Biosynthesis of the phagocyte NADPH oxidase cytochrome b558. Role of heme incorporation and heterodimer formation in maturation and stability of gp91phox and p22phox subunits.
The NADPH oxidase cytochrome b558 is a membrane heterodimer comprised of a glycosylated 91-kDa subunit, gp91(phox), and a nonglycosylated 22-kDa subunit, p22(phox). The role of heme in cytochrome b558 biosynthesis was studied using succinyl acetone, an inhibitor of heme synthesis, in PLB-985 myeloid cells undergoing granulocytic differentiation. Succinyl acetone markedly reduced expression of p22(phox) and the mature 91-kDa form of gp91(phox) but not its 65-kDa high mannose precursor, in association with a profound reduction in NADPH oxidase activity. Expression of non-heme-containing cytosolic oxidase components was unaffected. The reduction in cytochrome b558 expression and NADPH oxidase activity was prevented by adding exogenous heme and was reversible upon removal of succinyl acetone. Transgenic expression of gp91(phox) in monkey COS-7 and murine 3T3 cells, both of which lacked endogenous p22(phox) mRNA, demonstrated that p22(phox) was not required for maturation of gp91(phox) carbohydrate to complex oligosaccharides. However, coexpression of transgenic p22(phox) increased the abundance of the mature gp91(phox) glycoprotein. These results suggest that heme incorporation plays an important role in cytochrome b558 assembly and provide further support for the concept that stability of p22(phox) and the mature gp91(phox) subunit is increased by heterodimer formation.
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Herman B Wells Center for Pediatric Research, Department of Pediatrics (Hematology/Oncology), James Whitcomb Riley Hospital for Children, Indiana University Medical Center, Indianapolis, Indiana 46202, USA.
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Dinauer
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D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D002454:Cell Differentiation; D002462:Cell Membrane / Q000502:physiology; D003573:Cytochrome b Group / Q000096:biosynthesis* / Q000737:chemistry; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006098:Granulocytes / Q000166:cytology / Q000502:physiology*; D006105:Granulomatous Disease, Chronic; D018922:HL-60 Cells; D006418:Heme / Q000378:metabolism*; D006537:Heptanoates / Q000494:pharmacology; D006801:Humans; D007951:Leukemia, Myeloid; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry; D026901:Membrane Transport Proteins; D051379:Mice; D009252:NADPH Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000096:biosynthesis* / Q000737:chemistry; D010586:Phagocytes / Q000201:enzymology*; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013481:Superoxides / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D003573:Cytochrome b Group; D004791:Enzyme Inhibitors; D006537:Heptanoates; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D013481:Superoxides; D006418:Heme; C020804:succinylacetone; C036888:cytochrome b558; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase