title: Binding and internalization of Clostridium botulinum C2 toxin.
abstract: Clostridium botulinum C2 toxin is a binary toxin composed of an enzymatic component (C2I) and a binding component (C2II). The activated binding component (C2IIa) forms heptamers, and the oligomer with C2I is taken up by receptor-mediated endocytosis. We investigated the binding and internalization of C2IIa in cells. The C2IIa monomer formed oligomers on lipid rafts in membranes of MDCK cells. Methyl-beta-cyclodextrin inhibited the binding of C2IIa and the rounding of the cells induced by C2I plus C2IIa. C2I was localized to the rafts in the presence, but not the absence, of C2IIa. Surface plasmon resonance analysis revealed that C2I bound to the oligomer of C2IIa, but not the monomer of C2IIa. C2I and C2IIa were rapidly internalized in the cells. LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited the internalization of C2IIa in the cells and the rounding activity in the presence of C2I plus C2IIa. Incubation of the cells with C2I plus C2IIa resulted in the activation of PI3K and in phosphorylation of phosphoinositide-dependent kinase 1 and protein kinase B/Akt (Akt), but that with C2IIa alone did not. Akt inhibitor X, an Akt phosphorylation inhibitor, inhibited the rounding activity but not the internalization of C2IIa. The results suggest that the binding of C2I to the oligomer of C2IIa on rafts triggers the activation of the PI3K-Akt signaling pathway and, in turn, the initiation of endocytosis.
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nagahama
authors.forename: Tohko
authors.initials: T
authors.lastname: Hagiyama
authors.forename: Takashi
authors.initials: T
authors.lastname: Kojima
authors.forename: Kouhei
authors.initials: K
authors.lastname: Aoyanagi
authors.forename: Chihiro
authors.initials: C
authors.lastname: Takahashi
authors.forename: Masataka
authors.initials: M
authors.lastname: Oda
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Sakaguchi
authors.forename: Keiji
authors.initials: K
authors.lastname: Oguma
authors.forename: Jun
authors.initials: J
authors.lastname: Sakurai
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000378:metabolism*; D002460:Cell Line; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D021962:Membrane Microdomains / Q000378:metabolism*; D018613:Microscopy, Confocal; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D020349:Surface Plasmon Resonance
chemical_list: D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D001905:Botulinum Toxins; C096324:botulinum toxin type C