RESUMO
Clostridium perfringens iota-toxin is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). We investigated the role of the conserved Ca(2+)-binding motif of Ib in the cytotoxicity of iota-toxin. The cytotoxicity of iota-toxin increased with an increase in the concentration of extracellular Ca(2+). A surface plasmon resonance analysis showed that the binding of Ia to the oligomer of Ib is dependent on the concentration of Ca(2+). However, the addition of Ca(2+) had no effect on the binding of (125)I-labeled Ib to the cells. We replaced Asp-8, -10, and -12 in the Ca(2+)-binding motif of Ib with alanine. D8A, D10A, and D12A bound to the cell and formed an oligomer at about half of the wild-type Ib. The cytotoxicity of Ib variants in the presence of Ia was about 500-fold less than that of wild-type Ib. Immunofluorescence study showed that these variants were internalized in the early endosomes like wild-type Ib. However, wild-type Ib-induced internalization of Ia in the cells, but these variants did not. The result indicates that the conserved Ca(2+)-binding motif in the N-terminal region of Ib plays a role in the interaction of Ib with Ia in the presence of Ca(2+).
Assuntos
ADP Ribose Transferases/metabolismo , ADP Ribose Transferases/toxicidade , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Cálcio/metabolismo , Clostridium perfringens/metabolismo , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Sítios de Ligação , Chlorocebus aethiops , Sequência Conservada , Cães , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Alinhamento de Sequência , Ressonância de Plasmônio de Superfície , Células VeroRESUMO
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.
