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Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation
M R Popoff1, E Chaves-Olarte, E Lemichez
1Institut Pasteur, Unité des Toxines Microbiennes, 75724 Paris, Cedex 15, France.
Abstract:
Lethal toxin (LT) from Clostridium sordellii is one of the high molecular mass clostridial cytotoxins. On cultured cells, it causes a rounding of cell bodies and a disruption of actin stress fibers. We demonstrate that LT is a glucosyltransferase that uses UDP-Glc as a cofactor to covalently modify 21-kDa proteins both in vitro and in vivo. LT glucosylates Ras, Rap, and Rac. In Ras, threonine at position 35 was identified as the target amino acid glucosylated by LT. Other related members of the Ras GTPase superfamily, including RhoA, Cdc42, and Rab6, were not modified by LT. Incubation of serum-starved Swiss 3T3 cells with LT prevents the epidermal growth factor-induced phosphorylation of mitogen-activated protein kinases ERK1 and ERK2, indicating that the toxin blocks Ras function in vivo. We also demonstrate that LT acts inside the cell and that the glucosylation reaction is required to observe its dramatic effect on cell morphology. LT is thus a powerful tool to inhibit Ras function in vivo.
Insights
Clostridium sordellii lethal toxin (LT) glucosylates Ras, Rap, and Rac proteins, inhibiting Ras function. This toxin acts intracellularly, modifying key signaling proteins to alter cell morphology.
Area of Science:
- Microbiology
- Cell Biology
- Toxicology
Background:
- Clostridium sordellii lethal toxin (LT) is a high molecular mass cytotoxin.
- LT induces cell rounding and actin disruption in cultured cells.
- The precise molecular mechanism of LT's action was previously unclear.
Purpose of the Study:
- To elucidate the enzymatic activity and cellular targets of Clostridium sordellii lethal toxin (LT).
- To investigate the role of LT-mediated modification in cellular signaling pathways.
- To determine if LT can be utilized as a tool to inhibit specific cellular functions.
Main Methods:
- In vitro and in vivo biochemical assays to identify LT's enzymatic activity.
- Mass spectrometry to identify target proteins and specific amino acid modifications.
- Cell culture experiments using Swiss 3T3 cells treated with LT and epidermal growth factor (EGF).
- Analysis of mitogen-activated protein kinase (MAPK) phosphorylation (ERK1/ERK2).
Main Results:
- LT functions as a glucosyltransferase, utilizing UDP-Glc to modify 21-kDa proteins.
- LT specifically glucosylates Ras, Rap, and Rac proteins; Threonine 35 in Ras was identified as the target site.
- LT inhibits epidermal growth factor-induced phosphorylation of ERK1 and ERK2, demonstrating blockage of Ras function.
- LT's cytotoxic effects on cell morphology are dependent on its intracellular action and the glucosylation reaction.
Conclusions:
- Clostridium sordellii lethal toxin (LT) is an intracellular glucosyltransferase targeting Ras GTPase superfamily members.
- LT-mediated glucosylation of Ras proteins disrupts downstream signaling, including the MAPK pathway.
- LT serves as a valuable research tool for inhibiting Ras-dependent cellular processes.