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Review
. 2017 Apr;69(2):200-235.
doi: 10.1124/pr.116.012658.

Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology

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Free PMC article
Review

Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology

Marco Pirazzini et al. Pharmacol Rev. .
Free PMC article

Abstract

The study of botulinum neurotoxins (BoNT) is rapidly progressing in many aspects. Novel BoNTs are being discovered owing to next generation sequencing, but their biologic and pharmacological properties remain largely unknown. The molecular structure of the large protein complexes that the toxin forms with accessory proteins, which are included in some BoNT type A1 and B1 pharmacological preparations, have been determined. By far the largest effort has been dedicated to the testing and validation of BoNTs as therapeutic agents in an ever increasing number of applications, including pain therapy. BoNT type A1 has been also exploited in a variety of cosmetic treatments, alone or in combination with other agents, and this specific market has reached the size of the one dedicated to the treatment of medical syndromes. The pharmacological properties and mode of action of BoNTs have shed light on general principles of neuronal transport and protein-protein interactions and are stimulating basic science studies. Moreover, the wide array of BoNTs discovered and to be discovered and the production of recombinant BoNTs endowed with specific properties suggest novel uses in therapeutics with increasing disease/symptom specifity. These recent developments are reviewed here to provide an updated picture of the biologic mechanism of action of BoNTs, of their increasing use in pharmacology and in cosmetics, and of their toxicology.

Figures

Fig. 1.
Fig. 1.
Structure of BoNT/A1 and BoNT/B1 molecules. Crystal structures of BoNT/A1 (PDB ID: 3BTA) (Lacy et al., 1998) (A) and BoNT/B1 (PDB ID: 1EPW) (Swaminathan and Eswaramoorthy, 2000) (B) represented as space-filling models of the two opposite surfaces of each toxin molecule showing the organization of the three toxin domains: the neurospecific binding HC-C subdomain (green), the lectin-like HC-N subdomain (purple), the translocation HN domain (yellow), and the metalloprotease L domain (red). The pink cavity in the HC-C subdomains shown in the lower panels is the polysialoganglioside binding site. A peptide belt (shown in blue) surrounding the L domain and the interchain disulfide bond (white in the upper panels) linking the L and HN domain, which stabilize the structure, is also shown.
Fig. 2.
Fig. 2.
Structure of BoNTA1-NTNHA1 heterodimer and of the progenitor toxin complex (PTC). (A) Crystal structure of BoNT/A1 in complex with the NTNHA/A1 protein (PDB ID: 3V0B) (Gu et al., 2012) represented as space-filling models of the two opposite surfaces. For BoNT/A1, the L chain is in red, the HN domain is in yellow, and in green the HC domain. The BoNT/A1 protein binds “hand in hand” the NTNHA/A1 protein whose domain structure and organization are very similar to that of the toxin (see central inset for a simplified scheme). For NTNHA/A1 nL is in orange, nHN in pink, and nHC in light green. In blue and in light orange are the belts of toxin and NTNHA, respectively. Notice that NTNHA/A1 shields a large part of the BoNT surface. A similar structure has been determined for BoNT/E1 (PDB ID: 4ZKT) (Eswaramoorthy et al., 2015). (B) Space-filling representation of the large precursor toxin complex (PTC), which has a triskelion-like structure (Amatsu et al., 2013; Lee et al., 2013). BoNT/A1 (red) interacts with NTNHA/A1 (orange) but has no contacts with HA proteins. There are six HA33 proteins (blue), three HA17 proteins (light blue), and three HA70 proteins (pink) in each NTNHA/A1-BoNT/A1 complex. Because the HA proteins do not contact the BoNT/A1 molecule, they are unlikely to play any protective role on BoNT/A1, as previously proposed. Rather, the structure suggests a role as adhesin molecule (see text). Similar structures have been determined for BoNT/D and BoNT/B1 using single particle electron microscopy (Benefield et al., 2013; Hasegawa et al., 2007). The structure of (B) was assembled by joining the structure of the BoNT/A1-NTNHA/A1 heterodimer (PDB ID: 3V0B) and the structure of the triskelion (PDB ID: 3WIN).
Fig. 3.
Fig. 3.
Cleavage sites of the neuronal SNARE proteins by the different BoNT types and subtypes. The BoNT proteolytic activity is highly specific and directed toward unique peptide bonds within the sequence of their respective SNARE protein targets. VAMP of the synaptic vesicle (in blue, isoform 1 is shown here) or SNAP-25 (in green) or syntaxin (in red, isoform 1B is shown here) mainly localized on the cytosolic side of the presynaptic membrane. Available evidence indicate that all the toxin subtypes and chimeric toxins cleave the same SNARE substrate, although different subtypes may cleave different peptide bonds. For example BoNT/F5 and BoNT/FA, a chimeric toxin derived from a genetic recombination between BoNT/F2, /F5, and A1 neurotoxin genes, cleave VAMP at a peptide bond different from the one cleaved by BoNT/F1. Notice that tetanus neurotoxin and botulinum B1 neurotoxin cleave the same target at the same site proving that the different symptoms of tetanus and botulism are not due to a different target molecule, but to different neuronal targets: the Renshaw cells of the spinal cord for tetanus neurotoxin and peripheral nerve terminals for BoNT/B1.
Fig. 4.
Fig. 4.
The nerve terminal intoxication by botulinum neurotoxins is a multi-step process. The first step (1) is the binding of the HC domain (green) to a polysialoganglioside (PSG) receptor of the presynaptic membrane (gray and black), followed by binding to a protein receptor. The currently known protein receptors are i) synaptotagmin (Syt, gray) for BoNT/B1, /DC, and /G; ii) glycosylated SV2 (black with its attached N-glycan in pink) for BoNT/A1 and /E1. Syt may be located either within the exocytosed synaptic vesicle or on the presynaptic membrane. The BoNT is then internalized inside SVs, which are directly recycled (2a) or inside SVs that fuse with the synaptic endosome and re-enter SV cycle by budding from this intermediate compartment (2b). The acidification (orange) of the vesicle, operated by the v-ATPase (orange), drives the accumulation of neurotransmitter (blue dots) via the vescicular neurotransmitter transporter (light blue). The protonation of BoNT leads to the membrane translocation of the L chain into the cytosol (3), which is assisted by the HN domain (yellow). The L chain (red) is released from the HN domain by the action of the thioredoxin reductase-thioredoxin system (TrxR-Trx, blue and dark blue) and Hsp90 (mud color), which reduce the interchain disulfide bond (orange) and avoid the aggregation of the protease (4). In the cytosol, the L chain displays its metalloprotease activity: BoNT/B, /D, /F, /G cleave VAMP (blue); BoNT/A and BoNT/E cleave SNAP-25 (green); and BoNT/C cleaves both SNAP-25 and syntaxin (Stx, dark red) (5). Each of these proteolytic events is sufficient to cause a prolonged inhibition of neurotransmitter release with consequent neuroparalysis.
Fig. 5.
Fig. 5.
Close-up views of the binding interfaces between BoNT/A1 and BoNT/B1 to their synaptic vesicle protein receptors. (A) Two areas of interaction of BoNT/A1 with the synaptic vesicle protein glycosylated-SVC2 (PDB ID 5JLV). One main interaction is mediated by protein-protein contacts through the pairing of the backbones of two solvent-exposed β-strands (black dotted ellipsoid), one from each partner. Essential interactions are mediated by R1156 of BoNT/A1 making a cation-π stacking interaction with P563 of SV2C and also by R1294 of the toxin. The second main interaction is mediated by N559 whose side chain carries a N-glycan modification (shown as sticks), which fits within a crevice formed at the interface between HC-N (purple) and HC-C (green). Amino acids forming the groove are colored in cyan and labeled according to their location (P953, N954, S957, S1062, H1064, and R1065 from HC-N, in purple and T1145, Y1155, D1288, D1289, and G1292 from HC-C, in green). The cartoon also shows essential water molecules (black pellets) and the H bonds (yellow dotted lines), which mediate the interaction of BoNT/A1 with the N-glycan, suggesting the possibility that they serve to adapt the variety of N-glycans that are produced by different kind of neurons and/or by neurons of different individuals and animal species. (B) Interaction among BoNT/B1 and its synaptic vesicle protein receptors Synaptotagmin II (Syt-II) (PDB ID 4KBB). The interface of interaction is at the extreme bottom of BoNT/B and, at variance from BoNT/A1, involves exclusively HC-C (green). Syt-II is unstructured in solution but assumes an helical conformation upon binding to the toxin, forced by the interactions occurring at the level of two hydrophobic pockets. One pocket is formed by HC-C residues P1117, W1178, Y1181, P1194, A1196, P1197, F1204 with Syt-II residues M46, F47, and L50. The second pocket of HC-C is lined by residues K1113, S1116, P1117, V1118, Y1183, E1191, K1192, F1194, and F1204 with Syt-II residues F54, F55, E57, and I58. Only the most significant aminoacids involved in the interaction are shown and labeled. Note the H bond (black dotted line) formed by K1113 and E57, which may also interact electrostatically. The binding sites for the oligosaccharide portion of polysialoganglioside receptor are not shown, but in both BoNT/A1 and /B1 are located within the HC-C subdomain at a distance from the protein receptor binding sites in such a position that they do not interact with them (see text).

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