Characterization of botulinum C3-catalyzed ADP- ribosylation of rho proteins and identification of mammalian C3-like ADP-ribosyltransferase

  • Tomohiko Maehama
  • Nobuyuki Sekine
  • Hiroshi Nishina
  • Katsunobu Takahashi
  • Toshiaki Katada
Part of the Developments in Molecular and Cellular Biochemistry book series (DMCB, volume 12)


The exoenzyme C3 produced by Clostridium botulinum catalyzes ADP-ribosylation of rho gene products which belong to a family of small molecular-weight GTP-binding proteins. The C3 enzyme-catalyzed ADP-ribosylation of rho proteins partially purified from bovine brain was markedly activated by certain types of detergents or phospholipids and by endogenous factors present in the brain cytosol. Rho A protein that had been expressed in E. coli and subsequential purified was readily ADP-ribosylated by the C3 enzyme even in the absence of the activating factors. These results suggest that partially purified rho proteins contain an inhibitor, probably rho GDI (GDP-dissociation inhibitor for rho p21 ), of C3-catalyzed ADP-ribosylation. The activity of an endogenous enzyme, having the same substrate as botulinum C3 enzyme, was also found in brain cytosol. The enzyme activity was partially purified and characterized. The enzyme appeared to have a molecular mass of approximately 20,000 on a gel filtration and displayed unique properties similar to those observed with the botulinum C3 enzyme. The a-subunits of αβγ-trimeric G proteins which served as the substrates of cholera or pertussis toxin were not ADP-ribosylated by the brain enzyme. (Mol Cell Biochem 138: 135–140, 1994)

Key words

ADP-ribosylation botulinum ADP-ribosyltransferase C3 GTP-binding protein rho-gene product 


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  1. 1.
    Aktories K, Frevert J: ADP-ribosylation of a 21-24 kDa eukaryotic protein(s) by C3, a novel botulinum ADP-ribosyltransferase, is regulated by guanine nucleotide. Biochem J 247: 363–368, 1987PubMedGoogle Scholar
  2. 2.
    Sekine A, Fujiwara M, Narumiya S: Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase. J Biol Chem 264: 8602–8605, 1989PubMedGoogle Scholar
  3. 3.
    Rubin E J, Gill D M, Boquet P, Popoff M R: Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum. Mol Cell Biol 8: 418–426, 1988PubMedGoogle Scholar
  4. 4.
    Chardin P, Boquet P, Madaule P, Popoff M R, Rubin E J, Gill D M: The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells. EMBO J 8: 1087–1092, 1989PubMedGoogle Scholar
  5. 5.
    Paterson H F, Self A J, Garrett M D, Just I, Aktories K, Hall A: Microinjection of recombinant P21rho induces rapid changes in cell morphology. J Cell Biol 111: 1001–1007, 1990PubMedCrossRefGoogle Scholar
  6. 6.
    Nishiki T, Narumiya S, Morii N, Yamamoto M, Fujiwara M, Kamata Y, Sakaguchi G, Kozaki S: ADP-ribosylation of the rho/rac proteins induces growth inhibition, neurite outgrowth and acetylcholine esterase in cultured PC-12 cells. Biochem Biophys Res Commun 167: 265–272, 1990PubMedCrossRefGoogle Scholar
  7. 7.
    Mohr C, Just I, Hall A, Aktories K: Morphological alterations of Xenopus oocytes induced by valine-14 p21rho depend on isoprenylation and are inhibited by Clostridium botulinum C3 ADP-ribosyltransferase. FEBS Lett 275: 168–172, 1990PubMedCrossRefGoogle Scholar
  8. 8.
    Ridley A J, Hall A: The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389–399, 1992PubMedCrossRefGoogle Scholar
  9. 9.
    Morii N, Teru-uchi T, Tominaga T, Kumagai N, Kozaki S, Ushikubi F, Narumiya S: A rho gene product in human blood platelets. J Biol Chem 267: 20921–20926, 1992PubMedGoogle Scholar
  10. 10.
    Kishi K, Sasaki T, Kuroda S, Itoh T, Takai Y: Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI). J Cell Biol 120: 1113–1121, 1993CrossRefGoogle Scholar
  11. 11.
    Toratani S, KatadaT, Yokosawa H: Botulinum ADP-ribosyltransferase C3 induces elevation of the vitelline coat of ascidian eggs. Biochem Biophys Res Commun: in press.Google Scholar
  12. 12.
    Yost DA, Moss J: Amino acid-specificADP-ribosylation. J Biol Chem 258: 4926–4929, 1983PubMedGoogle Scholar
  13. 13.
    Peterson J E, Larew J S-A, Graves D J: Purification and partial characterization of arginine-specific ADP-ribosyltransferase from skeletal microsomal membranes. J Biol Chem 265: 17062–17069, 1990PubMedGoogle Scholar
  14. 14.
    Zolkiewska A, Nightingale M S, Moss J: Molecular characterization of NAD:arginine ADP-ribosyltransferase from rabbit skeletal muscle. Proc Natl Acad USA 89: 11352–11356, 1992CrossRefGoogle Scholar
  15. 15.
    Tanuma S, Kawashima K, Endo H: Eukaryotic mono(ADP-ribosyl)transferase that ADP-ribosylates GTP-binding regulatory Gi protein. J Biol Chem 263: 5485–5489, 1988PubMedGoogle Scholar
  16. 16.
    Maehama T, Ohoka Y, Ohtsuka T, Takahashi K, Nagata K, Nozawa Y, Ueno K, Ui M, Katada T: Botulinum ADP-ribosyltransferase activity as affected by detergents and phospholipids. FEBS Lett 263: 376–380, 1990PubMedCrossRefGoogle Scholar
  17. 17.
    Maehama T, Takahashi K, Ohoka Y, Ohtsuka T, Ui M, Katada T: Identification of a botulinum C3-like enzyme in bovine brain that catalyzes ADP-ribosylation of GTP-binding proteins. J Biol Chem 266: 10062–10065, 1991PubMedGoogle Scholar
  18. 18.
    Ohtsuka T, Nagata K, Iiri T, Nozawa Y, Ueno K, Ui M, Katada T: Activator protein supporting the botulinum ADP-ribosyltransferase reaction. J Biol Chem 264: 15000–15005, 1989PubMedGoogle Scholar
  19. 19.
    Kuroda S, Kikuchi A, Hirata K, Masuda T, Kishi K, Sasaki T, Takai Y: Cooperative function of rho GDS and rho GDI to regulate rho p21 activation in smooth muscle. Biochem Biophys Res Commun 185: 473–480, 1992PubMedCrossRefGoogle Scholar
  20. 20.
    Kikuchi A, Kuroda S, Sasaki T, Kotani K, Hirata K, Katayama M, Takai Y: Functional interactions of stimulatory and inhibitory GDP/ GTP exchange proteins and their common substrate small GTP-binding protein. J Biol Chem 267: 14611–14615, 1992PubMedGoogle Scholar
  21. 21.
    Regazzi R, Kikuchi A, Takai Y, Wollheim C B: The small GTP-binding proteins in the cytosol of insulin-secreting cells are complexed to GDP-dissociation inhibitor proteins. J Biol Chem 267: 17512–17519, 1992PubMedGoogle Scholar
  22. 22.
    Bourmeyster N, Stasia M-J, Garin J, Boquet P, Vignais P V: Copurification of rho protein and the rho-GDP dissociation inhibitor from bovine neutrophil cytosol. Effect of phosphoinositides on rho ADP-ribosylation by the C3 exoenzyme of Clostridium botulinum. Biochemistry 31: 12863–12869, 1992PubMedCrossRefGoogle Scholar
  23. 23.
    Hori Y, Kikuchi A, Isomura M, Katayama M, Miura Y, Fujioka H, Kaibuchi K, Takai Y: Post-translational modifications of the C-terminal region of the rho protein are important for its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins. Oncogene 6: 515–522, 1991PubMedGoogle Scholar
  24. 24.
    Kahn R A, Gilman A G: Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin. J Biol Chem 259: 6228–6234, 1984PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 1994

Authors and Affiliations

  • Tomohiko Maehama
    • 1
  • Nobuyuki Sekine
    • 1
  • Hiroshi Nishina
    • 1
  • Katsunobu Takahashi
    • 1
  • Toshiaki Katada
    • 1
  1. 1.Department of Life ScienceTokyo Institute of TechnologyYokohamaJapan

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