Gangliosides with O-Acetylated Sialic Acids in Tumors of Neuroectodermal Origin

Gangliosides, carrying an O-acetylated sialic acid in their carbohydrate moiety, are often found in growing and developing tissues, especially of neuro-ectodermal origin. The most prominent one is 9-O-Ac-GD3, which is considered as an oncofetal marker in animal and human tumors like neuronal tumors, melanoma, basalioma or breast cancer, as well as in psoriatic lesions. Also other gangliosides like GD2 or GT3 were found to be O-acetylated in their terminal sialic acid. In this review we are summarising the occurrence of such gangliosides in normal and transformed tissues and delineate a more general theory that O-acetylated sialic acids in gangliosides are a universal marker for growing cells and tissues.     

Gangliosides in the human brain development and aging.

In this study, brain gangliosides in prenatal and postnatal human life were analyzed. Immunohistochemically, the presence of "c"-pathway of gangliosides (GQ1c) in embryonic brain was only recorded at 5 weeks of gestation. Biochemical results indicated a twofold increase in human cortex ganglioside concentration between 16 and 22 weeks of gestation. The increasing ganglioside concentration was based on an increasing GD1a ganglioside fraction in all regions analyzed except cerebellar cortex, which was characterized by increasing GT1b. In this developmental period, GD3 was found to be localized in the ventricular zone of the cortical wall. After birth, GD1b ganglioside in neuropil of granular cell layer corresponding to growing mossy fibers was expressed in cerebellar cortex. Between birth and 20/30 years of age, a cerebral neocortical difference of ganglioside composition was observed, characterized by lowest GD1a in visual cortex. Analyzing the composition of gangliosides in cortical regions during aging, they were observed to follow region-specific alterations. In frontal cortex, there was a greater decrease in GD1a and GM1 than in GT1b and GD1b, but in occipital (visual) cortex there was no change in individual gangliosides. In hippocampus, GD1a moderately decreased, whereas other fractions were stable. In cerebellar cortex, GD1b and GT1b fractions decreased with aging.     

Differential distribution of major gangliosides in rat central nervous system detected by specific monoclonal antibodies

We investigated the localization of major gangliosides in adultrat brain by an immunofluorescence technique with mouse monoclonalantibodies (MAbs). Five MAbs (GMB16, GMR17, GGR12, GMR5 andGMR13) that specifically recognize gangliosides GM1, GD1a, GD1b,GT1b and GQ1b, respectively, were used. We have found that thereis a cell type-specific expression of the ganglioside in therat central nervous system. In cerebellar cortex, GM1 was expressedin myelin and some glial cells. GD1a was detected exclusivelyin the molecular layer. GD1b and GQ1b were present restrictedlyon the granular layer; GD1b was detected on the surface of thegranular cell bodies, whereas GQ1b was present in the cerebellarglomerulus. GT1b was distributed intensely in both the molecularlayer and the granular layer. In cerebral cortex, GM1 was detectedin some glial cells. Dense staining was limited to the whitematter. GD1a was distributed in layers I, II/III and Va, andthe upper part of layer VI, whereas GQ1b was localized in layersIV and Vb, and the lower part of layer VI. GD1b was detectedbeneath layer III. GT1b appeared to be distributed throughoutall layers. In other regions, such as hippocampal formationand spinal cord, the expression of the ganglioside was alsohighly localized to a specific cell type and layer. ganglioside monoclonal antibody rat brain     

GANGLIOSIDE1−COMPOSITION OF BRAIN IN TAY-SACHS DISEASE: INCREASED AMOUNTS OF GD2 AND N-ACETYL-β-D-GALACTOSAMINYL GD1a GANGLIOSIDE

Abstract— The ganglioside composition of the brain of a patient with Tay-Sachs disease (TS-brain) was determined by a newly developed ganglioside-mapping procedure and compared with that of an age-matched control brain. GM2 ganglioside was the predominant component in TS-brain and the following gangliosides were also found, GM1, GD1a, GD1b and GT1 (major gangliosides in normal brain), and GM3, GD3, GD2 and GD1a-GAN (minor or undetectable components of normal brain). Individual gangliosides were isolated by column chromatography using a combination of DEAE-Sepharose, Iatrobeads and Silica Gel 60 and their structures were confirmed by comparing them with authentic standards using TLC, analysing their carbohydrate compositions by gas-liquid chromatography and cleaving them sequentially with glycosidases. The amounts of individual components were measured by quantitative densitometric scanning of the thin-layer plates. As a reflection of myelin breakdown, no sialosylgalactosyl ceramide was detectable in TS-brain. Although the total amounts of all gangliosides except GM2 in TS-brain were low, there were normal molar ratios of the main gangliosides in normal brain, that is, GM1, GD1a, GD1b and GT1. In comparison with the amount of GDla ganglioside, the amounts of GM2, GD2 and GD1a-GAN, which contain N-acetylgalactosamine as a terminal carbohydrate residue, were all elevated in TS-brain. The long chain bases of individual gangliosides contained both C-18 and C-20 sphingosine in different ratios and the ratio of C-20 to C-18 increased in the gangliosides in the order: GM2 < GM1 < GD1a < GD1a-GAN < GD1b < GT1 in both normal brain and TS-brain. In contrast, GD2 and GD3 gangliosides consisted mainly of C-18 sphingosine. The C-20 to C-18 ratios of individual gangliosides in the TS-brain were lower than those of age-matched control brain. Hexosaminidase from Turbo cornutus showed the same specific activity and K_m value in catalysing the cleavage of terminal N-acetylgalactosaminyl residues from GM2, GD2 and GD1a-GAN, suggesting that the brain gangliosides that increase in Tay-Sachs disease may be cleaved by the same enzyme.     

Human brain gangliosides in development, aging and disease.

In this study, brain gangliosides in prenatal and postnatal human life and Alzheimer's disease were analyzed. Immunohistochemically, the presence of the "c"-series of gangliosides (GQ1c) was only registered in the embryonic brain at 5 weeks of gestation. Biochemical results indicated a two-fold increase in ganglioside concentration in the human cortex between 16 and 22 weeks of gestation. The increasing ganglioside concentration was based on an increasing GD1a ganglioside fraction in all regions analyzed except in the cerebellar cortex, which was characterized by increasing GT1b. During prenatal human development, regional differences in ganglioside composition could only be detected between the cerebrum ("a"-pathway) and the cerebellum ("b"-pathway). Between birth and 20-30 years of age, a cerebral neocortical difference of ganglioside composition occurred, characterized by the lowest GD1a in visual cortex. Analyzing the composition of gangliosides in cortical regions during aging, they were observed to follow region-specific alterations. In the frontal cortex, there was a greater decrease in GD1a and GM1 than in GT1b and GD1b, but in the occipital (visual) cortex there was no change in individual gangliosides. In hippocampus, GD1a moderately decreased, whereas other fractions were stable. In the cerebellar cortex, GD1b and GT1b fractions decreased with aging. In Alzheimer's disease, we found all ganglio-series gangliosides (GM1, GD1a, GD1b, GT1b) to be decreased in regions (temporal and frontal cortex and nucleus basalis of Meynert) involved in pathogenesis of disease. In addition, in Alzheimer's disease we found simple gangliosides (GN2, GM3) to be elevated in the frontal and parietal cortex, which might correlate accelerated lysosomal degradation of gangliosides and/or astrogliosis occurring during neuronal death.     

Involvement of 9-O-Acetyl GD3 Ganglioside in Mycobacterium leprae Infection of Schwann Cells

Mycobacterium leprae (ML), the etiologic agent of leprosy, mainly affects the skin and peripheral nerves, leading to demyelization and loss of axonal conductance. Schwann cells (SCs) are the main cell population infected by ML in the nerves, and infection triggers changes in the SC phenotype from a myelinated to a nonmyelinated state. In the present study, we show that expression of 9-O-acetyl GD3, a ganglioside involved in cellular anti-apoptotic signaling and nerve regeneration, increases in SCs following infection with ML. Observation by confocal microscopy together with coimmunoprecipitation suggested that this ganglioside participates in ML attachment and internalization by SC. Immunoblockage of 9-O-acetyl GD3 in vitro significantly reduced adhesion of ML to SC surfaces. Finally, we show that activation of the MAPK (ERK 1/2) pathway and SC proliferation, two known effects of ML on SCs that result in demyelization, are significantly reduced when the 9-O-acetyl GD3 ganglioside is immunoblocked. Taken together, these data suggest the involvement of 9-O-acetyl GD3 in ML infection on SCs.     

Gangliosides in various brain areas of three inbred strains of mice

The ganglioside patterns of cerebellum, cortex, pons-medulla, hypothalamus, hippocampus and caudate nucleus of three inbred strains of mice (C57BL/6J, DBA/2J and BALB/cJ) have been analysed. All brain areas contained both the simple and complex species of gangliosides. GD1a was the major ganglioside in cortex, hippocampus and caudate nucleus whereas GT1b was the major species in cerebellum, hypothalamus and pons-medulla. In hippocampus, the percentages of GT1b and GD1a were quite similar. Pons and medulla exhibited the highest levels of GM1 (which approaches the value of GT1b) and the lowest values of GD1a. A ganglioside, termed here GT1L, was located between GD1b and GT1b. This ganglioside, which was present in highest amounts in cerebellum disappeared after alkali treatment. Highly significant differences were observed in the amounts and patterns of gangliosides among brain areas of the three strains. Highly significant differences (p<0.001) were also found in the ganglioside distribution of various brain areas among the strains, especially for tri-and tetrasialogangliosides between Balb and DBA. A significant difference of GM1 was observed in the cerebellum when comparing DBA with the two other strains. It is likely that the differences might be related to their relative abundances in certain cell types and for defining synaptic circuits in brain areas of some strains.     

Gangliosides of rat eye lens: a severe reduction in the content of C-series gangliosides following streptozotocin treatment

Gangliosides of eye lenses from normal and experimentally induced diabetic rats were investigated by methods including glycolipid-overlay techniques. Adult rat eye lens showed a complex ganglioside pattern that consisted of six major ganglioside components. These gangliosides were identified as GM3, GD3, GD1a, GD1b, GT1b, and GQ1b based upon their reactivity to anti-GM1 antibody after in situ sialidase treatment and mobility on thin-layer chromatography (TLC). Gangliosides in eye lens were further characterized by TLC-immunostaining with A2B5, a specific monoclonal antibody directed toward c-series gangliosides. Eye lens contained GT3 as the main c-series ganglioside component. Unexpectedly, the relative concentration of GT3 in total gangliosides of eye lens was highest among neural and extra-neural tissues examined. Administration of streptozotocin to rats caused a severe reduction in the GT3 content in eye lenses as early as day 3 without apparent changes in the composition of major gangliosides. Alloxan failed to produce such an effect despite producing similar hyperglycemic conditions. These results suggest that rat eye lens probably contains a streptozotocin-susceptible cell type(s), which is highly enriched with c-series gangliosides.     

A monoclonal antibody reacting specifically with ganglioside GD1b in human brain

A mouse monoclonal antibody directed against one of the major human brain gangliosides, GD1b, has been produced. The antibody binds specifically to the carbohydrate structure of GD1b as it does not react with structurally related gangliosides like GM1, GD2, GT1b or Fuc-GM1, or any other ganglioside of human brain. The results further indicate that terminal galactose as well as the disialosyl group linked to the inner galactose moiety are involved in binding to the antibody.     

Specific Synthesis of Neurostatin and Gangliosides O-Acetylated in the Outer Sialic Acids Using a Sialate Transferase

Gangliosides are sialic acid containing glycosphingolipids, commonly found on the outer leaflet of the plasma membrane. O-acetylation of sialic acid hydroxyl groups is one of the most common modifications in gangliosides. Studies on the biological activity of O-acetylated gangliosides have been limited by their scarcity in nature. This comparatively small change in ganglioside structure causes major changes in their physiological properties. When the ganglioside GD1b was O-acetylated in the outer sialic acid, it became the potent inhibitor of astroblast and astrocytoma proliferation called Neurostatin. Although various chemical and enzymatic methods to O-acetylate commercial gangliosides have been described, O-acetylation was nonspecific and produced many side-products that reduced the yield. An enzyme with O-acetyltransferase activity (SOAT) has been previously cloned from the bacteria Campylobacter jejuni. This enzyme catalyzed the acetylation of oligosaccharide-bound sialic acid, with high specificity for terminal alpha-2,8-linked residues. Using this enzyme and commercial gangliosides as starting material, we have specifically O-acetylated the gangliosides’ outer sialic acids, to produce the corresponding gangliosides specifically O-acetylated in the sialic acid bound in alpha-2,3 and alpha-2,8 residues. We demonstrate here that O-acetylation occurred specifically in the C-9 position of the sialic acid. In summary, we present a new method of specific O-acetylation of ganglioside sialic acids that permits the large scale preparation of these modified glycosphingolipids, facilitating both, the study of their mechanism of antitumoral action and their use as therapeutic drugs for treating glioblastoma multiform (GBM) patients.     

Inhibitory effects of gangliosides on immune reactions of antibodies to neutral glycolipids in liposomes

Specific immune damage to liposomes containing Forssman or globoside glycolipid was inhibited when the liposomes also contained ganglioside. The activity of a human monoclonal Waldenstr?m macroglobulin antibody to Forssman glycolipid was inhibited by each of three gangliosides tested, GM3, GD1a and GD1b. Inhibition of the monoclonal antibody was dependent on the amount of ganglioside in the liposomes, and was diminished by reducing the relative amount of ganglioside. Inhibition also correlated positively with the number of ganglioside sialic acid groups, with inhibition by GT1b greater than GD1a greater than GM3. Naturally occurring human antibodies to globoside glycolipid were detected in 18% (9 out of 50) of normal human sera tested. Immune damage to liposomes induced by each of the three highest-reacting human anti-globoside sera was blocked by liposomal GM3. We conclude that gangliosides can strongly influence immune damage to membranes induced by antibody interactions with adjacent neutral glycolipids.     

Gangliosides in acetylcholine receptor-rich membranes fromTorpedo marmorata andDiscopyge tschudii

The ganglioside composition of membranes enriched in nicotinic acetylcholine receptor (AChR) from the electric raysDiscopyge tschudii andTorpedo marmorata has been determined, and compared to that of total electric organ. A ganglioside having the chromatographic mobility of GM2 constitutes the major ganglioside (60%) in totalD. tschudii electric organ, followed by a component with the mobility of GD3 (10%), and a component running just below GD1a (about 12%). Minor constituents running as GM3 (2%) and as polysialogangliosides (comprising 8–15%) were also observed. Purified native membranes ofD. tschudii andT. marmorata displayed a similar profile, except that they were richer in a GM1-like component, and the proportion of GM2-like gangliosides was lower than that in total electric organ. Using a¹²⁵I-cholera toxin overlay assay on neuraminidase-treated high-performance thin layer chromatograms, the presence of GM1, GD1a and trace amounts of GD1b and GT1 (or GQ) were detected inD. Tschudii total membranes. Immunocytochemical trechniques showed the co-localization of gangliosides GQ1c/GT1c/GP1c, recognized by the monoclonal antibody Q211, and the AChR at the ventral, innervated face of the electrocyte.     

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.     

Modification of sialic acids by 9-O-acetylation is detected in human leucocytes using the lectin property of influenza C virus

Influenza C virus spike glycoprotein HEF specifically recognizesglycoconjugates containing 9-O-acetyl-N-acetylneuraminic acid.The same protein also contains an esterase activity. Takingadvantage of these two properties, influenza C virus was usedas a very sensitive probe for the detection of traces of 9-O-acetyl-N-acetylneuraminicacid in human leucocytes. The binding of influenza C virus toleucocyte glycoproteins and gangliosides separated by sodiumdodecyl sulphate–polyacrylamide gel electrophoresis andthin-layer chromatography, respectively, was assayed using achromogenic esterase substrate. In this way, glycoproteins ofB-lymphocytes and T-lymphocytes were found to contain 9-O-acetylatedsialic acids. Of the various 9-O-acetylated gangliosides detected,one had the characteristics of 9-O-acetylated G^D3. The identificationof 9-O-acetylated sialic acids on distinct glycoproteins andglycolipids should be helpful in assigning a physiological roleto this sugar. O-acetylation gangliosides influenza C virus lymphocytes sialic acids     

Identification of Disialosyl Paragloboside and O-Acetyldisialosyl Paragloboside in Cerebellum and Embryonic Cerebrum

Abstract: The lacto series of glycolipids are only minor constituents in mammalian CNS and are found mostly during development. Expression of a significant amount (70 μg of neuraminic acid/g dry weight) of disialosyl-lacto- N -neotetraosylceramide (LD1) in adult mouse cerebellum is reported for the first time in the nervous system. The structure of this ganglioside was determined by hydrolysis with various glycosidases, immunochemical tests, sugar and fatty acid analyses after permethylation and capillary GLC-mass spectrometry, sugar linkage analysis of permethylated alditol acetates, and fast-atom bombardment-mass spectrometry of the native ganglioside. The structure of LD1 was determined to be NeuAc-NeuAc α 2-3Gal β 1-4GlcNAc β 1-3Gal β 1-4Glc β 1-1-ceramide. The major fatty acid was 18:0, and the long-chain base was C₁₈-sphingenine. Mouse cerebellum also contained O -acetyl-LD1 and several other O -acetylated gangliosides as recognized by monoclonal antibodies ME311 and 3G5. The levels of LD1 and O -acetyl-LD1 in cerebellum increased during postnatal development. During development of the Purkinje cell degeneration mutant, pcd/pcd , the levels of both of these gangliosides in the cerebellum declined with the loss of Purkinje cells, a finding indicating that these gangliosides are primarily associated with Purkinje cells. In the cortex, LD1, O -acetyl-LD1, and O -acetyl GD3, like GD3, are developmentally regulated antigens and are only expressed in the fetal cortex and not to any significant extent in the adult.     

Activities of Five Different Sialyltransferases in Fish and Rat Brains

Abstract: To investigate the role of Sialyltransferases in the metabolism of brain gangliosides, we examined activities of five different Sialyltransferases (GM3-, GD3-, GT3-, GD1a-, and GT1a-synthase) using total membrane preparations from cichlid fish and Sprague-Dawley rat brains, and analyzed the relationship between the enzyme activities and the ganglloside compositions. The patterns of sialyltransferase activities in fish and rat brains differed from each other. In fish brain, the GM3-synthase activity was lower than GD3-synthase activity, whereas the opposite relationship was observed in rat brain. The GT3-synthase reaction with fish brain membranes produced radiolabeled GM3, GD3, and a ganglioside that was identified as GT3 based on mobility on TLC using two different solvent systems. No GT3-synthase activity was detected in rat brain. The GD1a-and GT1a-synthase activities in fish brain were higher than those in rat brain. Although GT1a was a single radiolabeled ganglioside in fish GT1a-synthase reaction, this ganglioside could not be detected in rat brain. The ratios of GM3-, GD3-, GT3-, GD1a-, and GT1a-synthase activities in fish and rat brain were 23:31:4:28:14 and 61:21:0:18:0, respectively. Ganglioside analysis showed that fish brain was enriched with c-series gangliosides including GT3 and polysialo-species, whereas a-and b-se-ries gangliosides were major components in rat brain. These results suggest that the species-specific expression of gangliosides in brain tissues may be regulated, at least in part, at the level of sialyltransferase activities.     

Densitometric quantification of brain gangliosides separated by two-dimensional thin layer chromatography

A procedure for accurate densitometric quantification of gangliosides separated by two-dimensional thin layer chromatography is reported. The procedure was set up employing 9 different pure gangliosides and was applied to the analysis of calf and pig brain gangliosides. Silica gel high performance thin layer plates, 10 × 10 cm. were two-dimensionally developed at 18–20 C with the following solvents: chloroform methanol 0.2% aqueous CaCl₂, 50/40/10 by volume, for the first run; n-propanol 17 M NH₄OH/water, 6/2/1 by volume for the second run. Ganglioside spots were visualized by spraying with an Ehrlich reagent, which is specific for sialic acid, and heating at 120 C for 15 min. The spots were quantified by sequential scanning densitometry, linear responses being obtained for ganglioside amounts on the plate ranging from 0.1 to 6 nmol as bound sialic acid. The reproducibility of densitometric responses resulted to be acceptable since the standard deviation values were lower than ± 15% of the mean values also for those ganglioside species contained in minor proportions. The ganglioside mixtures of calf and pig brain were resolved in about 20 spots. Of these 9 corresponded to gangliosides GM3, GM2, GM1, Fuc-GM1, GD1a, GD1b, Fuc-GD1b, GT1b and GQ1b, which were identified with certainty and quantified. The identification of GM3 (carrying N-glycolylneuraminic acid), GD3, GD1a (carrying N-acetyl- and N-glycolyl-neuraminic acid) and GT1a was only tentative. All the other spots corresponded to unidentified gangliosides, some of them possibly new species.     

Immunoglobulin G-class mouse monoclonal antibodies to major brain gangliosides

Mice genetically engineered to lack complex gangliosides are improved hosts for raising antibodies against those gangliosides. We report the generation and characterization of nine immunoglobulin G (IgG)-class monoclonal antibodies (mAbs) raised against the four major brain gangliosides in mammals. These include (designated as ganglioside specificity-IgG subclass) two anti-GM1 mAbs (GM1-1, GM1-2b), three anti-GD1a mAbs (GD1a-1, GD1a-2a, GD1a-2b), one anti-GD1b mAb (GD1b-1), and three anti-GT1b mAbs (GT1b-1, GT1b-2a, GT1b-2b). Each mAb demonstrated high specificity, with little or no cross-reactivity with other major brain gangliosides. Enzyme-linked immunosorbent assay (ELISA) screening against 14 closely related synthetic and purified gangliosides confirmed the high specificity, with no significant cross-reactivity except that of the anti-GD1a mAbs for the closely related minor ganglioside GT1a alpha. All of the mAbs were useful for ELISA, TLC immunooverlay, and immunocytochemistry. Neural cells from wild-type rats and mice were immunostained to differing levels with the anti-ganglioside antibodies, whereas neural cells from mice engineered to lack complex gangliosides (lacking the ganglioside-specific biosynthetic enzyme UDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase) remained unstained, demonstrating that most of the mAbs react only with gangliosides and not with related structures on glycoproteins. These mAbs may provide useful tools for delineation of the expression and function of the major brain gangliosides and for probing the pathology of anti-ganglioside autoimmune diseases.     

Characterization of Sialyltransferase-IV Activity and Its Involvement in the c-Pathway of Brain Ganglioside Metabolism

Abstract: To characterize the sialyltransferase-IV activity in brain tissues, the activities of GM1b-, GD1a-, GT1b-, and GQ1c-synthases in adult cichlid fish and rat brains were examined using GA1, GM1, GD1b, or a cod brain ganglioside mixture as the substrate. The GD1a-synthase activity in the total membrane fraction from cichlid fish brain required divalent cations such as Mg²⁺ or Mn²⁺ and Triton CF-54 for its full activity. The V_max value was 1,340 pmol/mg of protein/h at an optimal pH of 6.5, whereas the apparent K_m values for CMP-sialic acid and GM1 were 172 and 78 µM, respectively. Cichlid fish and rat brains also contained GM1b-, GT1b-, and GQ1c-synthase activities. The ratio of GM1b-, GD1a-, and GT1b-synthase activities in fish brain was 1.00:0.89:1.13, respectively, and in rat brain 1.00:0.60:0.63. Incubation of fish brain membranes with a cod brain ganglioside mixture, which contains GT1c, and [³H]CMP-sialic acid produced radiolabeled GQ1c. It is interesting that the adult rat brain also contains an appreciable level of GQ1c-synthase activity despite its very low concentrations of c-series gangliosides. The GD1a- or GQ1c-synthase activity in fish and rat brain was inhibited specifically by coincubation with the glycolipids that serve as the substrates for other sialyltransferase-IV reactions. Thus, the GD1a-synthase activity was inhibited by GA1 and GD1b, but not by LacCer, GM3, or GD3. In a similar manner, the synthesis of GQ1c was suppressed by GA1, GM1, and GD1b, but not by LacCer, GM3, or GD3. The GD1a-synthase activity directed toward endogenous GM1 was inhibited by GA1 or GT1b, whereas the endogenous GT1b-synthase activity was suppressed by GA1 or GM1. GA1, GM1, and GD1b did not affect the endogenous GM3- and GD3-synthase activities. These results clearly demonstrate that sialyltransferase-IV in brain tissues catalyzes the reaction for GQ1c synthesis in the c-pathway as well as the corresponding steps in the asialo-, a-, and b-pathway in ganglioside biosynthesis.     

Age-related changes in GM1, GD1a, GT1b components of gangliosides in Wistar albino rats

In this study, age-related changes of GM1, GD1a, GT1b fractions of gangliosides were investigated in whole brain of male Wistar albino rats. Insignificant increases were detected in GM1 values from the third to the 24th month, whereas GD1a and GT1b concentrations of ganglioside in 24-month-old rats decreased significantly as compared to 6-month-old rats. Although there were no significant differences in the GD1a/GT1b ratio of any groups, GM1/GD1a and GM1/GT1b ratios were significantly increased as compared to 6-month-old rats. The increase in the ratios of gangliosides are not due to an increase of GM1 fractions; they result from a decrease of GD1a and GT1b fractions of gangliosides. In conclusion, the concentration of ganglioside decreased with ageing.