Two bacterial glycosphingolipid synthases responsible for the synthesis of glucuronosylceramide and α-galactosylceramide.

Bacterial glycosphingolipids such as glucuronosylceramide and galactosylceramide have been identified as ligands for invariant natural killer T cells and play important roles in host defense. However, the glycosphingolipid synthases required for production of these ceramides have not been well-characterized. Here, we report the identification and characterization of glucuronosylceramide synthase (ceramide UDP-glucuronosyltransferase [Cer-GlcAT]) in Zymomonas mobilis, a Gram-negative bacterium whose cellular membranes contain glucuronosylceramide. On comparing the gene sequences that encode the diacylglycerol GlcAT in bacteria and plants, we found a homologous gene that is widely distributed in the order Sphingomonadales in the Z. mobilis genome. We first cloned the gene and expressed it in Escherichia coli, followed by protein purification using nickel-Sepharose affinity and gel filtration chromatography. Using the highly enriched enzyme, we observed that it has high glycosyltransferase activity with UDP-glucuronic acid and ceramide as sugar donor and acceptor substrate, respectively. Cer-GlcAT deletion resulted in a loss of glucuronosylceramide and increased the levels of ceramide phosphoglycerol, which was expressed in WT cells only at very low levels. Furthermore, we found sequences homologous to Cer-GlcAT in Sphingobium yanoikuyae and Bacteroides fragilis, which have been reported to produce glucuronosylceramide and α-galactosylceramide, respectively. We expressed the two homologs of the cer-glcat gene in E. coli and found that each gene encodes Cer-GlcAT and Cer-galactosyltransferase, respectively. These results contribute to the understanding of the roles of bacterial glycosphingolipids in host-bacteria interactions and the function of bacterial glycosphingolipids in bacterial physiology.

Ceramide galactosyltransferase expression is regulated positively by Nkx2.2 and negatively by OLIG2.

Myelin, a multilamellar structure extended from oligodendrocytes or Schwann cells, plays a critical role in maintenance of neuronal function, and damage or loss of myelin causes demyelinating diseases such as multiple sclerosis. For precise alignment of the myelin sheath, there is a requirement for expression of galactosylceramide (GalCer), a major glycosphingolipid in myelin. Synthesis of GalCer is strictly limited in oligodendrocytes in a developmental stage-specific manner. Ceramide galactosyltransferase (CGT), a key enzyme for biosynthesis of GalCer, exhibits restricted expression in oligodendrocytes but the mechanism is poorly understood. Based on our assumption that particular oligodendrocyte-lineage-specific transcription factors regulate CGT expression, we co-expressed a series of candidate transcription factors with the human CGT promoter-driving luciferase expression in oligodendroglioma cells to measure the promoter activity. We found that Nkx2.2 strongly activated the CGT promoter. In addition, we identified a novel repressive DNA element in the first intron of CGT and OLIG2, an oligodendrocyte-specific transcription factor, as a binding protein of this element. Moreover, overexpression of OLIG2 completely canceled the activating effect of Nkx2.2 on CGT promoter activity. Expression of CGT mRNA was also upregulated by Nkx2.2, but this upregulation was cancelled by co-expression of OLIG2 with Nkx2.2. Our study suggests that CGT expression is controlled by balanced expression of the negative modulator OLIG2 and positive regulator Nkx2.2, providing new insights into how expression of GalCer is tightly regulated in cell-type- and stage-specific manners.

Inefficient fusion due to a lack of attachment receptor/co-receptor restricts productive human immunodeficiency virus type 1 infection in human hepatoma Huh7.5 cells.

Since the widespread use of the highly active antiretroviral therapy, the incidence of liver disease has increased to become a leading cause of death among human immunodeficiency virus type 1 (HIV-1)-infected individuals. It can be proposed that the ability of HIV-1 to infect hepatocytes could influence liver diseases. Although the presence of HIV-1 was identified in hepatocytes from HIV-1 seropositive patients, the susceptibility of hepatocytes to HIV-1 infection in vitro remains controversial. We present evidence here that human hepatoma cells are not productively infected with CD4-dependent HIV-1 strains because of inefficient fusion related to an absence of cell surface CD4 and CXCR4. However, these cells display an increased susceptibility to infection with a CD4-independent viral isolate through an interaction with galactosyl ceramide, an alternate receptor for HIV-1. This study provides further understanding of the susceptibility of human hepatocytes to HIV-1 infection. However, in vivo investigations are recommended to consolidate these data.

The PGRMC1 Antagonist AG-205 Inhibits Synthesis of Galactosylceramide and Sulfatide.

Sulfatide synthesis in the human renal cancer cell line SMKT-R3 was strongly inhibited in the presence of low µM concentrations of AG-205, a progesterone receptor membrane component 1 (PGRMC1) antagonist. This was also the case in Chinese hamster ovary (CHO) cells stably transfected with UDP-galactose: ceramide galactosyltransferase and cerebroside sulfotransferase, the two enzymes required for sulfatide synthesis. In CHO cells synthesizing galactosylceramide but not sulfatide, galactosylceramide was also strongly reduced, suggesting an effect at the level of galactolipid synthesis. Notably, AG-205 inhibited galactosylceramide synthesis to a similar extent in wild type CHO cells and cells that lack PGRMC1 and/or PGRMC2. In vitro enzyme activity assays showed that AG-205 is an inhibitor of UDP-galactose: ceramide galactosyltransferase, but not cerebroside sulfotransferase. This study shows that PGRMC1 is only one of several targets of AG-205 and should be used with caution, especially in studies using cells synthesizing galactosylceramide and sulfatide.

Myelin-specific proteins and glycolipids in rat Schwann cells and oligodendrocytes in culture.

We have used antibodies to identify Schwann cells and oligodendrocytes and to study the expression of myelin-specific glycolipids and proteins in these cells isolated from perinatal rats. Our findings suggest that only Schwann cells which have been induced to myelinate make detectable amounts of galactocerebroside (GC), sulfatide, myelin basic protein (BP), or the major peripheral myelin glycoprotein (P0). When rat Schwann cells were cultured, they stopped making detectable amounts of these myelin molecules, even when the cells were associated with neurites in short-term explant cultures of dorsal root ganglion. In contrast, oligodendrocytes in dissociated cell cultures of neonatal optic nerve, corpus callosum, or cerebellum continued to make GC, sulfatide and BP for many weeks, even in the absence of neurons. These findings suggest that while rat Schwann cells require a continuing signal from appropriate axons to make detectable amounts of myelin-specific glycolipids and proteins, oligodendrocytes do not. Schwann cells and oligodendrocytes also displayed very different morphologies in vitro which appeared to reflect their known differences in myelinating properties in vivo. Since these characteristic morphologies are maintained when Schwann cells and oligodendrocytes were grown together in mixed cultures and in the absence of neurons, we concluded that they are intrinsic properties of these two different myelin-forming cells.

Discrepancies of NKT cells expression in peripheral blood and in cerebrospinal fluid from Behçet's disease.

The precise role of natural killer T (NKT) cells in the pathogenesis of Behçet's disease (BD) remains unknown. The frequency, cytokine profile and heterogeneity of NKT cells were studied in peripheral blood mononuclear cells (PBMC) from 42BD patients and in cerebrospinal fluid (CSF) samples from 9 neuro-BD patients. Flow cytometry revealed a decreased frequency of NKT cells in PBMC from BD patients (median: 0.06%; range: 0%-0.3%) when compared to healthy controls (median: 0.23%; range: 0.1%-0.7%; P<0.01). NKT cells were biased toward a Th(1)-like phenotype, with a significant decrease of IL-4/IFN-gamma ratio in BD (median: 0.049; range: 0.01-0.13) vs. healthy controls (median: 0.82; range: 0.4-1.33; P<0.01). NKT cells were increased in CSF-BD samples (median: 0.18%; range: 0.1%-0.4%), when compared to CSF-NIND patients (median: 0.05%; range: 0.01%-0.09%; P<0.01). Based on the reactivity of PBMC-derived NKT cells toward alpha-galactosylceramide (alpha-GalCer), 80% of BD patients were non-responsive. At the opposite, the reactivity of NKT cells in CSF from BD patients was not impaired. BD-CSF NKT cells exhibited an increased expression of IFN-gamma-producing cells, demonstrating that CSF-NKT cells were functional, and biased toward a Th(1)-like phenotype. These data suggest that functional NKT cells are recruited into BD inflammatory sites contributing to BD pathogenesis.

A mammalian fatty acid hydroxylase responsible for the formation of alpha-hydroxylated galactosylceramide in myelin.

Hydroxylation is an abundant modification of the ceramides in brain, skin, intestinal tract and kidney. Hydroxylation occurs at the sphingosine base at C-4 or within the amide-linked fatty acid. In myelin, hydroxylation of ceramide is exclusively found at the alpha-C atom of the fatty acid moiety. alpha-Hydroxylated cerebrosides are the most abundant lipids in the myelin sheath. The functional role of this modification, however, is not known. On the basis of sequence similarity to a yeast C26 fatty acid hydroxylase, we have identified a murine cDNA encoding FA2H (fatty acid 2-hydroxylase). Transfection of FA2H cDNA in CHO cells (Chinese-hamster ovary cells) led to the formation of alpha-hydroxylated fatty acid containing hexosylceramide. An EGFP (enhanced green fluorescent protein)-FA2H fusion protein co-localized with calnexin, indicating that the enzyme resides in the endoplasmic reticulum. FA2H is expressed in brain, stomach, skin, kidney and testis, i.e. in tissues known to synthesize fatty acid alpha-hydroxylated sphingolipids. The time course of its expression in brain closely follows the expression of myelin-specific genes, reaching a maximum at 2-3 weeks of age. This is in agreement with the reported time course of fatty acid alpha-hydroxylase activity in the developing brain. In situ hybridization of brain sections showed expression of FA2H in the white matter. Our results thus strongly suggest that FA2H is the enzyme responsible for the formation of alpha-hydroxylated ceramide in oligodendrocytes of the mammalian brain. Its further characterization will provide insight into the functional role of alpha-hydroxylation modification in myelin, skin and other organs.

Neutral lipid precursors for gangliosides are not formed by rat liver homogenates or by purified cell fractions.

As part of an ongoing investigation into the ganglioside and neutral glycosphingolipid biosynthetic pathways in rodent liver, the synthesis of ceramide, glucosylceramide and lactosylceramide was examined in homogenates and purified membrane fractions of rat liver. Ceramide synthesis and its stimulation by exogenously added D-sphingosine was demonstrated in homogenates and isolated membrane fractions, with activity concentrated in the endoplasmic reticulum. In contrast, significant D-sphingosine- or ceramide-dependent formation of glucosylceramide, or glucosylceramide-dependent formation of lactosylceramide (in the presence of labelled UDP-[14C]glucose and UDP-[14C]galactose, respectively,) could not be demonstrated. The possibility is raised that liver may be dependent on extra-hepatic sources of neutral glycosphingolipids to support ganglioside biosynthesis.

Luteal phase-characteristic induction of I3SO3-GalCer in human cervical epithelia and uterine endometria, and follicular phase-characteristic formation of a ganglioside-derived negative charge gradient in different regions of fallopian tubes.

In a series of experiments on the hormone-dependent molecular alteration in the human genital tract during the menstrual cycle, we focused our attention on a change in the negative charge due to the sulfuric acid- and sialic acid-containing glycosphingolipids. Although a ganglioside-derived negative charge was maintained in the cervical epithelia and uterine endometria at a relatively constant concentration throughout the luteal and follicular phases, I3SO3GalCer in both tissues characteristically increased in the luteal phase, indicating that the synthesis of I3SO3-GalCer in both tissues is associated with the menstrual cycle. However, I3SO3-GalCer in mucosae of the fallopian tubes in both phases was present in a concentration similar to that in the uterine endometrium in the luteal phase, and the change in the concentration did not associated with the menstrual cycle. On the other hand, although the concentrations of I3SO3-GalCer and II3NeuAc-LacCer, a major ganglioside, were similar in different regions, that is, the isthmus, ampulla and fimbriae of the fallopian tubes in the luteal phase, II3NeuAc-LacCer was present in a gradually increasing concentration from the isthmus to the fimbriae in the follicular phase, giving a gradually decreasing ratio of I3SO3GalCer to ganglioside from the uterus to the fimbriae. These findings indicate that the metabolism of sulfo- and sialoglycosphingolipids in the human genital tract is strictly controlled by estrogen and progesterone.

Acetoacetate and glucose as substrates for lipid synthesis by rat brain oligodendrocytes and astrocytes in serum-free culture.

We have compared glucose and acetoacetate as precursors for lipogenesis and cholesterogenesis by oligodendrocytes and astrocytes, using mixed glial cultures enriched in oligodendrocytes. In order to differentiate between metabolic processes in oligodendrocytes and those in astrocytes, the other major cell type present in the mixed culture, we carried out parallel incubations with cultures from which the oligodendrocytes had been removed by treatment with anti-galactocerebroside serum and guinea-pig complement. The following results were obtained: 1. Both oligodendrocytes and astrocytes in culture actively utilize acetoacetate as a precursor for lipogenesis and cholesterogenesis. 2. In both cell types, the incorporation of acetoacetate into fatty acids and cholesterol exceeds that of glucose by a factor of 5-10 when the precursors are present at concentrations of 1 mM and higher. 3. Glucose stimulates acetoacetate incorporation into fatty acids and cholesterol, whereas acetoacetate reduces the entry of glucose into these lipids. This suggests that glucose is necessary for NADPH generation, but that otherwise the two precursors contribute to the same acetyl-CoA pool. 4. Both with acetoacetate and with glucose as precursor, oligodendrocytes are more active in cholesterol synthesis than astrocytes. 5. Using incorporation of 3H2O as an indicator for total lipid synthesis, we estimated that acetoacetate contributes one third of the acetyl groups and glucose one twentieth when saturating concentrations of both substrates are present.

Effect of dimethyl sulfoxide on transformed rat Schwann cells.

Cultured rat Schwann cells transformed by Simian Virus 40 (SV40) have previously been shown to retain their ability to synthesize myelin-associated galactosylceramide and sulfatide. Little is known about the mechanism regulating galactosphingolipid synthesis in Schwann cells. We have found that growing the transformed Schwann cells in the presence of dimethyl sulfoxide (DMSO) markedly inhibits the incorporation of [35S]sulfate into sulfatide, in a time- and dose-dependent manner. The concentration of DMSO which resulted in a half-maximal inhibition after 6 days of incubation was 0.5%, and the incubation time required for a half-maximal effect at 1.0% DMSO was approximately 4 days. In contrast, DMSC did not affect the incorporation of [35S]sulfate into glycosaminoglycans. In addition, DMSO treatment has little effect on the synthesis of cellular DNA, proteins and lipids. When transformed Schwann cells were treated with DMSO, a substantial decrease in the incorporation of [3H]galactose into galactosylceramide was observed. The concentration of DMSO which resulted in a half-maximal inhibition of galactosylceramide synthesis was approximately 0.5%, similar to the concentration required for a similar effect on sulfatide synthesis. However, the incubation time required for a half-maximal inhibitory effect on galactosylceramide synthesis at 1.0% DMSO was less than 1 day, which was substantially shorter than the time required for the inhibition of sulfatide synthesis at this concentration. This finding is consistent with the interpretation that treatment with DMSO inhibits the synthesis of galactosylceramide, a precursor of sulfatide, which results in a decrease in the synthesis of sulfatide during a prolonged incubation of DMSO.

Altered sphingolipid metabolism in multidrug-resistant ovarian cancer cells is due to uncoupling of glycolipid biosynthesis in the Golgi apparatus.

Multidrug-resistant tumor cells display enhanced levels of glucosylceramide. In this study, we investigated how this relates to the overall sphingolipid composition of multidrug-resistant ovarian carcinoma cells and which mechanisms are responsible for adapted sphingolipid metabolism. We found in multidrug-resistant cells substantially lower levels of lactosylceramide and gangliosides in sharp contrast to glucosylceramide, galactosylceramide, and sphingomyelin levels. This indicates a block in the glycolipid biosynthetic pathway at the level of lactosylceramide formation, with concomitant accumulation of glucosylceramide. A series of observations exclude regulation at the enzyme level as the underlying mechanism. First, reduced lactosylceramide formation occurred only in intact resistant cells whereas cell-free activity of lactosylceramide synthase was higher compared with the parental cells. Second, the level of lactosylceramide synthase gene expression was equal in both phenotypes. Third, glucosylceramide synthase (mRNA and protein) expression and activity were equal or lower in resistant cells. Based on the kinetics of sphingolipid metabolism, the observation that brefeldin A does not restore lactosylceramide synthesis, and altered localization of lactosylceramide synthase fused to green fluorescent protein, we conclude that lactosylceramide biosynthesis is highly uncoupled from glucosylceramide biosynthesis in the Golgi apparatus of resistant cells.

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.

Regulation of galactocerebroside and sulfatide synthesis by hormones in chick neural cultures.

Chick neural cultures were used to study effects of insulin, thyrotropin releasing hormone, growth hormone and glucagon on myelin lipid synthesis in vitro. The incorporation of [3H]galactose into myelin associated lipids such as cerebroside and sulfatide was used as an index for various hormonal effects on myelination. The data suggest that these hormones were effective on myelin lipid synthesis only in the central nervous cells, not in the peripheral nerve cells.

Heterogeneous induction of 72-kDa heat shock protein (HSP72) in cultured mouse oligodendrocytes and astrocytes.

The expression of 72-kDa heat shock protein (HSP72) in cultured mouse oligodendrocytes and astrocytes exposed to heat shock was investigated by double immunolabelling with anti-HSP72 monoclonal antibody (C92F3B-1) and antibodies against galactocerebroside (GalC) or glial fibrillary acidic protein (GFAP). After 3 h recovery from heat shock, an intermediate level of HSP72 immunolabelling was localized in the nucleolus and cytoplasm of astrocytes (less than 25%) and to a lesser extent in oligodendrocytes (less than 2%). After 8-48 h, HSP72 was expressed intensely in the cytoplasm and nuclear matrix of oligodendrocytes (20-40%), while weak/intermediate immunostaining was detectable in astrocytes (5-15%). The levels of HSP72 expressed in oligodendrocytes and astrocytes decreased around 72-120 h, but a few oligodendrocytes (4%) remained intensely immunolabelled. These results indicate that heat shock induces HSP72 in both oligodendrocytes and astrocytes. However, this response is heterogeneous.

Schwann cell responses to forskolin and cyclic AMP analogues: comparative study of mouse and rat Schwann cells.

Forskolin and cAMP analogues (8-bromo cAMP and dibutyryl cAMP) induced proliferation or surface galC expression, depending on the concentrations, in rat and mouse Schwann cells in vitro. However, rat Schwann cells required much higher concentrations of these agents than mouse Schwann cells in both proliferation and surface galC expression. The concentrations needed for cellular proliferation were 0.5 microM forskolin or 10 microM cAMP analogues in mice and 2.5 microM forskolin or 50 microM cAMP analogues in rats. Surface galC was expressed in mouse Schwann cells at concentrations of 10 microM forskolin or 100 microM cAMP analogues, while in rat Schwann cells, 50 microM forskolin or 500 microM cAMP analogues was needed for expression of surface galC. Rat Schwann cells transformed from an elongated spindle shape to flattened cells by the addition of these agents. However, mouse Schwann cells remained spindle shaped and their processes were apparently elongated at concentrations of more than 1.0 microM forskolin or 100 microM cAMP analogues. These results may reflect the differences in the cellular metabolism between mouse and rat cells. Moreover, the elongation of mouse Schwann cell processes appeared to be associated with surface galC expression, suggesting that elongation may be an initial signal for differentiation in mouse Schwann cells.

Age-related differences in mouse Schwann cell response to cyclic AMP.

An elevation of intracellular cyclic adenosine 3', 5'-monophosphate (cAMP) is known to stimulate proliferation and expression of surface galactocerebroside (galC) in cultured Schwann cells from newborn rats and mice. We investigated the age-related differences in proliferative and differentiating responses of Schwann cells to forskolin, an adenylate cyclase activator, in mice at postnatal days (P) 3, 10 and 30. The proportion of surface galC-positive cells decreased progressively with time in all ages during the initial three days in vitro (DIV) but the proportion of Schwann cells expressing surface galC at P30 was lower than that at P3 or P10. Administration of 50 microM forskolin in the media on the four DIV induced surface galC expression in Schwann cells from P3 and P10 mice but not from P30 mice. Forskolin also stimulated the proliferation of Schwann cells from P3 and P10 mice in media containing 2% fetal bovine serum (FBS) but not those from P30 mice, which, however, proliferated in media containing 10% FBS. These results suggest that Schwann cells in suckling mice are more sensitive to cAMP in both proliferative and differentiating responses than in adult. These different responses in vitro may reflect differences in the intrinsic metabolic status of Schwann cells in vivo since myelin-forming Schwann cells proliferate actively prior to myelination but cease or become less active in proliferation once myelination progresses.

Study of pathogenesis in twitcher mouse, an enzymatically authentic model of Krabbe's disease.

The twitcher mouse was investigated by examining in vivo synthesis of galactosylceramide (Galcer) and galactosylsphingosine (Galsph) in a sciatic nerve culture, and in vitro enzymic activities for synthesis of Galcer and Galsph in the spinal cord from normal and affected mice. For the in vivo study, the sciatic nerve was incubated for 24 h in medium containing [3H]galactose, or [3H]-sphingosine-labeled Galcer or Galsph. With [3H]galactose, reduced synthesis of Galcer was found as early as 1 week of age and synthesis decreased to about 15% of normal value at 4 weeks. Increased Galsph was detected after 7 days of feeding with galactose. In a study of [3H]sphingosine-labeled Galcer and Galsph feeding, Galcer did not induce Galsph synthesis in either normal or affected mice, and synthesis of Galcer from Galsph was found only in normal mice, suggesting that Galcer was synthesized from sphingosine after hydrolysis of Galsph. In vitro, the activities of UDP-galactose: ceramide galactosyltransferase and UDP-galactose: sphingosine galactosyltransferase were reduced to less than 50% of control after 2 weeks of age in affected mice. We conclude that (1) decreased Galcer was due to impaired synthesis of Galcer, (2) Galsph was synthesized from galactose and not from deacylation of Galcer, and (3) Galsph accumulation was due not to increased synthesis but to decreased hydrolysis.