Cloning of an alkaline ceramidase from Saccharomyces cerevisiae. An enzyme with reverse (CoA-independent) ceramide synthase activity

Ceramide is not only a core intermediate of sphingolipids but also an important modulator of many cellular events including apoptosis, cell cycle arrest, senescence, differentiation, and stress responses. Its turnover may be tightly regulated. However, little is known about the regulation of its metabolism because most enzymes responsible for its synthesis and breakdown have yet to be cloned. Here we report the cloning and characterization of the yeast gene YPC1 (YBR183w) by screening Saccharomyces cerevisiae genes whose overexpression bestows resistance to fumonisin B1. We demonstrate that the yeast gene YPC1 encodes an alkaline ceramidase activity responsible for the breakdown of dihydroceramide and phytoceramide but not unsaturated ceramide. YPC1 ceramidase activity was confirmed by in vitro studies using an Escherichia coli expression system. Importantly, YPC1p also has reverse activity, catalyzing synthesis of phytoceramide from palmitic acid and phytosphingosine. This ceramide synthase activity is CoA-independent and is resistant to fumonisin B1, thus explaining why YPC1 was cloned as a fumonisin B1-resistant gene.     

Cloning and characterization of a novel human alkaline ceramidase. A mammalian enzyme that hydrolyzes phytoceramide

Ceramidases are enzymes involved in regulating cellular levels of ceramides, sphingoid bases, and their phosphates. Based on sequence homology to the yeast alkaline ceramidases YPC1p (Mao, C., Xu, R., Bielawska, A., and Obeid, L. M. (2000) J. Biol. Chem. 275, 6876--6884) and YDC1p (Mao, C., Xu, R., Bielawska, A., Szulc, Z. M., and Obeid, L. M. (2000) J. Biol Chem. 275, 31369--31378), we report the identification and cloning of a cDNA encoding for a novel human alkaline ceramidase (aPHC) that hydrolyzes phytoceramide selectively. Northern blot analysis showed that aPHC was ubiquitously expressed, with the highest expression in placenta. Green fluorescent protein tagging showed that it was localized in both the Golgi apparatus and endoplasmic reticulum. Overexpression of aPHC in mammalian cells elevated in vitro ceramidase activity toward N-4-nitrobenz-2-oxa-1,3-diazole-C(12)-phytoceramide. Its expression in a yeast mutant strain devoid of any ceramidase activity restored the ceramidase activity and caused an increase in the hydrolysis of phytoceramide in yeast cells, thus leading to the decreased biosynthesis of sphingolipids. These data collectively suggest that, similar to the yeast phytoceramidase YPC1p, aPHC has phytoceramidase activity both in vitro and in cells; hence, it is a functional homolog of the yeast phytoceramidase YPC1p. However, in contrast to YPC1p, aPHC exhibited no reverse activity of ceramidase either in vitro or in cells. Biochemical characterization showed that aPHC had a pH optimum of 9.5, was activated by Ca(2+), but was inhibited by Zn(2+) and sphingosine. Substrate specificity showed that aPHC hydrolyzed phytoceramide preferentially. Together, these data demonstrate that aPHC is a novel human alkaline phytoceramidase, the first mammalian alkaline ceramidase to be identified as being specific for the hydrolysis of phytoceramide.     

Alkaline ceramidase family: The first two decades

Ceramidases are a group of enzymes that catalyze the hydrolysis of ceramide, dihydroceramide, and phytoceramide into sphingosine (SPH), dihydrosphingosine (DHS), and phytosphingosine (PHS), respectively, along with a free fatty acid. Ceramidases are classified into the acid, neutral, and alkaline ceramidase subtypes according to the pH optima for their catalytic activity. YPC1 and YDC1 were the first alkaline ceramidase genes to be identified and cloned from the yeast Saccharomyces cerevisiae two decades ago. Subsequently, alkaline ceramidase genes were identified from other species, including one Drosophila melanogaster ACER gene (Dacer), one Arabidopsis thaliana ACER gene (AtACER), three Mus musculus ACER genes (Acer1, Acer2, and Acer3), and three Homo sapiens ACER genes (ACER1, ACER2, and ACER3). The protein products of these genes constitute a large protein family, termed the alkaline ceramidase (ACER) family. All the biochemically characterized members of the ACER family are integral membrane proteins with seven transmembrane segments in the Golgi complex or endoplasmic reticulum, and they each have unique substrate specificity. An increasing number of studies suggest that the ACER family has diverse roles in regulating sphingolipid metabolism and biological processes. Here we discuss the discovery of the ACER family, the biochemical properties, structures, and catalytic mechanisms of its members, and its role in regulating sphingolipid metabolism and biological processes in yeast, insects, plants, and mammals.     

Molecular cloning and characterization of OsCDase, a ceramidase enzyme from rice

SUMMARY: Sphingolipids are a structurally diverse group of molecules based on long-chain sphingoid bases that are found in animal, fungal and plant cells. In contrast to the situation in animals and yeast, much less is known about the spectrum of sphingolipid species in plants and the roles they play in mediating cellular processes. Here, we report the cloning and characterization of a plant ceramidase from rice (Oryza sativa spp. Japonica cv. Nipponbare). Sequence analysis suggests that the rice ceramidase (OsCDase) is similar to mammalian neutral ceramidases. We demonstrate that OsCDase is a bona fide ceramidase by heterologous expression in the yeast double knockout mutant Deltaypc1Deltaydc1 that lacks the yeast ceramidases YPC1p and YDC1p. Biochemical characterization of OsCDase showed that it exhibited classical Michaelis-Menten kinetics, with optimum activity between pH 5.7 and 6.0. OsCDase activity was enhanced in the presence of Ca(2+), Mg(2+), Mn(2+) and Zn(2+), but inhibited in the presence of Fe(2+). OsCDase appears to use ceramide instead of phytoceramide as a substrate. Subcellular localization showed that OsCDase is localized to the endoplasmic reticulum and Golgi, suggesting that these organelles are sites of ceramide metabolism in plants.     

Lag1p and Lac1p Are Essential for the Acyl-CoA–dependent Ceramide Synthase Reaction in Saccharomyces cerevisae

Lag1p and Lac1p are two homologous transmembrane proteins of the endoplasmic reticulum in Saccharomyces cerevisiae. Homologous genes have been found in a wide variety of eukaryotes. In yeast, both genes, LAC1 and LAG1, are required for efficient endoplasmic reticulum-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins. In this study, we show that lag1 Delta lac1 Delta cells have reduced sphingolipid levels due to a block of the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction. The sphingolipid synthesis defect in lag1 Delta lac1 Delta cells can be partially corrected by overexpression of YPC1 or YDC1, encoding ceramidases that have been reported to have acyl-CoA-independent ceramide synthesis activity. Quadruple mutant cells (lag1 Delta lac1 Delta ypc1 Delta ydc1 Delta) do not make any sphingolipids, but are still viable probably because they produce novel lipids. Moreover, lag1 Delta lac1 Delta cells are resistant to aureobasidin A, an inhibitor of the inositolphosphorylceramide synthase, suggesting that aureobasidin A may be toxic because it leads to increased ceramide levels. Based on these data, LAG1 and LAC1 are the first genes to be identified that are required for the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction.     

Aureobasidin A arrests growth of yeast cells through both ceramide intoxication and deprivation of essential inositolphosphorylceramides

All mature Saccharomyces cerevisiae sphingolipids comprise inositolphosphorylceramides containing C26:0 or C24:0 fatty acids and either phytosphingosine or dihydrosphingosine. Here we analysed the lipid profile of lag1 Δ lac1 Δ mutants lacking acyl-CoA-dependent ceramide synthesis, which require the reverse ceramidase activity of overexpressed Ydc1p for sphingolipid biosynthesis and viability. These cells, termed 2Δ.YDC1, make sphingolipids containing exclusively dihydrosphingosine and an abnormally wide spectrum of fatty acids with between 18 and 26 carbon atoms. Like wild-type cells, 2Δ.YDC1 cells stop growing when exposed to Aureobasidin A (AbA), an inhibitor of the inositolphosphorylceramide synthase AUR1 , yet their ceramide levels remain very low. This finding argues against a current hypothesis saying that yeast cells do not require inositolphosphorylceramides and die in the presence of AbA only because ceramides build up to toxic concentrations. Moreover, W303 lag1 Δ lac1 Δ ypc1 Δ ydc1 Δ cells, reported to be AbA resistant, stop growing on AbA after a certain number of cell divisions, most likely because AbA blocks the biosynthesis of anomalous inositolphosphorylsphingosides. Thus, data argue that inositolphosphorylceramides of yeast, the equivalent of mammalian sphingomyelins, are essential for growth. Data also clearly confirm that wild-type strains, when exposed to AbA, immediately stop growing because of ceramide intoxication, long before inositolphosphorylceramide levels become subcritical.     

CDase is a pan-ceramidase in Drosophila

Ceramidases catalyze the conversion of ceramide to sphingosine. They are acylaminohydrolases that catalyze the deacylation of the amide-linked saturated fatty acid from ceramide to generate sphingosine. They also catalyze the reverse reaction of ceramide biosynthesis using sphingosine and fatty acid. In mammals, different proteins catalyze these reactions while individually exhibiting optimal activity over a narrow pH range and have been accordingly called acid, neutral, and alkaline ceramidases. Several genes encode for variants of alkaline ceramidase in mammals. Brainwashing (Bwa) is the only putative alkaline ceramidase homologue present in Drosophila. In this study we have demonstrated that BWA does not exhibit ceramidase activity and that bwa null mutants display no loss of ceramidase activity. Instead, the neutral ceramidase gene CDase encodes the protein that is responsible for all measurable ceramidase activity in Drosophila. Our studies show strong genetic interaction of Bwa with CDase and the Drosophila ceramide kinase gene (DCERK). We show that, although BWA is unlikely to be a ceramidase, it is a regulator of sphingolipid flux in Drosophila. Bwa exhibits strong genetic interaction with other genes coding for ceramide-metabolizing enzymes. This interaction might partly explain its original identification as a ceramidase.     

Purification and characterization of a membrane-bound nonlysosomal ceramidase from rat brain.

We have purified a membrane bound ceramidase 22,300-fold to apparent homogeneity. The purification scheme included Triton X-100 extraction of membranes followed by Q-Sepharose, blue Sepharose, phenyl-Sepharose, and MonoS column chromatography. The purified enzyme showed an apparent molecular mass of 90 kDa as estimated by SDS-polyacrylamide gel electrophoresis under reducing conditions and 95 kDa by chromatography on Superose 12. Using C(16)-ceramide as substrate, the enzyme showed a broad pH optimum in the neutral to alkaline range. A mixed micelle assay was developed, and using Triton X-100/ceramide mixed micelles, the enzyme exhibited classical Michaelis-Menten kinetics, with a K(m) of 1.29 mol % and a V(max) of 4.4 micromol/min/mg. When dihydroceramide was used as substrate, these values were 3.84 mol % and 1.2 micromol/min/mg, respectively, indicating that the enzyme hydrolyzes ceramides preferentially. The activity of the purified ceramidase did not require cations, and it was inhibited by reducing agents. Phosphatidylcholine and sphingomyelin were without effect on the enzyme activity, whereas phosphatidic acid and phosphatidylserine stimulated the activity 3-fold. Sphingosine acted as a competitive inhibitor with an IC(50) of 5-10 microM. These results indicate that the purified enzyme is a novel ceramidase.     

Isc1 regulates sphingolipid metabolism in yeast mitochondria

The Saccharomyces cerevisiae inositol sphingolipid phospholipase C (Isc1p), a homolog of mammalian neutral sphingomyelinases, hydrolyzes complex sphingolipids to produce ceramide in vitro. Epitope-tagged Isc1p associates with the mitochondria in the post-diauxic phase of yeast growth. In this report, the mitochondrial localization of Isc1p and its role in regulating sphingolipid metabolism were investigated. First, endogenous Isc1p activity was enriched in highly purified mitochondria, and western blots using highly purified mitochondrial membrane fractions demonstrated that epitope-tagged Isc1p localized to the outer mitochondrial membrane as an integral membrane protein. Next, LC/MS was employed to determine the sphingolipid composition of highly purified mitochondria which were found to be significantly enriched in alpha-hydroxylated phytoceramides (21.7 fold) relative to the whole cell. Mitochondria, on the other hand, were significantly depleted in sphingoid bases. Compared to the parental strain, mitochondria from isc1Delta in the post-diauxic phase showed drastic reduction in the levels of alpha-hydroxylated phytoceramide (93.1% loss compared to WT mitochondria with only 2.58 fold enrichment in mitochondria compared to whole cell). Functionally, isc1Delta showed a higher rate of respiratory-deficient cells after incubation at high temperature and was more sensitive to hydrogen peroxide and ethidium bromide, indicating that isc1Delta exhibits defects related to mitochondrial function. These results suggest that Isc1p generates ceramide in mitochondria, and the generated ceramide contributes to the normal function of mitochondria. This study provides a first insight into the specific composition of ceramides in mitochondria.     

Phytoceramide in Vertebrate Tissues: One Step Chromatography Separation for Molecular Characterization of Ceramide Species

Ceramide is a precursor for complex sphingolipids in vertebrates, while plants contain phytoceramide. By using a novel chromatography purification method we show that phytoceramide comprises a significant proportion of animal sphingolipids. Total ceramide including phytoceramide from mouse tissue (brain, heart, liver) lipid extracts and cell culture (mouse primary astrocytes, human oligodendroglioma cells) was eluted as a single homogenous fraction, and then analyzed by thin layer chromatography, and further characterized by gas chromatography-mass spectrometry (GC-MS). We detected a unique band that migrated between non-hydroxy fatty acyl ceramide and hydroxy fatty acyl ceramide, and identified it as phytoceramide. Using RT-PCR, we confirmed that mouse tissues expressed desaturase 2, an enzyme that has been reported to generate phytoceramide from dihydroceramide. Previously, only trace amounts of phytoceramide were reported in vertebrate intestine, kidney, and skin. While its function is still elusive, this is the first report of phytoceramide characterization in glial cells and vertebrate brain, heart, and liver.     

Hydroxylation state of fatty acid and long-chain base moieties of sphingolipid determine the sensitivity to growth inhibition due to AUR1 repression in Saccharomyces cerevisiae

The structures of ceramide found in the yeast Saccharomyces cerevisiae are classified into five groups according to the hydroxylation states of the long-chain base and fatty acid moieties. This diversity is created through the action of enzymes encoded by SUR2, SCS7, and as yet unidentified hydroxylation enzyme(s). Aur1p is an enzyme catalyzing the formation of inositol phosphorylceramide in the yeast, and the defect leads to strong growth inhibition due to accumulation of ceramide and reductions in complex sphingolipid levels. In this study, we found that the deletion of SCS7 results in the enhancement of growth inhibition due to repression of AUR1 expression under the control of a tetracycline-regulatable promoter, whereas the deletion of SUR2 attenuates the growth inhibition. Under AUR1-repressive conditions, SCS7 and SUR2 mutants showed reductions in the complex sphingolipid levels and the accumulation of ceramide, like wild-type cells. On the other hand, the deletion of SCS7 had no effect on the growth inhibition through reductions in the complex sphingolipid levels caused by repression of LIP1 encoding a ceramide synthase subunit. Furthermore, the deletion of SUR2 did not suppress the growth inhibition under LIP1-repressive conditions. Therefore, it is suggested that the deletion of sphingolipid hydroxylases changes the toxicity of ceramide under AUR1-repressive conditions.     

Specific and sensitive assay for alkaline and neutral ceramidases involving C12-NBD-ceramide

A fluorescent analogue of ceramide, C12-NBD-ceramide, was found to be hydrolyzed much faster than 14C-labeled ceramide by alkaline ceramidase from Pseudomonas aeruginosa and neutral ceramidase from mouse liver, while this substrate was relatively resistant to acid ceramidase from plasma of the horseshoe crab. The radioactive substrate was used more preferentially by the acid ceramidase. It should be noted that C6-NBD-ceramide, which is usually used for ceramidase assays, was hardly hydrolyzed by any of the enzymes examined, compared to C12-NBD-ceramide. For the alkaline and neutral enzymes, the Vmax and k (Vmax/Km) with C12-NBD-ceramide were much higher than those with 14C-ceramide. In contrast, for the acid enzyme these parameters with C12-NBD-ceramide were less than half those with the radioisotope-labeled substrate. It is noteworthy that the labeling of ceramide with NBD did not itself reduce the Km of the alkaline enzyme, but did that of the neutral enzyme. It was also found that C12-NBD-ceramide was preferentially hydrolyzed by the alkaline and neutral enzymes, but not the acid one, in several mammalian cell lines. This study clearly shows that the attachment of NBD, but not dansyl, increases the susceptibility of ceramide to alkaline and neutral enzyme, and decreases that to acid enzymes. Thus the use of this substrate provides a specific and sensitive assay for alkaline and neutral ceramidases.     

Identification of ISC1 (YER019w) as inositol phosphosphingolipid phospholipase C in Saccharomyces cerevisiae

Sphingolipids have emerged as novel bioactive mediators in eukaryotic cells including yeast. It has been proposed that sphingomyelin (SM) hydrolysis and the concomitant generation of ceramide are involved in various stress responses in mammalian cells. The yeast Saccharomyces cerevisiae has inositol phosphosphingolipids (IPS) instead of SM and glycolipids, and synthesis of IPS is indispensable to its growth. Although the genes responsible for the synthesis of IPS have been identified, the gene(s) for the degradation of IPS has not been reported. Here we show that ISC1 (YER019w), which has homology to bacterial neutral sphingomyelinase (SMase), encodes IPS phospholipase C (IPS-PLC). First, we observed that overexpression of ISC1 greatly increased neutral SMase activity, and this activity was dependent on the presence of phosphatidylserine. Cells deleted in ISC1 demonstrated negligible neutral SMase activity. Because yeast cells have IPS instead of SM, we investigated whether IPS are the physiologic substrates of this enzyme. Lysates of ISC1-overexpressing cells demonstrated very high PLC activities on IPS. Deletion of ISC1 eliminated endogenous IPS-PLC activities. Labeling yeast cells with [(3)H]dihydrosphingosine showed that IPS were increased in the deletion mutant cells. This study identifies the first enzyme involved in catabolism of complex sphingolipids in S. cerevisiae.     

Expression of LASS2 controlled by LAG1 or ADH1 promoters cannot functionally complement Lag1p

LAG1 contributes to the substrate specificity and catalytic activity of ceramide synthases in Saccharomyces cerevisiae. Double deletion of LAG1 and its yeast homologue LAC1 results in the slow growth defect of the cell under certain genetic backgrounds. LASS2, containing the conserved TLC domain and the specific HOX domain, is a human homologue of Lag1p. In this study, shuffling tests and tetrad analyses were carried out to examine the complementation between Lag1p and LASS2 or its fragment containing the TLC domain but lacking the HOX domain (LASS2DeltaHOX). Controlled by either the natural weak LAG1 promoter or the strong yeast ADH1 promoter, LASS2 and LASS2DeltaHOX could not rescue the slow growth defect of double mutant. The results indicated that LASS2 or LASS2DeltaHOX could not functionally complement Lag1p.     

Cell-nonautonomous function of ceramidase in photoreceptor homeostasis

Neutral ceramidase, a key enzyme of sphingolipid metabolism, hydrolyzes ceramide to sphingosine. These sphingolipids are critical structural components of cell membranes and act as second messengers in diverse signal transduction cascades. Here, we have isolated and characterized functional null mutants of Drosophila ceramidase. We show that secreted ceramidase functions in a cell-nonautonomous manner to maintain photoreceptor homeostasis. In the absence of ceramidase, photoreceptors degenerate in a light-dependent manner, are defective in normal endocytic turnover of rhodopsin, and do not respond to light stimulus. Consistent with a cell-nonautonomous function, overexpression of ceramidase in tissues distant from photoreceptors suppresses photoreceptor degeneration in an arrestin mutant and facilitates membrane turnover in a rhodopsin null mutant. Furthermore, our results show that secreted ceramidase is internalized and localizes to endosomes. Our findings establish a role for a secreted sphingolipid enzyme in the regulation of photoreceptor structure and function.     

Interleukin-1beta induces chronic activation and de novo synthesis of neutral ceramidase in renal mesangial cells.

The lipid signaling molecule ceramide is formed by the action of acid and neutral sphingomyelinases and degraded by acid and neutral ceramidases. Short-term stimulation of mesangial cells with the pro-inflammatory cytokine interleukin-1beta (IL-1beta) leads to a rapid and transient increase in neutral sphingomyelinase activity (Kaszkin, M., Huwiler, A., Scholz, K., van den Bosch, H., and Pfeilschifter, J. (1998) FEBS Lett. 440, 163-166). In this study, we report on a second delayed peak of activation occurring after hours of IL-1beta treatment. This second phase of activation was first detectable after 2 h of treatment and steadily increased over the next 2 h, reaching maximal values after 4 h. In parallel, a pronounced increase in neutral ceramidase activity was observed, accounting for a constant or even decreased level of ceramide after long-term IL-1beta treatment, despite continuous sphingomyelinase activation. The increase in neutral ceramidase activity was due to expressional up-regulation, as detected by an increase in mRNA levels and enhanced de novo protein synthesis. The increase in neutral ceramidase protein levels and activity could be blocked dose- dependently by the p38 MAPK inhibitor SB 202190, whereas the classical MAPK pathway inhibitor U0126 and the protein kinase C inhibitor Ro 318220 were ineffective. Moreover, cotreatment of cells for 24 h with IL-1beta and SB 202190 led to an increase in ceramide formation. Interestingly, IL-1beta-stimulated neutral ceramidase activation was not reduced in mesangial cells isolated from mice deficient in MAPK-activated protein kinase-2, which is a downstream substrate of p38 MAPK, thus suggesting that the p38 MAPK-mediated induction of neutral ceramidase occurs independently of the MAPK-activated protein kinase-2 pathway. In summary, our results suggest a biphasic regulation of sphingomyelin hydrolysis in cytokine-treated mesangial cells with delayed de novo synthesis of neutral ceramidase counteracting sphingomyelinase activity and apoptosis. Neutral ceramidase may thus represent a novel cytoprotective enzyme for mesangial cells exposed to inflammatory stress conditions.