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FUNCTION Botulinum toxin causes flaccid paralysis by inhibiting neurotransmitter (acetylcholine) release from the presynaptic membranes of nerve terminals of the eukaryotic host skeletal and autonomic nervous system, with frequent heart or respiratory failure (PubMed:15394302, PubMed:7578132). Precursor of botulinum neurotoxin A which has 2 coreceptors; complex polysialylated gangliosides found on neural tissue and specific membrane-anchored proteins of synaptic vesicles. Receptor proteins are exposed on host presynaptic cell membrane during neurotransmitter release, when the toxin heavy chain (HC) binds to them. Upon synaptic vesicle recycling the toxin is taken up via the endocytic pathway. When the pH of the toxin-containing endosome drops a structural rearrangement occurs so that the N-terminus of the HC forms pores that allows the light chain (LC) to translocate into the cytosol (PubMed:17666397, PubMed:19096517). Once in the cytosol the disulfide bond linking the 2 subunits is reduced and LC cleaves its target protein on synaptic vesicles, preventing their fusion with the cytoplasmic membrane and thus neurotransmitter release. Toxin activity requires polysialylated gangliosides; GT1b supports activity better than GD1a (PubMed:12089155). Binds to host peripheral neuronal presynaptic membranes via the synaptic vesicle glycoproteins SV2A, SV2B and SV2C (PubMed:16543415). It binds directly to the largest lumenal (intravesicular) loop of SV2A, SV2B and SV2C that is transiently exposed outside of cells during exocytosis; gangliosides enhance binding (PubMed:16543415, PubMed:16545378, PubMed:18815274). Recognizes an N-linked glycan on SV2 proteins (PubMed:18815274, PubMed:27294781). May also use FGFR3 as a receptor (PubMed:23696738). Toxin uptake into neural cells requires stimulation (incubation with K(+) to stimulate receptor exposure) to be internalized by receptor-mediated endocytosis (PubMed:16543415, PubMed:19650874, PubMed:21632541, PubMed:21832053). Subsequently the toxin colocalizes with its receptor in host cells (PubMed:16543415, PubMed:19650874). Toxin uptake can be blocked by the appropriate SV2 protein fragments in cell culture (PubMed:16543415).FUNCTION Has proteolytic activity (PubMed:7578132). After translocation into the eukaryotic host cytosol LC hydrolyzes the '197-Gln-|-Arg-198' bond in SNAP25, blocking neurotransmitter release (PubMed:10694409, PubMed:11700044, PubMed:11827515, PubMed:19351593, PubMed:7578132, PubMed:8243676, PubMed:9886085). Recognizes the '146-Met--Gly-155' region of SNAP25, which confers substrate specificity (PubMed:15592454, PubMed:9886085). Hydrolyzes the '202-Thr-|-Arg-203' bond of mouse SNAP23, but not in human which has a different sequence (PubMed:9886085). Reduction of the interchain disulfide bond occurs in the host cytosol and probably prevents retrotranslocation into the synaptic vesicle (PubMed:17666397). Has slow (occurs over 4 weeks) autocatalytic cleavage, however it is not clear if this is physiologically relevant (PubMed:11565902).FUNCTION Responsible for host epithelial cell transcytosis, host nerve cell targeting and translocation of botulinum neurotoxin A light chain (LC) into host cytosol. Composed of 3 subdomains; the translocation domain (TD), and N-terminus and C-terminus of the receptor-binding domain (RBD) (PubMed:19096517). The RBD is responsible for binding to host epithelial cells and transcytosis across them; this uses different receptors than those on nerve cells (PubMed:21106906). RBD is also responsible for adherence of toxin to host nerve cell surface; HC alone prevents uptake of whole toxin by neural cells, and delays paralysis onset by 75% (PubMed:10413679, PubMed:6694738). Isolated RBD also delays paralysis onset (PubMed:21106906). The N-terminus of the RBD binds to phosphatidylinositol, which might play a role in membrane-binding (PubMed:19161982). Binds to host protein receptor synaptic vesicle glycoproteins SV2A, SV2B and SV2C via lumenal loop 4 (PubMed:16545378, PubMed:19650874, PubMed:24240280, PubMed:27294781, PubMed:27313224, PubMed:6370252). Binding can be inhibited by protein fragments from either the HC or SV2C (PubMed:24240280). Isolated HC significantly decreases uptake and toxicity of whole BoNT/A, but also interferes with uptake of BoNT/E and to a lesser extent BoNT/F (PubMed:19650874). The RBD recognizes the N-linked glycan on 'Asn-559' of SV2A, SV2B and SV2C; hydrogen-bonding occurs via 10 well-defined water molecules and stacking of hydrophobic residues (PubMed:27294781). Binds one host GT1b ganglioside, which serves as a coreceptor (PubMed:14731268, PubMed:18704164, PubMed:27958736). Modeling shows the HC can bind both coreceptors (a ganglioside and SV2 protein) simultaneously at different sites (PubMed:24240280). Crystals of the RBD with a GT1b analog can be grown at pH 5.5, indicating the toxin-ganglioside complex could be stable within the endosome (PubMed:18704164). Isolated RBD binds NTNHA (a bacterial protein that protects toxin) with high affinity at pH 6.0 but not at pH 7.5 (PubMed:22363010). The N-terminal belt (residues 449-545) wraps around the perimeter of the LC, probably protecting Zn(2+) in the active site; it is not required for channel formation by the TD domain but may serve to prevent premature LC dissociation from the translocation channel and to protect toxin prior to translocation (PubMed:17907800, PubMed:19351593, PubMed:22158863). The isolated TD forms transmembrane channels of about 15 Angstroms in the absence of a pH gradient; LC translocation requires a pH and redox gradient (pH 5.0/oxidizing in the cis compartment, pH 7.0/reducing in the trans compartment), LC does not unfold unless the cis pH is 6.0 or less (PubMed:17666397, PubMed:19096517, PubMed:2446925). Pores are presumably made by 1-2 toxin molecules (PubMed:23471747). While interaction with the RBD modulates the pH threshold for membrane insertion, the RBD is not essential for toxin degradation of SNAP25 in neural cells (PubMed:19096517).CATALYTIC ACTIVITY Limited hydrolysis of proteins of the neuroexocytosis apparatus, synaptobrevins, SNAP25 or syntaxin. No detected action on small molecule substrates.COFACTOR Binds 1 zinc ion per subunit (PubMed:11700044, PubMed:1429690, PubMed:15592454, PubMed:19351593, PubMed:22363010).ACTIVITY REGULATION Toxin internalization is inhibited by azide or dinitrophenol or at 4 degrees Celsius (PubMed:6694738). Dynamin (DNM) inhibitors abolish toxin uptake (PubMed:21832053).BIOPHYSICOCHEMICAL PROPERTIES kcat is 140 min(-1) (PubMed:10694409). kcat is 1026 (-1) (PubMed:11827515).SUBUNIT Heterodimer; disulfide-linked heterodimer of a light chain (LC) and heavy chain (HC) (PubMed:7578132). Interacts with glycosylated host synaptic vesicle glycoproteins SV2A, SV2B and SV2C which serve as coreceptors (PubMed:16543415, PubMed:18815274, PubMed:19650874, PubMed:24240280, PubMed:27313224). Glycosylation of 'Asn-559' in SV2C contributes a 12-fold increase in affinity to this interaction (PubMed:27313224). Depolarization of target tissue with high levels of K(+) leads to greater levels of receptor exposure (PubMed:16543415). In vitro addition of gangliosides increases SV2-toxin interaction (PubMed:16543415). Forms a highly interlocked heterodimer with NTNHA at pH 6.0 but not at pH 7.5 called the minimally functional progenitor toxin complex (M-PTC) (PubMed:22363010). The PTC is thought to protect toxin in the host acidic gastrointestinal tract, facilitate transcytosis across the intestinal barrier and release at neutral pH as is found in the bloodstream (PubMed:22363010).INTERACTION Whole toxin may be released from the bacteria during cell wall exfoliation (PubMed:7592120). There are estimated to be 150-500 toxin molecules per um(2) of non-myelinated mouse hemidiaphragm nerve membrane (PubMed:6694738). In mouse hemidiaphragm binds only to nerve terminals, and not to muscle, blood vessels, connective tissue Schwann cells or myelin, toxin can be internalized by this preparation (PubMed:6694738).SUBCELLULAR LOCATION Whole toxin may be released from the bacteria during cell wall exfoliation (PubMed:7592120). Colocalizes with its receptor SV2C (synaptic vesicle glycoprotein 2C) and VGAT (vesicular inhibitory amino acid transporter) in neurons (PubMed:24240280). In neurons HC colocalizes with synaptophysin or VAMP2 probably in synaptic vesicles, a portion also colocalizes with RAB5 and may be in synaptic vesicle protein sorting endosomes (PubMed:21632541, PubMed:21832053). Therefore there may be more than one uptake pathway at nerve terminals. Uptake of HC and whole toxin is slowed by dynamin inhibitors (PubMed:21832053). 1-2 molecules of HC are found in the host synaptic vesicle lumen, uptake and subsequent release of LC is very rapid (PubMed:21832053, PubMed:23471747).INDUCTION In cultured bacteria, first detected in late exponential growth (17 hours), reaches maximal levels at 24-25 hours and remains nearly constant for 5 days (at protein level).DOMAIN Has protease activity (PubMed:7578132).DOMAIN Has 3 functional domains; the translocation domain (TD) and the receptor-binding domain (RBD) which is further subdivided into N- and C-terminal domains (N-RBD and C-RBD). Upon trypsin digestion the isolated TD forms channels in bilayers when the cis side is acidic/oxidizing and the trans side is pH 7.0/reducing (PubMed:17666397, PubMed:19096517, PubMed:2446925). The RBD rotates 140 degrees around the TD in the presence of NTNHA (PubMed:22363010). The 3 major domains each serve as a chaperone for the other 2 to ensure they act only in the correct host cell context (PubMed:19096517). In BoNT/A structures the LC is separated from the RBD by the TD; the belt wraps around the perimeter of the LC, protecting Zn(2+) in the active site (PubMed:18032388, PubMed:19351593, PubMed:22363010). The belt region (449-545) may be a pseudosubstrate inhibitor which serves as an intramolecular chaperone for the LC prior to its translocation into the host cytosol (PubMed:17907800).PTM In a bacterial culture the precursor chain is initally cleaved on the amino side of Gly-445 and is processed more slowly between Lys-448 and Ala-449 to give the final mature heavy chain sequence.PTM Has slow autocatalytic activity, cleaves 250-Tyr-Tyr-251, 266-Phe-Gly-267, 419-Phe-Thr-420, 423-Phe-Glu-424, 430-Cys-Val-431, 432-Arg-Gly-433, 438-Lys-Thr-439, and probably 429-Leu-Cys-430 over a period of 4 weeks. Catalysis of the '197-Gln-|-Arg-198' bond in SNAP25 is estimated to be 10(5) more efficient than autocatalysis, leaving the physiological importance of autocatalysis in doubt (PubMed:11565902).PTM Ubiquitinated by host HECD2. Deubiquitination by host VCPIP1 prevents degradation by the proteasome.BIOTECHNOLOGY Dynamin inhibitors slow toxin uptake by nerve cells, and might be used to prolong the treatment window for antitoxins.BIOTECHNOLOGY Replacement of the RBD by other proteins (such as wheat germ agglutinin) allows the rest of the toxin to be taken up by other cell types, and can be used for investigating synaptic vesicle docking-dependent processes in BoNT resistant cells (PubMed:10768948). LC retains protease activity (PubMed:10768948, PubMed:19351593).BIOTECHNOLOGY Isolated receptor-binding domain (RBD) can be used as a vaccine; a mutated form that is transcytosed into the general circulation but does not enter nerve cells (Leu-1266-1267-Ser) is as efficient as wild-type RBD (PubMed:21106906).PHARMACEUTICAL Available under the name Botox (onabotulinumtoxinA, Allergan), Dysport (abobotulinumtoxinA, Ipsen Biopharmaceuticals) and Xeomin (incobotulinumtoxinA, Merz Pharmaceuticals) for the treatment of strabismus and blepharospasm associated with dystonia and cervical dystonia. Also used for the treatment of hemifacial spasm and a number of other neurological disorders characterized by abnormal muscle contraction. It is also used cosmetically to smooth facial wrinkles.MISCELLANEOUS There are seven antigenically distinct forms of botulinum neurotoxin: Types A, B, C, D, E, F, and G; new subtypes are quite frequent.MISCELLANEOUS Types A, B and E are the most frequent cause of adult human foodborne botulism; type A is the most severe, while type E has the shortest incubation period (PubMed:1431246).MISCELLANEOUS Neurotoxin type A is released from bacteria in the two-chain form (PubMed:2126206, PubMed:6370252).MISCELLANEOUS Botulism poisoning is usually food-borne, either by ingesting toxin or bacterial-contaminated food, or less frequently by inhalation poisoning. In both cases the neurotoxin binds to the apical surface of epithelial cells in the gut or airway. Toxin undergoes receptor-mediated endocytosis (using a different receptor than on target nerve cells), transcytosis across the epithelial cells and release into the general circulation. Once in the general circulation it binds to its target cells.SIMILARITY Belongs to the peptidase M27 family.CAUTION Contradictory results show that only SV2C is the receptor; in these experiments gangliosides do not improve toxin-coreceptor interaction (PubMed:16545378).ONLINE INFORMATION From sausages to wrinkles - Issue 19 of February 2002
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