CYTOSOLIC GANGLIOSIDES: OCCURRENCE IN CALF BRAIN AS GANGLIOSIDE-PROTEIN COMPLEXES

—Calf brain was treated in order to prepare separately the cytosol from neuronal bodies and glial cells, and the cytosol from nerve endings. The first cytosol contained 29 μg of ganglioside bound sialic acid/g fresh tissue, the latter 3.1 μg. Upon addition of ammonium sulphate until saturation the gangliosides contained in the two cytosols precipitated and were totally recovered in the pellet. while, under the same conditions, pure gangliosides were completely soluble. After stepwise ammonium sulphate fractionation all the different fractions obtained contained gangliosides and carried an approximately constant ganglioside/protein ratio. Thus cytosolic gangliosides occur in calf brain as ganglioside-protein complexes. The qualitative and quantitative pattern of gangliosides appeared to be similar in the two cytosols and in the different ammonium sulphate fractions obtained from the same cytosols. In addition, the pattern of cytosolic gangliosides was similar to that of membrane bound gangliosides.     

GANGLIOSIDE COMPOSITION AND CONTENT OF RAT-BRAIN SUBCELLULAR FRACTIONS

Abstract— The composition and content of gangliosides from rat-brain microsomal, synaptosomal, mitochondrial and myelin fractions were studied. Outer membranes of synaptosomes were also isolated, separated into subfractions and investigated. Of all the fractions studied the outer membranes of synaptosomes are richest in gangliosides, in one of their sub-fractions the concentration of gangliosides per mg of protein is five times higher than in the homogenate. Microsomes are rich in gangliosides as well, but to a lesser degree, whereas the mitochondrial fraction contains considerably smaller amounts of gangliosides per mg of protein than does the homogenate. The ganglioside pattern of outer membranes of synaptosomes and of their subfractions is somewhat different from that of the homogenate; the outer membranes contain approximately one-third less monosialogangliosides. On the contrary a very high content of monosialogangliosides is characteristic of the ganglioside pattern of the myelin fraction. In this fraction monosialoganglioside G_MI (nomenclature of Svennerholm, 1963) constitutes 60–63 per cent of ganglioside sialic acid, or 75–80 molar per cent of gangliosides, the content of di- and trisialogangliosides being much lower than in other fractions. Fatty acid and long chain base composition of gangliosides from synaptosomal and microsomal fractions and homogenate is very similar, almost identical. In gangliosides from myelin fractions the relaitve content of palmitic and monoenoic acids is higher and that of arachinic acid and C₂₀-sphingosine—lower than in other fractions studied. The difference in ganglioside composition of synaptosomes and their outer membranes and on the other hand of myelin appears to reflect the difference in ganglioside composition of neuronal and oligodendroglial plasma membranes.     

An exposed carboxyl group on sialic acid is essential for gangliosides to inhibit calcium uptake via the sarco/endoplasmic reticulum Ca2+-ATPase: relevance to gangliosidoses

We previously observed that gangliosides GM2, GM1, and GM3 inhibit Ca²⁺-uptake via the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) in neurons and in brain microsomes. We now systematically examine the effect of various gangliosides and their analogs on Ca²⁺-uptake via SERCA and demonstrate that an exposed carboxyl group on the ganglioside sialic acid residue is required for inhibition. Thus, asialo-GM2 and asialo-GM1 have no inhibitory effect, and modifications of the carboxyl group of GM1 and GM2 into a hydroxymethyl residue (CH₂OH), a methyl ester (COOCH₃) or a taurine-conjugated amide (CONHCH₂CH₂SO₃H) drastically diminish their inhibitory activities. We also demonstrate that the saccharides must be attached to a ceramide backbone in order to inhibit SERCA as the ceramide-free ganglioside saccharides only inhibit SERCA to a minimal extent. Finally, we attempted to use the ceramide-free ganglioside saccharides to antagonize the effects of the gangliosides on SERCA; although some reversal was observed, the inhibitory effects of the gangliosides were not completely abolished.     

Myelin Gangliosides in Vertebrates

Abstract: A phylogenetic survey of brain myelin ganglioside patterns and concentrations has been carried out on 16 vertebrate species. Gangliosides were isolated from purified myelin and found to vary in concentration from 25 μg of sialic acid per 100 mg of myeh for goldfish to a value of 395 for turkey. The latter species had approximately equivalent amounts of G_M1 and G_M4 as the two major gangliosides. The 11 mammals studied all had G_M1 as the major ganglioside, with variable amounts of G_M4; rhesus monkey and human had 20-25% G_M4, whereas the others had less than 10%. Amphibia and fish myelin contained the least total ganglioside, with patterns that showed relatively little G_M1 and no detectable G_M4. Alligator myelin was unique in having a total concentration as high as the avian species, but a pattern with predominantly diand trisialo gangliosides.     

Developmental Changes in the Biosynthesis of Sialoglycoprotein Substrates for Intrinsic Synaptic Sialidase

Abstract: N′-Acetyl-d -[6-³H]mannosamine was administered to 13- and 28-day-old rats by intraventricular injection. At various time intervals following the injection, synaptic membranes were prepared and the incorporation of radiolabel into sialic acid residues released from endogenous glycoproteins and gangliosides by intrinsic sialidase determined. Radiolabel was incorporated into synaptic membrane gangliosides and glycoproteins, and at all times tested, >90% of the label was associated with sialic acid. Sialic acid released from endogenous glycoproteins by intrinsic sialidase present in 28-day membranes incorporated only 20–25% as much radiolabel per nmole as sialic acid released by mild acid hydrolysis or by exogenous neuraminidase. In contrast, sialic acid released from glycoproteins present in 13-day-old membranes by intrinsic sialidase, mild acid hydrolysis, or exogenous neuraminidase all were similarly labelled. At both ages the specific radioactivity (cpm/nmol) of sialic acid released from gangliosides by the intrinsic enzyme was similar to the total ganglioside sialic acid released by mild acid hydrolysis. The results identify glycoprotein substrates for intrinsic synaptic membrane sialidase as a distinct metabolic class in the mature brain and suggest the occurrence of a developmentally related change in the metabolism of these glycoproteins.     

Determination of N-acetyl- and N-glycolylneuraminic acids in gangliosides by combination of neuraminidase hydrolysis and fluorometric high-performance liquid chromatography using a GM3 derivative as an internal standard

A highly sensitive method for quantification of sialic acids in gangliosides was developed. The sialic acids, released by hydrolysis of gangliosides, were converted to fluorescent derivatives with 1,2-diamino-4,5-(methylenedioxy)benzene (DMB) and separated on a reversed-phase C18 column with an isocratic elution. As little as 0.1-1.0 nmol of sialic acid in ganglioside was quantified. The use of acetate buffer instead of water in the mobile phase could prevent damage on the column and reduce background peaks derived from the reagents. When gangliosides were subjected to acid hydrolysis, the velocity of hydrolysis varied depending on their structures and a part of the sialic acid liberated decomposed with prolonged heating time. Therefore gangliosides were hydrolyzed by Arthrobacter ureafaciens neuraminidase in the presence of sodium cholate after addition of an internal standard. For the internal standard, GM3 with N-propionylneuraminic acid (GM3(NeuPr)) was synthesized from GM3(NeuAc) by N-deacylation followed by N-propionylation. Folch partition was used to decrease lipophilic materials included in the sample, and the sialic acids released were recovered from the upper phase. The present method has a satisfactory sensitivity in the simultaneous quantification of NeuAc and NeuGc in purified gangliosides as well as in crude lipid fractions containing a variety of gangliosides.     

Isolation and partial characterization of human erythrocyte membrane NADH: (Acceptor) oxidoreductase

Gengliosides generally provide a small portion of the complex carbohydrate content of cell surfaces. An exception is the central nervous system where they comprise up to 5–10% of the total lipid of some membranes. This tissue is unique in that the quantity of lipid-bound sialic acid exceeds that of the protein-bound fraction. Over 30 different molecular species have been characterized to date. These range in complexity from sialosylgalactosyl ceramide with 2 sugars to the pentasialoganglioside of fish brain with 9 carbohydrate units. Virtually all cellular and subcellular fractions of brain that have been carefully examined contain gangliosides to one degree or another, but the majority of brain ganglioside is located in the neurons. Their mode of distribution within the neuron has not been entirely clarified by subcellular studies. Calculations based on reported values for axon terminal density and synaptosomal ganglioside concentration in the rat reveal that nerve endings contribute less than 12% of total cerebral cortical ganglioside. It is concluded that the plasma membranes of neuronal processes contain most of the neuronal ganglioside. These and other considerations suggest the possibility that gangliosides may be distributed over the entire neuronal surface.     

GANGLIOSIDES OF HUMAN MYELIN: SIALOSYLGALACTOSYLCERAMIDE (G7) AS A MAJOR COMPONENT

Abstract— Gangliosides were isolated from purified human myelin in a yield of 62 μg of lipid-bound sialic acid per 100 mg of dry myelin. Sialosylgalactosyl ceramide (G₇) was found to be a major component of the ganglioside fraction, amounting to 15 per cent of the total sialic acid. It accounted for 10 per cent of lipid-bound sialic acid in adult human white matter, making it the third most abundant ganglioside on a molar basis. These results were obtained with an improved method for isolating total gangliosides in high yield, by employing DEAE-Sephadex column chromatography. Myelin from other mammalian species had considerably less G₇, and there were also indications of maturational changes. Both 2-hydroxy and unsubstituted fatty acids were components of the ceramide unit, in a ratio of 3:2, respectively. The overall fatty acid pattern was very similar to that for myelin cerebroside and sulphatide. Long-chain bases included only C₁₈ species, with sphingosine predominating (>90 per cent). These observations suggest a metabolic relationship between G₇ and either cerebroside or sulphatide.     

THE ACTION OF CALF BRAIN SIALIDASE ON GANGLIOSIDES, SIALOGLYCOPROTEINS AND SIALOGLYCOPEPTIDES

Abstract— In agreement with other investigators it has been shown that endogenous as well as added gangliosides are a substrate for brain sialidase. The release of sialic acid was enhanced in the presence of Triton X-100; this might be due to the action of the detergent on the ganglioside micelles. The sialic acid release from endogenous gangliosides was observed over 48 h and compared with the effect of the sialidase on the endogenous glycoproteins. Though the hydrolysis of sialic acid from gangliosides is much faster in the first hours, after 48 h 40 per cent of the total bound sialic was released from both substrates at pH 4.0 and 37°C.
Sialoglycopeptides obtained from brain glycoproteins are also metabolized by the sialidase. No effect of Triton X-100 on this substrate has been observed. From sialoglycopeptides, fractions can be obtained by DEAE-Sephadex A-50 column chromatography with a sialic acid content from 8 to 26 per cent. The fractions with a high sialic acid content were about equally active towards brain sialidase as gangliosides. The results agree with the similar turnover rate observed for the carbohydrate chains from gangliosides and glycoproteins, but are in contrast to the observations of other investigators who have stated that glycoproteins are a poor substrate for brain sialidase. In our experiments bovine and ovine submaxillary mucins and sialyl-lactoses showed only slight activity compared to gangliosides and selected brain sialoglycopeptides.     

Myelin Gangliosides in Developing Rats: The Influence of Maternal Ethanol Consumption

Abstract: The present study examined myelin gangliosides in the developing offspring of rats that were pair-fed control or ethanol liquid diets prior to and during gestation. Between 17 and 31 days of age, we observed an increase in the proportion of G_M1 in myelin (from 15% to 38% of ganglioside sialic acid) and a decrease in the proportion of G_T1b (from 26% to 4%). G_M4 was detected at all ages examined. Between 17 and 31 days of age, there was an increase in the proportion of N -acetylman-nosamine-derived radioactivity associated with G_M1 (from 16% to 22%) and G_M4 (from 5% to 13%), and a decrease in that associated with G_T1b (from 24% to 4%). Small, but sygnificant (p < 0.05), developmentally related differences were found in G_D2 and G_D3. Detection of G_M4 in myelin of young rats in the present study appears to depend on the use of nonpartitioning methods of ganglioside extraction. Although the distribution of myelin gangliosides and radioactivity was near-normal in ethanol-treated pups, there was a consistent decrease in the proportion and radioactivity associated with the major myelin ganglioside, G_M1.     

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.     

DEVELOPMENTAL PATTERNS OF GANGLIOSIDES AND OF PHOSPHOLIPIDS IN CHICK RETINA AND BRAIN

Abstract— The changes in phospholipids and gangliosides during ontogenesis of chick retina have been compared with those in brain. Three phases of accumulation of ganglioside NeuNAc in the retina were detected. In contrast, brain NeuNAc rapidly increased during embryonic life until hatching, followed by a slower increase up to the adult stage. The phospholipid changes in retina and in brain occur in a-similar manner to the variations observed for gangliosides, however in retina the changes of phospholipid content are less marked than in brain, during embryonic life. There were marked changes in the retina and brain ganglioside patterns with age. G _{d 3} and G _{d 1b} decreased rapidly in per cent; correspondingly, G _{d 1a} increased during embryonic life and became the major ganglioside in place of G _{d 3}. There was a similarity between ganglioside patterns of chick retina and brain. Except for some slight variations during embryonic life, the retinal phospholipid pattern did not change noticeably.     

Modification of sialic acid carboxyl group of ganglioside

A simple and quantitative method for the modification of sialic acid carboxyl group in ganglioside is described. Methyl iodide was added to the ganglioside in dimethylsulfoxide. The reaction was completed quickly at room temperature, giving the methyl ester with practically no by-products. Reduction of the methyl ester was achieved by sodium borohydride. These modified gangliosides were chemically characterized. Reduced GM1 has a strong antigenicity compared with the original GM1, and it raised high titer antisera which did not crossreact with the original GM1 nor with its methyl ester. Peanut agglutinin, which binds strongly to asialo GM1 but weakly to GM1, bound to the methyl ester of GM1 and reduced GM1. Cholera toxin, which is specific for GM1, also reacted with the methyl ester of GM1 and reduced GM1 to a certain extent.     

Characterization of nuclear gangliosides in rat brain: concentration, composition, and developmental changes

Nuclear gangliosides were characterized using two distinct fractions of large (N1) and small (N2) nuclear populations from rat brain. The ganglioside concentration of N1 nuclei from adult rat brain was 0.92 microg sialic acid/mg protein, which was about 3.8 times higher than that of N2 nuclei. N1 and N2 nuclear gangliosides showed similar compositional profiles; they contained major gangliosides of GM1, GD1a, GD1b, and GT1b, with GM3 in lesser amounts. c-Series gangliosides such as GT3, GQ1c, and GP1c were also detected in both nuclear preparations. Nuclear localization of gangliosides was confirmed by immunofluorescence with anti-GM1 antibody, cholera toxin B subunit, and c-series ganglioside-specific monoclonal antibody A2B5. Developmental changes of nuclear gangliosides were examined using rats of different ages ranging from embryonic day 14 (E14) to postnatal 7 weeks. The concentration of N1 nuclear gangliosides changed only slightly during development and did not correlate with that of whole-brain gangliosides. The developmental pattern of ganglioside composition of N1 nuclei was also distinguished from that of microsomal membranes; the ganglioside changes in N1 nuclei included reduced expression of di- and polysialogangliosides at E16 and higher proportions of GM3 at early and late stages of the period. These findings suggest that gangliosides in nuclear membranes are developmentally regulated in a distinct manner in brain cells.     

Developmental profiles of gangliosides in trisomy 19 mice

The ganglioside composition of the cerebrum, cerebellum, brainstem, liver, heart, and spleen was analyzed quantitatively in trisomy 19 (Ts19) mice aged 4 to 12 days postpartum. The developmental profiles of cerebral gangliosides were similar in Ts19 mice and control littermates: Total ganglioside-sialic acid as well as the proportions of the individual gangliosides GD1a and GM1 increased with age, while the percentages of GQ1b and GT1b decreased during development. Both the accretion of the total ganglioside content and the development of the individual ganglioside fractions were delayed by 2-3 days in the Ts19 telencephalon. Likewise, the shift from the b- to the a-pathway of ganglioside synthesis was retarded. Ganglioside development was equally delayed in the cerebellum and the brainstem of Ts19 mice. Since in Ts19 mice, morphogenesis of several brain regions is similarly delayed by 2 days, these results confirm the usefulness of gangliosides as biochemical markers for brain maturation. In contrast to brain gangliosides, the ganglioside composition of the Ts19 livers was clearly distinguished from that of control livers. Total ganglioside-bound sialic acid was increased by 35-50% in Ts19 livers. This elevation in ganglioside content not explicable by a simple delay in development was mainly due to an increase in GD3 and fraction 2, which is likely to contain GD1a and GD1b. In contrast, GM2 which increased considerably with age in control mice persisted on a low level in Ts19 livers. Comparable alterations of the ganglioside pattern were neither observed in the spleen nor in the heart of Ts19 mice. The data presented give additional evidence that ganglioside synthesis in the liver is under a different regulation mechanism than that in the brain, heart, and spleen.     

Determination of brain gangliosides by determination of ganglioside stearic acid

A new method is described for the determination of brain gangliosides by measuring stearic acid, the chief acid of gangliosides, in an appropriately purified brain extract. The method includes extraction of tissue with chloroform-methanol, extraction of gangliosides from the extract with 0.1 M KCl, evaporation of the aqueous phase, methanolysis, and gas-liquid chromatography of the resultant methyl esters with a double internal standard. The method depends on the simple composition of ganglioside fatty acids (80% stearic acid) and allows determination of less than 0.05 micromole of gangliosides. Interfering lipids are removed from the ganglioside extract by washing with chloroform-methanol-water. The effects of contamination with nonlipid N-acetylneuraminic acid are avoided.     

Phorbol ester-associated changes in ganglioside metabolism

The effect of phorbol esters on ganglioside metabolism in contact-inhibited Chinese hamster V79 cells was examined. Three phorbol esters of varying structure and tumor-promoting activity were used. Treatment of cells with tumor-promoting phorbol esters resulted in accumulation of gangliosides and increased incorporation of [1-¹⁴C]palmitate and [9-³H]sialic acid into gangliosides. Moreover, the phorbol esters were found to increase the activity of CMP-sialic acid: lactosylceramide sialyltransferase, the enzyme catalysing the first step in ganglioside biosynthesis. The magnitude of phorbol ester effects on V79 cell ganglioside metabolism correlated with the in vivo phorbol ester tumor-promoting activity.     

GANGLIOSIDE ABNORMALITIES IN MULTIPLE SCLEROSIS

Abstract— Gangliosides were isolated from plaque tissue and normal appearing white matter of multiple sclerosis (MS) brain. All four plaques showed decreased ganglioside concn relative to normal human white matter on a wet wt basis, but significant elevation in terms of dry wt. The wet wt and dry wt concn of MS white matter gangliosides showed smaller but statistically significant decreases below normal. Thin-layer patterns of the plaques showed several departures from normal white matter, including decrease of G₄ and G₅, and complete loss of G₇ (sialosylgalactosylceramide). Most of the plaques had significant elevation of G_2A and G_3A along with increases of the slower-migrating polysialogangliosides. An additional ganglioside was present between G₂ and G_2A which was not seen in normal white matter. The TLC pattern of MS white matter gangliosides was essentially normal. The evidence for a general decrease of acidic lipids within normal appearing white matter is discussed.     

Density-Dependent Changes in Gangliosides and Sialidase Activity of Murine Neuroblastoma Cells

Abstract: Density-dependent changes in ganglioside composition, Vibrio cholerae neuraminidase (VCN)-susceptible sialyl residues, and membrane- associated sialidase activity were determined for the cholinergic murine neuroblastoma cell line S20Y. A decrease in total ganglioside sialic acid and VCN-releasable sialic acid was observed with increasing cell density. G^M3 was the major ganglioside component of preconfluent S20Y cells, whereas G_DIA was predominant in postconfluent cells. Sialidase activity increased in confluent and postconfluent cells and may account for the reduction in total ganglioside sialic acid observed with increasing cell density. In contrast, while adrenergic N115 cells showed a decrease in VCN-susceptible sialic acid residues with increasing cell density, there was no significant change in ganglioside composition or ganglioside sialic acid levels.     

The effect of cold stress on ganglioside fatty acid composition and ganglioside-bound sialic acid content of rat brain subcellular fractions

The effect of cold stress on the ganglioside fatty acid composition and sialic acid content of brain subcellular fractions and homogenate of rats was studied, the animals were kept in a cold room with 12h light-dark cycles at 3 and 10 degrees C for 2 weeks. (1) The rat brain homogenate, synaptosomes and myelin of rats exposed to 3 degrees C contained significantly higher amounts of ganglioside-bound sialic acid per mg of protein than these fractions of control rats kept at 23 degrees C; the differences were less pronounced in rats exposed to 10 degrees C. (2) A small, but significant, diminution of relative palmitic acid content and an increase of stearic acid content was found to take place in gangliosides from rat brain synaptosomes, synaptosomal plasma membranes and homogenate as a result of the exposure of animals to 3 degrees C and to a lesser extent to 10 degrees C. (3) The content of unsaturated fatty acids in gangliosides from brain subcellular fractions was approximately the same in cold exposed and control rats.