Functional reconstitution of the phagocyte NADPH oxidase by transfection of its multiple components in a heterologous system.
The phagocyte NADPH oxidase system, as previously defined by cell-free reconstitution, is comprised of five essential components, three of which are produced during late phagocytic differentiation--namely, two cytosolic proteins, p47- and p67-phox--and the large subunit of cytochrome b558, gp91-phox. To confirm that these are the only phagocyte-specific components necessary for oxidase activity in whole cells, the recombinant NADPH oxidase was reconstituted in a heterologous cell line. An undifferentiated multipotent leukemic cell line, K562, which expresses endogenous Rac and the small subunit of the flavocytochrome b558 (p22-phox), was cotransfected with episomal expression vectors containing cDNAs for the three other oxidase components. After 4 days of selection, the complete oxidase system was functionally reconstituted in transfected cells stimulated with phorbol myristate acetate or calcium ionophore. These easily transfected cells provide an ideal model system in which several oxidase components can be genetically manipulated and readily expressed. This system can be used to test the effects of mutations associated with any of the genes affected in chronic granulomatous disease and will facilitate studies on structure-function relationships within several oxidase components. This system will also aid in delineation of upstream regulators functioning through various signaling pathways.
de Mendez
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Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
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D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000096:biosynthesis* / Q000378:metabolism; D019255:NADPH Oxidases; D010586:Phagocytes / Q000201:enzymology*; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D010750:Phosphoproteins; D011994:Recombinant Proteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase