Ceramide Mono Research Articles

Ceramide glycosylation and fatty acid hydroxylation influence serological reactivity in Trypanosoma cruzi glycosphingolipids

Ceramide mono (CMH) or dihexoside (CDH) fractions from Trypanosoma cruzi (Dm28c clone) were identified as glucosyl and lactosylceramides containing non-hydroxylated fatty acids. The di-glycosylated form was much more efficiently recognized by sera from T. cruzi-immunized rabbits, indicating that glycosylation influences antigenicity. Fatty acid hydroxylation was also a determinant of serological reactivity, since an α-hydroxylated CMH, only present at the Y clone, was recognized by the hyperimmune sera. In summary, these data indicate that T. cruzi CMHs with non-hydroxylated fatty acids are unable to induce antibody responses in animal hosts, which is reverted by the addition of a sugar residue or an α-hydroxyl group.

Glycosphingolipids from Magnaporthe grisea cells: expression of a ceramide dihexoside presenting phytosphingosine as the long-chain base

Magnaporthe grisea is a fungal pathogen that infects rice leaves and causes rice blast, a devastating crop disease. M. grisea produces active elicitors of the hypersensitive response in rice that were previously identified as ceramide monohexosides (CMHs). Using several chromatographic approaches, mass spectrometry, and nuclear magnetic resonance, we identified ceramide mono- and dihexosides (CDH) in purified lipid extracts from M. grisea cells. As described by other authors, CMH consists of a ceramide moiety containing 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecenoic or 2-hydroxyhexadecenoic acids and a carbohydrate segment consisting of one residue of glucose. CDHs, however, contain β-galactose (1→4)-linked to β-glucose as sugar units and phytosphingosine as the long-chain base, bound to a C24 α-hydroxylated fatty acid. To our knowledge, this is the first report on the occurrence of CDH in a fungal species and illustrates the existence of an alternative path of ceramide glycosylation in fungal cells.

Neutral and Amphoteric Glycosphingolipids from Annelida (Lugworm, Tylorhynchus heterochaetus)

Gal β 1-Cer (CMS3) and cholinephosphoryl→6 Gal β 1-Cer (PGL) were found as the main neutral and amphoteric glycosphingolipids from the whole tissue of the lugworm, Tylorhynchus heterochaetus. These lipids were purified by successive column chromatography on DEAE- and QAE-Sephadex A-25, Florisil and silicic acid (Iatrobeads), and identified by gas-liquid chromatography, methylation analysis, enzymatic hydrolysis, chromium trioxide oxidation, hydrogen fluoride degradation and positive-ion fast atom bombardment mass spectrometry.The ceramide moieties of both glycolipids were virtually the same : fatty acids were saturated C16-, C17- and C18-acids ; sphingoids were octadeca-4-sphingenine and octadeca-4, 8-sphingadienine. The structural similarity of the ceramide moieties suggests PGL biosynthesis from CMS3.In addition, their thin-layer chromatographic behaviors and the results of sugar analysis suggest that the structures of at least three minor components of the ceramide mono-, di- and trisaccharide fractions are Glc-Cer, Man-Gal-Cer and Man-Gal-Gal-Cer, respectively.

Brefeldin A induced inhibition of de novo globo- and neolacto-series glycolipid core chain biosynthesis in human cells. Evidence for an effect on beta 1–>4galactosyltransferase activity.

De novo synthesis of neolacto-series glycolipids has been studied in human cell lines via metabolic labeling of ceramide with [3H]serine. Intense labeling of ceramide mono- and dihexoside glycolipids occurred with labeling of progressively longer chain derivatives with increasing time. Most of the label was recovered in neutral glycolipids with about 5% of the total labeling in the ganglioside fraction. Experiments done using cell treatment with 2.5 micrograms/ml brefeldin A resulted in a stimulation in the total amount of labeling, accumulation of a neutral glycolipid identified as Lc3 due to inhibited transfer of the neolacto-series core chain terminal beta-Gal residue, and a corresponding inhibition of labeling of longer chain neutral glycolipids in all cell lines. Brefeldin A also blocked synthesis of the globo-series precursor, Gb3, longer chain sialylated structures such as IV3NeuAcnLc4, but not de novo GM3 synthesis. Brefeldin A treatment had no effect on cellular beta 1-->3N-acetylglucosaminyl-, beta 1-->4galactosyl-, or alpha 1-->3fucosyltransferase specific activities, nor was it inhibitory in beta 1-->4galactosyltransferase assays in vitro. The results describe brefeldin A-induced blocks in globo- and neolacto-series glycolipid biosynthesis, consistent with differential localization of enzymes in intracellular membranes. In particular, the results suggest that the beta 1-->4galactosyltransferase in these cells is either not redistributed by brefeldin A or is otherwise rendered nonfunctional.

Specificity of lung surfactant protein SP-A for both the carbohydrate and the lipid moieties of certain neutral glycolipids.

Binding specificity of the major surfactant protein SP-A from human and dog lung has been investigated. Radiobinding experiments have shown that both proteins bind in a Ca(2+)-dependent manner to galactose, mannose, fucose, and glucose linked to bovine serum albumin. These results are in accord with a previous study in which monosaccharides were linked to agarose (Haagsman, H. P., Hawgood, S., Sargeant, T., Buckley, D., White, R. T., Drickamer, K., and Benson, B. J. (1987) J. Biol. Chem. 262, 13877-13880). Chromatogram overlays in conjunction with in situ liquid secondary ion mass spectrometry (TLC-LSIMS) of several purified glycosphingolipids and neoglycolipids as well as binding assays with glycolipids immobilized on plastic wells, demonstrate recognition of galactose (human and dog SP-A), glucose, and lactose (human SP-A) in association with specific lipids. In addition, the occurrence of several neutral and acidic glycosphingolipids in human and rat extracellular surfactants and rat alveolar type II cells is described. Selected components among the neutral glycolipids are bound by radiolabeled human SP-A; these are identified by TLC-LSIMS as predominantly ceramide mono- and disaccharides (human surfactant) and ceramide tri- and tetrasaccharides (rat surfactant and type II cells). A recombinant carbohydrate recognition domain (CRD) of human SP-A inhibits the binding of human SP-A to galactosyl ceramide and to galactose- and mannose-bovine serum albumin, indicating that the CRD is directly involved in the binding of SP-A to these ligands. These results provide evidence for a novel type of binding specificity for proteins that have Ca(2+)-dependent CRDs and raise the possibility that glycosphingolipids are endogenous ligands for SP-A.

Structural characterization of neutral glycosphingolipids from Trypanosoma cruzi

The major neutral glycosphingolipids from Trypanosoma cruzi ceramide mono- and dihexosides (CMH and CDH, respectively) were analysed after chromatographic purification using 1H 500 MHz nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. The ceramide monohexoside fraction (CMH) contained both glucosyl- and galactosylceramides. After peracetylation, the CMH fraction was separated into 2 subfractions, CMHC OH and CMHC n, containing either hydroxy fatty acids or n-fatty acids. In the CMHC OH fraction glucose and galactose were present in a ratio of 2:1, whereas this ratio was 1:1 in the CMHC n fraction. The CDH fraction was identified as lactosylceramide with sphingosine as the long chain base and 16:0, 18:0, and 24:0, 24:4 fatty acids as the major components.

Immunological recognition of larvalTaenia crassiceps glycolipids by sera from parasite-infected mice

The isolation and purification of a neutral glycolipid fraction from Taenia crassiceps metacestodes (KBS strain), harvested from both male and female NMRI mice at 70-80 days following intraperitoneal infection, revealed 24 thin-layer chromatography-designated glycolipid bands. The glycolipids were defined as ceramide mono- (n = 3), di- (n = 3), tri- (n = 4), tetra- (n = 5), and greater than tetrasaccharides (n = 9) according to their running properties as defined by thin-layer chromatography against standards of known structure. The defined glycolipids were tested for immunoreactivity with sera from noninfected and T. crassiceps-infected NMRI mice (intraperitoneal injection or implantation of 15 larvae/animal) using the enzyme-linked immunosorbent assay (ELISA) until day 33 p.i. (IgM and IgG reaction) and high-performance thin-layer chromatography (HPTLC) combined with immunostaining (IgG reaction) until day 7 p.i. ELISA-determined IgM and IgG titres were significantly elevated from day 5 p.i. Immunostaining revealed early reactivity for certain ceramide tetra- and greater than tetrasaccharides (n = 6) on day 3 p.i. From day 5 p.i. onwards, nearly all glycolipids, including ceramide mono- and disaccharides, were recognized by the sera from metacestode-challenged mice. On day 7 p.i., a total of 22 bands were serologically active; of these, a considerable number (n = 10) showed increased staining intensity. Remarkably, in many cases (10 of 20), 3 glycolipids (tetra- and greater than tetrasaccharides) were weakly recognized by mouse sera taken before infection.

The glycosphingolipids of human prostate tissue

Neutral glycolipids and gangliosides from surgical samples of benign human prostate tissue were analyzed by chemical, enzymatic and immunostaining procedures. The neutral glycolipids consisted of ceramide mono-, di-, tri- and tetrahexosides of the globo series plus paragloboside. The monosialoganglioside fraction contained G M3 and G M1 plus multiple species of monosialylated lactosamine-containing structures, including sialyl- α-2 → 3paragloboside plus at least two compounds having a non-reducing terminal sialyl α2 → 6Gal linkage. The disialoganglioside fraction contained G D3 as the major component plus G D1a, G D2 and G D1b. G T1b was the major trisialoganglioside.

Glycosphingolipids of cultured human colon carcinoma cells and their drug-resistant sublines

Human colon carcinoma cells were analyzed for lipid phosphorus, cholesterol and glycosphingolipids. Ceramide mono-, di- and trihexosides and sulfatides were isolated by column and thin-layer chromatography and determined quantitatively on the basis of their hexose content. The complex lipid fractions so isolated were only partially resolved with the material available. Gangliosides G M2 and 6 M2 and globoside were major components of the fraction and were determined on the basis of their hexose, hexosamine and neuraminic acid content. The HCT 116, 116a and 116b cells contained no fucolipids. Cell lines resistant to mitomycin C, teniposide and etoposide were developed and analyzed. Over the 5 year period of the study sulfatides declined to about one-fourth of their original amounts in both parent and drug-adapted cells. HCT 116 cells adapted to mitomycin C and teniposide had 30% less ceramide monohexoside and a 45% greater cholesterol to lipid phosphorus ratio than the parent cells. Reductions in ceramide dihexoside in the drug-adapted cells were greater than those of the ceramide monohexoside. Galabiosyl ceramide was the major ceramide dihexoside in all the cells and accumulated in HCT 116a to levels 4-6-fold greater than that of the other lines as the only dihexoside.

Retinoic acid alters the metabolic 3H-labelling of glycosphingolipids

Retinoic acid increases the incorporation of radioactivity from a mixture of [ 3H]-galactose and [ 3H]-glucosamine into glycosphingolipids of serum-starved quiescent human foreskin fibroblasts with a preferential labelling of ceramide mono- and dihexoside as compared to ceramide tri- and tetrahexoside. Under the conditions used, no similar change in the specific labelling of glycoprotein is observed. Alteration in [ 3H]-precursor uptake into glycolipids comparable to that seen under the influence of retinoic acid does not occur in the presence of phorbolester, colchicine, butyrate or after infection with cytomegylovirus.

Application of field desorption mass spectrometry for the analysis of sphingoglycolipids.

Simple molecular species of intact ceramide mono-, di-, tri-, tetra-, and pentasaccharides purified by reversed phase high-performance liquid chromatography (HPLC) were analyzed by field desorption mass spectrometry (FD-MS). The analysis of sphingoglycolipids without chemical derivatization by FD-MS not only provides molecular information but also significant characteristic fragments for structural determination due to the cleavage of glycosidic bonds. These ions, therefore, give information on the molecular species of sphingoglycolipids and sugar sequences of their oligosaccharides. Intact GL1a, GL2a, and an equimolar mixture of GL1a and GL2a were also analyzed by FD-MS. In the spectra, the ions, (M + H)+, (M + Na)+, and (M + H- H2O)+, were observed as high intensive ions and different molecular species ions thereafter could be identified in all spectra. The FD-MS method is particularly useful in structural studies of glycolipids from natural sources.

Acidic sphingoglycolipids in invertebrate nervous tissue: Presence of several kinds of sphingophosphonoglycolipids in the nervous tissue of Aplysia kurodai, a sea gastropod

Several hitherto unknown sphingophosphonoglycolipids were identified in the lipid extract from the nervous tissue, nerve fibers and ganglia of Aplysia kurodai, which has only a small amount of gangliosides, if any. The most abundant sphingophosphonoglycolipids of the nerve fiber (FGL-VII) and ganglion (GGL-V) are ceramide bis(2-aminoethylphosphono)-pentaosides. Their oligosaccharide moieties consist of 1 mol each of glucose, 3-O-methylgalactose and galactosamine and 2 mol of galactose. Another main glycolipid (FGL-II) may be ceramide mono(2-aminoethylphosphono)-pentaoside which contains 1 mol each of glucose, galactosamine and fucose and 2 mol of galactose as its oligosaccharide moiety.

Studies on the glycosphingolipids of the starfish, Asterina pectinifera. I. The isolation and characterization of ceramide mono- and di-hexosides.

Ceramide mono- and di-hexosides were isolated from the starfish, Asterina pectinifera. These glycolipids had both a novel ceramide which contained almost entirely phytosphingosines (long-chain bases) and a high content of 2-hydroxy fatty acids. The long-chain base composition of the glycolipids shows an unusual pattern with both branched bases (iso-C16, iso-C17, iso-C18, anteiso-C17, and anteiso-C18) and normal ones (C16, C17, and C18). Of these, iso-C16- and anteiso-C18-phytosphingosines had been undetected up to now. The glycolipids were identified as glucosyl and lactosyl ceramides. Lactosyl ceramide had not been previously reported in starfish, although the wide occurrence of this glycolipid has been demonstrated in other organisms.

Cytolipin R and Its Possible Precursor Ceramide Trisaccharide from Metastatic Rat Mammary Tumor

Ceramide mono, di-, and trihexoside, two globosides, and hematoside were isolated from metastatic rat mammary tumor (MRMT-1) by column chromatography on silicic acid and preparative thin-layer chrpmatography. Each glycolipid was analyzed for fatty acids, long chain bases, and sugars. A combination of periodate oxidation, specific enzymatic hydrolysis, permethylation, and gas chromatographic studies of the sugar moieties established the arrangement of sugars, their anomeric configurations, and positions of their anomeric linkages in the lipids. The main glycolipids were two globosides having structure identical with cytolipin R; N-acetylgalactosaminyl(β1→3)galactosyl(α1→3)galactosyl(β1→4)glucosyl-ceramide. The only structural differences were in their component fatty acid composition. The structure of ceramide trisaccharide was galactosyl(α1→3)galactosyl(β1→4)glucosyl-ceramide, which is assumed to be a possible precursor of cytolipin R. The hematoside from this tumor was N-acetylneuraminyl-lactosyl-ceramide.

Cell density dependent glycolipids in NIL 2 hamster cells, derived malignant and transformed cell lines

Incorporation of [1- 14C]palmitate has been used to study the glycolipid pattern of NIL 2 Syrian hamster cells, derived malignant and transformed cell lines. Eive neutral glycolipids from a ceramide mono to pentahexoside, plus the ganglioside sialyl dihexosylceramide are synthesised by the NIL 2 cell. After a 48-h labelling period with [1- 14C]palmitate the maximum variation in specific activity in the individual glycolipids was 1.8-fold indicating that the method broadly reflects the glycolipid pattern within the cell. We have found increased labelling of the ceramide tri-, tetra-, and pentahexoside in dense compared to sparse cultures of NIL 2 cells. These cells when transformed by tumour viruses synthesised little or none of the three compounds. Subclones of the NIL2 cell line showed little variation in glycolipid pattern suggesting that transformation was not simply selecting for a small percentage of cells already lacking the glycolipids. The kinetics of synthesis of the density dependent glycolipids suggest a possible correlation with cell contact, although two situations were noted in which non contacted cells synthesised the compounds. Of eleven tumour lines produced by injecting NIL 2 cells into hamsters, ten failed to incorporate label into two of the three glycolipids in dense culture. However, one tumour line retained the normal glycolipid pattern, which was modified in the expected way by viral transformation. The results are discussed in terms of the possible significance of the density dependent glycolipids in normal cellular physiology.

Thin-layer chromatographic separation of glycolipids in animal lipid mixtures

A method of thin-layer chromatography was used for the separation of glycolipids from lipid extracts of animal tissues. The technique of two-step development was applied. In the first step glycolipids are separated from phospholipids; the second step makes possible the separation of different glycolipid classes, viz. ceramide mono-, tri- and tetrahexosides, sulphatides and monogalactosyl diglycerides. Gluco- and galactolipids were separated on plates partially impregnated with sodium boarate. The separation of psychosine, sphingosine and ceramide is also described.