Cytotoxic effects of a chimeric protein consisting of tetanus toxin light chain and anthrax toxin lethal factor in non-neuronal cells

Abstract

The light chain of tetanus toxin is a zinc endoprotease that inhibits neurotransmitter release by selective proteolysis of the synaptic vesicle-associated protein synaptobrevin/vesicle-associated membrane protein. Cellubrevin is a homologue of synaptobrevin that is found in most cell types and is also a substrate for tetanus toxin. The lack of receptors for tetanus toxin on most cell types has made studies of tetanus toxin action in non-neuronal cells difficult. To characterize tetanus toxin effects in non-neuronal cells, a fusion protein consisting of the 254 amino-terminal amino acids of lethal factor (LF) of anthrax toxin and tetanus toxin light chain (LC) was prepared. This protein (LF-LC) inhibited evoked glycine release from primary spinal cord neurons at concentrations between 1.0 and 100 ng/ml. LF-LC was cytotoxic to RAW 264.7, ANA-1 cells (mouse macrophage cell lines), and Chinese hamster ovary cells in a dose-dependent manner. These effects required the presence of protective antigen, the receptor binding component of anthrax toxin. In contrast, LF-LC was not cytotoxic to RBL-2H3, Vero, or mouse hybridoma cell lines. Mutagenesis of conserved amino acids (His237 and Glu234) in the zinc-binding motif of LC resulted in fusion proteins having no biological activity. LF-LC did not inhibit regulated secretion of serotonin in RBL-2H3 cells or constitutive secretion in any non-neuronal cell lines as measured in several different assays. We suggest that the cytotoxic effects of LF-LC result from inhibition of a specific intracellular membrane fusion event mediated by cellubrevin.