Skip to main content
SpringerLink
Log in

Characteristics of NADPH oxidase genes (Nox2, p22, p47, and p67) and Nox4 gene expressed in blood cells of juvenile Ciona intestinalis

  • Original Paper
  • Published:
Immunogenetics Aims and scope Submit manuscript

Abstract

To illuminate the origins of NADPH oxidase (Nox), we identified cDNA clones encoding Nox2, Nox4, p22 phagocyte oxidase (phox), p47phox, and p67phox in a chordate phylogenetically distant to the vertebrates, the sea squirt Ciona intestinalis. We also examined the spatiotemporal expression of these genes in embryos and juveniles. The sequences of the Nox2, Nox4, p22phox, p47phox, and p67phox cDNAs contained open reading frames encoding 581, 811, 175, 461, and 515 amino acids, respectively. The level of identities between the deduced Nox2, Nox4, p22phox, p47phox, and p67phox amino acid sequences and their corresponding human components were 54.0, 31.0, 44.4, 36.0, and 26.2%, respectively. Despite these low identities, the functional domains of the C. intestinalis and human NADPH oxidase and Nox4 are highly conserved. The genomic organizations of the components of the NADPH oxidase gene except for p67phox (a single exon gene) and the Nox4 gene in C. intestinalis are highly similar to those of the corresponding human NADPH oxidase genes. Further, the analyzed part of the C. intestinalis genome and EST database do not seem to present p40phox and Nox5. The Nox2, p22phox, p47phox, and p67phox genes were specifically expressed in the blood cells of juveniles. The Nox4 gene was expressed in blood cells and endostyle of juveniles. These results suggest that C. intestinalis NADPH oxidase components possess potential functional activities similar to those of human, but the manner in which cytosolic phox proteins in C. intestinalis interact is different from that in human.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

Abbreviations

JGI:

Joint Genome Institute

ORF:

open reading frame

SH3:

Src homology 3

Phox:

phagocyte oxidase

PX:

phox homology domain

PRR:

proline-rich region

PC:

Phox and Cdc24p

Nox:

NADPH oxidase

WISH:

Whole-mount in situ hybridization

rboh:

respiratory burst oxidase homolog

ROS:

reactive oxygen species

References

  • Ago T, Nunoi H, Ito T, Sumimoto H (1999) Mechanism for phosphorylation-induced activation of the phagocyte NADPH oxidase protein p47(phox). Triple replacement of serines 303, 304, and 328 with aspartates disrupts the SH3 domain-mediated intramolecular interaction in p47(phox), thereby activating the oxidase. J Biol Chem 274:33644–33653

    Article  PubMed  CAS  Google Scholar 

  • Ago T, Kuribayashi F, Hiroaki H, Takeya R, Ito T, Kohda D, Sumimoto H (2003) Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation. Proc Natl Acad Sci U S A 100:4474–4479

    Article  PubMed  CAS  Google Scholar 

  • Ambasta RK, Kumar P, Griendling KK, Schmidt HH, Busse R, Brandes RP (2004) Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. J Biol Chem 279:45935–45941

    Article  PubMed  CAS  Google Scholar 

  • Azumi K, Satoh N, Yokosawa H (1993) Functional and structural characterization of hemocytes of the solitary ascidian, Halocynthia roretzi. J Exp Zool 265:309–349

    Article  CAS  Google Scholar 

  • Azumi K, Kuribayashi F, Kanegasaki S, Yokosawa H (2002) Zymosan induces production of superoxide anions by hemocytes of the solitary ascidian Halocynthia roretzi. Comp Biochem Physiol C Toxicol Pharmacol 133:567–574

    Article  PubMed  Google Scholar 

  • Baehner RL, Millar-Groff S, Bringas P (1999) Developmental expression of NADPH phagocytic oxidase components in mouse embryos. Pediatr Res 46:152–157

    Article  PubMed  CAS  Google Scholar 

  • Ballarin L, Cima F, Sabbadin A (1994) Phagocytosis in the colonial ascidian Botryllus schlosseri. Dev Comp Immunol 18:467–492

    Article  PubMed  CAS  Google Scholar 

  • Banfi B, Malgrange B, Knisz J, Steger K, Dubois-Dauphin M, Krause KH (2004) NOX3, a superoxide-generating NADPH oxidase of the inner ear. J Biol Chem 279:46065–46072

    Article  PubMed  CAS  Google Scholar 

  • 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 274:10451–10457

    Article  PubMed  CAS  Google Scholar 

  • Biberstine-Kinkade KJ, DeLeo FR, Epstein RI, LeRoy BA, Nauseef WM, Dinauer MC (2001) Heme-ligating histidines in flavocytochrome b(558): identification of specific histidines in gp91(phox). J Biol Chem 276:31105–31112

    Article  PubMed  CAS  Google Scholar 

  • Biberstine-Kinkade KJ, Yu L, Stull N, LeRoy B, Bennett S, Cross A, Dinauer MC (2002) Mutagenesis of p22(phox) histidine 94. A histidine in this position is not required for flavocytochrome b558 function. J Biol Chem 277:30368–30374

    Article  PubMed  CAS  Google Scholar 

  • Bionda C, Li XJ, van Bruggen R, Eppink M, Roos D, Morel F, Stasia MJ (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 115:418–427

    Article  PubMed  Google Scholar 

  • Cheng G, Cao Z, Xu X, van Meir EG, Lambeth JD (2001) Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene 269:131–140

    Article  PubMed  CAS  Google Scholar 

  • Dahan I, Issaeva I, Gorzalczany Y, Sigal N, Hirshberg M, Pick E (2002) Mapping of functional domains in the p22(phox) subunit of flavocytochrome b(559) participating in the assembly of the NADPH oxidase complex by “peptide walking.” J Biol Chem 277:8421–8432

    Article  PubMed  CAS  Google Scholar 

  • Dehal P, Satou Y, Campbell RK, Chapman J, Degnan B, De Tomaso A, Davidson B, Di Gregorio A, Gelpke M, Goodstein DM, Harafuji N, Hastings KE, Ho I, Hotta K, Huang W, Kawashima T, Lemaire P, Martinez D, Meinertzhagen IA, Necula S, Nonaka M, Putnam N, Rash S, Saiga H, Satake M, Terry A, Yamada L, Wang HG, Awazu S, Azumi K, Boore J, Branno M, Chin-Bow S, DeSantis R, Doyle S, Francino P, Keys DN, Haga S, Hayashi H, Hino K, Imai KS, Inaba K, Kano S, Kobayashi K, Kobayashi M, Lee BI, Makabe KW, Manohar C, Matassi G, Medina M, Mochizuki Y, Mount S, Morishita T, Miura S, Nakayama A, Nishizaka S, Nomoto H, Ohta F, Oishi K, Rigoutsos I, Sano M, Sasaki A, Sasakura Y, Shoguchi E, Shin-i T, Spagnuolo A, Stainier D, Suzuki MM, Tassy O, Takatori N, Tokuoka M, Yagi K, Yoshizaki F, Wada S, Zhang C, Hyatt PD, Larimer F, Detter C, Doggett N, Glavina T, Hawkins T, Richardson P, Lucas S, Kohara Y, Levine M, Satoh N, Rokhsar DS (2002) The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins. Science 298:2157–2167

    Article  PubMed  CAS  Google Scholar 

  • Deleo FR, Yu L, Burritt JB, Loetterle LR, Bond CW, Jesaitis AJ, Quinn MT (1995) Mapping sites of interaction of p47-phox and flavocytochrome b with random-sequence peptide phage display libraries. Proc Natl Acad Sci U S A 92:7110–7114

    Article  PubMed  CAS  Google Scholar 

  • Edens WA, Sharling L, Cheng G, Shapira R, Kinkade JM, Lee T, Edens HA, Tang X, Sullards C, Flaherty DB, Benian GM, Lambeth JD (2001) Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox. J Cell Biol 154:879–891

    Article  PubMed  CAS  Google Scholar 

  • el Benna J, Faust LP, Babior BM (1994) The phosphorylation of the respiratory burst oxidase component p47phox during neutrophil activation. Phosphorylation of sites recognized by protein kinase C and by proline-directed kinases. J Biol Chem 269:23431–23436

    PubMed  Google Scholar 

  • Finan P, Shimizu Y, Gout I, Hsuan J, Truong O, Butcher C, Bennett P, Waterfield MD, Kellie S (1994) An SH3 domain and proline-rich sequence mediate an interaction between two components of the phagocyte NADPH oxidase complex. J Biol Chem 269:13752–13755

    PubMed  CAS  Google Scholar 

  • Finegold AA, Shatwell KP, Segal AW, Klausner RD, Dancis A (1996) Intramembrane bis-heme motif for transmembrane electron transport conserved in a yeast iron reductase and the human NADPH oxidase. J Biol Chem 271:31021–31024

    Article  PubMed  CAS  Google Scholar 

  • Freeman JL, Lambeth JD (1996) NADPH oxidase activity is independent of p47phox in vitro. J Biol Chem 271:22578–22582

    Article  PubMed  CAS  Google Scholar 

  • Han CH, Freeman JL, Lee T, Motalebi SA, Lambeth JD (1998) Regulation of the neutrophil respiratory burst oxidase. Identification of an activation domain in p67(phox). J Biol Chem 273:16663–16668

    Article  PubMed  CAS  Google Scholar 

  • Hiroaki H, Ago T, Ito T, Sumimoto H, Kohda D (2001) Solution structure of the PX domain, a target of the SH3 domain. Nat Struct Biol 8:526–530

    Article  PubMed  CAS  Google Scholar 

  • Hua J, Hasebe T, Someya A, Nakamura S, Sugimoto K, Nagaoka I (2000) Evaluation of the expression of NADPH oxidase components during maturation of HL-60 cells to neutrophil lineage. J Leukoc Biol 68:216–224

    PubMed  CAS  Google Scholar 

  • Inoue Y, Suenaga Y, Yoshiura Y, Moritomo T, Ototake M, Nakanishi T (2004) Molecular cloning and sequencing of Japanese pufferfish (Takifugu rubripes) NADPH oxidase cDNAs. Dev Comp Immunol 28:911–925

    Article  PubMed  CAS  Google Scholar 

  • Ito T, Matsui Y, Ago T, Ota K, Sumimoto H (2001) Novel modular domain PB1 recognizes PC motif to mediate functional protein–protein interactions. EMBO J 20:3938–3946

    Article  PubMed  CAS  Google Scholar 

  • Kami K, Takeya R, Sumimoto H, Kohda D (2002) Diverse recognition of non-PxxP peptide ligands by the SH3 domains from p67(phox), Grb2 and Pex13p. EMBO J 21:4268–4276

    Article  PubMed  CAS  Google Scholar 

  • Koga H, Terasawa H, Nunoi H, Takeshige K, Inagaki F, Sumimoto H (1999) Tetratricopeptide repeat (TPR) motifs of p67(phox) participate in interaction with the small GTPase Rac and activation of the phagocyte NADPH oxidase. J Biol Chem 274:25051–25060

    Article  PubMed  CAS  Google Scholar 

  • Lambeth JD (2004) NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 4:181–189

    Article  PubMed  CAS  Google Scholar 

  • Lambeth JD, Cheng G, Arnold RS, Edens WA (2000) Novel homologs of gp91phox. Trends Biochem Sci 25:459–461

    Article  PubMed  CAS  Google Scholar 

  • Lara-Ortiz T, Riveros-Rosas H, Aguirre J (2003) Reactive oxygen species generated by microbial NADPH oxidase NoxA regulate sexual development in Aspergillus nidulans. Mol Microbiol 50:1241–1255

    Article  PubMed  CAS  Google Scholar 

  • Lassegue B, Sorescu D, Szocs K, Yin Q, Akers M, Zhang Y, Grant SL, Lambeth JD, Griendling KK (2001) Novel gp91(phox) homologues in vascular smooth muscle cells: nox1 mediates angiotensin II-induced superoxide formation and redox-sensitive signaling pathways. Circ Res 88:888–894

    Article  PubMed  CAS  Google Scholar 

  • Leusen JH, de Boer M, Bolscher BG, Hilarius PM, Weening RS, Ochs HD, Roos D, Verhoeven AJ (1994) 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 93:2120–2126

    Article  PubMed  CAS  Google Scholar 

  • Moreno JC, Bikker H, Kempers MJ, van Trotsenburg AS, Baas F, de Vijlder JJ, Vulsma T, Ris-Stalpers C (2002) Inactivating mutations in the gene for thyroid oxidase 2 (THOX2) and congenital hypothyroidism. N Engl J Med 347:95–102

    Article  PubMed  CAS  Google Scholar 

  • Ogasawara M, Minokawa T, Sasakura Y, Nishida H, Makabe KW (2001) A large-scale whole-mount in situ hybridization system: rapid one-tube, preparation of DIG-labeled RNA probes and high throughput hybridization using 96-well silent screen plates. Zool Sci 18:187–193

    Article  CAS  Google Scholar 

  • Ogasawara M, Sasaki A, Metoki H, Shin-i T, Kohara Y, Satoh N, Satou Y (2002) Gene expression profiles in young adult Ciona intestinalis. Dev Genes Evol 212:173–185

    Article  PubMed  Google Scholar 

  • Page RD (1996) TreeView: an application to display phylogenetic trees on personal computers. Comp Appl Biol Sci 12:357–358

    CAS  Google Scholar 

  • Price MO, McPhail LC, Lambeth JD, Han CH, Knaus UG, Dinauer MC (2002) Creation of a genetic system for analysis of the phagocyte respiratory burst: high-level reconstitution of the NADPH oxidase in a nonhematopoietic system. Blood 99:2653–2661

    Article  PubMed  CAS  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    PubMed  CAS  Google Scholar 

  • Satou Y, Takatori N, Fujiwara S, Nishikata T, Saiga H, Kusakabe T, Shin-i T, Kohara Y, Satoh N (2002) Ciona intestinalis cDNA projects: expressed sequence tag analyses and gene expression profiles during embryogenesis. Gene 287:83–96

    PubMed  CAS  Google Scholar 

  • Shiose A, Kuroda J, Tsuruya K, Hirai M, Hirakata H, Naito S, Hattori M, Sakaki Y, Sumimoto H (2001) A novel superoxide-producing NAD(P)H oxidase in kidney. J Biol Chem 276:1417–1423

    Article  PubMed  CAS  Google Scholar 

  • Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, Chung AB, Griendling KK, Lambeth JD (1999) Cell transformation by the superoxide-generating oxidase Mox1. Nature 401:79–82

    Article  PubMed  CAS  Google Scholar 

  • Sumimoto H, Sakamoto N, Nozaki M, Sakaki Y, Takeshige K, Minakami S (1992) Cytochrome b558, a component of the phagocyte NADPH oxidase, is a flavoprotein. Biochem Biophys Res Commun 186:1368–1375

    Article  PubMed  CAS  Google Scholar 

  • Takeya R, Ueno N, Kami K, Taura M, Kohjima M, Izaki T, Nunoi H, Sumimoto H (2003) Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases. J Biol Chem 278:25234–25246

    Article  PubMed  CAS  Google Scholar 

  • Taylor RM, Burritt JB, Baniulis D, Foubert TR, Lord CI, Dinauer MC, Parkos CA, Jesaitis AJ (2004) Site-specific inhibitors of NADPH oxidase activity and structural probes of flavocytochrome b: characterization of six monoclonal antibodies to the p22phox subunit. J Immunol 173:7349–7357

    PubMed  CAS  Google Scholar 

  • Teshima S, Kutsumi H, Kawahara T, Kishi K, Rokutan K (2000) Regulation of growth and apoptosis of cultured guinea pig gastric mucosal cells by mitogenic oxidase 1. Am J Physiol Gastrointest Liver Physiol 279:1169–1176

    Google Scholar 

  • Torres MA, Onouchi H, Hamada S, Machida C, Hammond-Kosack KE, Jones JD (1998) Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91phox). Plant J 14:365–370

    Article  PubMed  CAS  Google Scholar 

  • Vignais PV (2002) The superoxide-generating NADPH oxidase: structural aspects and activation mechanism. Cell Mol Life Sci 59:1428–1459

    Article  PubMed  CAS  Google Scholar 

  • Wientjes FB, Hsuan JJ, Totty NF, Segal AW (1993) p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains. Biochem J 15:557–561

    Google Scholar 

  • Yagisawa M, Yuo A, Yonemaru M, Imajoh-Ohmi S, Kanegasaki S, Yazaki Y, Takaku F (1996) Superoxide release and NADPH oxidase components in mature human phagocytes: correlation between functional capacity and amount of functional proteins. Biochem Biophys Res Commun 228:510–516

    Article  PubMed  CAS  Google Scholar 

  • Yu L, Zhen L, Dinauer MC (1997) Biosynthesis of the phagocyte NADPH oxidase cytochrome b558. Role of heme incorporation and heterodimer formation in maturation and stability of gp91phox and p22phox subunits. J Biol Chem 272:27288–27294

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgement

This study was supported in part by the Grants-in-Aid for Scientific Research (B) from the Ministry of Education, Science, Sports and Culture of Japan.

Author information

Authors and Affiliations

  1. Laboratory of Fish Pathology, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa, Kanagawa, 252-8510, Japan

    Yuuki Inoue, Takuma Moroi, Tadaaki Moritomo & Teruyuki Nakanishi

  2. Department of Biology, Faculty of Science, Chiba University, Yayoi 1-33, Inage, Chiba, 263-8522, Japan

    Michio Ogasawara

  3. Department of Molecular Immunology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-8575, Japan

    Masanobu Satake

  4. Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060-0812, Japan

    Kaoru Azumi

Authors
  1. Yuuki Inoue
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michio Ogasawara
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Takuma Moroi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Masanobu Satake
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kaoru Azumi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tadaaki Moritomo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Teruyuki Nakanishi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yuuki Inoue.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Inoue, Y., Ogasawara, M., Moroi, T. et al. Characteristics of NADPH oxidase genes (Nox2, p22, p47, and p67) and Nox4 gene expressed in blood cells of juvenile Ciona intestinalis . Immunogenetics 57, 520–534 (2005). https://doi.org/10.1007/s00251-005-0010-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00251-005-0010-4

Keywords

Search

Navigation