Single-chain Neurotoxin Research Articles

Clostridium butyricum neurotoxin: partial amino acid sequence of major proteolytic fragments and intracellular activity in PC12 cells

The ∼ 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 ∼ 50 kDa light and ∼ 100 kDa heavy chains, whose identities were established by amino acid sequence determination. In the ∼ 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 ∼ 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 ∼ 70% of the Ca 2+ 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.

Conformational changes associated with the nicking and activation of botulinum neurotoxin type E

Secondary and tertiary structural parameters of type E botulinum neurotoxin in the unactivated single-chain and activated two-chain (i.e., after proteolytic cleavage) forms were analyzed using circular dichroism, derivative absorption and fluorescence spectroscopy. The estimated secondary structures (22 and 20% alpha-helix, 44 and 44% beta-pleated sheets, and 34 and 36% random coils for the single- and two-chain neurotoxins, respectively) indicated that virtually no change occurred upon nicking of the single-chain neurotoxin. About 57% of the 70 Tyr residues were exposed in the single-chain form, which increased to 62% in the two-chain form. Fluorescence quenching experiments with neutral, anionic and cationic quenchers indicated that about 40% of the maximum accessible fluorescent Trp residues were exposed on the surface of the single-chain neurotoxin as compared to only 20% in the case of the two-chain neurotoxin. Acrylamide was the most effective quencher with a fraction accessibility of 0.56 and 0.48 of maximum accessible Trp fluorescence residues in the single and two-chain forms of the neurotoxin, respectively. Native polyacrylamide gel electrophoresis of the two forms of the neurotoxin revealed greater mobility for the two chain form. This indicates that the surface charges in the single-chain neurotoxin were altered upon nicking. These observations suggest that nicking of the single-chain type E neurotoxin results in refolding and redistribution of the surface charges of the neurotoxin.