Hyperexcitability disorders of cholinergically innervated muscles are treatable with botulinum neurotoxin (BoNT) A. The seven serotypes (A-G) potently block neurotransmission by binding to presynaptic receptors, undergoing endocytosis, transferring to the cytosol, and inactivating proteins essential for vesicle fusion. Although BoNT/A and BoNT/E cleave SNAP-25, albeit at distinct sites, BoNT/E blocks neurotransmission faster and more potently. To identify the domains responsible for these characteristics, the C-terminal heavy chain portions of BoNT/A and BoNT/E were exchanged to create chimeras AE and EA. After high yield expression in Escherichia coli, these single chain chimeras were purified by two-step chromatography and activated by conversion to disulfide-linked dichains. In vitro, each entered neurons, cleaved SNAP-25, and blocked neuromuscular transmission while causing flaccid paralysis in vivo. Acidification-dependent translocation of the light chain to the cytosol occurred more rapidly for BoNT/E and EA than for BoNT/A and AE because the latter pair remained susceptible for longer to inhibitors of the vesicular proton pump, and BoNT/A proved less sensitive. The receptor-binding and protease domains do not seem to be responsible for the speeds of intoxication; rather the N-terminal halves of their heavy chains are implicated, with dissimilar rates of cytosolic transfer of the light chains being due to differences in pH sensitivity. AE produced the most persistent muscle weakening and therefore has therapeutic potential. Thus, proof of principle is provided for tailoring the pharmacological properties of these toxins by protein engineering.

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 EBoTIM 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. Botulinum neurotoxins (BoNTs)3 inhibit transmitter release from peripheral cholinergic neurons causing the lifethreatening flaccid paralysis underlying botulism (1). The most potent biological substances, their estimated lethal doses (LD 50 ) in humans are between 0.1 and 1 ng/kg. Seven BoNT serotypes (A-G), produced by Clostridium botulinum, are synthesized as pro-form single chain proteins (SC, M r ϳ150,000) and converted by either Clostridial or tissue proteases into fully active dichain (DC) forms, consisting of a protease domain (LC; M r ϳ50,000) linked to a heavy chain (HC; M r ϳ100,000) through disulfide and noncovalent bonds. BoNT A preferentially enters cholinergic nerve endings by binding via the C-terminal half of their HC to a membrane acceptor, a lumenal domain of synaptic vesicle protein 2 (2, 3). On the other hand, type E only binds the glycosylated synaptic vesicle protein 2 A/B isoforms (4), which are sparsely expressed on sensory neurons, explaining its lack of effects on trigeminal ganglionic neurons (TGNs) (5). These toxins undergo acceptor-mediated endocytosis (6, 7) with translocation of the LCs into the cytosol through a channel formed by the N-terminal half...

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Botulinum neurotoxin (BoNT) A or E and tetanus toxin (TeTx) bind to motor-nerve endings and undergo distinct trafficking; their light-chain (LC) proteases cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) peripherally or centrally and cause flaccid or spastic paralysis, respectively. To seek protein domains responsible for local blockade of transmitter release (BoNTs) rather than retroaxonal transport to spinal neurons (TeTx), their acceptor-binding moieties (H(C))--or in one case, heavy chain (HC)--were exchanged by gene recombination. Each chimera, expressed and purified from Escherichia coli, entered rat cerebellar neurons to cleave their substrates, blocked in vitro nerve-induced muscle contractions, and produced only flaccid paralysis in mice. Thus, the local cytosolic delivery of BoNT/A or BoNT/E proteases and the contrasting retrograde transport of TeTx are not specified solely by their HC or H(C); BoNT/A LC translocated locally irrespective of being targeted by either of the latter TeTx domains. In contrast, BoNT/E protease fused to a TeTx enzymatically inactive mutant (TeTIM) caused spastic paralysis and cleaved SNAP-25 in spinal cord but not the injected muscle. Apparently, TeTIM precludes cytosolic release of BoNT/E protease at motor nerve endings. It is deduced that the LCs of the toxins, acting in conjunction with HC domains, dictate their local or distant destinations.

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