A SINGLE MUTATION IN THE RECOMBINANT LIGHT-CHAIN OF TETANUS TOXIN ABOLISHES ITS PROTEOLYTIC ACTIVITY AND REMOVES THE TOXICITY SEEN AFTER RECONSTITUTION WITH NATIVE HEAVY-CHAIN

Specific proteolysis by the tetanus toxin light chain of a vesicle-ass ociated membrane protein (VAMP) involved in exocytosis is thought to u nderlie its intracellular blockade of neurotransmitter release. To sub stantiate this mechanism, recombinant light chain was expressed as a m altose binding protein-light chain fusion product in Escherichia coli. After purification by affinity chromatography and cleavage with facto r Xa, the resultant light chain was isolated and its identity confirme d by Western blotting and N-terminal sequencing. It exhibited activity similar to that of the native light chain in proteolyzing its target in isolated bovine small synaptic vesicles and in hydrolyzing a 62-res idue synthetic polypeptide spanning the cleavage site of the substrate . The importance of Glu(234) in the catalytic activity of the light ch ain, possibly analogous to Glu(143) Of thermolysin, was examined using site-directed mutagenesis. Changing Glu(234) to Ala abolished the pro tease activity of the light chain, but its ability to bind the polypep tide substrate was retained. Each recombinant light chain could be rec onstituted with the heavy chain of tetanus toxin, yielding the same le vel of disulfide-linked species as the two native chains. Whereas the toxin formed with wild-type light chain exhibited appreciable neuromus cular paralysis activity and mouse lethality, the equivalent dichain m aterial containing the Ala(234) mutant lacked neurotoxicity in both th e in vitro and in vivo assays. Thus, these results demonstrate directl y, for the first time, that the lethality of tetanus toxin and its inh ibition of exocytosis in intact neurons are attributable largely, if n ot exclusively, to endoprotease activity.