Botulism, caused by botulinum neurotoxin (BoNT), is characterized by descending flaccid paralysis as a result of inhibition of neurotransmitter release at the neuromuscular junction - NMJ (Turton et al., 2002). According to their antigenic properties, BoNTs are classified into seven different toxin types (A, B, C1, D, E, F and G) although more than 50 sequences encoding 18 subtypes are known (Smith et al., 2005). The toxin is released as a 900 kDa complex containing some accessory proteins of unknown functions (Chen et al., 1998). The toxin types A, B and E are mainly involved in human botulism whereas C and D predominantly cause animal botulism (Poulain et al, 2006). The toxin is absorbed from the gut or other epithelium and reaches neuromuscular junctions by transcytosis (Park and Simpson, 2003). The binding sites for the toxins are distributed across the apical surface of the epithelium (Ahsan et al., 2005). It has been observed that the neurotoxin alone is capable of transcytosis across epithelial cells (Maksymowych and Simpson, 2004). Once internalized, the neurotoxin is dissociated from the non-toxic components of the progenitor toxin in endosome (Uotsu et al., 2006). The neurological inhibition is caused by the specific cleavage of a group of proteins integral to NMJ exocytosis, SNARE proteins (soluble NSF-attachment protein receptors). One or more SNARE proteins are cleaved by BoNT, blocking the release of synaptic vesicular contents like acetylcholine as in the case of motor neurons. BoNTs are synthesized as polypeptides of 150 kDa that are cleaved into heavy and light chains linked by a single disulfide bond. Cleavage takes place within a surface-exposed loop at the N-terminal of the Heavy chain subunit. Both bacterial and host endopeptidases can catalyze BoNT cleavage into heavy and light chains, but bacterial enzymes are thought to carry out this function in vivo.The Heavy Chain (HC) has two 50 kDa functional domains: the N-terminal translocation domain is capable of forming channels in lipid bilayers; the C-terminal ganglioside-binding domain is important for membrane binding and subsequent internalization of toxins by host neurons. The 50 kDa Light chain (LC) is a zinc-dependent endopeptidase specific for core components of neurotransmitter release complexes. BoNT action proceeds in the following steps: binding of cleaved toxin to the target cell membrane; transcytosis from epithelial membrane to target neuromuscular junction cells; release of BoNT Light chain into the target cell cytosol; and proteolytic cleavage of target cell proteins catalyzed by the BoNT Light chain.
Schiavo G, Malizio C, Trimble WS, Polverino de Laureto P, Milan G, Sugiyama H, Johnson EA, Montecucco C.; ''Botulinum G neurotoxin cleaves VAMP/synaptobrevin at a single Ala-Ala peptide bond.''; PubMedEurope PMCScholia
Kroken AR, Karalewitz AP, Fu Z, Kim JJ, Barbieri JT.; ''Novel ganglioside-mediated entry of botulinum neurotoxin serotype D into neurons.''; PubMedEurope PMCScholia
Barash JR, Arnon SS.; ''A novel strain of Clostridium botulinum that produces type B and type H botulinum toxins.''; PubMedEurope PMCScholia
Vaidyanathan VV, Yoshino K, Jahnz M, Dörries C, Bade S, Nauenburg S, Niemann H, Binz T.; ''Proteolysis of SNAP-25 isoforms by botulinum neurotoxin types A, C, and E: domains and amino acid residues controlling the formation of enzyme-substrate complexes and cleavage.''; PubMedEurope PMCScholia
Foran P, Lawrence GW, Shone CC, Foster KA, Dolly JO.; ''Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 in intact and permeabilized chromaffin cells: correlation with its blockade of catecholamine release.''; PubMedEurope PMCScholia
Dong M, Richards DA, Goodnough MC, Tepp WH, Johnson EA, Chapman ER.; ''Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells.''; PubMedEurope PMCScholia
Kozaki S, Kamata Y, Watarai S, Nishiki T, Mochida S.; ''Ganglioside GT1b as a complementary receptor component for Clostridium botulinum neurotoxins.''; PubMedEurope PMCScholia
Dong M, Liu H, Tepp WH, Johnson EA, Janz R, Chapman ER.; ''Glycosylated SV2A and SV2B mediate the entry of botulinum neurotoxin E into neurons.''; PubMedEurope PMCScholia
Karalewitz AP, Fu Z, Baldwin MR, Kim JJ, Barbieri JT.; ''Botulinum neurotoxin serotype C associates with dual ganglioside receptors to facilitate cell entry.''; PubMedEurope PMCScholia
Sun S, Tepp WH, Johnson EA, Chapman ER.; ''Botulinum neurotoxins B and E translocate at different rates and exhibit divergent responses to GT1b and low pH.''; PubMedEurope PMCScholia
Dasgupta BR, Datta A.; ''Botulinum neurotoxin type B (strain 657): partial sequence and similarity with tetanus toxin.''; PubMedEurope PMCScholia
Sathyamoorthy V, Dasgupta BR, Foley J, Niece RL.; ''Botulinum neurotoxin type A: cleavage of the heavy chain into two halves and their partial sequences.''; PubMedEurope PMCScholia
Link E, Edelmann L, Chou JH, Binz T, Yamasaki S, Eisel U, Baumert M, Südhof TC, Niemann H, Jahn R.; ''Tetanus toxin action: inhibition of neurotransmitter release linked to synaptobrevin proteolysis.''; PubMedEurope PMCScholia
Willjes G, Mahrhold S, Strotmeier J, Eichner T, Rummel A, Binz T.; ''Botulinum neurotoxin G binds synaptotagmin-II in a mode similar to that of serotype B: tyrosine 1186 and lysine 1191 cause its lower affinity.''; PubMedEurope PMCScholia
Lacy DB, Tepp W, Cohen AC, DasGupta BR, Stevens RC.; ''Crystal structure of botulinum neurotoxin type A and implications for toxicity.''; PubMedEurope PMCScholia
Schiavo G, Poulain B, Rossetto O, Benfenati F, Tauc L, Montecucco C.; ''Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc.''; PubMedEurope PMCScholia
Schmidt JJ, Sathyamoorthy V, DasGupta BR.; ''Partial amino acid sequence of the heavy and light chains of botulinum neurotoxin type A.''; PubMedEurope PMCScholia
Schiavo G, Rossetto O, Catsicas S, Polverino de Laureto P, DasGupta BR, Benfenati F, Montecucco C.; ''Identification of the nerve terminal targets of botulinum neurotoxin serotypes A, D, and E.''; PubMedEurope PMCScholia
Arndt JW, Chai Q, Christian T, Stevens RC.; ''Structure of botulinum neurotoxin type D light chain at 1.65 A resolution: repercussions for VAMP-2 substrate specificity.''; PubMedEurope PMCScholia
Binz T, Blasi J, Yamasaki S, Baumeister A, Link E, Südhof TC, Jahn R, Niemann H.; ''Proteolysis of SNAP-25 by types E and A botulinal neurotoxins.''; PubMedEurope PMCScholia
Koriazova LK, Montal M.; ''Translocation of botulinum neurotoxin light chain protease through the heavy chain channel.''; PubMedEurope PMCScholia
Kumaran D, Eswaramoorthy S, Furey W, Navaza J, Sax M, Swaminathan S.; ''Domain organization in Clostridium botulinum neurotoxin type E is unique: its implication in faster translocation.''; PubMedEurope PMCScholia
Deinhardt K, Berninghausen O, Willison HJ, Hopkins CR, Schiavo G.; ''Tetanus toxin is internalized by a sequential clathrin-dependent mechanism initiated within lipid microdomains and independent of epsin1.''; PubMedEurope PMCScholia
Turton K, Chaddock JA, Acharya KR.; ''Botulinum and tetanus neurotoxins: structure, function and therapeutic utility.''; PubMedEurope PMCScholia
Peng L, Tepp WH, Johnson EA, Dong M.; ''Botulinum neurotoxin D uses synaptic vesicle protein SV2 and gangliosides as receptors.''; PubMedEurope PMCScholia
Agarwal R, Eswaramoorthy S, Kumaran D, Binz T, Swaminathan S.; ''Structural analysis of botulinum neurotoxin type E catalytic domain and its mutant Glu212-->Gln reveals the pivotal role of the Glu212 carboxylate in the catalytic pathway.''; PubMedEurope PMCScholia
Mochida S, Poulain B, Weller U, Habermann E, Tauc L.; ''Light chain of tetanus toxin intracellularly inhibits acetylcholine release at neuro-neuronal synapses, and its internalization is mediated by heavy chain.''; PubMedEurope PMCScholia
Sun S, Suresh S, Liu H, Tepp WH, Johnson EA, Edwardson JM, Chapman ER.; ''Receptor binding enables botulinum neurotoxin B to sense low pH for translocation channel assembly.''; PubMedEurope PMCScholia
Lee K, Gu S, Jin L, Le TT, Cheng LW, Strotmeier J, Kruel AM, Yao G, Perry K, Rummel A, Jin R.; ''Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity.''; PubMedEurope PMCScholia
Blasi J, Chapman ER, Yamasaki S, Binz T, Niemann H, Jahn R.; ''Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin.''; PubMedEurope PMCScholia
Südhof TC, De Camilli P, Niemann H, Jahn R.; ''Membrane fusion machinery: insights from synaptic proteins.''; PubMedEurope PMCScholia
Yamasaki S, Baumeister A, Binz T, Blasi J, Link E, Cornille F, Roques B, Fykse EM, Südhof TC, Jahn R.; ''Cleavage of members of the synaptobrevin/VAMP family by types D and F botulinal neurotoxins and tetanus toxin.''; PubMedEurope PMCScholia
Dong M, Yeh F, Tepp WH, Dean C, Johnson EA, Janz R, Chapman ER.; ''SV2 is the protein receptor for botulinum neurotoxin A.''; PubMedEurope PMCScholia
Henderson I, Whelan SM, Davis TO, Minton NP.; ''Genetic characterisation of the botulinum toxin complex of Clostridium botulinum strain NCTC 2916.''; PubMedEurope PMCScholia
Chen C, Fu Z, Kim JJ, Barbieri JT, Baldwin MR.; ''Gangliosides as high affinity receptors for tetanus neurotoxin.''; PubMedEurope PMCScholia
Montecucco C, Schiavo G.; ''Mechanism of action of tetanus and botulinum neurotoxins.''; PubMedEurope PMCScholia
Benefield DA, Dessain SK, Shine N, Ohi MD, Lacy DB.; ''Molecular assembly of botulinum neurotoxin progenitor complexes.''; PubMedEurope PMCScholia
Giménez JA, DasGupta BR.; ''Botulinum neurotoxin type E fragmented with endoproteinase Lys-C reveals the site trypsin nicks and homology with tetanus neurotoxin.''; PubMedEurope PMCScholia
Swaminathan S, Eswaramoorthy S.; ''Structural analysis of the catalytic and binding sites of Clostridium botulinum neurotoxin B.''; PubMedEurope PMCScholia
Krieglstein K, Henschen A, Weller U, Habermann E.; ''Arrangement of disulfide bridges and positions of sulfhydryl groups in tetanus toxin.''; PubMedEurope PMCScholia
Simpson LL.; ''Identification of the major steps in botulinum toxin action.''; PubMedEurope PMCScholia
Schiavo G, Santucci A, Dasgupta BR, Mehta PP, Jontes J, Benfenati F, Wilson MC, Montecucco C.; ''Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds.''; PubMedEurope PMCScholia
Amatsu S, Sugawara Y, Matsumura T, Kitadokoro K, Fujinaga Y.; ''Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex.''; PubMedEurope PMCScholia
Peng L, Berntsson RP, Tepp WH, Pitkin RM, Johnson EA, Stenmark P, Dong M.; ''Botulinum neurotoxin D-C uses synaptotagmin I and II as receptors, and human synaptotagmin II is not an effective receptor for type B, D-C and G toxins.''; PubMedEurope PMCScholia
Foran P, Shone CC, Dolly JO.; ''Differences in the protease activities of tetanus and botulinum B toxins revealed by the cleavage of vesicle-associated membrane protein and various sized fragments.''; PubMedEurope PMCScholia
Fujinaga Y, Sugawara Y, Matsumura T.; ''Uptake of botulinum neurotoxin in the intestine.''; PubMedEurope PMCScholia
Lalli G, Bohnert S, Deinhardt K, Verastegui C, Schiavo G.; ''The journey of tetanus and botulinum neurotoxins in neurons.''; PubMedEurope PMCScholia
Rummel A, Häfner K, Mahrhold S, Darashchonak N, Holt M, Jahn R, Beermann S, Karnath T, Bigalke H, Binz T.; ''Botulinum neurotoxins C, E and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor.''; PubMedEurope PMCScholia
Schiavo G, Benfenati F, Poulain B, Rossetto O, Polverino de Laureto P, DasGupta BR, Montecucco C.; ''Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin.''; PubMedEurope PMCScholia
Fu Z, Chen C, Barbieri JT, Kim JJ, Baldwin MR.; ''Glycosylated SV2 and gangliosides as dual receptors for botulinum neurotoxin serotype F.''; PubMedEurope PMCScholia
Yamasaki S, Binz T, Hayashi T, Szabo E, Yamasaki N, Eklund M, Jahn R, Niemann H.; ''Botulinum neurotoxin type G proteolyses the Ala81-Ala82 bond of rat synaptobrevin 2.''; PubMedEurope PMCScholia
Botulinum neurotoxins (BoNTs) bind to polysialogangliosides, including GT1b, GD1b and GQ1b and synaptotagmin polypeptides on the neuronal plasma membrane (Verderio et al., 2006). In the body, this dual binding may have the effect of targeting BoNTs to specific regions of the neuromuscular junction for endocytosis. Different serotypes are known to bind to different receptors: Bont/A to SV2, Bont/B and G to Syt1 and Syt2 with different affinities.
Once BoNT molecules are bound to the host cell surface via their HC domains, they undergo transcytosis which include sorting and endocytosis into an acidic vesicular compartment within the cytosol. As a result of endocytosis, the toxin becomes resistant to neutralization by antisera. Endocytosis is temperature and energy-dependent. In the body, endocytosed BoNT molecules remain associated with the neuromuscular junction which they finally reach by transcytosis.
The N-terminal half of the BoNT Heavy Chain undergoes conformational changes effected by endosomal pH resulting in ion channel formation (Blaustein et al., 1987). This process has been demonstrated experimentally for BoNT serotypes A and B, but all serotypes are thought to have this property (Pellizzari et al. 1999).
Syntaxins are involved in the localization (docking) of both synaptic vesicles and calcium channels to the presynaptic active zone. Syntaxin 1A interacts with SNAP-25 in forming t-SNARE part of SNARE complex. BoNT Type C specifically cleaves Syntaxin 1A although a broader target spectrum is suspected.
Acidic pH triggers a conformation change in the Heavy chain N-terminal domain leading to its insertion into the lipid bilayer and formation of a trans-membrane channel large enough to accommodate the unfolded Light chain. It has been observed that in the closely related Diptheria toxin, a 10-aa motif is critical for pore formation. Ratts et al. identified this motif in some of the virulent BoNTs (Ratts et al., 2005).
The BoNT L chain traverses the H chain channel into the cytosol, refolds, and is released into the cytosol. The complete molecular details of cleavage of the L- H disulfide bond and L chain refolding are not yet known (Pellizzari et al.,1999). The cleavage of host proteins may require the toxins binding to specific recogntion sites as well as cleavage sites (Rossetto et al., 1994).
The neurological inhibition is caused by the specific cleavage of a group of proteins integral to NMJ exocytosis, SNARE proteins (soluble NSF-attachment protein receptors). One or more SNARE proteins are cleaved by BoNT, blocking the release of synaptic vesicular contents like acetylcholine as in the case of motor neurons.
BoNTs are synthesized as polypeptides of 150 kDa that are cleaved into heavy and light chains linked by a single disulfide bond. Cleavage takes place within a surface-exposed loop at the N-terminal of the Heavy chain subunit. Both bacterial and host endopeptidases can catalyze BoNT cleavage into heavy and light chains, but bacterial enzymes are thought to carry out this function in vivo.The Heavy Chain (HC) has two 50 kDa functional domains: the N-terminal translocation domain is capable of forming channels in lipid bilayers; the C-terminal ganglioside-binding domain is important for membrane binding and subsequent internalization of toxins by host neurons. The 50 kDa Light chain (LC) is a zinc-dependent endopeptidase specific for core components of neurotransmitter release complexes.
BoNT action proceeds in the following steps: binding of cleaved toxin to the target cell membrane; transcytosis from epithelial membrane to target neuromuscular junction cells; release of BoNT Light chain into the target cell cytosol; and proteolytic cleavage of target cell proteins catalyzed by the BoNT Light chain.
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