Cloning of murine gp91phox cDNA and functional expression in a human X-linked chronic granulomatous disease cell line.
The phagocyte cytochrome b558, a heterodimer comprised of gp91phox and p22phox, is a flavocytochrome that mediates the transfer of electrons from NADPH to molecular oxygen in the respiratory burst oxidase. The human gene encoding the glycosylated gp91phox subunit is the site of mutations in X-linked chronic granulomatous disease (CGD). Reverse transcriptase-polymerase chain reaction was used to obtain a full-length clone for the murine gp91phox cDNA, which was 87% identical to the human gp91phox cDNA. The encoded murine protein had 39 amino acids out of 570 that differed from the human, many of which were conservative substitutions. Nonconservative replacements occurred in hydrophilic regions outside of domains previously implicated in binding to NADPH, flavin, and the cytosolic oxidase subunit p47phox. Some substitutions altered potential N-glycosylation sites, which is likely to explain why the glycosylated murine protein migrates with an apparent molecular mass of 58 kD instead of 91 kD as seen for the human protein. Expression of murine gp91phox in a human myeloid cell line with a null gp91phox allele using a mammalian expression plasmid or a retroviral vector rescued stable expression of the p22phox subunit and fully reconstituted respiratory burst activity. This suggests that the murine gp91phox subunit forms a functional cytochrome b558 heterodimer with human oxidase subunits, consistent with the high degree of identity between the mouse and human proteins in domains implicated in cytochrome function.
Björgvinsdóttir
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Herman B Wells Center for Pediatric Research, Department of Pediatrics, James Whitcomb Riley Hospital for Children, Indiana University Medical Center, Indianapolis, USA.
Zhen
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Dinauer
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics*; D005816:Genetic Complementation Test; D006031:Glycosylation; D006105:Granulomatous Disease, Chronic / Q000201:enzymology / Q000235:genetics / Q000473:pathology*; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice / Q000235:genetics*; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D016897:Respiratory Burst; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014162:Transfection; D014960:X Chromosome / Q000235:genetics
D003573:Cytochrome b Group; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases