1887

Abstract

The 650 kDa large toxin complex (L-TC) produced by serotype D strain 4947 (D-4947) has a subunit structure composed of unnicked components, i.e. neurotoxin (NT), non-toxic non-haemagglutinin (NTNHA) and three haemagglutinin subcomponents (HA-70, HA-33 and HA-17). In this study, subunit interactions were investigated through the susceptibilities of the toxin components to limited trypsin proteolysis. Additionally, complex forms were reconstituted by various combinations of individual components. Trypsin treatment of intact D-4947 L-TC led to the formation of mature L-TC with nicks at specific sites of each component, which is usually observed in other strains of serotype D. NT, NTNHA and HA-17 were cleaved at their specific sites in either the single or complex forms, but HA-33 showed no sign of proteolysis. Unlike the other components, HA-70 was digested into random fragments as a single form, but it was cleaved into two fragments in the complex form. Based on the relative position of exposed or hidden regions of the individual components in the complex derived from their tryptic susceptibilities, an assembly model is proposed for the arrangement of individual subunits in the botulinum L-TC.

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2005-05-01
2020-04-09
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References

  1. Chen F., Kuziemko G. M., Amersdorfer P., Wong C., Marks J. D., Stevens R. C. 1997; Antibody mapping to domains of botulinum neurotoxin serotype A in the complexed and uncomplexed forms. Infect Immun65:1626–1630
    [Google Scholar]
  2. Davis B. J. 1964; Disc electrophoresis. II. Method and application to human serum proteins. Ann N Y Acad Sci121:404–427
    [Google Scholar]
  3. Fu F. N., Sharma S. K., Singh B. R. 1998; A protease-resistant novel hemagglutinin purified from type A Clostridium botulinum. J Protein Chem17:53–60[CrossRef]
    [Google Scholar]
  4. Fujinaga Y., Inoue K., Watanabe S., Yokota K., Hirai Y., Nagamachi E., Oguma K. 1997; The haemagglutinin of Clostridium botulinum type C progenitor toxin plays an essential role in binding of toxin to the epithelial cells of guinea pig small intestine, leading to the efficient absorption of the toxin. Microbiology143:3841–3847[CrossRef]
    [Google Scholar]
  5. Fujinaga Y., Inoue K., Nomura T., Sasaki J., Marvaud J. C., Popoff M. R., Kozaki S., Oguma K. 2000; Identification and characterization of functional subunits of Clostridium botulinum type A progenitor toxin involved in binding to intestinal microvilli and erythrocytes. FEBS Lett467:179–183[CrossRef]
    [Google Scholar]
  6. Fujinaga Y., Inoue K., Watarai S.. 10 other authors 2004; Molecular characterization of binding subcomponents of Clostridium botulinum type C progenitor toxin for intestinal epithelial cells and erythrocytes. Microbiology150:1529–1538[CrossRef]
    [Google Scholar]
  7. Hasegawa K., Watanabe T., Sato H.. 10 other authors 2004; Characterization of toxin complex produced by a unique strain of Clostridium botulinum serotype D 4947. Protein J23:371–378[CrossRef]
    [Google Scholar]
  8. Hirano H., Watanabe T. 1990; Microsequencing of proteins electrotransferred onto immobilizing matrices from polyacrylamide gel electrophoresis: application to an insoluble protein. Electrophoresis11:573–580[CrossRef]
    [Google Scholar]
  9. Inoue K., Sobhany M., Transue T. R., Oguma K., Pedersen L. C., Negishi M. 2003; Structural analysis by X-ray crystallography and calorimetry of a haemagglutinin component (HA1) of the progenitor toxin from Clostridium botulinum. Microbiology149:3361–3370[CrossRef]
    [Google Scholar]
  10. Kouguchi H., Watanabe T., Sagane Y., Ohyama T. 2001; Characterization and reconstitution of functional hemagglutinin of the Clostridium botulinum type C progenitor toxin. Eur J Biochem268:4019–4026[CrossRef]
    [Google Scholar]
  11. Kouguchi H., Watanabe T., Sagane Y., Sunagawa H., Ohyama T. 2002; In vitro reconstitution of the Clostridium botulinum type D progenitor toxin. J Biol Chem277:2650–2656[CrossRef]
    [Google Scholar]
  12. Krieglstein K. G., DasGupta B. R., Henschen A. H. 1994; Covalent structure of botulinum neurotoxin type A: location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains. J Protein Chem13:49–57[CrossRef]
    [Google Scholar]
  13. Lacy D. B., Tepp W., Cohen A. C., DasGupta B. R., Stevens R. C. 1998; Crystal structure of botulinum neurotoxin type A and implications for toxicity. Nat Struct Biol5:898–902[CrossRef]
    [Google Scholar]
  14. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227:680–685[CrossRef]
    [Google Scholar]
  15. Lee S. C., Whitaker J. R. 2004; Are molecular weights of proteins determined by Superose 12 column chromatography correct?. J Agric Food Chem52:4948–4952[CrossRef]
    [Google Scholar]
  16. Manavalan P., Johnson W. C.. Jr 1987; Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra. Anal Biochem167:76–85[CrossRef]
    [Google Scholar]
  17. Montecucco C., Schiavo G. 1993; Tetanus and botulism neurotoxins: a new group of zinc proteases. Trends Biochem Sci18:324–327[CrossRef]
    [Google Scholar]
  18. Mutoh S., Kouguchi H., Sagane Y., Suzuki T., Hasegawa K., Watanabe T., Ohyama T. 2003; Complete subunit structure of the Clostridium botulinum type D toxin complex via intermediate assembly with nontoxic components. Biochemistry42:10991–10997[CrossRef]
    [Google Scholar]
  19. Oguma K., Inoue K., Fujinaga Y., Yokota K., Watanabe T., Ohyama T., Takeshi K., Inoue K. 1999; Structure and function of Clostridium botulinum progenitor toxin. J Toxicol Toxin Rev18:17–34[CrossRef]
    [Google Scholar]
  20. Sagane Y., Watanabe T., Kouguchi H., Sunagawa H., Inoue K., Fujinaga Y., Oguma K., Ohyama T. 1999; Dichain structure of botulinum neurotoxin: identification of cleavage sites in types C, D, and F neurotoxin molecules. J Protein Chem18:885–892[CrossRef]
    [Google Scholar]
  21. Sagane Y., Watanabe T., Kouguchi H., Sunagawa H., Obata S., Oguma K., Ohyama T. 2002; Spontaneous nicking in the nontoxic-nonhemagglutinin component of the Clostridium botulinum toxin complex. Biochem Biophys Res Commun292:434–440[CrossRef]
    [Google Scholar]
  22. Sakaguchi G. 1982; Clostridium botulinum toxins. Pharmacol Ther19:165–194[CrossRef]
    [Google Scholar]
  23. Schiavo G., Matteoli M., Montecucco C. 2000; Neurotoxins affecting neuroexocytosis. Physiol Rev80:717–766
    [Google Scholar]
  24. Sharma S. K., Fu F. N., Singh B. R. 1999; Molecular properties of a hemagglutinin purified from type A Clostridium botulinum. J Protein Chem18:29–38[CrossRef]
    [Google Scholar]
  25. Shone C. C., Hambleton P., Melling J. 1985; Inactivation of Clostridium botulinum type A neurotoxin by trypsin and purification of two tryptic fragments. Proteolytic action near the COOH-terminus of the heavy subunit destroys toxin-binding activity. Eur J Biochem151:75–82[CrossRef]
    [Google Scholar]
  26. Simpson L. L. 2000; Identification of the characteristics that underlie botulinum toxin potency: implications for designing novel drugs. Biochimie82:943–953[CrossRef]
    [Google Scholar]
  27. Sugiyama H. 1980; Clostridium botulinum neurotoxin. Microbiol Rev44:419–448
    [Google Scholar]
  28. Swaminathan S., Eswaramoorthy S. 2000; Crystallization and preliminary X-ray analysis of Clostridium botulinum neurotoxin type B. Acta Crystallogr D Biol Crystallogr56:1024–1026[CrossRef]
    [Google Scholar]
  29. Watanabe T., Sagane Y., Kouguchi H., Sunagawa H., Inoue K., Fujinaga Y., Oguma K., Ohyama T. 1999; Molecular composition of progenitor toxin produced by Clostridium botulinum type C strain 6813. J Protein Chem18:753–760[CrossRef]
    [Google Scholar]
  30. Yang J. T., Wu C. S., Martinez H. M. 1986; Calculation of protein conformation from circular dichroism. Methods Enzymol130:208–269
    [Google Scholar]
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