The content and composition of gangliosides in brain subcellular fractions of the frog Rana temporaria

Ganglioside distribution in various frog brain subcellular fractions (myelin, microsomes, mitochondria, synaptosomes, plasma membranes of nerve endings and synaptic vesicles) was investigated. The synaptosomes and plasma membranes of nerve endings were found to be the main places of ganglioside localization, ganglioside concentration being 2.42 and 1.79 times higher than that in homogenates. Gangliosides were shown to be present in synaptic vesicles. The characteristic features of gangliosides from frog brain and its subcellular fractions are the predominance of polysialogangliosides with 3-5 sialic acid residues (up to 57.4%), low content of monosialogangliosides (not more than 7%) and the presence of disialogangliosides with short carbohydrate chain. The increase of ganglioside content per one nerve cell during phylogenetic development of vertebrates is discussed.     

Separation of brain monosialoganglioside molecular species by high-performance liquid chromatography

A method for the separation of molecular species of brain monosialogangliosides by high-performance liquid chromatography is described. GM4, GM3, GM2, and GM1 were purified from human brain and their individual molecular species were separated on a C18 reversed-phase column. Peaks were identified by mass spectrometry of the intact ganglioside, by gas-liquid chromatography of the fatty acids, and by high-performance liquid chromatography of the long chain bases. A characteristic elution sequence of molecular species permitted their identification based upon their retention times on the reversed-phase column.     

Absence of ganglioside GM1 in astroglial cells from 21-day old rat brain: Immunohistochemical,histochemical, and biochemical studies

A procedure was developed for the cultivation of cells derived from the cerebral hemispheres of the 21-day old rat. Approximately 98 percent of the cells in a 10 day culture are astrocytes that contain glial fibrillary acidic protein. Analysis of the extracted gangliosides by thin layer chromatography revealed that ganglioside GM1 was absent and that the predominant ganglioside was GM3. Very small amounts of the polysialogangliosides GD1a, GD1b, and GT1b were detected. The concentration of gangliosidic NeuNAc per mg protein in these astrocytes was only 3 percent that observed in the 5 day culture of a mixed cell preparation from newborn rat brain. Immunohistochemical and histochemical studies were performed on the mixed cell population of the minced tissue of 21-day old rat brain prior to cultivation. Astrocytes did not stain for hyaluronectin. These cells also did not provide a positive staining reaction for ganglioside GM1 utilizing the antiganglioside GM1 peroxidase-antiperoxidase procedure and the biotinylated choleragen-avidin-peroxidase procedure. These two histochemical methods for ganglioside GM1 also did not stain astrocytes that had been cultured for 5 days. Oligodendroglial cells, which were also present in the uncultured 21-day-old minced brain tissue, stained positively for ganglioside GM1 and hyaluronectin. Hyaluronectin had previously been shown to be a marker for oligodendroglia. Oligodendroglial cells which were present in the 5 day cultures of 21-day old brain tissue also provided a positive reaction for ganglioside GM1. It is concluded that ganglioside GM1 is absent in astroglia. The presence of small amounts of polysialogangliosides in the "pure" astrocyte preparation is discussed.     

GANGLIOSIDES IN DEVELOPING MOUSE BRAIN MYELIN

Abstract— Gangliosides were isolated from myelin prepared from mouse brains of different ages (23 to 490 days). Quantitative estimation of lipid-bound sialic acid levels indicated a gradual increase from 560 μg/g of myelin at 23 days to about 1200 μg/g of myelin at older ages. The major ganglioside in all myelin preparations was the monosialoganglioside G₄ (G_M1). However, considerable amounts of di- and trisialo species also were found in myelin from young animals. In contrast to human myelin in which the monosialoganglioside, sialosylgalactosylceramide (G₇) was highly enriched (L edeen et al. , 1973), a much smaller enrichment of this ganglioside was noticed in mouse brain myelin. Ganglioside G₇ was not detectable in myelin until the animals were 35 days old, and showed a slight increase with increasing age after that. The results strongly indicated that the concentration of G₇ in myelin is species specific and age dependent. The study also demonstrated that the ganglioside accretion in developing mouse brain myelin was attributable to the enrichment of monosialogangliosides G₄ (G_M1), G₅ (G_M2) and G₇ at the expense of polysialogangliosides.     

Ganglioside pattern and thermal tolerance of fish species

The brain ganglioside pattern of taxonomically close and distant related fish species living at different temperatures and the mammalian pattern were compared. The results demonstrate correlations between body temperature and brain ganglioside pattern: the higher the body temperature of animals, the lower is the relative proportion of polysialogangliosides in their brain.     

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.     

Preparation of GM1 ganglioside with sialidase-producing marine bacteria as a microbial biocatalyst.

This paper describes the preparation of monosialoganglioside GM1 with sialidase-producing marine bacteria as a microbial biocatalyst. A new sialidase-producing bacterium, identified tentatively as Pseudomonas sp. strain YF-2, was isolated from seawater by enrichment culture with ganglioside as the sole source of carbon. When YF-2 was cultured in a synthetic medium containing crude bovine brain gangliosides at 25 degrees C for 3 days, 80 to 90% of the gangliosides were converted to GM1. GM1 was then purified from the supernatant of YF-2 culture by C18 reverse-phased chromatography, followed by DEAE-Sephadex A25 anion-exchange chromatography. In a typical experiment, 178 mg of highly purified GM1 was obtained from 500 mg of the crude ganglioside fraction. The GM1 induced neurite outgrowth of neuroblastoma Neuro2a cells at a concentration of 33 to 100 microM in the presence of fetal calf serum. Sialidase was purified 33-fold with 13.3% recovery from the culture supernatant of YF-2. The purified enzyme hydrolyzed polysialogangliosides to produce GM1 but did not act on GM1. It was therefore concluded that polysialogangliosides in the culture of strain YF-2 were converted to GM1 by this sialidase.     

Brain ganglioside patterns of vertebrates

Abstract— The ganglioside content in brains of representatives of six vertebrate classes (lamprey, ray, sheat-fish, carp, frog, triton, tortoise, hen, pigeon, rabbit, rat and monkey) was determined. In most cases a correlation was found between the level of nervous organization and the ganglioside content of brain. In fish and amphibian brain ganglioside concentration is half to one third that in mammalian brain. Ganglioside composition of higher vertebrate brains (mammals, birds and reptiles) has many similar features. Four main gangliosides with 1-3 NANA residues in their molecules–G₁ * * Nomenclature of Korey and Gonatas (1963 ): G₁ trisialyl-hexosaminyl-trihexosyl-ceramide; G₂ and G₃, disialyl-hexosaminyl-trihexosyl-ceramides; G₄ monosialyl-hexosaminyl-trihexosyl-ceramide.
, G₂, G₃ and G₄–constitute 80-90 per cent of total ganglioside NANA. Fractions G_2a ? ? G_o, tetrasialyl-hexosaminyl-trihexosyl-ceramide; G_2a disialyl-hexosaminyl-dihexosyl-ceramide; G₅, monosialyl-hexosaminyl-dihexosyl-ceramide.
G_o and G₅ are present in much lesser amounts. Species peculiarities in distribution of NANA among different fractions were noted. The brain gangliosides of lower vertebrates–fish and amphibia–are unusual in having a high proportion of polysialogangliosides, containing 4 and 5 NANA residues, and a lower content of monosialogangliosides. In ray brain a considerable part of gangliosides has a reduced carbohydrate chain.     

Involvement of brain gangliosides in temperature adaptation of fish

The ganglioside pattern of goldfish brain was investigated after adaptation (acclimatization, acclimation) to different temperatures. Adaptation at lower ambient temperature causes a higher proportion of polysialogangliosides to be formed in fish brain.     

Rat brain microsomal gangliosides. Accessibility to a neuraminidase preparation and the possible existence of different pools in relation to their biosynthesis

1. Treatment of rat brain microsomal membranes with a neuraminidase preparation from Clostridium perfringens resulted in an almost complete conversion of polysialogangliosides into monosialogangliosides. 2. Neuraminidase treatment of the membranes did not increase the incorporation of N-[(3)H]acetylneuraminic acid from CMP-N-[(3)H]acetylneuraminic acid into the gangliosidic fraction, indicating that a monosialoganglioside is an acceptor of N-acetylneuraminic acid in these membranes only if, in addition to having the right chemical structure, it is in a proper position, probably in relation to the endogenous sialyltransferases. 3. These experiments also indicated that no independent turnover of the neuraminidase-labile N-acetylneuraminyl groups of gangliosides occurred in vitro. 4. N-[(3)H]Acetylneuraminic acid from endogenous polysialogangliosides labelled in vitro was released by neuraminidase at a slower rate than N-acetylneuraminic acid from unlabelled gangliosides of the same membranes. From this it was concluded that recently synthesized polysialogangliosides (completed in vitro) are in the membranes in a position less accessible to neuraminidase than are those synthesized earlier which were present in the membranes at the start of the labelling experiment.     

Intraventricular Sialidase Administration Enhances GM1 Ganglioside Expression and Is Partially Neuroprotective in a Mouse Model of Parkinson’s Disease

Background

Preclinical and clinical studies have previously shown that systemic administration of GM1 ganglioside has neuroprotective and neurorestorative properties in Parkinson’s disease (PD) models and in PD patients. However, the clinical development of GM1 for PD has been hampered by its animal origin (GM1 used in previous studies was extracted from bovine brains), limited bioavailability, and limited blood brain barrier penetrance following systemic administration.

Objective

To assess an alternative therapeutic approach to systemic administration of brain-derived GM1 to enhance GM1 levels in the brain via enzymatic conversion of polysialogangliosides into GM1 and to assess the neuroprotective potential of this approach.

Methods

We used sialidase from Vibrio cholerae (VCS) to convert GD1a, GD1b and GT1b gangliosides to GM1. VCS was infused by osmotic minipump into the dorsal third ventricle in mice over a 4-week period. After the first week of infusion, animals received MPTP injections (20 mg/kg, s.c., twice daily, 4 hours apart, for 5 consecutive days) and were euthanized 2 weeks after the last injection.

Results

VCS infusion resulted in the expected change in ganglioside expression with a significant increase in GM1 levels. VCS-treated animals showed significant sparing of striatal dopamine (DA) levels and substantia nigra DA neurons following MPTP administration, with the extent of sparing of DA neurons similar to that achieved with systemic GM1 administration.

Conclusion

The results suggest that enzymatic conversion of polysialogangliosides to GM1 may be a viable treatment strategy for increasing GM1 levels in the brain and exerting a neuroprotective effect on the damaged nigrostriatal DA system.     

Lipid content of brain, brain membrane lipid domains, and neurons from acid sphingomyelinase deficient mice

The cholesterol, sphingolipid, and glycerophospholipid content of total brain, of detergent-resistant membranes prepared from the total brain, and of cerebellar granule cells differentiated in culture from wild type (WT) and acid sphingomyelinase knockout (ASMKO) were studied. Brains derived from 7-month-old ASMKO animals showed a fivefold higher level of sphingomyelin and a significant increase in ganglioside content, mainly because of monosialogangliosides GM3 and GM2 accumulation, while the cholesterol and glycerophospholipid content was unchanged with respect to WT animals. An increase in sphingomyelin, but not in gangliosides, was also detected in cultured cerebellar granule neurons from ASMKO mice, indicating that ganglioside accumulation is not a direct consequence of the enzyme defect. When a detergent-resistant membrane fraction was prepared from ASMKO brains, we observed that a higher detergent-to-protein ratio was needed than in WT animals. This likely reflects a reduced fluidity in restricted membrane areas because of a higher enrichment in sphingolipids in the case of ASMKO brain.     

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.     

The lipid composition of axons from bovine brain

Abstract— Bovine axons derived from myelinated CNS axons were found to have 13.5% lipid. This lipid was composed of 20.1% cholesterol, 20.1% galactolipid, 14.6% ethanolarhine phosphatides (56.4% in the plasmalogen form), 18.3% choline phosphatides (10.0% in the plasmalogen form), 9.3% sphingomyelin, 5.6% phosphatidyl serine and 3.4% phosphatidyl inositol. The bovine axons had 0.33 μg ganglioside NeuNAc/mg dry weight. The axon gangliosides were found to contain the four major types of bovine gangliosides, as well as gangliosides G_M2 and G_D3. The latter two amounted to 20.9 and 15.8 mole per cent respectively, of total gangliosides. On a molar basis, about one half of the gangliosides were monosialogangliosides, with a decreased contribution by gangliosides G_T1 and GD_1b relative to ganglioside distributions which have been reported for most other CNS components. The relationship of the bovine axonal lipid composition to bovine white matter and its constituents, as well as to other CNS and PNS axonal preparations is discussed.     

Efficient conversion from polysialogangliosides to monosialotetrahexosylganglioside using Oerskovia xanthineolytica YZ-2

A new sialidase-producing strain isolated from soil was identified as Oerskovia xanthineolytica YZ-2. Sialidase was produced when Oerskovia xanthineolytica YZ-2 was exposed to polysialogangliosides. The sialidase of Oerskovia xanthineolytica YZ-2 hydrolyzed sialic acid linkages in polysialogangliosides, and released monosialotetrahexosylganglioside (GM1). The sialidase had the capability of product specificity because it did not attack the sialic acid linkage in GM1. Therefore, Oerskovia xanthineolytica YZ-2 was used for GM1 production from polysialogangliosides. In flasks cultivation phase, it was proved that Oerskovia xanthineolytica YZ-2 could convert polysialogangliosides to GM1 efficiently. Scaling-up the bioprocess with 8% crude ganglioside, polysialogangliosides was converted to GM1 by Oerskovia xanthineolytica YZ-2 in 30 L bioreactor after 18 h. The relative content of GM1 increased from 16.3% in crude ganglioside to 83.7% after Oerskovia xanthineolytica YZ-2 conversion. Therefore, a simple, large-scale conversion process for GM1 production from polysialogangliosides was achieved using Oerskovia xanthineolytica YZ-2 as a biocatalyst.     

Large scale purification of gangliosides GM3(Neu5Ac) and GM3(Neu5Gc) by trimethylaminoethyl-Fractogel high-performance liquid chromatography

A preparative anion-exchange high-performance liquid chromatographic method for the separation of the closely allied monosialogangliosides G_M3(Neu5Ac) and G_M3(Neu5Gc) has been developed. Hybridoma cells, readily available material derived from industrial monoclonal antibody production, were used as ganglioside source and led to fractions with pure G_M3(Neu5Ac) and G_M3(Neu5Gc) in high milligram quantities. The crude ganglioside extract was loaded onto columns filled with the strong anion-exchanger trimethylaminoethyl (TMAE)-Fractogel. Gangliosides were eluted from the stationary phase with a gradient system of ammonium acetate in methanol. The scaled-up approach ranged over more than one order of magnitude from 20 to 500 mg batches of G_M3 gangliosides. Thus, the high-resolution power of the strong anion-exchanger TMAE-Fractogel allowed the preparative isolation by one-step column chromatography of two G_M3 specimens which only differ in one hydroxyl group at position 5 of the neuraminic acid (N-acetyl- versus N-glycolylneuraminic acid).     

Calmodulin, a ganglioside-binding protein. Binding of gangliosides to calmodulin in the presence of calcium.

Ca(2+)-dependent ganglioside-binding protein was isolated from a soluble cytosol fraction of mouse brains using a ganglioside affinity column prepared with a mixture of bovine brain gangliosides. It was identified as calmodulin based on the following features identical with those of calmodulin: molecular weight, pI, chromatographic profile and amino acid sequences of lysyl-endopeptidase digests, and ability to activate cyclic nucleotide phosphodiesterase. Bovine brain calmodulin derivatized with 5-dimethylaminonaphthalene-1-sulfonyl (dansyl-calmodulin), tetramethylrhodamine isothiocyanate, or biotin was also shown to bind to the ganglioside affinity column Ca2+ dependently and elute with gangliosides GD1a, GD1b, GT1b, GQ1b, GM1, and GM2, melittin, and trifluoperazine but not with GgOse4Cer and oligosaccharides of GM1, GD1a, and GT1b. Modification of the Lys94 residue of calmodulin by biotinylation drastically reduced the capacity for ganglioside binding. Ganglioside GD1b caused a blue shift and increase in intensity of the fluorescence emission spectrum of dansyl-calmodulin in the presence of Ca2+. The increment in fluorescence was proportional to the amount of GD1b added and was maximal at the molar ratio of GD1b to calmodulin, approximately 7.8. Gangliosides are thus shown to specifically bind to calmodulin, and this binding may be a general mechanism for regulating calmodulin-dependent enzymes with consequent cellular response, such as cell differentiation.     

Gangliosides of Active and Inactive Neuroblastoma Clones

It is possible to divide neuroblastoma cells into clones able to synthesize neurotransmitters (active clones) or not (inactive clones).
The analysis of gangliosides of active and inactive clones shows that their total lipid sialic acids is markedly lower than that of neuron-enriched fractions prepared from brain. The ganglioside pattern of the cultured cells also differs notably from those obtained with neuronal fractions from brain. The absence of tri- and tetrasialogangliosides and the presence of appreciable amounts of the simplest monosialogangliosides are particularly noticeable in the neuroblastoma. Morphological differentiation obtained by serum deprivation, dibutyryl cyclic AMP or bromodeoxyuridine does not restore a true neuronal pattern. Gangliosides could not therefore be used as a marker of neuronal differentiation in this type of cell. No correlations can be found between the ganglioside pattern and the ability of cells to synthesize neurotransmitters.     

Gangliosides of active and inactive neuroblastoma clones.

It is possible to divide neuroblastoma cells into clones able to synthesize neurotransmitters (active clones) or not (inactive clones). The analysis of gangliosides of active and inactive clones shows that their total lipid sialic acids is markedly lower than that of neuron-enriched fractions prepared from brain. The ganglioside pattern of the cultured cells also differs notably from those obtained with neuronal fractions from brain. The absence of tri- and tetrasialogangliosides and the presence of appreciable amounts of the simplest monosialogangliosides are particularly noticeable in the neuroblastoma. Morphological differentiation obtained by serum deprivation, dibutyryl cyclic AMP or bromodeoxyuridine does not restore a true neuronal pattern. Gangliosides could not therefore be used as a marker of neuronal differentiation in this type of cell. No correlations can be found between the ganglioside pattern and the ability of cells to synthesize neurotransmitters.