Glucosylceramide Synthase Activity Research Articles

Glycosylation of Ceramide Potentiates Cellular Resistance to Tumor Necrosis Factor-α-Induced Apoptosis

Ceramide, as a second messenger, initiates one of the major signal transduction pathways in tumor necrosis factor-α (TNF-α)-induced apoptosis. Glucosylceramide synthase (GCS) catalyzes glycosylation of ceramide and produces glucosylceramide. By introduction of the GCS gene, cytotoxic resistance to TNF-α has been conferred in human breast cancer cells. MCF-7/GCS-transfected cells expressed 4.1-fold higher levels of GCS activity and exhibited a 15-fold (P < 0.0005) greater EC50 for TNF-α, compared with the parental MCF-7 cell line. DNA fragmentation and DNA synthesis studies showed that TNF-α had little influence on the induction of apoptosis or on growth arrest in MCF-7/GCS cells, compared to MCF-7 cells. These studies reveal that TNF-α resistance in MCF-7/GCS cells is closely related to ceramide hyperglycosylation, a hallmark of this transfected cell line, and resistance was not aligned with changes in TNF receptor 1 expression. This work demonstrates that GCS, which catalyzes ceramide glycosylation, potentiates cytotoxic resistance to TNF-α.

Histidine-193 of rat glucosylceramide synthase resides in a UDP-glucose- and inhibitor (D-threo-1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol)-binding region: a biochemical and mutational study

Glucosylceramide synthase (GCS) catalyses the transfer of glucose from UDP-glucose (UDP-Glc) to ceramide to form glucosylceramide, the common precursor of most higher-order glycosphingolipids. Inhibition of GCS activity has been proposed as a possible target of chemotherapeutic agents for a number of diseases, including cancer. Design of new GCS inhibitors with desirable pharmaceutical properties is hampered by lack of knowledge of the secondary structure or catalytic mechanism of the GCS protein. Thus we cloned the rat homologue of GCS to begin studies to identify its catalytic regions. The histidine-modifying agent diethyl pyrocarbonate (DEPC) inhibited recombinant rat GCS expressed in bacteria; this inhibition was rapidly reversible by hydroxylamine and could be diminished by preincubation of GCS with UDP-Glc. These data suggest that DEPC acts on histidine residues within or near the UDP-Glc-binding site of GCS. Mutant proteins were expressed in which the eight histidine residues in GCS were individually replaced by other amino acids. H193A (His193 → Ala) and H193N (His193 → Asn) mutants were unaffected by 0.1 mM DEPC, a concentration that inhibited other histidine mutants and the wild-type enzyme by at least 60%. These results indicate that His193 is the primary target of DEPC and is at, or near, the UDP-Glc-binding site of GCS. His193 mutants were also insensitive to the GCS inhibitor D - threo - 1 - phenyl - 2 - decanoylamino - 3 - morpholinopropan - 1 - ol, at concentrations which inhibited the wild-type enzyme by > 80%. These results have significance for both an understanding of the GCS active site and also for the possible design of new and specific inhibitors of GCS.

Histidine-193 of rat glucosylceramide synthase resides in a UDP-glucose- and inhibitor (D-threo-1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol)-binding region: a biochemical and mutational study

Glucosylceramide synthase (GCS) catalyses the transfer of glucose from UDP-glucose (UDP-Glc) to ceramide to form glucosylceramide, the common precursor of most higher-order glycosphingolipids. Inhibition of GCS activity has been proposed as a possible target of chemotherapeutic agents for a number of diseases, including cancer. Design of new GCS inhibitors with desirable pharmaceutical properties is hampered by lack of knowledge of the secondary structure or catalytic mechanism of the GCS protein. Thus we cloned the rat homologue of GCS to begin studies to identify its catalytic regions. The histidine-modifying agent diethyl pyrocarbonate (DEPC) inhibited recombinant rat GCS expressed in bacteria; this inhibition was rapidly reversible by hydroxylamine and could be diminished by preincubation of GCS with UDP-Glc. These data suggest that DEPC acts on histidine residues within or near the UDP-Glc-binding site of GCS. Mutant proteins were expressed in which the eight histidine residues in GCS were individually replaced by other amino acids. H193A (His193-->Ala) and H193N (His193-->Asn) mutants were unaffected by 0.1 mM DEPC, a concentration that inhibited other histidine mutants and the wild-type enzyme by at least 60%. These results indicate that His193 is the primary target of DEPC and is at, or near, the UDP-Glc-binding site of GCS. His193 mutants were also insensitive to the GCS inhibitor d-threo-1-phenyl-2- decanoylamino-3-morpholinopropan-1-ol, at concentrations which inhibited the wild-type enzyme by >80%. These results have significance for both an understanding of the GCS active site and also for the possible design of new and specific inhibitors of GCS.

Enzyme distributions in subcellular fractions of BHK cells infected with Semliki Forest virus: evidence for a major fraction of sphingomyelin synthase in the trans-Golgi network

BHK cells either untreated or infected with Semliki Forest virus have been fractionated on sucrose density gradients. Virus infection caused an increase in density of a membrane fraction enriched in sphingomyelin (SM), cholesterol, SM synthase and sialyltransferase activity. This increase in density was related to incorporation of viral proteins into this fraction, which is likely to contain trans-Golgi network (TGN) membranes. In contrast, glucosylceramide synthase and galactosyltransferase activities (markers for cis/medial and trans-Golgi respectively) underwent no density shift and alkaline phosphodiesterase, a plasma membrane marker, was only slightly density-shifted in infected cells. When cells were incubated with NBD-ceramide to enable them to synthesise NBD-SM and then washed with albumin to remove surface label, fluorescence in untreated cells was concentrated in a single juxtanuclear spot but in infected cells this region of bright fluorescence was larger and extended around the nucleus. After fractionation of these cells, NBD-SM (but only a small proportion of the NBD-ceramide) was found to be shifted into the higher density fraction in infected cells. This work provides further evidence that SM synthase is not mainly localised in the early Golgi cisternae as previously thought, but is associated more with a cholesterol-rich compartment which could be the TGN.

Improved Inhibitors of Glucosylceramide Synthase

Previous work has led to the identification of inhibitors of glucosylceramide synthase, the enzyme catalyzing the first glycosylation step in the synthesis of glucosylceramide-based glycosphingolipids. These inhibitors have two identified sites of action: the inhibition of glucosylceramide synthase, resulting in the depletion of cellular glycosphingolipids, and the inhibition of 1-O-acylceramide synthase, resulting in the elevation of cell ceramide levels. A new series of glucosylceramide synthase inhibitors based on substitutions in the phenyl ring of a parent compound, 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), was made. For substitutions of single functional groups, the potency of these inhibitors in blocking glucosylceramide synthase was primarily dependent upon the hydrophobic and electronic properties of the substituents. An exponential relationship was found between the IC50 of each inhibitor and the sum of derived hydrophobic (pi) and electronic (sigma) parameters. This relationship demonstrated that substitutions that increased the electron-donating characteristics and decreased the lipophilic characteristics of the homologues enhanced the potency of these compounds in blocking glucosylceramide formation. A novel compound was subsequently designed and observed to be even more active in blocking glucosylceramide formation. This compound, D-threo-4'-hydroxy-P4, inhibited glucosylceramide synthase at an IC50 of 90 nM. In addition, a series of dioxane substitutions was designed and tested. These included 3',4'-methylenedioxyphenyl-, 3',4'-ethylenedioxyphenyl-, and 3'4'-trimethylenedioxyphenyl-substituted homologues. D-threo-3', 4'-Ethylenedioxy-P4-inhibited glucosylceramide synthase was comparably active to the p-hydroxy homologue. 4'-Hydroxy-P4 and ethylenedioxy-P4 blocked glucosylceramide synthase activity at concentrations that had little effect on 1-O-acylceramide synthase activity. These novel inhibitors resulted in the inhibition of glycosphingolipid synthesis in cultured cells at concentrations that did not significantly raise intracellular ceramide levels or inhibit cell growth.

Oligomerization and Topology of the Golgi Membrane Protein Glucosylceramide Synthase

Glucosylceramide synthase (GCS) catalyzes the transfer of glucose from UDP-glucose to ceramide to form glucosylceramide, the precursor of most higher order glycosphingolipids. Recently, we characterized GCS activity in highly enriched fractions from rat liver Golgi membranes (Paul, P., Kamisaka, Y., Marks, D. L., and Pagano, R. E. (1996) J. Biol. Chem. 271, 2287-2293), and human GCS was cloned by others (Ichikawa, S., Sakiyama, H., Suzuki, G., Hidari, K. I.-P. J., and Hirabayashi, Y. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4638-4643). However, the polypeptide responsible for GCS activity has never been identified or characterized. In this study, we made polyclonal antibodies against peptides based on the predicted amino acid sequence of human GCS and used these antibodies to characterize the GCS polypeptide in rat liver Golgi membranes. Western blotting of rat liver Golgi membranes, human cells, and recombinant rat GCS expressed in bacteria showed that GCS migrates as an approximately 38-kDa protein on SDS-polyacrylamide gels. Trypsinization and immunoprecipitation studies with Golgi membranes showed that both the C terminus and a hydrophilic loop near the N terminus of GCS are accessible from the cytosolic face of the Golgi membrane. Treatment of Golgi membranes with N-hydroxysuccinimide ester-based cross-linking reagents yielded an approximately 50-kDa polypeptide recognized by anti-GCS antibodies; however, treatment of approximately 10,000-fold purified Golgi GCS with the same reagents did not yield cross-linked GCS forms. These results suggest that GCS forms a dimer or oligomer with another protein in the Golgi membrane. The migration of solubilized Golgi GCS in glycerol gradients was also consistent with a predominantly oligomeric organization of GCS.

Expression of Glucosylceramide Synthase, Converting Ceramide to Glucosylceramide, Confers Adriamycin Resistance in Human Breast Cancer Cells

Multidrug-resistant cancer cells display elevated levels of glucosylceramide (Lavie, Y., Cao, H. T., Volner, A., Lucci, A., Han, T. Y., Geffen, V., Giuliano, A. E., and Cabot, M. C. (1997) J. Biol. Chem. 272, 1682-1687). In this study, we have introduced glucosylceramide synthase (GCS) into wild type MCF-7 breast cancer cells using a retroviral tetracycline-on expression system, and we developed a cell line, MCF-7/GCS. MCF-7/GCS cells expressed an 11-fold higher level of GCS activity compared with the parental cell line. Interestingly, the transfected cells demonstrated strong resistance to adriamycin and to ceramide, whereas both agents were highly cytotoxic to MCF-7 cells. The EC50 values of adriamycin and ceramide were 11-fold (p < 0.0005) and 5-fold (p < 0.005) higher, respectively, in MCF-7/GCS cells compared with MCF-7 cells. Ceramide resistance displayed by MCF-7/GCS cells closely paralleled the activity of expressed GCS with a correlation coefficient of 0.99. In turn, cellular resistance and GCS activity were dependent upon the concentration of the expression mediator doxycycline. Adriamycin resistance in MCF-7/GCS cells was related to the hyperglycosylation of ceramide and was not related to shifts in the levels of either P-glycoprotein or Bcl-2. This work demonstrates that overexpression of GCS, which catalyzes ceramide glycosylation, induces resistance to adriamycin and ceramide in MCF-7 breast cancer cells.

Tumor Necrosis Factor Induces Ceramide Oscillations and Negatively Controls Sphingolipid Synthases by Caspases in Apoptotic Kym-1 Cells

The role, origin, and mode of action of the lipid messenger ceramide in programmed cell death and its linkage to receptor-associated apoptotic signal proteins is still unresolved. We show here in Kym-1 rhabdomyosarcoma cells that tumor necrosis factor (TNF)-induced apoptosis is preceded by a multiphasic increase in intracellular ceramide levels. Distinct enzymes were found to contribute to three waves of ceramide, neutral sphingomyelinase, ceramide synthase, and acid sphingomyelinase, with peak activities at 1-2, 40, and around 200 min, respectively, the latter coinciding with progression to irreversible damage. In parallel with ceramide generation, TNF-mediated inhibition of glucosylceramide and sphingomyelin (SM) synthase prevents the immediate metabolization of this lipid mediator. In the presence of benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) or benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethyl ketone (Z-DEVD-cmk), a broad spectrum and a caspase 3-selective inhibitor, respectively, glucosylceramide and SM synthase activity remains unaffected by TNF, and intracellular ceramide accumulation is not observed. Our results show that several lipid enzymes contribute to generation of ceramide in response to TNF and identify glucosylceramide and SM synthase as important regulators of the kinetics and magnitude of intracellular ceramide accumulation. As glucosylceramide and SM synthase activity is caspase-sensitive, our data suggest a novel functional link between caspase(s) and ceramide during apoptotic processes.

Up-regulation of Glucosylceramide Synthase Expression and Activity during Human Keratinocyte Differentiation

During keratinocyte differentiation, the glycolipid, glucosylceramide (GlcCer), is thought to be synthesized, stored in intracellular lamellar granules and eventually extruded into the intercellular space where GlcCer is hydrolyzed to ceramide, a major component of the epidermal permeability barrier. Previous studies showed that GlcCer synthase (GCS) activity increases during keratinocyte differentiation; however, the mechanism by which GCS activity is regulated was not established. In the present study, we prepared anti-peptide antibodies and amplified cDNA probes based on the cDNA sequence for human GCS (Ichikawa, S., Sakiyama, H., Suzuki, G., Hidari, K. I.-P. J., and Hirabayashi, Y. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4638-4643) in order to study GCS expression during keratinocyte differentiation. Confluent human keratinocytes in culture were induced to terminally differentiate by elevation of Ca+2 in the medium without exogenous hormones or growth factors. GlcCer synthesis assayed in situ using a fluorescent ceramide analog increased approximately 5-fold during keratinocyte differentiation, peaking at day 6. Fluorescence microscopy studies of living keratinocytes showed that fluorescent ceramide and/or its metabolites accumulated in the Golgi in undifferentiated cells but targeted to unique vesicular structures that may be derived from the trans-Golgi region. Expression of both GCS mRNA, a approximately 3. 8-kilobase transcript on Northern blots, and GCS protein, a approximately 38-kDa polypeptide detected by Western blotting, increased dramatically (approximately 5-fold) during differentiation, reaching a maximum at about day 8. These results suggest that GCS is up-regulated at the transcriptional level during keratinocyte differentiation and provide the first direct evidence for GCS up-regulation in any cell type.

Glucosylceramide synthase activity in murine epidermis: quantitation, localization, regulation, and requirement for barrier homeostasis

Ceramides, which derive from the hydrolysis of glucosylceramide (GlcCer), are the predominant lipid species in the stratum corneum and are critical for epidermal permeability barrier homeostasis. UDP-glucose:ceramide glucosyltransferase (GlcCer synthase) (EC 2.4.1.80) catalyzes the glucosylation of ceramide to form GlcCer. Recently, we demonstrated a progressive increase in GlcCer synthase expression during fetal barrier development, while others have reported increased GlcCer synthase activity with differentiation of cultured human keratinocytes. To further delineate the role of GlcCer synthase in barrier homeostasis, we determined GlcCer synthase activity and localization in hairless mouse epidermis, both under basal conditions and after acute barrier perturbation. Under basal conditions, GlcCer synthase activity localizes predominantly (∼80%) to the dithiothreitol-separated outer epidermis; i.e., 6.2 ± 0.6 versus 1.2 ± 0.1 pmol/min/mg for outer vs. lower epidermis, respectively (P < 0.0001). Although acute barrier disruption does not up-regulate epidermal GlcCer synthase activity at any time point up to 24 h, GlcCer synthase is required for barrier homeostasis: topical d,1-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (P4), a specific GlcCer synthase inhibitor, applied immediately after acute barrier disruption, causes a delay in barrier recovery attributable to specific enzyme inhibition. These findings demonstrate first, that GlcCer synthase activity predominates in the outer epidermis, consistent with an increased formation of GlcCer during barrier ontogenesis and maintenance. Second, GlcCer synthase activity is required for normal permeability barrier homeostasis. Third, baseline epidermal GlcCer synthase activity appears to accommodate acute challenges to the barrier.—Chujor, C. S. N., K. R. Feingold, P. M. Elias, and W. M. Holleran. Glucosylceramide synthase activity in murine epidermis: quantitation, localization, regulation, and requirement for barrier homeostasis.

Purification and Characterization of UDP-glucose:Ceramide Glucosyltransferase from Rat Liver Golgi Membranes

We present a method for solubilizing and purifying UDP-Glc:ceramide glucosyltransferase (EC 2.4.1.80; glucosylceramide synthase (GCS) from a rat liver and present data on its substrate specificity. A Golgi membrane fraction was isolated, washed with N-lauroylsarcosine, and subsequently treated with 3[3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate to solubilize the enzyme. GCS activity was monitored throughout purification using UDP-Glc and a fluorescent ceramide analog as substrates. Purification of GCS was achieved via a two-step dye-agarose chromatography procedure using UDP-Glc to elute the enzyme. This resulted in an enrichment > 10,000-fold relative to the starting homogenate. The enzyme was further characterized by sedimentation on a glycerol gradient, I labeling, and SDS-polyacrylamide gel electrophoresis. which demonstrated that two polypeptides (60-70 kDa) corresponded closely with GCS activity. Purified GCS was found to require exogenous phospholipids for activity, and optimal results were obtained using dioleoyl phosphatidylcholine. Studies of the substrate specificity of the purified enzyme demonstrated that it was stereospecific and dependent on the nature and chain length of the N-acyl-spingosine or -sphinganine substrate. UDP-Glc was the preferred hexose donor, but TDP-glucose and CDP-glucose were also efficiently used. This study provides a basis for molecular characterization of this key enzyme in glycosphingolipid biosynthesis.

Role of lactosylceramide and MAP kinase in the proliferation of proximal tubular cells in human polycystic kidney disease.

Polycystic kidney disease (PKD) is a common genetic disease characterized by the proliferation of epithelial cells, formation of cysts, and the progression of renal deficiency. We have investigated a possible role of glycosphingolipids in the proliferation of human kidney cells in this disease. The levels of glucosylceramide and lactosylceramide and the activity of glucosylceramide synthase (GlcT-1) and lactosylceramide synthase (GalT-2) were elevated 2-fold and 3-fold, respectively, in the PKD tissue compared to control. Lactosylceramide, but not glucosylceramide (10 microM) derived from PKD exerted a 4-fold stimulation in the proliferation of these cells. However, at a concentration of 40 microM, lactosylceramide and glucosylceramide both stimulated cell proliferation on the order of 10-fold and 2.5-fold, respectively, as compared to control. This phenomenon may be due to the enrichment of lactosylceramide containing shorter chain fatty acids (C16:0-C18:0). Lactosylceramide, but not glucosylceramide exerted a time-dependent stimulation in the phosphorylation of mitogen-activated protein kinase (p44 MAPK) in normal human kidney proximal tubular cells. Moreover, the kidneys and cultured cells from the PKD patients contained higher levels of the p44 MAPK as compared to normal human kidneys. In sum, our studies indicate that lactosylceramide present in the PKD kidney may stimulate cell proliferation via activation of the p44 MAPK, and contribute to the pathophysiology in this disease.

Abnormalities of glycosphingolipid, sulfatide, and ceramide in the polycystic (cpk/cpk) mouse.

Polycystic kidney disease is a disorder marked by aberrant renal tubular epithelial cell proliferation and transport abnormalities. Sphingolipids are ubiquitous membrane components implicated in several cellular functions including cell membrane sorting, signaling, growth, ion transport, and adhesion. To investigate a potential pathogenic role for sphingolipids in cystic kidney disease, we studied the sphingolipid content and associated enzymatic activities of the kidneys from cpk/cpk mice and their phenotypically normal litter mates. The neutral glycolipids, including glucosylceramide and lactosylceramide, displayed a striking increase in 3-week-old cpk/cpk mice as did the acidic lipid, ganglioside GM3. However, a correspondingly significant decrease in sulfoglycolipid and ceramide concentration was observed in the cpk/cpk kidneys. Glucosylceramide synthase activity was higher in the kidneys of the cpk/cpk mice than in those of the controls. Kinetic analysis of the glucosylceramide synthase revealed the presence of an endogenous activator in the cystic kidney. A marked decrease in sulfotransferase activity was observed in both whole kidney homogenates and in microsomal preparations that was consistent with the decrement in sulfolipid content. The increase in GM3, glucosyl- and lactosylceramide may therefore be the result of impaired sulfolipid synthesis at the 3-week time point. While sulfolipid and glucosylceramide concentrations are not different at 1 and 2 weeks of age, ceramide concentrations in cystic kidneys are significantly reduced compared to kidneys from phenotypically normal mice. These results suggest that sphingolipids may play a potential role in the proliferative and transport abnormalities associated with cystic renal disease and the development of azotemia.

Metabolism of D-[3H]threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthesis, and the synergistic action of an inhibitor of microsomal monooxygenase

D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) is an effective inhibitor of the glucosyltransferase that makes glucosylceramide. Virtually all of the hundreds of naturally occurring glycolipids are formed from this primary glycolipid, so the inhibitor acts to lower their concentrations by the process of attrition (hydrolytic catabolism). Trials with mice carrying ascites carcinoma cells showed that PDMP could produce a permanent cure in some of the animals and marked prolongation of life in the others (Inokuchi, J., I. Mason, and N.S. Radin. 1987. Cancer Lett. 38: 23-30). In order to maximize the effect, we studied the metabolism of PDMP by labeling it with [3H] on carbon one, using a labeling method that discriminated against the unwanted erythro-isomer. The active enantiomer of the inhibitor (D-) was isolated by chromatography of the camphanate esters, followed by methanolytic cleavage. Examination of the fate of the labeled drug after a single injection showed that it was very rapidly converted to several polar products that were rapidly excreted. The drug penetrated all of the organs readily and a small portion was oxidized at the C-1 position to yield 3H2O. From these findings it appeared likely that the amine is attacked by a mixed function oxidase based on cytochrome P450. This conclusion was confirmed by showing that the tissue levels of PDMP could be greatly elevated, for a much longer time, when the mice were pretreated with piperonyl butoxide or cimetidine. The amount of conversion to polar metabolites was substantially reduced and tissue levels of PDMP were maintained much longer. Analysis of mice injected with one or both drugs showed that piperonyl butoxide augmented the effects of PDMP on ceramide, glucosylceramide, and dihexosylceramide levels, as well as on the activity of glucosylceramide synthase. It is suggested that piperonyl butoxide be used as an adjuvant for the many useful drugs that are inactivated by the P450 system.