Clostridium perfringens iota toxin. Mapping of the Ia domain involved in docking with Ib and cellular internalization.
Clostridium perfringens iota toxin consists of two unlinked proteins. The binding component (Ib) is required to internalize into cells an enzymatic component (Ia) that ADP-ribosylates G-actin. To characterize the Ia domain that interacts with Ib, fusion proteins were constructed between the C. botulinum C3 enzyme, which ADP-ribosylates Rho, and various truncated versions of Ia. These chimeric molecules retained the wild type ADP-ribosyltransferase activity specific for Rho and were recognized by antibodies against C3 enzyme and Ia. Internalization of each chimera into Vero cells was assessed by measuring the disorganization of the actin cytoskeleton and intracellular ADP-ribosylation of Rho. Fusion proteins containing C3 linked to the C terminus of Ia were transported most efficiently into cells like wild type Ia in an Ib-dependent manner that was blocked by bafilomycin A1. The minimal Ia fragment that promoted translocation of Ia-C3 chimeras into cells consisted of 128 central residues (129-257). These findings revealed that iota toxin is a suitable system for mediating the entry of heterologous proteins such as C3 into cells.
Marvaud
Jean-Christophe
JC
CNR Anaérobies, Institut Pasteur, 75724 Paris Cedex 15, France.
Stiles
Bradley G
BG
Chenal
Alexandre
A
Gillet
Daniel
D
Gibert
Maryse
M
Smith
Leonard A
LA
Popoff
Michel R
MR
D036002:ADP Ribose Transferases / Q000737:chemistry*; D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001427:Bacterial Toxins / Q000737:chemistry*; D001905:Botulinum Toxins / Q000737:chemistry; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002469:Cell Separation; D002522:Chlorocebus aethiops; D002918:Chymotrypsin / Q000494:pharmacology; D002942:Circular Dichroism; D003599:Cytoskeleton / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D005982:Glutathione Transferase / Q000378:metabolism; D015151:Immunoblotting; D018942:Macrolides; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014466:Ultraviolet Rays; D014709:Vero Cells
D000199:Actins; D000900:Anti-Bacterial Agents; D001427:Bacterial Toxins; D018942:Macrolides; D011993:Recombinant Fusion Proteins; C050737:iota toxin, Clostridium perfringens; D000244:Adenosine Diphosphate; C040929:bafilomycin A1; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D005982:Glutathione Transferase; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D001905:Botulinum Toxins