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PubMed1991

Sphingolipid biosynthesis in cultured neurons. Down-regulation of serine palmitoyltransferase by sphingoid bases.

Addition of exogenous sphingosine homologues (D-erythro configuration) with different alkyl chain lengths (12 and 18 carbon atoms) to the medium of primary cultured cerebellar cells resulted in a decrease of serine palmitoyltransferase activity in a time- and concentration-dependent manner. This enzyme catalyzes the first committed step in sphingolipid biosynthesis. Half-maximal reduction of enzyme activity occurred after a 4-h treatment with 25 microM sphingoid bases. Maximal decrease (approx. 80%) was obtained after treating the cells for 4-8 h with 50 microM long-chain bases. When a biosynthetically inert sphingoid, azidosphingosine (10-50 microM), was fed to the cells, decrease of 3-ketosphinganine formation was much slower, reaching its maximum (approx. 80%) after 24 h. In contrast to D-erythro-sphingosine, L-threo-C18-sphingosine did not yield any decrease of serine palmitoyltransferase activity when added to the cells under identical experimental conditions. Decrease of serine palmitoyltransferase activity was fully reversible after removal of the long-chain bases from the culture medium. Activities of other enzymes of lipid metabolism, ceramide synthase, long-chain acyl-CoA synthase and choline phosphotransferase, were not affected by the addition of sphingoid bases, indicating that the down regulation of serine palmitoyltransferase is quite specific.

Acyltransferases↗
PubMed1994

Hydrolysis of lactosylceramide by human galactosylceramidase and GM1-beta-galactosidase in a detergent-free system and its stimulation by sphingolipid activator proteins, sap-B and sap-C. Activator proteins stimulate lactosylceramide hydrolysis.

Two exo-beta-galactosidases are involved in the lysosomal degradation of glycosphingolipids: GM1-beta-galactosidase (EC 3.2.1.23) and galactosylceramidase (EC 3.2.1.46). Analyses were performed with both enzymes, using lactosylceramides with varying acyl chain lengths as substrates that were inserted into unilamellar liposomes and naturally occurring sphingolipid activator proteins sap-B and sap-C, rather than detergents, to stimulate the reaction. While sap-B was a better activator for the reaction catalyzed by GM1-beta-galactosidase, sap-C preferentially stimulated lactosylceramide hydrolysis by galactosylceramidase. The enzymic hydrolysis of liposome-integrated lactosylceramides was significantly dependent on the structure of the lipophilic aglycon moiety of the lactosylceramide decreasing with increasing length of its fatty acyl chain (C2 > C4 > C6 > C8 > C10 > C18). However, in the presence of detergents the degradation rates were independent of the acyl chain length. Hydrolysis of liposomal lactosylceramide was compared with sap-B-stimulated hydrolysis of liposomal ganglioside GM1 by GM1-beta-galactosidase and sap-C-stimulated degradation of liposomal galactosylceramide by galactosylceramidase. Kinetic and dilution experiments indicated that sap-B forms water-soluble complexes with both lactosylceramide and GM1. These complexes were recognized by GM1-beta-galactosidase as optimal substrates in the same mode, as postulated for the hydrolysis of sulfatides by arylsulfatase A [Fischer, G. and Jatzkewitz, H. (1977) Biochim. Biophys. Acta 481, 561-572]. GM1-beta-galactosidase was more active on these complexes than on glycolipids (GM1 and lactosylceramides) still residing in liposomal membranes. On the other hand, dilution experiments indicated that degradation of galactosylceramide and lactosylceramide by galactosylceramidase proceeds almost exclusively on liposomal surfaces: both activators, sap-C and sap-B, stimulated the hydrolysis of lactosylceramide analogues with long acyl chains more than the hydrolysis of lactosylceramides with short acyl chains.

Antigens, CD↗
PubMed1996

Induction of programmed cell death and immunosuppression by exogenous sphingolipids are separate processes.

Gangliosides are highly immunosuppressive molecules but the mechanism(s) by which they act upon cells remains to be fully defined. Several metabolic products of exogenous gangliosides, including ceramide, have recently been suggested as second messengers in programmed cell death (PCD). Therefore, we have probed the role of gangliosides and ceramides in the induction of PCD and in the inhibition of in vitro lymphoproliferation. PCD was caused only by exogenous ceramides with short fatty acyl groups-d18:1-C2:0 (C2-ceramide, where d18:1 is sphingosine and C2:O is an acetyl group) and d18:1-C6:0 (C6-ceramide, where C6:O is a hexanoyl group). None of the gangliosides studied induced PCD, including naturally occurring GM3, synthetic d18:1-C18:0 GM3 (C18-Cer GM3, where C18:0 is a stearoyl group), or even d18:1-C2:0 GM3 (C2-Cer GM3), which itself contains a PCD-causing ceramide. However, these gangliosides were highly immunosuppressive, inhibiting antigen-induced lymphoproliferation at micromolar concentrations. We conclude that exogenous sphingolipids cause inhibition of lymphoproliferation and PCD by two separate and distinct mechanisms of action.

Apoptosis↗
PubMed1997

Human and murine serine-palmitoyl-CoA transferase--cloning, expression and characterization of the key enzyme in sphingolipid synthesis.

Serine palmitoyltransferase (SPT, EC 2.3.1.50) is the key enzyme in sphingolipid biosynthesis. It catalyzes the pyridoxal-5'-phosphate-dependent condensation of L-serine and palmitoyl-CoA to 3-oxosphinganine. Human expressed-sequence-tag (EST) clones are similar to the two yeast genes for synthesis of long-chain bases, LCB1 and LCB2, which are believed to encode two subunits of SPT [Buede, R., Pinto, W. J., Lester, R. L. & Dickson, R. C. (1991) J. Bacteriol. 173, 4325-5332; Nagiec, M. M., Baltisberger, J. A., Wells, G. B., Lester, R. L. & Dickson, R. C. (1994) Proc. Natl Acad. Sci. USA 91, 7899-7902]. We have cloned and characterized two complete human and murine cDNA sequences named hLCB1 & mLCB1 and hLCB2 & mLCB2, respectively, similar to the yeast LCB1 and LCB2 genes. Human embryonic kidney cells (HEK 293) transfected with murine sequences of LCB1 (mLCB1) and LCB2 (mLCB2) independently and in coexpression showed an overexpression of the transcripts on the mRNA and protein level. The enzymatic activity of cells expressing mLCB2 alone or coexpressed with mLCB1 was three times higher than the activity of untransfected HEK cells. mLCB1 expression was not required for the synthesis of 3-oxo-sphinganine in mammalian cells. Transcription/translation in vitro yielded mLCB1 (53 kDa) and mLCB2 (63 kDa). The two proteins do not contain a signal peptide nor are they glycosylated. The endogenous and overexpressed SPT activity were both sensitive to common SPT inhibitors. Labeling studies with [1-(14)C]palmitic acid indicated that cell lines transfected with mLCB2 preferentially use the excess sphingoid bases for glucocerebroside and galactocerebroside synthesis. Our results provide conclusive genetic and biochemical evidence that the human and murine LCB2 genes described here encode serine palmitoyltransferase. Further studies will be required to unravel the function of the LCB1 gene in mammalian cells.

Acyltransferases↗
PubMed2006

Cryptosporidium parvum infects human cholangiocytes via sphingolipid-enriched membrane microdomains.

Cryptosporidium parvum attaches to intestinal and biliary epithelial cells via specific molecules on host-cell surface membranes including Gal/GalNAc-associated glycoproteins. Subsequent cellular entry of this parasite depends on host-cell membrane alterations to form a parasitophorous vacuole via activation of phosphatidylinositol 3-kinase (PI-3K)/Cdc42-associated actin remodelling. How C. parvum hijacks these host-cell processes to facilitate its infection of target epithelia is unclear. Using specific probes to known components of sphingolipid-enriched membrane microdomains (SEMs), we detected aggregation of host-cell SEM components at infection sites during C. parvum infection of cultured human biliary epithelial cells (i.e. cholangiocytes). Activation and membrane translocation of acid-sphingomyelinase (ASM), an enzyme involved in SEM membrane aggregation, were also observed in infected cells. Pharmacological disruption of SEMs and knockdown of ASM via a specific small interfering RNA (siRNA) significantly decreased C. parvum attachment (by approximately 84%) and cellular invasion (by approximately 88%). Importantly, knockdown of ASM and disruption of SEMs significantly blocked C. parvum-induced accumulation of Gal/GalNAc-associated glycoproteins at infection sites by approximately 90%. Disruption of SEMs and knockdown of ASM also significantly blocked C. parvum-induced activation of host-cell PI-3K and subsequent accumulation of Cdc42 and actin by up to 75%. Our results suggest an important role of SEMs for C. parvum attachment to and entry of host cells, likely via clustering of membrane-binding molecules and facilitating of C. parvum-induced actin remodelling at infection sites through activation of the PI-3K/Cdc42 signalling pathway.

Actins↗
PubMed1984

Inhibition of sphingolipid synthesis by cycloserine in vitro and in vivo.

D- and L-cycloserine were shown to be irreversible inhibitors of the first enzyme of the sphingolipid pathway, 3-ketodihydrosphingosine synthetase, in a study using bacterial and brain microsomal enzymes. L-Cycloserine was shown to be 100 times more inhibitory than the D-isomer for the brain microsomal enzyme in vitro. In vivo, L-cycloserine caused a 70% inhibition of brain microsomal enzyme. Following one injection, enzyme activity recovered 80% of normal after 16 hours. Daily dosages of L-cycloserine on a regimen of intraperitoneal injection for 7 days caused a significant reduction in total brain ganglioside and cerebroside plus sulfatide levels.

Animals↗
PubMed1998

Use of BODIPY-labeled sphingolipids to study membrane traffic along the endocytic pathway.

In this chapter we discuss the use of BODIPY-labeled sphingolipids to study lipid transport along the endocytic pathway of cultured mammalian cells. The unique spectral properties of the BODIPY fluorophore allow the investigator to distinguish various populations of labeled endosome and lysosomes within the living cell by fluorescence microscopy, and in conjunction with quantitative fluorescence microscopy, to estimate the concentration of these lipids in different intracellular compartments. This methodology is particularly useful for visualizing the accumulation of lipids in the lysosomes of storage disease fibroblasts and may provide a useful method for screening various agents that abrogate this accumulation.

Animals↗
PubMed2000

Sphingolipid regulation of female gonadal cell apoptosis.

Ceramide and sphingosine-1-phosphate (S1P) are sphingosine-based lipid signaling molecules that have been implicated as key mediators of cellular growth, differentiation, and apoptosis. The cellular response depends on cell type, on the absence or presence of other signals initiated by the same or another stimulus, and on the subcellular location of sphingomyelin hydrolysis leading to ceramide generation. Consistent with mounting evidence implicating components of the sphingomyelin pathway as mediators of cellular life and death in nonreproductive tissues, recent data have indicated that sphingolipid-based signaling events are also prominent features of cellular development and apoptosis in the fetal and postnatal female gonads. This area of investigation represents a new research avenue of considerable significance for both basic biology and clinical medicine because of the massive levels of developmental death that occur normally in the female germ line, especially during gametogenesis, as well as of the central role of oocyte apoptosis in female gonadal failure resulting from pathologic insults.

Apoptosis↗
PubMed1988

Coding of two sphingolipid activator proteins (SAP-1 and SAP-2) by same genetic locus.

Several complementary DNAs (cDNAs) coding for sphingolipid activator protein-2 (SAP-2) were isolated from a lambda gt-11 human hepatoma library by means of polyclonal antibodies. The nucleotide sequence of the largest cDNA was colinear with the derived amino acid sequence of SAP-2 and with the nucleotide sequence of the cDNA coding for the 70-kilodalton precursor of SAP-1 (SAP precursor cDNA). The coding sequence for mature SAP-2 was located 3' to that coding for SAP-1 in the SAP precursor cDNA. Both SAP-1 and SAP-2 appeared to be derived by proteolytic processing from a common precursor that is coded by a genetic locus on human chromosome 10. Two other domains similar to SAP-1 and SAP-2 were also identified in SAP precursor protein. Each of the four domains was approximately 80 amino acid residues long, had nearly identical placement of cysteine residues, potential glycosylation sites, and proline residues. Each domain also contained internal amino acid sequences capable of forming amphipathic helices separated by helix breakers to give a cylindrical hydrophobic domain that is probably stabilized by disulfide bridges. Protein immunoblotting experiments indicated that SAP precursor protein (70 kilodaltons) as well as immunoreactive SAP-like proteins of intermediate sizes (65, 50, and 31 kilodaltons) are present in most human tissues.

Amino Acid Sequence↗
PubMed1999

Analysis of phosphorylated sphingolipid long-chain bases reveals potential roles in heat stress and growth control in Saccharomyces.

Sphingolipid long-chain bases and their phosphorylated derivatives, for example, sphingosine-1-phosphate in mammals, have been implicated as signaling molecules. The possibility that Saccharomyces cerevisiae cells also use long-chain-base phosphates to regulate cellular processes has only recently begun to be examined. Here we present a simple and sensitive procedure for analyzing and quantifying long-chain-base phosphates in S. cerevisiae cells. Our data show for the first time that phytosphingosine-1-phosphate (PHS-1-P) is present at a low but detectable level in cells grown on a fermentable carbon source at 25 degreesC, while dihydrosphingosine-1-phosphate (DHS-1-P) is only barely detectable. Shifting cells to 37 degreesC causes transient eight- and fivefold increases in levels of PHS-1-P and DHS-1-P, respectively, which peak after about 10 min. The amounts of both compounds return to the unstressed levels by 20 min after the temperature shift. These data are consistent with PHS-1-P and DHS-1-P being signaling molecules. Cells unable to break down long-chain-base phosphates, due to deletion of DPL1 and LCB3, show a 500-fold increase in PHS-1-P and DHS-1-P levels, grow slowly, and survive a 44 degreesC heat stress 10-fold better than parental cells. These and other data for dpl1 or lcb3 single-mutant strains suggest that DHS-1-P and/or PHS-1-P act as signals for resistance to heat stress. Our procedure should expedite experiments to determine how the synthesis and breakdown of these compounds is regulated and how the compounds mediate resistance to elevated temperature.

Heat Stress Disorders↗
PubMed2006

Sphingolipids in tumor metastases and angiogenesis.

This review article summarizes data on the involvement of sphingolipids (sphingosine-1-phosphate, sphingosine-1-phosphocholine, neutral glycosphingolipids, and gangliosides) in tumor metastases and angiogenesis.

Animals↗
PubMed1996

"Cross talk" between the bioactive glycerolipids and sphingolipids in signal transduction.

Hydrolysis of phosphatidylcholine via receptor-mediated stimulation of phospholipase D produces phosphatidate that can be converted to lysophosphatidate and diacylglycerol. Diacylglycerol is an activator of protein kinase C, whereas phosphatidate and lysophosphatidate stimulate tyrosine kinases and activate the Ras-Raf-mitogen-activated protein kinase pathway. These three lipids can stimulate cell division. Conversely, activation of sphingomyelinase by agonists (e.g., tumor necrosis factor-alpha) causes ceramide production that inhibits cell division and produces apoptosis. If ceramides are metabolized to sphingosine and sphingosine 1-phosphate, then these lipids can stimulate phospholipase D and are also mitogenic. By contrast, ceramides inhibit the activation of phospholipase D by decreasing its interaction with the G-proteins, ARF and Rho, which are necessary for its activation. In whole cells, ceramides also stimulate the degradation of phosphatidate, lysophosphatidate, ceramide 1-phosphate, and sphingosine 1-phosphate through a multifunctional phosphohydrolase (the Mg(2+)-independent phosphatidate phosphohydrolase), whereas sphingosine inhibits phosphatidate phosphohydrolase. Tumor necrosis factor-alpha causes insulin resistance, which may be partly explained by ceramide production. Cell-permeable ceramides decrease insulin-stimulated glucose uptake in 3T3-L1 adipocytes after 2-24 h, whereas they stimulate basal glucose uptake. These effects do not depend on decreased tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 or the interaction of insulin receptor substrate-1 with phosphatidylinositol 3-kinase. They appear to rely on the differential effects of ceramides on the translocation of GLUT1-and GLUT4-containing vesicles. It is concluded that there is a significant interaction and "cross-talk" between the sphingolipid and glycerolipid pathways that modifies signal transduction to control vesicle movement, cell division, and cell death.

Animals↗
PubMed2006

Potential new antimalarial chemotherapeutics based on sphingolipid metabolism.

The discovery of new antimalarial drugs is mandatory to improve the effectiveness of antimalarial prophylaxis and treatment. In this review, we focused on sphingolipids as potential new targets for antimalarial drugs. Inhibition of sphingomyelin and/or glucosylceramide synthases leads to increased intracellular concentrations of ceramide and results in growth inhibition of Plasmodium falciparum. In mammalian cells, ceramide mediates death by chemotherapeutic drugs. We demonstrated that ceramide mediates the antimalarial effect of artemisinin and mefloquine by depletion of glutathione levels. Furthermore, ceramide and artemisinin activated p38 mitogen-activated protein kinase in P. falciparum, thus inhibiting its growth, apparently by a non-apoptotic mechanism. In summary, we propose novel options of antimalarials based on ceramide cytotoxic activity.

Animals↗
PubMed1996

Tumor necrosis factor alpha-induced apoptosis in cardiac myocytes. Involvement of the sphingolipid signaling cascade in cardiac cell death.

In the present study, it was shown that physiologically relevant levels of the proinflammatory cytokine TNFalpha induced apoptosis in rat cardiomyocytes in vitro, as quantified by single cell microgel electrophoresis of nuclei ("cardiac comets") as well as by morphological and biochemical criteria. It was also shown that TNFalpha stimulated production of the endogenous second messenger, sphingosine, suggesting sphingolipid involvement in TNFalpha-mediated cardiomyocyte apoptosis. Consistent with this hypothesis, sphingosine strongly induced cardiomyocyte apoptosis. The ability of the appropriate stimulus to drive cardiomyocytes into apoptosis indicated that these cells were primed for apoptosis and were susceptible to clinically relevant apoptotic triggers, such as TNFalpha. These findings suggest that the elevated TNFalpha levels seen in a variety of clinical conditions, including sepsis and ischemic myocardial disorders, may contribute to TNFalpha-induced cardiac cell death. Cardiomyocyte apoptosis is also discussed in terms of its potential beneficial role in limiting the area of cardiac cell involvement as a consequence of myocardial infarction, viral infection, and primary cardiac tumors.

Analysis of Variance↗
PubMedDate not supplied

Sphingolipid microdomains mediate CD38 internalization: topography of the endocytosis.

Plasma membranes of several cell types contain specialized microdomains (or lipid rafts) enriched in sphingolipids, cholesterol, sphingomyelin, and glycosyl-phosphatidylinositol-anchored proteins. These membrane domains are characterized by detergent insolubility at low temperatures and low buoyant density. Human CD38 is the prototype of a gene family encoding surface molecules endowed with multiple functional activities. The endocytosis of the human CD38 molecule has been investigated in normal lymphocytes and in a number of leukemia- and lymphoma-derived cell lines demonstrating that internalization after CD38 ligation is a reproducible event involving only a fraction of the whole amount of the surface molecule. This study reports the results obtained by conventional, confocal, and electron microscopy on the effects induced by the engagement of the molecule with agonistic mAb, reproducing the signals mediated by its natural ligand. The results demonstrate that the endocytosis induced as consequence of CD38 ligation is preceded by a thorough rearrangement of the cell surface with formation of glycosphingolipid- and cholesterol-rich plasma membrane microdomains. These data suggest that specialized raft microdomains might be the plasma membrane structure through which CD38 translocates at intracellular level. The CD38/lipid interactions during the coated pit formation trigger a process that generate membrane curvature, considered as the first step of CD38 endocytosis. Moreover, ultrastructural studies show that early CD38(+) endosomes are pleiomorphic and contain cisternal and vesicular regions. Late endosomes exhibit a complex organisation, containing uncoupled CD38-ligand multivesicular- or multilamellar-regions.

ADP-ribosyl Cyclase↗
PubMed2005

LASS5 is the predominant ceramide synthase isoform involved in de novo sphingolipid synthesis in lung epithelia.

Ceramide is a key bioactive mediator that inhibits surfactant phosphatidylcholine (PtdCho) synthesis in lung epithelia. Ceramide availability is governed by sphingomyelin (SM) hydrolysis, but less is known regarding its de novo synthesis. In this study, we observed that ceramide synthesis within murine lung epithelia was associated with high-level ceramide synthase (dihydroceramide synthase) activity. Longevity assurance homolog 5 (LASS5) was the predominant ceramide synthase isoform detected in lung epithelia, whereas relatively lower level expression was detected for the other five mammalian homologs. Pulmonary LASS5 was developmentally regulated, but its expression was spatially and gender nonspecific. Exogenously expressed LASS5 in lung epithelia was membrane-associated, triggering increased ceramide synthesis, whereas knockdown studies using fumonisin B1 or LASS5 small, interfering RNA reduced ceramide synthase activity by 78% or 45%, respectively. Overexpression of LASS5 also reduced PtdCho synthesis, but maximal inhibition was achieved when LASS5 was coexpressed with a plasmid encoding a neutral sphingomyelinase involved in SM hydrolysis. These results demonstrate that LASS5 is the major ceramide synthase gene product involved in sphingolipid production that may also regulate PtdCho metabolism in pulmonary epithelia.

Animals↗
PubMed2004

Dysregulation of sphingolipid and sterol metabolism by ApoE4 in HIV dementia.

BACKGROUND: Polymorphisms in apolipoprotein E have been associated with worse prognoses in numerous neurodegenerative conditions, including HIV dementia (HIVD). Despite these correlative observations, there has been little evidence suggesting a mechanism whereby the expression of ApoE4 renders neurons susceptible to insult. METHODS: Electrospray ionization tandem mass spectrometry was used to quantify levels of sphingolipids and sterols in brains of HIVD patients. Data were stratified according to APOE genotype. RESULTS: The authors found evidence of dysregulated lipid and sterol metabolism in HIVD patients with an APOE4 genotype. They also found elevations of sphingomyelin, ceramide, and cholesterol in the medial frontal cortex, parietal cortex, and cerebellum of HIVD patients with an APOE3/4 or APOE4/4 genotype compared with HIVD patients with an APOE3/3 genotype. There was no difference in the number of astrocytes or activated microglia in any brain region of the two patient populations, suggesting that modification of lipid metabolism in HIVD patients with an APOE4 genotype was not the result of increased CNS inflammation. CONCLUSIONS: HIV dementia patients with an APOE4 genotype may be sensitized to neural insults because of dysregulations in lipid metabolism.

AIDS Dementia Complex↗
PubMed1994

Oral administration of semisynthetic sphingolipids promotes recovery of striatal dopamine concentrations in a murine model of parkinsonism.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administered to young C57/B16J black mice caused a striatal dopamine depletion that could be at least partially reversed by chronic intraperitoneal administration of GM1 ganglioside. The present study shows that the semisynthetic sphingolipids LIGA 4 (II3Neu-5-AcGgOse4-2-d-erythro-1,3-dihydroxy-2-acetamide-4-t rans-octadacene) and LIGA 20 (II3Neu-5-AcGgOse4-2-d-erythro-1,3-dihydroxy-2-chloro-acetam ide-4- trans-octadacene) are also effective in at least partially reversing MPTP-induced striatal dopamine depletions in mice after oral administration. These results suggest that semisynthetic ganglioside derivatives may be superior to the parent GM1 ganglioside for human clinical use.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗