A dileucine in the protease of botulinum toxin A underlies its long-lived neuroparalysis: Transfer of longevity to a novel potential therapeutic

Blockade of neurotransmitter release by botulinum neurotoxin type A (BoNT_A) underlies the severe neuroparalytic symptoms of human botulism, which can last a few years. The structural basis for this remarkable persistence remains unclear. Herein, recombinant BoNT_A was found to match the neurotoxicity of that from Clostridium botulinum, producing persistent cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) and neuromuscular paralysis. When two leucines near the C terminus of the protease light chain of A (LC_A) were mutated, its inhibition of exocytosis was followed by fast recovery of intact SNAP-25 in cerebellar neurons and neuromuscular transmission in vivo. Deletion of 6-7 N terminus residues diminished BoNT_A activity but did not alter the longevity of its SNAP-25 cleavage and neuromuscular paralysis. Furthermore, genetically fusing LC_E to a BoNT_A enzymically inactive mutant (BoTIM _A) yielded a novel LC_E-BoTIM_A protein that targets neurons, and the BoTIM_A moiety also delivers and stabilizes the inhibitory LC_E, giving a potent and persistent cleavage of SNAP-25 with associated neuromuscular paralysis. Moreover, its neurotropism was extended to sensory neurons normally insensitive to BoNT_E. LC _E-BoTIM_A(AA) with the above-identified dileucine mutated gave transient neuromuscular paralysis similar to BoNT_E, reaffirming that these residues are critical for the persistent action of LC _E-BoTIM_A as well as BoNT_A. LC _E-BoTIM_A inhibited release of calcitonin gene-related peptide from sensory neurons mediated by transient receptor potential vanilloid type 1 and attenuated capsaicin-evoked nociceptive behavior in rats, following intraplantar injection. Thus, a long acting, versatile composite toxin has been developed with therapeutic potential for pain and conditions caused by overactive cholinergic nerves.