Cellular uptake of Clostridium botulinum C2 toxin requires oligomerization and acidification.
The actin-ADP-ribosylating binary Clostridium botulinum C2 toxin consists of two individual proteins, the binding/translocation component C2II and the enzyme component C2I. To elicit its cytotoxic action, C2II binds to a receptor on the cell surface and mediates cell entry of C2I via receptor-mediated endocytosis. Here we report that binding of C2II to the surface of target cells requires cleavage of C2II by trypsin. Trypsin cleavage causes oligomerization of the activated C2II (C2IIa) to give SDS-stable heptameric structures, which exhibit a characteristic annular or horseshoe shape and form channels in lipid bilayer membranes. Cytosolic delivery of the enzyme component C2I is blocked by bafilomycin but not by brefeldin A or nocodazole, indicating uptake from an endosomal compartment and requirement of endosomal acidification for cell entry. In the presence of C2IIa and C2I, short term acidification of the extracellular medium (pH 5.4) allows C2I to enter the cytosol directly. Our data indicate that entry of C2 toxin into cells involves (i) activation of C2II by trypsin-cleavage, (ii) oligomerization of cleaved C2IIa to heptamers, (iii) binding of the C2IIa oligomers to the carbohydrate receptor on the cell surface and assembly with C2I, (iv) receptor-mediated endocytosis of both C2 components into endosomes, and finally (v) translocation and release of C2I into the cytosol after acidification of the endosomal compartment.
Barth
H
H
Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Str. 5, D-79104 Freiburg, Germany.
Blocker
D
D
Behlke
J
J
Bergsma-Schutter
W
W
Brisson
A
A
Benz
R
R
Aktories
K
K
D000143:Acids / Q000378:metabolism*; D000818:Animals; D001704:Biopolymers; D001905:Botulinum Toxins / Q000378:metabolism*; D002451:Cell Compartmentation; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D014357:Trypsin / Q000378:metabolism
D000143:Acids; D001704:Biopolymers; D014357:Trypsin; D001905:Botulinum Toxins; C096324:botulinum toxin type C