Quantification of gangliotetraose gangliosides with cholera toxin

A procedure is described for assay of GM1 and other gangliotetraose-type gangliosides at the picomole level. The gangliosides are absorbed onto polystyrene microwells and treated with neuraminidase and then with cholera toxin B subunits conjugated to horseradish peroxidase. Color is developed and quantified spectrophotometrically. Omission of neuraminidase gives a measure of GM1 alone. Linearity was obtained between 0.5 and 3 pmol. This procedure was applied to ganglioside mixtures isolated fron neuro-2A neuroblastoma and PC12 pheochromocytoma cells. For the latter, an additional step involving reaction with fucosidase increased the yield of GM1 due to the presence of fucosylated gangliosides. Application of the same reagents as a TLC overlay procedure to the gangliosides from neuro-2A cells revealed the presence of GD1a, GD1b, and GT1b in addition to GM1, thus confirming the presence of a family of gangliotetraose gangliosides.     

Detection of gangliosides of the GM1b type on high-performance thin-layer chromatography plates by immunostaining after neuraminidase treatment

A method for the detection of GM1b-type gangliosides in complex mixtures of gangliosides was developed. The procedure involves separation of gangliosides on high-performance thin-layer chromatography plates, fixation of the silica gel, treatment with neuraminidase from Vibrio cholerae in the absence of detergent, and incubation of the plates with GgOse4Cer-specific antibodies. Alkaline phosphatase-conjugated second antibodies are used to visualize bound first antibodies by generating a blue dye from 5-bromo-4-chloro-3-indolylphosphate. The procedure is capable of detecting as little as 30 ng of gangliosides. Gangliosides from murine T lymphocytes and from human brain served as examples. Besides GM1b, GD1 alpha is also detectable by this method, whereas the human brain gangliosides GM1a, GD1a, GD1b, and GT1b are not, because they are neuraminidase resistant. Since terminally sialylated gangliosides such as GM1b were described as virus receptors, and certain other terminally sialylated gangliosides are discussed as tumor markers, this method should be useful to screen gangliosides from different tissues or cell lines for the presence of such components, especially if only small amounts of material are available.     

Simple micro-method for the isolation of gangliosides by reversed-phase chromatography

A simple and convenient technique has been developed for the isolation of gangliosides from small amounts of tissues or cells. A ganglioside fraction obtained by chromatography of the total lipid extract of DEAE-Sephadex was subjected to alkaline hydrolysis and salts and other non-lipid contaminants were removed by reversed-phase chromatography on a C₁₈ Sep-Pak cartridge. The purified gangliosides were then obtained by chromatography on a small Iatrobeads or Unisil column. This procedure yields a quantitative recovery of gangliosides that are free of contaminants which interfere with thin-layer chromatographic analysis. The procedure was used for the quantitative isolation of gangliosides from human brain white matter and human erythrocytes.     

Quantitative analysis of brain gangliosides by high performance liquid chromatography of their perbenzoyl derivatives

This report describes a convenient, highly sensitive, and reproducible HPLC procedure for the quantitative analysis of gangliosides from brain tissues. The procedure involves the conversion of gangliosides to their perbenzoyl derivatives, isolation of the derivatives on a C18-reversed-phase cartridge, separation of the derivatives on a column (3-micron silica) maintained at an elevated temperature, and UV detection of the derivatives at 230 nm. The convenience of the procedure, its sensitivity, reproducibility, and application to the analysis of gangliosides from tissue sources make it the method of choice for ganglioside quantification in our laboratories. Three aspects of the procedure contribute to its convenience: reaction conditions that lead to single products, a convenient isolation procedure for the derivatives, and chromatographic conditions that provide resolution of the derivatives.     

Separation of gangliosides on a new type of anion-exchange resin

A new anion exchange resin, Spherosil-DEAE-Dextran, consisting of porous glass beads covered with cross-linked DEAE-Dextran, was used in the separation of gangliosides. The gangliosides were eluted from the resin with a discontinuous gradient of potassium acetate in methanol, separating the gangliosides quantitatively into mono-, di, tri-, tetra- and pentasialoganglioside fractions. The new resin was found to have higher binding capacity, to show less unspecific adsorption and to give a better separation of the higher oligosialogangliosides than did DEA-Spherosil, QAE-Sephadex, DEAE-Sephadex and DEAE-Sepharose. Anion exchange chromatography improved discrimination between closely allied gangliosides as well as quantification and identification of individual gangliosides, especially the minor ones. The new procedure was used in the separation of the gangliosides in human infant forebrain and cerebellum.     

Retention of gangliosides in serum delipidated by diisopropyl ether-1-butanol extraction

Extraction with diisopropyl ether-1-butanol is a rapid method for the delipidation of serum without protein denaturation. We sought to confirm that this solvent system would also extract the highly polar acidic glycosphingolipids, gangliosides. In fact, however, both endogenous serum gangliosides and radiolabeled rat brain gangliosides added to serum were nearly quantitatively retained (87.5% and 98.7%, respectively) in the aqueous phase after two extractions. Therefore, while useful for the extraction of most lipids, this delipidation procedure cannot be used to remove gangliosides from serum or plasma.     

Molecular parameters of gangliosides in monolayers: comparative evaluation of suitable purification procedures

The molecular area, collapse pressure, and surface potential of gangliosides obtained by different methods were systematically compared in monolayers at the air-water interface. Different values of these parameters are obtained depending on the purification procedure employed for the isolation of pure gangliosides. This is due to impurities (such as peptidaceous material) that remain in different amounts in the various preparations and that modify the ganglioside surface behavior. Routine purity checking by HPTLC analysis of gangliosides usually fails to reveal these impurities. On the other hand, even if the monolayer technique cannot identify the nature or amount of contaminants, it is extremely sensitive to reveal alterations of the surface molecular parameters caused by relatively small amounts of other components coextracted with the ganglioside or adventitiously introduced with the solvents or subphases employed. This is a serious problem for the obtention of correct and reproducible values of such important parameters as the molecular area of gangliosides, their electrostatic potential in oriented interfaces, and their interactions with other lipids and proteins. A procedure leading to consistent molecular parameters that remain reproducible after several repurification cycles is to perform an alkaline treatment on previously purified gangliosides species with NaOH, this is followed by dialysis against bidistilled water, rechromatography on DEAE-Sephadex A25, silicic acid or Iatrobeads, and Sephadex LH-20 columns; repurified gangliosides are stored in chloroform-methanol-0.01 M NaOH (60:30:4.5).     

In situ immunological determination of basic carbohydrate structures of gangliosides on thin-layer plates

An immunological method for the determination of the basic carbohydrate structure of gangliosides by using a thin-layer chromatographic immunostaining technique was developed. After high-performance thin-layer chromatography of gangliosides, the chromatogram is treated with a 0.4% polyisobutylmethacrylate solution. Arthrobacter ureafaciens neuraminidase is then applied to the separated gangliosides in situ on the chromatographic plate. This procedure will remove both external and internal sialic acid residues from the core oligosaccharide backbone. The resulting glycolipid products are then incubated with anti-Gg4 serum and 125I-staphylococcal protein A, successively, and exposed to an X-ray film. Through a highly specific binding, the anti-Gg4 antibody detects only those gangliosides having the oligosaccharide backbone of Gg4.     

Study of ganglioside patterns with two-dimensional thin-layer chromatography and radioautography; Detection of new fucogangliosides and other minor species in rabbit brain

Brain gangliosides are shown to be highly complex mixtures of major and minor species through the use of two-dimensional thin-layer chromatography combined with radioautography. When applied to ¹⁴C-labeled gangliosides, radioautography could be employed as a sensitive detection procedure capable of revealing minor species that were not visualized with a conventional spray. The brain ganglioside pattern of neonatal rabbits was studied in this manner following intracranial injection of ¹⁴C-labeled N-acetylmannosamine and fucose. Numerous minor components representing previously uncharacterized gangliosides were detected, including several new fucose-containing gangliosides.     

Recovery of gangliosides from aqueous solutions on styrene-divinylbenzene copolymer columns

An investigation was made using a styrene-divinylbenzene copolymer as a solid phase sorbent to recover gangliosides from aqueous solutions. A comparison with octadecyl-bonded (C18) silica gel showed that the general procedure used to purify gangliosides on C18 silica gel could be used with the copolymer. The yield of gangliosides depended on various parameters such as the composition of the conditioning solution, the salt concentration of the loading solution, and the amount of applied gangliosides per gram of copolymer. In optimal conditions, the recovery of gangliosides and other lipids present in the upper phases of partition was higher than 95%. Using radiolabeled gangliosides, it was found that gangliosides present in serum-containing medium could also be quantitatively recovered on copolymer, provided the medium was diluted with an equal volume of methanol prior to its application onto the column. The major advantage of the copolymer is its high stability in acidic or alkaline conditions that allows multiple cycles of cleaning and reconditioning of the sorbent without alteration of its chromatographic properties.     

Incorporation of N-Acetylmannosamine into Rat Brain Subcellular Gangliosides: Effect of Pentylenetetrazol-Induced Convulsions on Brain Gangliosides1

Abstract: The labeling pattern of the major individual gangliosides from the microsomal and synaptosomal fractions of rat brain was determined following intracerebral injection of the radioactive sialic acid precursor, N-acetylmannosamine. Microsomal gangliosides initially had a higher specific radioactivity than synaptosomal gangliosides, with both fractions reaching similar specific radioactivities 18 h after precursor injection. In both subcellular fractions, the polysialogangliosides G_T1b and G_Q1b were initially more highly labeled than all other gangliosides. With the establishment of the labeling pattern, the effect of the convulsant pentylenetetrazol on brain gangliosides was examined in detail. Significant decreases in radioactive label were noted in the polysialogangliosides, G_T1b and G_Q1b, from the synaptosomal and microsomal fractions of the convulsed animals. The decreases may be due to activation of the membrane-bound neuraminidase present with the gangliosides in neuronal tissue. Prior to experimentation, a methodology was developed to insure quantitative isolation of small amounts of ganglioside free of other lipids and water-soluble contaminants. Combination of this isolation procedure with quantitative densitometry of thin-layer chromatograms permits accurate distributional analyses for individual gangliosides. In applications involving radioactive gangliosides, the method allows the determination of both radioactivity and sialic acid distributions from the same thin-layer chromatogram.     

Preparation of radioactive Tay-Sachs ganglioside labeled in the sialic acid moiety

A procedure is described for the preparation of Tay-Sachs ganglioside specifically labeled in the sialic acid portion of the molecule. Rat brain gangliosides were labeled biosynthetically by the intracranial injection of N-acetyl-(3)H-D-mannosamine. Radioactive gangliosides were isolated and selectively degraded with bacterial neuraminidase and rat liver beta-galactosidase to Tay-Sachs ganglioside-(3)H. Radioactivity in the labeled product was confined to the N-acetyl-neuraminic acid portion of the molecule.     

Acetonitrile-hydrochloric acid hydrolysis of gangliosides for high performance liquid chromatographic analysis of their long chain bases

An improved method for the hydrolysis of long chain bases from gangliosides with aqueous acetonitrile-HCl is described. The long chain bases released from brain gangliosides were derivatized with biphenylcarbonylchloride and resolved by high performance liquid chromatography on a C18 reversed-phase column. Components of individual peaks were identified by gas-liquid chromatography-mass spectrometry as their trimethylsilyl derivatives. The acetonitrile-HCL hydrolysis procedure resulted in no formation of O-methyl ethers of long chain bases and a significant decrease in the level of secondary products.     

A novel carbohydrate, differentiation antigen on fucogangliosides of human myeloid cells recognized by monoclonal antibody VIM-2

A mouse monoclonal antibody, VIM-2, specific for human blood cells of myelomonocytic lineage, was found to bind to a series of minor gangliosides isolated from the cells of patients with chronic myelogenous leukemia (Uemura, K., Macher, B.A., DeGregorio, M., Scudder, P., Buehler, J., Knapp, W., and Feizi, T. (1985) Biochim. Biophys. Acta 846, 26-36). TLC immunostaining studies with the VIM-2 antibody of gangliosides from normal human neutrophils, acute myeloid leukemia, and chronic myelogenous leukemia cells showed that the total amount and the ratio of the VIM-2 gangliosides varies among these different myeloid cells and appears to be related to the level of cellular differentiation. Purification of these gangliosides from chronic myelogenous leukemia cells was aided by a sensitive enzyme-linked immunosorbent assay procedure used in conjunction with high performance liquid chromatography. Structures for two of the immunoreactive gangliosides (a ceramide decasaccharide, VIII3NeuAcV3-Fuc-nLc8Cer and a ceramide dodecasaccharide X3-NeuAcVII3Fuc-nLc10Cer) are proposed from negative ion fast atom bombardment mass spectrometry of the native gangliosides, methylation analysis, and the combined use of glycosidase treatment and TLC immunostaining with carbohydrate sequence specific antibodies. The VIM-2 antigen was thus characterized as involving the sialofucooligosaccharide sequence.     

A new thin-layer chromatographic approach for separation of multisialogangliosides: Novel gangliosides fractions in the embryonic chicken brain

A novel thin-layer chromatographic procedure has been developed that permits rapid, high-resolution separation of complex ganglioside mixtures and direct densitometric quantification. A special advantage of the new procedure, performed by two different consecutive runs on high-performance thin-layer chromatography plates, is an excellent separation of multisialogangliosides containing more than three sialic acid residues. Using the new procedure, 10 unidentified fractions were detected in embryonic chick brains. These gangliosides were clearly distinguishable from the known gangliosides, GM1, GD3, GD1a, GD2, GD1b, GT1b, and GQ1b. Eight of these “additional” fractions were also found in the brains of rays. From published data on the cod fish brain, 6 of the novel fractions are suggested to correspond to GT3, GT2, GT1c, GQ1c, GP1c, and GP1b. Four fractions, moving on thin-layer chromatography plates below the suggested GP1c have not been reported previously in any vertebrate. Due to their very slow migration rates they may contain gangliosides with six, seven, or more sialic acid residues. During development of the chicken, the relative amounts of the newly detected fractions decrease in favor of GT1b and GD1a.     

Simple method for large-scale isolation and purification of gangliosides by non-ionic adsorption chromatography

A type of non-ionic adsorption resin, X-5, was used in the isolation and purification of brain gangliosides. Hydrolysis with base treatment was carried out for the purpose of eliminating contaminants. The major advantages of the new procedure, compared to conventional methods, were the shorter separation time, higher loading capacity (80 micromol LBSA per gram resin), and recovery (98%) of separated ganglioside fractions with little solvent. It is a practical way for large-scale isolation and purification of gangliosides.     

Improved preparation method for lysogangliosides.

Lyso-GM3 and -GM1 gangliosides were prepared from the corresponding N,N'-dideacylated gangliosides using N-trifluoroacetylation at the sphingosine moiety, followed by N-acetylation and mild saponification. The blocking reaction was performed using a water-ether bilayer system at alkaline medium, in which the N-trifluoroacetylation occurred predominantly at the lipid moiety. Through the procedure, lysoGM3 and lysoGM1 were obtained with higher yields from the corresponding dideacylated gangliosides than through the previous method using 9-fluorenylmethoxycarbonyl chloride as a blocking group or of direct N-acetylation of it on liposomes containing starting ganglioside and other lipid. Chemical structures of the lysogangliosides and the synthetic intermediates were confirmed by the proton nuclear magnetic resonance spectrometry and negative fast atom bombardment-mass spectrometry.     

Synthesis of lysogangliosides

The synthesis of gangliosides GM3, GM2, GM1, and GD1a solely lacking the fatty acid moiety, and thus called lysogangliosides in analogy to lysophospholipids, is described. Since a selective elimination of the fatty acid residue has not been achieved as yet, the gangliosides were first subjected to alkaline hydrolysis. By this procedure the fatty acyl as well as the acetyl groups of the sialic acid residue(s) were completely removed. The acetamido group of the N-acetylgalactosamine moiety of the gangliosides GM2, GM1, and GD1a was very little (congruent to 10%) hydrolyzed. In a two-phase system composed of water and ether, the selective protection of the sphingoid amino group was accomplished with a hydrophobic protective group (9-fluorenylmethoxycarbonyl). Lysogangliosides were obtained after re-N-acetylation of the sialooligosaccharide amino group(s) followed by removal of the protecting group. The overall yield was about 30%. The structures of the lysogangliosides were confirmed by chemical analysis as well as negative ion FAB mass spectrometry and 1H NMR spectroscopy. By simple re-N-acylation of lysogangliosides with any labeled fatty acid, labeled gangliosides are now obtainable that are identical with their parent gangliosides except for their labeled fatty acid residue. This has been demonstrated by the synthesis of GM1 with a [1-13C]palmitic acid moiety in its ceramide portion. If desired, double-labeled gangliosides may be obtained by use of labeled acetic anhydride in the synthesis of the lysogangliosides.     

High performance liquid chromatography preparation of the molecular species of GM1 and GD1a gangliosides with homogeneous long chain base composition

A semi-preparative, analytical high performance liquid chromatographic (HPLC) procedure is described for the isolation of molecular species of GM1 and GD1a gangliosides containing a single long chain base, C18 or C20 sphingosine, C18 or C20 sphinganine, each in its natural erythro or unnatural threo form. The threo forms were obtained from 2,3-dichloro-5,6-dicyanobenzoquinone/NaBH4 -treated gangliosides. The ganglioside molecular species separated by HPLC were analyzed for carbohydrate, fatty acid, and long chain base composition. In particular, long chain bases were submitted to gas-liquid chromatographic-mass spectrometric analyses as their trimethylsilyl (TMS) or N-acetyl-TMS derivatives, and chain length, presence or absence of C4-C5 double bond, and C-3 steric configuration were ascertained. The final preparations of individual molecular species of GM1 and GD1a gangliosides were more than 99% homogeneous in their saccharide moiety, contained a single long chain base (homogeneity higher than 99%), and had a fatty acid composition primarily of stearic acid (92 to 97%). All the individual molecular species of GM1 and GD1a gangliosides were also prepared in radioactive form by selective tritiation at C-3 of the long chain base. Their specific radioactivity ranged from 1.3 to 1.45 Ci/mmol. The availability of these molecular species of gangliosides is expected to facilitate studies aimed at ascertaining the role played by the hydrophobic portion in the functional behavior of gangliosides.     

Gangliosides Inhibit Glucosylceramide Synthase: A Possible Role in Ganglioside Therapy

Gangliosides stimulate the hydrolysis of glucosylceramide (GlcCer), their precursor, and therefore may lower the level of cellular GlcCer and exert a feedback control effect to slow the formation of gangliosides. Tests were made to see if a similar effect on GlcCer levels can be exerted by the action of gangliosides on GlcCer synthesis. Using a new assay procedure, we showed that gangliosides do inhibit the synthase in brain membranes quite effectively, the most active being those lipids with more sugar and sialic acid moieties. Mice injected with a mixture of brain gangliosides for 5 days were found to have a lower level of ceramide:UDP-Glc glucosyltransferase activity in brain, liver, and kidney. The inhibition seems to be exerted by competition for the active site and binding to effector site(s) on the enzyme. It is possible that the reported therapeutic actions of gangliosides on the nervous system are, in part, the result of lowered levels of GlcCer. Malignant tumors shed gangliosides into the extracellular fluid, which are believed to block the generation of antibodies by the host's immunodefense system; this effect also may be due, in part, to reduction in the GlcCer level of immunogenic cells. A new finding is that a ceramide containing phytosphingosine is a markedly better substrate for GlcCer synthase than one containing the more common base.