Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase

Abstract

Glucosylceramide synthase (GCS or GlcT-1), converting ceramide to glucosylceramide, is a key enzyme for the synthesis of glycosphingolipids. Due to its diverse roles in physiology and diseases, GCS may be a disease marker and drug target. Current assays for enzymes including GCS are based on reactions conducted in a test tube using enzyme preparations. Measurement of enzyme activity in laboratory-made conditions cannot directly evaluate the role of GCS in cells. Here, we introduce a new approach to determine GCS cellular activity using fluorescent NBD C6-ceramide in vivo. Cellular GCS transfers UDP-glucose to NBD C6-ceramide and produces NBD C6-glucosylceramide. C6-glucosylceramide is then separated from C6-ceramide by thin-layer chromatography and both are then quantitated by spectrophotometer. This cell-based method is able to quantitate glucosylceramide in pmol range, produced by approximately 50,000 cells or 1.0 mg tissue. This method has been used successfully to evaluate the degrees of GCS enzyme in cells and in tumors subjected to gene manipulation and chemical inhibition. These data indicate that this cell-based fluorescent method is direct, reproducible, and simple for assessing ceramide glycosylation. It is applicable to validate GCS activity in drug-resistant cancers and in other disorders.

Fig. 1.

Quantitation of NBD C6-Cer and C6-GlcCer after TLC separation. Increasing amounts of mixture of NBD C6-Cer and NBD C6-GlcCer (1:1, mol/mol) were resolved by TLC. The identified C6-Cer and C6-GlcCer were quantitated by fluorescent spectrophotometer. Each data point represents mean ± SD of three independent experiments. The correlation coefficient for Cer (dash line) is 0.991 ± 0.002, and for GlcCer (solid line) is 0.989 ± 0.002.

Fig. 2.

Determination of Cer glycosylation in cells. NCI/ADR-RES cells were grown in 10% FBS medium for 24 h and switched to 1% BSA RPMI-1640 medium containing NBD C6-Cer for enzymatic reaction. A: Fluorescent chromatogram and (B) spectrophotometry measurements of GlcCer after glycosylation with increasing concentrations of NBD C6-Cer for 2 h. The correlation coefficient for cellular C6-Cer (dashed line) to NBD C6-Cer in medium is 0.97, and for cellular C6-GlcCer (solid line) to NBD C6-Cer in medium is 0.99. C: Fluorescent chromatogram and (D) spectrophotometry measurement of time-dependent Cer glycosylation. NCI/ADR-RES cells were incubated with NBD C6-Cer (100 µM) in 500 µl medium for indicated periods. Results represent the mean ± SD of three independent enzyme reactions in triplicate.

Fig. 3.

Cer glycosylation in cell lines that express different degrees of GCS. Human GCS gene and its antisense sequence were introduced into NCI/ADR-RES cells (CTL, control) and established NCI/ADR-RES (GCS) and NCI/ADR-RES (asGCS) cell lines. A: Western blot. The detergent-soluble proteins (50 µg protein/lane) were resolved using 4–20% gradient SDS-PAGE. The transferred membranes were immunoblotted with GCS antiserum (1:1000) and detected by ECL. β-actin was used as endpoint control; GCS protein levels were represented by ratios of GCS/actin densities. * P > 0.01 compared with NCI/ADR-RES cells (CTL). B: Immunostaining. GCS protein was recognized by anti-GCS serum and Alexa 488 conjugated second antibody (green). DAPI nuclear-counter staining (blue) was used as endpoint control. C: Fluorescent chromatogram and (D) measurement of Cer glycosylation in GCS transfectants. Cells were grown overnight in 10% FBS medium then incubated with NBD C6-Cer (100 µM) in 1% BSA RPMI-1640 medium at 37°C for 2 h. Fluorescent C6-Cer and C6-GlcCer were identified by commercial standard (Std). * P < 0.001 compared with NCI/ADR-RES parental cells (CTL). Results are the mean ± SD of three independent experiments.

Fig. 4.

Comparison of Cer glycosylation in cells. A: GalCer generated in cellular glycosylation of NBD C6-Cer. After 4 h D-threo-PDMP treatments (D-PDMP, 2.5, 10 µM), NCI/ADR-RES cells were incubated with NBD C6-Cer (100 µM, 2 h). Sphingolipids were resolved on borate-impregnated HPTLC plates in solvent system of chloroform/methanol/water (100:30:4, v/v/v) and visualized under UV exposure (left) or with anisaldehyde (right). Std, standards of NBD C6-sphingolipids. B: Ratio of GlcCer/Cer versus GalCer/Cer in cells treated with D-threo-PDMP. The intensities of Cer, GlcCer, and GalCer were measured by using fluorescent spectrophotometer after TLC separation as described above. ** P < 0.001 as compared with vehicle treatment. C: GCS radioenzymatic assay. Proteins for enzyme reactions were extracted from NCI/ADR-RES (CTL, control), NCI/ADR-RES/GCS (GCS), and NCI/ADR/RES/asGCS (asGCS) cells. ** P < 0.01 compared with CTL). D: Endogenous total GlcCers and C18-GlcCer analyzed using LC/MS. Lipids were extracted from NCI/ADR-RES (CTL, control), NCI/ADR-RES/GCS (GCS), and NCI/ADR-RES/asGCS (asGCS) cells. * P < 0.01 compared with CTL). * P < 0.05, ** P < 0.01 compared with NCI/ADR-RES cells (CTL). Results are the mean ± SD of three independent experiments.

Fig. 5.

Quantitation of Cer glycosylation in tumors. A: Sphingolipid accumulation in tumor cells. NCI/ADR-RES tumors suspended were incubated with NBD C6-Cer (400 µM) and observed under microscope in fluorescent (left panel, green) and visible light (right panel). B: Chromatogram of Cer glycosylation in tumors suspended. The suspensions of NCI/ADR-RES tumors (200 µl in 0.3 mg/µl) were incubated with increasing concentrations of NBD C6-Cer for 120 min. C: Quantitation of Cer glycosylation in tumors suspended. Intensities of GlcCer and Cer described above were measured using spectrophotometer. Each data point represents mean ± SD of three independent enzyme reactions in triplicate. The correlation coefficient for the cellular C6-Cer (open circle) to NBD C6-Cer in medium is 0.96 and for cellular C6-GlcCer (open square) is 0.95.

Fig. 6.

GCS activity in tumors. A: GlcCer and GalCer generated in glycosylation of NBD C6-Cer. After D-threo-PDMP administration (intratumoral injection two times per day), NCI/ADR-RES tumors suspended were incubated with NBD C6-Cer (400 µM in 200 µl) for 2 h. Sphingolipids were subjected to borate-impregnated TLC separation and visualized under UV exposure (left) or with anisaldehyde (right). Std, standards of NBD C6-sphingolipids. B: Ratio of GlcCer/Cer versus GalCer/Cer after D-threo PDMP treatments and glycosylation described in A. C: GCS protein levels in tumors. Detergent-soluble proteins (50 µg protein/lane) of tumors were subjected to Western blot analysis. GAPDH was used as endpoint control. D: GCS activities in tumor tissues. Cer glycosylation was performed by incubating cell suspension with NBD C6-Cer (400 µM in 200 µl) at 37°C for 2 h. Suspensions were prepared from tumors (50 mg) of NCI/ADR-RES/GCS (GCS), NCI/ADR-RES (CTL, control), NCI/ADR-RES treated with MBO-asGCS (MBO-asGCS), SW620Ad (CTL), and SW620Ad treated with MBO-asGCS (MBO-asGCS) (3–5 mice per each group), respectively. Results are the mean ± SD of three independent experiments in triplicate. * P < 0.001 compared with NCI/ADR-RES or SW620Ad treated with medium (CTL), respectively.