Formation of a biologically active toxin complex of the binary Clostridium botulinum C2 toxin without cell membrane interaction.
Clostridium botulinum produces a binary toxin, which is composed of two separate proteins. The enzyme component, C2I, is an ADP-ribosyltransferase which modifies G-actin of eukaryotic cells. The proteolytically activated binding/translocation component, C2IIa, forms ring-shaped heptamers, which bind to cell receptors and mediate the transport of C2I into the cytosol of target cells. According to the current model, receptor-bound C2IIa serves as a docking platform for C2I on the cell surface. Following assembly of C2I, the toxin complex is taken up via receptor-mediated endocytosis, and finally, C2IIa facilitates translocation of C2I from acidic endosomes into the cytosol. Our data support an alternative scenario for the early steps of interaction of the C2 toxin and eukaryotic cells, due to the fact that C2IIa and C2I can interact prior to binding of the toxin to the cell surface. The C2IIa-C2I complex, which was formed in a cell-free system, was detected by native gel electrophoresis and subsequent immunoblot analysis or radiolabeling methods. The preformed C2 toxin complex ADP-ribosylated actin in vitro and induced cell rounding. The interaction of C2I with C2IIa did not enhance the binding of C2IIa to the cellular receptor. Intoxication of Vero cells and of human colon carcinoma cells (CaCo-2) was significantly enhanced when the preformed toxin complex was added to cultured cells as compared to addition of the single components.
Kaiser
Eva
E
Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, D-89081 Ulm, Germany.
Haug
Gerd
G
Hliscs
Marion
M
Aktories
Klaus
K
Barth
Holger
H
D000199:Actins / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D019274:Botulinum Toxins, Type A; D002462:Cell Membrane / Q000187:drug effects; D002522:Chlorocebus aethiops; D003014:Clostridium botulinum / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D011485:Protein Binding; D014709:Vero Cells
D000199:Actins; C096323:rimabotulinumtoxinB; D000246:Adenosine Diphosphate Ribose; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A