1. Longer-acting and highly potent chimaeric inhibitors of excessive exocytosis created with domains from botulinum neurotoxin A and B
- Author
-
J. Oliver Dolly, MacDara Bodeker, Tomas H. Zurawski, Jiafu Wang, Sanjay V. Boddul, K. Roger Aoki, and Jianghui Meng
- Subjects
Botulinum Toxins ,Synaptosomal-Associated Protein 25 ,Vesicle-Associated Membrane Protein 2 ,Recombinant Fusion Proteins ,medicine.medical_treatment ,Urinary Bladder ,Neuromuscular Junction ,In Vitro Techniques ,Immunoglobulin light chain ,Synaptic Transmission ,Biochemistry ,Synaptic vesicle ,Exocytosis ,Synaptotagmin 1 ,Green fluorescent protein ,Rats, Sprague-Dawley ,Mice ,medicine ,Animals ,Paralysis ,Botulinum Toxins, Type A ,Receptor ,Molecular Biology ,Cells, Cultured ,Neurons ,Protease ,Chemistry ,Synovial Membrane ,Muscle, Smooth ,Cell Biology ,Fibroblasts ,Molecular biology ,Protein Structure, Tertiary ,Rats ,Phrenic Nerve ,Membrane protein ,Female ,Peptide Hydrolases - Abstract
Various human neurogenic hyper-excitability disorders are successfully treated with type A or B BoNT (botulinum neurotoxin). The BoNT/A complex is widely used because of its longer-lasting benefits; also, autonomic side-effects are more often reported for BoNT/B. To establish if this distinct effect of BoNT/B could be exploited therapeutically, BoNT/A was modified so that it would bind the more abundant BoNT/B acceptor in rodents while retaining its desirable persistent action. The advantageous protease and translocation domain of BoNT/A were recombinantly combined with the acceptor-binding moiety of type B [HC/B (C-terminal half of BoNT/B heavy chain)], creating the chimaera AB. This purified protein bound the BoNT/B acceptor, displayed enhanced capability relative to type A for intraneuronally delivering its protease, cleaved SNAP-25 (synaptosome-associated protein of 25 kDa) and induced a more prolonged neuromuscular paralysis than BoNT/A in mice. The BA chimaera, generated by substituting HC/A (C-terminal half of BoNT/A heavy chain) into BoNT/B, exhibited an extremely high specific activity, delivered the BoNT/B protease via the BoNT/A acceptor into neurons, or fibroblast-like synoviocytes that lack SNAP-25, cleaving the requisite isoforms of VAMP (vesicle-associated membrane protein). Both chimaeras inhibited neurotransmission in murine bladder smooth muscle. BA has the unique ability to reduce exocytosis from non-neuronal cells expressing the BoNT/A-acceptor and utilising VAMP, but not SNAP-25, in exocytosis. Abbreviations: BoNT, botulinum neurotoxin; CGN, cerebellar granule neuron; DAS, digit abduction score; DC, di-chain; DTT, dithiothreitol; GFP, green fluorescent protein; GST, glutathione transferase; HC, heavy chain; HC, C-terminal half of HC; HN, N-terminal half of HC; IMAC, immobilized metal-affinity chromatography; LC, light chain; mLD50, mouse LD50; SC, single chain; SNAP-23, synaptosome-associated protein of 23 kDa; SNAP-25, synaptosome-associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SV2, synaptic vesicle protein 2; Syt, synaptotagmin; TDmax., maximal tolerated dose; VAMP, vesicle-associated membrane protein
- Published
- 2012