Cleavage of a C-terminal peptide is essential for heptamerization of Clostridium perfringens epsilon-toxin in the synaptosomal membrane.

Activation of Clostridium perfringens epsilon-protoxin by tryptic digestion is accompanied by removal of the 13 N-terminal and 22 C-terminal amino acid residues. In this study, we examined the toxicity of four constructs: an epsilon-protoxin derivative (PD), in which a factor Xa cleavage site was generated at the C-terminal trypsin-sensitive site; PD without the 13 N-terminal residues (DeltaN-PD); PD without the 23 C-terminal residues (DeltaC-PD); and PD without either the N- or C-terminal residues (DeltaNC-PD). A mouse lethality test showed that DeltaN-PD was inactive, as is PD, whereas DeltaC-PD and DeltaNC-PD were equally active. DeltaC-PD and DeltaNC-PD, but not the other constructs formed a large SDS-resistant complex in rat synaptosomal membranes as demonstrated by SDS-polyacrylamide gel electrophoresis. When DeltaNC-PD and DeltaC-PD, both labeled with (32)P and mixed in various ratios, were incubated with membranes, eight distinct high molecular weight bands corresponding to six heteropolymers and two homopolymers were detected on a SDS-polyacrylamide gel, indicating the active toxin forms a heptameric complex. These results indicate that C-terminal processing is responsible for activation of the toxin and that it is essential for its heptamerization within the membrane.