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)     

Glycosphingolipid composition of human semen

Glycosphingolipids were extracted from human semen and purified. Based on the fluorometric assay of sphingosine, in spermatozoa a content of 4.4 +/- 0.9 nmol/10(8) cells of gangliosides and 22.1 +/- 1.7 nmol/10(8) cells of neutral glycosphingolipids was determined. Seminal plasma contained 4.1 +/- 0.6 nmol gangliosides and 29.3 +/- 1.5 nmol neutral glycosphingolipids per milliliter. The glycosphingolipid component patterns of human spermatozoa and seminal plasma were determined by thin-layer chromatography. Four neutral glycolipids were isolated and their carbohydrate moieties were characterized. All of these glycolipid components belonged to the globo-series. Gas chromatography, combined gas chromatography/mass fragmentography, and exoglycosidase treatments revealed the following structures for the glycosphingolipids of human semen: Glc1-Cer, Gal beta 1-4Glc1-Cer, Gal alpha 1-4Gal beta 1-4Glc1-Cer, and Gal-NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc1-Cer. In addition, the occurrence of trace amounts of lactoneotetraosyl- and lactoneohexaosylceramide was detected by immunostaining after thin-layer chromatographic separation. Human spermatozoa, as well as seminal plasma, contained the gangliosides Glac1,Glac2, a sialolactoneotetraosylceramide, and a sialolactoneohexaosylceramide. The gangliosides were identified on the basis of their running characteristics by high-performance thin-layer chromatography, exoglycosidase treatment, and immunostaining after thin-layer chromatography. The ceramide composition of the glycolipids in human spermatozoa, as well as in seminal plasma, was dominated by C22:0-behenic acid and the saturated sphingoid d18:0, sphinganine.     

Glycosphingolipids of human plasma

A number of glycosphingolipids, including 10 gangliosides, not previously identified in human plasma have been characterized. The plasma contains 2 micrograms of lipid-bound sialic acid/ml plasma and 54% of the gangliosides are monosialo, 30% disialo, 10% trisialo, and 6% tetrasialo. Individual glycosphingolipids were purified by high-performance liquid chromatography and thin-layer chromatography, and were characterized on the basis of their chromatographic mobility, carbohydrate composition, hydrolysis by glycosidases, methylation analysis, and immunostaining with anti-glycosphingolipid antibodies. The monosialogangliosides were identified as GM3, GM2, sialosyl(2-3)- and sialosyl(2-6)lactoneotetraosylceramides, sialosyllacto-N-nor-hexaosylceramide, and sialosyllacto-N-isooctaosylceramide. The major gangliosides in the polysialo fractions contained a ganglio-N-tetraose backbone and were identified as GD3, GD1a, GD1b, and GQ1b. The most abundant neutral glycosphingolipids were glucosyl, lactosyl, globotriaosyl, globotetraosyl and lactoneotetraosylceramides. The other neutral glycosphingolipids, tentatively identified by immunostaining with monoclonal antibodies, contained H1, Lea, Leb, and lacto-N-fucopentose III (X hapten) structures.     

Gangliosides and neutral glycosphingolipids of normal tissue and oat cell carcinoma of human lung

Concentration and composition of gangliosides and neutral glycosphingolipids of adult human lung, and lung small cell carcinoma were studied. The structures of the glycolipids were determined by quantitative component determination, enzymic degradation, permethylation and fast atom bombardment mass spectrometry. Adult human lung contained mainly gangliosides with lactosylceramide as the basic core, GM3, GD3 and GT3, and approx. equal proportions (10%) of gangliosides of the gangliotetraosyl- and lactotetraosylceramide series. 18 gangliosides with different carbohydrate moieties were identified: four of them were only found in the tumor tissue. The adult human lung contained 85 nmol (77-120) gangliosides and 140 nmol neutral glycosphingolipids per g wet weight. Globoside was the major neutral glycolipid and there were only minor amounts of glycolipids of the lactotetraose series. In small cell carcinoma tissue the concentration of neutral glycosphingolipids was approximately twice as high than in normal lung tissue, and there was a markedly larger concentration of both lactosylceramide and glycolipids of the lactotetraose series and fucose derivatives of these. The concentration of gangliosides varied between 202 and 415 nmol per g wet weight. Compared to normal lung tissue, the tumor tissue had a lower proportion of GD3, and a higher proportion of complex gangliosides, and they contained five tumor-associated gangliosides: Fuc-GM1, Fuc-GD1b, 3'-LM1, Fuc-3'-LM1 and 6'-nLM1.     

Critical micelle concentrations of gangliosides

The micellar properties of mixed, bovine gangliosides and purified galactosyl-N-acetylacetylgalactosaminyl (N-acetylneuraminyl) galactosylglucosylceramide were studied by gel filtration, equilibrium dialysis, and band and boundary centrifugation in sucrose gradients. The dissociation of micelles is very slow (days) in water and required us to approach equilibrium by association of monomers rather than by the dissociation of micelles. The gangliosides were therefore first converted into very low molecular weight aggregates (1-3 molecules) by dissolving them in Me2SO. Galactosyl-N-acetylgalactosaminyl(N-acetylneuraminyl)galactosylglucosylceramide was then diluted into aqueous sucrose gradients and sedimented by the boundary centrifugation technique. This gave a sedimenting micelle and a nonsedimenting monomer concentration of (1-2) x 10-10 M (or less) which corresponds to the critical micelle concentration value. The mixed gangliosides revealed two micellar sizes (i.e., 10 and 4.5 S), the slower sedimenting species being formed from the larger one with time (days). The critical micelle concentration of the mixed gangliosides was found to be approximately 10-8 M by a gel filtration, equilibrium dialysis, and band centrifugation.     

Application of a simple methylation procedure for the analyses of glycosphingolipids

Acid and neutral glycosphingolipids (0.01-1 mumol) were completely methylated in high yields and with little formation of by-products in 10 min at room temperature, using methyl sulphoxide, methyl iodide, and powdered NaOH. Re-methylation of methylated and LiAlH4-reduced gangliosides gave a new derivative that was useful for the analysis of gangliosides by mass spectrometry.     

Effect of calcium ions on the thermotropic behaviour of neutral and anionic glycosphingolipids

In the concentration range of 10(-5) to 10(-1) M Ca2+ modulates the thermotropic properties of several neutral and anionic glycosphingolipids (galactosylceramide, asialo-GM1, sulfatide, GM1, GD1a, GT1b) and of their mixtures with dipalmitoylphosphatidylcholine. The transition temperature of gangliosides is not appreciably changed while the transition enthalpy increases by 20% in the presence of Ca2+. The more marked effect of Ca2+ is on the thermotropic behavior of systems containing sulfatide. Increasing concentrations of Ca2+ between 10(-5) and 10(-3) M (up to a molar ratio of Ca2+/sulfatide 1:2) induce a progressive increase of both the transition temperature and enthalpy. Further increases up to 10(-1) M Ca2+ induce a new phase transition at a lower temperature. No evidence is found for induction of phase separation of pure glycosphingolipid-Ca2+ domains in mixtures of any of the glycosphingolipids with dipalmitoylphosphatidylcholine. The modification of the phase behavior of anionic glycosphingolipids by Ca2+ does not involve detectable variations of the intermolecular packing but is accompanied by marked modifications of the dipolar properties of the polar head group region.     

Glycosphingolipids of normal bovine and enzootic bovine leukosis lymph node cells

We analyzed glycosphingolipids from normal lymph node cells of seven cattle and lymph node cells of eight cattle with enzootic bovine leukosis. The neutral glycosphingolipids and gangliosides were analyzed by thin-layer chromatography. Both normal and tumorous lymph node cells had GlcCer, LacCer, and GbOse3Cer as major neutral glycosphingolipids. In the ganglioside fraction, GM3 was the predominant component in both normal and tumorous lymph node cells, and another component, ganglioside Gx fraction, was also prominent in tumorous lymph node cells. The structure of this ganglioside Gx fraction was elucidated by thin-layer chromatography, sugar analysis, neuraminidase digestion, and permethylation studies. This ganglioside Gx fraction was found to be a mixture of four ganglioside species. The structures of individual gangliosides Gx (1 to 4) were characterized as follows. 1: GD3, NeuAc alpha 2-8NeuAc alpha 2-3Gal1-4Glc-Cer. 2: GD3, NeuAc alpha 2-8NeuGc alpha 2-3Gal1-4Glc-Cer. 3: GD3, NeuGc alpha 2-8NeuAc alpha 2-3Gal1-Glc-Cer. 4: GD3, NeuGc alpha 2-8NeuGc alpha 2-3Gal1-4Glc-Cer. These GD3 species may be formed as a result of the induced synthesis inassociation with malignant transformation.     

Comparative study of acidic glycosphingolipids by field desorption and secondary ion mass spectrometry

Acidic glycosphingolipids were analyzed by field desorption (FD-MS) and secondary ion mass spectrometry (SI-MS) using the primary ion Xe+ with a glycerol matrix. In the analysis of underivatized gangliosides by FD-MS, the fragment corresponding to the asialo residue resulting from the cationized cluster ion (M + Na)+ was the base peak, and ions due to cleavage at the glycosidic linkages were detected, as in the neutral glycosphingolipids. In the case of sulfatide, the ceramide fragment showed the highest intensity in the spectrum. In SI-MS spectra of acidic glycosphingolipids, (M + Na)+, (M + 2Na-H)+, and (M + K)+ were continuously detected as relatively high intensity ions during analysis of gangliosides and sulfatide. Other ions were mostly similar to those obtained by FD-MS. In FD-MS spectra of permethylated gangliosides, the cationized molecular ion (M + Na)+ was the base peak, and fragment ions due to asialo gangliosides were prominent. Other peaks were hard to detect. In SI-MS, molecular ions (M + H)+ and (M + H-32)+ and other ions due to cleavage of the glycosidic linkages were clearly detected. In this case, the sensitivity was greatly improved. Ions due to the non reducing end sugars were clearly detected, because of the relatively low intensity of ion peaks due to the glycerol matrix. It is concluded that the combination with FD-MS and SI-MS is particularly useful for the determination of molecular weight, sugar sequence and ceramide structure with sample amounting to only a few micrograms order.     

Uptake and metabolism of exogenous glycosphingolipids by cultured cells

Exogenous glycosphingolipids, especially gangliosides, are used to study transport and metabolism of their endogenous counterparts as well as their role in cell adhesion, cell recognition and signal transduction. Unlike monodispersed solutes, in aqueous media ganglioside molecules aggregate into micelles (or bilayer structures) with a very low critical micellar concentration. Upon addition to cells in culture, exogenous gangliosides bind to the cell surface in three operationally defined modes: loosely associated micelles removable by serum; tightly attached micelles removable by proteases such as trypsin; and ganglioside molecules inserted into the outer leaflet of the plasma membrane. As shown by a biotin-labeled derivative of the ganglioside GM1 these inserted molecules are endocytosed and transported to intralysosomal membranes for catabolism. The benefit from using (partially) nondegradable as well as semi-truncated glycosphingolipids in transport studies is discussed.     

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.     

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.     

Biosynthesis of proteins, nucleic acids and glycosphingolipids by synchronized KB cells

The biosynthesis of glycosphingolipids and various types of proteins and nucleic acids at specific periods of the cell cycle was studied by using synchronized KB cells. Maximum incorporation of radioactive galactose, leucine and thymidine into several proteins and nucleic acids occurred as has been reported previously (6,11). Maximum incorporation of $\text{D}$-1[¹⁴C] galactose into glycosphingolipids was observed during the M and G-1 phases. There was a 5 fold increase in the levels of gangliosides and combined neutral glycosphingolipids during the M and G-1 phases. Thus, regulated biosynthesis of glycosphingolipids and macromolecules might be important in the cyclic expression of some of the functional properties which are characteristic of these compounds.     

Characterization of a soluble glycolipid galactosyltransferase which occurs in bovine milk.

Bovine milk was found to contain, in soluble form, an enzyme which transfers galactose from UDPgalactose to glucosylceramide. This enzyme was partially purified by the same procedure used to isolate the galactosyltransferase of lactose synthetase. The partially purified enzyme required detergents for activity, had a pH optimum of 7.2--7.3 and required Mn2+. The apparent Km calculated for glucosylceramide was 1.33 . 10(-4) M. With glucosylceramide as acceptor the product of the reaction was identified as lactosylceramide by autoradiography on thin-layer chromatograms. Lactosylceramide was also an effective acceptor for the transferase reaction but neutral glycosphingolipids or gangliosides with terminal galactose of N-acetylgalactosamine residues were ineffective or poorly effective as acceptors. Addition of alpha-lactalbumin inhibited the transferase reaction.     

Glycosphingolipids of human aorta

The structures of the main gangliosides of human aorta (intima and media) were elucidated. The main component (67%) was identified as N-acetylneuraminosyl-lactosylceramide (ganglioside GM3). The aorta tissue contained also gangliosides GM1, GD3, GD1a, and GT1. All sialic acid residues in gangliosides were present as N-acetyl-neuraminosyl derivatives. Among neutral glycosphingolipids of human aorta, the main components were identified as glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. The preliminary data suggest that the composition of the investigated glycosphingolipids in tissue might vary upon atherosclerosis lesions of aorta.     

Identification of the neutral glycosphingolipids of murine mast cells: expression of Forssman glycolipid by the serosal but not the bone marrow-derived subclass

The expression of neutral glycosphingolipids by mouse T cell-dependent, bone marrow-derived mast cells (BMMC) obtained in vitro was determined by chromatographic and immunochemical criteria. Neutral glycosphingolipids were isolated from BMMC by extraction of 3 to 5 X 10(8) cells in chloroform/methanol (1/1, v/v) and chromatography on DEAE-Sephadex, and were analyzed by thin layer chromatography with orcinol staining. The predominant neutral glycosphingolipids of BMMC were glucosylceramide (CMH), lactosylceramide (CDH), globotriosylceramide (CTH), globotetraosylceramide (globoside), and a molecule migrating slightly faster than gangliotetraosylceramide (asialo GM1) and slower than globopentaosylceramide (Forssman glycolipid). The profiles on thin layer chromatograms of the neutral glycosphingolipids were the same for BMMC derived from BALB/c, C57BL/6, WBBF1-W/Wv, and WBBF1-+/+ mice, and for cells differentiated in either WEHI-3 conditioned medium or concanavalin A-splenocyte conditioned medium. High performance liquid chromatography of benzoylated neutral glycosphingolipids of BMMC on a Zipax column confirmed the identity of the four neutral glycosphingolipids identified by thin layer chromatography. The fifth major glycosphingolipid had an elution time greater than that of globotetraosylceramide and did not co-elute with any of the standards tested. Direct biochemical analyses of the neutral glycosphingolipids of mouse serosal mast cells (SMC) were not feasible because only 2 X 10(6) SMC could be isolated per 100 mice. However, mouse SMC bound a rat monoclonal anti-globopentaosylceramide antibody (M1/87.27.7) and rat monoclonal B1.1 antibody, as assessed by indirect immunofluorescence and flow cytometry, whereas mouse BMMC did not. The binding of B1.1 antibody to SMC could be blocked by the anti-globopentaosylceramide antibody, and the specificity of B1.1 antibody for globopentaosylceramide was confirmed immunochemically with the use of a solid phase radioimmunoassay. As estimated immunochemically, the amount of globopentaosylceramide in mouse SMC was 62 ng/10(6) cells, whereas BMMC contained less than 8 ng/10(6) cells. Thus, the expression of globopentaosylceramide is a characteristic of the mouse SMC that is lacking in the T cell-dependent BMMC.     

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.     

Helicobacter pylori-binding gangliosides of human gastric adenocarcinoma.

Acidic and neutral glycosphingolipids were isolated from a human gastric adenocarcinoma, and binding of Helicobacter pylori to the isolated glycosphingolipids was assessed using the chromatogram binding assay. The isolated glycosphingolipids were characterized using fast atom bombardment mass spectrometry and by binding of antibodies and lectins. The predominating neutral glycosphingolipids were found to migrate in the di- to tetraglycosylceramide regions as revealed by anisaldehyde staining and detection with lectins. No binding of H. pylori to these compounds was obtained. The most abundant acidic glycosphingolipids, migrating as the GM3 ganglioside and sialyl-neolactotetraosylceramide, were not recognized by the bacteria. Instead, H. pylori selectively interacted with slow-migrating, low abundant gangliosides not detected by anisaldehyde staining. Binding-active gangliosides were isolated and characterized by mass spectrometry, proton nuclear magnetic resonance, and lectin binding as sialyl-neolactohexaosylceramide (NeuAcalpha3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and sialyl-neolactooctaosylceramide (NeuAcalpha3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer).     

Glycosphingolipids of leukemic cells in adult T-cell leukemia-lymphoma

We analyzed lipids from leukemic cells of two patients with adult T-cell leukemia and compared them with those from T-cell lymphocytes of normal subjects. The neutral glycosphingolipids and gangliosides which were isolated were characterized by thin-layer chromatography and neuraminidase treatment. Both leukemic cells and normal lymphocytes had monoglycosylceramide and diglycosylceramide as major neutral glycosphingolipids. In one patient, diglycosylceramide was markedly increased. II3NeuAc-LacCer (GM3) and more complex gangliosides were detected in both cells. The most characteristic finding in leukemic cells was the occurrence of a disialylated ganglioside, II3(NeuAc)2-LacCer (GD3), which is not found in normal lymphocytes and neutrophils. This ganglioside may be due to the induced synthesis in association with malignant transformation.     

Molecular parameters and physical state of neutral glycosphingolipids and gangliosides in monolayers at different temperatures

The effect of temperature on the behaviour of four different gangliosides (GM3, GM1, GD1a and GT1b), sulphatide, ceramide (Cer) and three neutral glycosphingolipids (GalCer, Gg3Cer, Gg4Cer) was investigated in monolayers at the air-NaCl (145 mM) interface. GM1, GD1a and GT1b are liquid-expanded in the range of temperatures studied (5-65 degrees C). GM3, sulphatide, Cer and neutral glycosphingolipids show isothermal liquid-expanded----liquid-condensed transitions. The collapse pressure of ganglioside monolayers decreases with temperature, whereas neutral glycosphingolipids may show some maximum values at particular temperatures. The reduction of the molecular area of liquid-expanded glycosphingolipids under compression occurs with a favorable positive entropy change and an unfavorable negative enthalpy. By contrast, the compression of interfaces with a two-dimensional phase transition occurs with an unfavorable entropy but a favorable enthalpy change. From the temperature dependence of the surface pressure at which the two-dimensional phase transition takes place, a minimal temperature above which the isotherm becomes totally liquid-expanded can be obtained. For the different glycosphingolipids this temperature decreases in the order Cer greater than GalCer greater than sulphatide greater than Gg3Cer greater than Gg4Cer greater than GM3 greater than GM1 greater than GD1a greater than GT1b. This sequence is similar to that found for the calorimetrically determined transition temperatures (cf. Maggio, B., Ariga, T., Sturtevant, J.M. and Yu, R.K. (1985) Biochemistry 24, 1084-1092).