Article Figures & Data
Figures
- 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.
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.
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.
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.
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).
Tables
- TABLE 1
Sequence alignment of mouse, rat, and human VAMP isoforms and cleavage sites of the different BoNTs
The SNARE motifs of mouse, rat, and human VAMP isoforms have been aligned using http://www.uniprot.org/align/. The conserved cleavage sites of VAMP isoforms targeted by specific BoNT types and subtypes are in the same color of the respective toxins. The cleavage site of the newly identified BoNT-like metalloprotease of Weissella oryzae (WO) is also shown (purple). Conserved proteolytic sites whose susceptibility to cleavage is predicted, but not experimentally proven, are underlined with the color of the respective BoNT. Nonconserved cleavage sites are underlined in black as well as conserved cleavage sites, which were experimentally found to be noncleavable. When known, the recognition motif, used by the toxin to bind the substrate in addition to the cleavage sites (Rossetto et al., 1994), is underlined in red.
- TABLE 2
Sequence alignment of mouse, rat, and human SNAP25 isoforms and cleavage sites of the different BoNTs
The C-terminal SNARE motif of mouse, rat, and human SNAP isoforms have been aligned using http://www.uniprot.org/align/. The conserved cleavage sites targeted by specific BoNT types and subtypes are colored like the respective toxin. Conserved cleavage sites whose susceptibility to cleavage is predicted, but not experimentally proven, are underlined with the color of the respective BoNT. Nonconserved cleavage sites are underlined in black as well as conserved cleavage sites that were experimentally found to be noncleavable. Notice that mouse SNAP-23 has a cleavage site for BoNT/E different from the one present in SNAP-25, which is anyhow cleaved, whereas human SNAP-23, which has the same proteolytic site as SNAP-25, is not cleaved (Vaidyanathan et al., 1999). Similarly, mouse SNAP-23 was reported to be cleaved by BoNT/A, although the peptide bond is not conserved. At the same time, BoNT/C, whose cleavage site is conserved (also in human SNAP-23) does not cleave mouse SNAP-23. The cleavability of rat SNAP-23 was predicted on these premises. When known, the recognition motif used by the toxin to bind the substrate in addition to the cleavage sites (Rossetto et al., 1994) is underlined in red.
- TABLE 3
Sequence alignment of mouse, rat, and human Syntaxin isoforms and cleavage site of BoNTs
The C-terminal SNARE motifs of mouse, rat, and human syntaxin isoforms are aligned using http://www.uniprot.org/align/. BoNT/C conserved cleavage sites are shown in purple. Conserved cleavage sites whose susceptibility to cleavage is predicted, but not experimentally proven, are underlined in purple. Nonconserved cleavage sites are underlined in black.
- TABLE 4
Comparison of botulinum neurotoxin products marketed in Europe and North America
Units are manufacturer specific and are not interchangeable.
Botox/Vistabel Dysport/Azzalure Xeomin/Bocouture Neurobloc/Myobloc Generic name OnabotulinumtoxinA AbobotulinumtoxinA IncobotulinumtoxinA RimabotulinumtoxinB Manufacturer Allergan (USA) Ipsen Pharmaceuticals (France) Merz Pharmaceuticals (Germany) US WorldMeds (USA) C. botulinum strain Hall A-hyper Hall A Hall A (ATCC 3502) Bean Toxin type A1 A1 A1 B1 MW 900 kDa complex MW not reported 150 kDa MW not reported (PTCs) (Yes) (Yes) None (Yes) Pharmaceutical form Vacuum-dried powder for reconstitution Freeze-dried powder for reconstitution Freeze-dried powder for reconstitution Ready-to-use solution Shelf life 2–8°C 2–8°C Room temperature 2–8°C 36 months 24 months 36 months 24 months pH (reconstituted) 7.4 7.4 7.4 5.6 Excipients In 100 U vial: In 500 U vial: In 100 U vial: HSA 500 μg/ml HSA 500 μg HSA 125 μg HSA 1000 μg Succinate 10 mM NaCl (900 μg/vial) Lactose (2.5 mg/vial) Sucrose (4.7 mg/vial) NaCl 100 mM Unit/vial 100 U or 200 U Botox 300 U or 500 U Dysport 100 U or 200 U Xeomin 2500 U/0.5 ml 50 U Vistabel 125 U Azzalure 50 U Bocouture 5000 U/1 ml 10,000 U/2 ml Protein load/vial 5 ng/100 U 4.35 ng/500 U 0.44 ng/100 Ua 55 ng/2500 U Clinical activity in relation to Botox 1 1:2–1:3 1 1:40–1:50 HSA, human serum albumin; PTC, progenitor toxin complex.
↵a Neurotoxin concentration measured by ELISA (Frevert, 2010).
Ophtalmology Strabismusa,b,c Nistagmus Neurology Focal Dystonias Blepharospasma,b,c Cervical dystoniaa,b,c (Torticollis, anterocollis, laterocollis) Occupational dystonias (writer’s crampb, musician’s cramps) Laryngeal Dysphoniac Oromandibular dystonia Lingual dystonia Nondystonic disorders Hemifacial spasma,b,c Tremor (essential, parkinsonism) Tics Bruxism Spasticity (poststroke, multiple sclerosis, brain or spinal cord injury) Focal spasticitya,b,c: Upper and lower limb spasticity Nonfocal: hemispasticity, paraspasticity, tetraspasticity Cerebral palsyab Hyperhidrosisa,b,c Focal: axillary, palmar, plantar Diffuse Hypersalivation Sialorrheab (motoneuron diseases/amyotrophic lateral sclerosis) Droolingb (Parkinsonian syndromes) Frey’s syndrome/gustatory sweating Aesthetic (muscle) Glabellar rythidesa,b,c Pain Muscular Dystonia Spasticity Chronic myofascial pain Temporomandibular disorders Low back pain Nonmuscular Migraine (chronica and tension type migraine) Neuropathic pain Trigeminal pain Pelvic pain Urology Detrusor sphincter dyssynergia Overactive bladdera,b,c (Idiopathic or neurogenic detrusor overactivity) Urinary retention Bladder pain syndrome Pelvic floor spasms Benign prostate hyperplasia Gastroenterology Achalasia Chronic anal fissures Psychiatry Depressiond Toxin Type LD50 Reference BoNT/A1 1.15a Duff, et al., 1957a 0.45 Nakamura et al., 2010 0.38 Pier et al., 2011 0.40 Whitemarsh et al., 2013 0.40 Pellett et al., 2015b 0.25 Azarnia Tehran et al., 2015 BoNT/A2 0.11 Pier et al., 2011 0.39 Whitemarsh et al., 2013 BoNT/A3 0.35 Whitemarsh et al., 2013 BoNT/A4 400–500 Whitemarsh et al., 2013 BoNT/A5 0.35 Whitemarsh et al., 2013 BoNT/B1 1.93a Lamanna and Glassman, 1947 1.23a Duff, et al., 1957b 0.41 Nakamura et al., 2010 0.45b Azarnia Tehran et al., 2015 BoNT/B2 0.40 Fan et al., 2016 BoNT/C1 5.00 Notermans et al., 1982 2.30 Tsukamoto et al., 2005 1.65 Morbiato et al., 2007 0.92 Nakamura et al., 2010 BoNT/CD 1.42 Notermans et al., 1982 1.80 Matsuda et al., 1986 1.92 Tsukamoto et al., 2005 0.80 Nakamura et al., 2010 BoNT/D 0.15 Tsukamoto et al., 2005 0.18 Nakamura et al., 2010 0.83 Eleopra et al., 2013 0.43 Pellett et al., 2015b 0.02b Azarnia Tehran et al., 2015 BoNT/DC 0.05 Nakamura et al., 2010 BoNT/E1 0.65 Pier et al., 2011 0.84 Chatla et al., 2012 1.00 Meunier et al., 2003 BoNT/F1 2.50 Oishi and Sakaguchi, 1974 10.0 Meunier et al., 2003 BoNT/FA 2.20 Fan et al., 2016 1.30 Maslanka et al., 2016 BoNT/G 5.00 Schiavo et al., 1994
