Studies on the specificity and sensitivity of the influenza C virus binding assay for 9-O-acetylated sialic acids and its application to human melanomas

The sensitivity and specificity of two influenza C virus assays, solid-phase and overlay assays, were investigated using naturally occurring 9-O-acetylated GD(3), rat serum glycoproteins containing 60% of N-acetyl-9-O-acetylneuraminic acid, and synthetically O-acetylated sialylated compounds. The sensitivity of the solid-phase assay was higher for glycoproteins containing N-acetyl-9-O-acetylneuraminic acid than for gangliosides, and also differed for various 9-O-acetylated gangliosides. The overlay assay was less sensitive for all glycoconjugates tested. For virus recognition the presentation of the sialic acid within the molecule and the structure of the sialic acid are essential. Investigation of gangliosides from human melanomas and normal skin with the influenza C virus assay showed an increase of O-acetylation of sialic acids in most tumour samples and the occurrence of several O-acetylated gangliosides.     

Isolation and characterization of gangliosides from pig lymphocytes

Two major gangliosides from pig spleen lymphocytes, accounting for 57% of the total lipid-bound sialic acids, were isolated and purified to homogeneity by column chromatography on DEAE-Sephadex and silica gel. They were identified as GM3 (II3Neu5GcLacCer), and GD3 (II3(Neu5Gc)2LacCer), by thin-layer chromatography in comparison with standards and by analysis of the constituent sugars. The major fatty acids of these gangliosides were stearic acid and myristic acid, respectively. In addition to these gangliosides, GD2 and bands comigrating on thin-layer chromatography with authentic GM2, GM1, GD1a and GD1b were found. These compounds also occur in pig peripheral blood lymphocytes, where, however, GD3 represents about 70% of the total lipid-bound sialic acid.     

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.     

Analytical detection of 9(4)-O-acetylated sialoglycoproteins and gangliosides using influenza C virus.

The unique glycoprotein of influenza C virus, designated hemagglutinin (HEF), exhibits three functions: hemagglutination, esterase activity, and fusion factor. As the virus uses 9-O-acetylated sialic acid as a high-affinity receptor determinant for attachment to cells, its binding activity was used to reveal O-acetylated sialic acid residues after polyacrylamide gel electrophoresis and transfer onto nitrocellulose sheets of proteins and thin-layer chromatography of lipids. The specificity of the binding for O-acetylated sialoglycoconjugates was investigated. Our results showed that influenza C virus could detect the different forms of the two murine glycophorins which are known to be O-acetylated sialoglycoconjugates. The virus also bound to O-acetylated gangliosides isolated from embryonic chicken brain such as purified O-acetylated NeuAc alpha (2-8)NeuAc alpha (2-8)NeuAc alpha (2-3)Gal beta (1-4)Glc beta (1-1)ceramide (GT3). The esterase activity of the HEF protein of influenza C virus was used to unmask the sialic acid. After its deacetylation by the virus enzyme, the O-acetylated GT3 was recognized by a monoclonal antibody which binds only to the nonacetylated derivative. The results presented here show that influenza C virus is a discriminating analytical probe for identifying O-acetylated sialoglycoconjugates directly after Western blotting of proteins and thin-layer chromatography of lipids, thus providing a new analytical tool.     

Composition of gangliosides from ovine testis and spermatozoa

Gangliosides were extracted and purified from ovine testis and ejaculated spermatozoa which contained, respectively, 57 and 9 nmol lipid-bound sialic acid per gram wet weight. Fourteen gangliosides were resolved by thin-layer chromatography of testicular gangliosides, of which eleven were purified in sufficient quantity to enable a complete compositional analysis of the carbohydrate residues to be performed. None of the gangliosides contained fucose, but several contained N-glycolylneuraminic acid as a component of the sialic acid species. Relative migration on thin-layer chromatograms relative to known standards, compositional analysis, and selective degradation by specific enzymes were used as the basis for identification. Testis contained members of the ganglio series (GM1, GD1a, GD1b, GT1b, GQ1b), hematoside series (GM3, GD3), and sialosylparagloboside in the molar ratio of 54:40:6, respectively. Testicular GM3, GM1, GD3, GD1a, GD1b and GT1b ran as double bands on thin-layer chromatography which could be accounted for by observed differences in the fatty acid moiety. In addition, the slower migrating band of each pair contained some or all of its sialic acid residues as N-glycolylneuraminic acid, whereas the faster migrating band contained exclusively N-acetylneuraminic acid, except for GM3 where N-acetylneuraminic acid was the sole species in both bands. Thin-layer chromatography of sperm gangliosides revealed seven bands comigrating with equivalent testicular gangliosides. These coincided with the slower migrating bands of testicular GM3, GM1, GD3, GD1a, both bands of GD1b, and possibly both bands of GT1b. Sperm contained only trace amounts of sialosylparagloboside but, in addition, two unidentified bands which were absent from testis were also observed. The molar ratio of the ganglio series to the hematoside series in sperm was 42:58 with GM3 accounting for 42% of total gangliosides.     

Purification and structural characterization of de-N-acetylated form of GD3 ganglioside present in human melanoma tumors

The presence of gangliosides containing de-N-acetylated sialic acids in human tissues has been so far shown by using mouse monoclonal antibodies specific for the de-N-acetylated forms, but the isolation and chemical characterization of such compounds have not yet been performed. Since indirect evidence suggested that de-N-acetylGD3 ganglioside could be present in human melanoma tumors, we analyzed the gangliosides purified from a 500-g pool of those tumors. The de-N-acetylGD3 that was found to migrate just below GD2 in thin-layer chromatography was isolated from the disialogangliosides by high-pressure liquid chromatography using the specific antibody SGR37 to monitor the elution. The amount of antigen was found to be 320 ng per gram of fresh tumor or 0.1% of total gangliosides. Gas chromatography-mass spectrometry analysis of the antibody-positive ganglioside showed that sialic acids were formed of one molecule of N-acetylneuraminic acid and one molecule of neuraminic acid. Radioactive re-N-acetylation of the antigen yielded a GD3-like ganglioside with the radioactive label on the external sialic acid. The constitutive fatty acids were found to differ markedly from those of GD3 and 9-O-acetylGD3 isolated from the same pool of tumors. The major fatty acids were C16:0 and C18:0 in de-N-acetylGD3, whereas GD3 and its 9-O-acetylated derivative contained a large amount of C24:1. These data show that de-N-acetylGD3 ganglioside is indeed present in human melanoma tumors, and the fatty acid content suggests the existence of a de-N-acetylase mostly active on the molecular species of gangliosides with short-chain fatty acids.     

Biosynthesis and turnover of O-acetyl and N-acetyl groups in the gangliosides of human melanoma cells

We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with [3H]acetate and [14C]glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with [acetyl-3H]acetyl-coenzyme A, the major labeled products were disialogangliosides. [Acetyl-3H]O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in [3H]N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from [3H]acetate was exclusively in the form of [3H]N-acetyl groups, whereas the 14C-label was at the 4-position. Pulse-chase analysis of the 3H/14C ratio showed that the N-acetyl groups of both GD3 and of the monosialoganglioside GM3 were turning over faster than the parent molecules. Selective periodate oxidation showed that both the inner and outer sialic acid residues of GD3 incorporated 3H-label in the in vitro reaction, and showed similar turnover of N-acetylation in the pulse-chase study. Taken together, these results indicate that both the O- and N-acetyl groups of the sialic acid residues of gangliosides turn over faster than the parent molecules. They also demonstrate a novel re-N-acetylation reaction that predicts the existence of de-N-acetyl gangliosides in melanoma cells.     

Monoclonal antibody R24 distinguishes between different N-acetyl- and N-glycolylneuraminic acid derivatives of ganglioside GD3

Monoclonal antibody (MAb) R24 was previously shown to be directed toward ganglioside GD3 [Pukel, C. S., Lloyd, K. O., Travassos, L. R., Dippold, W. G., Oettgen, H. F., and Old, L. J. (1982) J. Exp. Med. 155, 1133-1147]. The structural specificity of the MAb has now been further characterized based on binding to structurally related glycolipids, including four GD3 derivatives with different N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc) substituents. Three assay systems (enzyme immunostaining on thin-layer chromatography, enzyme-linked immunosorbent assay, and immune adherence inhibition assay) were used. MAb R24 was found to react with (NeuAc-NeuAc-)GD3 and (NeuAc-NeuGc-)GD3 but not with (NeuGc-NeuAc-)GD3 or (NeuGc-NeuGc-)GD3. These results clearly indicate that the outer sialic acid (Sia) moiety of GD3 is crucial and must be a NeuAc residue, while the inner sialic acid is less involved in binding to the MAb and can be either NeuAc or NeuGc. The MAb was also found to cross-react weakly with two gangliosides, GT1a and GQ1b, but none of other gangliosides nor neutral glycolipids tested reacted. These findings suggest that the epitope detected by MAb R24 is the trisaccharide structure NeuAc alpha 2----8Sia alpha 2----3Gal-, which must be in a terminal position.     

Evidence for sialidase hydrolyzing gangliosides GM2 and GM1 in rat liver plasma membrane

Rat liver plasma membrane removed sialic acid from mixed bovine brain gangliosides more efficiently than from sialyllactose and orosomucoid with an optimal pH of 4.5. When individual gangliosides, each labeled with [14C]sialic acid or [3H]sphingosine, were tested, not only GD1a and GM3 but also GM2 and GM1, both of which had been considered to resist mammalian sialidases, were desialylated. The products of GM2 and GM1 hydrolysis were identified as asialo-GM2 and asialo-GM1, respectively, by thin-layer chromatography.     

Glycosphingolipid binding specificities of rotavirus: identification of a sialic acid-binding epitope

The glycosphingolipid binding specificities of neuraminidase-sensitive (simian SA11 and bovine NCDV) and neuraminidase-insensitive (bovine UK) rotavirus strains were investigated using the thin-layer chromatogram binding assay. Both triple-layered and double-layered viral particles of SA11, NCDV, and UK bound to nonacid glycosphingolipids, including gangliotetraosylceramide (GA1; also called asialo-GM1) and gangliotriaosylceramide (GA2; also called asialo-GM2). Binding to gangliosides was observed with triple-layered particles but not with double-layered particles. The neuraminidase-sensitive and neuraminidase-insensitive rotavirus strains showed distinct ganglioside binding specificities. All three strains bound to sialylneolactotetraosylceramide and GM2 and GD1a gangliosides. However, NeuAc-GM3 and the GM1 ganglioside were recognized by rotavirus strain UK but not by strains SA11 and NCDV. Conversely, NeuGc-GM3 was bound by rotaviruses SA11 and NCDV but not by rotavirus UK. Thus, neuraminidase-sensitive strains bind to external sialic acid residues in gangliosides, while neuraminidase-insensitive strains recognize gangliosides with internal sialic acids, which are resistant to neuraminidase treatment. By testing a panel of gangliosides with triple-layered particles of SA11 and NCDV, the terminal sequence sialyl-galactose (NeuGc/NeuAcalpha3-Galbeta) was identified as the minimal structural element required for the binding of these strains. The binding of triple-layered particles of SA11 and NCDV to NeuGc-GM3, but not to NeuAc-GM3, suggested that the sequence NeuGcalpha3Galbeta is preferred to NeuAcalpha3Galbeta. Further dissection of this binding epitope showed that the carboxyl group and glycerol side chain of sialic acid played an important role in the binding of such triple-layered particles.     

Gangliosides in rat kidney: composition, distribution, and developmental changes

Gangliosides in rat kidney were analyzed for their composition, regional distribution, and developmental changes. Renal tissue from 7-week-old rats showed a GM3-dominant pattern with GD3 and several minor ganglioside components including GM4, GM2, GD1a, and an unknown ganglioside (ganglioside X). The tissue also contained c-series gangliosides that included GT3 as the main component with GT2 in a lesser amount. Ganglioside analysis of cortical and medullary regions of renal tissue suggested the restricted localization of some gangliosides. While GM4 and GD3 were enriched in the cortical region, GM2 was distributed mainly in the medullary area. Renal gangliosides showed unique developmental profiles during a period from Embryonic Day 20 (E20) to 7 weeks postnatal. The content of renal gangliosides increased from E20, reached the highest around Postnatal Day 1, and thereafter, decreased rapidly to the adult level. The ratio of N-glycolylneuraminic acid to total sialic acids in gangliosides tended to change in inverse proportion to the amount of total sialic acids. The composition of major gangliosides in renal tissues shifted from GD3-dominant to GM3-dominant patterns with advancing ages. While GM1 was expressed only at early stages of the development, GM4, GM2, and ganglioside X appeared after Postnatal Day 3. The expression of c-series gangliosides was less affected through the period examined. These results suggest that gangliosides may be implicated with development and function of rat kidney.     

Gangliosides modulate the activity of the plasma membrane Ca(2+)-ATPase from porcine brain synaptosomes

We systematically examined the effects of gangliosides on the plasma membrane Ca(2+)-ATPase (PMCA) from porcine brain synaptosomes. Our results showed that GD1b (two sialic acid residues) stimulated the activity, GM1 (one sialic acid residue) slightly reduced the activity, while asialo-GM1 (no sialic acid residue) markedly inhibited it, suggesting that sialic acid residues of gangliosides are important in the modulation of the PMCA. We also examined the oligosaccharide effects by using GM1, GM2, and GM3 whose only difference was in the length of their oligosaccharide chain. GM1, GM2, and GM3 reduced the enzyme activities, whereas GM2 and GM3 were potent inhibitors. Gangliosides affect both affinity for Ca(2+) and the Vmax of enzyme. It was observed that GD1b and GM2 increased the affinity of the enzyme for Ca(2+). GD1b, GM2 affected the Vmax with an increase of GD1b, but decreases of GM2. The study of the affinity for ATP and the Vmax of enzyme in the presence of gangliosides showed that GD1b and GM2 had little effect on the ATP binding to the enzyme, but the Vmax was apparently changed. Moreover, the effects of gangliosides are additive to that of calmodulin, suggesting that the modulation of PMCA by gangliosides should be through a different mechanism. The conformational changes induced by gangliosides were probed by fluorescence quenching. We found that fluorescent quenchers (I(-) and Cs(+)) with opposite charges had different accessibility to the IAEDANS binding to the PMCA in the presence of gangliosides. An apparent red shift (25nm) with increased maximum of fluorescence spectrum was also observed in the presence of GD1b.     

Natural occurrence and preparation of O-acylated 2,3-unsaturated sialic acids

Three O-acylated, unsaturated sialic acids, N-acetyl-9-O-acetyl-, N-acetyl-9-O-lactoyl-, and 2-deoxy-N-glycoloyl-9-O-lactoyl-2,3-didehydroneuraminic acid (5-acetamido-9-O-acetyl-, 5-acetamido-9-O-lactoyl-, and 2,6-anhydro-3,5-dideoxy-5-glycoloylamido-9-O-lactoyl-D-glycero-D-g alacto-non-2- enonic acid) were isolated from urine or submandibular glands of rat, pig, and cow. Mass spectrometric evidence for the existence of 2,3-unsaturated 9-O-acetyl-N-glycoloylneuraminic acid in porcine urine was also obtained. The sialic acids were purified by dialysis, gel- and ion-exchange chromatography, and preparative thin-layer chromatography. They were analyzed by thin-layer chromatography, high-pressure liquid chromatography, and capillary gas-liquid chromatography-mass spectrometry. For comparison, O-acetylated unsaturated sialic acids were synthesized.     

Generation of murine monoclonal antibodies specific for N-glycolylneuraminic acid-containing gangliosides.

We generated two murine monoclonal antibodies (MAbs) specific for mono- and disialylgangliosides having N-glycolylneuraminic acid (NeuGc) as their sialic acid moiety, respectively, by immunizing C3H/HeN mice with these purified gangliosides adsorbed to Salmonella minnesota followed by fusion with mouse myeloma cells. By use of a wide variety of glycolipids, including NeuGc-containing gangliosides, the precise structures recognized by these two antibodies were elucidated through enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatography. One MAb, GMR8, which was generated by immunizing the mice with purified GM3(NeuGc), reacted specifically with gangliosides having NeuGc alpha 2----3Gal- terminal structures, such as GM3(NeuGc), IV3NeuGc alpha-Gg4Cer, IV3NeuGc alpha-nLc4Cer, V3NeuGc alpha-Gb5Cer, and GD1a(NeuGc, NeuGc). None of the other gangliosides having internal NeuGc alpha2----3Gal- sequences, such as GM2(NeuGc) and GM1(NeuGc), nor corresponding gangliosides having NeuAc alpha 2----3Gal- sequences, nor neutral glycolipids were recognized. Thus, the epitope structures recognized by the MAb were found to be strictly NeuGc alpha 2----3Gal- terminal structures. In contrast, the other MAb, GMR3, which was generated by immunizing the mice with purified GD3(NeuGc-NeuGc-) adsorbed to the bacteria, reacted specifically with gangliosides having NeuGc alpha 2----8NeuGc alpha 2----3Gal- terminal sequences, such as GD3(NeuGc-NeuGc-), IV3NeuGc alpha 2-Gg4Cer, IV3NeuGc alpha 2-nLc4Cer, and V3NeuGc alpha 2-Gb5Cer, but did not react with corresponding gangliosides having NeuAc as their sialic acid moiety or with the neutral glycolipids tested. The epitope structures recognized by the MAb were suggested to be NeuGc alpha 2----8NeuGc alpha 2----3Gal- terminal structures. Using these MAbs, we determined the distribution of such gangliosides in the spleen, kidney, and liver of several mice strains. Novel gangliosides reactive with these MAbs were detected in these tissues.     

Rabbit muscle gangliosides

Four ganglioside fractions were isolated from rabbit muscle: one hematoside and three hexosamine-containing species. They were analyzed for hexoses, hexosamine, sialic acid, fatty acids, and long-chain base content. The molar ratios of sphingosine-hexose-hexosamine-sialic acid were: for hematoside, 1:2:0:1; for the disialogangliosides, 1:3:1:2; and for trisialoganglioside, 1:3:1:3. The carbohydrates were studied by thin-layer and paper chromatography. The hexoses were glucose and galactose; the hexosamine was N-acetylgalactosamine and the sialic acid was N-acetylneuraminic acid. Fatty acids and long-chain bases were analyzed by gas-liquid chromatography. The fatty acid composition was similar in all of the four gangliosides. The most abundant fatty acids were 16:0 and 18:0, but significant amounts of 16:1, 18:1, 20:0, and 22:0 were also found. Hydroxy fatty acids were not detected. In all of the muscle gangliosides the main long-chain bases were C(18)-sphingenine and C(20)-sphingenine. In hematoside there were also measurable amounts of C(18)-sphinganine and C(20)-sphinganine, whereas in the major gangliosides only traces of C(18)-sphinganine were detected.     

Major gangliosides in normal and pathological human thyroids

The major gangliosides of human thyroids were extracted, purified and then analyzed by gas-liquid chromatography. In normal thyroid, GM3 and GD3 represented about 80% of lipid-bound sialic acid. GM3 contained more than 50% of long-chain fatty acids, whereas GD3 contained mostly short ones. 4D hydroxy sphinganine represented 20% of long-chain base content in both cases. In pathological thyroids (Graves' disease, cancer, toxic adenoma), GM3 represented about 60% of lipid-bound sialic acid; its fatty acid content was mostly short chain fatty acids, as in normal GD3. 4D hydroxy sphinganine proportion was decreased.     

Enhanced binding of the neural siglecs, myelin-associated glycoprotein and Schwann cell myelin protein, to Chol-1 (alpha-series) gangliosides and novel sulfated Chol-1 analogs

Extended glycoconjugate binding specificities of three sialic acid-dependent immunoglobulin-like family member lectins (siglecs), myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), and sialoadhesin, were compared by measuring siglec-mediated cell adhesion to immobilized gangliosides. Synthetic gangliosides bearing the alpha-series determinant (NeuAc alpha2,6-linked to GalNAc on a gangliotetraose core) were tested, including GD1alpha (IV(3)NeuAc, III(6)NeuAc-Gg(4)OseCer), GD1alpha with modified sialic acid residues at the III(6)-position, and the "Chol-1" gangliosides GT1aalpha (IV(3)NeuAc, III(6)NeuAc, II(3)NeuAc-Gg(4)OseCer) and GQ1balpha (IV(3)NeuAc, III(6)NeuAc, II(3)(NeuAc)(2)-Gg(4)OseCer). The alpha-series gangliosides displayed enhanced potency for MAG- and SMP-mediated cell adhesion (GQ1balpha > GT1aalpha, GD1alpha > GT1b, GD1a > GM1 (nonbinding)), whereas sialoadhesin-mediated adhesion was comparable with alpha-series and non-alpha-series gangliosides. GD1alpha derivatives with modified sialic acids (7-, 8-, or 9-deoxy) or sulfate (instead of sialic acid) at the III(6)-position supported adhesion comparable with that of GD1alpha. Notably, a novel GT1aalpha analog with sulfates at two internal sites of sialylation (NeuAcalpha2,3Galbeta1,4GalNAc-6-sulfatebeta1, 4Gal3-sulfatebeta1,4Glcbeta1,1'ceramide) was the most potent siglec-binding structure tested to date (10-fold more potent than GT1aalpha in supporting MAG and SMP binding). Together with prior studies, these data indicate that MAG and SMP display an extended structural specificity with a requirement for a terminal alpha2, 3-linked NeuAc and great enhancement by nearby precisely spaced anionic charges.     

O-acetylated gangliosides in bovine buttermilk. Characterization of 7-O-acetyl, 9-O-acetyl, and 7,9-di-O-acetyl GD3.

Three O-acetylated gangliosides, G1, G2, and G3, were purified from bovine buttermilk by using chloroform/methanol extraction, Folch partitioning, chromatography on DEAE-Sephadex A-25, and Iatrobeads columns. The final yields of gangliosides G1, G2, and G3 were 2 mg, 37 mg, and 40 mg per 1.7 kg of the buttermilk powder, respectively. On the basis of immunostaining on high performance thin layer chromatography with specific monoclonal antibodies, mild alkaline treatment, gas-liquid chromatographic analysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance studies, G1 and G2 are characterized as O-acetylated GD3 and G3 as O-acetylated GT3, and the structures of these gangliosides are as follows: [formula: see text] The major fatty acids of these gangliosides were C18:0, C22:0, C23:0, and C24:0, and the long chain base was C18-sphingosine.     

Gangliosides of human thyroid gland

The ganglioside composition of adult human thyroid gland was examined in autopsy material obtained from patients who died of circulatory diseases but who showed no signs of thyroid disorders. The concentrations of phospholipids, cholesterol and gangliosides (lipid-bound sialic acid) in the whole glands were 5.2, 4.3 and 0.12 mmol/kg fresh tissue weight and, in dissected follicular material, 7.0, 3.4 and 0.24 mmol/kg tissue, respectively. The molar ratio of phospholipids/cholesterol/gangliosides in the follicular material was 1.00:0.49:0.034. Twelve molecular species of gangliosides were isolated and identified. Gangliosides GM3 and GD3 were most abundant, but GD1a, GD1b, GT1b and 3'-LM1 were also present in quantities greater than 5% of the total gangliosides. N-Acetylneuraminic acid and an alkali labile sialic acid, probably N-acetyl-9-O-acetylneuraminic acid, were found to occur in human thyroid.     

Acidic glycosphingolipids of cock testis elucidated by mass spectrometry and NMR spectroscopy

The main acidic glycosphingolipids (GSLs) of cock testis were identified as GalCer I³-sulfate and gangliosides GM4, GM3, GD3 and GT3. They contained N-acetylneuraminic acid as the major sialic acid, and ceramides composed mainly of sphingosine (dl8:1) and C18–24 non-hydroxy fatty acids. Appreciable amounts of hydroxy fatty acids were detected only in the GM4 preparation.