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.
Highlights
Ras proteins, with their major isoforms H, K- and N-Ras, are molecular switches, which are regulated by cycling between an inactive GDP and an active GTPbound form [1,2,3]
We studied the GlcNAcylation catalyzed by the glucosyltransferase domain of TpeL in vitro with Ras loaded with GDP, GTPγS and GDP plus aluminium trifluoride (AlF3) to mimic the active and inactive states of Ras
TpeL causes GlcNAcylation of Ras proteins at threonine35 thereby inactivating these important switch proteins, which are involved in several fundamental cellular processes controlling proliferation, differentiation and survival [3]
Summary
With their major isoforms H-, K- and N-Ras, are molecular switches, which are regulated by cycling between an inactive GDP and an active GTPbound form [1,2,3]. They are controlled by guanine nucleotide exchange factors (GEFs; e.g. son of sevenless and others), which activate Ras proteins, and by GTPase activating proteins (GAPs, e.g., neurofibromin 1 and others), which terminate the activated state of Ras proteins [4]. Ras is a target of Clostridium perfringens toxin TpeL, which inactivates the switch protein by attachment of N-acetylglucosamine (GlcNAc) [8,9,10]
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