title: Binding and internalization of Clostridium perfringens iota-toxin in lipid rafts.
abstract: Clostridium perfringens iota-toxin is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). The oligomer of Ib formed in membranes induces endocytosis. We examined the binding and internalization of Ib by using Cy3-labeled Ib. Labeled Ib was retained at the membranes of MDCK cells for 60 min of incubation at 37 degrees C, and later it was detected in cytoplasmic vesicles. To determine whether Ib associates with lipid rafts, we incubated MDCK cells with Ib at 4 or 37 degrees C and fractionated the Triton-insoluble membranes. An Ib complex of 500 kDa was localized at 37 degrees C to the insoluble fractions that fulfilled the criteria of lipid rafts, but it did not form at 4 degrees C. The amount of complex in the raft fraction reached a maximum after 60 min of incubation at 37 degrees C. When the cells that were preincubated with Ib at 4 degrees C were incubated at 37 degrees C, the complex was detected in the raft fraction. The treatment of MDCK cells with methyl-beta-cyclodextrin reduced the localization of the Ib complex to the rafts and the rounding of the cells induced by Ia plus Ib. When 125I-labeled Ia was incubated with the cells in the presence of Ib at 37 degrees C, it was localized in the raft fraction. Surface plasmon resonance analysis revealed that Ia binds to the oligomer of Ib. We conclude that Ib binds to a receptor in membranes and then moves to rafts and that Ia bound to the oligomer of Ib formed in the rafts is internalized.
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima 770-8514, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nagahama
authors.forename: Akiwo
authors.initials: A
authors.lastname: Yamaguchi
authors.forename: Tohko
authors.initials: T
authors.lastname: Hagiyama
authors.forename: Noriko
authors.initials: N
authors.lastname: Ohkubo
authors.forename: Keiko
authors.initials: K
authors.lastname: Kobayashi
authors.forename: Jun
authors.initials: J
authors.lastname: Sakurai
mesh_terms: D036002:ADP Ribose Transferases / Q000378:metabolism* / Q000633:toxicity; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism* / Q000633:toxicity; D001665:Binding Sites; D002460:Cell Line; D003016:Clostridium perfringens / Q000472:pathogenicity*; D003505:Cyclodextrins / Q000494:pharmacology; D004705:Endocytosis; D021962:Membrane Microdomains / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D047392:beta-Cyclodextrins
chemical_list: D001427:Bacterial Toxins; D003505:Cyclodextrins; D047392:beta-Cyclodextrins; C050737:iota toxin, Clostridium perfringens; C108732:methyl-beta-cyclodextrin; D036002:ADP Ribose Transferases