STRUCTURE-FUNCTION ANALYSIS OF PHAGOCYTE PROTEINS

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A phagocyte-specific cytochrome-b is a critical component of the phagocyte oxidase complex which generates the superoxide radical. Inherited defects in this important host defense pathway result in chronic granulomatous disease (CGD). Cytochrome-b is a heterodimer of a 91 kD membrane glycoprotein encoded by the gene mutated in the classic X-linked form of CGD, and a non-glycosylated 22 kD polypeptide that derives from an autosomal locus mutated in a subgroup of autosomal recessive CGD. The primary structures of the two subunits, deduced from their corresponding cDNAs, have no obvious similarities to known proteins. In the proposed research, the assembly, structure and function of cytochrome-b will be investigated, taking advantage of molecular and biochemical reagents developed in previous studies. The specific genetic lesions in selected cases of CGD will be characterized, with the aim of identifying important functional domains in the cytochrome heterodimer. Antibodies raised to specific regions of each subunit will be used as reagents to study biosynthesis and assembly of cytochrome-b and to probe its functional interactions. In addition, a potential role will be explored for Rap1, a Ras-related protein that copurifies with cytochrome-b, by modulation of its expression using mutated derivatives and antisense oligonucleotides. Finally, an important goal of this proposal is to develop systems suitable for the study of the cytochrome-b heterodimer by gene transfer of specifically mutated cDNAs. Whether non-phagocytic cell lines can express each subunit and assemble a stable heterodimer will be investigated. For functional studies, an X-CGD-like phagocytic cell line will be developed using targeted homologous recombination to inactivate the gene for the kD cytochrome subunit in the myeloid PLB cell line. The proposed research should provide insight into the functional basis of CGD, and more broadly, extend knowledge about the superoxide generating system of the phagocyte.