title: Light chain of botulinum A neurotoxin expressed as an inclusion body from a synthetic gene is catalytically and functionally active.
abstract: Botulinum neurotoxins, the most potent of all toxins, induce lethal neuromuscular paralysis by inhibiting exocytosis at the neuromuscular junction. The light chains (LC) of these dichain neurotoxins are a new class of zinc-endopeptidases that specifically cleave the synaptosomal proteins, SNAP-25, VAMP, or syntaxin at discrete sites. To facilitate the structural and functional characterization of these unique endopeptidases, we constructed a synthetic gene for the LC of the botulinum neurotoxin serotype A (BoNT/A), overexpressed it in Escherichia coli, and purified the gene product from inclusion bodies. Our procedure can provide 1.1 g of the LC from 1 L of culture. The LC product was stable in solution at 4 degrees C for at least 6 months. This rBoNT/A LC was proteolytically active, specifically cleaving the Glu-Arg bond in a 17-residue synthetic peptide of SNAP-25, the reported cleavage site of BoNT/A. Its calculated catalytic efficiency kcat/Km was higher than that reported for the native BoNT/A dichain. Treating the rBoNT/A LC with mercuric compounds completely abolished its activity, most probably by modifying the cysteine-164 residue located in the vicinity of the active site. About 70% activity of the LC was restored by adding Zn2+ to a Zn2+-free, apo-LC preparation. The LC was nontoxic to mice and failed to elicit neutralizing epitope(s) when the animals were vaccinated with this protein. In addition, injecting rBoNT/A LC into sea urchin eggs inhibited exocytosis-dependent plasma membrane resealing. For the first time, results of our study make available a large amount of the biologically active toxin fragment in a soluble and stable form.
authors.affiliation: Department of Immunology and Molecualr Biology, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702.
authors.forename: S A
authors.initials: SA
authors.lastname: Ahmed
authors.forename: L A
authors.initials: LA
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002021:Buffers; D002384:Catalysis / Q000187:drug effects; D003545:Cysteine / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005089:Exocytosis / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D018583:Pliability; D012492:Salts; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014162:Transfection
chemical_list: D002021:Buffers; D008670:Metals; D012492:Salts; D013439:Sulfhydryl Reagents; D019274:Botulinum Toxins, Type A; D003545:Cysteine