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Proc Natl Acad Sci U S A. Sep 1989; 86(18): 7195–7199.
PMCID: PMC298023

Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase.

Abstract

Neutrophil NADPH oxidase is a multicomponent enzyme that is activated to generate superoxide anion and is defective in the cells of patients with chronic granulomatous disease. It requires both membrane and cytosolic components, the latter including 47- and 67-kDa proteins recognized by the polyclonal antiserum B-1. Immunoscreening of an induced HL-60 lambda ZAP cDNA library yielded seven cross-hybridizing cDNAs encoding the 47-kDa component. Fusion proteins of 22-50 kDa were recognized by B-1. Antiserum against a fusion protein recognized a 47-kDa protein in normal neutrophils but not in those from patients with autosomal chronic granulomatous disease who lack the 47-kDa cytosolic oxidase component. In a cell-free NADPH oxidase system full-length and C-terminal fusion proteins augmented superoxide generation and reconstituted the cytosolic defect of a patient missing the 47-kDa protein. The cDNA hybridized with a 1.4-kilobase mRNA from induced HL-60 cells. The longest cDNA contained an open reading frame encoding a protein of 41,440 Da with a calculated pI of 10.4, an N-terminal glycine, sites favorable for phosphorylation, a nucleotide binding domain, and a region of homology to the src protein kinases, phospholipase C, and alpha-fodrin. These structural features are pertinent to proposed functional roles of the protein in the respiratory burst oxidase.

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  • Segal AW. The electron transport chain of the microbicidal oxidase of phagocytic cells and its involvement in the molecular pathology of chronic granulomatous disease. J Clin Invest. 1989 Jun;83(6):1785–1793. [PMC free article] [PubMed]
  • Bromberg Y, Pick E. Unsaturated fatty acids stimulate NADPH-dependent superoxide production by cell-free system derived from macrophages. Cell Immunol. 1984 Oct 1;88(1):213–221. [PubMed]
  • Curnutte JT. Activation of human neutrophil nicotinamide adenine dinucleotide phosphate, reduced (triphosphopyridine nucleotide, reduced) oxidase by arachidonic acid in a cell-free system. J Clin Invest. 1985 May;75(5):1740–1743. [PMC free article] [PubMed]
  • McPhail LC, Shirley PS, Clayton CC, Snyderman R. Activation of the respiratory burst enzyme from human neutrophils in a cell-free system. Evidence for a soluble cofactor. J Clin Invest. 1985 May;75(5):1735–1739. [PMC free article] [PubMed]
  • Gabig TG, English D, Akard LP, Schell MJ. Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein. J Biol Chem. 1987 Feb 5;262(4):1685–1690. [PubMed]
  • Clark RA, Leidal KG, Pearson DW, Nauseef WM. NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate-activatable superoxide-generating system. J Biol Chem. 1987 Mar 25;262(9):4065–4074. [PubMed]
  • Volpp BD, Nauseef WM, Clark RA. Two cytosolic neutrophil oxidase components absent in autosomal chronic granulomatous disease. Science. 1988 Dec 2;242(4883):1295–1297. [PubMed]
  • Orkin SH. Molecular genetics of chronic granulomatous disease. Annu Rev Immunol. 1989;7:277–307. [PubMed]
  • Young RA, Davis RW. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. [PMC free article] [PubMed]
  • Short JM, Fernandez JM, Sorge JA, Huse WD. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. [PMC free article] [PubMed]
  • Borregaard N, Heiple JM, Simons ER, Clark RA. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. J Cell Biol. 1983 Jul;97(1):52–61. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Babior BM, Kuver R, Curnutte JT. Kinetics of activation of the respiratory burst oxidase in a fully soluble system from human neutrophils. J Biol Chem. 1988 Feb 5;263(4):1713–1718. [PubMed]
  • White BA, Bancroft FC. Cytoplasmic dot hybridization. Simple analysis of relative mRNA levels in multiple small cell or tissue samples. J Biol Chem. 1982 Aug 10;257(15):8569–8572. [PubMed]
  • Johnson KR, Nauseef WM, Care A, Wheelock MJ, Shane S, Hudson S, Koeffler HP, Selsted M, Miller C, Rovera G. Characterization of cDNA clones for human myeloperoxidase: predicted amino acid sequence and evidence for multiple mRNA species. Nucleic Acids Res. 1987 Mar 11;15(5):2013–2028. [PMC free article] [PubMed]
  • Feinberg AP, Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. [PMC free article] [PubMed]
  • Okamura N, Curnutte JT, Roberts RL, Babior BM. Relationship of protein phosphorylation to the activation of the respiratory burst in human neutrophils. Defects in the phosphorylation of a group of closely related 48-kDa proteins in two forms of chronic granulomatous disease. J Biol Chem. 1988 May 15;263(14):6777–6782. [PubMed]
  • Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. [PMC free article] [PubMed]
  • Towler DA, Gordon JI, Adams SP, Glaser L. The biology and enzymology of eukaryotic protein acylation. Annu Rev Biochem. 1988;57:69–99. [PubMed]
  • Edelman AM, Blumenthal DK, Krebs EG. Protein serine/threonine kinases. Annu Rev Biochem. 1987;56:567–613. [PubMed]
  • Segal AW, Heyworth PG, Cockcroft S, Barrowman MM. Stimulated neutrophils from patients with autosomal recessive chronic granulomatous disease fail to phosphorylate a Mr-44,000 protein. Nature. 1985 Aug 8;316(6028):547–549. [PubMed]
  • Caldwell SE, McCall CE, Hendricks CL, Leone PA, Bass DA, McPhail LC. Coregulation of NADPH oxidase activation and phosphorylation of a 48-kD protein(s) by a cytosolic factor defective in autosomal recessive chronic granulomatous disease. J Clin Invest. 1988 May;81(5):1485–1496. [PMC free article] [PubMed]
  • Bolscher BG, van Zwieten R, Kramer IM, Weening RS, Verhoeven AJ, Roos D. A phosphoprotein of Mr 47,000, defective in autosomal chronic granulomatous disease, copurifies with one of two soluble components required for NADPH:O2 oxidoreductase activity in human neutrophils. J Clin Invest. 1989 Mar;83(3):757–763. [PMC free article] [PubMed]
  • Ohtsuka T, Ozawa M, Okamoto T, Uchida M, Okamura N, Ishibashi S. Significance of phosphorylation/dephosphorylation of 46K protein(s) in regulation of superoxide anion production in intact guinea pig polymorphonuclear leukocytes. J Biochem. 1987 Apr;101(4):897–903. [PubMed]
  • Ohtsuka T, Ozawa M, Katayama T, Ishibashi S. Synergism of phosphorylation of 46K protein(s) and arachidonate release in the induction of superoxide anion production in guinea pig polymorphonuclear leukocytes. Arch Biochem Biophys. 1988 May 1;262(2):416–421. [PubMed]
  • Curnutte JT, Scott PJ, Mayo LA. Cytosolic components of the respiratory burst oxidase: resolution of four components, two of which are missing in complementing types of chronic granulomatous disease. Proc Natl Acad Sci U S A. 1989 Feb;86(3):825–829. [PMC free article] [PubMed]
  • Dever TE, Glynias MJ, Merrick WC. GTP-binding domain: three consensus sequence elements with distinct spacing. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1814–1818. [PMC free article] [PubMed]
  • Jurnak F. Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins. Science. 1985 Oct 4;230(4721):32–36. [PubMed]
  • Suh PG, Ryu SH, Moon KH, Suh HW, Rhee SG. Inositol phospholipid-specific phospholipase C: complete cDNA and protein sequences and sequence homology to tyrosine kinase-related oncogene products. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5419–5423. [PMC free article] [PubMed]
  • Stahl ML, Ferenz CR, Kelleher KL, Kriz RW, Knopf JL. Sequence similarity of phospholipase C with the non-catalytic region of src. Nature. 1988 Mar 17;332(6161):269–272. [PubMed]
  • Mayer BJ, Hamaguchi M, Hanafusa H. A novel viral oncogene with structural similarity to phospholipase C. Nature. 1988 Mar 17;332(6161):272–275. [PubMed]
  • Lehto VP, Wasenius VM, Salvén P, Saraste M. Transforming and membrane proteins. Nature. 1988 Aug 4;334(6181):388–388. [PubMed]
  • Rhee SG, Suh PG, Ryu SH, Lee SY. Studies of inositol phospholipid-specific phospholipase C. Science. 1989 May 5;244(4904):546–550. [PubMed]
  • Pawson T. Non-catalytic domains of cytoplasmic protein-tyrosine kinases: regulatory elements in signal transduction. Oncogene. 1988 Nov;3(5):491–495. [PubMed]
  • Katan M, Parker PJ. Oncogenes and cell control. Nature. 1988 Mar 17;332(6161):203–203. [PubMed]

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