Inhibitor Of Glycosphingolipid Biosynthesis Research Articles

Treatment with miglustat reverses the lipid-trafficking defect in Niemann–Pick disease type C

Niemann–Pick disease type C (NP-C) is a hereditary neurovisceral lipid storage disorder. Although traditionally considered a primary cholesterol storage disorder, a variety of glycolipids accumulate in NP-C cells, which resemble those from glycosphingolipidosis patients. Substrate reduction therapy (SRT) with miglustat, an inhibitor of glycosphingolipid biosynthesis, is a novel therapy for the glycosphingolipidoses. We report the use of SRT in a patient with NP-C. We show that depletion of glycosphingolipids by miglustat treatment reduces pathological lipid storage, improves endosomal uptake and normalises lipid trafficking in peripheral blood B lymphocytes. The demonstration that treatment with miglustat, which has no direct effect on cholesterol metabolism, corrects the abnormal lipid trafficking seen in B lymphocytes in NP-C indicates that glycosphingolipid accumulation is the primary pathogenetic event in NP-C. These observations support the use of SRT in patients with this devastating neurodegenerative disease.

Pneumocystis carinii Cell Wall β-Glucan Induces Release of Macrophage Inflammatory Protein-2 from Alveolar Epithelial Cells via a Lactosylceramide-mediated Mechanism

Infiltration of the lungs with neutrophils promotes respiratory failure during severe Pneumocystis carinii (PC) pneumonia. Recent studies have shown that alveolar epithelial cells (AECs), in addition to promoting PC attachment, also participate in lung inflammation by the release of cytokines and chemokines. Herein, we demonstrate that a PC beta-glucan rich cell wall isolate (PCBG) stimulates the release of macrophage inflammatory protein-2 (MIP-2) from isolated AECs through a lactosylceramide-dependent mechanism. The results demonstrate that MIP-2 mRNA and protein production is significantly increased at both early and late time points after PCBG challenge. Although CD11b/CD18 (Mac-1, CR3) is the most widely studied beta-glucan receptor, we demonstrate that CD11b/CD18 is not present on AECs. This study instead demonstrates that preincubation of AECs with an antibody directed against the membrane glycosphingolipid lactosylceramide (CDw17) results in a significant decrease in MIP-2 secretion. Preincubation of the anti-CDw17 antibody with solubilized lactosylceramide reverses this effect. Furthermore, incubation of AECs with inhibitors of glycosphingolipid biosynthesis, including N-butyldeoxyno jirimycin and d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol-HCl, also results in a significant decrease in AEC MIP-2 production following challenge with PCBG. These data demonstrate that PC beta-glucan induces significant production of MIP-2 from AECs and that CDw17 participates in the glucan-induced inflammatory signaling in lung epithelial cells during PC infection.

Apoptosis of human carcinoma cells in the presence of inhibitors of glycosphingolipid biosynthesis: I. Treatment of Colo-205 and SKBR3 cells with isomers of PDMP and PPMP.

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells by anti-cancer drugs and biosynthetic inhibitors of cells surface glycolipids in the human colon carcinoma cells (Colo-205) are of interest in recent years. In our present studies, we have employed different stereoisomers of PPMP and PDMP (inhibit GlcT-glycosyltransferase (GlcT-GLT)) to initiate apoptosis in Colo-205 cells grown in culture in the presence of (3)H-TdR and (3)H/or (14)C-L-Serine. Our analysis showed that the above reagents (between 1 to 20 microM) initiated apoptosis with induction of Caspase-3 activities and phenotypic morphological changes in a dose-dependent manner. We have observed an increase of radioactive ceramide formation in the presence of a low concentration (1-4 microM) of these reagents in these cell lines. However, high concentrations (4-20 microM) inhibited incorporation of radioactive serine in the higher glycolipids. Colo-205 cells were treated with L-threo-PPMP (0-20 microM) and activities of different GSL: GLTs were estimated in total Golgi-pellets. The cells contained high activity of GalT-4 (UDP-Gal: LcOse3Cer beta 1-4galactosyltransferase), whereas negligible activity of GalT-3 (UDP-Gal: GM2 beta 1-3galactosyltransferase) or GM2-synthase activity of the ganglioside pathway was detected. Previously, GLTs involved in the biosynthetic pathway of SA-Le(x) formation had been detected in these colon carcinoma (or Colo-205) cells (Basu M et al. Glycobiology 1, 527-35 (1991)). However, during progression of apoptosis in Colo-205 cells with increasing concentrations of L-PPMP, the GalT-4 activity was decreased significantly. These changes in the specific activity of GalT-4 in the total Golgi-membranes could be the resultant of decreased gene expression of the enzyme.

Inhibitors of glycosphingolipid biosynthesis reduce transepithelial electrical resistance in MDCK I and FRT cells.

Madin-Darby canine kidney (MDCK) I and Fisher rat thyroid (FRT) cells exhibit transepithelial electrical resistance (TER) values in excess of 5,000 Omega. cm(2). When these cells were incubated in the presence of various inhibitors of sphingolipid biosynthesis, a >5-fold reduction of TER was observed without changes in the gate function for uncharged solutes or the fence function for apically applied fluorescent lipids. The localization of ZO-1 and occludin was not altered between control and inhibitor-treated cells, indicating that the tight junction was still intact. Furthermore, the complexity of tight junction strands, analyzed by freeze-fracture microscopy, was not reduced. Once the inhibitor was removed and the cells were allowed to synthesize sphingolipids, a gradual recovery of the TER was observed. Interestingly, these inhibitors did not attenuate the TER of MDCK II cells, a cell line that typically exhibits values below 800 omega x cm(2.) These results suggest that glycosphingolipids play a role in regulating the electrical properties of epithelial cells.

Glucosylceramide modulates membrane traffic along the endocytic pathway

Glycosphingolipids are endocytosed and targeted to the Golgi apparatus, but are mistargeted to lysosomes in numerous sphingolipidoses. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Gaucher disease is a hereditary deficiency in glucocerebrosidase leading to glucosylceramide accumulation; however, Gaucher fibroblasts exhibited normal Golgi transport of lactosylceramide. To better understand the effects of glycosphingolipid accumulation on intracellular trafficking and the use of imino sugar inhibitors, we studied sphingolipid endocytosis in fibroblast and macrophage models for Gaucher disease. Treatment of fibroblasts or RAW macrophages with conduritol B epoxide, an inhibitor of lysosomal glucocerebrosidase, resulted in a change in the endocytic targeting of lactosylceramide from the Golgi to the lysosomes. Co-treatment of macrophages with conduritol B-epoxide and 12-25 microM N-butyldeoxygalactonojirimycin, an inhibitor of glycosphingolipid biosynthesis, prevented the mistargeting of lactosylceramide to the lysosomes and restored trafficking to the Golgi. Surprisingly, higher doses (>25 microM) of NB-DGJ induced targeting of lactosylceramide to the lysosomes, even in the absence of conduritol B-epoxide. These data demonstrate that both increases and decreases in glucosylceramide levels can dramatically alter the endocytic targeting of lactosylceramide and suggest a role for glucosylceramide in regulation of membrane transport.

Differential effects of three inhibitors of glycosphingolipid biosynthesis on neuronal differentiation of embryonal carcinoma stem cells.

Gangliosides have been implicated in having important roles in neural development. It has been shown that disruption of ganglioside biosynthesis inhibits neurite outgrowth. However, many contradictory results have been reported. The inconsistency of these reports may result from the differential use of neuronal cell lines and inhibitors for ganglioside biosynthesis. In order to clarify the inconsistency in these studies, we utilized an in vitro neuronal differentiation model using an embryonic caricinoma (EC) stem cell line to elucidate the relationship between ganglioside expression and neural development. These cells were exposed to three different inhibitors of glucosylceramide synthase, the first enzyme committed for the biosynthesis of most of the brain gangliosides. All three inhibitors, D-threo-1-phenyl-2-decanoylamino-3-morphlino-1-propanol (D-PDMP), D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (D-PPPP), and N-butydeoxynojirimycin (NB-DNJ) can inhibit greater than 90% of ganglioside biosynthesis at certain concentrations, respectively. D-PDMP significantly slowed down cellular proliferation in undifferentiated P19 EC cells, inhibited neurite outgrowth, and eventually caused cell death in differentiated cells. However, no retardation in cell growth, neuronal differentiation, and neurite outgrowth was observed in cultures treated with D-PPPP or NB-DNJ despite the depletion of gangliosides. These results indicate that the effect of D-PDMP on cellular proliferation, neurite outgrowth, and survival of differentiated cells is independent of the inhibition of ganglioside biosynthesis.

3′-Azidothymidine Potently Inhibits the Biosynthesis of Highly Branched N-Linked Oligosaccharides and Poly-N-acetyllactosamine Chains in Cells

3′-Azidothymidine Potently Inhibits the Biosynthesis of Highly Branched N-Linked Oligosaccharides and Poly-N-acetyllactosamine Chains in Cells

3'-Azidothymidine potently inhibits the biosynthesis of highly branched N-linked oligosaccharides and poly-N-acetyllactosamine chains in cells.

Previous studies in our laboratory have characterized 3'-azido-3'-deoxythymidine (AZT) as a potent inhibitor of glycosphingolipid biosynthesis in cultured cells (Steet, R., Alizadeh, M., Melançon, P., and Kuchta, R. D. (1999) Glycoconj. J. 16, 237-245; Yan, J.-P., Ilsley, D. D., Frohlick, C., Steet, R., Hall, E. T., Kuchta, R. D., and Melançon, P. (1995) J. Biol. Chem. 270, 22836-22841). Here, we report that AZT treatment of K562 cells results in significant alterations in the profile of N-linked oligosaccharides. Fractionation of [(3)H]mannose-labeled oligosaccharides from AZT-treated K562 cells using lectin affinity chromatography revealed striking changes in the branching and processing of N-linked glycoconjugates. AZT treatment resulted in the production of fewer highly branched complex glycans (60% of control at 20 micrometer AZT) and a significant accumulation of core-fucosylated biantennary oligosaccharides. In addition, extension of branched oligosaccharides with multiple poly-N-acetyllactosamine repeats is nearly abolished by AZT concentrations as low as 2 micrometer. A shift from multiantennary to moderately branched oligosaccharides was also apparent in the melanoma cell line SK-MEL-30 upon AZT treatment. N-Linked glycans from both cell lines exhibited increased affinity for the beta-galactoside-binding lectin RCA-I in the presence of AZT, suggesting that the addition of terminal sialic acid is sensitive to the drug. These results demonstrate the ability of AZT to modulate strongly the processing of asparagine-linked glycoconjugates in whole cells and reveal a novel mechanism by which AZT treatment may cause anemia.

N-butyldeoxygalactonojirimycin: a more selective inhibitor of glycosphingolipid biosynthesis than N-butyldeoxynojirimycin, in vitro and in vivo

N-Butyldeoxynojirimycin ( NB-DNJ) inhibits the ceramide glucosyltransferase which catalyses the first step in glycosphingolipid (GSL) biosynthesis. It has the potential to be used for the treatment of the GSL lysosomal storage diseases and is currently in clinical trials for the treatment of type 1 Gaucher’s disease. However, NB-DNJ is also a potent inhibitor of other enzymes, including α-glucosidase I and II, which could potentially cause side effects in patients receiving life-long therapy. We therefore evaluated a potentially more selective GSL biosynthesis inhibitor, N-butyldeoxygalactonojirimycin ( NB-DGJ), in vitro and in vivo. The distribution and degree of GSL depletion in the liver of mice treated with NB-DGJ or NB-DNJ were equivalent. Mice treated with NB-DGJ had normal body weights and lymphoid organ sizes, whereas NB-DNJ-treated mice showed weight loss and partial lymphoid organ shrinkage. NB-DNJ inhibited glycogen catabolism in the liver, whereas NB-DGJ did not. NB-DNJ was also a potent inhibitor of sucrase and maltase in vitro but not of lactase, while NB-DGJ inhibited lactase but not sucrase or maltase. NB-DGJ is therefore more selective than NB-DNJ, and deserves to be evaluated for human therapy.

Delayed symptom onset and increased life expectancy in Sandhoff disease mice treated with N-butyldeoxynojirimycin.

Sandhoff disease is a neurodegenerative disorder resulting from the autosomal recessive inheritance of mutations in the HEXB gene, which encodes the beta-subunit of beta-hexosaminidase. GM2 ganglioside fails to be degraded and accumulates within lysosomes in cells of the periphery and the central nervous system (CNS). There are currently no therapies for the glycosphingolipid lysosomal storage diseases that involve CNS pathology, including the GM2 gangliosidoses. One strategy for treating this and related diseases is substrate deprivation. This would utilize an inhibitor of glycosphingolipid biosynthesis to balance synthesis with the impaired rate of catabolism, thus preventing storage. One such inhibitor is N-butyldeoxynojirimycin, which currently is in clinical trials for the potential treatment of type 1 Gaucher disease, a related disease that involves glycosphingolipid storage in peripheral tissues, but not in the CNS. In this study, we have evaluated whether this drug also could be applied to the treatment of diseases with CNS storage and pathology. We therefore have treated a mouse model of Sandhoff disease with the inhibitor N-butyldeoxynojirimycin. The treated mice have delayed symptom onset, reduced storage in the brain and peripheral tissues, and increased life expectancy. Substrate deprivation therefore offers a potentially general therapy for this family of lysosomal storage diseases, including those with CNS disease.