title: The binary Clostridium botulinum C2 toxin as a protein delivery system: identification of the minimal protein region necessary for interaction of toxin components.
abstract: The binary Clostridium botulinum C2 toxin is composed of the enzyme component C2I and the binding component C2II, which are individual and non-linked proteins. Activated C2IIa mediates cell binding and translocation of C2I into the cytoplasm. C2I ADP-ribosylates G-actin at Arg-177 to depolymerize actin filaments. A fusion toxin containing the N-terminal domain of C2I (residues 1-225) transports C3 ADP-ribosyltransferase from Clostridium limosum into cells (Barth, H., Hofmann, F., Olenik, C., Just, I., and Aktories, K. (1998) Infect. Immun. 66, 1364-1369). We characterized the adaptor function of C2I and its interaction with C2IIa. The fusion toxin GST-C2I(1-225)-C3 was efficiently transported by C2IIa, indicating that C2IIa translocates proteins into the cytosol even when the C2I(1-225) adaptor was positioned in the middle of a fusion protein. Amino acid residues 1-87 of C2I were sufficient for interaction with C2IIa and for translocation of C2I fusion toxins into HeLa cells. Residues 1-87 were the minimal part of C2I to bind to C2IIa on the cell surface, as detected by fluorescence-activated cytometry. An excess of C2I(1-87) (but not of further truncated C2I fragments) competed with Alexa488-labeled C2I for binding to C2IIa. Also, the fragment C2I(30-431) and the fusion toxin C2I(30-225)-C3 competed with C2I-Alexa488 for binding to C2IIa. C2I(30-225)-C3 did not induce cytotoxic effects on cells when applied together with C2IIa, indicating that amino acid residues 1-29 are involved in translocation of C2I but are not absolutely essential for binding to C2IIa.
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, D-79104 Freiburg Germany. barthh@uni-freiburg.de
authors.forename: Holger
authors.initials: H
authors.lastname: Barth
authors.forename: Robert
authors.initials: R
authors.lastname: Roebling
authors.forename: Michaela
authors.initials: M
authors.lastname: Fritz
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity; D002469:Cell Separation; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014709:Vero Cells
chemical_list: D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D001905:Botulinum Toxins; C096324:botulinum toxin type C