The glycosphingolipid composition and glycosyltransferase activities of the small intestinal mucosa of testosterone-treated rats

Prior studies have demonstrated that sex hormones can influence the glycosphingolipid composition of different organs, including small intestine. However, to date, the effects of testosterone on glycosphingolipids of rat small intestinal mucosa have not been examined. Experiments were conducted to examine the effect of subcutaneous administration of synthetic testosterone (500 micrograms/100 g body wt.) on the gangliosides and neutral glycosphingolipids of rat small intestinal mucosa. Their results demonstrated that testosterone administrations: (i) increased the ganglioside content including hematoside (GM3); (ii) increased the total content of neutral glycosphingolipids, which was due to the increases in glucosylceramide and globotriaosylceramide; (iii) increased the activities of cytidine 5'-monophosphate-N-acetylneuraminic acid: lactosylceramide sialyltransferase, and UDPgalactose: lactosylceramide galactosyltransferase; (iv) increased the percentage of the long chain base phytosphingosine in hematoside, glucosyl-, and globotriaosylceramide; and (v) significantly altered the fatty acid composition of each of these glycosphingolipids. These results demonstrate that administration of testosterone induces alterations in glycosphingolipid composition and glycosyltransferases activities in rat small intestinal mucosa.     

Estrogen-induced alterations of the acidic and neutral glycosphingolipids of rat kidney

In order to determine whether female sex hormones could influence the glycosphingolipid composition of the rat kidney, male albino rats of the Sherman strain were subcutaneously administered the synthetic estrogen, ethinylestradiol (5 mg/kg body wt. per day) or vehicle for 5 days, and the ganglioside, ceramide and neutral glycosphingolipid compositions of the kidneys of these animals were analyzed and compared. The results of these experiments demonstrate that estrogen treatment: (1) increased the ceramide, acidic and neutral glycosphingolipid contents of this tissue; (2) decreased the relative percentages of glucosyl- and globotetraosylceramide and hematoside (GM3), but increased the relative percentage of globotriaosylceramide and 'other' gangliosides; (3) increased the relative percentage of N-acetyl- to N-glycolylneuraminic acid in GM3; and (4) altered the long-chain bases of GM3, glucosyl- and globotetraosylceramide in this organ. These data, therefore, demonstrate that estrogen administration induces quantitative and qualitative alterations in the gangliosides, neutral glycosphingolipids and ceramide of the rat kidney. This data as well as a discussion of the possible physiological consequences of these estrogen-induced alterations in kidney glycosphingolipids serve as the basis for this report.     

Estrogen-induced alterations of hematoside of rat small intestinal mucosa

Prior studies have suggested that sex hormones could influence the ganglioside and/or neutral glycosphingolipid composition of various organs. To date, the effects of sex hormones on the glycosphingolipid composition of the rat small intestinal mucosa, however, have not been examined. In the present studies, male albino rats of the Sherman strain were subcutaneously administered the synthetic estrogen, ethinylestradiol (5 mg/kg body wt. per day), or diluent for 5 days, and the ganglioside, neutral glycosphingolipid and ceramide composition of the small intestinal mucosa of these animals were analyzed and compared. The results of these experiments demonstrate that estrogen administration: increased the ganglioside concentration of this tissue, including hematoside (Gm3); increased the percentage of the long-chain base phytosphingosine of hematoside; and did not appear to significantly influence the concentration or composition of the neutral glycosphingolipids or ceramide in this tissue. These data, therefore, indicate that estrogen administration induces quantitative and qualitative alterations in the gangliosides but not in the neutral glycosphingolipids or ceramide of rat small intestinal mucosa.     

Effect of Differentiation and Cell Density on Glycosphingolipid Class and Molecular Species Composition of Mouse Neuroblastoma NB2a Cells

The effects of cell density and retinoic acid-induced differentiation on the class and molecular species composition of mouse neuroblastoma NB2a cell glycosphingolipids were examined under conditions where the period of culture was controlled. The total amount of neutral glycosphingolipids per cell decreased both with differentiation and as the cells became confluent. The relative amount of the neutral glycosphingolipid classes was not affected by differentiation, whereas there were small but significant changes in the relative amount of the neutral glycosphingolipid classes as the cells became confluent. The total amount of the gangliosides was unaffected by either differentiation or cell density, but there were significant changes in the ganglioside class composition as a result of both cell density and differentiation, and the effects were additive. The molecular species of all the major neutral glycosphingolipid and ganglioside classes were essentially identical, and were altered only slightly by either differentiation or cell density.     

Degradation of dilauroylphosphatidylcholine by phospholipase A2 in monolayers containing glycosphingolipids

The ability of phospholipase A2 from porcine pancreas to degrade all of the available dilauroylphosphatidylcholine in mixed monolayers with galactocerebroside, sulfatide, or ganglioside GM1 was investigated at different constant surface pressures. Under the conditions used the interfacial glycosphingolipid composition was continuously enriched as the enzyme action proceeded. The total percentage of phospholipid degradation depends on the surface pressure and on the type of glycosphingolipid. The presence of sulfatide activates the enzyme while galactocerebroside and ganglioside GM1 are inhibitory. The extent of phospholipid hydrolysis is independent of the effect of glycosphingolipids on the enzyme velocity. This is so when the latter is measured either in conditions of constant glycosphingolipid composition and zero-order kinetics [Bianco, I.D., Fidelio, G.D., & Maggio, B. (1989) Biochem. J. 258, 95-99] or under variable surface composition as in the present work. The modulation of phospholipase A2 activity by glycosphingolipids operates at two independent levels. One controls the rate of enzyme activity, and the other modulates the total extent of substrate degradation. This depends on the initial interaction of the enzyme with the interface. The glycosphingolipid effect on the activity is different depending on whether the enzyme has access to the substrate from the subphase or is already adsorbed to the lipid interface.     

Glycolipids support E-selectin-specific strong cell tethering under flow

This study provides functional evidence that glycosphingolipids constitute ligands for E-selectin but not P-selectin. Chinese hamster ovary (CHO) cells expressing E-selectin (CHO-E) or P-selectin (CHO-P) were perfused over alpha2,3-sialyl Lewis X (alpha2,3-sLe(x)) presented as the hexaosylceramide glycosphingolipid adsorbed in a monolayer containing phosphatidylcholine and cholesterol. CHO-E cells tethered extensively and formed slow, stable rolling interactions with alpha2,3-sLe(x) glycosphingolipid but not with the comparable alpha2,6-sLe(x) glycosphingolipid. Tethering/rolling varied with wall shear stress, selectin density, and ligand density. In contrast, alpha2,3-sLe(x) glycosphingolipid supported only limited, fast CHO-P cell rolling. As calculated from a stochastic model of cell rolling, the step size between successive bond releases from the alpha2,3-sLe(x) glycosphingolipid was smaller for CHO-E than CHO-P cells, whereas the opposite effect was observed for the waiting time between these events. Detachment assays revealed stronger adhesive interactions of CHO-E than CHO-P cells with alpha2,3-sLe(x) glycosphingolipid. These findings indicate that glycosphingolipids expressing an appropriate oligosaccharide mediate cell tethering/rolling via E-selectin but not P-selectin.     

Mast cell heterogeneity. Differential synthesis and expression of glycosphingolipids by mouse serosal mast cells as compared to IL-3-dependent bone marrow culture-derived mast cells before or after coculture with 3T3 fibroblasts

The synthesis and intracellular expression of glycosphingolipids by mouse serosal mast cells (SMC) have been characterized by radiolabeling and TLC and by immunodetection in situ. Chromatographic analysis of purified glycosphingolipids from SMC intrinsically labeled with [14C]galactose and [14C]glucosamine hydrochloride revealed the predominant synthesis of only the simplest neutral glycosphingolipid and ganglioside, glucosylceramide and ganglioside GM3, respectively. Intracellular indirect immunofluorescence staining of permeabilized SMC demonstrated the absence of the more complex neutral glycosphingolipids lactosylceramide, globotriosylceramide, globotetraosylceramide, and globopentaosylceramide, the absence of ganglioside GM1, and the presence of ganglioside GM3. By contrast, permeabilized mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) and mast cells recovered after 21 days of coculture of BMMC with mouse 3T3 fibroblasts expressed lactosylceramide, globotriosylceramide, globotetraosylceramide, ganglioside GM1, and ganglioside GM3, but not globopentaosylceramide intracellularly as determined by immunofluorescence. The findings indicate a loss of biosynthetic capacity and epitope maintenance for glycosphingolipids with in vivo differentiation of SMC from IL-3-dependent BMMC progenitors. Thus, although mast cells derived after coculture of these progenitors for 21 days with fibroblasts assume multiple SMC-like properties in terms of their histochemical staining and their secretory granule proteoglycan and neutral protease constituents, they do not lose the ability to express complex glycosphingolipids. The finding that glycosphingolipid composition does not change coordinately with other secretory granule markers defines a new stage of mouse mast cell development between the BMMC and SMC and provides evidence that mast cell development is more complex than previously appreciated.     

Egghead and brainiac are essential for glycosphingolipid biosynthesis in vivo

The Drosophila genes, brainiac and egghead, encode glycosyltransferases predicted to act sequentially in early steps of glycosphingolipid biosynthesis, and both genes are required for development in Drosophila. egghead encodes a beta4-mannosyltransferase, and brainiac encodes a beta3-N-acetylglucosaminyltransferase predicted by in vitro analysis to control synthesis of the glycosphingolipid core structure, GlcNAcbeta1-3Manbeta1-4Glcbeta1-Cer, found widely in invertebrates but not vertebrates. In this report we present direct in vivo evidence for this hypothesis. egghead and brainiac mutants lack elongated glycosphingolipids and exhibit accumulation of the truncated precursor glycosphingolipids. Furthermore, we demonstrate that despite fundamental differences in the core structure of mammalian and Drosophila glycosphingolipids, the Drosophila egghead mutant can be rescued by introduction of the mammalian lactosylceramide glycosphingolipid biosynthetic pathway (Galbeta1-4Glcbeta1-Cer) using a human beta4-galactosyltransferase (beta4Gal-T6) transgene. Conversely, introduction of egghead in vertebrate cells (Chinese hamster ovary) resulted in near complete blockage of biosynthesis of glycosphingolipids and accumulation of Manbeta1-4Glcbeta1-Cer. The study demonstrates that glycosphingolipids are essential for development of complex organisms and suggests that the function of the Drosophila glycosphingolipids in development does not depend on the core structure.     

Inhibition of glycosphingolipid biosynthesis reduces secretion of the beta-amyloid precursor protein and amyloid beta-peptide

Alzheimer disease is associated with extracellular deposits of amyloid beta-peptides in the brain. Amyloid beta-peptides are generated by proteolytic processing of the beta-amyloid precursor protein by beta- and gamma-secretases. The cleavage by secretases occurs predominantly in post-Golgi secretory and endocytic compartments and is influenced by cholesterol, indicating a role of the membrane lipid composition in proteolytic processing of the beta-amyloid precursor protein. To analyze the role of glycosphingolipids in these processes we inhibited glycosyl ceramide synthase, which catalyzes the first step in glycosphingolipid biosynthesis. The depletion of glycosphingolipids markedly reduced the secretion of endogenous beta-amyloid precursor protein in different cell types, including human neuroblastoma SH-SY5Y cells. Importantly, secretion of amyloid beta-peptides was also strongly decreased by inhibition of glycosphingolipid biosynthesis. Conversely, the addition of exogenous brain gangliosides to cultured cells reversed these effects. Biochemical and cell biological experiments demonstrate that the pharmacological reduction of cellular glycosphingolipid levels inhibited maturation and cell surface transport of the beta-amyloid precursor protein. In the glycosphingolipid-deficient cell line GM95, cellular levels and maturation of beta-amyloid precursor protein were also significantly reduced as compared with normal B16 cells. Together, these data demonstrate that glycosphingolipids are implicated in the regulation of the subcellular transport of the beta-amyloid precursor protein in the secretory pathway and its proteolytic processing. Thus, enzymes involved in glycosphingolipid metabolism might represent targets to inhibit the production of amyloid beta-peptides.     

Neutral glycolipids of atherosclerotic plaques and unaffected human aorta tissue

The composition, structure and localization of neutral glycosphingolipids of human aorta taken from subjects who had died after myocardial infarction were studied. Individual glycosphingolipids were purified by high-performance liquid chromatography and were characterized on the basis of their chromatographic mobility, carbohydrate composition, methylation analysis and by 1H-NMR spectroscopy. The main aortic glycosphingolipids were identified as glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. Significant differences in the neutral glycosphingolipid composition of intima and media were detected. The neutral glycosphingolipid profile of medial plaques resembled that of unaffected media; however, significant differences were detected between intimal plaques and unaffected intima. Whereas the latter contained trihexosylceramide and globoside as the only neutral glycolipids, the intimal plaque glycolipids consisted mainly of glucosylceramide and also contained appreciable amounts of lactosylceramide which were completely absent in the unaffected intima. In comparison to intimal plaques, unaffected intima is characterized by a much higher content of cerebrosides terminating by beta-galactosyl residues which are known to interact with growth factors and other external stimuli. It thus seems possible that the proliferative activity of smooth muscle cells in atherosclerotic diseases is to some extent associated with their neutral glycolipid profile.     

Fluorimetric determination of sphingosine and its application to natural mixtures of glycosphingolipids

A sensitive estimation of sphingosine, by measurement of the fluorescence of a complex formed with 1-naphthylamino-4-sulfonic acid, is described. The practical range is 5-35 nmoles sphingosine. The method is used to estimate, in terms of sphingosine, amounts of ceramide and glycosphingolipids. The isolation of microamounts (5-30 micro g) of individual glycosphingolipids from a mixture, and their quantitative estimation is described. The percentage composition of a glycosphingolipid mixture from the kidneys of adult C57/BL male mice is given.     

Cell density-dependent changes of glycosphingolipid biosynthesis in cultured human skin fibroblasts

In this study, the glycosphingolipid biosynthesis was investigated in the sparse and the confluent cell populations of cultured human skin fibroblasts.The human skin fibroblast cell populations were metabolically pulse labeled with ¹⁴C-galactose (48 h). The amounts of ¹⁴C-radioactivity (cpm) incorporated into extracted and purified total cellular glycosphingolipid fractions were counted by -scintillation and the individual glycosphingolipid species were separated by high performance thin layer chromatography and visualized by autoradiography. The relative labeling (%) of individual newly synthesized glycosphingolipid species was detected by densitometric scanning of autoradiographic glycosphingolipid patterns.The incorporation of ¹⁴C-label into total glycosphingolipids per cell increased significantly as the cell-density increased, referring to five fold higher rate of glycosphingolipid biosynthesis de novo in cells at confluency vs. sparse populations. The total newly synthesized glycosphingolipid pattern (100%) of sparse cell populations showed a significant predominance of the gangliosides (70%) over the neutral glycosphingolipids (30%), with ganglioside GM2 as the major species followed by monohexosyl-ceramide. Oppositely, the newly synthesized neutral glycosphingolipids (67%) predominated over the gangliosides (33%) in cells at confluency (contact inhibition). Cells reaching confluency were characterized by: (a) a dramatic increase of absolute amount of all newly synthesized neutral glycosphingolipid species, particularly the most abundant monohexosyl-ceramide and trihexosyl-ceramide, but also of the ganglioside GM3; (b) a drastic decrease of absolute amount of newly synthesized ganglioside GM2. The specific shift in newly synthesized glycosphingolipid pattern in cells reaching confluency suggests a down-regulation of biosynthetic pathway primarily at the level of N-acetylgalactosaminyl-transferase. A possible involvement of glycosphingolipids in cell density-dependent regulation of cell growth through establishment of the direct intermolecular intermembrane interactions is discussed.     

Use of the enzyme-linked immunoadsorbent assay to monitor the purification of glycosphingolipid antigens by high-performance liquid chromatography

An enzyme-linked immunoadsorbent assay (ELISA) technique has been applied to the analysis of glycosphingolipid fractions separated by high-performance liquid chromatography. Nanogram amounts of selected fractions were placed in microtiter wells and analyzed for glycosphingolipids carrying carbohydrate epitopes recognized by monoclonal antibodies using an avidin-biotin enzyme system (ABC reagents). A large number of fractions (more than 100) can be conveniently evaluated for the presence of glycosphingolipids recognized by one or more monoclonal antibodies in a single analysis. This method is a rapid and sensitive procedure for monitoring the purification of glycosphingolipid antigens and can be used in conjunction with immunostaining of glycosphingolipids separated by thin-layer chromatography.     

Separation of derivatized glycosphingolipids into individual molecular species by high performance liquid chromatography

The high performance liquid chromatography separation of the perbenzoyl derivatives of the neutral glycosphingolipids (GlcCer, LacCer, GbOse3Cer, GbOse4Cer, and GgOse3Cer) and the p-bromophenacyl and 2,4-dinitrophenyl hydrazide derivatives of the gangliosides (GM4, GM3, GM2, GM1, GD1a) into individual molecular species on a C18 reversed-phase column is described. Peaks were identified by comparing their relative retention times to the relative retention time of the corresponding glycosphingolipid of known molecular species composition. As little as 5 to 10 pmol of each molecular species of neutral glycosphingolipids and 3 to 5 pmol of the gangliosides can be detected. The effects of changes in the proportion of acetonitrile, methanol, and water in the mobile phase and of column temperature on the molecular species separation are described. A procedure for the tentative identification of glycosphingolipid molecular species based on their relative retention times is presented.     

Analysis of underivatized glycosphingolipids by high-performance liquid chromatography/atmospheric pressure ionization mass spectrometry

Analytical conditions for underivatized glycosphingolipids by using high-performance liquid chromatography atmospheric pressure ionization mass spectrometry (HPLC/API-MS) were investigated. The analysis was performed by using an ordinary reversed-phase column (4.6 X 150 or 4.6 X 250 mm) at a flow rate of 1 ml/min. The glycosphingolipids could be characterized from the HPLC/API-MS in terms of molecular weight, ceramide composition, and partial oligosaccharide sequence. In order to obtain an adequate spectrum the amount of material needed is in the range of a few micrograms of lipid. By selected ion monitoring the sensitivity of the method allowed characterization of only 60 ng of glycosphingolipid. The method will be very useful in the characterization of small quantities of glycosphingolipids from biological samples.     

Glycosphingolipids as Potential Diagnostic Markers and/or Antigens in Neurological Disorders

Glycosphingolipids are most abundant in the nervous system within which there are developmental, regional, structural and cellular differences regarding their composition. They are shedded to the cerebrospinal fluid and thus potential markers for pathogenic alterations in the brain, such as developmental abnormalities, demyelination, gliosis, neuronal cell destruction. The glycosphingolipids have also been found to be antigens in autoimmune processes involving the nervous system, in particular in peripheral neuropathies like Guillain Barré syndrome, multifocal motor neuropathy etc. The immune response might have been triggered by infectious agents with an antigen epitope which mimic the glycosphingolipid or by a primary nerve tissue damage leading to release of glycosphingolipids. There is a series of support for a clinical significance of cerebrospinal fluid glycosphingolipid determinations and the presence of anti-glycosphingolipid antibodies but this has to be further explored. This paper is a mini review of the state of the art and discuss methodological aspects and improvements that might help to explore the relevance of glycosphingolipids in neurological disorders.     

The glycosphingolipid composition of the human hepatoma cell line,Hep-G2

The origin of plasma glycosphingolipids in normal individuals and the mechanisms by which tumor-associated glycosphingolipid antigens enter the plasma in patients with cancer are largely unknown. The Hep-G2 human hepatoma cell line retains many of the characteristics of differentiated hepatocytes including the ability to synthesize and secrete lipoproteins. Preliminary results indicated that newly synthesized Hep-G2 cell glycosphingolipids are coupled to the secreted lipoproteins. This suggests that this cell line may offer an interesting model for studying glycosphingolipid secretion, transfer, and shedding. We now report on the chemical and immunological characterization of Hep-G2 cell glycosphingolipids. Five major glycosphingolipids were purified and biochemically characterized: glycosylceramide, lactosyl ceramide, ceramide trihexoside, ganglioside GM3, and lactosyl sulfatide. Four additional minor components (3-fucosyl-lactosamine containing glycolipids, asialo GM2, galactosylgloboside, and ganglioside GM1) were identified using a combination of exoglycosidase digestion and immunostaining of thin-layer chromatography plates with specific carbohydrate binding proteins. This demonstrates that although this cell line synthesizes a limited number of major glycosphingolipids, it retains the ability to produce at least small amounts of structures in the lactoneo, globo, and ganglio series of glycosphingolipids. These studies show that it will be possible to investigate the mechanisms of secretion by Hep-G2 cells of different classes of these molecules such as neutral glycosphingolipids, gangliosides, and sulfatides.     

Carbohydrate recognition by enterohemorrhagic Escherichia coli: characterization of a novel glycosphingolipid from cat small intestine

A key virulence trait of pathogenic bacteria is the ability to bind to receptors on mucosal cells. Here the potential glycosphingolipid receptors of enterohemorrhagic Escherichia coli were examined by binding of 35S-labeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby a selective interaction with two nonacid glycosphingolipids of cat small intestinal epithelium was found. The binding-active glycosphingolipids were isolated and, on the basis of mass spectrometry, proton NMR spectroscopy, and degradation studies, identified as Galalpha3Galbeta4Glcbeta1Cer (isoglobotriaosylceramide) and Galalpha3Galalpha3Galbeta4Glcbeta1Cer. The latter glycosphingolipid has not been described before. The interaction was not based on terminal Galalpha3 because the bacteria did not recognize the structurally related glycosphingolipids Galalpha3Galalpha4Galbeta4Glcbeta1Cer and Galalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer (B5 glycosphingolipid). However, further binding assays using reference glycosphingolipids showed that the enterohemorrhagic E. coli also bound to lactosylceramide with phytosphingosine and/or hydroxy fatty acids, suggesting that the minimal structural element recognized is a correctly presented lactosyl unit. Further binding of neolactotetraosylceramide, lactotetraosylceramide, the Le(a)-5 glycosphingolipid, as well as a weak binding to gangliotriaosylceramide and gangliotetraosylceramide, was found in analogy with binding patterns that previously have been described for other bacteria classified as lactosylceramide-binding.     

Fast atom bombardment-mass spectrometry of glycosphingolipids extracted from thin-layer chromatography plates

A method is described for analysis of glycosphingolipids extracted from thin-layer chromatography plates. Mixtures of glycolipids and gangliosides were separated by thin-layer chromatography and the individual bands were eluted, permethylated, and, after purification, analyzed by fast atom bombardment-mass spectrometry. The glycosphingolipids could be characterized from their fast atom bombardment mass spectra in terms of partial monosaccharide sequence, ceramide composition, and molecular weight. The sensitivity of the method allows characterization of 1-5 micrograms of glycosphingolipid.     

Lipid composition of lymphocyte plasma membrane from pig mesenteric lymph node.

1. The lipid fraction of the plasma membrane of pig mesenteric lymph-node lymphocytes contained primarily (94%) neutral lipids and phospholipids in about equal weights. The remianing lipid comprised glycosphingolipids (1.8%), gangliosides (o.27%)and probably ceramides (1.3%).The major phospholipid was phosphatidylcholine (46% of the total), and mono- and tri-hexosylceramides accounted for 72% of the glycosphingolipids. Haematoside was distributed between the glycosphingolipid and ganglioside fractions. The major ganglioside was monosialoganglioside. About 90% of the sialic acid was N-glycollylated. 2. A comparision of the lipid composition of the plasma-membrane fraction with that of the initial lymph-node homogenate showed that the purified membrane contained increased proportions of phospholipids, especially sphingomyelin, glycosphingolipids and gangliosides. 3. Fatty acid analyses showed that the membrane phosphatidylcholine was rich in palmitic acid, that the sphingomeyelin and phosphatidylethanolamine were high in myristic acid and that the glycosphingolipids were rich in oleic acid.