title: Enhancement of the endopeptidase activity of purified botulinum neurotoxins A and E by an isolated component of the native neurotoxin associated proteins.
abstract: In botulism disease, neurotransmitter release is blocked by a group of structurally related neurotoxin proteins produced by Clostridium botulinum. Botulinum neurotoxins (BoNT, A-G) enter nerve terminals and irreversibly inhibit exocytosis via their endopeptidase activities against synaptic proteins SNAP-25, VAMP, and Syntaxin. Type A C. botulinum secretes the neurotoxin along with 5 other proteins called neurotoxin associated proteins (NAPs). Here, we report that hemagglutinin-33 (Hn-33), one of the NAP components, enhances the endopeptidase activity of not only BoNT/A but also that of BoNT/E, both under in vitro conditions and in rat synaptosomes. BoNT/A endopeptidase activity in vitro is about twice as high as that of BoNT/E under disulfide-reduced conditions. Addition of Hn-33 separately to nonreduced BoNT/A and BoNT/E (which otherwise have only residual endopeptidase activity) enhanced their in vitro endopeptidase activity by 21- and 25-fold, respectively. Cleavage of rat-brain synaptosome SNAP-25 by BoNTs was used to assay endopeptidase activity under nerve-cell conditions. Reduced BoNT/A and BoNT/E cleaved synaptosomal SNAP-25 by 20% and 15%, respectively. Addition of Hn-33 separately to nonreduced BoNT/A and BoNT/E enhanced their endopeptidase activities by 13-fold for the cleavage of SNAP-25 in synaptosomes, suggesting a possible functional role of Hn-33 in association with BoNTs. We believe that Hn-33 could be used as an activator in the formulation of the neurotoxin for therapeutic use.
authors.affiliation: Department of Chemistry and Biochemistry, and Center for Marine Science and Technology, University of Massachusetts Dartmouth, Dartmouth, Massachusetts 02747, USA.
authors.forename: Shashi Kant
authors.initials: SK
authors.lastname: Sharma
authors.forename: B R
authors.initials: BR
authors.lastname: Singh
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology*; D001905:Botulinum Toxins / Q000302:isolation & purification* / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000302:isolation & purification* / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D006388:Hemagglutinins / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology*; D006868:Hydrolysis; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D013574:Synaptosomes / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D006388:Hemagglutinins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; C118973:hemagglutinin 33 protein, Clostridium botulinum; D010450:Endopeptidases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E