The Clostridium botulinum C2 Toxin Subunit C2IIa Delivers Enzymes with Positively Charged N-Termini into the Cytosol of Target Cells

Abstract

The binary Clostridium (C.) botulinum C2 toxin consists of two non-linked proteins. The proteolytically activated binding/transport subunit C2IIa forms barrel-shaped homoheptamers, which bind to cell surface receptors, mediate endocytosis, and translocate the enzyme subunit C2I into the cytosol of target cells. Here, we investigate whether C2IIa can be harnessed as a transporter for proteins/enzymes fused to polycationic tags, as earlier demonstrated for the related anthrax toxin transport subunit PA63. To test C2IIa-mediated transport in cultured cells, reporter enzymes are generated by fusing different polycationic tags to the N- or C-terminus of other bacterial toxins’ catalytic A subunits. C2IIa as well as PA63 deliver N-terminally polyhistidine-tagged proteins more efficiently compared to C-terminally tagged ones. However, in contrast to PA63, C2IIa does not efficiently deliver polylysine-tagged proteins into the cytosol of target cells. Moreover, untagged enzymes with a native cationic N-terminus are efficiently transported by both C2IIa and PA63. In conclusion, the C2IIa-transporter serves as a transport system for enzymes that harbor positively charged amino acids at their N-terminus. The charge distribution at the N-terminus of cargo proteins and their ability to unfold in the endosome and subsequently refold in the cytosol determine transport feasibility and efficiency.

Assessment of genetically modified maize MON 95275 (application GMFF‐2022‐5890)

“…lethal (LF) or oedema factors (EF) for PA and C2‐I subunit in the case of C2‐II, to cause cellular toxicity (Schleberger et al., 2006 ; Sherer et al., 2007 ). In agreement with this, several studies have shown that both PA and C2‐II proteins are non‐toxic in the absence of their respective binary partners (Collier & Young, 2003 ; Friedlander, 1986 ; Heber et al., 2023 ). Moreover, both PA and C2‐II proteins possess a conserved phenylalanine residue at position 425, which is a critical functional amino acid of the φ‐clamp to catalyse the translocation of their corresponding enzymatic components (i.e.…”

Some Examples of Bacterial Toxins as Tools

“…C2-I is an ADP ribosyltransferase that modifies actin leading to the breakdown of the actin cytoskeleton and cell rounding. Comparable to anthrax toxin, the N-terminal part of C2-I or a polycationic tag sufficiently mediates the binding and transport of cargo proteins through the C2-II pore [29]. Using this method, the C2 toxin also has been engineered to deliver proteins into mammalian cells [30].…”