Clostridium butyricum neurotoxin: partial amino acid sequence of major proteolytic fragments and intracellular activity in PC12 cells.
The approximately 150 kDa single-chain neurotoxin produced by Clostridium butyricum, reported to be similar to C. botulinum neurotoxin serotype E (Giménez and Sugiyama, Infect. Immun. 54, 926-929, 1988), was probed with trypsin and endoproteinase Lys-C. The two proteases cleaved the butyricum neurotoxin between residues Arg 421-Lys 422 and Lys 418-Gly 419, respectively. Cleavage at this region, highly susceptible to proteolysis, generated approximately 50 kDa light and approximately 100 kDa heavy chains, whose identities were established by amino acid sequence determination. In the approximately 150 kDa dichain neurotoxin these two chains remained linked by an interchain disulfide bond. The endoproteinase Lys-C also cleaved the heavy chain between residues Lys 594-Ile 595, yielding a approximately 73 kDa fragment. A total of 77 amino acid residues was identified by Edman degradation of the major fragments generated by proteolysis. By analogy with other botulinum neurotoxin serotypes, the light chain contains the intracellular inhibitory domain and the heavy chain the receptor-binding and channel-forming domains. Butyricum neurotoxin, whether in single-chain or dichain form, inhibited approximately 70% of the Ca2+ stimulated [3H]norepinephrine release from permeabilized PC12 cells. Reduction with 5 mM dithiothreitol enhanced the intracellular inhibitory activity of dichain neurotoxin about 30-fold and increased the extent of inhibition to about 80%, while the activity of single-chain neurotoxin was slightly affected. Increase in the intracellular inhibitory activity of butyricum neurotoxin following mild treatment with trypsin (i.e. conversion of the single-chain protein to the dichain form) was virtually complete within 6 min.
Giménez
J
J
Department of Food Microbiology and Toxicology, University of Wisconsin, Madison 53706.
Lomneth
R
R
DasGupta
B R
BR
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002463:Cell Membrane Permeability; D003013:Clostridium / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009498:Neurotoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity; D009638:Norepinephrine / Q000378:metabolism; D010084:Oxidation-Reduction; D016716:PC12 Cells / Q000187:drug effects; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity; D051381:Rats
D004220:Disulfides; D009498:Neurotoxins; D010446:Peptide Fragments; D002118:Calcium; D004229:Dithiothreitol; D009638:Norepinephrine