title: Enhancement of the endopeptidase activity of botulinum neurotoxin by its associated proteins and dithiothreitol.
abstract: Botulinum neurotoxins type A (BoNT/A), the most toxic substance known to man, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs), possibly through a polycistronic expression of a clustered group of genes. The botulinum neurotoxin complex is the only known example of a protein complex where a group of proteins (NAPs) protect another protein (BoNT) against acidity and proteases of the GI tract. We now report that NAPs also potentiate the Zn2+ endopeptidase activity of BoNT/A in both in vitro and in vivo assays against its known intracellular target protein, 25 kDa synaptosomal associated protein (SNAP-25). While BoNT/A exhibited no protease activity prior to reduction with dithiothreitol (DTT), the BoNT/A complex exhibited a high protease activity even in its nonreduced form. Our results suggest that the bacterial production of NAPs along with BoNT is designed for the NAPs to play an accessory role in the neurotoxin function, in contrast to their previously known limited role in protecting the neurotoxin in the GI tract and in the external environment. Structural features of BoNT/A change considerably upon disulfide reduction, as revealed by near-UV circular dichroism spectroscopy. BoNT/A in the reduced form adopts a more flexible structure than in the unreduced form, as also indicated by large differences in DeltaH values (155 vs 248 kJ mol-1) of temperature-induced unfolding of BoNT/A.
authors.affiliation: Department of Chemistry and Biochemistry, Center for Marine Science and Technology, University of Massachusetts at Dartmouth 02747, USA.
authors.forename: S
authors.initials: S
authors.lastname: Cai
authors.forename: H K
authors.initials: HK
authors.lastname: Sarkar
authors.forename: B R
authors.initials: BR
authors.lastname: Singh
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D050825:Synaptosomal-Associated Protein 25; D013574:Synaptosomes / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D008666:Metalloendopeptidases; C044194:microbial metalloproteinases; D019274:Botulinum Toxins, Type A; D004229:Dithiothreitol