Variations of ganglioside biosynthetic pathways in the phenotype conversion from myofibroblasts to lipocytes in murine hepatic stellate cell line

GRX cell line represents hepatic stellate cell and can be transformed from an actively proliferation myofibroblast phenotype into a quiescent fat-storing lipocyte phenotype. Both express the same gangliosides (GM3, GM2, GM1 and GD1a), which are resolved as doublets on HPTLC. Upper/lower band ratio is increased in lipocyte-like cells and the upper band is composed by ceramides with long-chain fatty acids. This study evaluated the contribution of de novo synthesis, sphingosine and Golgi recycling pathways on ganglioside biosynthesis, in both phenotypes. Cells were preincubated with 5 mM β-chloroalanine (SPT: serine palmitoyltransferase inhibitor) or with 25 μM fumonisin B1 (ceramide synthase inhibitor) and then radiolabeled with [U-¹⁴C]galactose in the continued presence of inhibitors. Gangliosides were extracted, purified and analyzed by HPTLC. In myofibroblast-like cells, simple gangliosides use the de novo pathway while complex gangliosides are mainly synthesized by recycling pathways. In lipocyte-like cells, de novo pathway has a lesser contribution and this is in agreement with the lower activity of the committed enzyme of sphingolipid synthesis (SPT) detected in this phenotype. SPT mRNA has an identical expression in both phenotypes. It was also observed that gangliosides doublets from myofibroblast-like cells have the same distribution between triton soluble and insoluble fractions (upper band > lower band) while the gangliosides doublets from lipocyte-like cells show an inversion in the insoluble fraction (lower band > upper band) in comparison to soluble fraction. These results indicate that myofibroblast- and lipocyte-like cells have important differences between the glycosphingolipid biosynthetic pathways, which could contribute with the respective glycosphingolipid-enriched membrane microdomain’s composition.     

Recycling of glucosylceramide and sphingosine for the biosynthesis of gangliosides and sphingomyelin in rat liver.

It was previously shown that sphingomyelin and gangliosides can be biosynthesized starting from sphingosine or sphingosine-containing fragments which originated in the course of GM1 ganglioside catabolism. In the present paper we investigated which fragments were specifically re-used for sphingomyelin and ganglioside biosynthesis in rat liver. At 30 h after intravenous injection of GM1 labelled at the level of the fatty acid ([stearoyl-14C]GM1) or of the sphingosine ([Sph-3H]) moiety, it was observed that radioactive sphingomyelin was formed almost exclusively after the sphingosine-labelled-GM1 administration. This permitted the recognition of sphingosine as the metabolite re-used for sphingomyelin biosynthesis. Conversely, gangliosides more complex than GM1 were similarly radiolabelled after the two treatments, thus ruling out sphingosine re-utilization for ganglioside biosynthesis. For the identification of the lipid fragment re-used for ganglioside biosynthesis, we administered to rats neutral glycosphingolipids (galactosylceramide, glucosylceramide and lactosylceramide) each radiolabelled in the sphingosine moiety or in the terminal sugar residue. Thereafter we compared the formation of radiolabelled gangliosides in the liver with respect to the species administered and the label location. After galactosylceramide was injected, no radiolabelled gangliosides were formed. After the administration of differently labelled glucosylceramide, radiolabelled gangliosides were formed, regardless of the position of the label. After lactosylceramide administration, the ganglioside fraction became more radioactive when the long-chain-base-labelled precursors were used. These results suggest that glucosylceramide, derived from glycosphingolipid and ganglioside catabolism, is recycled for ganglioside biosynthesis.     

Isolation and characterization of major gangliosides from frog liver

Four major gangliosides isolated from frog liver were characterized by compositional analysis involving GLC and GC-MS, methylation analysis, chromium trioxide oxidation, and enzymatic hydrolysis. The results revealed that the most major ganglioside in the tissue was GM4 containing N-acetylneuraminic acid and the others were GM4 containing N-glycolylneuraminic acid, GD1a, and a fucosyl ganglioside which was tentatively assigned to be alpha-galactosyl alpha-fucosyl GM1. This is the first report describing the presence of GM4 containing N-glycolylneuraminic acid. The fatty acids in both GM4 were mainly alpha-hydroxylated, and those in the fucosyl ganglioside were exclusively nonhydroxy fatty acids. The GD1a contained both nonhydroxy and alpha-hydroxy fatty acids in a ratio of about 3:2. The predominant species were 22:0, 23:0, 24:0, and 24:1 in both species of the fatty acids. The long-chain bases of these four gangliosides consisted of C18-sphingosine and C18-phytosphingosine together with significant amounts of C16 to C19 dihydroxy and trihydroxy bases with iso and anteiso structures.     

Incorporation of Very-Long-Chain Fatty Acids into Sphingolipids of Cultured Neural Cells

We examined effects of exogenous very-long-chain fatty acids on lipids of cultured chick neurons and astrocytes. When chick neurons were incubated in chemically defined medium containing 10 microM nervonic acid (C24:1) for 7 days, it was found that a major fatty acid moiety of gangliosides and sphingomyelin was nervonic acid itself, which was not normally detected in the sphingolipid fraction. This alteration in the fatty acid composition apparently occurred in each ganglioside species. Under these experimental conditions, nervonic acid was not found in the glycerophospholipid fraction, and the amounts of triacylglycerol and free nervonic acid increased. Addition of behenic acid (C22:0) or erucic acid (C22:1) also induced changes in the fatty acid composition of gangliosides. When chick astrocytes were incubated in the presence of 10 microM nervonic acid for 7 days, no significant change was observed in the fatty acid composition of gangliosides. These studies indicate that the manipulation of the fatty acid moiety of sphingolipids in cultured neurons is possible.     

C18:3-GM1a induces apoptosis in Neuro2a cells: enzymatic remodeling of fatty acyl chains of glycosphingolipids

GM1a [Gal beta1-3GalNAc beta1-4(NeuAc alpha2-3)Gal beta1-4Glc beta1-1Cer] is known to support and protect neuronal functions. However, we report that alpha-linolenic acid-containing GM1a (C18:3-GM1a), which was prepared using the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase, induced apoptosis in neuronal cells. Intranucleosomal DNA fragmentation, chromatin condensation, and caspase activation, all typical features of apoptosis, were observed when mouse neuroblastoma Neuro2a cells were cultured with C18:3-GM1a but not GM1a containing stearic acid (C18:0) or oleic acid (C18:1). The phenotype of Neuro2a cells induced by C18:3-GM1a was similar to that evoked by lyso-GM1a. However, lyso-GM1a caused a complete disruption of lipid microdomains of Neuro2a cells and hemolysis of sheep erythrocytes, whereas C18:3-GM1a did neither. C18:3-GM1a, but not lyso-GM1a, was found to be abundant in lipid microdomains after the removal of loosely bound GM1a by BSA. The activation of stress-activated protein kinase/c-Jun N-terminal kinase in Neuro2a cells was observed with lyso-GM1a but not C18:3-GM1a. These results indicate that the mechanism of apoptosis induced by C18:3-GM1a is distinct from that caused by lyso-GM1a. This study also clearly shows that fatty acid composition of gangliosides significantly affected their pharmacological activities when added to the cell cultures and suggests why naturally occurring gangliosides do not possess polyunsaturated fatty acids as a major constituent.     

Different Ceramide Compositions of Gangliosides Between Human Motor and Sensory Nerves

Ganglioside analysis of human motor and sensory nerves revealed that ceramide compositions of sensory nerve GD1a, GD1b, and GM1 differed apparently from those in the motor nerve. These gangliosides from sensory nerve contained a large amount of long-chain fatty acids and d18:1 as a major long chain base. On the contrary, the motor nerve gangliosides contained C16-18 fatty acids and a large amount of d20:1 besides d18:1. Furthermore, these gangliosides were enriched more in the axon fraction than in the myelin fraction. LM1, which was a major ganglioside in myelin from human peripheral nerve, was composed of similar ceramide compositions in the two nerves. The present findings suggest that the characteristic ceramide species of nerve gangliosides may reflect in part properties of their own neurons.     

Sphingolipidomics of A2780 human ovarian carcinoma cells treated with synthetic retinoids

The dihydroceramide, ceramide, sphingomyelin, lactosylceramide, and ganglioside species of A2780 human ovarian carcinoma cells treated with the synthetic retinoids N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) in culture were characterized by ESI-MS. We characterized 32 species of ceramide and dihydroceramide, 15 of sphingomyelin, 12 of lactosylceramide, 9 of ganglioside GM2, and 6 of ganglioside GM3 differing for the long-chain base and fatty acid structures. Our results indicated that treatment with both 4-HPR and 4-oxo-4-HPR led to a marked increase in dihydroceramide species, while only 4-oxo-4-HPR led to a minor increase of ceramide species. Dihydroceramides generated in A2780 cells in response to 4-HPR or 4-oxo-4-HPR differed for their fatty acid content, suggesting that the two drugs differentially affect the early steps of sphingolipid synthesis. Dihydroceramides produced upon treatments with the drugs were further used for the synthesis of complex dihydrosphingolipids, whose levels dramatically increased in drug-treated cells.     

Cell type specific localization of sphingomyelin biosynthesis

We have studied the incorporation of [(14)C]serine and of [(3)H]sphingosine into sphingomyelin in the presence or absence of brefeldin A (BFA) in three different cell types. Administration of BFA (1 microgram/ml) to fibroblasts for 24 h increased the incorporation of label into sphingomyelin 1.5-3 fold compared with untreated controls. In contrast, BFA strongly decreased sphingomyelin biosynthesis (4-5 fold) in cerebellar neurons as well as in neuroblastoma cells. The effect of BFA on glycosphingolipid formation, however, was similar in all three cell types studied: an increased labeling of the precursor glycolipids GlcCer, LacCer, GM3 and GD3 was paralleled by a decreased formation of complex gangliosides, GM1, GD1a, GT1b and GQ1b. Our data therefore suggest that in neuronal cells sphingomyelin synthesis, like the formation of complex gangliosides, is localized primarily distal to the BFA block, in a post-Golgi compartment, most probably the trans-Golgi network, whereas in fibroblasts sphingomyelin biosynthesis is mainly localized prior to the BFA block, in the Golgi apparatus, as has been shown for LacCer, GlcCer, GM3 and GD3 synthases.     

Isolation and structural elucidation of a novel type of ganglioside, deaminated neuraminic acid (KDN)-containing glycosphingolipid, from rainbow trout sperm. The first example of the natural occurrence of KDN-ganglioside, (KDN)GM3

Rainbow trout sperm contained almost exclusively monoanionic ganglioside fraction as a major acidic glycosphingolipid. Two monoacidic gangliosides were isolated and purified in this study and designated as sperm ganglioside 1 and 2 (sg-1 and sg-2). The two gangliosides, sg-1 and sg-2, contained the same neutral sugars, galactose and glucose in molar ratio of 1:1 and no GalNAc except for the presence of N-acetyl-neuraminic acid (NeuAc) in sg-1 and deaminated neuraminic acid (KDN; 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) in sg-2. The complete structures of these gangliosides were determined by a combination of methylation analysis, fast atom bombardment mass spectrometry, 400-MHz one- and two-dimensional 1H nuclear magnetic resonance spectroscopy, fatty acid analysis, and endoglycoceramidase digestion NeuAc alpha 2----3Gal beta 1----4Glc beta 1----Cer sg-1 [(NeuAc)GM3] KDN alpha 2----3Gal beta 1----4Glc beta 1----Cer sg-2 [(KDN)GM3] where, for both sg-1 and sg-2, the ceramide moieties (Cer) were found to be made up of 4-sphingenine and mainly C16:0 fatty acid (palmitate; 95%) with a minor amount of C24:1 fatty acyl chain (nervonate, 5%). The structure of sg-2 is novel and represents the first example of a new class of gangliosides, i.e. KDN-gangliosides.     

Physiological relevance of sphingolipid activator proteins in cultured human fibroblasts

The physiological degradation of several membrane-bound glycosphingolipids (GSLs) by water-soluble lysosomal exohydrolases requires the assistance of sphingolipid activator proteins (SAPs). Four of these SAPs are synthesized from a single precursor protein (prosaposin). Inherited deficiency of this precursor results in a rare disease in humans with an accumulation of ceramide (Cer) and glycolipids such as glucosylceramide and lactosylceramide (LacCer). In a previous study, we have shown that human SAP-D stimulates the lysosomal degradation of Cer in precursor deficient cells. In order to study the role of SAPs (or saposins) A-D in cellular GSL catabolism, we recently investigated the catabolism of exogenously added [(3)H]labeled ganglioside GM1, Forssman lipid, and endogenously [(14)C]labeled GSLs in SAP-precursor deficient human fibroblasts after the addition of recombinant SAP-A, -B, -C and -D. We found that activator protein deficient cells are still able to slowly degrade gangliosides GM1 and GM3, Forssman lipid and globotriaosylceramide to a significant extent, while LacCer catabolism critically depends on the presence of SAPs. The addition of either of the SAPs, SAP-A, SAP-B or SAP-C, resulted in an efficient hydrolysis of LacCer.     

Incorporation and metabolism of exogenous GM1 ganglioside in rat liver.

The pathways of metabolic processing of exogenously administered GM1 ganglioside in rat liver was investigated at the subcellular level. The GM1 used was 3H-labelled at the level of long-chain base ([Sph(sphingosine)-3H]GM1) or of terminal galactose ([Gal-3H]GM1). The following radioactive compounds, derived from exogenous GM1, were isolated and chemically characterized: gangliosides GM2, GM3, GD1a and GD1b (nomenclature of Svennerholm [(1964) J. Lipid Res. 5, 145-155] and IUPAC-IUB Recommendations [(1977) Lipids 12, 455-468]); lactosylceramide, glucosylceramide and ceramide; sphingomyelin. GM2, GM3, lactosylceramide, glucosylceramide and ceramide, relatively more abundant shortly after GM1 administration, were mainly present in the lysosomal fraction and reflected the occurrence of a degradation process. 3H2O was also produced in relevant amounts, indicating complete degradation of GM1, although no free long-chain bases could be detected. GD1a and GD1b, relatively more abundant later on after administration, were preponderant in the Golgi-apparatus fraction and originated from a biosynthetic process. More GD1a was produced starting from [Sph-3H]GM1 than from [Gal-3H]GM1, and radioactive GD1b was present only after [Sph-3H]GM1 injection. This indicates the use of two biosynthetic routes, one starting from a by-product of GM1 degradation, the other implicating direct sialylation of GM1. Both routes were used to produce GD1a, but only the first one for producing GD1b. Sphingomyelin was the major product of GM1 processing, especially at the longer times after injection, and arose from a by-product of GM1 degradation, most likely ceramide.     

Characterization of nuclear gangliosides in rat brain: concentration, composition, and developmental changes

Nuclear gangliosides were characterized using two distinct fractions of large (N1) and small (N2) nuclear populations from rat brain. The ganglioside concentration of N1 nuclei from adult rat brain was 0.92 microg sialic acid/mg protein, which was about 3.8 times higher than that of N2 nuclei. N1 and N2 nuclear gangliosides showed similar compositional profiles; they contained major gangliosides of GM1, GD1a, GD1b, and GT1b, with GM3 in lesser amounts. c-Series gangliosides such as GT3, GQ1c, and GP1c were also detected in both nuclear preparations. Nuclear localization of gangliosides was confirmed by immunofluorescence with anti-GM1 antibody, cholera toxin B subunit, and c-series ganglioside-specific monoclonal antibody A2B5. Developmental changes of nuclear gangliosides were examined using rats of different ages ranging from embryonic day 14 (E14) to postnatal 7 weeks. The concentration of N1 nuclear gangliosides changed only slightly during development and did not correlate with that of whole-brain gangliosides. The developmental pattern of ganglioside composition of N1 nuclei was also distinguished from that of microsomal membranes; the ganglioside changes in N1 nuclei included reduced expression of di- and polysialogangliosides at E16 and higher proportions of GM3 at early and late stages of the period. These findings suggest that gangliosides in nuclear membranes are developmentally regulated in a distinct manner in brain cells.     

Age-Related Changes in the Ceramide Composition of the Major Gangliosides Present in Rat Brain Subcellular Fractions Enriched in Plasma Membranes of Neuronal and Myelin Origin

Abstract: Age-related changes of the ceramide composition of gangliosides were studied in the synaptosomal and myelin fractions from rat brain, carrying plasma membranes of neuronal and glial origin, respectively. The five major gangliosides (GM1, GD1 a, GD1 b, GT1 b, and GQ1 b) present in these fractions were separated and quantitated by normal-phase HPLC. Each ganglioside was then fractionated by reverse-phase HPLC into the molecular species carrying a single long-chain base (LCB). The largely preponderant LCBs in the synaptosomal and myelin fractions were the C18:1 and C20:1. The content of C20.1 LCB, generally low at 1 month, increased with age in all analyzed gangliosides and in all subcellular fractions and was greater in the "b series" than in the "a series" gangliosides. Remarkably, GM1 was the only ganglioside where the proportion of LCB 20:1 was higher in the synaptosomal fraction than in the myelin fraction. The fatty acid composition of the C18:1 or C20:1 LCB species of the different gangliosides in the synaptosomal and myelin fractions did not undergo appreciable changes with age. Stearic acid was largely predominant in all the gangliosides of the synaptosomal fraction, more in the C18:1 than in the C20:1 LCB species (80–90% vs. 60–70%). The gangliosides of the myelin fraction were characterized by a lower content of 18:0 and a much higher content of 16:0 and 18:1 fatty acids than those of the synaptosomal fraction. Thus, the ceramide composition is different in the gangliosides of neuronal and myelin origin and appears to be subjected to an age-related control.     

Brain Gangliosides of Quaking and Shiverer Mutants: Qualitative and Quantitative Changes of Monosialogangliosides in the Quaking Brain

Ganglioside compositions in the brains of the mutant mice quaking and shiverer were compared with those of their littermate controls, C57BL/6 and C3HSWV. Neither ganglioside content nor composition of shiverer brains differed from those of the control brains. Change in the ganglioside composition of the mutant brain from that of the control was observed only in the quaking mutant brain, in which monosialoganglioside GM1 was significantly reduced and GM4 was completely absent. The structures of the gangliosides were determined by negative ion fast atom bombardment mass spectrometry, and the GM3 and GM4 gangliosides in the quaking brain were found to be altered in regard to their long-chain base and fatty acid compositions when compared to the normal C57BL/6 brain.     

Anabolic sialosylation of gangliosides in situ in rat brain cortical slices

Radiolabeling of the sialic acid residues of gangliosides was examined in thin slices of rat brain cerebral cortex incubated under physiologic conditions in the presence of either [14C]N-acetyl-mannosamine (ManNAc) or cytidine 5'-monophosphoryl-[14C]N-acetyl-neuraminic acid (CMP-NeuAc). CMP-NeuAc is the direct donor substrate in the transfer of sialic acid to gangliosides by sialosyl transferases (SATs), including ectosialosyl transferases at the cell surface. ManNAc must be internalized by the neural cells (neuronal or glial) where it serves as an obligate precursor for the biosynthesis of the NeuAc moiety of intracellular CMP-NeuAc, via multiple reactions in the cytosol and nucleus. When exogenous [14C]ManNAc was supplied, there appeared to be a 2-h lag period before label was incorporated measurably into ganglioside sialic acid. That was followed by rapid ganglioside labeling continuing up to 6 h. There was high incorporation into ganglioside GM1. Labeling by ManNAc was inhibited by monensin, a monovalent cationophore that blocks anabolic transport in medial and trans Golgi. Extracellular CMP-NeuAc was not internalized by the cells. CMP-[14C]NeuAc labeling of gangliosides had no lag period, reached a maximum within 2 h, and then began to level. The label distribution among gangliosides was high in GD3, but quite low in GM1. CMP-NeuAc labeling was not inhibited by 10(-7) M monensin. These findings support a model in which ManNAc labels gangliosides by an intracellular route involving monensin-sensitive, Golgi-associated SATs. In this intracellular system, the major labeled products are gangliosides of the gangliotetraosyl series (GM1, GD1a, etc.).(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of Exogenous Gangliosides in Cerebellar Granule Cells, Differentiated in Culture

The metabolism of exogenous gangliosides in the CNS has been investigated using cerebellar granule cells in culture as a model. For this purpose, GM2 and GM1, both isotopically radiolabeled at the level of the terminal sugar residue or of the long chain base moiety, were administered to differentiated cells for a 15-h pulse, and their metabolic fate was followed in a time course protocol. At each time investigated (1, 2, and 4 days after the pulse), several compounds, besides the ones administered, were detected: (a) GM2 (only after GM1 was given), GM3, lactosylceramide, glucosylceramide, and ceramide, all products of ganglioside stepwise catabolism; (b) GM1 (only after GM2 was given), GD1a, GD1b, O-Ac-GT1b, and GT1b, that is, gangliosides more complex than the one administered; and (c) sphingomyelin. The compounds derived from ganglioside catabolism and sphingomyelin were detected only after administration of long chain base-labeled precursors, whereas the others were found regardless of the labeling position of the precursor. In addition, radioactivity was incorporated in the delipidized residue when sugar-labeled gangliosides were given to cells. Besides qualitative differences, quantitative ones were found after administration of the different precursors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for Nonrandom Distribution of GD1 a Ganglioside in Rabbit Brain Microsomal Membranes

GD1a is the major ganglioside of rabbit brain microsomal membranes and occurs mainly with two molecular species, containing the C18:1 (62.3%) and C20:1 (37.7%) long-chain bases. The membranes were exposed to Vibrio cholerae (VC) sialidase under conditions where the enzyme hydrolyzed only GD1a (approximately 9%), producing GM1 ganglioside, whereas the other gangliosides remained virtually unaffected. The long-chain-base analysis showed that newly-formed GM1 contained approximately 68% of the C20:1 molecular species. This indicates that VC sialidase did not randomly affect the two molecular species of GD1a but hydrolyzed preferentially the C20:1 one. In similar experiments, GD1a was inserted into the external layer of phosphatidylcholine vesicles and incubated with VC sialidase under conditions producing approximately 10% hydrolysis. Long-chain-base analysis showed that the proportion of C20:1 species in GM1 was 25.1% using vesicles composed of dipalmitoylphosphatidylcholine and 42.3% with egg phosphatidylcholine, whereas it was 39.2% in the starting GD1a. Therefore, in artificial membranes, VC sialidase acted preferentially on the C18:1 or C20:1 molecular species, depending on the length and unsaturation of the phospholipid fatty acids. Because VC sialidase is known to affect molecular dispersions more easily than packed aggregations of the gangliosidic substrate, the data suggest that in rabbit brain microsomal membranes the GD1a ganglioside molecular species carrying C20:1 long-chain base are more molecularly dispersed than those containing C18:1 long-chain base.     

Lipid components of sialosylgalactosylceramide of human brain

A ganglioside, previously designated HG-B in our laboratory, was isolated from mixed human brain ganglioside preparations and shown to contain equimolar quantities of sialic acid, galactose, and sphingosine. Treatment of this material with neuraminidase yielded a galactosylceramide. The ganglioside, now referred to as sialosylgalactosylceramide, thus appears to be identical with G(gal) reported by Kuhn and Wiegandt. The fatty acids and long-chain bases of this material were analyzed by gas-liquid chromatography. Approximately equal amounts of normal and hydroxy acids were found. Oleic, palmitic, and stearic acids were the only normal fatty acids present. In the hydroxy series, the C(24) and C(23) saturated acids were the major components. The ratio of C(20) to C(18) long-chain base was approximately 5:3. These data suggest that sialosylgalactosylceramide has no direct metabolic relationship with either the major brain gangliosides or adult brain cerebroside.     

The characterization of sphingolipids from neurons and astroglia of immature rat brain

Abstract— Isolated neuronal cell bodies and astroglia of young (15–20-day-old) rat brains were both found to contain small concentrations of a variety of glycosphingolipids, including glucosylceramide, galactosylceramide, sulphatide, dihexosylceramide and gangliosides. These sphingolipids, plus sphingomyelin, were isolated, quantitated and their fatty acid and long chain base patterns determined. These data were compared to similar data obtained on these lipids isolated from whole brain and myelin of rats of the same age range. Glucosylceramide was found in an amount equal to galactosylceramide in neurons, and accounted for 35 per cent of the total monohexosylceramide in astroglia. Dihexosylceramide was present in nearly the same amount as sulphatide in both cell types. The sphingolipids of each cell type had characteristic fatty acid patterns. Generally the whole brain fatty acid patterns resembled those of astroglial lipids rather than neuronal lipids. In no case did the cell sphingolipid fatty acids resemble those of myelin. However, the galactosylceramide and sulphatides of both cells had unsubstituted and α-hydroxy acids, both of which had appreciable quantities of C₂₄ acids. The ganglioside fatty acids of each cell type were similar and not unusual, but were quite different from those of glucosylceramide and dihexosylceramide; the latter having appreciable quantities of 16:0 and acids longer than 18:0. The ganglioside patterns of these cells were similar and only slightly different from that of whole brain. Long chain bases of sphingolipids were mainly C₁₈-sphingosine in both cell types, and those of ganglioside and sphingomyelin contained small amounts of C₂₀-sphingosine.     

Changes in the Ganglioside Long-Chain Base Composition of Rat Cerebellar Granule Cells During Differentiation and Aging in Culture

Abstract: Changes in the ganglioside long-chain base (LCB) composition in rat cerebellar granule cells in culture were studied during differentiation and aging. The total native ganglioside mixtures, extracted from the cells maintained in culture up to 22 days, were fractionated by reversed-phase HPLC, each ganglioside homogeneous in the oligosaccharide chain as well as in the LCB being quantified. Two main LCBs were components of the ganglioside species of cultured cells, the C18:1 LCB and the C20:1 LCB. The content of C20:1 ganglioside molecular species was low and quite constant during differentiation, comprising ∼8% of the total ganglioside species content, the C20:1 LCB appearing to be represented more in the ganglioside of the "b series" (GD1b, GT1b, and GQ1b) than in the "a series" (GM1 and GD1a). During aging in culture, for 8–22 days, the content of the C20:1 species of all gangliosides increased, being more pronounced for GM1 and GD1a.