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PubMed1998

Cerebrosides A and C, sphingolipid elicitors of hypersensitive cell death and phytoalexin accumulation in rice plants.

When plants interact with certain pathogens, they protect themselves by generating various chemical and physical barriers called the hypersensitive response. These barriers are induced by molecules called elicitors that are produced by pathogens. In the present study, the most active elicitors of the hypersensitive response in rice were isolated from the rice pathogenic fungus Magnaporthe grisea, and their structures were identified as cerebrosides A and C, sphingolipids that were previously isolated as inducers of cell differentiation in the fungus Schizophyllum commune. Treatment of rice leaves with cerebroside A induced the accumulation of antimicrobial compounds (phytoalexins), cell death, and increased resistance to subsequent infection by compatible pathogens. The degradation products of cerebroside A (fatty acid methyl ester, sphingoid base, and glucosyl sphingoid base) showed no elicitor activity. Hydrogenation of the 8E-double bond in the sphingoid base moiety or the 3E-double bond in the fatty acid moiety of cerebroside A did not alter the elicitor activity, whereas hydrogenation of the 4E-double bond in the sphingoid base moiety led to a 12-fold decrease in elicitor activity. Furthermore, glucocerebrosides from Gaucher's spleen consisting of (E)-4-sphingenine and cerebrosides from rice bran mainly consisting of (4E,8E)-4,8-sphingadienine and (4E,8Z)-4,8-sphingadienine showed no elicitor activity. These results indicate that the methyl group at C-9 and the 4E-double bond in the sphingoid base moiety of cerebrosides A and C are the key elements determining the elicitor activity of these compounds. This study is the first to show that sphingolipids have elicitor activity in plants.

Anti-Infective Agents↗
PubMed1999

SEC14-dependent secretion in Saccharomyces cerevisiae. Nondependence on sphingolipid synthesis-coupled diacylglycerol production.

The SEC14 gene in Saccharomyces cerevisiae encodes a phosphatidylinositol transfer protein required for secretory protein movement from the Golgi. Mutation of SAC1, a gene of unknown function, restores secretory flow in sec14-1(ts) strains. The existing model for the bypass of the sec14-1(ts) defect by sac1-22 involves stimulation of sphingolipid biosynthesis and, in particular, the synthesis of mannosyl-diinositolphosphoryl-ceramide with concomitant increases in Golgi diacylglycerol levels. To test this model, we disrupted IPT1, the mannosyl-diinositolphosphoryl-ceramide synthase of S. cerevisiae. Disruption of the IPT1 gene had no effect on the ability of sac1-22 to bypass sec14-1(ts). Furthermore, sphingolipid analysis of sec14-1(ts) and sec14-1(ts) sac1-22 strains showed that mannosyl-diinositolphosphoryl-ceramide synthesis was not stimulated in the bypass mutant. However, the sec14-1(ts) strain had elevated mannosyl-monoinositolphosphoryl-ceramide levels, and the sec14-1(ts) sac1-22 strain showed an 8-fold increase in phosphatidylinositol 4-phosphate along with a decrease in phosphatidylinositol 4,5-bisphosphate. Cellular diacylglycerol levels, measured by [14C]acetate incorporation, did not differ between the sec14-1(ts) and the sec14-1 sac1-22 bypass strains, although disruption of IPT1 in the bypass strain resulted in reduced levels. These data indicate that phosphatidylinositol 4-phosphate, rather than mannosyl-diinositolphosphoryl-ceramide, accumulates in the sec14-1(ts) sac1-22 bypass strain, and that Golgi diacylglycerol accumulation is not required for bypass of the sec14-1(ts) growth and secretory phenotypes.

Carrier Proteins↗
PubMed1999

Activation of the de novo biosynthesis of sphingolipids mediates angiotensin II type 2 receptor-induced apoptosis.

This study examines the role of sphingolipids in mediating the apoptosis of PC12W cells induced by the angiotensin II type 2 (AT2) receptor. PC12W cells express abundant AT2 receptor but not angiotensin II type 1 receptor and undergo apoptosis when stimulated by angiotensin II. AT2 receptor-induced ceramide accumulation preceded the onset of caspase 3 activation and DNA fragmentation. AT2 receptor-induced ceramide accumulation did not result from the degradation of complex sphingolipids (SL) such as sphingomyelin or glycosphingolipids, as no changes in neutral or acidic sphingomyelinase activities, sphingomyelin level, nor in cellular glycolipid composition were observed. AT2 receptor activated serine palmitoyltransferase with a maximum time of 24 h after angiotensin II stimulation. The AT2 receptor-induced accumulation of ceramide was blocked by inhibitors of the de novo pathway of SL synthesis, beta-chloro-L-alanine and fumonisin B1. Inhibition of the de novo biosynthesis of SLs by fumonisin B1 and beta-chloro-L-alanine completely abrogated the AT2 receptor-mediated apoptosis. Pertussis toxin and orthovanadate blocked AT2 receptor-mediated ceramide production. Taken together our data demonstrate that in PC12W cells the stimulation of AT2 receptor induces the activation of de novo pathway, and a metabolite of this pathway, possibly ceramide, mediates AT2 receptor-induced apoptosis.

Acyltransferases↗
PubMed2000

Interfacial regulation of acid ceramidase activity. Stimulation of ceramide degradation by lysosomal lipids and sphingolipid activator proteins.

The lysosomal degradation of ceramide is catalyzed by acid ceramidase and requires sphingolipid activator proteins (SAP) as cofactors in vivo. The aim of this study was to investigate how ceramide is hydrolyzed by acid ceramidase at the water-membrane interface in the presence of sphingolipid activator proteins in a liposomal assay system. The degradation of membrane-bound ceramide was significantly increased both in the absence and presence of SAP-D when anionic lysosomal phospholipids such as bis(monoacylglycero)phosphate, phosphatidylinositol, and dolichol phosphate were incorporated into substrate-bearing liposomes. Higher ceramide degradation rates were observed in vesicles with increased membrane curvature. Dilution assays indicated that acid ceramidase remained bound to the liposomal surface during catalysis. Not only SAP-D, but also SAP-C and SAP-A, were found to be stimulators of ceramide hydrolysis in the presence of anionic phospholipids. This finding was confirmed by cell culture studies, in which SAP-A, -C, and -D reduced the amount of ceramide storage observed in fibroblasts of a patient suffering from prosaposin deficiency. Strong protein-lipid interactions were observed for both SAP-D and acid ceramidase in surface plasmon resonance experiments. Maximum binding of SAP-D and acid ceramidase to lipid bilayers occurred at pH 4.0. Our results demonstrate that anionic, lysosomal lipids are required for efficient hydrolysis of ceramide by acid ceramidase.

Anions↗
PubMed2002

Altered sphingolipid metabolism in N-(4-hydroxyphenyl)-retinamide-resistant A2780 human ovarian carcinoma cells.

In the present work, we studied the effects of fenretinide (N-(4-hydroxyphenyl)retinamide (HPR)), a hydroxyphenyl derivative of all-trans-retinoic acid, on sphingolipid metabolism and expression in human ovarian carcinoma A2780 cells. A2780 cells, which are sensitive to a pharmacologically achievable HPR concentration, become 10-fold more resistant after exposure to increasing HPR concentrations. Our results showed that HPR was able to induce a dose- and time-dependent increase in cellular ceramide levels in sensitive but not in resistant cells. This form of resistance in A2780 cells was not accompanied by the overexpression of multidrug resistance-specific proteins MDR1 P-glycoprotein and multidrug resistance-associated protein, whose mRNA levels did not differ in sensitive and resistant A2780 cells. HPR-resistant cells were characterized by an overall altered sphingolipid metabolism. The overall content in glycosphingolipids was similar in both cell types, but the expression of specific glycosphingolipids was different. Specifically, our findings indicated that glucosylceramide levels were similar in sensitive and resistant cells, but resistant cells were characterized by a 6-fold lower expression of lactosylceramide levels and by a 6-fold higher expression of ganglioside levels than sensitive cells. The main gangliosides from resistant A2780 cells were identified as GM3 and GM2. The possible metabolic mechanisms leading to this difference were investigated. Interestingly, the mRNA levels of glucosylceramide and lactosylceramide synthases were similar in sensitive and resistant cells, whereas GM3 synthase mRNA level and GM3 synthase activity were remarkably higher in resistant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Trans-Golgi network and subapical compartment of HepG2 cells display different properties in sorting and exiting of sphingolipids.

In HepG2 cells, the subapical compartment (SAC) is involved in the biogenesis of membrane polarity. By contrast, direct apical transport originating from the trans-Golgi network (TGN), which may contribute to polarity establishment, has been poorly defined in these cells. Thus, although newly synthesized sphingolipids can be directly transported from the TGN to the apical membrane, numerous apical resident proteins are traveling via the transcytotic route. Here, we developed an in vitro transport assay and compared the molecular sorting of 6-[N-(7-nitrobenz-2-oxa-1,3 diazol-4-yl)amino] hexanoyl-sphingomyelin (C(6)NBD-SM) and C(6)NBD-glucosylceramide (C(6)NBD-GlcCer) in TGN and SAC. SM is released from both TGN and SAC in the lumenal leaflet of transport vesicles. This holds also for GlcCer released from the SAC but not for a substantial fraction that departed from the Golgi. Distinct transport vesicles, enriched in either SM or GlcCer are released from SAC, consistent with their rigid sorting in this compartment. Different vesicle populations could not be recovered from TGN, although in situ experiments reveal that GlcCer is preferentially transported to the apical membrane, reflecting different transport mechanisms. The results indicate that in HepG2 cells sphingolipids are mainly sorted in the SAC membrane and that the release of SM from SAC and TGN is differentially regulated.

4-Chloro-7-nitrobenzofurazan↗
PubMed2003

Protein sorting in the late Golgi of Saccharomyces cerevisiae does not require mannosylated sphingolipids.

Glycosphingolipids are widely viewed as integral components of the Golgi-based machinery by which membrane proteins are targeted to compartments of the endosomal/lysosomal system and to the surface domains of polarized cells. The yeast Saccharomyces cerevisiae creates glycosphingolipids by transferring mannose to the head group of inositol phosphorylceramide (IPC), yielding mannosyl-IPC (MIPC). Addition of an extra phosphoinositol group onto MIPC generates mannosyldi-IPC (M(IP)2C), the final and most abundant sphingolipid in yeast. Mannosylation of IPC is partially dependent on CSG1, a gene encoding a putative sphingolipidmannosyltransferase. Here we show that open reading frame YBR161w, renamed CSH1, is functionally homologous to CSG1 and that deletion of both genes abolishes MIPC and M(IP)2C synthesis without affecting protein mannosylation. Csg1p and Csh1p are closely related polytopic membrane proteins that co-localize with IPC synthase in the medial-Golgi. Loss of Csg1p and Csh1p has no effect on clathrin- or AP-3 adaptor-mediated protein transport from the Golgi to the vacuole. Moreover, segregation of the periplasmic enzyme invertase, the plasma membrane ATPase Pma1p and the glycosylphosphatidylinositol-anchored protein Gas1p into distinct classes of secretory vesicles occurs independently of Csg1p and Csh1p. Our results indicate that protein sorting in the late Golgi of yeast does not require production of mannosylated sphingolipids.

Adenosine Triphosphatases↗
PubMed2004

The sphingolipid pathway regulates Pkc1 through the formation of diacylglycerol in Cryptococcus neoformans.

The sphingolipid biosynthetic pathway generates bioactive molecules crucial to the regulation of mammalian and fungal physiological and pathobiological processes. In previous studies (Luberto, C., Toffaletti, D. L., Wills, E. A., Tucker, S. C., Casadevall, A., Perfect, J. R., Hannun, Y. A., and Del Poeta, M. (2001) Genes Dev. 15, 201-212), we demonstrated that an enzyme of the fungal sphingolipid pathway, Ipc1 (inositol-phosphorylceramide synthase-1), regulates melanin, a pigment required for the pathogenic fungus Cryptococcus neoformans to cause disease. In this study, we investigated the mechanism by which Ipc1 regulates melanin production. Because Ipc1 also catalyzes the production of diacylglycerol (DAG), a physiological activator of the classical and novel isoforms of mammalian protein kinase C (PKC), and because it has been suggested that PKC is required for melanogenesis in mammalian cells, we investigated whether Ipc1 regulates melanin in C. neoformans through the production of DAG and the subsequent activation of Pkc1, the fungal homolog of mammalian PKC. The results show that modulation of Ipc1 regulates the levels of DAG in C. neoformans cells. Next, we demonstrated that C. neoformans Pkc1 is a DAG-activated serine/threonine kinase and that the C1 domain of Pkc1 is necessary for this activation. Finally, through both pharmacological and genetic approaches, we found that inhibition of Pkc1 abolishes melanin formation in C. neoformans. This study identifies a novel signaling pathway in which C. neoformans Ipc1 plays a key role in the activation of Pkc1 through the formation of DAG. Importantly, this pathway is essential for melanin production with implications for the pathogenicity of C. neoformans.

Amino Acid Sequence↗
PubMed2004

Bimodal effect of advanced glycation end products on mesangial cell proliferation is mediated by neutral ceramidase regulation and endogenous sphingolipids.

Advanced glycation end-products (AGE) are generated by chronic hyperglycaemia and may cause diabetic microvascular complications such as diabetic nephropathy. Many factors influence the development of diabetic nephropathy; however, dysregulation of mesangial cell (MC) proliferation appears to play an early and crucial role. In this study, we investigated the effects of AGE on rat MC proliferation and the involvement of sphingolipids in the AGE response. Results show a bimodal effect of AGE on MC proliferation. Thus, low AGE concentrations (<1 microm) induced a significant increase (+26%) of MC proliferation, whereas higher concentrations (10 microm) markedly reduced it (-24%). In parallel, AGE exerted biphasic effects on neutral ceramidase expression and activity. Low AGE concentrations increased neutral ceramidase activity and expression, whereas high AGE concentrations showed opposite effects. Surprisingly, neutral ceramidase modulation did not result in changes of ceramide levels. However, the AGE (10 microm)-inhibitory effect on MC proliferation was associated with accumulation of sphingosine and was specifically prevented by blocking glucosylceramide synthesis, suggesting that the high AGE concentration effects are mediated by sphingosine and/or glycolipids. On the other hand, treatment of cells with low AGE concentrations led to an increase of sphingosine kinase activity and sphingosine-1-phosphate production that drove the increase of MC proliferation. Interestingly, in glomeruli isolated from streptozotocin-diabetic rats, a time-dependent modulation of ceramidase activity was observed as compared with controls. These results suggest that AGE regulate MC growth by modulating neutral ceramidase and endogenous sphingolipids.

Amidohydrolases↗
PubMed2005

Synthesis of sphingolipids with very long chain fatty acids but not ergosterol is required for routing of newly synthesized plasma membrane ATPase to the cell surface of yeast.

The proton pumping H(+)-ATPase, Pma1p, is an abundant and very long-lived polytopic protein of the Saccharomyces cerevisiae plasma membrane. Pma1p constitutes a major cargo of the secretory pathway and thus serves as an excellent model to study plasma membrane biogenesis. We have previously shown that newly synthesized Pma1p is mistargeted to the vacuole in an elo3Delta mutant that affects the synthesis of the ceramide-bound C26 very long chain fatty acid (Eisenkolb, M., Zenzmaier, C., Leitner, E., and Schneiter, R. (2002) Mol. Biol. Cell 13, 4414-4428) and now describe a more detailed analysis of the role of lipids in Pma1p biogenesis. Remarkably, a block at various steps of sterol biosynthesis, a complete block in sterol synthesis, or the substitution of internally synthesized ergosterol by externally supplied ergosterol or even by cholesterol does not affect Pma1p biogenesis or its association with detergent-resistant membrane domains (lipid "rafts"). However, a block in sphingolipid synthesis or any perturbation in the synthesis of the ceramide-bound C26 very long chain fatty acid results in mistargeting of newly synthesized Pma1p to the vacuole. Mistargeting correlates with a lack of newly synthesized Pma1p to acquire detergent resistance, suggesting that sphingolipids with very long acyl chains affect sorting of Pma1p to the cell surface.

Calcium-Transporting ATPases↗
PubMed1991

Sorting of sphingolipids in the endocytic pathway of HT29 cells.

The intracellular flow and fate of two fluorescently labeled sphingolipids, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoyl glucosyl sphingosine (C6-NBD-glucosylceramide) and C6-NBD-sphingomyelin, was examined in the human colon adenocarcinoma cell line HT29. After their insertion into the plasma membrane at low temperature and subsequent warming of the cells to 37 degrees C, both sphingolipid analogues were internalized by endocytosis, but their intracellular site of destination differed. After 30 min of internalization, C6-NBD-glucosylceramide was localized in the Golgi apparatus, as demonstrated by colocalization with fluorescently labeled ceramide, a Golgi complex marker, and by showing that monensin-induced disruption of the Golgi structure was paralleled by a similar perturbation of the fluorescence distribution. By contrast, C6-NBD-sphingomyelin does not colocalize with the tagged ceramide. Rather, a colocalization with ricin, which is internalized by endocytosis and predominantly reaches the lysosomes, was observed, indicating that the site of delivery of this lipid is restricted to endosomal/lysosomal compartments. Also, in monensin-treated cells no change in the distribution of fluorescence was observed. Thus, these results demonstrate that (sphingo)lipid sorting can occur in the endocytic pathway. Interestingly, the observed sorting phenomenon was specific for glucosylceramide, when compared to other glycolipids, while only undifferentiated HT29 cells displayed the different routing of the two lipids. In differentiated HT29 cells the internalization pathway of sphingomyelin and glucosylceramide was indistinguishable from that of transferrin.

Adenocarcinoma↗
PubMed1996

Sphingolipids and glycoproteins are differentially trafficked to the Chlamydia trachomatis inclusion.

Chlamydia trachomatis is an obligate intracellular pathogen that multiples within the confines of a membrane-bound vacuole called an inclusion. Approximately 40-50% of the sphingomyelin synthesized from exogenously added NBD-ceramide is specifically transported from the Golgi apparatus to the chlamydial inclusion (Hackstadt, T., M.A. Scidmore, and D.D. Rockey. 1995. Proc. Natl. Acad. Sci. USA. 92: 4877-4881). Given this major disruption of a cellular exocytic pathway and the similarities between glycolipid and glycoprotein exocytosis, we wished to determine whether the processing and trafficking of glycoproteins through the Golgi apparatus to the plasma membrane in chlamydia-infected cells was also disrupted. We analyzed the processing of several model glycoproteins including vesicular stomatitis virus G-protein, transferrin receptor, and human histocompatibility leukocyte class I antigen. In infected cells, the posttranslational processing and trafficking of these specific proteins through the Golgi apparatus and subsequent transport to the plasma membrane was not significantly impaired, nor were these glycoproteins found associated with the chlamydial inclusion membrane. Studies of receptor recycling from endocytic vesicles employing fluorescently and HRP-tagged transferrin and anti-transferrin receptor antibody revealed an increased local concentration of transferrin and transferrin receptor around but never within the chlamydial inclusion. However, Scatchard analysis failed to show either an increased intracellular accumulation of transferrin receptor or a decreased number of plasma membrane receptors in infected cells. Furthermore, the rate of exocytosis from the recycling endosomes to the plasma membrane was not altered in chlamydia-infected cells. Thus, although C. trachomatis disrupts the exocytosis of sphingolipids and the Golgi apparatus appears physically distorted, glycosylation and exocytosis of representative secreted and endocytosed proteins are not disrupted. These results suggest the existence of a previously unrecognized sorting of sphingolipids and glycoproteins in C. trachomatis-infected cells.

4-Chloro-7-nitrobenzofurazan↗
PubMed1998

PDMP blocks brefeldin A-induced retrograde membrane transport from golgi to ER: evidence for involvement of calcium homeostasis and dissociation from sphingolipid metabolism.

In this study, we show that an inhibitor of sphingolipid biosynthesis, D,L-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when BFA was allowed to exert its effect before PDMP addition, PDMP could not "rescue" the Golgi compartment. Evidence is presented showing that this action of PDMP is indirect, which means that the direct target is not sphingolipid metabolism at the Golgi apparatus. A fluorescent analogue of PDMP, 6-(N-[7-nitro-2,1, 3-benzoxadiazol-4-yl]amino)hexanoyl-PDMP (C6-NBD-PDMP), did not localize in the Golgi apparatus. Moreover, the effect of PDMP on membrane flow did not correlate with impaired C6-NBD-sphingomyelin biosynthesis and was not mimicked by exogenous C6-ceramide addition or counteracted by exogenous C6-glucosylceramide addition. On the other hand, the PDMP effect was mimicked by the multidrug resistance protein inhibitor MK571. The effect of PDMP on membrane transport correlated with modulation of calcium homeostasis, which occurred in a similar concentration range. PDMP released calcium from at least two independent calcium stores and blocked calcium influx induced by either extracellular ATP or thapsigargin. Thus, the biological effects of PDMP revealed a relation between three important physiological processes of multidrug resistance, calcium homeostasis, and membrane flow in the ER/ Golgi system.

Animals↗
PubMed2000

Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids.

Whereas the physiological significance of microsomal fatty acid elongation is generally appreciated, its molecular nature is poorly understood. Here, we describe tissue-specific regulation of a novel mouse gene family encoding components implicated in the synthesis of very long chain fatty acids. The Ssc1 gene appears to be ubiquitously expressed, whereas Ssc2 and Cig30 show a restricted expression pattern. Their translation products are all integral membrane proteins with five putative transmembrane domains. By complementing the homologous yeast mutants, we found that Ssc1 could rescue normal sphingolipid synthesis in the sur4/elo3 mutant lacking the ability to synthesize cerotic acid (C(26:0)). Similarly, Cig30 reverted the phenotype of the fen1/elo2 mutant that has reduced levels of fatty acids in the C(20)-C(24) range. Further, we show that Ssc1 mRNA levels were markedly decreased in the brains of myelin-deficient mouse mutants known to have very low fatty acid chain elongation activity. Conversely, the dramatic induction of Cig30 expression during brown fat recruitment coincided with elevated elongation activity. Our results strongly implicate this new mammalian gene family in tissue-specific synthesis of very long chain fatty acids and sphingolipids.

Acetyltransferases↗
PubMed2002

Sphingosine-1-phosphate phosphohydrolase in regulation of sphingolipid metabolism and apoptosis.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biological processes by binding to a family of G protein-coupled receptors or as an intracellular second messenger. Mammalian S1P phosphatase (SPP-1), which degrades S1P to terminate its actions, was recently cloned based on homology to a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast. Confocal microscopy surprisingly revealed that epitope-tagged SPP-1 is intracellular and colocalized with the ER marker calnexin. Moreover, SPP-1 activity and protein appeared to be mainly enriched in the intracellular membranes with lower expression in the plasma membrane. Treatment of SPP-1 transfectants with S1P markedly increased ceramide levels, predominantly in the intracellular membranes, diminished survival, and enhanced apoptosis. Remarkably, dihydro-S1P, although a good substrate for SPP-1 in situ, did not cause significant ceramide accumulation or increase apoptosis. Ceramide accumulation induced by S1P was completely blocked by fumonisin B1, an inhibitor of ceramide synthase, but only partially reduced by myriocin, an inhibitor of serine palmitoyltransferase, the first committed step in de novo synthesis of ceramide. Furthermore, S1P, but not dihydro-S1P, stimulated incorporation of [3H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide. Collectively, our results suggest that SPP-1 functions in an unprecedented manner to regulate sphingolipid biosynthesis and is poised to influence cell fate.

3T3 Cells↗
PubMed2001

Niemann-Pick C variant detection by altered sphingolipid trafficking and correlation with mutations within a specific domain of NPC1.

Niemann-Pick disease type C (NPC) is a fatal, autosomal recessive lipidosis characterized by lysosomal accumulation of unesterified cholesterol and multiple neurological symptoms, such as vertical supranuclear ophthalmoplegia, progressive ataxia, and dementia. More than 90% of cases of NPC are due to a defect in Niemann-Pick C1 (NPC1), a late endosomal, integral membrane protein that plays a role in cholesterol transport or homeostasis. Biochemical diagnosis of NPC has relied on the use of patient skin fibroblasts in an assay to demonstrate delayed low-density lipoprotein (LDL)-derived cholesterol esterification and a cytological technique-filipin staining-to demonstrate the intracellular accumulation of cholesterol. A small percentage of patients, referred to as "NPC variants," present with clinical symptoms of NPC but show near-normal results of these biochemical tests, making laboratory confirmation of NPC disease problematic. Here, we demonstrate that NPC-variant fibroblast samples can be detected as sphingolipid storage disease cells, using a fluorescent sphingolipid analog, BODIPY-lactosylceramide. This lipid accumulated in endosomes/lysosomes in variant cells preincubated with LDL cholesterol but targeted to the Golgi complex in normal cells under these conditions. The reproducibility of this technique was validated in a blinded study. In addition, we performed mutation analysis of the NPC1 gene in NPC variant and "classical" NPC cell samples and found a high incidence of specific mutations within the cysteine-rich region of NPC1 in variants. We also found that 5 of the 12 variant cell samples had no apparent defect in NPC1 but were otherwise indistinguishable from other variant cells. This is a surprising result, since, in general, approximately 90% of patients with NPC possess defects in NPC1. Our findings should be useful for the detection of NPC variants and also may provide significant new insight regarding NPC1 genotype/phenotype correlations.

Alleles↗
PubMed2001

Depletion of acyl-coenzyme A-binding protein affects sphingolipid synthesis and causes vesicle accumulation and membrane defects in Saccharomyces cerevisiae.

Deletion of the yeast gene ACB1 encoding Acb1p, the yeast homologue of the acyl-CoA-binding protein (ACBP), resulted in a slower growing phenotype that adapted into a faster growing phenotype with a frequency >1:10(5). A conditional knockout strain (Y700pGAL1-ACB1) with the ACB1 gene under control of the GAL1 promoter exhibited an altered acyl-CoA profile with a threefold increase in the relative content of C18:0-CoA, without affecting total acyl-CoA level as previously reported for an adapted acb1Delta strain. Depletion of Acb1p did not affect the general phospholipid pattern, the rate of phospholipid synthesis, or the turnover of individual phospholipid classes, indicating that Acb1p is not required for general glycerolipid synthesis. In contrast, cells depleted for Acb1p showed a dramatically reduced content of C26:0 in total fatty acids and the sphingolipid synthesis was reduced by 50-70%. The reduced incorporation of [(3)H]myo-inositol into sphingolipids was due to a reduced incorporation into inositol-phosphoceramide and mannose-inositol-phosphoceramide only, a pattern that is characteristic for cells with aberrant endoplasmic reticulum to Golgi transport. The plasma membrane of the Acb1p-depleted strain contained increased levels of inositol-phosphoceramide and mannose-inositol-phosphoceramide and lysophospholipids. Acb1p-depleted cells accumulated 50- to 60-nm vesicles and autophagocytotic like bodies and showed strongly perturbed plasma membrane structures. The present results strongly suggest that Acb1p plays an important role in fatty acid elongation and membrane assembly and organization.

Carrier Proteins↗
PubMed2004

Cross talk between sphingolipids and glycerophospholipids in the establishment of plasma membrane asymmetry.

Glycerophospholipids and sphingolipids are distributed asymmetrically between the two leaflets of the lipid bilayer. Recent studies revealed that certain P-type ATPases and ATP-binding cassette (ABC) transporters are involved in the inward movement (flip) and outward movement (flop) of glycerophospholipids, respectively. In this study of phytosphingosine (PHS)-resistant yeast mutants, we isolated mutants for PDR5, an ABC transporter involved in drug efflux as well as in the flop of phosphatidylethanolamine. The pdr5 mutants exhibited an increase in the efflux of sphingoid long-chain bases (LCBs). Genetic analysis revealed that the PHS-resistant phenotypes exhibited by the pdr5 mutants were dependent on Rsb1p, a putative LCB-specific transporter/translocase. We found that the expression of Rsb1p was increased in the pdr5 mutants. We also demonstrated that expression of RSB1 is under the control of the transcriptional factor Pdr1p. Expression of Rsb1p also was enhanced in mutants for the genes involved in the flip of glycerophospholipids, including ROS3, DNF1, and DNF2. These results suggest that altered glycerophospholipid asymmetry induces the expression of Rsb1p. Conversely, overexpression of Rsb1p resulted in increased flip and decreased flop of fluorescence-labeled glycerophospholipids. Thus, there seems to be cross talk between sphingolipids and glycerophospholipids in maintaining the functional lipid asymmetry of the plasma membrane.

4-Chloro-7-nitrobenzofurazan↗