Clostridium perfringens ε-toxin forms a heptameric pore within the detergent-insoluble microdomains of Madin-Darby canine kidney cells and rat synaptosomes

Shigeru Miyata, Junzaburo Minami, Eiji Tamai, Osamu Matsushita, Seiko Shimamoto, Akinobu Okabe

Research output: Contribution to journalArticle

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Abstract

Clostridium perfringens ε-toxin, which is responsible for enterotoxaemia in ungulates, forms a heptamer in rat synaptosomal and Madin-Darby canine kidney (MDCK) cell membranes, leading to membrane permealization. Thus, the toxin may target the detergent-resistant membrane domains (DRMs) of these membranes, in analogy to aerolysin, a heptameric pore-forming toxin that associates with DRMs. To test this idea, we examined the distribution of radiolabeled E-toxin in DRM and detergent-soluble membrane fractions of MDCK cells and rat synaptosomal membranes. When MDCK cells and synaptosomal membranes were incubated with the toxin and then fractionated by cold Triton X-100 extraction and flotation on sucrose gradients, the heptameric toxin was detected almost exclusively in DRMs. The results of a toxin overlay assay revealed that the toxin preferentially bound to and heptamerized in the isolated DRMs. Furthermore, cholesterol depletion by methyl-β-cyclodextrin abrogated their association and lowered the cytotoxicity of the toxin toward MDCK cells. When ε-protoxin, an inactive precursor able to bind to but unable to heptamerize in the membrane, was incubated with MDCK cell membranes, it was detected mainly in their DRMs. These results suggest that the toxin is concentrated and induced to heptamerize on binding to a putative receptor located preferentially in DRMs, with all steps from initial binding through pore formation completed within the same DRMs.

Original languageEnglish
Pages (from-to)39463-39468
Number of pages6
JournalJournal of Biological Chemistry
Volume277
Issue number42
DOIs
Publication statusPublished - Oct 18 2002
Externally publishedYes

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Clostridium
Clostridium perfringens
Madin Darby Canine Kidney Cells
Synaptosomes
Detergents
Rats
Membranes
Cell Membrane
Cell membranes
Enterotoxemia
Octoxynol
Cyclodextrins
Cytotoxicity
Sucrose
Flotation
Cholesterol
Assays
Association reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Clostridium perfringens ε-toxin forms a heptameric pore within the detergent-insoluble microdomains of Madin-Darby canine kidney cells and rat synaptosomes. / Miyata, Shigeru; Minami, Junzaburo; Tamai, Eiji; Matsushita, Osamu; Shimamoto, Seiko; Okabe, Akinobu.

In: Journal of Biological Chemistry, Vol. 277, No. 42, 18.10.2002, p. 39463-39468.

Research output: Contribution to journalArticle

Miyata, Shigeru ; Minami, Junzaburo ; Tamai, Eiji ; Matsushita, Osamu ; Shimamoto, Seiko ; Okabe, Akinobu. / Clostridium perfringens ε-toxin forms a heptameric pore within the detergent-insoluble microdomains of Madin-Darby canine kidney cells and rat synaptosomes. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 42. pp. 39463-39468.
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