Dileucine in the Protease of Botulinum Toxin A Underlies Its Long-lived Neuroparalysis: TRANSFER OF LONGEVITY TO A NOVEL POTENTIAL THERAPEUTIC

• Published in: The Journal of biological chemistry 2011-02, Vol.286 (8), p.6375-6385
• Main Authors: Wang, Jiafu, Zurawski, Tomas H, Meng, Jianghui, Lawrence, Gary, Olango, Weredeselam M, Finn, David P, Wheeler, Larry, Dolly, J. Oliver
• Format: Article
• Language: English
• Subjects: Animals; Botulinum toxin; Botulinum Toxins, Type A - genetics; Botulinum Toxins, Type A - pharmacology; Botulism; Calcitonin - genetics; Calcitonin - metabolism; Calcitonin gene-related peptide; Cerebellum; Cerebellum - cytology; Cerebellum - metabolism; Cholinergic nerves; Clostridium botulinum; Exocytosis; Female; Leucine; Light chains; Longevity; Male; Mice; Mutation; Neurobiology; Neuromuscular Agents - pharmacology; Neuromuscular junctions; Neurotoxicity; Neurotransmitter release; Pain perception; Paralysis; Protein Structure, Tertiary; Proteinase; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - pharmacology; Sensory neurons; Sensory Receptor Cells - cytology; Sensory Receptor Cells - metabolism; SNAP-25 protein; Synaptic Transmission - drug effects; Synaptosomal-Associated Protein 25 - genetics; Synaptosomal-Associated Protein 25 - metabolism; Time Factors; transient receptor potential proteins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.181784

Blockade of neurotransmitter release by botulinum neurotoxin type A (BoNTA) 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 BoNTA 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 (LCA) 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 BoNTA activity but did not alter the longevity of its SNAP-25 cleavage and neuromuscular paralysis. Furthermore, genetically fusing LCE to a BoNTA enzymically inactive mutant (BoTIMA) yielded a novel LCE-BoTIMA protein that targets neurons, and the BoTIMA moiety also delivers and stabilizes the inhibitory LCE, giving a potent and persistent cleavage of SNAP-25 with associated neuromuscular paralysis. Moreover, its neurotropism was extended to sensory neurons normally insensitive to BoNTE. LCE₋BoTIMA(AA) with the above-identified dileucine mutated gave transient neuromuscular paralysis similar to BoNTE, reaffirming that these residues are critical for the persistent action of LCE-BoTIMA as well as BoNTA. LCE-BoTIMA 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.