Glycosphingolipid Synthesis Research Articles

Comprehensive characterization of sphingolipid ceramide N-deacylase for the synthesis and fatty acid remodeling of glycosphingolipids.

Sphingolipid ceramide N-deacylase (SCDase) catalyzes reversible reactions in which the amide linkage in glycosphingolipids is hydrolyzed or synthesized. While SCDases show great value for the enzymatic synthesis of glycosphingolipids, they are relatively poorly characterized enzymes. In this work, the enzymatic properties of SCDase from Shewanella alga G8 (SA_SCD) were systematically characterized and compared with the commercially available SCDase from Pseudomonas sp. TK4 (PS_SCD). The optimal pH values for the hydrolytic and synthetic activity of SA_SCD were pH 6.0 and pH 7.5, respectively. Both activities were strongly inhibited by Zn(2+) and Cu(2+), while Fe(2+), Co(2+), Ni(2+), Mn(2+), Ca(2+), and Mg(2+) promoted the hydrolytic activity but inhibited the synthetic activity. SA_SCD showed very broad substrate specificity both in hydrolysis and synthesis. Importantly, SA_SCD has a broader specificity for acyl donor acceptance than does PS_SCD, especially for unsaturated fatty acids and fatty acids with very short or long acyl chains. Further kinetic analysis revealed that the k cat/K M value for the hydrolytic activity of SA_SCD was 8.9-fold higher than that of PS_SCD for GM1a, while the values for the synthetic activity were 38-fold higher for stearic acid and 23-fold higher for lyso-GM1a (d18:1) than those of PS_SCD, respectively. The broad fatty acid specificity and high catalytic efficiency, together with the ease of expression of SA_SCD in Escherichia coli, make it a better biocatalyst than is PS_SCD for the synthesis and structural remodeling of glycosphingolipids.

Simplifying complexity: genetically resculpting glycosphingolipid synthesis pathways in mice to reveal function.

Glycosphingolipids (GSLs) are a group of plasma-membrane lipids notable for their extremely diverse glycan head groups. The metabolic pathways for GSLs, including the identity of the biosynthetic enzymes needed for synthesis of their glycans, are now well understood. Many of their cellular functions, which include plasma-membrane organization, regulation of cell signaling, endocytosis, and serving as binding sites for pathogens and endogenous receptors, have also been established. However, an understanding of their functions in vivo had been lagging. Studies employing genetic manipulations of the GSL synthesis pathways in mice have been used to systematically reduce the large numbers and complexity of GSL glycan structures, allowing the in vivo functions of GSLs to be revealed from analysis of the resulting phenotypes. Findings from these studies have produced a clearer picture of the role of GSLs in mammalian physiology, which is the topic of this review.

Human genetic disorders of sphingolipid biosynthesis.

Monogenic defects of sphingolipid biosynthesis have been recently identified in human patients. These enzyme deficiencies affect the synthesis of sphingolipid precursors, ceramides or complex glycosphingolipids. They are transmitted as autosomal recessive or dominant traits, and their resulting phenotypes often replicate the abnormalities seen in murine models deficient for the corresponding enzymes. In quite good agreement with the known critical roles of sphingolipids in cells from the nervous system and the epidermis, these genetic defects clinically manifest as neurological disorders, including paraplegia, epilepsy or peripheral neuropathies, or present with ichthyosis. The present review summarizes the genetic alterations, biochemical changes and clinical symptoms of this new group of inherited metabolic disorders. Hypotheses regarding the molecular pathophysiology and potential treatments of these diseases are also discussed.

The Effect of Heme Oxygenase on Ganglioside Redistribution Within Hepatocytes in Experimental Estrogen-Induced Cholestasis

Cholestasis is characterized by the elevation of serum total bile acids (TBA), which leads to the production of both free radicals and oxidative stress. Although they do not share the same mechanisms, membrane glycosphingolipids (GSL) and the antioxidant enzyme heme oxygenase-1 (HMOX1) both act against the pro-oxidative effect of TBA. The aim of the study was to assess the role of HMOX on GSL redistribution and composition within hepatocytes in the rat model of estrogen-induced cholestasis. Compared to the controls, an increase of total gangliosides in the liver homogenates of the cholestatic group (P=0.001) was detected; further, it paralleled along with the activation of their biosynthetic b-branch pathway (P<0.01). These effects were partially prevented by HMOX activation. Cholestasis was accompanied by a redistribution of GM1 ganglioside from the cytoplasm to the sinusoids; while HMOX activation led to the retention of GM1 in the cytoplasm (P=0.014). Our study shows that estrogen-induced cholestasis is followed by changes in the synthesis and/or distribution of GSL. These changes are not only triggered by the detergent power of accumulated TBA, but also by their pro-oxidant action. Increases in the antioxidant defenses might represent an important supportive therapeutic measure for patients with cholestatic liver disease.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Inhibition of Glycosphingolipid Synthesis Ameliorates Atherosclerosis and Arterial Stiffness in Apolipoprotein E −/− Mice and Rabbits Fed a High-Fat and -Cholesterol Diet

Glycosphingolipids, integral components of the cell membrane, have been shown to serve as messengers, transducing growth factor-initiated phenotypes. Here, we have examined whether inhibition of glycosphingolipid synthesis could ameliorate atherosclerosis and arterial stiffness in transgenic mice and rabbits. Apolipoprotein E(-/-) mice (12 weeks of age; n=6) were fed regular chow or a Western diet (1.25% cholesterol, 2% fat). Mice were fed 5 or 10 mg/kg of an inhibitor of glycosphingolipid synthesis, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), solubilized in vehicle (5% Tween-80 in PBS); the placebo group received vehicle only. At 20 and 36 weeks of age, serial echocardiography was performed to measure aortic intima-media thickening. Aortic pulse-wave velocity measured vascular stiffness. Feeding mice a Western diet markedly increased aortic pulse-wave velocity, intima-media thickening, oxidized low-density lipoprotein, Ca(2+) deposits, and glucosylceramide and lactosylceramide synthase activity. These were dose-dependently decreased by feeding D-PDMP. In liver, D-PDMP decreased cholesterol and triglyceride levels by raising the expression of SREBP2, low-density lipoprotein receptor, HMGCo-A reductase, and the cholesterol efflux genes (eg, ABCG5, ABCG8). D-PDMP affected very-low-density lipoprotein catabolism by increasing the gene expression for lipoprotein lipase and very-low-density lipoprotein receptor. Rabbits fed a Western diet for 90 days had extensive atherosclerosis accompanied by a 17.5-fold increase in total cholesterol levels and a 3-fold increase in lactosylceramide levels. This was completely prevented by feeding D-PDMP. Inhibition of glycosphingolipid synthesis ameliorates atherosclerosis and arterial stiffness in apolipoprotein E(-/-) mice and rabbits. Thus, inhibition of glycosphingolipid synthesis may be a novel approach to ameliorate atherosclerosis and arterial stiffness.

Inhibition of glycosphingolipid synthesis ameliorates atherosclerosis and arterial stiffness in Apo E‐/‐ mice and rabbits fed a high fat and cholesterol diet (607.11)

Objective ‐‐ Glycosphingolipids are integral components of the cell membrane and have been shown to serve as messengers, transducing growth factor initiated phenotypes. Here we have examined whether inhibition of glycosphingolipid synthesis could ameliorate atherosclerosis and arterial stiffness in transgenic mice and in rabbits.Methods and Results ‐‐ Apolipoprotein E‐/‐ (apoE‐/‐) mice (12 weeks of age, n = 6) were fed regular chow or a western diet (1.25% cholesterol, 2% fat). Mice were fed 5mg/kg (mpk) or 10 mpk of an inhibitor of glycosphingolipid synthesis inhibitor D‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (D‐PDMP) solubilized in vehicle (5% Tween‐80 in PBS) and the placebo group received vehicle only. At 20 weeks and 36 weeks of age, serial echocardiography was performed without sedation to measure aortic pulse wave velocity (PWV). Feeding mice a western diet markedly increased aortic PWV, aortic intima medial thickening (IMT), oxidized LDL, Ca2+ deposits, and glucosyl‐ and lactosylceramide synthase activity. These were dose‐dependently decreased by feeding D‐PDMP. In liver, D‐PDMP decreased cholesterol and triglyceride levels by raising the expression of SREBP‐1, LDL‐r, HMGCo‐A reductase, and cholesterol efflux genes (e.g., ABCG5, ABCG8). D‐PDMP affected VLDL catabolism by increasing the gene expression for LPL and VLDLr. Rabbits fed a western diet for 90 days had extensive atherosclerosis accompanied by a 17.5‐fold increase in total cholesterol levels and a 3‐fold increase in lactosylceramide levels. This was completely reversed by feeding D‐PDMP.Conclusions ‐‐ Inhibition of glycosphingolipid synthesis ameliorates atherosclerosis and arterial stiffness in ApoE‐/‐ mice and rabbits. Thus, inhibition of glycosphingolipid synthesis may be a novel approach to treating atherosclerosis and arterial stiffness.Grant Funding Source: Supported by P01HL107153

Miglustat: A Review of Its Use in Niemann-Pick Disease Type C

Miglustat (Zavesca®, Brazaves®), a small iminosugar molecule that reversibly inhibits glycosphingolipid synthesis, is the only disease-specific drug approved for the treatment of progressive neurological manifestations of Niemann-Pick disease type C (NP-C) in adult and paediatric patients. NP-C is a rare, autosomal-recessive lipid storage disorder characterized by impaired intracellular lipid trafficking and progressive neurological symptoms leading to premature death. In a randomized clinical trial, long-term extension studies and a retrospective observational cohort study, treatment with oral miglustat stabilized key neurological manifestations of NP-C (including horizontal saccadic eye movement peak velocity, ambulation, manipulation, language and swallowing) in paediatric and adult patients with the disease. The therapeutic effects of miglustat in stabilizing or slowing disease progression have been confirmed in other reports in the clinical experience setting. The primary tolerability issues associated with miglustat are mild to moderate gastrointestinal effects (e.g. diarrhoea, flatulence and abdominal pain/discomfort) and weight loss, which usually occur during initial therapy and are generally manageable. In the absence of a cure, miglustat is a valuable agent to reduce the progression of clinically relevant neurological symptoms in paediatric and adult patients with NP-C, which is considered a significant achievement in the treatment of this disease.

Correction: Glycolipid Transfer Protein Expression Is Affected by Glycosphingolipid Synthesis.

[This corrects the article DOI: 10.1371/journal.pone.0070283.].

A Glycosphingolipid Binding Domain Controls Trafficking and Activity of the Mammalian Notch Ligand Delta-Like 1

The activity of Notch ligands is tightly regulated by trafficking events occurring both before and after ligand-receptor interaction. In particular endocytosis and recycling have been shown to be required for full signaling activity of the ligands before they encounter the Notch receptor. However little is known about the precise endocytic processes that contribute to ligand internalization. Here we demonstrate that endocytosis contributes to Dll1 signaling activity by preserving the ligand from shedding and degradation. We further show that the glycosphingolipid-binding motif originally identified in Drosophila Notch ligands is conserved in mammals and is necessary for Dll1 internalization. Mutation of its conserved tryptophan residue results in a Dll1 molecule which is rapidly inactivated by shedding and degradation, does not recycle to the cell surface and does not activate Notch signaling. Finally, silencing in the signal-sending cells of glucosylceramide synthase, the enzyme implicated in the initial phase of glycosphingolipid synthesis, down-regulates Notch activation. Our data indicate that glycosphingolipids, by interacting with Dll1, may act as functional co-factors to promote its biological activity.

Glycolipid Transfer Protein Expression Is Affected by Glycosphingolipid Synthesis

Members of the glycolipid transfer protein superfamily (GLTP) are found from animals and fungi to plants and red micro-alga. Eukaryotes that encode the glucosylceramide synthase responsible for the synthesis of glucosylceramide, the precursor for most glycosphingolipids, also produce GLTPs. Cells that does not synthesize glucosylceramide neither express GLTPs. Based on this genetic relationship there must be a strong correlation between the synthesis of glucosylceramide and GLTPs. To regulate the levels of glycolipids we have used inhibitors of intracellular trafficking, glycosphingolipid synthesis and degradation, and small interfering RNA to down-regulate the activity of glucosylceramide synthase activity. We found that GLTP expression, both at the mRNA and protein levels, is elevated in cells that accumulate glucosylceramide. Monensin and brefeldin A block intracellular vesicular transport mechanisms. Brefeldin A treatment leads to accumulation of newly synthesized glucosylceramide, galactosylceramide and lactosylceramide in a fused endoplasmic reticulum-Golgi complex. On the other hand, inhibiting glycosphingolipid degradation with conduritol-B-epoxide, that generates glucosylceramide accumulation in the lysosomes, did not affect the levels of GLTP. However, glycosphingolipid synthesis inhibitors like PDMP, NB-DNJ and myriocin, all decreased glucosylceramide and GLTP below normal levels. We also found that an 80% loss of glucosylceramide due to glucosylceramide synthase knockdown resulted in a significant reduction in the expression of GLTP. We show here that interfering with membrane trafficking events and simple neutral glycosphingolipid synthesis will affect the expression of GLTP. We postulate that a change in the glucosylceramide balance causes a response in the GLTP expression, and put forward that GLTP might play a role in lipid directing and sensing of glucosylceramide at the ER-Golgi interface.

Use of a Glycolipid Inhibitor to Ameliorate Renal Cancer in a Mouse Model

In a xenograft model wherein, live renal cancer cells were implanted under the kidney capsule in mice, revealed a 30-fold increase in tumor volume over a period of 26 days and this was accompanied with a 32-fold increase in the level of lactosylceramide (LacCer). Mice fed D- threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of glucosylceramide synthase and lactosylceramide synthase (LCS: β-1,4-GalT-V), showed marked reduction in tumor volume. This was accompanied by a decrease in the mass of lactosylceramide and an increase in glucosylceramide (GlcCer) level. Mechanistic studies revealed that D-PDMP inhibited cell proliferation and angiogenesis by inhibiting p44MAPK, p-AKT-1 pathway and mammalian target for rapamycin (mTOR). By linking glycosphingolipid synthesis with tumor growth, renal cancer progression and regression can be evaluated. Thus inhibiting glycosphingolipid synthesis can be a bonafide target to prevent the progression of other types of cancer.

CPC-086 Miglustat Off-Label in a Paediatric Formulation For a Rare Metabolic Disease: Early Infantile GM1 Gangliosidosis

BackgroundGM1 gangliosidosis, a rare metabolic disease, is an autosomal recessive lysosomal storage disorder caused by a deficiency of beta-galactosidase, and characterised by the generalised accumulation of GM1 ganglioside. The most...

A small molecule inhibitor of Bcl-2, HA14-1, also inhibits ceramide glucosyltransferase

HA14-1 is a Bcl-2 inhibitor that is widely used for studies of apoptosis. In the course of searching for a ceramide glucosyltransferase inhibitor that catalyzes the first glycosylation step of glycosphingolipid synthesis, we unexpectedly found that HA-14-1 also has the ability to inhibit ceramide glucosyltransferase. The IC50 value of HA14-1 against ceramide glucosyltransferase is 4.5μM, which is lower than that reported for Bcl-2 in vitro. Kinetic analyses revealed that HA14-1 is a competitive and mixed-type inhibitor with respect to C6-NBD-ceramide and UDP-glucose, respectively.

Home infusions: One center's experience with ERT and self-infusion in the home setting

4-one) is an isoflavone which has been proposed as a potential drug for the use in substrate reduction therapy (SRT) in mucopolysaccharidoses and other lysosmal storage diseases (LSD). It was proposed that the effect of genistein to reduce the lysosomal storage is due to inhibition of phosphorylation of epidermal growth factor receptor, thus, impairing the signal transduction mechanism that normally leads to activation of expression of genes coding for enzymes necessary in biochemical reactions leading to production of glycosaminoglycans (GAG) and possibly other compounds. However, to learn about the exact mechanism(s) of action of genistein, we have performed transcriptomic analyses. Cultures of human dermal fibroblasts were treated with genistein and transcriptomic signatures were determined. Time points assessed for 0.03, 0.06 and 0.1 mM genistein included 1, 24 and 48 h. The microarray analysis was performedwith the use of HumanHT-12 Expression BeadChip. Significant differences in expression of between 259 and 631 genes, depending on genistein concentration and the time of treatment, were detected. Among the affected genes, 74 coded for proteins with known functions in lysosomal biogenesis and/or function. Of these proteins, nearly 50% are associated with human lysosomal diseases. Particularly, genistein regulated expression of some genes coding for enzymes required for synthesis or degradation of GAG and glycosphingolipids. Moreover, expression of many genes involved in secondary effects of LSD was found to be modulated by genistein. Therefore, it appears that the mechanism of genistein-mediated SRT is significantly more complicated than just reduction in GAG synthesis.

Property based designed inhibitors of glycosphingolipid synthesis lower ganglioside GM2 in the Sandhoff mouse

Property based designed inhibitors of glycosphingolipid synthesis lower ganglioside GM2 in the Sandhoff mouse

Programmed Death Machines All at Sea

Environmental Microbiology The interaction between viruses and phytoplankton blooms can influence marine carbon export. In culture, it has been established that giant double-stranded DNA coccolithoviruses invoke glycosphingolipid synthesis to trigger programmed cell death in neighboring uninfected

PS15 - 72. In vivo targeting of glycosphingolipid catabolism: will it work for obesity/type 2 diabetes?

Glycosphingolipids (GSL) are implicated to play an important role in insulin resistance. We have recently described (using several rodent models of type 2 diabetes) that lowering of GSL synthesis by pharmacological inhibition of glucosylceramide synthase with the iminosugar AMPDNM, improves insulin sensitivity in adipocytes, liver and muscle.

Quantitative Proteomic Analysis of Niemann-Pick Disease, Type C1 Cerebellum Identifies Protein Biomarkers and Provides Pathological Insight

Niemann-Pick disease, type C1 (NPC1) is a fatal, neurodegenerative disorder for which there is no definitive therapy. In NPC1, a pathological cascade including neuroinflammation, oxidative stress and neuronal apoptosis likely contribute to the clinical phenotype. While the genetic cause of NPC1 is known, we sought to gain a further understanding into the pathophysiology by identifying differentially expressed proteins in Npc1 mutant mouse cerebella. Using two-dimensional gel electrophoresis and mass spectrometry, 77 differentially expressed proteins were identified in Npc1 mutant mice cerebella compared to controls. These include proteins involved in glucose metabolism, detoxification/oxidative stress and Alzheimer disease-related proteins. Furthermore, members of the fatty acid binding protein family, including FABP3, FABP5 and FABP7, were found to have altered expression in the Npc1 mutant cerebellum relative to control. Translating our findings from the murine model to patients, we confirm altered expression of glutathione s-transferase α, superoxide dismutase, and FABP3 in cerebrospinal fluid of NPC1 patients relative to pediatric controls. A subset of NPC1 patients on miglustat, a glycosphingolipid synthesis inhibitor, showed significantly decreased levels of FABP3 compared to patients not on miglustat therapy. This study provides an initial report of dysregulated proteins in NPC1 which will assist with further investigation of NPC1 pathology and facilitate implementation of therapeutic trials.

Correction of Liver Steatosis by a Hydrophobic Iminosugar Modulating Glycosphingolipids Metabolism

The iminosugar N-(5′-adamantane-1′-yl-methoxy)-pentyl-1-deoxynoijirimycin (AMP-DNM), an inhibitor of glycosphingolipid (GSL) biosynthesis is known to ameliorate diabetes, insulin sensitivity and to prevent liver steatosis in ob/ob mice. Thus far the effect of GSL synthesis inhibition on pre-existing NASH has not yet been assessed. To investigate it, LDLR(−/−) mice were kept on a western-type diet for 12 weeks to induce NASH. Next, the diet was continued for 6 weeks in presence or not of AMP-DNM in the diet. AMP-DNM treated mice showed less liver steatosis, inflammation and fibrosis. Induction of fatty acid beta-oxydation was observed, as well as a reduction of plasma lipids. Our study demonstrates that AMP-DNM treatment is able to significantly correct pre-existing NASH, suggesting that inhibiting GSL synthesis may represent a novel strategy for the treatment of this pathology.