Enhancement of the endopeptidase activity of purified botulinum neurotoxins A and E by an isolated component of the native neurotoxin associated proteins.
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.
Sharma
Shashi Kant
SK
Department of Chemistry and Biochemistry, and Center for Marine Science and Technology, University of Massachusetts Dartmouth, Dartmouth, Massachusetts 02747, USA.
Singh
B R
BR
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
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