Recombinant forms of tetanus toxin engineered for examining and exploitingneuronal trafficking pathways

Tetanus toxin is a fascinating, multifunctional protein that binds to perip heral neurons, undergoes retrograde transport and trans-synaptic transfer t o central inhibitory neurons where it blocks transmitter release, thereby, causing spastic paralysis. As a pre-requisite for exploiting its unique tra fficking properties, a novel recombinant single chain was expressed at a hi gh level in Eacherichia coli as a soluble, easily purifiable protein. It co uld be activated with enterokinase to produce a di-chain that matched nativ e toxin in terms of proteolytic and neuroinhibitory activities, as well as induction of spastic paralysis in mice. Importantly, nicking was not essent ial for protease activity. Substitution of GIU234 by Ala created a protease -deficient atoxic form, which blocked the neuroparalytic action of tetanus toxin in vitro, with equal potency to its heavy chain; but, the mutant prov ed > 30-fold more potent in preventing tetanus in mice. This observation un veils differences between the intoxication processes resulting from retrogr ade transport of toxin in vivo and its local uptake into peripheral or cent ral nerves in vitro, dispelling a popularly held belief that the heavy chai n is the sole determinant for efficient trafficking. Thus, this innocuous m utant may be a useful vehicle, superior to the heavy chain, for drug delive ry to central neurons.