Abstract
Type E botulinum neurotoxin is produced byClostridium botulinum along with a neurotoxin binding protein which helps protect the neurotoxin from adversepH, temperature, and proteolytic conditions. The neurotoxin binding protein has been purified as a 118-kDa protein. Secondary structure content of the neurotoxin binding protein as revealed by far-UV circular dichroism spectroscopy was 19% α-helix, 50%β-sheets, 28% random coils, and 3%β-turns. This compared to 22% α-helix, 44%β-sheets, 34% random coils, and noβ-turns of the type E botulinum neurotoxin. The complex of the two proteins revealed 25%α-helix, 45%β-sheets, 27% random coils, and 3%β-turns, suggesting a significant alteration at least in theα-helical folding of the two proteins upon their interaction. Tyrosine topography is altered considerably (28%) when the neurotoxin and its binding protein are separated, indicating strong interaction between the two proteins. Gel filtration results suggested that type E neurotoxin binding protein clearly complexes with type E neurotoxin. The interaction is favored at lowpH as indicated by an initial binding rate of 8.4 min−1 atpH 5.7 compared to 4.0 min−1 atpH 7.5 as determined using a fiber optic-based biosensor. The neurotoxin and its binding protein apparently are of equivalent antigenicity, as both reacted equally on enzyme-linked immunosorbent assay to polyclonal antibodies raised against the toxoid of their complex.
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Binz, T., Kurazono, H., Popoff, M. R., Eklund, M. W., Sakaguchi, G., Kozaki, S., Krieglstein, K., Henschen, A., Gill, D. M., and Niemann, H. (1990a).Nucleic Acids Res. 18, 5556.
Binz, T., Kurazono, H., Popoff, M. W., Frevert, J., Wernars, K., and Niemann, H. (1990b).J. Biol. Chem. 265, 9153–9158.
Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. S., Brennan, R. G., and Scott, J. C. (1991).J. Biol. Chem. 266, 14188–14192.
Chang, T. C., Wu, C. S. C., and Yang, J. T. (1978).Anal. Biochem. 91, 13–31.
Eisenberg, D., Wilcox, W., and Eshita, S. (1987). InProteins: Structure and Function (L'Italien, J. J., ed.), Plenum Press, New York, pp. 425–436.
Fujii, N., Kimura, K., Yokosawa, N., Yashiki, T., Tsuzuki, K., and Oguma, K. (1993).J. Gen. Microbiol. 139, 79–83.
Gimenez, J. A., and Sugiyama, H. (1987).Appl. Environ. Microbiol. 53, 2827–2830.
Hall, J. A., and Sugiyama, H. (1987).Appl. Environ. Microbiol. 53, 2827–2830.
Hall, J. D., McCroskey, L. M., Pincomb, B. J., and Hatheway, C. L. (1985).J. Clin. Microbiol. 21, 654–655.
Hauser, D., Eklund, M. W., Kurazono, H., Binz, T., Niemann, H., Gill, D. M., Bouquet, P., and Popoff, M. R. (1990).Nucleic Acids Res. 18, 4924.
Laemmli, U. K. (1970).Nature 227, 680–685.
McCroskey, L. M., Hatheway, C. L., Fenicia, L., Pasolini, B., and Aureli, P. (1986).J. Clin. Microbiol. 23, 201–202.
Middlebrook, J. L. (1989). InBotulinum Neurotoxin and Tetamus Toxin (Simpson, L. L., ed.), Academic Press, San Diego, pp. 95–119.
Ogert, R. A., Brown, J. E., Singh, B. R., Shriver-Lake, L. C., and Ligler, F. S. (1992).Anal. Biochem. 205, 306–312.
Ohishi, L., and Sakaguchi, G. (1980).Infect. Immunol.,28, 303–309.
Ohishi, I., Sugii, S., and Sakaguchi, G. (1977).Infect. Immunol. 16, 107–109.
Patel, L., Abate, C., and Curran, T. (1990).Nature 347, 572–574.
Ragone, R., Colonna, G., Balestrieri, C., Servillo, L., and Trace, G. (1984).Biochemistry 23, 1871–1875.
Sakaguchi, G. (1983).Pharmac. Ther. 19, 165–194.
Sathyamoorthy, V., and DasGupta, B. R. (1985).J. Biol. Chem. 260, 10461–10466.
Schantz, E. J., and Johnson, E. A. (1992).Microbiol. Rev. 56, 80–99.
Schuermann, M., Hunter, J. B., Hennig, G., and Muller, R. (1991).Nucleic Acids Res. 19, 739–746.
Simpson, L. L. (1989). InBotulinum Neurotoxin and Tetanus Toxin (Simpson, L. L., ed.), Academic Press, San Diego, pp. 153–178.
Singh, B. R., and Be, X. (1992). InTechniques in Protein Chemistry III (Angeletti, R. H., ed.), Academic Press, Orlando, Florida, pp. 373–383.
Singh, B. R., and DasGupta, B. R. (1989).Mol. Cell. Biochem. 85, 67–73.
Singh, B. R., and DasGupta, B. R. (1990).Biophysical Chem. 38, 123–170.
Singh, B. R., and Poirier, M. P. (1993). InProceedings of Fiber Optic Sensors in Medical Diagnostics, SPIE Vol. 1886, pp. 27–34.
Singh, B. R., Kokan-Moore, N. P., and Bergdoll, M. S. (1988).Biochemistry 27, 8730–8735.
Strynadka, N. C. J., and James, M. N. G. (1990).Protein Struct. Funct. Genet. 7, 234–348.
Sugii, S., and Sakaguchi, G. (1975).Infect. Immunol. 12, 1262–1270.
Teale, F. W., and Weber, G. (1957).Biochem. J. 65, 476–482.
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.
Whelan, S. M., Elmore, M. J., Bodsworth, N. J., Atkinson, T., and Minton, N. P. (1992a).Eur. J. Biochem. 204, 657–667.
Whelan, S. M., Elmore, M. J., Bodsworth, N. J., Brehm, J. K., Atkinson, T., and Minton, N. P. (1992b).Appl. Environ. Microbiol. 58, 2345–2354.
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Singh, B.R., Foley, J. & Lafontaine, C. Physicochemical and immunological characterization of the type E botulinum neurotoxin binding protein purified fromClostridium botulinum. J Protein Chem 14, 7–18 (1995). https://doi.org/10.1007/BF01902839
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DOI: https://doi.org/10.1007/BF01902839