Cytotoxic effects of a chimeric protein consisting of tetanus toxin light chain and anthrax toxin lethal factor in non-neuronal cells.
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.
Arora
N
N
Laboratory of Microbial Ecology, NIDR, National Institutes of Health, Bethesda, Maryland 20892.
Williamson
L C
LC
Leppla
S H
SH
Halpern
J L
JL
D000818:Animals; D000942:Antigens, Bacterial; D001427:Bacterial Toxins / Q000633:toxicity*; D001483:Base Sequence; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000633:toxicity*; D013744:Tetanus Toxin / Q000633:toxicity*; D015032:Zinc / Q000378:metabolism
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; C030325:anthrax toxin; D015032:Zinc