Related Experiment Videos
Destruction of cholera toxin receptor on HeLa cell membrane using microbial endoglycoceramidase
K Yamamoto1, T Nagano, H Kumagai
1Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
Abstract:
The sensitivity of HeLa cells to cholera toxin decreased by Corynebacterium sp. endoglycoceramidase treatment. This endo-enzyme destroyed the cholera toxin receptor, ganglioside G(M1), on the cell surface membrane by liberating intact oligosaccharide from it, which was confirmed by the decrease of intracellular cAMP accumulation and the results of the analysis of released oligosaccharide with a combination of pyridylamination method and HPLC. Fluorescence microscopy using the immunofluorescence method revealed that the amount of cholera toxin attached to the cells decreased in endoglycoceramidase-treated cells. The enzyme acted on cellular glycosphingolipids without addition of any activator protein which is required by other similar enzymes. Corynebacterium endoglycoceramidase is a useful tool to elucidate the function of glycosphingolipids on the cell surface in situ.
Insights
Corynebacterium sp. endoglycoceramidase treatment reduced HeLa cell sensitivity to cholera toxin by degrading the ganglioside G(M1) receptor. This enzyme offers a novel tool for studying cell surface glycosphingolipid functions in situ.
Area of Science:
- Cell Biology
- Biochemistry
- Enzymology
Background:
- Cholera toxin binds to ganglioside G(M1) receptors on cell surfaces, triggering cellular responses.
- Understanding the role of cell surface glycosphingolipids in toxin interactions is crucial.
Purpose of the Study:
- To investigate the effect of Corynebacterium sp. endoglycoceramidase on cholera toxin sensitivity in HeLa cells.
- To explore the enzyme's mechanism in degrading ganglioside G(M1) and its implications for cell surface function.
Main Methods:
- Treatment of HeLa cells with Corynebacterium sp. endoglycoceramidase.
- Measurement of intracellular cAMP accumulation to assess cholera toxin activity.
- Analysis of released oligosaccharides using pyridylamination and High-Performance Liquid Chromatography (HPLC).
- Fluorescence microscopy with immunofluorescence to quantify cholera toxin binding.
Main Results:
- Endoglycoceramidase treatment significantly decreased HeLa cell sensitivity to cholera toxin.
- The enzyme degraded ganglioside G(M1) receptors on the cell surface.
- Reduced intracellular cAMP accumulation confirmed the loss of functional receptors.
- Fluorescence microscopy showed decreased cholera toxin attachment to treated cells.
- Corynebacterium endoglycoceramidase functions without requiring activator proteins.
Conclusions:
- Corynebacterium sp. endoglycoceramidase effectively degrades cell surface ganglioside G(M1), reducing cholera toxin binding and cellular response.
- This enzyme is a valuable tool for in situ studies of glycosphingolipid function in cell surface interactions.