PMID:20693440

Citation	Wang, HH, Riding, S, Lindo, P and Singh, BR (2010) Endopeptidase activities of botulinum neurotoxin type B complex, holotoxin, and light chain. Appl. Environ. Microbiol. 76:6658-63
Abstract	Botulinum neurotoxin (BoNT) serotype B (BoNT/B) is one of the serotypes of BoNT that causes deadly human botulism, though it is used clinically for treatment of many neuromuscular diseases. BoNT/B is produced by Clostridium botulinum, and it is secreted along with a group of neurotoxin-associated proteins (NAPs) in the form of a BoNT/B complex. The complex dissociates into a 150-kDa holotoxin and NAPs at alkaline pHs. The 150-kDa BoNT/B holotoxin can be nicked to produce a 50-kDa domain referred to as the light chain (LC) and a 100-kDa heavy chain, with the former possessing a unique endopeptidase activity. The two chains remain linked through a disulfide bond that can be reduced to separate the two chains. The endopeptidase activity is present in all three forms of the toxin (complex, purified BoNT/B holotoxin, and separated light chain), which are used by different researchers to develop detection methods and screen for inhibitors. In this research, the endopeptidase activities of the three forms, for the first time, were compared under the same conditions. The results show that enzyme activities of the three forms differ significantly and are largely dependent on nicking and disulfide reduction conditions. Under the conditions used, LC had the highest level of activity, and the complex had the lowest. The activity was enhanced by nicking of BoNT/B holotoxin and was enhanced even more by dithiothreitol (DTT) reduction after nicking. This information is useful for understanding the properties of BoNT endopeptidases and for comparing the efficacies of different inhibitors when they are tested with different forms of BoNT endopeptidase.
Links	PubMed PMC2950459 Online version:10.1128/AEM.00731-10
Keywords	Botulinum Toxins/chemistry; Botulinum Toxins/metabolism; Clostridium botulinum/enzymology; Disulfides/metabolism; Endopeptidases/chemistry; Endopeptidases/metabolism; Glycosides/chemistry; Glycosides/metabolism; Neurotoxins/chemistry; Neurotoxins/metabolism; Oxidation-Reduction; Protein Subunits/chemistry; Protein Subunits/metabolism; Triterpenes/chemistry; Triterpenes/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
CLOBO:Q08077		GO:0004175: endopeptidase activity	ECO:0000314:		F		Figure 3 shows the endopeptidase activities of Bont/b. Cleavage of VAMPtide was measured when exposed to various forms of the protein.	complete

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