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 digestio n is accompanied by removal of the 13 N-terminal and 22 C-terminal amino ac id 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-ter minal residues (DeltaN-PD); PD without the 23 C-terminal residues (DeltaC-P D); and PD without either the N- or C-terminal residues (Delta NC-PD). A mo use lethality test showed that DeltaN-PD was inactive, as is PD, whereas De ltaC-PD and Delta NC-PD were equally active. DeltaC-PD and Delta NC-PD, but not the other constructs formed a large SDS-resistant complex in rat synap tosomal membranes as demonstrated by SDS-polyacrylamide gel electrophoresis . When Delta NC-PD and DeltaC-PD, both labeled with P-32 and mixed in vario us ratios, were incubated with membranes, eight distinct high molecular wei ght bands corresponding to six heteropolymers and two homopolymers were det ected on a SDS-polyacrylamide gel, indicating the active toxin forms a hept americ complex. These results indicate that C-terminal processing is respon sible for activation of the toxin and that it is essential for its heptamer ization within the membrane.