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PubMed2000

The skin of atopic dermatitis patients contains a novel enzyme, glucosylceramide sphingomyelin deacylase, which cleaves the N-acyl linkage of sphingomyelin and glucosylceramide.

We have demonstrated previously that there is an abnormal expression of sphingomyelin (SM) deacylase in the epidermis of patients with atopic dermatitis (ADe). In the present study, we have prepared N-[palmitic acid-1-(14)C]SM and N-[palmitic acid-1-(14)C]glucosylceramide (GCer) to use as substrates and have quantified SM deacylase activity by detecting the release of [(14)C]palmitic acid in extracts of the stratum corneum or the epidermis of ADe patients. In studies using [palmitic acid-1-(14)C]SM as a substrate, a pH dependency of catalytic activity with a peak at pH 5.0 was found. Preparative SDS/PAGE using an extract of ADe epidermis revealed that the molecular mass of SM deacylase is 40000 Da, which is consistent with its apparent molecular mass of 42000 Da estimated by gel-filtration analysis of stratum corneum extracts. Analytical isoelectric focusing (IEF) chromatography demonstrated that the pI values of SM deacylase, beta-glucocerebrosidase (GlcCDase), sphingomyelinase (SMase) and acid ceramidase were 4.2, 7.4, 7.0 and 5.7, respectively. In enzymic analysis using pI-4.2 SM deacylase partially purified by IEF, which had no detectable contamination with acid ceramidase, GlcCDase or SMase, radio-TLC analysis revealed that radiolabelled sphingosylphosphocholine or [1-(14)C]palmitic acid was enzymically liberated from [choline-methyl-(14)C]SM or N-[palmitoyl-1-(14)C]GCer, respectively, used as substrates. Further the pI-4.2 protein purified from extracts of the stratum corneum of ADe patients was able to hydrolyse N-[palmitoyl-1-(14)C]SM and GCer, but not N-[palmitoyl-1-(14)C]ceramide. These results indicate that a hitherto undiscovered epidermal enzyme, termed here glucosylceramide sphingomyelin deacylase, is expressed in the skin of ADe patients, which plays an important role in ceramide deficiency (including acylceramides) in the stratum corneum.

Carboxylic Ester Hydrolases↗
PubMed1994

Identification of a distinct pool of sphingomyelin involved in the sphingomyelin cycle.

Sphingomyelin (SM) is a membrane phosphosphingolipid that has recently been identified as a key component of the SM cycle. In this signal transduction pathway, extracellular inducers such as tumor necrosis factor alpha cause hydrolysis of membrane SM, resulting in the generation of the lipid second messenger ceramide. Only 10-20% of cellular SM appears to be involved in the SM cycle, raising the possibility of the existence of a unique "signaling" pool of SM. The existence and subcellular location of such a pool were investigated. Using bacterial sphingomyelinase from Staphylococcus aureus (bSMase), we first characterized two pools of SM, identified as an outer leaflet bSMase-sensitive pool and a distinct bSMase-resistant pool. These pools were further characterized by their differential solubility in Triton X-100 and by their kinetics of labeling. The signaling pool of SM was distinguished by the following: 1) resistance to bSMase, 2) solubility in Triton X-100, and 3) delayed labeling kinetics. In subfractionation studies, the signaling pool of SM co-fractionated with the plasma membrane. Since the SM cycle involves a cytosolic sphingomyelinase and the intracellular release of choline phosphate, this pool of SM appears to localize to the inner leaflet of the plasma membrane (or to a closely related compartment). These results identify a unique signaling pool of SM that undergoes significant hydrolysis (20-40%) in response to inducers of the SM cycle.

Calcitriol↗
PubMed1996

Opposite effects of tumor necrosis factor alpha on the sphingomyelin-ceramide pathway in two myeloid leukemia cell lines: role of transverse sphingomyelin distribution in the plasma membrane.

Tumor necrosis factor alpha (TNF alpha) mediates proliferation, functional activation, and apoptotic cell death depending on the target cell type. Although sphingomyelin (SPM) hydrolysis and ceramide generation may function as an important mediator in TNF alpha signaling, the molecular mechanisms of the signaling pathway(s) are still not well understood. The aim of the present study is to compare the effect of TNF alpha on SPM metabolism and cell growth in two myeloid leukemic cell lines (U937 and KG1a) that differ in their sensitivity to TNF alpha. Our results show that TNF alpha induced apoptosis in U937 but not in KG1a cells. TNF alpha triggered in KG1a cells neither SPM hydrolysis nor ceramide generation, but induced SPM synthesis and ceramide breakdown as well as dose-dependent cell proliferation. In contrast, TNF alpha induced in U937 SPM hydrolysis and ceramide generation as well as dose-dependent cell death. Synthetic cell permeant ceramide, as well as natural ceramide, generated by treatment with bacterial sphingomyelinase (SPMase), all induced apoptosis in both U937 and KG1a cells. These findings indicate that the SPM-ceramide pathway is altered in KG1a cells upstream of the ceramide generation. Analysis of the transverse distribution of SPM in the plasma membrane showed that the SPM pool involved in cell signaling (inner leaflet) was markedly reduced in KG1a cells; it is 7-fold lower than that found in the inner leaflet of U937 cells. Therefore, our study strongly suggests that the different responses induced by TNF alpha in myeloid cells are dependent on the SPM plasma membrane transverse asymmetry.

Animals↗
PubMed2002

Phosphatidylinositol transfer protein beta displays minimal sphingomyelin transfer activity and is not required for biosynthesis and trafficking of sphingomyelin.

Mammalian phosphatidylinositol transfer proteins (PITPs) alpha and beta, which share 77% identity, have been shown to exhibit distinct lipid-transfer activities. In addition to transferring phosphatidylinositol (PI) and phosphatidylcholine (PC), PITPbeta has been shown to transfer sphingomyelin (SM), and this has led to the suggestion that PITPbeta is important for the regulation of SM metabolism. In the present study, we have analysed the ability of human PITPbeta to transfer and regulate the metabolism of cellular SM. We report that, in vitro, the two PITP isoforms were comparable in mediating PI, PC or SM transfer. Using permeabilized HL-60 cells as the donor compartment, both PITP isoforms efficiently transferred PI and PC, and were slightly active towards SM, with the activity of PITPbeta being slightly greater. To identify which cellular lipids were selected by PITPs, PITPalpha and PITPbeta were exposed to permeabilized HL-60 cells, and subsequently repurified and analysed for their bound lipids. Both PITPs were able to select only PI and PC, but not SM. SM synthesis takes place at the Golgi, and PITPbeta was shown to localize in that compartment. To examine the role of PITPbeta in SM biosynthesis, Golgi membranes were used. Purified Golgi membranes had lost their endogenous PITPbeta, but were able to recruit PITPbeta when added exogenously. However, PITPbeta did not enhance the activities of either SM synthase or glucosylceramide synthase. Further analysis in COS-7 cells overexpressing PITPbeta showed no effects on (a) SM and glucosylceramide biosynthesis, (b) diacylglycerol or ceramide levels, (c) SM transport from the Golgi to the plasma membrane, or (d) resynthesis of SM after exogenous sphingomyelinase treatment. Altogether, these observations do not support the suggestion that PITPbeta participates in the transfer of SM, the regulation of SM biosynthesis or its intracellular trafficking.

Animals↗
PubMed2007

Resistance to alkyl-lysophospholipid-induced apoptosis due to downregulated sphingomyelin synthase 1 expression with consequent sphingomyelin- and cholesterol-deficiency in lipid rafts.

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; Et-18-OCH3) induces apoptosis in S49 mouse lymphoma cells. To this end, ALP is internalized by lipid raft-dependent endocytosis and inhibits phosphatidylcholine synthesis. A variant cell-line, S49AR, which is resistant to ALP, was shown previously to be unable to internalize ALP via this lipid raft pathway. The reason for this uptake failure is not understood. In the present study, we show that S49AR cells are unable to synthesize SM (sphingomyelin) due to down-regulated SMS1 (SM synthase 1) expression. In parental S49 cells, resistance to ALP could be mimicked by small interfering RNA-induced SMS1 suppression, resulting in SM deficiency and blockage of raft-dependent internalization of ALP and induction of apoptosis. Similar results were obtained by treatment of the cells with myriocin/ISP-1, an inhibitor of general sphingolipid synthesis, or with U18666A, a cholesterol homoeostasis perturbing agent. U18666A is known to inhibit Niemann-Pick C1 protein-dependent vesicular transport of cholesterol from endosomal compartments to the trans-Golgi network and the plasma membrane. U18666A reduced cholesterol partitioning in detergent-resistant lipid rafts and inhibited SM synthesis in S49 cells, causing ALP resistance similar to that observed in S49AR cells. The results are explained by the strong physical interaction between (newly synthesized) SM and available cholesterol at the Golgi, where they facilitate lipid raft formation. We propose that ALP internalization by lipid-raft-dependent endocytosis represents the retrograde route of a constitutive SMS1- and lipid-raft-dependent membrane vesicular recycling process.

Androstenes↗
PubMed2004

Expression cloning of a human cDNA restoring sphingomyelin synthesis and cell growth in sphingomyelin synthase-defective lymphoid cells.

Sphingomyelin (SM) synthase has been assumed to be involved in both cell death and survival by regulating pro-apoptotic mediator ceramide and pro-survival mediator diacylglycerol. However, its precise functions are ambiguous due to the lack of molecular cloning of SM synthase gene(s). We isolated WR19L/Fas-SM(-) mouse lymphoid cells, which show a defect of SM at the plasma membrane due to the lack of SM synthase activity and resistance to cell death induced by an SM-directed cytolytic protein lysenin. WR19L/Fas-SM(-) cells were also highly susceptible to methyl-beta-cyclodextrin (MbetaCD) as compared with the WR19L/Fas-SM(+) cells, which are capable of SM synthesis. By expression cloning method using WR19L/Fas-SM(-) cells and MbetaCD-based selection, we have succeeded in cloning of a human cDNA responsible for SM synthase activity. The cDNA encodes a peptide of 413 amino acids named SMS1 (putative molecular mass, 48.6 kDa), which contains a sterile alpha motif domain near the N-terminal region and four predicted transmembrane domains. WR19L/Fas-SM(-) cells expressing SMS1 cDNA (WR19L/Fas-SMS1) restored the resistance against MbetaCD, the accumulation of SM at the plasma membrane, and SM synthesis by transferring phosphocholine from phosphatidylcholine to ceramide. Furthermore, WR19L/Fas-SMS1 cells, as well as WR19L/Fas-SM(-) cells supplemented with exogenous SM, restored cell growth ability in serum-free conditions, where the growth of WR19L/Fas-SM(-) cells was severely inhibited. The results suggest that SMS1 is responsible for SM synthase activity in mammalian cells and plays a critical role in cell growth of mouse lymphoid cells.

Animals↗
PubMed2004

Milk sphingomyelin is more effective than egg sphingomyelin in inhibiting intestinal absorption of cholesterol and fat in rats.

We reported previously that egg sphingomyelin (SM) inhibits the intestinal absorption of cholesterol and fat in rats. This study was conducted to compare the relative efficiencies of milk and egg SM in inhibiting intestinal absorption of cholesterol and other lipids. Adult male rats with lymph cannulae were infused at 3.0 mL/h for 8 h via a duodenal catheter with a lipid emulsion (451.7 micromol triolein, 20.7 micromol cholesterol, 33.3 kBq (14)C-cholesterol, 3.1 micromol alpha-tocopherol, and 396.0 micromol sodium taurocholate in 24 mL PBS, pH, 6.5), without SM (controls), or with 80.0 micromol egg SM or milk SM. The lymphatic absorptions of (14)C-cholesterol were significantly lower in rats infused with milk SM (19.5 +/- 1.4% dose) and egg SM (24.4 +/- 1.9% dose) than in those infused with no SM (37.6 +/- 1.8% dose). In addition, the lymphatic outputs of fatty acids and phospholipid were significantly lowered by milk and egg SM. Similarly, the absorption of alpha-tocopherol also was decreased by milk SM (13.6 +/- 1.7% dose) and egg SM (18.3 +/- 2.4% dose) compared with controls (27.0 +/- 1.8% dose). Total lymphatic SM output was not affected by egg SM, but markedly decreased by milk SM, relative to controls. The results indicate that both milk and egg SM markedly inhibit the absorption of cholesterol, fat, and other lipids. However, milk SM is a more potent inhibitor than egg SM. The strong inhibitory effect of milk SM may be associated with the higher degree of saturation and longer chain length of its fatty acyl groups, which may slow the rate of luminal lipolysis, micellar solubilization, and transfer of micellar lipids to the enterocyte.

Animals↗
PubMed1984

Lysosomal involvement in cellular turnover of plasma membrane sphingomyelin.

At least two isoenzymes of sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12), including lysosomal acid sphingomyelinase and nonlysosomal magnesium-dependent neutral sphingomyelinase, catalyse the degradation of sphingomyelin in cultured human skin fibroblasts. A genetically determined disorder of sphingomyelin metabolism, type A Niemann-Pick disease, is characterized by a deficiency of lysosomal acid sphingomyelinase. To investigate the involvement of lysosomes in the degradation of cellular membrane sphingomyelin, we have undertaken studies to compare the turnover of plasma membrane sphingomyelin in fibroblasts from a patient with type A Niemann-Pick disease, which completely lack acid sphingomyelinase activity but retain nonlysosomal neutral sphingomyelinase activity, with turnover in fibroblasts from normal individuals. Plasma membrane sphingomyelin was labeled by incubating cells at low temperature with phosphatidylcholine vesicles containing radioactive sphingomyelin. A fluorescent analog of sphingomyelin, N-4-nitrobenzo-2-oxa-1,3-diazoleaminocaproyl sphingosylphosphorylcholine (NBD-sphingomyelin) is seen to be readily transferred at low temperature from phosphatidylcholine liposomes to the plasma membranes of cultured human fibroblasts. Moreover, when kinetic studies were done in parallel, a constant ratio of [14C]oleoylsphingosylphosphorylcholine ( [14C]sphingomyelin) to NBD-sphingomyelin was taken up at low temperature by the fibroblast cells, suggesting that [14C]sphingomyelin undergoes a similar transfer. The comparison of sphingomyelin turnover at 37 degrees C in normal fibroblasts compared to Niemann-Pick diseased fibroblasts shows that a rapid turnover of plasma membrane-associated sphingomyelin within the first 30 min appears to be similar in both normal and Niemann-Pick diseased cells. This rapid turnover appears to be primarily due to rapid removal of the [14C]sphingomyelin from the cell surface into the incubation medium. During long-term incubation, an increase in the formation of [14C]ceramide correlating with the degradation of [14C]sphingomyelin is observed in normal fibroblasts. In contrast, the level of [14C]ceramide remains constant in Niemann-Pick diseased cells, which correlates with a higher level of intact [14C]sphingomyelin remaining in these cells compared to normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloro-7-nitrobenzofurazan↗
PubMed1989

Divergent fate of unsaturated and saturated ceramides and sphingomyelins in rat liver and cells in culture.

The metabolism of sphingomyelins and ceramides with defined labeled fatty acids was compared after injection in vivo or incubation with cultured cells. The liver was the major site of uptake of sphingomyelins and ceramides with 18:2 or 16:0 fatty acids, but with both sphingolipids a higher recovery of radioactivity was found with 16:0 species. The distribution of radioactivity among liver lipids showed that 1.5 h after injection of 18:2 sphingomyelin, only 21% of the label was found as sphingomyelin, and this value was 37% in the case of 16:0 sphingomyelin. There was a very marked difference in the metabolism of 18:2 and 16:0 ceramides. After injection of 18:2 ceramide only 14% of the radioactivity was recovered as sphingomyelin, and this value was more than 50% with 16:0 ceramide. [14C]18:2 ceramide was converted also to glucoceramide and hydrolyzed more extensively than 16:0 ceramide. These observations were extended to sphingomyelins and ceramides with other fatty acids, using Hep-G2 cells in culture. Significantly more radioactivity was recovered as labeled sphingomyelin after incubation with 16:0, 18:0, 20:0 and 24:0 sphingomyelins than with 18:1 and 18:2 sphingomyelins, while more labeled phosphatidylcholine and phosphatidylethanolamine were found with the unsaturated sphingomyelins. In analogy to the findings in vivo, in the Hep-G2 cells more 16:0, 18:0 and 24:0 ceramides were converted to sphingomyelin than 18:1 or 18:2 ceramides. These differences were also seen with cultured macrophages, in which a more marked reutilization for sphingomyelin formation was found with the saturated ceramide series. The sphingomyelin liposomes were tested also for their capacity to mobilize cholesterol, and a rise in plasma unesterified cholesterol occurred after injection of 18:2 sphingomyelin. Marked enhancement of cholesterol efflux from cholesterol ester-loaded macrophages was also seen with 18:1 and 18:2, 20:0 sphingomyelin in the presence of delipidated high-density lipoprotein. The present results demonstrate that the metabolic fate of sphingolipids is related to their fatty acid composition. While ceramides with saturated fatty acids are predominantly reutilized for sphingomyelin formation, those with unsaturated fatty acids undergo probably more rapid hydrolysis with liberation of fatty acids and channeling into glycerolipids.

Animals↗
PubMed1998

Lysenin, a novel sphingomyelin-specific binding protein.

Lysenin, a novel 41-kDa protein purified from coelomic fluid of the earthworm Eisenia foetida, induced erythrocyte lysis. Preincubation of lysenin with vesicles containing sphingomyelin inhibited lysenin-induced hemolysis completely, whereas vesicles containing phospholipids other than sphingomyelin showed no inhibitory activity, suggesting that lysenin bound specifically to sphingomyelin on erythrocyte membranes. The specific binding of lysenin to sphingomyelin was confirmed by enzyme-linked immunosorbent assay, TLC immunostaining, and liposome lysis assay. In these assays, lysenin bound specifically to sphingomyelin and did not show any cross-reaction with other phospholipids including sphingomyelin analogs such as sphingosine, ceramide, and sphingosylphosphorylcholine, indicating that it recognized a precise molecular structure of sphingomyelin. Kinetic analysis of the lysenin-sphingomyelin interaction by surface plasmon resonance measurements using BIAcoreTM system showed that lysenin associated with membrane surfaces composed of sphingomyelin (kon = 3.2 x 10(4) M-1 s-1) and dissociated extremely slowly (koff = 1.7 x 10(-4) s-1), giving a low dissociation constant (KD = 5.3 x 10(-9) M). Incorporation of cholesterol into the sphingomyelin membrane significantly increased the total amount of lysenin bound to the membrane, whereas it did not change the kinetic parameters of the lysenin-membrane interaction, suggesting that lysenin specifically recognized sphingomyelin and cholesterol incorporation changed the topological distribution of sphingomyelin in the membranes, thereby increasing the accessibility of sphingomyelin to lysenin. Immunofluorescence staining of fibroblasts derived from a patient with Niemann-Pick disease type A showed that lysenin stained the surfaces of the fibroblasts uniformly, whereas intense lysosomal staining was observed when the cells were permeabilized by digitonin treatment. Preincubation of lysenin with vesicles containing sphingomyelin abolished lysenin immunostaining. This study demonstrated that lysenin bound specifically to sphingomyelin on cellular membranes and should be a useful tool to probe the molecular motion and function of sphingomyelin in biological membranes.

Animals↗
PubMed1993

Sphingomyelin transport to the cell surface occurs independently of protein secretion in rat hepatocytes.

The transport of sphingomyelin from its site of synthesis, in the cis/medial-Golgi, to the plasma membrane has been investigated in monolayer cultures of rat hepatocytes. Sphingomyelin was synthesized intracellularly from [methyl-3H]choline, and the arrival of sphingomyelin at the plasma membrane was monitored by treatment of the cells with sphingomyelinase, which hydrolyzed only sphingomyelin at the cell surface. When hepatocytes were maintained at temperatures from 5 to 37 degrees C, both protein secretion and sphingomyelin trafficking to the cell surface were inhibited at low temperatures, especially at temperatures below 25 degrees C. Treatment of cells with the energy poison potassium cyanide prevented protein secretion and reduced the incorporation of [3H]choline into sphingomyelin, but did not affect the percentage of newly synthesized sphingomyelin that was transported to the cell surface. Brefeldin A and monensin, both of which disrupt protein movement through the Golgi apparatus, were used to evaluate the mechanism of transport of sphingomyelin to the plasma membrane. Although brefeldin A and monensin each effectively blocked protein secretion, sphingomyelin movement to the cell surface was not inhibited, even though sphingomyelin synthesis normally occurs in the Golgi. We conclude that the most likely mechanism of sphingomyelin transport to the cell surface is a vesicle-mediated process (because of the known topology of sphingomyelin synthesis and distribution). However, the putative vesicles that are involved in the movement of sphingomyelin to the plasma membrane are distinct from those that transport proteins for secretion. In addition, the route of trafficking of sphingomyelin to the plasma membrane is not one that requires an obligatory passage through the Golgi apparatus.

Albumins↗