Abstract
Botulinum neurotoxins, the most potent of all toxins, induce lethal neuromuscular paralysis by inhibiting exocytosis at the neuromuscular junction. The light chains (LC) of these dichain neurotoxins are a new class of zinc-endopeptidases that specifically cleave the synaptosomal proteins, SNAP-25, VAMP, or syntaxin at discrete sites. To facilitate the structural and functional characterization of these unique endopeptidases, we constructed a synthetic gene for the LC of the botulinum neurotoxin serotype A (BoNT/A), overexpressed it in Escherichia coli, and purified the gene product from inclusion bodies. Our procedure can provide 1.1 g of the LC from 1 L of culture. The LC product was stable in solution at 4°C for at least 6 months. This rBoNT/A LC was proteolytically active, specifically cleaving the Glu-Arg bond in a 17-residue synthetic peptide of SNAP-25, the reported cleavage site of BoNT/A. Its calculated catalytic efficiency k cat/K m was higher than that reported for the native BoNT/A dichain. Treating the rBoNT/A LC with mercuric compounds completely abolished its activity, most probably by modifying the cysteine-164 residue located in the vicinity of the active site. About 70% activity of the LC was restored by adding Zn2+ to a Zn2+-free, apo-LC preparation. The LC was nontoxic to mice and failed to elicit neutralizing epitope(s) when the animals were vaccinated with this protein. In addition, injecting rBoNT/A LC into sea urchin eggs inhibited exocytosis-dependent plasma membrane resealing. For the first time, results of our study make available a large amount of the biologically active toxin fragment in a soluble and stable form.
Article PDF
Similar content being viewed by others
REFERENCES
Andersson, S. G. and Kurland, C. G. (1990). Microbiol. Rev. 54, 198–210.
Auld, D. S. (1995). Meth. Enzymol. 248, 228–242.
Bi, G. Q., Alderton, J. M., and Steinhardt, R. A. (1995). J. Cell Biol. 131, 1747–1758.
Bittner, M. A., DasGupta, B. R., and Holz, R. W. (1989). J. Biol. Chem. 264, 10354–10360.
Black, J. D. and Dolly, J. O. (1986). J. Cell Biol. 103, 535–544.
Blasi, J., Chapman, E. R., Link, E., Binz, T., Yamasaki, S., De Camilli, P., Sudhof, T. C., Niemann, H., and Jahn, R. (1993). Nature 365, 160–163.
Cai, S., Sarkar, H. K., and Singh, B. R. (1999). Biochemistry 38, 6903–6910.
Chen, F., Kuziemko, G. M., Amersdorfer, P., Wong, C., Marks, J. D., and Stevens, R. C. (1997). Infect. Immun. 65, 1626–1630.
Creighton, T. E. (1984). Proteins, Structures and Molecular Properties, Freeman, New York.
DasGupta, B. R. and Dekleva, M. L. (1990). Biochimie 72, 661–664.
DasGupta, B. R. and Foley, J., Jr. (1989). Biochimie 71, 1193–1200.
de Paiva, A., Poulain, B., Lawrence, G. W., Shone, C. C., Tauc, L., and Dolly, J. O. (1993). J. Biol. Chem. 268, 20838–20844.
Dertzbaugh, M. T. and West, M. W. (1996). Vaccine 14, 1538–1544.
Foran, P., Shone, C. C., and Dolly, J. O. (1994). Biochemistry 33, 15365–15374.
Foran, P., Lawrence, G. W., Shone, C. C., Foster, K. A., and Dolly, J. O. (1996). Biochemistry 35, 2630–2636.
Fu, F. N., Lomneth, R. B., Cai, S., and Singh, B. R. (1998). Biochemistry 37, 5267–5278.
Kiyatkin, N., Maksymowych, A. B., and Simpson, L. L. (1997). Infect. Immun. 65, 4586–4591.
Kurazono, H., Mochida, S., Binz, T., Eisel, U., Quanz, M., Grebenstein, O., Wernars, K., Poulain, B., Tauc, L., and Niemann, H. (1992). J. Biol. Chem. 267, 14721–14729.
Lacy, D. B. and Stevens, R. C. (1999). J. Mol. Biol. 291, 1091–1104.
Lacy, D. B., Tepp, W., Cohen, A. C., DasGupta, B. R., and Stevens, R. C. (1998). Nature Struct. Biol. 5, 898–902.
Laemmli, U. K. (1970). Nature 227, 680–685.
Lebeda, F. J. and Olson, M. A. (1994). Proteins 20, 293–300.
Li, L. and Singh, B. R. (1999). Protein Expr. Purif. 17, 339–344.
Li, Y., Foran, P., Fairweather, N. F., de Paiva, A., Weller, U., Dougan, G., and Dolly, J. O. (1994). Biochemistry 33, 7014–7020.
Maisey, E. A., Wadsworth, J. D., Poulain, B., Shone, C. C., Melling, J., Gibbs, P., Tauc, L., and Dolly, J. O. (1988). Eur. J. Biochem. 177, 683–691.
Makoff, A. J., Oxer, M. D., Romanos, M. A., Fairweather, N. F., and Ballantine, S. (1989). Nucleic Acids Res. 17, 10191–10202.
Montal, M. S., Blewitt, R., Tomich, J. M., and Montal, M. (1992). FEBS Lett. 313, 12–18.
Montecucco, C. and Schiavo, G. (1994). Mol. Microbiol. 13, 1–8.
Montecucco, C. and Schiavo, G. (1995). Q. Rev. Biophys. 28, 423–472.
Nowakowski, J. L., Courtney, B. C., Bing, Q. A., and Adler, M. (1998). J. Protein Chem. 17, 453–462.
Rossetto, O., Schiavo, G., Montecucco, C., Poulain, B., Deloye, F., Lozzi, L., and Shone, C. C. (1994). Nature 372, 415–416.
Schiavo, G., Rossetto, O., Catsicas, S., Polverino de Laureto, P., Das-Gupta, B. R., Benfenati, F., and Montecucco, C. (1993). J. Biol. Chem. 268, 23784–23787.
Schiavo, G., Malizio, C., Trimble, W. S., Polverino de Laureto, P., Milan, G., Sugiyama, H., Johnson, E. A., and Montecucco, C. (1994). J. Biol. Chem. 269, 20213–20216.
Schiavo, G., Rossetto, Tonello, F., and Montecucco, C. (1995). Intracellular targets and metalloprotease activity of tetanus and botulinum neurotoxins. In Clostridial Neurotoxins: The Molecular Pathogenesis of Tetanus and Botulism (Montecucco, C., ed.), Springer, New York, pp. 257–273.
Schmidt, J. J. and Bostian, K. A. (1995). J. Protein Chem. 14, 703–708.
Schmidt, J. J. and Bostian, K. A. (1997). J. Protein Chem. 16, 19–26.
Shone, C. C. and Roberts, A. K. (1994). Eur. J. Biochem. 225, 263–270.
Shone, C. C. and Tranter, H. S. (1995). Curr. Top. Microbiol. Immunol. 195, 143–160.
Shone, C. C., Quinn, C. P., Wait, R., Hallis, B., Fooks, S. G., and Hambleton, P. (1993). Eur. J. Biochem. 217, 965–971.
Smith, L. A. (1998). Toxicon 36, 1539–1548.
Steinhardt, R. A., Bi, G., and Alderton, J. M. (1994). Science 263, 390–393.
Syuto, B. and Kubo, S. (1981). J. Biol. Chem. 256, 3712–3717.
Thompson, D. E., Brehm, J. K., Oultram, J. D., Swinfield, T. J., Shone, C. C., Atkinson, T., Melling, J., and Minton, N. P. (1990). Eur. J. Biochem. 189, 73–81.
Washbourne, P., Pellizzari, R., Baldini, G., Wilson, M. C., and Montecucco, C. (1997). FEBS Lett. 418, 1–5.
Winkler, H. H. and Wood, D. O. (1988). Biochimie 70, 977–986.
Zhou, L., de Paiva, A., Liu, D., Aoki, R., and Dolly, J. O. (1995). Biochemistry 34, 15175–15181.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Ahmed, S.A., Smith, L.A. Light Chain of Botulinum A Neurotoxin Expressed as an Inclusion Body from a Synthetic Gene Is Catalytically and Functionally Active. J Protein Chem 19, 475–487 (2000). https://doi.org/10.1023/A:1026549431380
Published:
Issue Date:
DOI: https://doi.org/10.1023/A:1026549431380