title: Compositional and stoichiometric analysis of Clostridium perfringens enterotoxin complexes in Caco-2 cells and claudin 4 fibroblast transfectants.
abstract: Clostridium perfringens enterotoxin (CPE) binds to host cell receptors, forming a small complex precursor for two large complexes reportedly having molecular masses of approximately 155 or approximately 200 kDa. Formation of the approximately 155 kDa complex causes a Ca(2+) influx that leads to apoptosis or oncosis. CPE complex composition is currently poorly understood, although occludin was identified in the approximately 200 kDa complex. The current study used heteromer gel shift analysis to show both CPE large complexes contain six CPE molecules. Ferguson plots and size exclusion chromatography re-sized the approximately 155 and approximately 200 kDa complexes as approximately 425-500 kDa and approximately 550-660 kDa respectively. Co-immunoprecipitation and electroelution studies demonstrated both CPE-binding and non-CPE-binding claudins are associated with all three CPE complexes in Caco-2 cells and with small complex and approximately 425-500 kDa complex of claudin 4 transfectants. Fibroblast transfectants expressing claudin 4 or C-terminal truncated claudin 4 were CPE-sensitive and formed the approximately 425 kDa complex, indicating claudin-induced cell signalling is not required for CPE action and that expression of a single receptor claudin suffices for approximately 425-500 kDa CPE complex formation. These results identify CPE as a unique toxin that combines with tight junction proteins to form high-molecular-mass hexameric pores and alter membrane permeability.
authors.affiliation: Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Susan L
authors.initials: SL
authors.lastname: Robertson
authors.forename: James G
authors.initials: JG
authors.lastname: Smedley
authors.forename: Usha
authors.initials: U
authors.lastname: Singh
authors.forename: Ganes
authors.initials: G
authors.lastname: Chakrabarti
authors.forename: Christina M
authors.initials: CM
authors.lastname: Van Itallie
authors.forename: James M
authors.initials: JM
authors.lastname: Anderson
authors.forename: Bruce A
authors.initials: BA
authors.lastname: McClane
mesh_terms: D015153:Blotting, Western; D018938:Caco-2 Cells; D002850:Chromatography, Gel; D062445:Claudin-1; D062465:Claudin-3; D062506:Claudin-4; D003016:Clostridium perfringens / Q000235:genetics / Q000378:metabolism*; D004768:Enterotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: C568108:CLDN1 protein, human; C568122:CLDN3 protein, human; C568157:CLDN4 protein, human; D062445:Claudin-1; D062465:Claudin-3; D062506:Claudin-4; D004768:Enterotoxins; D008565:Membrane Proteins; C035598:enterotoxin, Clostridium