Sphingolipid Fatty Acids Research Articles

A Sphingolipid Fatty Acid Constituent Made by Alkyne trans‐Hydrogenation: Total Synthesis of Symbioramide

AbstractA scalable approach to (2R)‐hydroxyoctadec‐3‐enoic acid ((−)‐2) is reported, which is the fatty acid constituent of numerous sphingolipids of biological significance. Key to success was the chemoselective trans‐hydrogenation of a functionalized propargylic alcohol precursor with [Cp*RuCl]4 as the catalyst, followed by lipase‐catalyzed resolution of the racemic product. (−)‐2 then served the shortest total synthesis of the marine natural product symbioramide known to date.magnified image

Convergence of sphingolipid desaturation across over 500 million years of plant evolution.

For plants, acclimation to low temperatures is fundamental to survival. This process involves the modification of lipids to maintain membrane fluidity. We previously identified a new cold-induced putative desaturase in Physcomitrium (Physcomitrella) patens. Lipid profiles of null mutants of this gene lack sphingolipids containing monounsaturated C24 fatty acids, classifying the new protein as sphingolipid fatty acid denaturase (PpSFD). PpSFD mutants showed a cold-sensitive phenotype as well as higher susceptibility to the oomycete Pythium, assigning functions in stress tolerance for PpSFD. Ectopic expression of PpSFD in the Atads2.1 (acyl coenzyme A desaturase-like 2) Arabidopsis thaliana mutant functionally complemented its cold-sensitive phenotype. While these two enzymes catalyse a similar reaction, their evolutionary origin is clearly different since AtADS2 is a methyl-end desaturase whereas PpSFD is a cytochrome b5 fusion desaturase. Altogether, we suggest that adjustment of membrane fluidity evolved independently in mosses and seed plants, which diverged more than 500 million years ago.

Arabidopsis Bax inhibitor-1 promotes sphingolipid synthesis during cold stress by interacting with ceramide-modifying enzymes

Bax inhibitor-1 (BI-1) is a widely conserved cell death suppressor localized in the endoplasmic reticulum membrane. Our previous results revealed that Arabidopsis BI-1 (AtBI-1) interacts with not only Arabidopsis cytochrome b 5 (Cb5), an electron transfer protein, but also a Cb5-like domain (Cb5LD)-containing protein, Saccharomyces cerevisiae fatty acid 2-hydroxylase 1, which 2-hydroxylates sphingolipid fatty acids. We have now found that AtBI-1 binds Arabidopsis sphingolipid Δ8 long-chain base (LCB) desaturases AtSLD1 and AtSLD2, which are Cb5LD-containing proteins. The expression of both AtBI-1 and AtSLD1 was increased by cold exposure. However, different phenotypes were observed in response to cold treatment between an atbi-1 mutant and a sld1sld2 double mutant. To elucidate the reasons behind the difference, we analyzed sphingolipids and found that unsaturated LCBs in atbi-1 were not altered compared to wild type, whereas almost all LCBs in sld1sld2 were saturated, suggesting that AtBI-1 may not be necessary for the desaturation of LCBs. On the other hand, the sphingolipid content in wild type increased in response to low temperature, whereas total sphingolipid levels in atbi-1 were unaltered. In addition, the ceramide-modifying enzymes AtFAH1, sphingolipid base hydroxylase 2 (AtSBH2), acyl lipid desaturase 2 (AtADS2) and AtSLD1 were highly expressed under cold stress, and all are likely to be related to AtBI-1 function. These findings suggest that AtBI-1 contributes to synthesis of sphingolipids during cold stress by interacting with AtSLD1, AtFAH1, AtSBH2 and AtADS2.

Sustained activation of sphingomyelin synthase by 2-hydroxyoleic acid induces sphingolipidosis in tumor cells

The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor drug, involves the rapid and specific activation of sphingomyelin synthase (SMS), leading to a 4-fold increase in SM mass in tumor cells. In the present study, we investigated the source of the ceramides required to sustain this dramatic increase in SM. Through radioactive and fluorescent labeling, we demonstrated that sphingolipid metabolism was altered by a 24 h exposure to 2OHOA, and we observed a consistent increase in the number of lysosomes and the presence of unidentified storage materials in treated cells. Mass spectroscopy revealed that different sphingolipid classes accumulated in human glioma U118 cells after exposure to 2OHOA, demonstrating a specific effect on C16-, C20-, and C22-containing sphingolipids. Based on these findings, we propose that the demand for ceramides required to sustain the SMS activation (ca. 200-fold higher than the basal level) profoundly modifies both sphingolipid and phospholipid metabolism. As the treatment is prolonged, tumor cells fail to adequately metabolize sphingolipids, leading to a situation resembling sphingolipidosis, whereby cell viability is compromised.

Arabidopsis mutants of sphingolipid fatty acid α‐hydroxylases accumulate ceramides and salicylates

In Arabidopsis, the fatty acid moiety of sphingolipids is mainly α-hydroxylated. The consequences of a reduction in this modification were analysed. Mutants of both Fatty Acid Hydroxylase genes (AtFAH1 and AtFAH2) were analysed for sphingolipid profiles. To elucidate further consequences of the mutations, metabolic analyses were performed and the influence on pathogen defence was determined. Ceramide and glucosylceramide profiles of double-mutant plants showed a reduction in sphingolipids with α-hydroxylated fatty acid moieties, and an accumulation of sphingolipids without these moieties. In addition, the free trihydroxylated long-chain bases and ceramides were increased by five- and ten-fold, respectively, whereas the amount of glucosylceramides was decreased by 25%. Metabolite analysis of the double mutant revealed salicylates as enriched metabolites. Infection experiments supported the metabolic changes, as the double mutant showed an enhanced disease-resistant phenotype for infection with the obligate biotrophic pathogen Golovinomyces cichoracearum. In summary, these results suggest that fatty acid hydroxylation of ceramides is important for the biosynthesis of complex sphingolipids. Its absence leads to the accumulation of long-chain bases and ceramides as their precursors. This increases salicylate levels and resistance towards obligate biotrophic fungal pathogens, confirming a role of sphingolipids in salicylic acid-dependent defence reactions.

Arabidopsis Sphingolipid Fatty Acid 2-Hydroxylases (AtFAH1 and AtFAH2) Are Functionally Differentiated in Fatty Acid 2-Hydroxylation and Stress Responses

2-Hydroxy fatty acids (2-HFAs) are predominantly present in sphingolipids and have important physicochemical and physiological functions in eukaryotic cells. Recent studies from our group demonstrated that sphingolipid fatty acid 2-hydroxylase (FAH) is required for the function of Arabidopsis (Arabidopsis thaliana) Bax inhibitor-1 (AtBI-1), which is an endoplasmic reticulum membrane-localized cell death suppressor. However, little is known about the function of two Arabidopsis FAH homologs (AtFAH1 and AtFAH2), and it remains unclear whether 2-HFAs participate in cell death regulation. In this study, we found that both AtFAH1 and AtFAH2 had FAH activity, and the interaction with Arabidopsis cytochrome b₅ was needed for the sufficient activity. 2-HFA analysis of AtFAH1 knockdown lines and atfah2 mutant showed that AtFAH1 mainly 2-hydroxylated very-long-chain fatty acid (VLCFA), whereas AtFAH2 selectively 2-hydroxylated palmitic acid in Arabidopsis. In addition, 2-HFAs were related to resistance to oxidative stress, and AtFAH1 or 2-hydroxy VLCFA showed particularly strong responses to oxidative stress. Furthermore, AtFAH1 interacted with AtBI-1 via cytochrome b₅ more preferentially than AtFAH2. Our results suggest that AtFAH1 and AtFAH2 are functionally different FAHs, and that AtFAH1 or 2-hydroxy VLCFA is a key factor in AtBI-1-mediated cell death suppression.

Functional association of cell death suppressor, Arabidopsis Bax inhibitor‐1, with fatty acid 2‐hydroxylation through cytochrome b5

Bax inhibitor-1 (BI-1) is a widely conserved cytoprotective protein localized in the endoplasmic reticulum (ER) membrane. We identified Arabidopsis cytochrome b(5) (AtCb5) as an interactor of Arabidopsis BI-1 (AtBI-1) by screening the Arabidopsis cDNA library with the split-ubiquitin yeast two-hybrid (suY2H) system. Cb5 is an electron transfer protein localized mainly in the ER membrane. In addition, a bimolecular fluorescence complementation (BiFC) assay and fluorescence resonance energy transfer (FRET) analysis confirmed that AtBI-1 interacted with AtCb5 in plants. On the other hand, we found that the AtBI-1-mediated suppression of cell death in yeast requires Saccharomyces cerevisiae fatty acid hydroxylase 1 (ScFAH1), which had a Cb5-like domain at the N terminus and interacted with AtBI-1. ScFAH1 is a sphingolipid fatty acid 2-hydroxylase localized in the ER membrane. In contrast, AtFAH1 and AtFAH2, which are functional ScFAH1 homologues in Arabidopsis, had no Cb5-like domain, and instead interacted with AtCb5 in plants. These results suggest that AtBI-1 interacts with AtFAHs via AtCb5 in plant cells. Furthermore, the overexpression of AtBI-1 increased the level of 2-hydroxy fatty acids in Arabidopsis, indicating that AtBI-1 is involved in fatty acid 2-hydroxylation.

Selective Substrate Supply in the Regulation of Yeast de Novo Sphingolipid Synthesis

The heat stress response of Saccharomyces cerevisiae is characterized by transient cell cycle arrest, altered gene expression, degradation of nutrient permeases, trehalose accumulation, and translation initiation of heat shock proteins. Importantly heat stress also induces de novo sphingolipid synthesis upon which many of these subprograms of the heat stress response depend. Despite extensive data addressing the roles for sphingolipids in heat stress, the mechanism(s) by which heat induces sphingolipid synthesis remains unknown. This study was undertaken to determine the events and/or factors required for heat stress-induced sphingolipid synthesis. Data presented indicate that heat does not directly alter the in vitro activity of serine palmitoyltransferase (SPT), the enzyme responsible for initiating de novo sphingolipid synthesis. Moreover deletion of the small peptide Tsc3p, which is thought to maximize SPT activity, specifically reduced production of C(20) sphingolipid species by over 70% but did not significantly decrease overall sphingoid base production. In contrast, the fatty-acid synthase inhibitor cerulenin nearly completely blocked sphingoid base production after heat, indicating a requirement for endogenous fatty acids for heat-mediated sphingoid base synthesis. Consistent with this, genetic studies show that fatty acid import does not contribute to heat-induced de novo synthesis under normal conditions. Interestingly the absence of medium serine also ameliorated heat-induced sphingoid base production, indicating a requirement for exogenous serine for the response, and consistent with this finding, disruption of synthesis of endogenous serine did not affect heat-induced sphingolipid synthesis. Serine uptake assays indicated that heat increased serine uptake from medium by 100% during the first 10 min of heat stress. Moreover treatments that increase serine uptake in the absence of heat including acute medium acidification and glucose treatment also enhanced de novo sphingoid base synthesis equivalent to that induced by heat stress. These data agree with findings from mammalian systems that availability of substrates is a key determinant of flux through sphingolipid synthesis. Moreover data presented here indicate that SPT activity can be driven by several factors that increase serine uptake in the absence of heat. These findings may provide insights into the many systems in which de novo synthesis is increased in the absence of elevated in vitro SPT activity.

Analyses of Pneumocystis Fatty Acids

The major ester-linked fatty acids of the total lipids extracted from Pneumocystis carinii isolated from the lungs of corticosteroid-treated rats were 16:0, 18:0, 18:1, 18:2 and 20:4. Others detected included 14:0, 16:1 and 22:4. The major sphingolipid fatty acids were 16:0, 18:0, 22:0, 24:0 and 24:1; others included 14:0, 18:1, 20:0, 23:0, 24:2 and 26:0. The total lipid fatty acid compositions of preparations from appropriate lung controls were similar to those of the organism.

Analyses of Pneumocystis Fatty Acids

ABSTRACTThe major ester‐linked fatty acids of the total lipids extracted from Pneumocystis carinii isolated from the lungs of corticosteroid‐trcaicd rats were 16:0. 18:0, 18:1, 18:2 and 20:4. Others detected included 14:0, 16:1 and 22:4. The major sphingolipid fatty acids were 16:0, 18:0,22:0,24:0 and 24:1; others included 14:0, 18:1, 20:0, 23:0, 24:2 and 26:0. The total lipid fatty acid compositions of preparations from appropriate lung controls were similar to those of the organism.

Lipid composition and sphingolipid fatty acids of myelin in fruit bat brain

Myelin was isolated from the brain of adult fruit bats ( Rousettus aegyptiacus) in a discontinuous sucrose gradient. Cholesterol comprised 189.0 mol/100 mol lipid phosphorus, galactolipids 60.3 mol/100 mol phosphorus and plasmalogens 32.5 mol/100 mol phosphorus. Choline and ethanolamine glycerophosphatide were present in nearly equal amounts followed by serine glycerophosphatide, sphingomyelin and inositol glycerophosphatides. The fatty acid composition of sphingomyelin and non-hydroxy cerebroside was determined by gas-liquid chromatography. Fatty acids were mainly saturated or mono-unsaturated with a small percentage of polyunsaturated fatty acids present. The lipid composition and sphingolipid fatty acid distribution in bat myelin was fairly similar to that of other species.

Prepartion of sphingolipid fatty acid methyl esters for determination by gas—liquid chromatography

Sphingolipid faty acids are first convrted to a mixture of free acids and their n-butyl esters by heating the specimen at 82°C in aqueous butanolic hydrogen chloride; the butyl esters are then saponified with methanolic potassium hydroxide. After acidification and extraction into hexane, the fatty acids are extracted into a very small volume of aqueous trimethyl( m-trifluorotoylyl)ammonium hydroxide (TMTFTH) injection of an aliquot of the TMTFTH extract into the gas chromatograph yields the fatty acid methyl esters by pyrolytic methylation of the quartenary ammonium salts of the fatty acids. The preparation of a specimen ready for gas—liquid chromatographic(GLC) analysis with quantitative recovery of teh sphingolipid fatty acids can be accoomplished in less than 2 h. By comparison, none of a number of well-accepted techniques for the release of sphingomyelin fatty acids by hydrolysis or methanolysis released the fatty acids quantitatively in less than 3 h, and all required additional manipulations before GLC analysis.

Delta 15,18-tetracosadienoic acid content of sphingolipids from platelets and erythrocytes of animals fed diets high in saturated or polyunsaturated fats.

AbstractThe effect of diets high (15%) in saturated (beef tallow) or polyunsaturated (corn or cottonseed oil) fatty acids on the fatty acid composition of sphingomyelin from canine erythrocytes and platelets and sphingomyelin and neutral glycosphingolipids of swine erythrocytes was determined. Sphingolipids of platelets and erythrocytes from animals fed high levels of corn or cottonseed oil exhibited a dramatic alteration in their fatty acid composition, most notable of which was a 50% reduction in nervonic acid (24∶1ω9) as compared to levels observed in control or tallow fed animals. This decrease was compensated for by a quantitatively similar increase in a C24 dienoic acid. The long chain dienoic acid was isolated by silver nitrate thin layer chromatography and determined by analysis of its oxidation products to be Δ15, 18‐tetracosadienoic acid (24∶2ω6). When the animals were fed the diets high in polyunsaturates, the 24∶2ω6 represented 13, 20, and 9% of the sphingomyelin fatty acids from canine erythrocytes, platelets, and swine erythrocytes, respectively, and 5% of the neutral glycosphingolipid fatty acids of swine erythrocytes. In contrast, the 24∶2ω6 represented less than 4% of the total cellular sphingolipid fatty acids in animals fed the control or high beef tallow diets. The 24∶1ω9 in the sphingolipids of the animals fed the polyunsaturated diet was roughly equal to that of 24∶2ω6, whereas in the sphingolipids of animals fed the control or saturated fat (beef tallow) diet, the 24∶1ω9 was twice these values. Since sphingomyelin is a membrane component, the increase in unsaturation (24∶2ω6) in its fatty acid moiety induced by dietary polyunsaturates may affect membrane fluidity and may alter membrane properties.

In vivo metabolism of [G-3H]lignoceric acid in developing rat brain.

Abstract— [G‐3H]Lignoceric acid (tetracosanoic acid) was injected into the brains of 20‐day‐old rats, and the animals were killed after 8, 24, or 72 h. Various lipids were isolated from these brains, and the distribution of radioactivity was determined. The injected free acid rapidly disappeared, and the radioactivity was incorporated into varying chain‐length nonhydroxy‐ and hydroxy saturated fatty acids of sphingolipids and phospholipids. Little radioactivity was found in unsaturated acids, sphingo‐sine, and cholesterol. A time‐dependent shift of the label among various fatty acids was relatively small 8 h after injection, probably because of the metabolic stability of the brain sphingolipids. In cerebrosides, the radioactivity was equally distributed between nonhydroxy and x‐hydroxy fatty acids of all chain lengths. C23 and C22 fatty acids contained equal total radioactivities; C23 and C24 fatty acids contained similar specific activities. These results confirm the significant role of a‐hydroxylation and 2‐oxidation in the synthesis of very long‐chain fatty acids in brain. In total lipid fatty acids, docosanoic acid (22:0) contained more radioactivity than its α‐oxidation precursor, α‐hydroxytricosanoic acid (23h:0) at all times. In sphingolipid fatty acids, the specific activity of 21:0 was always higher than that of its ct‐oxidation precursor 22:0. These observations indicate that part of the 22:0 and 21:0 was derived by β‐oxidation from the injected lignoceric acid or its α‐oxidation product, respectively.

Effect of sterol replacement in vivo on the fatty acid composition of Tetrahymena.

The addition of ergosterol to cultures of Tetrahymena pyriformis results in (a) the accumulation of the sterol by the cells; (b) the inhibition of the synthesis of the pentacyclic triterpenoid alcohol, tetrahymanol; (c) the replacement of tetrahymanol by ergosterol in the ciliate membranes. The dry weight and lipid content of sterol-supplemented ciliates did not differ from the controls. Examination of the lipid classes revealed no change in composition except for a higher content of ergosterol in supplemented cells than tetrahymanol in control cultures. The relative proportions of triglycerides, the major classes of polar lipids, 1-alkyl phospholipids and phosphonolipids, appeared unaltered. A complex array of fatty acids is found in this ciliate. Several acids not reported previously in this organism were isolated and identified, and the novel fatty acid 18:2 delta 6, 11, was found in substantial amounts. Ergosterol supplementation altered the proportions of the fatty acids, although not all lipid classes were affected to the same extent. The changes noted were of three general types: (a) a shortening of the fatty acyl chain length in the acids of the normal series; (b) a lowering in the degree of unsaturation; (c) a discrimination between two isomers of lionoleate, 18:2 delta 6, 11 and 18:2 delta 9, 12. The former is elevated in the presence of ergosterol while the latter is depressed. Each class of polar lipids has a distinctive fatty acid composition. Among the glycerophospholipids, cardiolipin and phosphatidylcholine were least affected, while the mixture of 1-alkyl-2-acyl-sn-glycero-3-(2-aminoethyl)-phosphonate and 1,2-diacyl-sn-glycero-3-(2-aminoethyl)-phosphonate was most markedly altered. Sphingolipid fatty acid composition was influenced by ergosterol supplementation. Two changes were noted: (a) a reduction in the length of the hydrocarbon chain; (b) an increase in the proportion of alpha-hydroxy acids. The impact of ergosterol on the fatty acid composition of the polar lipids may be on fatty acid biosynthesis, on incorporation of fatty acids, or on the turnover rates of the fatty acyl groups. Ergosterol is concentrated in the ciliary (limiting) membrane, as are the polar lipids most affected. This localization allows the speculation that the change in fatty acid composition may be related to the maintenance of optimal membrane properties upon the introduction of the sterol.

Sphingolipid fatty acid composition of rat brain "membranous sacs".

AbstractA fraction enriched in “membranous sacs” was isolated from the brains of young rats. Ceramide, glucosylceramide, galactosylceramide, lactosylceramide, sulfatide, sphingomyelin and ganglioside were purified. The fatty acids of the individual compounds were identified after hydrolysis. Certain similarities and dissimilarities were observed.

Long-chain bases in the sphingolipids of bovine milk and kidney, rumen bacteria, rumen protozoa, hay and concentrate

It has been suggested that many of the unusual long-chain bases in bovine milk and kidney sphingomyelins are derived from sphingolipids of dietary plant material and rumen protozoa. The long-chain bases in the rumen and food of cows were therefore studied to determined whether they could be the source of these unusual bases. Milk and kidney sphingomyelins from cows on normal and protein-free diets had almost identical base compositions. Since the rumen of the cows on the protein-free diet contained no protozoa, it was concluded that the unusual bases in the bovine sphingomyelins were not derived from protozoan sphingolipids. The bases could, however, have been derived from bacterial sphingolipids. A comparison was then made of sphingolipids from the milk and rumen of two cows on a hay and concentrate diet. Gross differences in the sphingolipids and sphingolipid fatty acids showed that intact rumen sphingolipids or their ceramide moieties were not absorbed or retained to any extent. The long-chain bases in the dietary plant material were mostly cis and trans isomers of t18:1 8 with small amounts of t18:0, d18:0, d18:1 4, d18:1 * and d18:2 4.*. The rumen bacterial and protozoan sphingolipids contained mostly n-, iso-andanteiso-d17:0 to d20:0 and t17:0 to t20:0 bases together with some t18:1 possibly derived from plant sphingolipids. About 70% of the trihydorxy bases and almost all of the dihydroxy bases in the rumen contents were from bacterial and protozoan sphingolipids. There were several points of difference between the long-chain bases in the rumen contents and those in milk sphingomyelin. It was concluded that little or none of the bases in milk and kidney sphingomyelins were of dietary origin. The digestive metabolism of sphingolipids in the cow is therefore probably similar to that of the rat [Nilsson, A. (1968) Biochim. Biophys. Acta 164, 575–584 and Nilsson, A. (1969) Biochim. Biophys. Acta 187, 113–121]. From these conclusions it follows that bovine tissues can synthesise straight and branched d:0, d:1 41 and t:0 long-chain bases.

Abnormality in sphingolipid fatty acids from sciatic nerve and brain of Quaking mice.

Abnormality in sphingolipid fatty acids from sciatic nerve and brain of Quaking mice.

In vivo conversion of labeled fatty acids to the sphingolipid fatty acids in rat brain

Emulsions of carboxyl-labeled palmitate, stearate, lignocerate, and dl -cerebronate were injected into the brains of weanling rats. After various time intervals, the rats were sacrificed, and the saturated fatty acids of the brain sphingolipids were isolated and decarboxylated. All four acids had been incorporated into the sphingolipids. The palmitate and stearate gave rise to radioactive lignocerate and cerebronate in which the carboxyl groups were relatively nonradioactive, indicating the latter acids are made by chain lengthening of the shorter acids. The lignocerate-C14 gave rise to labeled cerebronate in which the carboxyl group contained nearly all the activity, showing that the normal acid is converted fairly directly into the hydroxy acid. Some radioactivity was found also in the shorter acids, apparently by the intermediate degradation of lignocerate to acetate. The injected cerebronate-C14 underwent little or no conversion to normal acids.