Targeting oncogenic Ras by the Clostridium perfringens toxin TpeL

Abstract

Clostridium perfringens toxin TpeL belongs to the family of large clostridial glycosylating toxins. The toxin causes N-acetylglucosaminylation of Ras proteins at threonine35 thereby inactivating the small GTPases. Here, we show that all main types of oncogenic Ras proteins (H-Ras, K-Ras and N-Ras) are modified by the toxin in vitro and in vivo. Toxin-catalyzed modification of Ras was accompanied by inhibition of the MAP kinase pathway. Importantly, TpeL inhibited the paradoxical activation of the MAP kinase pathway induced by the BRAF inhibitor Vemurafenib in the human melanoma cell line SBCL2. The toxin also blocked Ras signaling in a zebrafish embryo model expressing oncogenic H-RasG12V, resulting in a reduction of melanocyte number. By using the binding and translocation component of anthrax toxin (protective antigen), the glucosyltransferase domain of TpeL was effectively introduced into target cells that were not sensitive to native TpeL toxin. To reach a higher specificity towards cancer cells, a chimeric TpeL toxin was engineered that possessed the knob region of adenovirus serotype 35 fiber, which interacts with CD46 of target cells frequently overexpressed in cancer cells. The chimeric TpeL fusion toxin efficiently inhibited Ras and MAP kinases in human pancreatic cancer Capan-2 cells, which were insensitive to the wild-type toxin. The data reveal that TpeL and TpeL-related immunotoxins provide a new toolset as Ras-inactivating agents.

Homogeneous Dual-Parametric-Coupled Assay for Simultaneous Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring

“…In the assay with GMPPNP-KRAS, TpeL could not completely inhibit the nucleotide exchange with either one of the used UDP-sugars, but the level of glycosylation with UDP-GlcNAc was again higher. This observed GDP-KRAS-preferring behavior of TpeL is similar as reported with TpeL-related glycosylating toxin, the lethal toxin of Clostridium sordellii. , As TpeL KRAS glycosylation is reported as a KRAS/RAF-RBD interaction blocker and not for nucleotide exchange, QTR-FRET results from the nucleotide exchange were further confirmed with a conventional QRET nucleotide exchange assay. QRET assay results were in line with the ones from QTR-FRET and confirmed the enzyme preference to UDP-GlcNAc and GDP-KRAS (Figure S10, Table S1).…”

The Binary Toxin CDT of Clostridium difficile as a Tool for Intracellular Delivery of Bacterial Glucosyltransferase Domains

“…The binary toxin systems additionally bear the advantage of possible retargeting to cell-specific receptors by exchange of the receptor binding domain of the B subunits of toxins. Likewise, the LF/PA system from B. anthracis was used in combination with the pathogenic domain of TpeL to target oncogenic Ras [ 44 ]. These applications illustrate even a pharmacological potential of binary toxins.…”

Ras Isoforms from Lab Benches to Lives—What Are We Missing and How Far Are We?

“…Large cytotoxin TpeL from C. perfringens catalyzes N -acetylglucosamination of the same Thr35 residue in all main types of oncogenic Ras. Again, the toxin modification resulted in a loss of MapK activity and cell death [ 197 ]. Pseudomonas ExoS is an effector protein that is transferred into host cells via a type III secretion system.…”

“…Thus, this toxin might be employed to specifically delete Ras and kill tumor cells. Indeed, such an approach was later employed by Schorch et al, where the catalytically active domain of TpeL was coupled to the binding domain of the anthrax toxin binding and translocation component PA (protective antigen) [ 197 ]. To increase the specificity for tumor cells, a CD46 interaction domain was engineered into the binding region, as CD46 is often overexpressed on cancer cells.…”