NADPH Oxidases pp 153-171 | Cite as

The X-CGD PLB-985 Cell Model for NOX2 Structure-Function Analysis

  • Sylvain Beaumel
  • Marie José StasiaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1982)


Structure-function analysis of specific regions of NOX2 can be carried out after stable expression of site-directed mutagenesis-modified NOX2 in the X0-CGD PLB-985 cell model. Indeed, the generation of this human cellular model by Prof. MC Dinauer’s team gave researchers the opportunity to gain a deeper understanding of functional regions of NOX2. With this model cell line, the functional impact of X+-CGD or of new mutations in NOX2 can be highlighted, as the biological material is not limited. PLB-985 cells transfected with various NOX2 mutations can be easily cultured and differentiated into neutrophils or monocytes/macrophages. Several measurements in intact mutated NOX2 PLB-985 cells can be carried out such as NOX2 expression, cytochrome b558 spectrum, enzymatic activity, and assembly of the NADPH oxidase complex. Purified membranes or purified cytochrome b558 from mutated NOX2 PLB-985 cells can be used for the study of the impact of specific mutations on NADPH oxidase or diaphorase activity, FAD incorporation, and NADPH or NADH binding in a cell-free assay system. Here, we describe a method to generate mutated NOX2 PLB-985 cells in order to analyze NOX2 structure-function relationships.

Key words

PLB-985 cell line Chronic granulomatous disease NOX2 deficiency NADPH oxidase activity Cellular model Structure-function analysis 


  1. 1.
    Bedard K, Krause KH (2007) The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87:245–313CrossRefGoogle Scholar
  2. 2.
    Van den Berg JM, van Koppen E, Ahlin A et al (2009) Chronic granulomatous disease: the European experience. PLoS 4:e5234CrossRefGoogle Scholar
  3. 3.
    Stasia MJ, Li XJ (2008) Genetics and immunopathology of chronic granulomatous disease. Semin Immunopathol 30:209–235CrossRefGoogle Scholar
  4. 4.
    Tucker KA, Lilly MB, Heck L Jr, Rado TA (1987) Characterization of a new human diploid myeloid leukemia cell line (PLB-985) with granulocytic and monocytic differentiating capacity. Blood 70:372–378PubMedGoogle Scholar
  5. 5.
    Zhen L, King AA, Xiao Y, Chanock SJ, Orkin SH, Dinauer MC (1993) Gene targeting of X chromosome-linked chronic granulomatous disease locus in a human myeloid leukemia cell line and rescue by expression of recombinant gp91phox. Proc Natl Acad Sci U S A 90:9832–9836CrossRefGoogle Scholar
  6. 6.
    Yu L, Cross AR, Zhen L, Dinauer MC (1999) Functional analysis of NADPH oxidase in granulocytic cells expressing a delta 488-497 gp91(phox) deletion mutant. Blood 94:2497–2504PubMedGoogle Scholar
  7. 7.
    Bionda C, Li XJ, van Bruggen R et al (2004) Functional analysis of two-amino acid substitutions in gp91 phox in a patient with X-linked flavocytochrome b558-positive chronic granulomatous disease by means of transgenic PLB-985 cells. Hum Genet 115:418–427CrossRefGoogle Scholar
  8. 8.
    Li XJ, Grunwald D, Mathieu J, Morel F, Stasia MJ (2005) Crucial role of two potential cytosolic regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH oxidase activation. J Biol Chem 280:14962–14973CrossRefGoogle Scholar
  9. 9.
    Li XJ, Fieschi F, Paclet MH et al (2007) Leu505 of Nox2 is crucial for optimal p67phox-dependent activation of the flavocytochrome b558 during phagocytic NADPH oxidase assembly. J Leukoc Biol 81:238–249CrossRefGoogle Scholar
  10. 10.
    Debeurme F, Picciocchi A, Dagher MC et al (2010) Regulation of NADPH oxidase activity in phagocytes: relationship between FAD/NADPH binding and oxidase complex assembly. J Biol Chem 285:33197–33208CrossRefGoogle Scholar
  11. 11.
    Picciocchi A, Debeurme F, Beaumel S et al (2011) Role of putative second transmembrane region of Nox2 protein in the structural stability and electron transfer of the phagocytic NADPH oxidase. J Biol Chem 286:28357–28369CrossRefGoogle Scholar
  12. 12.
    Carrichon L, Picciocchi A, Debeurme F et al (2011) Characterization of superoxide overproduction by the D-Loop(Nox4)-Nox2 cytochrome b(558) in phagocytes-Differential sensitivity to calcium and phosphorylation events. Biochim Biophys Acta 1808:78–90CrossRefGoogle Scholar
  13. 13.
    Zhen L, Yu L, Dinauer MC (1998) Probing the role of the carboxyl terminus of the gp91phox subunit of neutrophil flavocytochrome b558 using site-directed mutagenesis. J Biol Chem 273:6575–6581CrossRefGoogle Scholar
  14. 14.
    Beaumel S, Picciocchi A, Debeurme F et al (2017) Down-regulation of NOX2 activity in phagocytes mediated by ATM-kinase dependent phosphorylation. Free Radic Biol Med 113:1–15CrossRefGoogle Scholar
  15. 15.
    Magnani F, Nenci S, Millana Fananas E et al (2017) Crystal structures and atomic model of NADPH oxidase. Proc Natl Acad Sci U S A 114:6764–6769CrossRefGoogle Scholar
  16. 16.
    Biberstine-Kinkade KJ, Yu L, Dinauer MC (1999) Mutagenesis of an arginine- and lysine-rich domain in the gp91(phox) subunit of the phagocyte NADPH-oxidase flavocytochrome b558. J Biol Chem 274:10451–10457CrossRefGoogle Scholar
  17. 17.
    Jackson HM, Kawahara T, Nisimoto Y, Smith SM, Lambeth JD (2010) Nox4 B-loop creates an interface between the transmembrane and dehydrogenase domains. J Biol Chem 285:10281–10290CrossRefGoogle Scholar
  18. 18.
    Taylor WR, Jones DT, Segal AW (1993) A structural model for the nucleotide binding domains of the flavocytochrome b-245 beta-chain. Protein Sci 2:1675–1685CrossRefGoogle Scholar
  19. 19.
    Kean KM, Carpenter RA, Pandini V et al (2017) High resolution studies of hydride transfer in the ferredoxin: NADP(+) reductase superfamily. FEBS J 284:3302–3319CrossRefGoogle Scholar
  20. 20.
    Beaumel S, Grunwald D, Fieschi F, Stasia MJ (2014) Identification of NOX2 regions for normal biosynthesis of cytochrome b558 in phagocytes highlighting essential residues for p22phox binding. Biochem J 464:425–437CrossRefGoogle Scholar
  21. 21.
    Ding C, Kume A, Björgvinsdóttir H, Hawley RG, Pech N, Dinauer MC (1996) High-level reconstitution of respiratory burst activity in a human X-linked chronic granulomatous disease (X-CGD) cell line and correction of murine X-CGD bone marrow cells by retroviral-mediated gene transfer of human gp91phox. Blood 88:1834–1840PubMedGoogle Scholar
  22. 22.
    Wrona D, Siler U, Reichenbach J (2017) CRISPR/Cas9-generated p47(phox)-deficient cell line for Chronic Granulomatous Disease gene therapy vector development. Sci Rep 7:44187CrossRefGoogle Scholar
  23. 23.
    Pedruzzi E, Fay M, Elbim C, Gaudry M, Gougerot-Pocidalo MA (2002) Differentiation of PLB-985 myeloid cells into mature neutrophils, shown by degranulation of terminally differentiated compartments in response to N-formyl peptide and priming of superoxide anion production by granulocyte-macrophage colony-stimulating factor. Br J Haematol 117:719–726CrossRefGoogle Scholar
  24. 24.
    Pivot-Pajot C, Chouinard FC, El Azreq MA, Harbour D, Bourgoin SG (2010) Characterisation of degranulation and phagocytic capacity of a human neutrophilic cellular model, PLB-985 cells. Immunobiology 215:38–52CrossRefGoogle Scholar
  25. 25.
    Yamauchi A, Yu L, Pötgens AJ et al (2001) Location of the epitope for 7D5, a monoclonal antibody raised against human flavocytochrome b558, to the extracellular peptide portion of primate gp91phox. Microbiol Immunol 45:249–257CrossRefGoogle Scholar
  26. 26.
    Lord CI, Riesselman MH, Gripentrog JM, Burritt JB, Jesaitis AJ, Taylor RM (2008) Single-step immunoaffinity purification and functional reconstitution of human phagocyte flavocytochrome b. J Immunol Methods 329:201–207CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Centre Diagnostic et Recherche CGD (CDiReC), Pôle Biologie, CHU Grenoble AlpesGrenobleFrance
  2. 2.Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie StructuraleGrenobleFrance

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