Summary
The primary interaction of tetanus toxin and toxoid with mouse neuroblastoma cells (C 1300, clone NB2A) in tissue culture was studied using direct immunofluorescence. Experiments were done in standard routine cultures and also those influenced by chemical modulators.
There is a difference in the characteristic binding response between the growth culture cells (grown in presence of fetal calf serum) and differentiating culture cells (grown in absence of serum). Exposure to the toxin gives no visible effect on the cell division or viability in growth cultures; whereas in differentiating cells the processes are shortened and the adherence to the glass is diminished without involving significant cell death. The toxoid did not bind at all under the same experimental conditions.
Since there was no biological effect in growth cultures we have called this binding ineffective, and in the case of the differentiating cells, effective binding. Stimulation of pinocytosis increases the uptake of toxin in both cultures. Presence of some surface bound toxin still remaining on the differentiating cells indicates the possibility of another sort of mechanism for internalization. Pre-treatment of the cells with neuraminidase of β-galactosidase to alter the membrane gangliosides eliminates binding in growth cultures but not in differentiating cultures.
From these results we suggest that even though the toxin may well bind to gangliosides, at least in the differentiating cultures they are not solely responsible for the fixation. The morphologically observed effective binding is probably that not related to gangliosides.
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References
Augusti-Tocco, G., Sato, G.: Establishment of functional clonal lines of neurons from mouse neuroblastoma. Proc. nat. Acad. Sci. (Wash.) 64, 311–315 (1969)
Barton, N. W., Rosenberg, A.: Action of Vibrio cholerae neuraminidase (sialidase) upon the surface of intact cells and their isolated sialolipid components. J. biol. Chem. 248, 7353–7358 (1973)
Bizzini, B., Blass, J., Turpin, A., Raynaud, M.: Chemical characterization of tetanus toxin and toxoid. Amino acid composition, number of SH and S-S groups and N-terminal amino acid. Europ. J. Biochem. 17, 100–105 (1970)
Bizzini, B., Turpin, A., Raynaud, M.: Immunochemistry of tetanus toxin. The nitration of tyrosyl residues in tetanus toxin. Europ. J. Biochem. 39, 171–181 (1973)
Bonventre, P. F., Saelinger, C. B., Ivins, B., Wascinski, C., Amorini, M.: Interaction of cultured mammalian cells with (125I) diphtheria toxin. Inf. Immun. 11, 675–684 (1975)
Breakefield, Xandra, O.: Minireview. Neurotransmitter metabolism in murine neuroblastoma cells. Life Sci. 18, 267–278 (1976)
Cohn, Z. A., Parks, Eileen: The regulation of pinocytosis in mouse marcrophages. II. Factors inducing vesicle formation. J. exp. Med. 125, 213–230 (1967)
Craven, C. J., Dawson, D. S.: The chain composition of tetanus toxin. Biochim. biophys. Acta (Amst.) 317, 277–285 (1973)
Craven, G. R., Steers, E., Jr., Anginsen, C. B.: Purification, composition and molecular weight of the β-galactosidase of Escherichia coli K12. J. biol. Chem. 240, 2468–2477 (1965)
Cuatrecasas, P.: Interaction of Vibrio cholerae enterotoxin with cell membranes. Biochemistry 12, 3547–3557 (1973)
Erdmann, G., Wiegand, H., Wellhöner, H. H.: Intraaxonal and extraaxonal transport of 125I-tetanus toxin in early local tetanus. Naunyn-Schmiedeberg's Arch. Pharmacol. 290, 357–373 (1975)
Habermann, E., Dimpfel, W.: Distribution of 125I-tetanus toxin and 125I-toxoid in rats with generalized tetanus, as influenced by antitoxin. Naunyn-Schmiedeberg's Arch. Pharmacol. 276, 327–340 (1973)
Haksar, A., Maudsley, D. V., Peron, F. G.: Neuraminidase treatment of adrenal cells increases their response to cholera enterotoxin. Nature (Lond.) 251, 514–515 (1974)
Helting, T., Zwisler, O.: Enzymatic breakdown of tetanus toxin. Biochem. biophys. Res. Commun. 57, 1263–1270 (1974)
King, L. E., Jr., Fedinec, A. A.: Pathogenesis of local tetanus in rats: neural ascent of tetanus toxin. Naunyn-Schmiedeberg's Arch. Pharmacol. 281, 391–401 (1974)
Kryzhanovsky, G. N.: The mechanism of action of tetanus toxin: effect on synaptic processes and some particular features of toxin binding by the nervous tissue. Naunyn-Schmiedeberg's Arch. Pharmacol. 276, 247–270 (1973)
Kryhanovsky, G. N., Rozanov, A. Ya., Kirilenko, O. A., Bondarchuk, N. G.: Competitive relations between the tetanus toxoid and toxin. Bull. exp. Biol. Med. 78, 17–20 (1975)
Matsuda, M., Yoneda, M.: Dissociation of tetanus neurotoxin into two polypeptide fragments. Biochem. biophys. Res. Commun. 57, 1257–1262 (1974)
Nelson, P., Ruffner, W., Nirenberg, M.: Neuronal tumor cells with excitable membranes grown in vitro. Proc. nat. Acad. Sci. (Wash.) 64, 1004–1010 (1969)
Price, D. L., Griffin, J., Young, A., Peck, K., Stocks, A.: Tetanus toxin: direct evidence for retrograde intraaxonal transport. Science 188, 945–947 (1975)
Rubin, C. S., Erlichman, J., Rosen, O. M.: Cyclic adenosine 3′, 5′-monophosphate-dependent protein kinase of human erythrocyte membranes. J. biol. Chem. 247, 6135–6139 (1972)
Ryser, H. J.-P.: The uptake of foreign proteins by mammalian cells and the functions of pinocytosis. Bull. Acad. Suisse Sci. Med. 24, 363–385 (1968)
Seeds, N. W., Gilman, A. G., Amano, T., Nirenberg, M. W.: Regulation of axon formation by clonal lines of a neural tumor. Proc. nat. Acad. Sci. (Wash.) 66, 160–167 (1970)
Snennerholm, L.: Chromatographic separation of human brain gangliosides. J. Neurochem. 10, 613–623 (1963)
Stöckel, K., Schwab, M., Thoenen, H.: Comparison between the retrograde axonal transport of nerve growth factor and tetanus toxin in motor, sensory and andrenergic neurons. Brain Res. 99, 1–16 (1975)
Thé, T. H., Feltkamp, T. E. W.: Conjugation of fluorescein isothiocyanate to antibodies. I. Experiments on the conditions of conjugation. Immunology 18, 865–873 (1970)_
van Heyningen, W. E.: Chemical assay of the tetanus toxin receptor in nervous tissue. J. gen. Microbiol. 20, 301–309 (1959)
van Heyningen, W. E.: Gangliosides as membrane receptors for tetanus toxin, cholera toxin and serotonin. Nature (Lond.) 249, 415–417 (1974)
van Heyningen, W. E., Mellanby, J.: Tetanus toxin; in: Microbial toxins, vol. IIA: Bacterial protein toxins, pp. 69–108 (S. Kadis, Th. C. Montie, and S. S. Ajl, eds). London: Acad. Press 1971
Weir, D. M. (ed.) Handbook of experimental immunology. 2nd ed. London: Blackwell 1973
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Zimmerman, J.M., Piffaretti, J.C. Interaction of tetanus toxin and toxoid with cultured neuroblastoma cells. Naunyn-Schmiedeberg's Arch. Pharmacol. 296, 271–277 (1977). https://doi.org/10.1007/BF00498693
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DOI: https://doi.org/10.1007/BF00498693