This project is directed toward characterizing the interaction of tetanus toxin with eukaryotic cells and defining how this toxin interferes with the targeting and fusion of intracellular transport vesicles. The toxicity of tetanus toxin results from its proteolytic cleavage of certain members of the family of VAMP (vesicle-associated membrane protein) proteins and subsequent inhibition of vesicle fusion. In order to study tetanus toxin action in non-neuronal cells (which lack receptors for tetanus toxin) a fusion protein consisting of the enzymatically active domain of tetanus toxin and the receptor binding domain of anthrax toxin was prepared. This protein, LF-LC, can gain entry into most mammalian cells by receptor-mediated endocytosis. LF-LC inhibits cell growth and transferrin receptor recycling in several different cell lines. The predicted mechanism for this inhibition of cell growth would be cleavage of the non-neuronal VAMP, cellubrevin. However, while LF-LC is fully active against neuronal VAMPs, it had little or no effect on cellubrevin. Possible explanations for these results include, the existence of cellubrevin analogues resistant to tetanus toxin, inaccessibility of cellubrevin to cleavage by tetanus toxin in vivo, or stimulation of cellubrevin synthesis in tetanus treated cells. Cell lines that over-express different forms of cellubrevin have been prepared to distinguish among these possibilities. Publications: Bartels, F., Bergel, H., Bigalke, H., Frevert, J., Halpern, J., and Middlebrook, J. (1994) Specific antibodies against the zinc-binding domain of clostridial neurotoxins restore exocytosis in chromaffin cells treated with tetanus or botulinum A neurotoxin. J. Biol. Chem. 269:8122-8127. Arora, N., Williamson, L.C., Leppla, S.H., and Halpern, J.L. (1994) Cytotoxic effects of a chimeric protein consisting of tetanus toxin light chain and anthrax toxin lethal factor in non-neuronal cells. J. Biol. Chem. (in press).
