Abstract
Tetanus and botulinum A neurotoxins were introduced into the cytosol of chromaffin cells by means of an electric field in which the plasma membrane is forced to form pores of approximately 1 μm at the sites facing the electrodes. As demonstrated by electron microscopy, both [125I] and gold-labelled tetanus toxin (TeTx) diffuse through these transient openings. Dichain TeTx, with its light chain linked to the heavy chain by means of a disulfide bond, causes the block of exocytosis to develop more slowly than does the purified light chain. The disulfide bonds, which in both toxins hold the subunits together, were cleaved by the intrinsic thioredoxin-reductase system. Single chain TeTx, in which the heavy and light chains are interconnected by an additional peptide bond, was far less effective than dichain TeTx at blocking exocytosis, which indicates that proteolysis is the rate-limiting step. The toxins were degraded further to low-molecular weight fragments which, together with intact toxins and subunits, were released by the cells. The intracellular half-life of [125 I] dichain TeTx was approximately three days. The number of light-chain molecules required to maintain exocytosis block in a single cell, as calculated by two different methods, was less than 10. The long duration of tetanus poisoning may result from the persistence of intracellular toxin due to a scarcity of free cytosolic proteases. This may also hold for the slow recovery from botulism.
Similar content being viewed by others
References
Aguilera J, Ahnert-Hilger G, Bigalke H, DasGupta BR, Dolly O, Habermann E, Halpern J, Heyningen van S, Middlebrook J, Mochida S, Montecucco C, Niemann H, Oguma K, Popoff M, Poulain B, Simpson L, Shone CC, Thompson DE, Weller U, Wellhöner HH, Whelan SM (1992) Clostridial neurotoxins —Proposal of a common nomenclature. FEMS Microbiol Lett 90:99–100
Ahnert-Hilger G, Weller U, Dauzenroth ME, Habermann E, Gratzl M (1989) The tetanus toxin light chain inhibits exocytosis. FEBS Lett 242:245–248
Bartels F, Bigalke H (1992) Restoration of exocytosis occurs after inactivation of intracellular tetanus toxin. Infection and Immunity 60:302–307
Bartels F, Bergel H, Bigalke H, Frevert J, Halpern J, Middlebrook J (1994) Specific antibodies against the Zn-binding domain of clostridial neurotoxins restore exocytosis in chromaffin cells treated with tetanus or botulinum A neurotoxin. J Biol Chem 269:8122–8127
Bittner MA, Holz RW (1988) Effects of tetanus toxin on catecholamine release from intact and digitonin permeabilized chromaffin cells. J Neurochem 51:451–456
Bittner MA, DasGupta BR, Holz RW (1989) Isolated light chains of botulinum neurotoxins inhibit exocytosis. J Biol Chem 264:10354–10360
Blasi J, Chapman ER, Link EP, Binz T, Yamasaki S, De Camill P, Südhof TC, Niemann H, Jahn R (1993) Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature 365:160–163
DasGupta BR, Sathyamoorthy V (1984) Purification and amino acid composition of type A botulinum neurotoxin. Toxicon 22:415–424
Fairweather NF, Sanders D, Slater D, Hudel M, Habermann E, Weller U (1993) Production of biologically active light chain of tetanus toxin in Escherichia coli. FEBS Lett 323:218–222
Habermann E, Dimpfel W, Räker KO (1973) Interaction of labelled tetanus toxin with substructures of rat spinal cord in vivo. Naunyn-Schmiedeberg's Arch Pharmacol 276:361–373
Habermann E, Weller U, Hudel M (1991) Limited proteolysis of single-chain tetanus toxin by tissue enzymes in cultured brain tissue and during retrograde axonal to the spinal cord. Naunyn-Schmiedeberg's Arch Pharmacol 343:323–329
Habig W, Bigalke H, Bergey G, Neale EA, Hardegree C, Nelson PG (1986) Tetanus toxin in dissociated spinal cord cultures: Long-term characterization of form and action. J Neurochem 47:930–937
Holmgren A (1977) Bovine thioredoxin system. Purification of thioredoxin reductase from calf liver and thymus and studies of its function in disulfide reduction. J Biol Chem 252:4600–4606
Kistner A, Habermann E (1992) Reductive cleavage of tetanus toxin and botulinum neurotoxin A by the thioredoxin system from brain. Naunyn-Schmiedeberg's Arch Pharmacol 345:227–234
Kistner A, Sanders D, Habermann E (1993) Disulfide formation in reduced tetanus toxin by thioredoxin: The pharmacological role of interchain covalent and noncovalent bonds. Toxicon 31:1423–1434
Knight DE, Baker PF (1982) Calcium-dependence of catecholamine release from adrenal medullary cells after exposure to intense electric fields. J Membr Biol 68:107–140
Kozaki S, Togashi S, Sakaguchi G (1981) Separation of clostridium botulinum type A derivative toxin into two fragments. Jpn J Med Sci Biol 34:61–68
Krieglstein KG, Henschen AH, Weller U, Habermann E (1991) Limited proteolysis of tetanus toxin. Relation to activity and identification of cleavage sites. Eur J Biochem 202:41–51
Krieglstein KG, DasGupta BR, Henschen AH (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 Chem 13:49–57
Kurazono H, Mochida S, Binz T, Eisel U, Quanz M, Grebenstein O, Wernars K, Poulain B, Time L, Niemann H (1992) Minimal essential domains specifying the toxicity of the light chains of tetanus toxin and botulinum neurotoxin type A. J Biol Chem 267:14721–14729
Kyse-Anderson J (1984) Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 10:203–209
Lambert R, Pankov R, Gauthier J, Hancock R (1990) Electroporation-mediated uptake of proteins into mammalian cells. Biochem. Cell Biol 68:729–734
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lindau M (1991) Time-resolved capacitance-measurements: monitoring exocytosis in single cells. Quart Rev Biophys 24:75–101
Lindau M, Neher E (1988) Patch-clamp techniques for time resolved capacitance measurements. Pflüger's Arch 411:137–146
Livett BG (1984) Adrenal medullary chromaffin cells in vitro. Physiol Rev 64:1103–1161
Marxen P, Bigalke H (1989) Tetanus toxin: inhibitory action in chromaffin cells is initiated by specific types of gangliosides and promoted in low ionic strength solution. Neuroscience Lett 107:261–266
Marxen P, Fuhrmann U, Bigalke H (1989) Gangliosides mediate inhibitory effects of tetanus and botulinum A neurotoxin in chromaffin cells. Toxicon 27:849–859
Marxen P, Erdmann G, Bigalke H (1991) The translocation of botulinum A neurotoxin by chromaffin cells is promoted in low ionic strength solution and is insensitive to trypsin. Toxicon 29:181–189
Neher E (1988) The use of the patch clamp technique to study second messenger-mediated cellular events Neuroscience 26:727–734
Role LW, Perlman RL (1980) Purification of adrenal medullary chromaffin cells by density gradient centrifugation. J Neurosci Methods 2:253–265
Rozell B, Hansson HA, Luthman M, Holmgren A (1985) Immunohistochemical localization of thioredoxin and thioredoxin reductase in adult rats. Eur J Cell Biol 38:79–86
Sanders D, Habermann E (1992) Evidence for a link between proteolysis and inhibition of [3H] noradrenaline release by the light chain of tetanus toxin. Naunyn-Schmiedeberg's Arch Pharmacol 346:358–361
Schiavo G, Papini E, Gerna G, Montecucco C (1990) An intact interchain disulfide bond is required for the neurotoxicity of tetanus toxin. Infect Immunol 58:4136–4141
Schiavo G, Benfenati F, Poulain B, Rosetto O, Polverino de Laureto P, DasGupta BR, Montecucco C (1992) Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359:832–835
Shigekawa K, Dower WJ (1988) Electroporation of eukaryotes and prokaryotes: a general approach to the introduction of macromolecules into cells. BioTechniques 6:742–751
Slot JW, Geuze HJ (1985) A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol 38:87–93
Stecher B, Gratzl M, Ahnert-Hilger G (1989) Reductive chain separation of botulinum A toxin is a prerequisite to its inhibitory action on exocytosis in chromaffin cells. FEBS Lett 248:23–27
Stemme S, Hansson HA, Holmgren A, Rozell B (1985) Axoplasmic transport of thioredoxin and thioredoxin reductase in rat sciatic nerve. Brain Res 359:140–146
Weller U, Mauler F, Habermann E (1988) Tetanus toxin: biochemical and pharmacological comparison between its protoxin and some isotoxins obtained by limited proteolysis. Naunyn-Schmiedeberg's Arch Pharmacol 338:99–106
Weller U, Dauzenroth ME, Meyer zu Heringsdorf D, Habermann E (1989) Chains and fragments of tetanus toxin. Separation, reassociation and pharmacological properties. Eur J Biochem 182:649–656
Wiegand H, Wellhoner HH (1977) The action of botulinum A neurotoxin on the inhibition by antidromic stimulation of the lumbar monosynaptic reflex. Naunyn-Schmiedeberg's Arch Pharmacol 298:235–238
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Erdal, E., Bartels, F., Binscheck, T. et al. Processing of tetanus and botulinum A neurotoxins in isolated chromaffin cells. Naunyn-Schmiedeberg's Arch Pharmacol 351, 67–78 (1995). https://doi.org/10.1007/BF00169066
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00169066