Studies on the glycosphingolipids of the starfish, Asterina pectinifera. II. Isolation and characterization of a novel ganglioside with an internal sialic acid residue.

1. Three gangliosides, provisionally named Gangliosides 1, 2, and 3, were obtained from the lipid extract of the starfish, Asterina pectinifera by silicic acid, DEAE-Sephadex, and Iatrobeads column chromatography. The most abundant, Ganglioside 3 (37.7 microgram/g wet weight of starfish) was isolated in the pure state and its chemical structure was studied. 2. The sugar composition of Ganglioside 3 consisted of arabinose, glucose, galactose, and sialic acid (as N-glycolylneuraminic acid) in a molar ratio of 1:1:3:1. Three sialic acid-containing oligosaccharide fragments were isolated from partial acid hydrolysates of the ganglioside by Dowex 1 X 8 (acetate form) column chromatography and preparative paper chromatography, and identified as Gal leads to NeuGc, Gal (1 leads to 4)[Gal(1 leads to 8)]NeuGc and Ara-(1 leads to 6)Gal(1 leads to 4)[Gal(1 leads to 8)]NeuGc. 3. The structure of Ganglioside 3 was postulated to be: Araf,p(1 leads to 6)Galpbeta(1 leads to 4)[Galpbeta(1 leads to 8)]NeuGc(2 leads to 3)Galpbeta(1 leads to 4)Glcpbeta(1 leads to 1)-ceramide. This is a unique structure with the sialic acid residue internally located in the sugar chain. 4. The ganglioside contained saturated 2-hydroxy fatty acids ranging in length from C16 to C24, among which C22, C23, and C24 acids were predominant. The long-chain bases consisted exclusively of C16, C17, and C18 phytosphingosines of iso and anteiso types.     

Gangliosides in Human Fetal Brain

The ganglioside concentration and composition were determined in 42 human fetal brains from gestational week 10 to 22, a period that is morphologically characterized by rapid neuroblast proliferation and migration. The ganglioside concentration was constant during this period, approximately 1 mumol of ganglioside sialic acid/g of fresh tissue weight. At gestational week 10 the ganglioside pattern was dominated by gangliosides of the ganglio b series, with the major ganglioside being GT1b, contributing 40% of total ganglioside sialic acid, whereas GD1b and GD3 contributed only 15 and 10%, respectively. The proportion of b series ganglioside decreased to gestational week 22, with the most pronounced relative reduction affecting GD3, but also GT1b and GD1b to a lesser extent. The ganglioside GQ1b increased in content from gestational week 10 and peaked around week 16. The proportion of GD1a increased markedly between gestational week 12 and 14 and slowly between week 14 and 18 and then increased rapidly from week 20. Ganglioside GM1 underwent a similar change. Gangliosides of the lacto series contributed 6-10% of ganglioside sialic acid between gestational week 10 and 15, and thereafter the proportion slowly decreased. 3'-isoLM1 decreased rapidly in content from gestational week 10 (20 nmol/g of fresh weight) to week 22 (less than 0.5 nmol/g of fresh weight), whereas the gangliosides of the neolacto series (3'-LM1 and 3',8'-LD1) showed a slower and less marked decline in level. The biological significance of the ganglioside changes is discussed.     

GANGLTOSIDES AND THE ARCHITECTURE OF HUMAN FRONTAL AND RAT SOMATOSENSORY ISOCORTEX

Ganglioside sialic acid was determined in the layers of human frontal association cortex and rat somatosensory cortex by microchemical methods of sampling and analysis. In both cortices the distribution per unit dry weight showed three main peaks or inflections: (1) at the junction of layers II and III; (2) in the lower part of layer III at the junction with IV; and (3) at the junction of layers V and VI. These distributions parallel the occurrence of high concentrations of dendritic and axonal plexuses and their synaptic articulations. In human cortex, the concentration of ganglioside sialic acid per unit dry weight was slightly greater, the amount per cell was twice as great. and the amount per neuron present was 2- to 6-fold greater than in rat somatosensory cortex. The ganglioside sialic acid per cell was 120- to 200-fold greater in human association cortex than in rat retina, which is a CNS region with sparse neuropil. The results support the validity of ganglioside sialic acid as an index of the relative mass of neuronal plasma membranes in neural tissues and its usefulness in chemoanatomic quantitation of axodendritic interrelationships established by synaptic contacts with local and distant neurons.     

Gangliosides in normal human serum. Concentration, pattern and transport by lipoproteins

The total content and pattern of gangliosides were determined in the unfractionated sera of 11 healthy human adults and in isolated lipoproteins. The total content of lipid-bound sialic acid was 10.5 +/- 3.2 nmol/ml serum. The ganglioside profile consisted of more than ten different components. The major ganglioside was GM3, followed by GD3, GD1a, GM2, GT1b, MG-3 (sialosyllactoneotetraosylceramide), GD1b and GQ1b. Traces of four additional gangliosides could not be quantified reliably. Ganglioside patterns did not vary in sera taken from healthy adults of different age and sex. Approximately 98% of human serum gangliosides were transported by serum lipoproteins, predominantly by LDL (66%), followed by HDL (25%) and VLDL (7%). The quantitative distribution of individual gangliosides in VLDL and LDL was almost the same as that in the unfractionated serum; some differences existed with the ganglioside profile in HDL.     

Subcellular distribution and biosynthesis of rat liver gangliosides

Gangliosides have generally been assumed to be localized primarily in the plasma membrane. Analysis of gangliosides from isolated subcellular membrane fractions of rat liver indicated that 76% of the total ganglioside sialic acid was present in the plasma membrane. Mitochondria and endoplasmic reticulum fractions, while containing only low levels of gangliosides on a protein basis, each contained approx. 10% of total ganglioside sialic acid. Gangliosides also were present in the Golgi apparatus and nuclear membrane fractions, and soluble gangliosides were in the supernatant. Individual gangliosides were non-homogeneously distributed and each membrane fraction was characterized by a unique ganglioside composition. Plasma membrane contained only 14 and 28% of the total GD1a and GD3, respectively, but 80-90% of the GM1, GD1b, GT1b and GQ1b. Endoplasmic reticulum, when corrected for plasma membrane contamination, contained only trace amounts of GM1, GD1b, GT1b and GQ1b, but 11 and 5% of the total GD1a and GD3, respectively. The ganglioside composition of highly purified endoplasmic reticulum was similar. Ganglioside biosynthetic enzymes were concentrated in the Golgi apparatus. However, low levels of these enzymes were present in the highly purified endoplasmic reticulum fractions. Pulse-chase experiments with [3H]galactose revealed that total gangliosides were labeled first in the Golgi apparatus, mitochondria and supernatant within 10 min. Labeled gangliosides were next observed at 30 min in the endoplasmic reticulum, plasma membrane and nuclear membrane fractions. Analysis of the individual gangliosides also revealed that GM3, GM1, GD1a and GD1b were labeled first in the Golgi apparatus at 10 min. These studies indicate that gangliosides synthesized in the Golgi apparatus may be transported not only to the plasma membrane, but to the endoplasmic reticulum and to other internal endomembranes as well.     

Substrate specificities of a bacterial sialidase and rat liver ganglioside GM3 sialyltransferase

Sialidases cleave off sialic acid residues from the oligosaccharide chain of gangliosides in their catabolic pathway while sialyltransferases transfer sialic acid to the growing oligosaccharide moiety in ganglioside biosynthesis. Ganglioside G_M3 is a common substrate for both types of enzymes, for sialidase acting on ganglioside G_M3 as well as for ganglioside G_D3 synthase. Therefore, it is possible that both enzymes recognize similar structural features of the sialic acid moiety of their common substrate, ganglioside G_M3. Based on this idea we used a variety of G_M3 derivatives as glycolipid substrates for a bacterial sialidase (Clostridium perfringens) and for G_D3 synthase (of rat liver Golgi vesicles). This study revealed that those G_M3 derivatives that were poorly degraded by sialidase also were hardly recognized by sialyltransferase (G_D3 synthase). This may indicate similarities in the substrate binding sites of these enzymes.     

Alkali-Labile, Sodium Borohydride-Reducible Ganglioside Sialic Acid Residues in Brain

Abstract: We have shown that ganglioside internal esters, reduced with sodium borohydride and hydrolyzed with mild acid, form nonulosamine and glycosan, whereas ester-free gangliosides yield only sialic acid when similarly treated. In an effort to demonstrate the occurrence of ganglioside internal esters in brain tissue, brain homogenates and brain ganglioside fractions were treated with NaB³H₄. The gangliosides were then hydrolyzed with mild acid and unlabeled carrier nonulosamine and its glycosan were added. The nonulosamine was purified to constant specific radioactivity. Homogenates and ganglioside fractions, initially treated with alkali and then similarly reduced and analyzed, provided control values. Ganglioside fractions directly reduced consistently gave nonulosamine with higher specific radioactivities than controls. A larger quantity of tissue was processed to allow the isolation of chemically measurable amounts of nonulosamine. The amount of nonulosamine formed by reduction of the crude ganglioside fraction was estimated by isotope dilution analysis. The quantity of nonulosamine formed from reduced untreated ganglioside fractions was about sevenfold that formed from alkali-treated fractions. These data provide evidence for the existence in brain tissue of ganglioside sialic acid residues in which the carboxyl group is bound in a structure that is alkali-labile and reducible with sodium borohydride.     

Characterization, distribution and biosynthesis of the major ganglioside of rat intestinal mucosa.

The major sialic acid containing glycolipid has been isolated from rat intestinal mucosa. Characterization of this ganglioside by thin layer and gas chromatographic analysis indicates that it is an hematoside (GM3) with the major portion of the sialic acid in the N-glycolyl form. The distribution of this ganglioside was determined in villus and crypt cells isolated from rat intestine. The hematoside content of crypt cells was found to be significantly decreased when compared to villus cells. CMP-sialic acid:lactosylceramide sialyltransferase, responsible for the sialylation of lactosylceramide, was measured in differentiated villus and undifferentiated crypt cells and found to be greatly reduced in the crypt cell fraction. The present study demonstrates that marked differences in ganglioside content and biosynthesis occur in contiguous populations of cells in varying states of differentiation when isolated from normal rat intestine.     

A NeuGc-containing trisialoganglioside of bovine brain.

A N-glycolyneuraminic acid containing trisialoganglioside was isolated from bovine brains ganglioside mixture using Q-Sepharose. Its chemical structure was characterized as IV3NeuAc, II3NeuAc-NeuGc, Gg4Cer by gas-liquid chromatography, a permethylation study, sialidase degradation, TLC/enzyme-immunostaining, fast atom bombardment-mass spectrometry, fluorometric HPLC and proton nuclear magnetic resonance spectroscopy. This was unique in the mixed sialic acid constituents. (formula; see text) This accounted for 0.78% of the gangliosides. The ceramide structure was almost identical with those of major bovine brain ganglioside, as mainly composed of 18:0 fatty acid (90.9%) and d20:0 sphingosine base.     

Gangliosides in subacute sclerosing leukoencephalitis: isolation and fatty acid composition of nine fractions

Gangliosides from brain of an 8 yr old boy with subacute sclerosing leukoencephalitis have been studied in terms of pattern and structure. Thin-layer chromatography showed that both gray and white matter have a highly abnormal pattern, with elevation of the relative proportion of four gangliosides corresponding to minor species in normal brain. The total level of lipid-bound sialic acid, however, was not increased, which indicated a compensating loss of other gangliosides. Two of the proliferating species were monosialogangliosides (G(5) and G(6)) (Korey nomenclature), and two were disialo types (G(2A) and G(3A)). Studies of their carbohydrate structures are described. Nine ganglioside fractions were isolated by preparative TLC in combination with column chromatography, and the fatty acid compositions were determined. Seven contained stearate as the major component, while two (G(3A) and G(6)) had relatively large proportions of oleate and palmitate. Five of the fractions contained two fatty acids of long chain-length and unknown structure.     

Expression of Mouse Sialic Acid on Gangliosides of a Human Glioma Grown as a Xenograft in SCID Mice

Ganglioside sialic acid content was examined in the U87-MG human glioma grown as cultured cells and as a xenograft in severe combined immunodeficiency (SCID) mice. The cultured cells and the xenograft possessed N-glycolylneuraminic acid (NeuGc)-containing gangliosides, despite the inability of human cells to synthesize NeuGc. Human cells express only N-acetylneuraminic acid (NeuAc)-containing gangliosides, whereas mouse cells express both NeuAc- and NeuGc-containing gangliosides. Small amounts of NeuGc ganglioside sialic acid (2-3% of total ganglioside sialic acid) were detected in the cultured cells, whereas large amounts (66% of total ganglioside sialic acid) were detected in the xenograft. The NeuGc in gangliosides of the cultured cells was derived from gangliosides in the fetal bovine serum of the culture medium, whereas that in the U87-MG xenograft was derived from gangliosides of the SCID host. The chromatographic distribution of U87-MG gangliosides differed markedly between the in vitro and in vivo growth environments. The neutral glycosphingolipids in the U87-MG cells consisted largely of glucosylceramide, galactosylceramide, and lactosylceramide, and their distribution also differed in the two growth environments. Asialo-GM1 (Gg4Cer) was not present in the cultured tumor cells but was expressed in the xenograft, suggesting an origin from infiltrating cells (macrophages) from the SCID host. The infiltration of mouse host cells and the expression of mouse sialic acid on human tumor cell glycoconjugates may alter the biochemical and immunogenic properties of xenografts.     

A trisialoganglioside containing a sialyl alpha 2-6 N-acetylgalactosamine residue is a cholinergic-specific antigen, Chol-1 alpha.

Cholinergic-specific antigens termed the Chol-1 family have been suggested to be of a ganglioside nature by Richardson et al. (J. Neurochem. 38, 1605-1614, 1982). Two molecular species of polysialogangliosides among bovine brain gangliosides were found to react with anti-Chol-1 alpha antiserum. One of them, Chol-1 alpha-a, was isolated and characterized as a trisialoganglioside containing the gangliotetraose backbone in which 1 mol of sialic acid was attached to each of the reducing end galactose, N-acetylgalactosamine and internal galactose residues, respectively. The chemical structure of Chol-1 alpha-a was determined for the first time, being as follows: IV3NeuAc III6NeuAc II3NeuAc-GgOse4 Cer.     

High-performance thin-layer chromatography and densitometric determination of brain ganglioside compositions of several species.

Improved resolution of complex brain ganglioside mixtures was achieved by high-performance thin-layer chromatography. The percentage distribution of individual gangliosides was then determined by direct densitometric seanning, employing a transmittance mode, of the resorcinol-positive spots on the plate. As little as 90 pmol (29 ng) of lipid-bound sialic acid could be detected with a good signal-to-noise ratio. A linear detector response was observed up to 3.0 μg of lipid-bound sialic acid. The brain white matter ganglioside patterns of eight animal species, including human, chimpanzee, monkey, chicken, bovine, sheep, and pig, were examined in detail. In addition, human brain gray matter, rat cerebral, rat brain gray matter, and rat cerebellar ganglioside patterns were also studied. Ganglioside G_M4 (G₇) was found to be one of the major components in primate and chicken brain white matter, but it represented only a minor ganglioside in other species. Other major gangliosides in all brain samples studied were G_M1, G_D1a, G_D1b, and G_T1b. G_M1 was more abundant in white matter than in gray matter. G_T1a, a recently discovered ganglioside species, was found in all species examined, but was most abundant in the rat cerebellum. The latter source also contained high proportions of G_T1b and G_Q1b.     

High-resolution proton NMR studies of gangliosides. III. Elucidation of the structure of ganglioside GM3 lactone

Ganglioside GM3 lactone (1) was prepared in 95% yield from the parent ganglioside by incubation at 25 degrees C for 4 days in glacial acetic acid. Inspection of the 500 MHz proton NMR spectra of 1 and its precursor in dimethylsulfoxide-d6-deuterium oxide at 30 degrees C revealed a large deshielding (+1.42 ppm) of the H-2 resonance of the galactosyl residue. This suggests that 1 must be the lactone formed by esterification of the sialic acid carboxyl group with the C-2 hydroxyl of the galactosyl residue. Consideration of all the NMR data leads to a specific structure proposal in which 1 has a highly rigid structure. Interesting features of the structure include a hydrophobic inner surface and a semicircular outer edge of seven-oxygen atoms, which may have physiological importance.     

Preparation of lyso-GM1 (II3Neu5AcGgOse4-long chain bases) by a one-pot reaction.

A simple procedure is described for preparing lyso-GM1, a GM1 derivative that lacks the fatty acid moiety, starting from GM1 ganglioside using a one-pot reaction. Ganglioside deacylation was carried out in KOH/propan-1-ol in the absence of oxygen. The yield of lyso-GM1 under optimal conditions (6 h, 90 degrees C, 0.2 N KOH, 1 mM GM1) was 54%. The chemical structure of lyso-GM1 was determined by 1H-NMR and FAB-MS analyses, thus proving that the acetamide groups of galactosamine and sialic acid units were not affected during the deacylation reaction.     

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.     

A model for ganglioside behaviour in cell membranes.

Gangliosides from beef brain have been spin-labeled using two different attaching groups and employed to investigate the physical nature of ganglioside behaviour in membranes. Results obtained using EPR spectroscopy indicate that, in phosphatidylcholine bilayers at physiological pH, ganglioside oligosaccharide chains are quite mobile and show a measurable tendency towards cooperative interaction amongst themselves. We suggest that the source of this interaction is the formation of H-bonds between sugar residues in adjacent ganglioside molecules. We present evidence that physiological (extracellular fluid) levels of Ca2+ and Mg2+ lead to cross-linking and condensing of ganglioside headgroups by complexing sialic acid carboxyl residues. Ganglioside headgroup interactions are not very sensitive to changes in the buffer ionic strength, suggesting that ionic interactions are of minor importance. We have found no measurable tendency for headgroup carbohydrate to penetrate hydrophobic regions of lipid bilayers. EPR spectroscopy was also used to follow the interaction of spin-labeled gangliosides with the glycoprotein, glycophorin, and with intact BHK cells. We suggest that these carbohydrate-based interactions should contribute significantly to the properties of the eucaryotic cell glycocalyx. We predict that laterally mobile carbohydrate-bearing components of cell surface will show a tendency to cluster about complex glycoprotein arrays, especially if the species involved bear accessible carboxylic acid functions.     

Characterization of two molecular species GD3 ganglioside from bovine buttermilk

Two gangliosides, representing 85% of total lipid-bound sialic acid, have been isolated from bovine buttermilk and characterized. Both contained long-chain base, glucose, galactose and sialic acid in the molar ratio 1:1:1:2, and gave, upon sialidase treatment, a neutral glycolipid, characterized as lactosylceramide. Partial acid hydrolysis, permethylation analysis and chromium trioxide oxidation indicated their basic oligosaccharide portion to be NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4Glc. The difference between the two forms was exclusively in the ceramide moiety of the molecule, one containing mainly long-chain (C22-C25) fatty acids and an equimolar proportion of C16 and C18 long-chain bases, and the other mainly palmitic acid and C18 long-chain base.     

Receptor ganglioside content of three hosts for Sendai virus. MDBK, HeLa, and MDCK cells

Specific gangliosides GD1a, GT1b and GQ1b isolated from brain have been shown to function as receptors for Sendai virus by conferring susceptibility to infection when they are incorporated into receptor-deficient cells (Markwell, M.A.K., Svennerholm, L. and Paulson, J.C. (1981) Proc. Natl. Acad. Sci. USA 78, 5406-5410). The endogenous gangliosides of three commonly used hosts for Sendai virus: MDBK, HeLa, and MDCK cells were analyzed to determine the amount and type of receptor gangliosides present. In all three cell lines, GM3 was the major ganglioside component. The presence of GM1, GD1a and the more complex homologs of the gangliotetraose series was also established. In cell lines derived from normal tissue, MDBK and MDCK cells, gangliosides contributed 47-65% of the total sialic acid. In HeLa cells, gangliosides contributed substantially less (17% of the total sialic acid). The ganglioside content of each cell line was shown not to be immutable but instead to depend on the state of differentiation, passage number, and surface the cells were grown on. Thus, the ganglioside concentration of undifferentiated MDCK cells was found to be substantially greater than that of MDBK or HeLa cells, but decreased as the MDCK cells underwent differentiation. Changes in culture conditions that were shown to decrease the receptor ganglioside content of the cells resulted in a corresponding decrease in susceptibility to infection. The endogenous oligosialogangliosides present in susceptible host cells were shown to function as receptors for Sendai virus.     

ON THE STRUCTURE OF TWO NEW GANGLIOSIDES FROM BEEF BRAIN

Abstract— Two new gangliosides were isolated in pure form from beef brain. They were provisionally named ganglioside G5a and G5b. Ganglioside GSa is a monosialoganglioside containing fucose. Its basic neutral glycosphingolipid core is the gangliotetraose ceramide: Gal (1 —> 3) GalNac (1—> 4) Gal (1 —> 4) Glc (1—>) ceramide, most likely with β-linkages. Fucose is linked to the 2-position of external galactose, N -acetylneuraminic acid to the 3- position of internal galactose. Ganglioside G5b is a mixture of at least two isomeric disialogangliosides containing N -acetylneuraminic acid and N -glycolylneura-minic acid. The major isomer has the following structure: NeuNac (α,2—>3) Gal (β,1—>3) GalNac (β, 1 —> 4) (NeuNglα, 2 —> 3) gal (β,1—>4) Glc (β,1 —>)-ceramide. The minor isomer contains N -acetylneur-aminic acid and N -glycolylneuraminic acid in an inverted linkage position.