Enhancement of the Endopeptidase Activity of Purified Botulinum Neurotoxins A and E by an Isolated Component of the Native Neurotoxin Associated Proteins

• Published in: Biochemistry (Easton) Vol. 43; no. 16; pp. 4791 - 4798
• Main Authors: Sharma, Shashi Kant, Singh, B. R
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 27.04.2004
• Subjects: Animals; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Bacterial Proteins - physiology; Botulinum Toxins - isolation & purification; Botulinum Toxins - metabolism; Botulinum Toxins, Type A - isolation & purification; Botulinum Toxins, Type A - metabolism; Clostridium botulinum; Endopeptidases - metabolism; Enzyme Activation; Hemagglutinins - isolation & purification; Hemagglutinins - metabolism; Hemagglutinins - physiology; Hydrolysis; Membrane Proteins - metabolism; Nerve Tissue Proteins - metabolism; Rats; Recombinant Fusion Proteins - metabolism; Synaptosomal-Associated Protein 25; Synaptosomes - enzymology
• DOI: 10.1021/bi0355544
• ISSN: 0006-2960

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.