Studies on the Interaction between Subunits of the Botulinum Toxin Complex and X-Ray Crystallography

• Project/Area Number: 15570103
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: Tokyo University of Agriculture
• Principal Investigator: OHYAMA Tohru Tokyo Univ.of Agriculture, Dept.of Food Science and Technology, Fac.of Bioindustry, Professor, 生物産業学部, 教授 (60318178)
• Co-Investigator(Kenkyū-buntansha): WATANABE Toshihiro Tokyo Univ.of Agriculture, Dept.of Food Science and Technology, Fac.of Bioindustry, Professor, 生物産業学部, 教授 (80175695) ; NAKAGAWA Tomoyuki Tokyo Univ.of Agriculture, Dept.of Food Science and Technology, Fac.of Bioindustry, Assistant Professor, 生物産業学部, 講師 (70318179)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥3,700,000 (Direct Cost: ¥3,700,000); Fiscal Year 2004: ¥1,600,000 (Direct Cost: ¥1,600,000); Fiscal Year 2003: ¥2,100,000 (Direct Cost: ¥2,100,000)
• Keywords: Botulinum toxin / Subunit structure / X-ray crystallography / Protein complex / Protein-protein interaction / タンパク質問相互作用 / タンパク間相互作用

Research Abstract

Botulinum toxin is produced as a 650 kDa toxin complex(TC) with subunit structure that non-covalently associated with five different protein components, 150 kDa neurotoxin(NT), nontoxic non-hemagglutinin(NTNHA) and three hemagglutinin subcomponents, in culture supernatant. In this study, we have clarified molecular ration of subunit components in the TC. Type D strain 4947(D-4947) produces a large amount of TC species (650 kDa L-TC,610 kDa and 540 kDa TC). They revealed same banding patterns on SDS-PAGE, but less number of HA-33/17 complexes in the TC. Based on titration experiments with HA-33/17 complex, we have concluded that complete subunit composition of the TC is a dodecamer composed of a single NT, a single NTNHA, two HA-70,four HA-33 and four HA-17 molecules. Accordingly, we have preliminary information on the analysis of the three-dimensional structure of botulinum TC by X-ray crystallography. Of the TC species, recombination of the components was found in 610 and 540 kDa TCs depending upon storage condition such as temperature, and was thus depressed by the addition of glycerol leading supply of stable TC for the experiments. Additionally, we have established new method to separate considerable amount of the NT from the D-4947 TC, which has been technically difficult to separate. Although we have attempted to crystallize TCs (M and L-TC) that were examined using dynamic light scattering from serotype C and D strains by 130 combinations of experimental condition, it has not been yet obtain large crystals for X-ray analysis.

Research Products

• [Journal Article] Structure and function of the Clostridium botulinum serotypes C and D toxin complex (2005)
  • Author(s): Watanabe, T.
  • Journal Title: Research Signpost Pages: 17-33
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Characterization of the interaction between subunits of the botulinum toxin complex produced by serotype D through tryptic susceptibility of the isolated components and complex forms. (2005)
  • Author(s): Suzuki T, Watanabe T, Mutoh S, Hasegawa K, Kouguchi H, Fujinaga F, Oguma K, Ohyama, T.
  • Journal Title: Microbiology -UK 151
• [Journal Article] Characterization of toxin complex produced by a uniqe strain of Clostridium botulinum serotype D4947 (2004)
  • Author(s): Hasegawa, K.
  • Journal Title: The Protein Journal 23 Pages: 371-378
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells (2004)
  • Author(s): Nishikawa, A.
  • Journal Title: Biochemical and Biophysical Research Communications 25 Pages: 327-333
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Molecular characterization of binding subcomponents of Clostridium botulinum type C progenitor toxin for intestinal epithelial cells and erythrocytes (2004)
  • Author(s): Fujinaga, Y.
  • Journal Title: Microbiology 150 Pages: 1529-1538
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Molecular characterization of binding subcomponents of Clostridium botulinum type C progenitor toxin for intestinal cells and erythrocytes (2004)
  • Author(s): Fujinaga, Y., Inoue, K., Watarai, S., Sakaguchi, Y., Arimitsu, H., Lee, J.C., Jin, Y., Matsumura, T., Kabumoto, Y., Watanabe, T., Ohyama, T., Nishikawa, A., Oguma, K.
  • Journal Title: Microbiology Vol.150 Pages: 1529-1538
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells (2004)
  • Author(s): Nishikawa, A., Uotsu, N., Arimitsu, H., Lee, J.C., Miura, Y., Fujinaga, Y., Nakada, H., Watanabe, T., Ohyama, T., Sakano, Y., Oguma, K.
  • Journal Title: Biochem.Biophys.Res.Commun. Vol.319 Pages: 327-333
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Characterization of Toxin Complex Produced by a Unique Strain of Clostridium botulinum type D 4947 (2004)
  • Author(s): Hasegawa, K., Watanabe, T., Sato, H., Sagane, Y., Mutoh, S., Suzuki, T., Yamano, Y., Kouguchi, H., Takeshi, K., Kamaguchi, A., Fujinaga, Y., Oguma, K., Ohyama, T.
  • Journal Title: Protein J. Vol.23 Pages: 371-378
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Characterization of Toxin Complex Produced by a Unique Strain of Clostridium botulinum type D 4947. (2004)
  • Author(s): Hasegawa K, Watanabe T, Sato H, Sagane Y, Mutoh S, Suzuki T, Yamano Y, Kouguchi H, Takeshi K, Kamaguchi A, Fujinaga Y, Oguma K, Ohyama T.
  • Journal Title: Protein J. 23 Pages: 371-378
• [Journal Article] Molecular characterization of GroES and Gro EL Homologues from Clostridium botulinum (2003)
  • Author(s): Sagane, S.
  • Journal Title: Journal of Protein Chemistry 22 Pages: 99-108
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Complete subunit structure of the Clostridium botulinum type D toxin complex via intermediate assembly with nontoxic components (2003)
  • Author(s): Mutoh, S.
  • Journal Title: Biochemistry 42 Pages: 10991-10997
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Purification of fully activated Clostridium botulinum serotype B toxin for treatment of patients with dystonia (2003)
  • Author(s): Arimitsu, H.
  • Journal Title: Infection and Immunity 71 Pages: 1599-1603
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Molecular Characterization of GroES and GroEL Homologues from Clostridium botulinum (2003)
  • Author(s): Sagane, Y., Hasegawa, K., Mutoh, S., Kouguchi, K., Suzuki, T., Sunagawa, H., Nakagawa, T., Kamaguchi, A., Okasaki, S., Nakayama, K., Watanabe, T., Oguma K., Ohyama, T.
  • Journal Title: J.Protein Chem. Vol.22 Pages: 99-108
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Purification of Fully Activated Clostridium botulinum Serotype B Toxin for Treatment of Patients with Dystonia (2003)
  • Author(s): Arimitsu, H., Inoue, K., Sakaguchi, Y., Lee, J.C., Fujinaga, Y., Watanabe, T., Ohyama, T., Hirst, R., Oguma, K.
  • Journal Title: Infect.Immun. Vol.71 Pages: 1599-1603
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Complete Subunit Structure of Clostridium botulinum Type D Toxin Complex via Intermediate Assembly with Nontoxic Components (2003)
  • Author(s): Mutoh, S., Kouguchi, H., Sagane, Y., Suzuki, T., Hasegawa, K., Watanabe, T., Ohyama, T.
  • Journal Title: Biochemistry Vol.37 Pages: 10991-10997
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Complete Subunit Structure of Clostridium botulinum Type D Toxin Complex via Intermediate Assembly with Nontoxic Components. (2003)
  • Author(s): Mutoh S, Kouguchi H, Sagane Y, Suzuki T, Hasegawa K, Watanabe T, Ohyama T.
  • Journal Title: Biochemistry 37 Pages: 10991-10997
• [Book] Structure and function of the Clostridium botulinum serotypes C and D toxin complex (2005)
  • Author(s): Watanabe T., Sagane, Y., Mutoh, S., Suzuki, T., Hasegawa, K., Kouguchi, H., Ohyama, T.
  • Publisher: Research Signpost
  • Description: 「研究成果報告書概要(欧文)」より
• [Book] Structure and Function of the Clostridium botulinum Serotypes C and D Toxin Complex. (2005)
  • Author(s): Watanabe T, Sagane Y, Mutoh S, Suzuki T, Hasegawa K, Kouguchi H, Ohyama T.
  • Total Pages: 17
  • Publisher: Research Signpost
• [Publications] Mutoh, S., Kouguchi, H., Sagane, Y., Suzuki, T., Hasegawa, K., Watanabe, T., Ohyama, T.: "Complete Subunit Structure of the Clostridium botulinum Type D Toxin Complex via Intermediate Assembly with Nontoxic Components"Biochemistry. 42. 10991-10997 (2003)