title: Identification of a claudin-4 residue important for mediating the host cell binding and action of Clostridium perfringens enterotoxin.
abstract: The 24-member claudin protein family plays a key role in maintaining the normal structure and function of epithelial tight junctions. Previous studies with fibroblast transfectants and naturally sensitive Caco-2 cells have also implicated certain claudins (e.g., Claudin-4) as receptors for Clostridium perfringens enterotoxin (CPE). The present study first provided evidence that the second extracellular loop (ECL-2) of claudins is specifically important for mediating the host cell binding and cytotoxicity of native CPE. Rat fibroblast transfectants expressing a Claudin-4 chimera, where the natural ECL-2 was replaced by ECL-2 from Claudin-2, exhibited no CPE-induced cytotoxicity. Conversely, CPE bound to, and killed, CPE-treated transfectants expressing a Claudin-2 chimera with a substituted ECL-2 from Claudin-4. Site-directed mutagenesis was then used to alter an ECL-2 residue that invariably aligns as N in claudins known to bind native CPE but as D or S in claudins that cannot bind CPE. Transfectants expressing a Claudin-4(N149D) mutant lost the ability to bind or respond to CPE, while transfectants expressing a Claudin-1 mutant with the corresponding ECL-2 residue changed from D to N acquired CPE binding and sensitivity. Identifying carriage of this N residue in ECL-2 as being important for native CPE binding helps to explain why only certain claudins can serve as CPE receptors. Finally, preincubating CPE with soluble recombinant Claudin-4, or Claudin-4 fragments containing ECL-2 specifically blocked the cytotoxicity on Caco-2 cells. This result opens the possibility of using receptor claudins as therapeutic decoys to ameliorate CPE-mediated intestinal disease.
authors.affiliation: University of Pittsburgh School of Medicine, E1240 BSTWR, Pittsburgh, PA 15261, USA.
authors.forename: Susan L
authors.initials: SL
authors.lastname: Robertson
authors.forename: James G
authors.initials: JG
authors.lastname: Smedley
authors.forename: Bruce A
authors.initials: BA
authors.lastname: McClane
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018938:Caco-2 Cells; D062506:Claudin-4; D003016:Clostridium perfringens / Q000378:metabolism*; D004768:Enterotoxins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000633:toxicity*; D005347:Fibroblasts; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: C568157:CLDN4 protein, human; D062506:Claudin-4; D004768:Enterotoxins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C035598:enterotoxin, Clostridium