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. 1994 Dec 1;180(6):2329–2334. doi: 10.1084/jem.180.6.2329

156Pro-->Gln substitution in the light chain of cytochrome b558 of the human NADPH oxidase (p22-phox) leads to defective translocation of the cytosolic proteins p47-phox and p67-phox

PMCID: PMC2191792  PMID: 7964505

Abstract

Src homology 3 (SH3) domains have been suggested to play an important role in the assembly of the superoxide-forming nicotinamide adenine dinucleotide phosphate (NADPH) oxidase upon activation of phagocytes, which involves the association of membrane-bound and cytosolic components. We studied the translocation of the cytosolic proteins to the plasma membrane in neutrophils of a patient with a point mutation in the gene encoding the light chain of cytochrome b558. This mutation leads to a substitution at residue 156 of a proline into a glutamine in a putative SH3 binding domain of p22-phox (Dinauer, M., E. A. Pierce, R. W. Erickson, T. Muhlebach, H. Messner, R. A. Seger, S. H. Orkin, and J. T. Curnutte. 1991. Proc. Natl. Acad. Sci. 88:11231). In PMA- stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient's neutrophils was virtually absent. In contrast, when solubilized membranes of the patient's neutrophils were activated with phospholipids in the absence of cytosol (Koshkin, V., and E. Pick. 1993. FEBS [Fed. Eur. Biochem. Soc.] Lett. 327:57), the rate of NADPH- dependent oxygen uptake was observed at a rate similar to that of control membranes. We suggest that the binding of an SH3 domain of p47- phox to p22-phox, and thus activation of the oxidase, does not occur in the neutrophils of this patient, although under artificial conditions, electron flow from NADPH to oxygen in cytochrome b558 is possible.

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Selected References

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  1. Abo A., Pick E., Hall A., Totty N., Teahan C. G., Segal A. W. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature. 1991 Oct 17;353(6345):668–670. doi: 10.1038/353668a0. [DOI] [PubMed] [Google Scholar]
  2. Ambruso D. R., Bolscher B. G., Stokman P. M., Verhoeven A. J., Roos D. Assembly and activation of the NADPH:O2 oxidoreductase in human neutrophils after stimulation with phorbol myristate acetate. J Biol Chem. 1990 Jan 15;265(2):924–930. [PubMed] [Google Scholar]
  3. Bolscher B. G., Denis S. W., Verhoeven A. J., Roos D. The activity of one soluble component of the cell-free NADPH:O2 oxidoreductase of human neutrophils depends on guanosine 5'-O-(3-thio)triphosphate. J Biol Chem. 1990 Sep 15;265(26):15782–15787. [PubMed] [Google Scholar]
  4. Clark R. A., Volpp B. D., Leidal K. G., Nauseef W. M. Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation. J Clin Invest. 1990 Mar;85(3):714–721. doi: 10.1172/JCI114496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dinauer M. C., Pierce E. A., Erickson R. W., Muhlebach T. J., Messner H., Orkin S. H., Seger R. A., Curnutte J. T. Point mutation in the cytoplasmic domain of the neutrophil p22-phox cytochrome b subunit is associated with a nonfunctional NADPH oxidase and chronic granulomatous disease. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11231–11235. doi: 10.1073/pnas.88.24.11231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Heyworth P. G., Curnutte J. T., Nauseef W. M., Volpp B. D., Pearson D. W., Rosen H., Clark R. A. Neutrophil nicotinamide adenine dinucleotide phosphate oxidase assembly. Translocation of p47-phox and p67-phox requires interaction between p47-phox and cytochrome b558. J Clin Invest. 1991 Jan;87(1):352–356. doi: 10.1172/JCI114993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Imajoh-Ohmi S., Tokita K., Ochiai H., Nakamura M., Kanegasaki S. Topology of cytochrome b558 in neutrophil membrane analyzed by anti-peptide antibodies and proteolysis. J Biol Chem. 1992 Jan 5;267(1):180–184. [PubMed] [Google Scholar]
  8. Kleinberg M. E., Malech H. L., Rotrosen D. The phagocyte 47-kilodalton cytosolic oxidase protein is an early reactant in activation of the respiratory burst. J Biol Chem. 1990 Sep 15;265(26):15577–15583. [PubMed] [Google Scholar]
  9. Knaus U. G., Heyworth P. G., Kinsella B. T., Curnutte J. T., Bokoch G. M. Purification and characterization of Rac 2. A cytosolic GTP-binding protein that regulates human neutrophil NADPH oxidase. J Biol Chem. 1992 Nov 25;267(33):23575–23582. [PubMed] [Google Scholar]
  10. Koch C. A., Anderson D., Moran M. F., Ellis C., Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. doi: 10.1126/science.1708916. [DOI] [PubMed] [Google Scholar]
  11. Koshkin V., Pick E. Superoxide production by cytochrome b559. Mechanism of cytosol-independent activation. FEBS Lett. 1994 Feb 7;338(3):285–289. doi: 10.1016/0014-5793(94)80285-8. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Leto T. L., Lomax K. J., Volpp B. D., Nunoi H., Sechler J. M., Nauseef W. M., Clark R. A., Gallin J. I., Malech H. L. Cloning of a 67-kD neutrophil oxidase factor with similarity to a noncatalytic region of p60c-src. Science. 1990 May 11;248(4956):727–730. doi: 10.1126/science.1692159. [DOI] [PubMed] [Google Scholar]
  14. Leusen J. H., de Boer M., Bolscher B. G., Hilarius P. M., Weening R. S., Ochs H. D., Roos D., Verhoeven A. J. A point mutation in gp91-phox of cytochrome b558 of the human NADPH oxidase leading to defective translocation of the cytosolic proteins p47-phox and p67-phox. J Clin Invest. 1994 May;93(5):2120–2126. doi: 10.1172/JCI117207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Orkin S. H. Molecular genetics of chronic granulomatous disease. Annu Rev Immunol. 1989;7:277–307. doi: 10.1146/annurev.iy.07.040189.001425. [DOI] [PubMed] [Google Scholar]
  16. Parkos C. A., Dinauer M. C., Jesaitis A. J., Orkin S. H., Curnutte J. T. Absence of both the 91kD and 22kD subunits of human neutrophil cytochrome b in two genetic forms of chronic granulomatous disease. Blood. 1989 May 1;73(6):1416–1420. [PubMed] [Google Scholar]
  17. Parkos C. A., Dinauer M. C., Walker L. E., Allen R. A., Jesaitis A. J., Orkin S. H. Primary structure and unique expression of the 22-kilodalton light chain of human neutrophil cytochrome b. Proc Natl Acad Sci U S A. 1988 May;85(10):3319–3323. doi: 10.1073/pnas.85.10.3319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ren R., Mayer B. J., Cicchetti P., Baltimore D. Identification of a ten-amino acid proline-rich SH3 binding site. Science. 1993 Feb 19;259(5098):1157–1161. doi: 10.1126/science.8438166. [DOI] [PubMed] [Google Scholar]
  19. Roos D. The genetic basis of chronic granulomatous disease. Immunol Rev. 1994 Apr;138:121–157. doi: 10.1111/j.1600-065x.1994.tb00850.x. [DOI] [PubMed] [Google Scholar]
  20. Roos D., de Boer M. Purification and cryopreservation of phagocytes from human blood. Methods Enzymol. 1986;132:225–243. doi: 10.1016/s0076-6879(86)32010-x. [DOI] [PubMed] [Google Scholar]
  21. Segal A. W., Cross A. R., Garcia R. C., Borregaard N., Valerius N. H., Soothill J. F., Jones O. T. Absence of cytochrome b-245 in chronic granulomatous disease. A multicenter European evaluation of its incidence and relevance. N Engl J Med. 1983 Feb 3;308(5):245–251. doi: 10.1056/NEJM198302033080503. [DOI] [PubMed] [Google Scholar]
  22. Smith R. M., Curnutte J. T. Molecular basis of chronic granulomatous disease. Blood. 1991 Feb 15;77(4):673–686. [PubMed] [Google Scholar]
  23. Tsunawaki S., Mizunari H., Nagata M., Tatsuzawa O., Kuratsuji T. A novel cytosolic component, p40phox, of respiratory burst oxidase associates with p67phox and is absent in patients with chronic granulomatous disease who lack p67phox. Biochem Biophys Res Commun. 1994 Mar 30;199(3):1378–1387. doi: 10.1006/bbrc.1994.1383. [DOI] [PubMed] [Google Scholar]
  24. Verhoeven A. J., Bolscher B. G., Meerhof L. J., van Zwieten R., Keijer J., Weening R. S., Roos D. Characterization of two monoclonal antibodies against cytochrome b558 of human neutrophils. Blood. 1989 May 1;73(6):1686–1694. [PubMed] [Google Scholar]
  25. Verhoeven A. J., Leusen J. H., Kessels G. C., Hilarius P. M., de Bont D. B., Liskamp R. M. Inhibition of neutrophil NADPH oxidase assembly by a myristoylated pseudosubstrate of protein kinase C. J Biol Chem. 1993 Sep 5;268(25):18593–18598. [PubMed] [Google Scholar]
  26. Volpp B. D., Nauseef W. M., Donelson J. E., Moser D. R., Clark R. A. Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7195–7199. doi: 10.1073/pnas.86.18.7195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wientjes F. B., Hsuan J. J., Totty N. F., Segal A. W. p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains. Biochem J. 1993 Dec 15;296(Pt 3):557–561. doi: 10.1042/bj2960557. [DOI] [PMC free article] [PubMed] [Google Scholar]

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