Preparation of homogenous oligosaccharide chains from glycosphingolipids

After the discovery of glycosphingolipid (GSL) glycan detaching enzymes, Rhodococcal endoglycoceramidase (EGCase) and leech ceramide glycanase (CGase), the method for enzymatically releasing glycans from GSLs has become the method of choice for preparing intact ceramide-free oligosaccharide chains from GSLs. This paper describes (1) the preparation of the intact oligosaccharides from GM1 (II³NeuAcGgOse₄Cer) and GbOse₄Cer as examples to show the use of CGase to prepare intact glycan chains from GSLs, and (2) the specificity and detergent requirements of Rhodococcal EGCases for the release of glycan chains from different GSLs.     

Substrate-specificities of acid and alkaline ceramidases in fibroblasts from patients with Farber disease and controls

The specific activity of acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) was measured at pH4.5 in normal fibroblasts and in fibroblasts from patients with Farber disease and obligate heterozygotes. Greater activity was found when the synthetically made ceramide substrates contained shorter-chain fatty acids or higher content of double bonds. Acid ceramidase activities towards N-lauroyl- (C_12:0), N-myristoyl- (C_14:0) and N-palmitoyl- (C_16:0) sphingosine (C_18:1) were respectively about 38, 26 and 6 times higher than the activity towards the N-stearoyl (C_18:0) substrate. The activity towards N-linolenoylsphingosine (C_18:3/C_18:1), N-linoleoylsphingosine (C_18:2/C_18:1) and N-oleoylsphingosine (C_18:1/C_18:1) were respectively about 5, 4 and 3 times higher than the activity towards N-stearoylsphingosine (C_18:0/C_18:1). The activity towards N-stearoyldihydrosphingosine (C_18:0/C_18:0) was about 40% of that towards N-stearoylsphingosine. Fibroblast alkaline ceramidase possessed significant activity only towards ceramides of unsaturated fatty acids, with a pH optimum of about 9.0. Deficiency of acid ceramidase activity in fibroblasts from patients with Farber disease and intermediate activities in obligate heterozygotes were demonstrated with all ceramides examined except for N-hexanoylsphingosine (C_6:0/C_18:1), whereas alkaline ceramidase activity was unaffected. Comparative kinetic studies of acid ceramidase activity with N-lauroylsphingosine and N-oleoylsphingosine demonstrated about 5 (2–12)-fold and 7 (4–17)-fold higher K_m values in fibroblasts from patients with Farber disease as compared with normal controls. N-Lauroylsphingosine, towards which acid ceramidase activity in control fibroblasts was about 10 times higher than that towards N-oleoylsphingosine, may serve as a better substrate for enzymic diagnosis of Farber disease as well as for further characterization of the catalytically defective acid ceramidase.     

Omega-3 fatty acids concentrate production by enzyme-catalyzed ethanolysis of supercritical CO<Subscript>2</Subscript> extracted oyster oil

The separation of oil by a suitable technique from the Pacific oyster muscle is important for the utilization of the oil as a ω-3 polyunsaturated fatty acids (ω-3 PUFAs) source and production of bio-functional peptides/ oligosaccharides from oil-free residue. This study was conducted to prepare ω-3 PUFAs concentrate from supercritical carbon dioxide (SC-CO₂) extracted Pacific oyster oil by enzyme-catalyzed ethanolysis reactions. SC-CO₂ extractions were done at different temperatures and pressures to optimize suitable extraction conditions and extracted oils were compared with Soxhlet (n-hexane) extracted oil to evaluate the yield and quality. Oil extracted by SC-CO₂ at optimized conditions was used for ethanolysis reaction catalyzed by immobilized sn-1,3 specific lipases, namely Novozymes-435, Lipozyme TLIM, and Lipozyme RMIM to produce 2-monoacylglycerols (2-MAG) rich in ω-3 PUFAs. The optimum temperature and pressure for SC-CO₂ extractions of oyster oil was 50°C and 30 MPa. In this condition, the yield of oil was 5.96% and the acid, peroxide, free fatty acid, and p-anisidine values were 4.49 mg KOH/g, 4.72 meq/kg, 3.42%, and 10.03, respectively. The ω-3 PUFAs content significantly increased in 2-MAG obtained from Novozymes 435, Lipozyme TLIM, and Lipozyme RMIM to 43.03 ± 0.36, 45.95 ± 0.29, and 40.50 ± 0.77%, respectively (p < 0.05). A thin layer chromatography (TLC) analysis confirmed the production and separation of 2-MAG in the ethanolysis process. The ratio of total ω-3 to ω-6 fatty acids was almost twice in 2-MAG of SC-CO₂ extracted oyster oil. SC-CO₂ extracted Pacific oyster oil can be used for sn-1,3 specific lipases catalyzed ethanolysis to produce ω-3 PUFAs rich in 2-MAG.     

Molecular cloning and characterization of neutral ceramidase homologue from the red flour beetle, Tribolium castaneum

Ceramidase plays an important role in regulating the metabolism of sphingolipids, such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P), by controlling the hydrolysis of ceramide. Here we report the cloning and biochemical characterization of a neutral ceramidase from the red flour beetle Tribolium castaneum which is an important storage pest. The Tribolium castaneum neutral ceramidase (Tncer) is a protein of 696 amino acids. It shares a high degree of similarity in protein sequence to neutral ceramidases from various species. Tncer mRNA levels are higher in the adult stage than in pre-adult stages, and they are higher in the reproductive organs than in head, thorax, and midgut. The mature ovary has higher mRNA levels than the immature ovary. Tncer is localized to the plasma membrane. It uses various ceramides (D-erythro-C₆, C₁₂, C₁₆, C_18:1, and C_24:1-ceramide) as substrates and has an abroad pH optimum for its in vitro activity. Tncer has an optimal temperature of 37 °C for its in vitro activity. Its activity is inhibited by Fe²⁺. These results suggest that Tncer has distinct biochemical properties from neutral ceramidases from other species.     

Brain sphingoglycolipids in Krabbe's globoid cell leucodystrophy

Abstract— Seven sphingoglycolipids were isolated from the white matter of a patient with globoid cell leucodystrophy (Krabbe's disease). After purification by saponification and column and preparative thin-layer chromatography, these compounds were analysed for the carbohydrate composition and sequence and for fatty acid composition by paper and gas-liquid chromatography. The compounds were identified as gluco- and galactocerebrosides, lactosyl-ceramide, digalactosy I-glucosyl-ceramide, two types of tetrahexosyl-ceramides (asialo-ganglioside and globoside), and sulphatide. Glucocerebrosideconstituted 13 percent of total cerebroside in white matter, but sulphatide contained only galactose. Galactocere-broside and sulphatide exhibited compositions of fatty acids similar to those in normal white matter, with only minor abnormalities. Other sphingoglycolipids showed fatty acid patterns with relatively high proportions of longer-chain fatty acids, rather than the predominant C_18:0 acid usually found in ceramide hexosides of the brain. Hematoside, also found in the white matter in a significant amount, similarly contained a large proportion of longer-chain fatty acids, whereas other gangliosides contained predominantly C_18:0 acid. The abnormal ceramide hexoside pattern was restricted mostly to white matter except for glucocerebroside, which constituted 32 per cent of grey matter cerebroside. We postulate that the visceral type of sphingoglycolipids may be constituents of globoid cells, abundantly present in white matter and considered to be cells of mesenchymal origin.     

Optimization of <Emphasis Type="Italic">Prochlorothrix hollandica</Emphasis> cyanobacteria culturing for obtaining myristoleic acid

Plankton filament cyanobacteria Prochlorothrix hollandica is characterized by a very high content of C₁₄ and C₁₆ fatty acids (FA) in the lipid membranes. Depending on culturing conditions of the cyanobacteria, total concentrations of myristic and myristoleic acids can reach 35% and those of palmitic and palmitoleic acids can reach 60% of all esterified FA cells. In P. hollandica, a variety of monounsaturated FA is represented by myristoleic and palmitic acids, and by hexadecenoic (C_16:1) acid with olefin bond of cis-configuration, located in the Δ4 position. The process of intensive culturing for P. hollandica cells to yield a maximal biomass in order to isolate the pure drug of myristoleic acid derivative has been optimized. The use of a threestage purification gives 30 mg of chromatographically pure myristoleic acid methyl ester from 17 g of P. hollandica raw biomass (dry mass is 3 g), which is 1% of dry cell mass.     

Characterization of genes involved in ceramide metabolism in the Pacific oyster (Crassostrea gigas)

ABSTRACT: BACKGROUND: The lipid signaling molecule, ceramide, is a key component of the vertebrate stress response, however, there is limited information concerning its role in invertebrate species. In order to identify genes involved in ceramide metabolism in bivalve molluscs, Pacific oyster genomic resources were examined for genes associated with ceramide metabolism and signaling. RESULTS: Several genes were identified including full-length sequences characterized for serine palmitoyltransferase-1, 3-ketodihydrosphingosine reductase, acid ceramidase, and ceramide glucosyltransferase. Genes involved in ceramide synthesis and metabolism are conserved across taxa in both form and function. Expression analysis as assessed by quantitative PCR indicated all genes were expressed at high levels in gill tissue. The role of the ceramide pathway genes in the invertebrate stress response was also explored by measuring expression levels in adult oysters exposed to Vibrio vulnificus. Two genes demonstrated increased expression during the bacterial challenge: a gene involved in hydrolytic breakdown of ceramide (acid ceramidase) and a gene involved in de novo generation of ceramide (3-ketodihydrosphingosine reductase), suggesting a possible role of ceramide in the invertebrate stress and immune responses. CONCLUSIONS: In silico and laboratory results support that Pacific oysters have the basic components of the ceramide metabolism pathway. These results also indicate that ceramide may have analogous functions in vertebrates and invertebrates. The gene expression pattern of acid ceramidase and 3-kethodihydrosphingosine reductase in response to bacterial exposure especially supports that ceramide and sphingolipid metabolism may be involved in the oyster's stress and/or immune responses.     

Changes in the composition of winter rape oil during seed maturation

The course of biosynthesis of fatty acids in the seeds of winter rape (Brassica napus L. ssp.oleifera, f.biennis cv. T?ebí?ská) was investigated. After the termination of flowering seed samples were taken at five intervals, the seeds were divided into 4 fractions according to size, and their weight, water content, oil content and fatty acid composition were determined. The oil content was found to increase in all size categories with time, with the exception of a minute drop when complete maturity is reached. Larger seeds contained more oil. The fatty acid composition changes with time in the individual size fractions almost continuously. The same holds for differences between seed sizes of the same sample. The main change in oil composition consists in the decrease of C₁₈ acids in favour of C₂₂ acids. Greatest decrements during maturation were found with oleic acid, less with linoleic acid. In absolute amounts the quantity of all synthesized acids rises, the greatest rise being observed with C₂₂ acids (i.e. predominantly erucic acid). It follows from the mean rates of synthesis of the individual groups (C₁₆, C₁₈, C₂₀, C₂₂) of fatty acids that the fraction of C₂₂ rate of synthesis increases, while that of the C₁₈ acids decreases with the same speed. The results indicate that the fatty acid synthesis is most intense during the second half of seed maturation, the main role being played by accelerating the synthesis of higher acids, especially of erucic acid.     

Systematic analyses of free ceramide species and ceramide species comprising neutral glycosphingolipids by MALDI-TOF MS with high-energy CID

Free ceramides and glycosphingolipids (GSLs) are important components of the membrane microdomain and play significant roles in cell survival. Recent studies have revealed that both fatty acids and long-chain bases (LCBs) are more diverse than expected, in terms of i) alkyl chain length, ii) hydroxylation and iii) the presence or absence of double bonds. Electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been well utilized to characterize sphingolipids with high throughput, but reports to date have not fully characterized various types of ceramide species such as hydroxyl fatty acids and/or trihydroxy-LCBs of both free ceramides and the constituent ceramides in neutral GSLs. We performed a systematic analysis of both ceramide species, including LCBs with nona-octadeca lengths using MALDI-TOF MS with high-energy collision-induced dissociation (CID) at 20 keV. Using both protonated and sodiated ions, this technique enabled us to propose general rules to discriminate between isomeric and isobaric ceramide species, unrelated to the presence or absence of sugar chains. In addition, this high-energy CID generated ^3,5A ions, indicating Hex1-4Hex linkage in the sugar chains. Using this method, we demonstrated distinct differences among ceramide species, including free ceramides, sphingomyelins, and neutral GSLs of glucosylceramides, galactosylceramides, lactosylceramides, globotriaosylceramides and Forssman glycolipids in the equine kidneys.     

The activity of sphingomyelin cycle enzymes and concentration of sphingomyelin degradation products in rat liver in dynamics of acute toxic hepatitis

Activity of key enzymes of the sphingomyelin cycle and the content of its components (sphingomyelin, ceramide and sphingosine-1-phosphate) have been studied in livers of rats in dynamics of acute toxic hepatitis induced by subcutaneous administration of oil solution of CCl₄. Sphingomyelinase activity significantly increased already on early terms and remained increased over the whole period of observation. Ceramidase activity insignificantly differed from the control level. The levels of sphingomyelin and sphingosine-1-phosphate did not undergo marked changes while ceramide content significantly increased. The balance between liver content of ceramide (proapoptotic) and sphingosine-1-phosphate (the antiapoptotic factor) was shifted towards ceramide over the whole observation period. In sphingomyelin molecules there was a significant decrease in the content of the fatty acids C_18:1 and C_22:2, while in ceramide molecules and sphingosine-1-phosphate only the fatty acid C_22:2 changed. In spite of a significant decrease in the content of some unsaturated fatty acids, calculated unsaturation coefficients of the fatty acid component of the sphingomyelin cycle metabolites insignificantly differed from control. Taking into consideration literature data our results suggest involvement of ceramide-mediated apoptosis in the pathogenesis of acute toxic hepatitis. Elimination of damaged hepatocytes permits realization of repair processes and promotes optimization of cellular community in the liver.     

Purification and characterization of human intestinal neutral ceramidase

Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut to ceramide and sphingosine, which may inhibit cell proliferation and induce apoptosis, and thus have anti-tumour effects in the gut. Although previous rodent studies including experiments on knockout mice indicate a role of neutral ceramidase in ceramide digestion, the human enzyme has never been purified and characterized in its purified form. We here report the purification and characterization of neutral ceramidase from human ileostomy content, using octanoyl-[(14)C]sphingosine as substrate. After four chromatographic steps, a homogeneous protein band with 116kDa was obtained. MALDI mass spectrometry identified 16 peptide masses similar to human ceramidase previously cloned by El Bawab et al. [Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513] and Hwang et al. [Subcellular localization of human neutral ceramidase expressed in HEK293 cells, Biochem. Biophys. Res. Commun. 331 (2005) 37-42]. By RT-PCR and 5'-RACE methods, a predicted partial nucleotide sequence of neutral ceramidase was obtained from a human duodenum biopsy sample, which was homologous to that of known neutral/alkaline ceramidases. The enzyme has neutral pH optimum and catalyses both hydrolysis and formation of ceramide without distinct bile salt dependence. It is inhibited by Cu(2+) and Zn(2+) ions and by low concentrations of cholesterol. The enzyme is a glycoprotein but deglycosylation does not affect its activity. Our study indicates that neutral ceramidase is expressed in human intestine, released in the intestinal lumen and plays a major role in ceramide metabolism in the human gut.     

Lipid and Fatty Acid Composition in the Flesh of an Edible Marine Fish: Amadi (Coilia reynaldi)

Amadi is a small sized edible marine fish species (Coilia reynaldi) under the order-Clupeiformes. It is important for principal lipids and in particular for highly unsaturated fatty acids which have potential biomedical benefits. Among the lipid classes, phospholipids were found to be the most predominant constituents than the glycolipid and neutral lipid in Amadi. Twenty six fatty acids were quantified by open tube gas–liquid chromatography. Dominant fatty acids in this fish are Palmitic acid (C_16:0), Stearic acid (C_18:0), Oleic acid (C_18:1n?9), Myristic acid (C_14:0), Palmitoleic acid (C_16:1), Docosahexanoic acid (C_22:6n?3), Pentadecanoic acid (C_15:0), and Eicosatetraenoic acid (C_20:4n?3). Fatty acid deficiency in fish species is indicated by the presence of C_20:3n?9 acid. It is absent in this fish.The content of DHA and EPA are maximum in amount in neutral lipid than other lipid classes.     

Isolation and Characterization of a Neutral Glycosphingolipid from the Epimastigote Form of Trypanosoma mega1

ABSTRACT. A glycosphingolipid fraction from Trypanosoma mega was isolated after acetylation and was further purified on a silicic acid column. Final purification was by preparative thin-layer chromatography. The carbohydrate components of the glycolipid were fucose and galactose in approximately equimolar amounts. The neutral glycolipid of T. mega has a sphingosine base composition that consists of sphingosine and traces of dihydrosphingosine. Fatty acids forming amide groups with the sphingosine bases were analyzed by gas-liquid chromatography-mass spectrometry and are a mixture of normal and α-hydroxy fatty acids. Normal C_16:0, C_18:0, and 2-hydroxy C_18:0 are the predominant fatty acids.     

Direct analysis of sialylated or sulfated glycosphingolipids and other polar and neutral lipids using TLC-MS interfaces

Gangliosides and sulfatides (STs) are acidic glycosphingolipids (GSLs) that have one or more sialic acids or sulfate substituents, in addition to neutral sugars, attached to the C-1 hydroxyl group of the ceramide long chain base. TLC is a widely employed and convenient technique for separation and characterization of GSLs. When TLC is directly coupled to MS, it provides both the molecular mass and structural information without further purification. Here, after development of the TLC plates, the structural analyses of acidic GSLs, including gangliosides and STs, were investigated using the liquid extraction surface analysis (LESA™) and CAMAG TLC-MS interfaces coupled to an ESI QSTAR Pulsar i quadrupole orthogonal TOF mass spectrometer. Coupling TLC with ESI-MS allowed the acquisition of high resolution mass spectra of the acidic GSLs with high sensitivity and mass accuracy, without the loss of sialic acid residues that frequently occurs during low-pressure MALDI MS. These systems were then applied to the analysis of total lipid extracts from bovine brain. This allowed profiling of many different lipid classes, not only gangliosides and STs, but also SMs, neutral GSLs, and phospholipids.     

Ceramidase and ceramide synthesis in human kidney and cerebellum. Description of a new alkaline ceramidase.

It has been shown that tissues of patients with Farber's disease characteristically lack acid (pH 4.0) ceramidase. In normal cerebellum, however, ceramide cleavage and the reverse reaction, free fatty acid-dependent ceramide synthesis, both occur not only at pH 4.0 but also at pH 9.0, although normal kidney exhibits these activities only at pH 4.0. Both tissues are capable of snythesizing ceramide via an acyl-COA-dependent pathway at neutral pH. The synthetic analog of ceramide, N-oleoyl-ethanolamine, is a potent inhibitor of ceramidase.     

Extractive compounds of birch buds (<Emphasis Type="Italic">Betula pendula</Emphasis> Roth.): I. Composition of fatty acids,hydrocarbons, and esters

This is the first report devoted to study of the hydrocarbon composition of the extract of buds of European birch Betula pendula (family Betulacea). We have identified saturated (C₁₆ to C₂₈, even number of carbon atoms) and unsaturated (linoleic and linolenic) fatty acids, β-caryophyllene, α-humulene, and the components of epicuticular waxes of cover scales, such as n-alkanes (C₂₁ to C₂₆), esters of fatty acids (C₁₆ to C₂₈, even number of carbon atoms), and fatty alcohols (C₁₈ to C₃₀, even number of carbon atoms). The gas chromatographic retention indices of all identified compounds have been determined.     

Purification and characterization of a neutral ceramidase from mouse liver. A single protein catalyzes the reversible reaction in which ceramide is both hydrolyzed and synthesized

We report here a novel ceramidase that was purified more than 150, 000-fold from the membrane fraction of mouse liver. The enzyme was a monomeric polypeptide having a molecular mass of 94 kDa and was highly glycosylated with N-glycans. The amino acid sequence of a fragment obtained from the purified enzyme was homologous to those deduced from the genes encoding an alkaline ceramidase of Pseudomonas aeruginosa and a hypotheical protein of the slime mold Dictyostelium discoideum. However, no significant sequence similarities were found in other known functional proteins including acid ceramidases of humans and mice. The enzyme hydrolyzed various N-acylsphingosines but not galactosylceramide, sulfatide, GM1a, or sphingomyelin. The enzyme exhibited the highest activity around pH 7.5 and was thus identified as a type of neutral ceramidase. The apparent K(m) and V(max) values for C12-4-nitrobenzo-2-oxa-1, 3-diazole-ceramide and C16-(14)C-ceramide were 22.3 microM and 29.1 micromol/min/mg and 72.4 microM and 3.6 micromol/min/mg, respectively. This study also clearly demonstrated that the purified 94-kDa ceramidase catalyzed the condensation of fatty acid to sphingosine to generate ceramide, but did not catalyze acyl-CoA-dependent acyl-transfer reaction.     

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.     

Content of fatty acids ofChlorella kessleri in a deep tank fermentation

Chlorella kessleri cultivated in a deep tank contained 4.8% of non-polar lipid; 51% of this fraction represents saturated fatty acids, 7% unsaturated fatty acids. Our investigation of the fatty acids profile demonstrated even- and odd-numbered saturated and unsaturated fatty acids ranging from C₁₂ to C₂₀. Unlike in otherChlorella species, stearic acid was the dominant fatty acid found. Also shown was an elevated C_16:0 fatty acid content and a reduced level of unsaturated fatty acids.