Abstract
A unique strain of Clostridium botulinum, serotype D 4947 (D-4947), produces a considerable amount of a 650 kDa toxin complex (L-TC) and a small amount of a 280 kDa M-TC, a 540 kDa TC, and a 610 kDa TC. The complexes are composed of only un-nicked components, including neurotoxin (NT), nontoxic nonhemagglutinin (NTNHA) and hemagglutinin subcomponents (HA-70, HA-33 and HA-17). Unlike other NTs from all serotype strains, separation of D-4947 NT from L-TC, except for M-TC, during chromatography required highly alkaline conditions around pH 8.8. The separated NT and NTNHA/HAs complex can be reconstituted to L-TC that is indistinguishable from the parent L-TC with respect to toxicity, hemagglutination activity and gel filtration profile. The isoelectric points of NT and NTNHA/HAs were close together depending on the number of HA-33/17 molecules. We have established a new method to separate the unique D-4947 NT from the complex, which will yield valuable information on structure of botulinum toxin.
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REFERENCES
Chen, F., Kuziemko, G. M., Amersdorfer, P., Wong, C., Marks, J. D., and Stevens, R. C. (1997). Infect. Immun. 65: 1626–1630.
Chen, F., Kuziemko, G. M., and Stevens, R. C. (1998). Infect. Immun. 66: 2420–2425.
Fu, F. N., Sharma, S. K., and Singh, B. R. (1998). J. Protein Chem. 17: 53–60.
Gessler, F., and Bohnel, H. (1999). FEMS Immunol. Med. Microbiol. 24: 361–367.
Halouzka, J., and Hubalek, Z. (1992). Folia Microbiol. 37: 157–158.
Kouguchi, H., Watanabe, T., Sagane, Y., Sunagawa, H., and Ohyama, T. (2002). J. Biol. Chem. 277: 2650–2656.
Laemmli, U. K. (1970). Nature 227: 680–685.
Montecucco, C., and Schiavo, G. (1994). Mol. Microbiol. 13: 1–8.
Mutoh, S., Kouguchi, H., Sagane, Y., Suzuki, T., Hasegawa, K., Watanabe, T., and Ohyama, T. (2003). Biochemistry 42: 10991–10997.
Ohyama, T., Watanabe, T., Fujinaga, Y., Inoue, K., Sunagawa, H., Fujii, N., Inoue, K., and Oguma, K. (1995). Microbiol. Immunol. 39: 457–465.
Prabakaran, S., Tepp, W., and DasGupta, B. R. (2001). Toxicon 39: 1515–1531.
Sagane, Y., Watanabe, T., Kouguchi, H., Sunagawa, H., Inoue, K., Fujinaga, Y., Oguma, K., and Ohyama, T. (1999). J. Protein Chem. 18: 885–892.
Sagane, Y., Watanabe, T., Kouguchi, H., Sunagawa, H., Inoue, K., Fujinaga, Y., Oguma, K., and Ohyama, T. (2000). J. Protein Chem. 19: 575–581.
Sagane, Y., Watanabe, T., Kouguchi, H., Sunagawa, H., Obata, S., Oguma, K., and Ohyama, T. (2002). Biochem. Biophys. Res. Commun. 292: 434–440.
Sakaguchi, G. (1982). Pharmacol. Ther. 19: 165–194.
Sakaguchi, G., Kozaki, S., and Ohishi, I. (1984). Structure and function of botulinum toxins, In Bacterial Protein Toxins (Alouf, J. E., Fehrenbach, F. J., Freer, J. H., and Jeljiaszewiz, J. (eds.), Academic Press, London, pp. 435–443.
Schiavo, G., Rossetto, O., Catsicas, S., Polverino de Laureto, P., DasGupta, B. R., Benfenati, F., and Montecucco, C. (1993). J. Biol. Chem. 268: 23784–23787.
Schiavo, G., Matteoli, M., and Montecucco, C. (2000). Physiol. Rev. 80: 717–766.
Simpson, L. L. (2000). Biochimie 82: 943–953.
Watanabe, T., Sagane, Y., Kouguchi, H., Sunagawa, H., Inoue, K., Fujinaga, Y., Oguma, K., and Ohyama, T. (1999). J. Protein Chem. 18: 753–760.
Wilbrandt, W. (1952). Arzneimittelforschung 2: 501–503.
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Hasegawa, K., Watanabe, T., Sato, H. et al. Characterization of Toxin Complex Produced by a Unique Strain of Clostridium botulinum Serotype D 4947. J Protein Chem 23, 371–378 (2004). https://doi.org/10.1023/B:JOPC.0000039551.42676.4c
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DOI: https://doi.org/10.1023/B:JOPC.0000039551.42676.4c