Abstract
Glucosylceramide synthase (GCS) is a rate-limiting enzyme catalyzing ceramide glycosylation, thereby regulating cellular ceramide levels and the synthesis of glycosphingolipids (GSLs) in cellular membranes. Alterations of GCS not only affect membrane integrity, but also closely correlate with stem cell pluripotency, cancer drug resistance, GSL storage disorders and other diseases. Enzyme activities measured conventionally with currently available ex-vivo methods do not enable reliable assessment of the roles played by GCS in vivo. We report herein a substrate-incorporation method enabling rapid and efficient assessment of GCS in-vivo activity. Upon nanoparticle-based delivery, fluorescent NBD C6-ceramide was efficiently converted to NBD C6-glucosylceramide in live cells or in mouse tissues, whereupon an HPLC assay enabled detection and quantification of NBD C6-glucosylceramide in the low-femtomolar range. The enzyme kinetics of GCS in live cells and mouse liver were well-described by the Michaelis-Menten model. GCS activities were significantly higher in drug-resistant cancer cells and in tumors overexpressing GCS, but reduced after silencing GCS expression or inhibiting this enzyme. Our studies indicate that this rapid and efficient method provides a valuable means for accurately assessing the roles played by GCS in normal vs. pathological states, including ones involving cancer drug resistance.
Highlights
Quantitative assessment of Glucosylceramide synthase (GCS) activity is essential for evaluating the roles Cer glycosylation plays in cell functions, as well as in the therapeutic efficacies of relevant disease treatments
GCS converts NBD C6-Cer to NBD C6-glucosylceramide (C6-GlcCer), accompanying glycosylation of endogenous ceramide in the Golgi apparatus
Protein levels of GCS and Gb3S are represented as ratios of GCS/Glyceraldehyde3-phosphate dehydrogenase (GAPDH) optical densities averaged from three blots. *p < 0.001, compared to A2780 cells; **p < 0.001 compared to vehicle. (e) Intracellular GlcCer speciation by ESI/MS/MS
Summary
Quantitative assessment of GCS activity is essential for evaluating the roles Cer glycosylation plays in cell functions, as well as in the therapeutic efficacies of relevant disease treatments. After Basu’s work[1], several additional methods have been reported[2, 31,32,33] Besides those assays relying on the radioactivity of UDP-[3H]glucose[31, 34, 35] for detection, N-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoyl]-d-erythro-sphingosine (NBD C6-Cer) www.nature.com/scientificreports/. The HPLC method based on NBD C6-Cer has proven to be a highly sensitive and reproducible assay for assessing GCS activity in vitro with optimal conditions[2, 32]. With nanoparticle based delivery of NBD C6-Cer, we developed a rapid, efficient, and fully quantitative substrate incorporation HPLC analysis for assessing GCS in-vivo activity in live cells and in living mice
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