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Stability and Endopeptidase Activity of Botulinum Neurotoxin Type A Light Chain
Author(s) -
Wang Haihong,
Singh Bal Ram
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1006-c
Subject(s) - proteases , endopeptidase , botulinum neurotoxin , clostridium botulinum , chemistry , protease , biochemistry , trypsin , enzyme , toxin
Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum can block the release of neurotransmitter, resulting in muscular paralysis. As the most deadly poison, they also have therapeutic functions against some neuromuscular disorders. Both the toxicity and therapeutic effects of BoNTs highly depend on their endopeptidase activity, which resides exclusively in their light chain (LC) part. The objective of this study was to investigate the stability and endopeptidase activity of BoNT type A (BoNT/A) LC (LCA). Previous study showed that Hn‐33, a 33 kDa protein secreted together with BoNTs, can protect BoNT/A from some proteases and enhance the endopeptidase activity of BoNT/A and E. Attempts were also made in this study to analyze if Hn‐33 exerts similar protection on LCA. Cleavage of LCA by proteases was carried out at 25 o C and pH 7.8. The reaction was terminated by adding protease inhibitors at 0, 30, 60, and 90 min. The proteolytic profiles were monitored using SDS‐PAGE. The endopeptidase activity of LCA before and after exposure to proteases was determined using fluorescent spectroscopy. The results show that LCA is considerably cleaved right after trypsin was added, though the endopeptidase activity of LCA largely retained. Extended exposure of LCA to trypsin resulted in further cleavage and decrease of endopeptidase activity. Hn‐33 did not show protection on LCA against proteases. This research is funded by National Institute of Health through New England Regional Centre of Excellence for Biodefense (1‐U54AI057159‐03) and Department of Defense/US Army (W911NF‐06 1‐0095).