A rapid method for the preparation of ganglioside Glac2 (GD3)

A method is described for the preparation of ganglioside G_lac2 [(II³(NeuAc)₂-LacCer, GD3] from cream of bovine milk using liquid-phase extraction with methanol or ethanol followed by anion exchange chromatography. The method is rapid and inexpensive; 1 kg cream, centrifuged from 14–15 L of bovine milk, yields approximately 70 mg of pure ganglioside G_lac2. The sialic acid constituent of ganglioside G_lac2 isolated from bovine milk cream consists solely of theN-acetylneuraminic acid derivative. The major components of its ceramide consist of octadecasphing-4-enine and the 22∶0 (behenic acid) and 23∶0 fatty acids. Short hand notations for gangliosides are according to Wiegandt (Ref. 1). G_lac2, (II³(NeuAc)₂-LacCer) is NeuAcα,8NeuAcα,-3Galβ,4Glcβ,1Cer; G_tet1, (II³NeuAc-Gg₄Cer) is Gal\,3GalNAcβ,-4(NeuAcα,3)Galβ,4Glcβ,1Cer.     

Gb3 Glycosphingolipids with Fluorescent Oligoene Fatty Acids: Synthesis and Phase Behavior in Model Membranes

Glycosphingolipids are involved in a number of physiological and pathophysiological processes, and they serve as receptors for a variety of bacterial toxins and viruses. To investigate their function in lipid membranes, fluorescently labeled glycosphingolipids are highly desirable. Herein, a synthetic route to access Gb₃ glycosphingolipids with fluorescently labeled fatty acids, consisting of pentaene and hexaene moieties either at the terminus or in the middle of the acyl chain, has been developed. The fluorescent properties of the Gb₃ derivatives were investigated in small unilamellar vesicles composed of a raft-like mixture. Phase-separated giant unilamellar vesicles (GUVs) allowed the quantification of the apparent partitioning coefficients of the Gb₃ compounds by means of confocal fluorescence laser scanning microscopy. The determined partition coefficients demonstrate that the Gb₃ derivatives are preferentially localized in the liquid-disordered (l_d) phase. To analyze whether the compounds behave like their physiological counterparts, Cy3-labeled (Cy: cyanine) Shiga toxin B subunits (STxB) were specifically bound to Gb₃-doped GUVs. However, the protein was favorably localized in the l_d phase, in contrast to results reported for STxB bound to naturally occurring Gb₃, which is discussed in terms of the packing density of the lipids in the liquid-ordered (l_o) phase.     

Dexamethasone-induced alterations in the glycosphingolipids of rat kidney

To determine whether glucocorticoids would influence the glycosphingolipid composition of the rat kidney, male albino rats of the Sherman strain were subcutaneously administered dexamethasone (100 μg/100 g body wt/day) or diluent for four days. The compositions of ceramide and of acidic and neutral glycosphingolipids of the kidneys of these animals were then examined and compared. The results demonstrated that dexamethasone administration: 1) increased the content of ceramide and of acidic and neutral glycosphingolipids in kidney; 2) increased the relative percentage of globotriaosyl- and globotetraosylceramide, but decreased the relative percentages of glucosylceramide; 3) decreased the relative percentages of G_M3 and increased other gangliosides; 4) increased the relative percentages ofN-glycolylneuraminic acid in G_M3; 5) did not appear to influence significantly the long-chain bases of the major glycosphingolipids; and 6) altered the relative percentages and chain length of the hydroxy and nonhydroxy fatty acids of the major acidic and neutral glycosphingolipids in this tissue. The data show that dexamethasone administration induces quantitative and qualitative changes in the glycosphingolipids of the rat kidney.     

Distribution of glycosphingolipids of monkey small and large intestinal mucosa

The ganglioside and neutral glycosphingolipid composition of adult monkey small and large intestinal mucosa were characterized and compared. GM₃, GM₂ and GD_1A were found to be the principal gangliosides in each of these tissues. Dihexosylceramide was the major neutral glycosphingolipid of both organs. The total content of gangliosides and neutral glycolipids/ceramide, however, was ca. four-fold and two-fold higher, respectively, in small intestinal than colonic mucosa. While all glycosphingolipids examined contained hydroxy and nonhydroxy fatty acids, the former fatty acids accounted for 60–90% of the total fatty acids in both organs. Sphingosine was the predominant long chain base of ceramide, mono-, di-, tri- and tetrahexosylceramide, whereas phytosphingosine was the major base of GM₃ in both tissues. The results of these studies demonstrate that while many similarities of monkey small and large intestinal glycosphingolipids exist, qualitative and quantitative differences are present along the length of the monkey gut. These differences may be at least partially responsible for certain of the well-recognized variations in normal physiological and pathological processes that occur in these organs.     

Glycosphingolipids of fetal and adult sheep colonic mucosa

The ganglioside and neutral glycosphingolipid composition of fetal and adult sheep colonic mucosa were characterized and compared. Mono- and tetrahexosylceramide were the major neutral glycolipids of both fetal and adult colons. Adult, but not fetal, mucosa also possessed di- and trihexosylceramide. Similarly, GD_1a, GM₃ and GM₂ were found to be the principal gangliosides in fetal and adult tissue. Adult colonic mucosa possessed significant amounts of GT_1a not present in fetal tissue. Analysis of the hydroxy and nonhydroxyfatty acids as well as of the long chain bases of the major glycosphingolipids revealed differences between these lipophilic components of glycolipids in fetal and adult colonic mucosa. The present results, therefore, indicate that both quantitative and qualitative differences in glycosphingolipid composition exist between fetal and adult sheep colonic mucosa.     

Neutral glycolipids of human and bovine milk

The neutral glycolipids of milk, a small fraction of the total lipids, are of potential biological importance. The simultaneous quantitation of the simple (less than five sugars) glycosphingolipids of human milk samples was achieved by high-pressure liquid chromatography. The samples, representing various stages of lactation, parity of the nursing child, and age of the mother, contained similar glycolipid patterns, but with varying individual glycolipid concentrations. The cerebrosides are major glycosphingolipids of human milk: the non-hydroxylated fatty acid (NFA)-containing species are present at 1.8 μM, and the hydroxylated and/or short-chain fatty acid-containing species (HFA) are present at 1.7 μM; NFA lactosylceramide is present at 931 nM. The cerebrosides appear to be primarily galactosylceramides (galactocerebrosides); glucosylceramides (glucocerebrosides) are a minor component. Globotriaosylceramide (Gb₃) is found at 50 nM and 73 nM for the NFA and HFA species, respectively, while globoside (Gb₄) is found at 45 nM and 46 nM for the NFA and HFA species. Bovine milk glycosphingolipids differ from those of human milk, with bovine milk containing mainly NFA glucosylceramide (8 μM) and NFA lactosylceramide (17 μM); bovine milk contains little Gb₃ or Gb₄. Based on a paper presented at the Symposium on Milk Lipids held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

Glycolipids of a human glioma cell line bearing receptors for platelet-derived growth factor (PDGF)

Glycolipids of U-1242 MG were characterized because results of previous studies showed that exogenous gangliosides, especially G_M3, inhibit PDGF-stimulated growth of this human glioma cell line. G_M3 and G_M2 are the major gangliosides; both separate as doublets with thin-layer chromatography. The major neutral glycolipid is glucocerebroside with nonhydroxy fatty acids, but paragloboside, ceramide dihexoside, globoside, and asialoG_M2 (G_A2) are also present. The coexistence in U-1242 MG of these gangliosides and the PDGF receptor, whose mitogenic signal is modulated by G_M3 in these cells, suggests a possible functional relationship among them with respect to growth regulation.     

Sialylation of lacto-N-neotetraosyl ceramide by a solubilized sialyltransferase(s) from chicken skeletal muscle: Effect of phosphatidylcholine and sphingomyelin

The sialytransferase(s) that transfers sialic acid to lacto-N-neotetraosylceramide and other glycosphingolipids with a galactose nonreducing terminus has been successfully solubilized from embryonic chicken skeletal muscle. The enzyme can be stored in 50 mM HEPES (pH 6.8), 1% Triton CF-54, and 20% glycerol at −70°C for as long as six months. Addition of phosphatidylcholine or sphingomyelin (0.167%) readily reactivates the stored inactive enzymes and such activity persists for about two weeks at 0°–4°C with the peak activity occurring at 1 to 2 days. Sphingomyelin from chicken muscle, which contains mainly C16∶0 and C18∶0, is 2.1-fold more effective than bovine brain sphingomyelin at the same concentration (0.4%). Abbreviations: GM₃, GM₁, GD_1a, GD_1b, GT_1b were designated according to Svennerholm, L. (1963)J. Neurochem. 10, 613–623. GgOse₄Cer and nLcOse₄Cer are short designations recommended by the IUPAC-IUB Commission on Lipid Nomenclature (1977)Eur. J. Biochem. 79, 11–21. Other abbreviations used are IV³ NeuAcnLcOse₄Cer; nLcOse₆Cer, V⁴Gal, IV³ GlcNAc-nLcOse₄Cer; NeuAc-nLcOse₆Cer, VI³ NeuAc, V⁴ Gal, IV³ GlcNAc-nLcOse₄Cer; PC, phosphatidylcholine; SM, sphingomyelin, SAT, sialyltransferase; Hepes, 4-2(hydroxyethyl)1-piperazineethane sulfanic acid. Structures of glycolipids: nLcOse₄Cer, Gal(β1→4)GlcNAc(β1→3)Gal(β1→4)-GlcCer; GgOse₄Cer, Gal(β1→3)GalNAc(β1→4)Galβ(1→4)GlcCer.     

The major gangliosides of the bovine pineal body

Acetone powders of fresh-frozen pineals were extracted with chloroform/methanol mixtures. By column chromatography on silicic acid, mild alkaline methanolysis, ion-exchange high performance liquid chromatography and a final thin layer chromatography on silicic acid, the major glycosphingolipids were purified from the extracts of a total of 300 bovine pineal bodies. Chromatographically purified fractions were characterized by gas chromatographic analysis. The most prominent glycosphingolipid appeared to be cerebroside. In addition, five different gangliosides were found in detectable levels. The two major gangliosides have the chromatographic and component characteristics of GD₃ and GM₃, with disialoganglioside predominating. Gangliosides indistinguishable from purchased standards of GM₁ and GD_1a were third and fourth, respectively, in amount. The fatty acid profiles of the two lactosyl gangliosides are similar and significantly different from those of the two gangliotetraose gangliosides. The fifth most prominent ganglioside, present at a level of 1.09% of total recovered ganglioside sialic acid, appears to be a novel trisialoganglioside, called GT_x. This new molecule has a component ratio of gal:glc:sialic acid:amino sugar of approximately 1∶2∶3∶1. Similarities between bovine pineal and rod outer segments are discussed.     

Distribution of glycosphingolipids and ceramide of rat small intestinal mucosa

Previous studies have suggested that glycosphingolipids may be involved in a number of physiological functions of the small intestinal mucosa. Regional variations in many of these processes exist along the length of this organ. In the present studies, the glycosphingolipid and ceramide composition of the proximal, middle and distal thirds of the rat small intestine were characterized and compared. Mono- and trihexosylceramide were the major neutral glycolipids and hematoside (GM₃), the principal ganglioside of this organ. Monohexosylceramide was the major glycolipid of the proximal segment, whereas trihexosylceramide predominated in the distal segments. The total content of neutral glycolipids, ceramide and gangliosides as well as the content of the individual glycosphingolipids and ceramide were highest in the distal segment, intermediate in the middle and lowest in the proximal segment. Additionally, regional variations were noted in the fatty acid composition of the major glycosphingolipids. These differences in the composition of glycolipids and ceramide along the length of the intestine may be responsible, at least partially, for the regional functional specialization seen in this organ.     

A convenient method for the preparation of asialo-GM1

A convenient and efficient procedure has been devised for the large-scale preparation of asialo-G_M1 from bovine brain gangliosides. The procedure relies on the complete desialylation of brain ganglio-sides, consisting primarily of G_M1, G_D1a, G_D1b and G_T1b, by mild formic acid hydrolysis (0.1 N, 100 C; 2 hr). Following the hydrolysis step, asialo-G_M1 can be isolated and purified by Folch partitioning and Iatrobeads column chromatography, with an overall yield of more than 50%. The ganglioside nomenclature used is that of Svennerholm (1).     

Liquid Chromatography–High-Resolution Mass Spectrometry for Quantitative Analysis of Gangliosides

Gangliosides are a large family of glycosphingolipids that are abundant in the brain, and have been shown to affect neuronal plasticity during development, adulthood, and aging. We developed a fast, efficient, and sensitive liquid chromatography–mass spectrometry method to quantify eight different classes of gangliosides (GM₁, GM₂, GM₃, GD₃, GD_1a, GD_1b, GT_1b, GQ_1b) in the brains of 2-day-old and 80-day-old Wistar rats. The gangliosides were extracted from rat brain using a modified Svennerholm and Fredman method. After ganglioside class separation using a hydrophilic high performance liquid chromatography (HPLC) column, the resolving power of the LTQ-Orbitrap™ mass spectrometer was used to extract and sum the major species of each ganglioside class, generating fully resolved extracted ion current peaks for both standards and samples. The flexibility and the specificity of this method are such that it can be applied to the analysis of other ganglioside species/classes not discussed in this paper, provided appropriate standards are available. The method had good repeatability (coefficient of variation 4.8–12.3%) and mean recoveries in the range 92–107%.     

Premalignant alterations in the glycosphingolipids of small intestinal mucosa of rats treated with 1,2-dimethylhydrazine

1,2-Dimethylhydrazine is a procarcinogen with selectivity for the colon and proximal small intestine. In weekly subcutaneous (s.c.) doses of 20 mg/kg body weight, this agent produces colonic and proximal small intestinal tumors in a high percentage of rodents with a latency period of approximately six months. To determine whether alterations in the glycosphingolipid content of rat proximal and/or distal small intestinal mucose existed before the development of dimethylhydrazine-induced cancer, rat were given s.c. injections of this agent (20 mg/kg body weight per wk) or diluent for five wk. Animals were killed at this time, and mucosa was isolated from each small intestinal segment of both groups. Glycosphingolipids then were extracted from these tissues and analyzed by high performance thin layer chromatography and gas liquid chromatography. The results of these studies demonstrated that (1) the content of neutral and acidic glycosphingolipids was significantly decreased (approximately 20%) in the proximal small intestine of treated rats compared with their control counterparts; (2) no significant difference in the glycosphingolipid content was seen, however, in the distal small intestinal mucosa of control and treated rats; and (3) while significant differences were noted in the majority of fatty acids of G_M3, glucosyl- and globotriaosylceramide in the proximal small intestine of control and treated animals, differences in the fatty acids of these glycosphingolipids in the distal segment of these groups were confined to stearic (18∶0) acid and/or arachidic (20∶0) acid.     

Characterization of gangliosides of porcine erythrocyte membranes: Occurrence of ganglioside GD3 as major ganglioside

Four major ganglioside species were isolated from porcine erythrocyte membranes by DEAE-Sephadex and Iatrobeads column chromatography. Treatment of the lipids with graded neuraminidase and β-galactosidase, gas chromatographic analysis of their carboyhydrates, sphingosine bases and molecular species of sialic acid revealed that the structure of these gangliosides were G_M3(NeuAc), G_M3(NeuGc), G_D3(NeuAc) and G_D3(NeuGc), each of which was 16±2 μg, 304±42 μg, 30±3 μg and 240±26 μg, respectively, per gram of the dry erythrocyte stroma. The amount of G_M3 and G_D3 accounted for more than 95% of total gangliosides of the erythrocytes. Porcine erythrocytes may provide a good source for large scale preparation of ganglioside G_D3 which recently was identified as a human melanoma-associated antigen. Gangliosides are named according to Svennerholm (1) and the recommendation of the IUPAC-IUB Commission on Biochemical Nomenclature (2).     

Membrane lipids in bromodeoxyuridine-differentiated astroglial cells in culture

Embryonic hamster astroblasts (NN strain) grown in continuous line were cultivated in the presence of bromodeoxyuridine (BrdU). A decrease in the growth rate of the cells and striking changes in their morphology were observed, the morphology of the cells resembling that of mature astrocytes. Membrane lipids of BrdU-differentiated and standard cells were compared. No modification of the lipid/protein ratio was observed. Phospholipids and cholesterol were increased in the same proprotions in the cells, and no modification of the phospholipid distribution was observed. Ganglioside sialic acid remained at the same level, but the ganglioside distribution was highly modified. Complex gangliosides appeared (G_M1 and G_D1a), while the proportion of simple gangliosides (G_M3 and G_D3) decreased. However, neither G_T1 nor G_Q1 were detected in differentiated cells. The distribution of phosphoglyceride acyl groups was highly modified, the proportion of arachidonic and docosapentaenoic acids being 2 to 3 times higher in BrdU-treated cells than in proliferating ones. These results were compared to those obtained with another clonal line of glial cells (C6) which exhibited no morphological differentiation in the presence of BrdU; the lipids of these cells were not modified by such a treatment.     

Characterization of Glycosphingolipids in the Human Parathyroid and Thyroid Glands

As part of a systematic investigation of the glycosphingolipids in human tissues, acid and non-acid glycosphingolipids from human thyroid and parathyroid glands were isolated and characterized with mass spectrometry and binding of carbohydrate-recognizing ligands, with a focus on complex compounds. The glycosphingolipid patterns of the human parathyroid and thyroid glands were very similar. The major acid glycosphingolipids were sulfatide and the gangliosides GM3, GD3, GD1a, GD1b, GT1b and Neu5Ac-neolactotetraosylceramide, and the major non-acid glycosphingolipids were globotriaosylceramide and globoside. We also found neolactotetra- and neolactohexaosylceramide, the x₂ glycosphingolipid, and complex glycosphingolipids with terminal blood group O and A determinants in both tissues. A glycosphingolipid with blood group Le^b determinant was identified in the thyroid gland, and the parathyroid sample had a glycosphingolipid with terminal blood group B determinant. Immunohistochemistry demonstrated the expression of blood group A antigens in both the thyroid and parathyroid glands. A weak cytoplasmatic expression of the GD1a ganglioside was present in the thyroid, while the parathyroid gland had a strong GD1a expression on the cell surface. Thus, the glycosylation of human thyroid and parathyroid glands is more complex than previously appreciated. Our findings provide a platform for further studies of alterations of cell surface glycosphingolipids in thyroid and parathyroid cancers.     

GM3 Ganglioside Linked to Neurofibrillary Pathology in a Transgenic Rat Model for Tauopathy

Glycosphingolipids (GSLs) are amphipathic lipids composed of a sphingoid base and a fatty acyl attached to a saccharide moiety. GSLs play an important role in signal transduction, directing proteins within the membrane, cell recognition, and modulation of cell adhesion. Gangliosides and sulfatides belong to a group of acidic GSLs, and numerous studies report their involvement in neurodevelopment, aging, and neurodegeneration. In this study, we used an approach based on hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry (HRMS/MS) to characterize the glycosphingolipid profile in rat brain tissue. Then, we screened characterized lipids aiming to identify changes in glycosphingolipid profiles in the normal aging process and tau pathology. Thorough screening of acidic glycosphingolipids in rat brain tissue revealed 117 ganglioside and 36 sulfatide species. Moreover, we found two ganglioside subclasses that were not previously characterized—GT1b-Ac2 and GQ1b-Ac2. The semi-targeted screening revealed significant changes in the levels of sulfatides and GM1a gangliosides during the aging process. In the transgenic SHR24 rat model for tauopathies, we found elevated levels of GM3 gangliosides which may indicate a higher rate of apoptotic processes.     

Gangliosides of bovine optic nerve

Gangliosides from bovine optic nerve were analyzed. The optic nerve contained 129, 98, 97, 80, 31 and 12 μg of GM₁, GD_1a, GD_1b, GT_1b, GD₃ and GQ₁ gangliosides, respectively, per g of tissue wet wt. These 6 gangliosides altogether contributed 97% of the total sialic acid. GM₃ and GM₂ gangliosides contributed the remaining 3% of total sialic acid. Stearoyl (18∶0) was the predominant acyl group (61–76%) in all gangliosides. There was a marked variation in acyl group composition between GT₁ and most of the other major gangliosides except GD₃. In comparison to the other gangliosides, GT₁ contained a lower proportion of the stearoyl group and a higher proportion of the oleoyl, nervonoyl and the long chain acyl groups. Both GT₁ and GD₃ gangliosides contained proportionately higher levels of long chain acyl groups (20∶0→24∶0) than did other gangliosides. GD₃ gangliosides showed 2 bands on thin layer chromatography, and the upper band was more distinct than the lower band.     

Isolation and partial characterization of the neutral glycosphingolipids and gangliosides of the human heart

The glycosphingolipids (GLS) of the human heart muscle have been isolated from total lipids by column and thin layer chromatography and their sugars and fatty acids analyzed by gas liquid chromatography. Hearts from traffic victims were obtained at autopsy between 12 and 16 hr after death and dissected into parts (left and right ventricular walls, intraventricular septum and papillary muscle). The neutral GSL content for those parts of the hearts of two males aged 22 and one female aged 14 ranged from about 90 to 160 nmoles/g wet weight. Trihexosyl ceramide and globoside were the most abundant neutral GSL. Total ganglioside content was about 50 nmoles/g wet weight, and the most abundant gangliosides were partially characterized as GM₃ and GM₁; other mono-, di- and trisialogangliosides were also present. Differences in the content and composition of neutral GSL and gangliosides between the heart and other human tissues are discussed. It is concluded that the patterns of these two GSL fractions of the heart are more complex than those of most of the extraneural human tissues.     

Phospholipid hydroperoxides are detoxified by phospholipase A<Subscript>2</Subscript> and GSH peroxidase in rat gastric mucosa

The aim of this study was to determine the metabolic fate of phospholipid hydroperoxides (PLOOH) in rat gastric mucosa. Here we report evidence concering the mechanism for PLOOH detoxification in gastric mucosa homogenate. Analysis by the TLC blot technique showed that the gastric mucosa has the highest potential to eliminate 1-palmitoyl-2-linoleoyl-phosphatidylcholine hydroperoxides (PL-PtdChoOOH) compared with the intestinal mucosa and liver. Major products detected after incubation with gastric mucosa were the partially reduced linoleic acid hydroperoxides (LAOOH) and lysophosphatidylcholine, indicating the involvement of phospholipase A₂ (PLA₂) in the elimination pathway. Using unilamellar vesicles, we demonstrated that gastric mucosal PLA₂ does not distinguish between PLOOH and intact phospholipids. Although gastric mucosal PLA₂ activity efficiently eliminated excess amounts of PLOOH, the complete reduction of LAOOH was dependent on the supply of exogenous GSH. In a separate experiment, administration of egg yolk PtdChoOOH to rats for 6 d significantly elevated GSH peroxidase (GPx) activity in the gastric mucosa. We concluded that excess amounts of PLOOH are efficiently eliminated through the hydrolysis by PLA₂, and the subsequent reduction of FA hydroperoxide by GPx is the critical step for complete detoxification of oxidized phospholipids in the stomach.