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J. Biol. Chem., Vol. 275, Issue 24, 18407-18417, June 16, 2000
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From the The previous model for the action of
Clostridium perfringens enterotoxin (CPE) proposed that (i)
CPE binds to host cell receptor(s), forming a small (~90 kDa)
complex, (ii) the small complex interacts with other eucaryotic
protein(s), forming a large (~160 kDa) complex, and (iii) the large
complex triggers massive permeability changes, thereby inducing
enterocyte death. In the current study, Western immunoblot analysis
demonstrated that CPE bound to CaCo-2 human intestinal cells at
37 °C forms multiple large complex species, with apparent sizes of
~200, ~155, and ~135 kDa. These immunoblot experiments also
revealed that occludin, an ~65-kDa tight junction protein, is present
in the ~200-kDa large complex but absent from the other large complex
species. Immunoprecipitation studies confirmed that occludin physically
associates with CPE in large complex material and also indicated that
occludin is absent from small complex. These results strongly suggest
that occludin becomes associated with CPE during formation of the
~200-kDa large complex. A postbinding association between CPE and
occludin is consistent with the failure of rat fibroblast transfectants
expressing occludin to bind CPE in the current study. Those occludin
transfectants were also insensitive to CPE, strongly suggesting that
occludin expression is not sufficient to confer CPE sensitivity.
However, the occludin-containing, ~200-kDa large complex may
contribute to CPE-induced cytotoxicity, because nontoxic CPE point
mutants did not form any large complex species. By showing that large complex material is comprised of several species (one containing occludin), the current studies indicate that CPE action is more complicated than previously appreciated and also provide additional evidence for CPE interactions with tight junction proteins, which could
be important for CPE-induced pathophysiology.
CaCo-2 Cells Treated with Clostridium perfringens
Enterotoxin Form Multiple Large Complex Species, One of Which Contains
the Tight Junction Protein Occludin*
,
Department of Molecular Genetics and
Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania 15261, the § Department of Internal Medicine,
Yale University School of Medicine, New Haven, Connecticut 06511, and
the ¶ Department of Cell Biology, Yale University School of
Medicine, New Haven, Connecticut 06511
*
This work was supported by Public Health Service Grant
AI-19844-17 and U.S. Department of Agriculture Grant 9802822 (to
B. A. M.) and by Public Health Service Grants DK45134 and
DK34988 (to J. M. A.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: E1240 BSTWR, Dept.
of Molecular Genetics and Biochemistry, University of Pittsburgh School
of Medicine, Pittsburgh, PA 15261. Tel.: 412-648-9022; Fax:
412-624-1401; E-mail: bamcc@pop.pitt.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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