Cleavage of a C-terminal Peptide Is Essential for Heptamerization of Clostridium perfringens ε-Toxin in the Synaptosomal Membrane

Shigeru Miyata, Osamu Matsushita, Junzaburo Minami, Seiichi Katayama, Seiko Shimamoto, Akinobu Okabe

Research output: Contribution to journalArticle

91 Citations (Scopus)

Abstract

Activation of Clostridium perfringens ε-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 ε-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 (ΔN-PD); PD without the 23 C-terminal residues (ΔC-PD); and PD without either the N- or C-terminal residues (ΔNC-PD). A mouse lethality test showed that ΔN-PD was inactive, as is PD, whereas ΔC-PD and ΔNC-PD were equally active. ΔC-PD and ΔNC-PD, but not the other constructs formed a large SDS-resistant complex in rat synaptosomal membranes as demonstrated by SDS-polyacrylamide gel electrophoresis. When ΔNC-PD and ΔC-PD, both labeled with 32P 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.

Original languageEnglish
Pages (from-to)13778-13783
Number of pages6
JournalJournal of Biological Chemistry
Volume276
Issue number17
DOIs
Publication statusPublished - Apr 27 2001
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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