December 2009

A Cell-based Glycosylation Assay

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Fluorescent (left panel) and visible light (right) imaging of NCI/ADR-RE tumor cells incubated with NBD C6-Cer, revealing the accumulation of sphingolipid in the cells.

Glucosylceramide synthase (GCS) catalyzes the first step of glycosphingolipid synthesis by adding glucose residues to ceramide (Cer) to produce glucosylceramide (GlcCer). As GlcCer is a core component of more than 300 different glycolipids, which have diverse roles in physiology and disease, GCS may be a potential biomarker and drug target. However, current enzyme assays typically involve in vitro reactions using prepared enzyme samples, which may not directly correlate with enzyme activity in a cell. In this study, the researchers introduced an approach to determine direct GCS cellular activity using fluorescent NBD C6-ceramide. They were able to separate C6-glucosylceramide from C6-ceramide in cellular extracts using thin-layer chromatography and then quantified the levels by spectrophotometer. This cell-based method is highly sensitive, being able to quantitate values from as little as 1 mg of tissue (~50,000 cells), and the researchers successfully evaluated GCS enzyme activity in multiple cell and tumor samples. The results suggest that this cell-based fluorescent approach would be a simple, reliable and direct means to evaluate GCS in cells and tissues for applications such as predicting drug resistance for cancer.  

Direct Quantitative Determination of Ceramide Glycosylation in Vivo: A New Approach to Evaluate Cellular Enzyme Activity of Glucosylceramide Synthase 

Vineet Gupta, Gauri A. Patwardhan, Qian-Jin Zhang, Myles C. Cabot, S. Michal Jazwinski and Yong-Yu Liu

J. Lipid Res., published online Oct. 13, 2009 


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