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Originally published In Press as doi:10.1074/jbc.M001530200 on April 3, 2000

J. Biol. Chem., Vol. 275, Issue 24, 18407-18417, June 16, 2000
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CaCo-2 Cells Treated with Clostridium perfringens Enterotoxin Form Multiple Large Complex Species, One of Which Contains the Tight Junction Protein Occludin*

Usha SinghDagger , Christina M. Van Itallie§, Laura L. Mitic, James M. Anderson§, and Bruce A. McClaneDagger ||

From the Dagger  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

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.

* 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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