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.
Miyata
S
S
Department of Microbiology, Faculty of Medicine, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan.
Matsushita
O
O
Minami
J
J
Katayama
S
S
Shimamoto
S
S
Okabe
A
A
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002942:Circular Dichroism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D051381:Rats; D013574:Synaptosomes / Q000378:metabolism*
D000596:Amino Acids; D001427:Bacterial Toxins; C039811:Clostridium perfringens epsilon-toxin; D010455:Peptides; D011108:Polymers