Light chain of botulinum A neurotoxin expressed as an inclusion body from a synthetic gene is catalytically and functionally active.
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.
Ahmed
S A
SA
Department of Immunology and Molecualr Biology, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702.
Smith
L A
LA
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
D002021:Buffers; D008670:Metals; D012492:Salts; D013439:Sulfhydryl Reagents; D019274:Botulinum Toxins, Type A; D003545:Cysteine