Role of Ca2+-binding motif in cytotoxicity induced by Clostridium perfringens iota-toxin.
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+).
Kobayashi
Keiko
K
Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima 7708514, Japan.
Nagahama
Masahiro
M
Ohkubo
Noriko
N
Kojima
Takashi
T
Shirai
Hidenori
H
Iwamoto
Shinobu
S
Oda
Masataka
M
Sakurai
Jun
J
D036002:ADP Ribose Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003016:Clostridium perfringens / Q000378:metabolism*; D017124:Conserved Sequence; D004285:Dogs; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance; D014709:Vero Cells
D001427:Bacterial Toxins; C050737:iota toxin, Clostridium perfringens; D036002:ADP Ribose Transferases; D002118:Calcium