Endogenous pore-forming protein complex targets acidic glycosphingolipids in lipid rafts to initiate endolysosome regulation.

Bacterial pore-forming toxin aerolysin-like proteins (ALPs) are widely distributed in animals and plants. However, functional studies on these ALPs remain in their infancy. ßγ-CAT is the first example of a secreted pore-forming protein that functions to modulate the endolysosome pathway via endocytosis and pore formation on endolysosomes. However, the specific cell surface molecules mediating the action of ßγ-CAT remain elusive. Here, the actions of ßγ-CAT were largely attenuated by either addition or elimination of acidic glycosphingolipids (AGSLs). Further study revealed that the ALP and trefoil factor (TFF) subunits of ßγ-CAT bind to gangliosides and sulfatides, respectively. Additionally, disruption of lipid rafts largely impaired the actions of ßγ-CAT. Finally, the ability of ßγ-CAT to clear pathogens was attenuated in AGSL-eliminated frogs. These findings revealed a previously unknown double binding pattern of an animal-secreted ALP in complex with TFF that initiates ALP-induced endolysosomal pathway regulation, ultimately leading to effective antimicrobial responses.

Δ10(E)-Sphingolipid Desaturase Involved in Fusaruside Mycosynthesis and Stress Adaptation in Fusarium graminearum.

Sphingolipids are biologically important and structurally distinct cell membrane components. Fusaruside (1) is a 10,11-unsaturated immunosuppressive fungal sphingolipid with medical potentials for treating liver injury and colitis, but its poor natural abundance bottlenecks its druggability. Here, fusaruside is clarified biosynthetically, and its efficacy-related 10,11-double bond can be generated under the regioselective catalysis of an unprecedented Δ10(E)-sphingolipid desaturase (Δ10(E)-SD). Δ10(E)-SD shares 17.7% amino acid sequence similarity with a C9-unmethylated Δ10-sphingolipid desaturase derived from a marine diatom, and 55.7% with Δ8(E)-SD from Fusarium graminearum. Heterologous expression of Δ10(E)-SD in Pichia pastoris has been established to facilitate a reliable generation of 1 through the Δ10(E)-SD catalyzed desaturation of cerebroside B (2), an abundant fungal sphingolipid. Site directed mutageneses show that the conserved histidines of Δ10(E)-SD are essential for the 10,11-desaturation catalysis, which is also preconditioned by the C9-methylation of the substrate. Moreover, Δ10(E)-SD confers improved survival and faster growth to fungal strains at low temperature and high salinity, in parallel with to higher contents of 1 in the mycelia. Collectively, the investigation describes a new Δ10(E)-sphingolipid desaturase with its heterologous expression fundamentalizing a biotechnological supply of 1, and eases the follow-up clarification of the immunosuppression and stress-tolerance mechanism.

The preclinical activity of lenalidomide in indolent urothelial carcinoma.

BACKGROUND: Lenalidomide is an IMiD® immunomodulatory drug, which may warrant evaluation in urothelial carcinoma (UC). MATERIALS AND METHODS: The in vitro and in vivo activity of lenalidomide was evaluated in human and murine UC cell lines. Tumors were evaluated by immunohistochemistry for (CD31), cleaved caspase-3 (CC3) and CD3+/CD20+ lymphocyte infiltration. Cereblon, a molecular target of lenalidomide was analyzed by immunohistochemistry. RESULTS: Significant pro-apoptotic activity, and reduction of cell viability was seen at low micromolar concentrations of lenalidomide against indolent human RT4 UC cells in vitro. Cereblon expression was quantitatively lower in sensitive RT4 cells compared to resistant 5637 cells. In RT4 xenografts, lenalidomide significantly reduced tumor size and CD31 expression, and increased expression of CC3 (p<0.05). Cereblon expression increased in lenalidomide-treated RT4 xenografts (p<0.05). CONCLUSION: Lenalidomide demonstrated preclinical activity against superficially-invasive low-grade UC cells attributable to direct tumor cell apoptosis and anti-angiogenic activity. Clinical trials are warranted in patients with indolent UC.

Modification of sphingoglycolipids and sulfolipids in kidney cell lines under heat stress: activation of monohexosylceramide synthesis as a ceramide scavenger.

Heat stress on Madin-Darby canine kidney cells increased ceramide content to 187% at 40 degrees C for 24 h, and the de novo synthesis from serine increased to 146%. Glucosylceramide (GlcCer) and galactosylceramide (GalCer) synthesis from ceramide, the first glycosylation step of sphingolipid metabolism in kidney cells, increased to 290% (GalCer) and 143% (GlcCer) after metabolic labeling with (14)C-glucose at 42 degrees C for 20 h. The more complex glycolipid lactosylceramide also increased to 151%, whereas sulfatide and ganglioside GM3 decreased to 21% and 43%, respectively. Sulfatide (SM4s) showed optimal sulfation at 37 degrees C, whereas cholesterol sulfate was optimally sulfated at 40 degrees C. The gene expression of ceramide glucosyltransferase (GluT), ceramide galactosyltransferase, and GalCer sulfotransferase (GST) after 24 h culture at 42 degrees C significantly increased to 714%, 221%, and 174%, respectively. Another kidney cell line, COS7, showed less activation of these transferases by heat stress. Although GST gene expression was higher under heat stress, SM4s synthesis decreased, which may have been due to increased GST sensitivity to a temperature higher than 37 degrees C. When we introduced the HSP70 gene into the expression vector and transfected the plasmid (pCDM-dHSP70) into kidney cells, GlcCer synthesis increased significantly. From these results, we speculated that HSP70 may play a role in GluT gene expression to increase GlcCer and decrease intracellular ceramide level.

Oleamide: a fatty acid amide signaling molecule in the cardiovascular system?

Oleamide (cis-9,10-octadecenoamide), a fatty acid primary amide discovered in the cerebrospinal fluid of sleep-deprived cats, has a variety of actions that give it potential as a signaling molecule, although these actions have not been extensively investigated in the cardiovascular system. The synthetic pathway probably involves synthesis of oleoylglycine and then conversion to oleamide by peptidylglycine alpha-amidating monooxygenase (PAM); breakdown of oleamide is by fatty acid amide hydrolase (FAAH). Oleamide interacts with voltage-gated Na(+) channels and allosterically with GABA(A) and 5-HT(7) receptors as well as having cannabinoid-like actions. The latter have been suggested to be due to potentiation of the effects of endocannabinoids such as anandamide by inhibiting FAAH-mediated hydrolysis. This might underlie an "entourage effect" whereby co-released endogenous nonagonist congeners of endocannabinoids protect the active molecule from hydrolysis by FAAH. However, oleamide has direct agonist actions at CB(1) cannabinoid receptors and also activates the TRPV1 vanilloid receptor. Other actions include inhibition of gap-junctional communication, and this might give oleamide a role in myocardial development. Many of these actions are absent from the trans isomer of 9,10-octadecenoamide. One of the most potent actions of oleamide is vasodilation. In rat small mesenteric artery the response does not involve CB(1) cannabinoid receptors but another pertussis toxin-sensitive, G protein-coupled receptor, as yet unidentified. This receptor is sensitive to rimonabant and O-1918, an antagonist at the putative "abnormal-cannabidiol" or endothelial "anandamide" receptors. Vasodilation is mediated by endothelium-derived nitric oxide, endothelium-dependent hyperpolarization, and also through activation of TRPV1 receptors. A physiological role for oleamide in the heart and circulation has yet to be demonstrated, as has production by cells of the cardiovascular system, but this molecule has a range of actions that could give it considerable modulatory power.

Isolation and characterization of cerebrosides of the hydrocarbon-assimilating yeast Yarrowia lipolytica.

Yarrowia lipolytica yeast was grown batchwise on n-hexadecane as the carbon and energy source. Two cerebroside species were quantitatively isolated from sphingolipid fractions of total lipids by a combination of column chromatography and preparative high-performance thin-layer chromatography. The cerebroside content accounted for 1.3% of the total cell lipids. Glucose was detected as the sole sugar constituent in cerebrosides. The fatty acid composition of cerebrosides was characterized by a high proportion of hydroxylated long-chain saturated fatty acids. The major fatty acids were h16:0 and 16:0. The long-chain bases composition shows a preponderance of trihydroxy bases and a small amount of dihydroxy bases. The striking finding was a high proportion of 19-phytosphingosine.

The cerebrosides.

This review covers distribution, structural determination, syntheses and biological activities of the cerebrosides reported so far. Those with absolute stereochemistry assigned are marked herein with '*'. The literature from 1969 to mid-2003 is reviewed and 189 references cited.

Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc:ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth.

The opportunistic mycopathogen Aspergillus fumigatus expresses both glucosylceramide and galactosylceramide (GlcCer and GalCer), but their functional significance in Aspergillus species is unknown. We here identified and characterized a GlcCer from Aspergillus nidulans, a non-pathogenic model fungus. Involvement of GlcCer in fungal development was tested on both species using a family of compounds known to inhibit GlcCer synthase in mammals. Two analogs, D-threo-1-phenyl-2-palmitoyl-3-pyrrolidinopropanol (P4) and D-threo-3',4'-ethylenedioxy-P4, strongly inhibited germination and hyphal growth. Neutral lipids from A. fumigatus cultured in the presence of these inhibitors displayed a significantly reduced GlcCer/GalCer ratio. These results suggest that synthesis of GlcCer is essential for normal development of A. fumigatus and A. nidulans.

Cerebroside synthesis as a measure of the rate of remyelination following cuprizone-induced demyelination in brain.

We studied markers of myelin content and of the rate of myelination in brains of mice between 8 and 20 weeks of age. During the 12-week time-course, control animals showed slight increases in the content of oligodendroglial-specific cerebroside, as well as cholesterol (enriched in, but not specific to, myelin). In contrast, synthesis of these lipids, as assayed by in vivo incorporation of (3)H(2)O, was substantial, indicating turnover of 0.4% and 0.7% of total brain cerebroside and cholesterol, respectively, each day. We also studied mice exposed to a diet containing 0.2% of the copper chelator, cuprizone. After 6 weeks 20%, and by 12 weeks, over 30% of brain cerebroside was gone. Demyelination was accompanied by down-regulation of mRNA expression for enzymes controlling myelin lipid synthesis (ceramide galactosyl transferase for cerebroside; hydroxymethylglutaryl-CoA reductase for cholesterol), and for myelin basic protein. Synthesis of myelin lipids was also greatly depressed. The 20% cerebroside deficit consequent to 6 weeks of cuprizone exposure was restored 6 weeks after return to a control diet. During remyelination, expression of myelin-related mRNA species, as well as cerebroside and cholesterol synthesis were restored to normal. However, in contrast to the steady state metabolic turnover in the control situation, all the cerebroside and cholesterol made were accumulated. To the extent that accumulating cerebroside is targeted for eventual inclusion in myelin (discussed) the rate of its synthesis is proportional to remyelination. With our assay, in vivo rates of cerebroside synthesis can be determined for a time window of the order of hours. This offers greater temporal resolution and accuracy relative to classical methods assaying accumulation of myelin components at time intervals of several days. We propose this experimental design, and the reproducible cuprizone model, as appropriate for studies of how to promote remyelination.

Parameters related to lipid metabolism as markers of myelination in mouse brain.

Myelination, during both normal development and with respect to disorders of myelination, is commonly studied by morphological and/or biochemical techniques that assay as their end-points the extent of myelination. The rate of myelination is potentially a more useful parameter, but it is difficult and time-consuming to establish, requiring a complete developmental study with labor-intensive methodology. We report herein development of methodology to assay the absolute rate of myelination at any desired time during development. This involves intraperitoneal injection of (3)H(2)O to label body water pools, followed by determination of label in the myelin-specific lipid, cerebroside. The absolute amount of cerebroside synthesized can then be calculated from the specific radioactivity of body water and knowledge of the number of hydrogens from water incorporated into cerebroside. During development, the rate of cerebroside synthesis correlated well with the rate of accumulation of the myelin-specific components, myelin basic protein and cerebroside. For purposes of control, we also tested other putative, albeit less quantitative, indices of the rate of myelination. Levels of mRNA for ceramide galactosyltransferase (rate-limiting enzyme in cerebroside synthesis) and for myelin basic protein did not closely correlate with myelination at all times. Cholesterol synthesis closely matched the rate of cholesterol accumulation but did not track well with myelination. Synthesis of fatty acids did not correlate well with accumulation of either fatty acids (phospholipids) or myelin markers. We conclude that measurement of cerebroside synthesis rates provides a good measure of the rate of myelination. This approach may be useful as an additional parameter for examining the effects of environmental or genetic alterations on the rate of myelination.

Stage-associated expression of ceramide structures in glycosphingolipids from the human trematode parasite Schistosoma mansoni.

Glycosphingolipids of Schistosoma mansoni adults, cercariae and eggs comprise ceramide monohexosides (CMH) with glucose or galactose and ceramide dihexosides (CDH) with the schistosome-specific structure GalNAc(beta1-4)Glc(1-1)ceramide. Ceramide analysis revealed C18- and C20-phytosphingosines in egg CMH, C18-sphinganine as well as C18-, C19- and C20-phytosphingosines in cercarial CMH, and C18- and C20-phytosphingosines as well as C18-sphingosine and C18-sphinganine in adult CMH. For all three life cycle stages, the predominant fatty acid was C16h:0. As a characteristic feature, a range of saturated, unsaturated and hydroxylated long-chain fatty acids with 24-28 carbon atoms were additionally found in minor cercarial CMH species. The corresponding ceramides represented major constituents in cercarial CDH, while adult and egg CDH were dominated by ceramides with short fatty acid chains. The resultant ceramide patterns could be correlated with the differential expression of carbohydrate antigens on schistosomal glycolipids at various stages. A possible impact of ceramide structure on the biosynthesis of the carbohydrate moieties is discussed.

Cerebroside elicitors found in diverse phytopathogens activate defense responses in rice plants.

Cerebrosides A and C, compounds categorized as glycosphingolipids, were isolated in our previous study from the rice blast fungus (Magnaporthe grisea) as novel elicitors which induce the synthesis of rice phytoalexins. In this paper, these cerebroside elicitors showed phytoalexin-inducing activity when applied to plants by spray treatment and also induced the expression of pathogenesis-related (PR) proteins in rice leaves. This elicitor activity of the cerebrosides showed the structural specificity as that for the induction of phytoalexins. Ceramides prepared from the cerebrosides by removal of glucose also showed the elicitor activity even in lower level compared to the cerebrosides. In field experiments, the cerebroside elicitors effectively protected rice plants against the rice blast fungus, an economically devastating agent of disease of rice in Japan. The cerebrosides elicitors protected rice plants from other disease as well and were found to occur in a wide range of different phytopathogens, indicating that cerebrosides function as general elicitors in a wide variety of rice-pathogen interactions.

Inhibition of insulin-specific autoreactive T-cells by sulphatide which is variably expressed in beta cells.

AIMS/HYPOTHESIS: Sulphatide and insulin are present in the secretory granules and at the surface of beta cells in islets of Langerhans. Insulin autoantibodies and T-cell reactivity against insulin exist during the development of Type I (insulin-dependent) diabetes during which active beta cells may be more vulnerable than passive. Our aims were to examine the presence of sulphatide in active and passive beta cells and to clarify whether sulphatide influences the direct autoimmunity against insulin. METHODS: We incubated rat islets in 2.8, 11.0 or 20.0 mmol/l glucose for 24 h and did an electron microscopic evaluation after labelling with a specific anti-sulphatide monoclonal antibody. The reactivity of an insulin-specific T-cell clone isolated from a patient with Type I diabetes, was examined using insulin or insulin B-chain (B11-27) peptide incubated together with sulphatide. RESULTS: We detected lower amounts of sulphatide per insulin secretory granule in active compared with passive beta cells (p = 0.003). The presence of sulphatide in vitro at doses of 43-8.3 micromol/l resulted in greatly reduced proliferation (median 3.4 % of control value, p = 0. 0004) of the insulin-specific T-cell clone. No inhibition was found using the precursor of sulphatide, galactosylceramide, or GM1. Sulphatide did not reduce non-aspecific proliferation (induced by phorbol myristate acetate plus anti-CD3) or specific proliferation induced by insulin peptide. CONCLUSION/INTERPRETATION: These results imply that sulphatide possibly affect processing of the insulin molecule. Sulphatide which has been reported to interfere with phagosome-lysosome fusion, conceivably interacts with insulin. We hypothesize a (patho)physiological role of sulphatide, variably expressed in beta cells, by reducing the antigenicity of insulin. [Diabetologia (1999) 42: 1212-1218]

Control of cholesterol biosynthesis in Schwann cells.

Cholesterol accounts for over one-fourth of total myelin lipids. We found that, during development of the rat sciatic nerve, expression of mRNA for hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis, was up-regulated in parallel with mRNA for P0, the major structural protein of PNS myelin, and with ceramide galactosyltransferase (CGT), the rate-limiting enzyme in cerebroside biosynthesis. To help establish the nature of this coordinate regulation of myelin-related genes, we examined their steady-state mRNA levels in cultured primary Schwann cells. We also assayed synthesis of cholesterol and cerebroside to distinguish how much control of synthetic activity for these two myelin lipids involved mRNA levels for HMG-CoA reductase and CGT, and how much involved post-mRNA control mechanisms. Addition of forskolin to cells cultured in media supplemented with normal calf serum resulted in up-regulation of P0 and CGT mRNA expression and cerebroside synthesis, without corresponding increases in HMG-CoA reductase mRNA or cholesterol synthesis. Cholesterol synthesis increased approximately threefold in Schwann cells cultured with lipoprotein-deficient serum, without any increase in HMG-CoA reductase mRNA. Furthermore, addition of either serum lipoproteins or 25-hydroxycholesterol decreased cholesterol synthesis without altering HMG-CoA reductase mRNA levels. We conclude that, as in other tissues, cholesterol synthesis in Schwann cells is regulated primarily by intracellular sterol levels. Much of this regulation occurs at posttranscriptional levels. Thus, the in vivo coordinate up-regulation of HMG-CoA reductase gene expression in myelinating Schwann cells is secondary to intracellular depletion of cholesterol, as it is compartmentalized within the myelin. It is probably not due to coordinate control at the level of mRNA expression.

Expression of neolactoglycolipids: sialosyl-, disialosyl-, O-acetyldisialosyl- and fucosyl- derivatives of neolactotetraosyl ceramide and neolactohexaosyl ceramide in the developing cerebral cortex and cerebellum.

The following neolacto glycolipids were identified and their developmental expression was studied in the rat cerebral cortex and cerebellum: Fuc alpha 1-3IIInLcOse4Cer,Fuc alpha 1-3VnLcOse6Cer and (Fuc)2 alpha 1-3III,3VnLcOse6Cer, as well as acidic glycolipids, NeuAc alpha 2-3IVnLcOse4Cer [nLM1], (NeuAc)2 alpha 2-3IVnLcOse4Cer [nLD1], O-acetyl (NeuAc)2 alpha 2-3IVnLcOse4Cer [OAc-nLD1] and their higher neolactosaminyl homologues NeuAc alpha 2-3VlnLcOse6Cer [nHM1] and (NeuAc)2 alpha 2-3VlnLcOse6Cer [nHD1]. These glycolipids were expressed in the cerebral cortex only during embryonic stages and disappeared postnatally. This loss was ascribed to the down regulation of the synthesis of the key precursor LcOse3Cer which is synthesized by the enzyme lactosylceramide: N-acetylglucosaminyl transferase. On the other hand in the cerebellum, these glycolipids increased with postnatal development due to increasing availability of LcOse3Cer. In the cerebellum, only nLM1 and fucosyl-neolactoglycolipids declined after postnatal day 10-15, perhaps due to regulation by other glycosyltransferases. Also, in the cerebellum, nLD1 and nHD1 were shown to be specifically associated with Purkinje cells and their dendrites in the molecular layer and with their axon terminals in the deep cerebellar nuclei, similar to other neolactoglycolipids shown previously.

Cerebroside formation in the peripheral nervous system of normal and Trembler mice.

The formation of cerebrosides by the galactosylation of ceramides was investigated in a microsomal fraction prepared from sciatic nerves of normal and Trembler mice. In the control, cerebroside synthesis is observed in the presence of uridine-diphosphate-galactose both from endogenously synthesized [1-14C]stearoyl-sphingosine (C18-ceramide), and from [1-14C]lignoceroyl-sphingosine (C24-ceramide). Cerebroside formation is also demonstrated by studying the galactosylation of exogenous ceramides with UDP[1-14C]-galactose. In the mutant, only trace amounts of labeled cerebrosides are formed from labeled stearoyl-sphingosine, whereas with lignoceroyl-sphingosine, no cerebroside synthesis is detected under conditions allowing their formation in the control. However, a higher rate of synthesis of short acyl chain-glucosyl ceramides is observed in the Trembler samples.

Expression of glycolipids and myelin-associated glycoprotein during the differentiation of oligodendrocytes: comparison of the CG-4 glial cell line to primary cultures.

The expression of cerebrosides, sulfatides, gangliosides and the myelin-associated glycoprotein (MAG) during differentiation of the CG-4 line of oligodendrocyte progenitors [Louis et al.: J. Neurosci Res 31: 193, 1992] was compared with their expression in primary cultures of oligodendrocyte precursors [McCarthy and de Vellis: J Cell Biol 85: 890, 1980]. When the CG-4 cells differentiated from bipolar progenitors to oligodendrocytes, there was a decrease of glucosylcerebroside synthesis and an increase in galactosylcerebroside and sulfatide synthesis. However, even after differentiation, the incorporation of [3H]galactose into these glycolipids, the amounts of galactosylcerebroside and sulfatide, and the galactocerebroside/sulfatide ratio were all much less than in primary cultures of differentiating oligodendrocytes. The major gangliosides in differentiated primary oligodendrocyte cultures were GM3 and GD3, and GD3 was also a major ganglioside in the CG-4 line. However, unlike primary cultures of O-2A lineage cells in which GM3 synthesis increased dramatically during differentiation to oligodendrocytes, the CG-4 cells expressed very little GM3. Also, the CG-4 cells expressed larger amounts of more complex gangliosides, e.g. GD1b and GT1b, which were almost entirely restricted to the b-series. The amount of MAG expressed by the CG-4 cells increased substantially when they differentiated to oligodendrocytes, and it was almost all the large immature isoform. However, even after differentiation, the amount expressed was less than in differentiated primary oligodendrocyte cultures. Overall, the lower expression of myelin-related glycolipids and MAG by the CG-4 line suggests a lesser degree of differentiation in comparison to primary oligodendrocytes under the culture conditions of these experiments, but the larger amounts of cells available from the CG-4 line should be useful for investigating glycolipid and MAG function related to the early stages of myelinogenesis.

Peripheral nerve sphingomyelin and cerebroside are both formed via two metabolically and kinetically distinct pathways in vivo.

We have studied the labeling kinetics of peripheral nerve sphingolipids in vivo. The kinetic analysis of the labeling profiles observed for the various sphingolipids demonstrated that 90% of cerebrosides, but only 30% of sphingomyelin, were synthesized via a de novo synthesized ceramide intermediate following the injection of 1-4 pmol [3H]palmitate into mouse sciatic nerves. The remaining sphingolipid labeling (30% of the total) was due to direct acylation events, using free fatty acids originating from a pool different from those implicated in the de novo ceramide pathway. Direct acylation events ceased within 1 h following substrate administration, while labeling via the ceramide pathway continued through 5 h. The results provide the first in vivo demonstration that the formation of cerebrosides and sphingomyelin in peripheral nerves in situ can be simultaneously assured via two metabolically and kinetically distinct pathways that employ different fatty acid pools.

The synthetic pathway for glucosylsphingosine in cultured fibroblasts.

The synthesis of glucosylsphingosine (GlcSph), a glucosylceramide (GlcCer) analogue devoid of fatty acids, in cultured fibroblasts was studied by using conduritol beta epoxide (CBE), an inhibitor of beta-glucosidase, and 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glucosylceramide (GlcCer) synthase (glucosyltransferase). When CBE was added to the culture medium, the intracellular beta-glucosidase activity decreased, and both GlcCer and GlcSph accumulated in the cells. After the addition of PDMP, the concentration of GlcCer decreased, while the content of GlcSph increased. When CBE and PDMP were added together, the intracellular accumulation of GlcSph to decreased to less than when CBE alone was added. Based on these results, the synthetic pathway for GlcSph was thus considered to not only be through the glucosylation of sphingosine, but also through the deacylation of GlcCer. When GlcCer (d18:1, C12:0) was added to the culture medium, the intracellular accumulation of GlcSph (d18:1) was evident, and it was also more pronounced in the presence of CBE. In addition, when GlcCer (d18:0, C12:0) was used, apparent accumulation of GlcSph (d18:0) was also observed. In order to determine whether or not the deacylase of GlcCer is identical to acid ceramidase, a deacylase of ceramide, the same experiments were carried out using fibroblasts from two patients with Farber disease, in which acid ceramidase is genetically deficient. The accumulation of GlcSph in the Farber disease fibroblasts after the loading of GlcCer for 7 days was found to be one-fifth of the control level.(ABSTRACT TRUNCATED AT 250 WORDS)

The human blood fluke Schistosoma mansoni synthesizes a novel type of glycosphingolipid.

Previous studies have shown that the glycoprotein oligosaccharides synthesized by adult Schistosoma mansoni, the organism responsible for human schistosomiasis, are unusual in that they contain terminal beta-GalNAc residues and lack sialic acid. These observations and other studies indicating that schistosome glycoproteins and glycolipids are antigenic in infected animals led us to investigate the structures of the glycosphingolipids synthesized by these organisms and to determine whether they are structurally related to those synthesized by their vertebrate hosts. For our studies, adult schistosomes were metabolically radiolabeled with either [3H]galactose or [3H]glucosamine, and the newly synthesized glycosphingolipids were isolated and characterized. The major glycosphingolipids synthesized by adult schistosomes were found to be galactosylceramide and glucosylceramide. The adult worms synthesized no lactosylceramide (Gal beta 1-4Glc-ceramide), a common constituent of vertebrate cells; however, another disaccharide-containing glycosphingolipid cleavable by ceramide glycanase was found. The results of compositional and methylation analyses and exoglycosidase treatments demonstrated that this ceramide-disaccharide has the structure GalNAc beta 1-4Glc-ceramide. We also found that extracts of adult schistosomes are unable to transfer Gal from UDP-Gal to glucosylceramide, whereas extracts of Chinese hamster ovary cells, as a control, are able to do so, confirming that schistosomes are unable to synthesize lactosylceramide. Low levels of higher molecular weight glycosphingolipids were also found to be synthesized by adult schistosomes, and although their levels were too small to allow definitive characterization, compositional analyses indicated that they also contained GalNAc. We have tentatively designated the new disaccharide structure GalNAc beta 1, 4Glc- the "schistocore", which may represent a new type of glycosphingolipid core series.