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-C2I1–225-C3 was efficiently transported by C2IIa, indicating that C2IIa translocates proteins into the cytosol even when the C2I1–225 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 C2I1–87(but not of further truncated C2I fragments) competed with Alexa488-labeled C2I for binding to C2IIa. Also, the fragment C2I30–431 and the fusion toxin C2I30–225-C3 competed with C2I-Alexa488 for binding to C2IIa. C2I30–225-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.