C-series Gangliosides Research Articles

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 Distinct Ganglioside Composition of Rat Pancreatic Islets

Gangliosides in rat pancreatic tissue and isolated pancreatic islets were examined by methods including glycolipid-overlay techniques. The content of gangliosides in isolated pancreatic islets was approximately 6-fold higher than that in pancreatic tissue when compared on a protein basis. While N-glycolylneuraminic acid amounted to 7.2% of total lipid-bound sialic acids of pancreatic tissue, this molecular species was not detected in that of pancreatic islets. A remarkable difference in ganglioside composition was observed between pancreatic tissue and pancreatic islets. Pancreatic tissue showed a complex ganglioside pattern with GM3 as the largest ganglioside component, whereas isolated pancreatic islets had a simpler ganglioside profile without detectable amounts of GM3 and some other components. Pancreatic gangliosides were further examined by thin-layer chromatographic immunostaining with a monoclonal antibody A2B5 that reacts specifically with c-series gangliosides. Pancreatic tissue and pancreatic islets showed almost identical ganglioside patterns consisting of GT3, GT2, GQ1c, and GP1c. The concentration of c-series gangliosides in pancreatic islets was calculated to be more than 250-fold higher than that of pancreatic tissue. These results shows that pancreatic islet cells have a distinct ganglioside composition in rat pancreas.

Tissue-specific expression of c-series gangliosides in the extraneural system

c-Series gangliosides in extraneural tissues from young and adult rats were examined using thin-layer chromatographic (TLC) immunostaining with a specific monoclonal antibody A2B5. The composition of c-series gangliosides significantly differed among tissues. In adult rats, while liver tissue contained GT1c, GQ1c, and GP1c, renal tissue had GT3 as the major c-series ganglioside with GT2 in a lesser amount. Pancreatic tissue expressed c-series gangliosides that consisted of GT3, GT2, GQ1c, and GP1c. In other tissues including adrenal, thyroid, and eye lens, GT3 constituted the main c-series ganglioside species. While total ganglioside contents of extraneural tissues were much lower than that of brain tissue, the proportions of c-series gangliosides to total gangliosides were higher in many extraneural tissues. Interestingly, eye lens had the highest GT3 content among rat tissues examined. The compositions and concentrations of c-series gangliosides in liver and kidney significantly differed between 5-day-old and 7-week-old rats, suggesting the development-dependent expression of c-series gangliosides in these tissues. These results suggest that the expression of c-series gangliosides in extraneural tissues is regulated in a tissue-specific manner.

Expression of gangliosides in neuronal development of P19 embryonal carcinoma stem cells.

Gangliosides are constituents of the cell membrane and are known to have important functions in neuronal differentiation. We employed an embryonal carcinoma stem cell line P19 as an in vitro model to investigate the expression of gangliosides during neuronal development. After treatment with retinoic acid, these cells differentiate synchronously into neuron-like cells by a series of well-defined events of development. We examined several aspects of ganglioside metabolism, including the changes of ganglioside pattern, the activities and gene expression of several enzymes at different stages of differentiation, and the distribution of gangliosides in differentiating neurons. Undifferentiated P19 cells express mainly GM3 and GD3. After P19 cells were committed to differentiation, the synthesis of complex gangliosides was elevated more than 20-fold, coinciding with the stage of neurite outgrowth. During the maturation of differentiated cells, the expression of c-series gangliosides was downregulated concomitantly with upregulation of the expression of a- and b-series gangliosides. We also examined the distribution of gangliosides in differentiating neurons by confocal and transmission electron microscopy after cholera toxin B subunit and sialidase treatment. Confocal microscopic studies showed that gangliosides were distributed on the growth cones and exhibited a punctate localization on neurites and soma. Electron microscopic studies indicated that they also are enriched on the plasma membranes of neurites and the filopodia as well as on the lamellipodia of growth cones during the early stage of neurite outgrowth. Our data demonstrate that the expression of gangliosides in P19 cells during RA-induced neuronal differentiation resembles that of the in vivo development of the vertebrate brain, and hence validates it as an in vitro model for investigating the function of gangliosides in neuronal development.

Variation of the ganglioside compositions of human milk, cow's milk and infant formulas.

The ganglioside compositions of human milk, cow's milk and infant formulas were compared. The results showed that there was a drastic change in the ganglioside composition from the colostrum to later human milk, and that both the patterns and contents of gangliosides in human milk, cow's milk and infant formulas differed markedly. In human milk, the total lipid-bound sialic acid level was two times higher than those in cow's milk and infant formulas. The major ganglioside in the later human milk, GM3 (27.7%), was only a minor component in the colostrum, cow's milk and infant formulas (3.3, 2.8 and 0.4-2.6%, respectively). GD3 represented 49.0, 61.0 and 72.4-86.6%, respectively, of the colostrum, cow's milk and infant formulas, compared to 31.8% of the later human milk gangliosides. Another four gangliosides, which were assumed to be c-series gangliosides, were detected in the colostrum and the later human milk. They represented 33-38% of total lipid-bound sialic acid, and were tentatively designated as GX1, GX2, GX3 and GX4, respectively. However, only GX1 and GX2 were observed in cow's milk and infant formulas. The variation of the gangliosides in human and cow's milk, and infant formulas might have some biological significance regarding neonatal brain development, allergies, infant growth and non-immunoglobulin prophylactic activities against some bacterial toxins.

Expression of c-series gangliosides in rat hepatocytes and liver tissues

C-series gangliosides in rat hepatocytes and liver tissues were analyzed by thin-layer chromatographic (TLC) immunostaining with the specific monoclonal antibody A2B5. Primary cultures of hepatocytes isolated from adult rats were immunostained positively by A2B5. TLC immunostaining with A2B5 of gangliosides from the cells suggested that rat hepatocytes express c-series gangliosides including GT3, GT1c, GQ1c, and GP1c. Expression of c-series gangliosides in cultured hepatocytes was modulated by growth conditions of cells. The amount of GT3 was increased significantly by epidermal growth factor, while the contents of polysialo species such as GT1c, GQ1c, and GP1c were enhanced by higher cell density in culture. Examination of c-series gangliosides in rat liver tissues showed a unique developmental profile with a shift from GT3-dominant to polysialo species-dominant composition in late embryonic stages. These results suggest that the expression of c-series gangliosides in rat hepatocytes is regulated in a growth- and development-dependent manner.

A specific loss of c-series gangliosides in pancreas of streptozotocin-induced diabetic rats

Gangliosides in pancreas, kidney, and liver tissues from streptozotocin-induced diabetic rats were analyzed by methods including thin-layer Chromatographic (TLC) immunostaining with a specific monoclonal antibody to c-series gangliosides. In rats suffering diabetes for one month, the composition of major gangliosides in pancreatic tissue was almost identical to control, except for a slight increase in the content of GM3. Though c-series gangliosides such as GT3, GT2, GQ1c, and CP1c were expressed in normal pancreatic tissue, they were practically lost in pancreas of diabetic animals. A specific loss of c-series gangliosides was also observed in pancreatic tissue from rats suffering diabetes only for three days. While the composition of major gangliosides in the kidney did not change, streptozotocin-induced diabetic conditions brought about significant increases in contents of practically all major ganglioside species in liver tissue. No change was observed in the amount and composition of c-series gangliosides in both tissues. These results strongly suggest that c-series gangliosides are specifically localized in pancreatic B cells.

Immunoreactivity for glial cell markers in the human testis.

Previous studies provide evidence for the neuroendocrine nature of Leydig cells of the human testis (Schulze et al., 1987; Davidoff et al., 1993). In a comparative study of the testes of different vertebrates and man, Maunoury et al. (1991) established immunoreactivity for glial fibrillary acidic protein (GFAP) in Leydig cells and adrenal cortex cells of the Syrian hamster. Holesh et al. (1993) found GFAP in rat Leydig cells situated in the vicinity of microvessels. The fact that Leydig cells of the testis may contain glial cell antigens encouraged us to search for GFAP (found in astrocytes and cell types of non-neural tissues), galactocerebroside (Ga1C; expressed in oligodendrocytes and peripheral Schwann cells), cyclic 2’,3’-nucleotide-3’-phosphodiesterase (CNPase; expressed in oligodendrocytes and peripheral Schwann cells), and A2B5-antigen (recognizing GT3 and other c-series gangliosides expressed by oligodendrocytes, type II astrocyte progenitors, some neurons and neuroendocrine cells) in structures of the human testis.

Effects of brefeldin A on synthesis and intracellular transport of ganglioside GT3 by chick embryo retina cells.

Ganglioside GT3 is the precursor of c-series gangliosides. It is synthesized by sialylation of GD3 and is expressed in nervous tissue of birds and mammals at early stages of development. In this study we examined the sub-Golgi location of GT3 synthesis and the mechanism of its transport from the site of synthesis to the plasma membrane in chicken embryo retina cells in culture. Neural retina cells from 10-day-old chick embryo were cultured with [3H]galactose in the absence (control cells) or in the presence of 1 micrograms/ml brefeldin A (BFA). At the end of the labeling period, the fraction of labeled gangliosides transported to the plasma membrane was determined. For this, cells were treated with C. perfringens neuraminidase in conditions to desialylate only those gangliosides that were transported to the plasma membrane and consequently accessible to the enzyme. After neuraminidase treatment of cells, gangliosides were isolated, purified, and the pattern of radioactivity analyzed by HPTLC-fluorography. It was found that BFA blocked the synthesis of complex gangliosides without affecting the synthesis of GM3, GD3, and GT3. Furthermore, in BFA-treated cells, GM3, GD3, and GT3 were protected from the action of added neuraminidase, indicating an intracellular localization and, hence, an inhibition of their transport to the plasma membrane. The results indicate that synthesis of the first intermediates of a-, b-, and c- series gangliosides occurs in a proximal Golgi compartment and that the proximal Golgi-synthesized gangliosides (GM3, GD3, and GT3) use a transport mechanism that is dependent on ADP ribosylation factor and coatomer proteins.

Characterization of sialyltransferase-IV activity and its involvement in the c-pathway of brain ganglioside metabolism.

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 Vmax value was 1,340 pmol/mg of protein/h at an optimal pH of 6.5, whereas the apparent Km values for CMP-sialic acid and GM1 were 172 and 78 microM, 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.(ABSTRACT TRUNCATED AT 250 WORDS)

The c-series gangliosides GT3, GT2 and GP1c are formed in rat liver Golgi by the same set of glycosyltransferases that catalyse the biosynthesis of asialo-, a- and b-series gangliosides.

Biosynthesis of the c-series gangliosides GT3, GT2 and GP1c was studied in Golgi derived from rat liver. Competition experiments show that the synthesis of ganglioside GT2 (GalNAc beta 1----4-(NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal- beta 1----4Glc beta 1----1Cer) from GT3 (NeuAc alpha 2----8NeuAc alpha 2----8-NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) seems to be catalysed by the same N-acetylgalactosaminyl-transferase (GalNAc-T), which converts GM3 (NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) to GM2 (GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1Cer). Similar competition experiments suggest moreover that the sialytransferase V (SAT V), which catalyses the synthesis of GT1a (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4- (NeuAc alpha 2----3)-Gal beta 1----4Glc beta 1----1Cer) from GD1a (NeuAc alpha-2----3Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1-Cer) appears to be identical to the enzyme that catalyses the synthesis of GP1c (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3-GalNAc beta 1----4(NeuAc alpha 2----8-NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta-1----4Glc beta 1----4Glc beta 1----1Cer) from GQ1c (NeuAc alpha 2----3Gal beta 1----3Gal-NAc beta 1----4 (NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4-Glc beta 1----1Cer).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistological study on brains of Alzheimer's disease using antibodies to fetal antigens, C-series gangliosides and microtubule-associated protein 5

An immunohistological study of Alzheimer's brains was performed using antibodies to C-series gangliosides and microtubule-associated protein 5 (MAP5), and their staining patterns were compared with those of antibodies to tau and beta-amyloid precursor protein. Antibodies to C-series gangliosides and MAP5, both of which are known to preferentially expressed in the fetal brains, immunostained dystrophic neurites of senile plaques, neurofibrillary tangles and neuropil threads abundant in 3rd and 5th layers in the cerebral cortex, all of which are considered to be pathological hallmarks of Alzheimer's disease. The immunostaining patterns of these structures by antibodies to C-series gangliosides and MAP5 were similar to those by the antibody to tau. These three antibodies also immunostained some neurons in Alzheimer's brain, although their staining patterns were slightly different from one another; i.e., both diffuse and granular patterns were seen by the antibody to tau, but only granular pattern by the antibodies to C-series gangliosides and MAP5. These neurons immunostained by these three types of antibodies appeared to be the precursors of the classical neurofibrillary tangles, as positively stained neurons were not seen in the brains of non-demented cases. The presence of fetal antigens such as the C-series gangliosides and MAP5 in Alzheimer's brain may suggest that regeneration or sprouting of neurons is ongoing in association with the re-induction of gene expression characteristic for the brain in the early stage of development.

Differences in lipid composition between isolated growth cones from the forebrain and those from the brainstem in the fetal rat

The lipid composition of nerve growth cone membranes isolated from rat fetal forebrain or brainstem by the sucrose density gradient method was analyzed biochemically and immunochemically. In the forebrain, growth cone membrane (GCM) contained lower levels of gangliosides than those from other heavier fractions, but it was not the case in the fetal brainstem at the same developmental stage. The distinctive features in the ganglioside composition of GCM are the predominance of GD3 and the presence of c-series gangliosides that are due to fetal expression in mammals. A unique acidic glycolipid, sulfoglucuronylparagloboside (SGPG), which is not present in adult brains, was first detected in both forebrain and brainstem GCM. Including such minor species, the ganglioside composition in forebrain or brainstem GCM was almost identical to other membrane fractions from the forebrain or brainstem. The compositional ratios of the major lipid classes in membranes, cholesterol and phospholipids, seemed to be common to forebrain GCM and brainstem GCM, as indicated by the identical values of phospholipid-to-protein (PL/Pr), cholesterol-to-protein (Ch/Pr), and cholesterol-to-phospholipid (Ch/PL) ratios for both. This study has revealed that GCM isolated from forebrain which is supposed to be at an earlier stage of neuronal differentiation than brainstem has less amounts of total gangliosides, high proportion of GD3 to GD1a and enriched c-series gangliosides as compared to brainstem GCM.

Developmentally expressed O-acetyl ganglioside GT3 in fetal rat cerebral cortex

Monoclonal antibody M6704, established against the chick neural tube, was shown to recognize a trisialosyl residue, NeuAcα2-8NeuAcα2-8NeuAcα2-3-R of C-series gangliosides. Using this antibody, the developmental changes of C-series gangliosides in fetal rat cerebral cortex have been examined. Two dimensional thin layer chromatography (TLC) enzyme-immunostaining analysis revealed that alkali treatment resulted in a great increase in GT3 that amounted to more than 85% of the total GT3 detected. The alkalilabile form was easily degraded to form GT3 by the action of the receptor-destroying enzyme of influenza C virus, sialate O-acetylesterase, indicating that the antigen was most probably 9- O-acetyl-NeuAc containing GT3. The ganglioside was highly enriched at the 14th gestation day, gradually decreased, and was not detected in adult rat cerebral cortex.

S-4-1 - Expression of C-series gangliosides in nervous tissue during development and regeneration.

S-4-1 - Expression of C-series gangliosides in nervous tissue during development and regeneration.

Developmental Changes of C-Series Polysialogangliosides in Chick Brains Revealed by Mouse Monoclonal Antibodies M6704 and M7103 with Different Epitope Specificities1

We have established many mouse monoclonal antibodies detecting developmentally regulated antigens in chicken embryonic neural tissues of the otic vesicles and neural tube by immunizing mice with the membrane fraction of the neural tube and somite prepared from 3-d chick embryos. Among them, three monoclonal antibodies (MAbs) M6703, M6704, and M7103 were shown to react with the gangliosides isolated from chicken embryonic brains. The precise specificity of the antibodies was determined mainly by enzyme-immunostaining on thin layer plates. MAbs M6703 and M6704 bound to C-series polysialogangliosides including GT3, GT2, GT1c, GQ1c, and GP1c isolated from cod fish brains, but never to A-series, B-series, or X-series gangliosides. On the other hand, MAb M7103 antibody has a rather narrow specificity, reacting with GT1c, GQ1c, and GP1c, but not with ganglioside GT3. This indicates that the epitopes defined by these two MAbs are not with ganglioside GT3. This indicates that the epitopes defined by these two MAbs are different from each other. MAbs M6703 and M6704 recognize a trisialosyl residue, NeuAc alpha 2-8NeuAc alpha 2-8NeuAc alpha 2-3, while M7103 requires both a trisialosyl residue and the gangliotetraosy backbone structure for binding. As compared to similar MAbs which have been reported in the literature. M6703 and M6704 are unique in that they react equally with all of the C-series gangliosides and belong to the IgG3 subclass.(ABSTRACT TRUNCATED AT 250 WORDS)

P-521 - C-series gangliosides are present in the mammalian nervous tissue and promote the neurite outgrowth from cultured cells

P-521 - C-series gangliosides are present in the mammalian nervous tissue and promote the neurite outgrowth from cultured cells