Production of recombinant cytochrome b558 allows reconstitution of the phagocyte NADPH oxidase solely from recombinant proteins.
Phagocytic white blood cells contain a multicomponent oxidase that generates microbicidal products by catalyzing electron transfer from NADPH to molecular oxygen. Activation of this oxidase requires interactions of a unique membrane flavocytochrome with the cytosolic proteins p47phox, p67phox, and p21Rac. This flavocytochrome, designated cytochrome b558, is a heteromer comprising a 22-kDa alpha-subunit (p22phox) and a glycosylated approximately 91-kDa beta-subunit (gp91phox). Cytochrome b558 was expressed in Sf9 insect cells coinfected with recombinant baculoviruses carrying cDNAs for p22phox and gp91phox. Membranes of these cells contained a b-type cytochrome with a dithionite-reduced minus oxidized difference spectrum similar to that of neutrophil cytochrome b558. The recombinant cytochrome b558 beta-subunit was heterogeneously N-glycosylated as demonstrated by its susceptibility to cleavage with endoglycosidases F and H. In contrast to the neutrophil cytochrome b558, a portion of the N-linked oligosaccharide was of the high mannose type. Recombinant cytochrome b558 supported superoxide production in a cell-free assay containing recombinant p47phox, p67phox, and p21Rac. The enzymatic turnover of the partially purified recombinant cytochrome b558 and neutrophil cytochrome b558 were similar (approximately 100-160 mol of superoxide generated/s/mol of cytochrome heme, range of two experiments) and the native and recombinant cytochromes showed similar requirements for NADPH and exogenous FAD. These studies represent the first reconstitution of the NADPH oxidase solely from recombinant proteins and define a model system to explore the structure and function of cytochrome b558.
Rotrosen
D
D
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Yeung
C L
CL
Katkin
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004227:Dithionite / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009036:Moths; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D016366:Open Reading Frames; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D013053:Spectrophotometry; D014162:Transfection; D020744:rac GTP-Binding Proteins
D003573:Cytochrome b Group; D046911:Macromolecular Substances; D010750:Phosphoproteins; D011994:Recombinant Proteins; C079789:neutrophil cytosol factor 67K; D004227:Dithionite; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins