High-performance liquid chromatography of long-chain neutral glycosphingolipids and gangliosides

We describe a method for analyzing the perbenzoyl derivatives of both neutral glycosphingolipids and gangliosides with a single high-performance liquid chromatography system. Use of this system, combined with endo- and/or exoglycosidase treatment of glycosphingolipids, provides a sensitive method for obtaining structural information on these compounds. This system has two advantages over previously published chromatography procedures: (i) it uses a commercially available column, and (ii) this single column can be used to analyze gangliosides and their neutral glycosphingolipid products generated by neuraminidase treatment. With this method, we have studied 24 different glycosphingolipids, containing one to ten sugars and one or two sialic acid residues, and have demonstrated its usefulness in evaluating the gangliosides present in human leukocytes.     

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.     

Glycosphingolipids of normal and leukemic human leukocytes

Summary We have reviewed the studies on neutral glycosphingolipids and gangliosides of normal and leukemia human leukocytes. In this review, we examine (a) the glycosphingolipid composition of various leukocyte populations, (b) the differences in glycosphingolipids found among subsets of these cells, (c) the possible use of these compounds as markers of differentiation, and (d) the changes in glycosphingolipid composition that occur with leukemogenesis.     

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.     

Glycosphingolipid metabolic reprogramming drives neural differentiation

Neural development is accomplished by differentiation events leading to metabolic reprogramming. Glycosphingolipid metabolism is reprogrammed during neural development with a switch from globo‐ to ganglio‐series glycosphingolipid production. Failure to execute this glycosphingolipid switch leads to neurodevelopmental disorders in humans, indicating that glycosphingolipids are key players in this process. Nevertheless, both the molecular mechanisms that control the glycosphingolipid switch and its function in neurodevelopment are poorly understood. Here, we describe a self‐contained circuit that controls glycosphingolipid reprogramming and neural differentiation. We find that globo‐series glycosphingolipids repress the epigenetic regulator of neuronal gene expression AUTS2. AUTS2 in turn binds and activates the promoter of the first and rate‐limiting ganglioside‐producing enzyme GM3 synthase, thus fostering the synthesis of gangliosides. By this mechanism, the globo–AUTS2 axis controls glycosphingolipid reprogramming and neural gene expression during neural differentiation, which involves this circuit in neurodevelopment and its defects in neuropathology.     

Micellar properties of glycosphingolipids in aqueous media

The aggregation properties of neutral glycosphingolipids and gangliosides were investigated by ultracentrifugal sedimentation and gel permeation chromatography. Initially, glycosphingolipids in buffer formed high molecular aggregates. On standing, the glycosphingolipids produced smaller and stable micelles in equilibrium with their monomers at the critical micellar concentration (cmc). The cmc's of monohexaosyl- to tetrahexaosylceramides were on the order of 10(-8) to 10(-7)M. In contrast, values of 10(-8)M for monosialogangliosides and 10(-6)-10(-5)M for di- and trisialogangliosides were found. From estimations of hydrodynamic radii, Svedberg coefficients, and partial specific volumes, relative masses of glycosphingolipid micelles were calculated to be in the range of 300 to 1000 for the neutral glycosphingolipids and 100 to 350 for the gangliosides.     

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.     

Influence of monovalent cation transport on anabolism of glycosphingolipids in cultured human fibroblasts

We have reported [Saito, M., Saito, M., & Rosenberg, A. (1984) Biochemistry 23, 1043-1046] that the monovalent cationic ionophore monensin reduced the incorporation of labeled galactose into oligosaccharidyl glycosphingolipids (globotriaosylceramide, globotetraosylceramide, and gangliosides) and induced a cellular accumulation of glucosyl- and lactosylceramide in cultured diploid human fibroblasts. We have undertaken further studies on the effects of monensin and made comparison with the effects of related monovalent cation transporters on plasma membrane glycosphingolipid anabolism in human fibroblasts. Our results demonstrate that ionic flux can markedly influence glycosphingolipid synthesis, and they indicate that, like glycoprotein, the sites of glycosylation of the initial, precursor glycosphingolipids are different from the sites of higher glycosylation. At a concentration of 10(-7) M, monensin induced the maximum inhibition of incorporation of labeled galactose into polyglycosyl sphingolipids: globotriaosylceramide, globotetraosylceramide, and gangliosides; increased incorporation of labeled galactose into glucosyl- and lactosylceramide was clearly evident, and their content rose measurably in the cell at concentrations of monensin as low as 10(-8) M. These effects of monensin were reversible. Incorporation of labeled galactose into higher glycosylated neutral glycosphingolipids and gangliosides slowly resumed, and the accumulated glycosylceramide diminished after removal of monensin from the culture medium. Ouabain (plasma membrane Na+,K+-ATPase inhibitor) and A23187 (Ca2+ ionophore) also caused a rapid increase in incorporation of labeled hexose into glucosylceramide and decreased its incorporation into higher neutral glycosphingolipids and into gangliosides.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Selective enrichment of phytosphingosine in glycosphingolipids of isolated human thyrocytes as compared to the whole thyroid

The glycosphingolipids of isolated human thyrocytes have been analyzed. As compared to the total thyroid gland, the pattern of gangliosides was found to be similar, whereas the neutral glycolipid profile was quite different, with glucosylceramide as the major glycosphingolipid of thyrocytes. Moreover, this glucosylceramide contains almost exclusively phytosphingosine (4-D-hydroxy-sphinganine) which is only a minor component in the long-chain bases of the glycosphingolipids extracted from the whole thyroid gland.     

Neutral glycosphingolipids and gangliosides of human lung and lung tumours.

In order to help determine whether alterations of the profiles of glycosphingolipids occur consistently in human tumours, the neutral glycosphingolipids and gangliosides of nine lung tumours (one adenocarcinoma, four squamous cell, two mixed adeno-squamous cell, one large cell and one oat-cell carcinomata) were analysed. The control tissue consisted of adjacent lung; it contained neutral glycosphingolipids corresponding in properties to glucosyl-, lactosyl-, globotriaosyl- and globotetraosyl-ceramides. All of the tumours also contained these four neutral glycosphingolipids. However, in addition, five of the tumours (two of the squamous, the large cell and the two mixed adeno-squamous cell carcinomata) contained neutral glycosphingolipids corresponding in properties to lactotriaosyl- and neolactotetraosyl-ceramides; these same tumours also exhibited higher amounts of lactosylceramide than the other tumours analysed. Both of the two former neutral glycosphingolipids and very substantial amounts of the latter neutral glycosphingolipid were detected in pneumonic lung and in polymorphonuclear leucocytes; it thus appears possible that these particular compounds were derived from these latter cells rather than from the tumour cells. The ganglioside patterns of the tumours were almost equivalent in complexity to that exhibited by the control lung tissue. This study shows that the profiles of two major classes of glycosphingolipids (neutral glycosphingolipids and gangliosides) occurring in lung tumours are almost as complex as those of the parent tissue, a finding in contrast with the notably simplified patterns of these lipids found in many cancer cells grown in vitro. It also suggests that when lactotriaosyl- and neolactotetraosyl-ceramides and high amounts of lactosylceramide are detected in human tumours, the possibility must be considered that these compounds are derived from polymorphonuclear leucocytes.     

Occurrence of glycosphingolipids in chicken egg yolk.

Chicken egg yolk was found to contain a unique glycosphingolipid pattern not seen in other types of tissue or cell. These glycosphingolipids were isolated in pure form and their structures established by sequential enzymic hydrolysis and permethylation analysis. The major gangliosides in chicken egg yolk are N-acetylneuraminosylgalactosylceramide, N-acetylneuraminosyl-lactosylceramide and di-N-acetylneuraminosyl-lactosylceramide. The only neutral glycosphingolipid found in chicken egg yolk is galactosylceramide.     

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.     

Immunochemical detection of glycosphingolipids on thin-layer chromatograms.

A sensitive immunochemical method was developed for the detection of glycosphingolipids on thin-layer chromatograms. The procedure involves oxidation of diol groups of glycosphingolipids with sodium periodate, derivatization of the formed aldehyde groups with digoxigenin-hydrazide, and reaction of the bound digoxigenin with an alkaline phosphatase-labeled polyclonal anti-digoxigenin antibody. The latter is detected by an insoluble indigo-like dye as a result of dephosphorylation of 5-bromo-4-chloro-3-indolyl phosphate. The detectability of all glycosphingolipid species was improved over that of the orcinol and resorcinol staining methods. Two nanograms of the standard gangliosides GM1, GD1A, and GT1 was detected, whereas the detection limit for short-chain neutral glycosphingolipids was in the range of 20-50 ng. Long-chain glycosphingolipids were detectable with a particularly high sensitivity. Selective staining of the gangliosides could be achieved by the use of low periodate concentrations.     

A new approach to the modification of cell membrane glycosphingolipids: Ganglioside composition of JTC-12 P3 cells altered by feeding with galactose as a sole carbohydrate source in protein- and lipid-free synthetic medium

A significant difference in the glycosphingolipid composition of JTC-12 P3 cells established from monkey kidney tissue was observed when cells cultured in a protein- and lipid-free synthetic medium containing glucose (DM-160) as a sole carbohydrate source were transferred and cultured in the same medium containing galactose and pyruvic acid (DM-170) in place of glucose. In particular, the amounts of gangliosides GM3, GM2, and GD3 in the cells cultured in DM-170 were 5.3-, 17.8-, and more than 8-fold those in the cells cultured in DM-160, respectively, indicating that anabolism of gangliosides is greatly enhanced in cells cultured in the presence of galactose and pyruvic acid, as compared with cells cultured in the presence of glucose. In fact, after cultivation of cells in the medium with N-acetyl-D-[14C]mannosamine for 96 h, the radioactivity incorporated into the gangliosides of the cells in DM-170 was 10-fold that of the cells in DM-160. Among the gangliosides of the cells in DM-170, highly sialylated molecules such as GD3, GD1a, GD1b, and GT1b were preferentially labeled, indicating that the sialyltransferases responsible for the synthesis of gangliosides are significantly more activated in cells cultured in DM-170 than in DM-160. These observations reveal that the glycosphingolipid composition of the plasma membrane can be modified epigenetically under well-defined conditions and provide important clues for clarifying the roles of glycosphingolipids associated with particular cell functions.     

Glycosphingolipids from bovine milk and milk fat globule membranes: a comparative study. Adhesion to enterotoxigenic Escherichia coli strains

Several components of milk fat globule membranes (MFGMs) have been reported to display beneficial health properties and some of them have been implicated in the defense of newborns against pathogens. These observations prompted us to determine the glycosphingolipid content of MFGMs and their interaction with pathogens. A comparative study with whole milk components was also carried out. Milk fat globules and MFGMs were isolated from milk. Gangliosides and neutral glycosphingolipids were obtained from MFGMs and whole milk and their fatty acid contents were determined by gas chromatography-mass spectrometry (GC-MS). MFGMs and whole milk showed similar ganglioside and neutral glycosphingolipid contents, with whole milk having more GM3 and glucosylceramide and less GD3, O-acetyl GD3, O-acetyl GT3, and lactosylceramide. The fatty acid content of gangliosides from both sources showed a similar composition. However, the neutral glycosphingolipid fatty acid content seemed to be quite different. Whole milk had fewer very-long-chain fatty acids (18.1% vs. 46.4% in MFGMs) and more medium-chain and unsaturated C18:1 and C18:2 fatty acids. Milk fat globules, MFGMs, lactosylceramide, and gangliosides GM3 and GD3 were observed to bind enterotoxigenic Escherichia coli strains. Furthermore, bacterial hemagglutination was inhibited by MFGMs and glycosphingolipids.     

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.     

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.     

Effect of puromycin and cycloheximide on glycosphingolipid biosynthesis by PHA stimulated human lymphocytes

The effect of puromycin and cycloheximide on the biosynthesis of neutral glycosphingolipids and gangliosides by PHA stimulated lymphocytes is studied. Under conditions of permanent inhibition of protein synthesis, and depending on the time of lymphocyte stimulation, inhibition of the biosynthesis of neutral glycosphingolipids was either restricted only to certain species or was very low for all glycosphingolipid species. The degree of inhibition of the various ganglioside species was affected by the time of incubation. After transient inhibition of protein synthesis and at times when protein biosynthesis had recovered, neutral glycosphingolipid and ganglioside biosynthesis inhibition was very prominent and did not recover. The possibility is discussed that glycosphingolipid biosynthesis does not depend directly on concurrent nascent peptide formation and it is proposed that inhibition of glycosphingolipid biosynthesis is related primarily to impairment of the endoplasmic reticulum and to inhibition of galactose transferases, secondary to the binding of the inhibitors.