRNA and protein in order to define the genetic basis for the deficiencies. These proposed studies will contribute to a fundamental understanding of the human neutrophil NADPH oxidase system at a molecular level and to a definition of the genetic basis for the microbicidal defect in patients with autosomal CGD.
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