肝癌中CMP-乙酰神经氨酸:GM_3(α2→8)唾液酸转移酶(GD_3合成酶)的研究及纯化的初步报告

本实验室曾报道在所检测的不同种族来源与不同致癌剂所诱发的肝细胞肝癌中均有神经节苷脂GD_3组份的明显增高,本文就这一现象的机制进行了探讨。实验结果表明在人肝癌手术标本、人肝癌细胞株SMMC,3′-甲基奶油黄(3′Me-DAB)和二乙基亚硝胺(DENA)所诱发的大鼠肝癌以及大鼠肝癌株BERH-2中GD_3合成酶的活性均有不程度的增高,同对GD_3前体的合成酶(GM_3合成酶)的活性也有所增高。这就提示肝癌中GD_3增高的原因之一在于GD_3合成酶的活性增高与前体供应充足的结果。另外,本文还对GD_3合成酶的提纯做了初步尝试。主要采用Tritonx-100抽提和CDP-hydrazide Sepharose 4B亲合层析的方法从二乙基亚硝胺诱发的大鼠肝癌中提纯了GD_3合成酶。提纯倍数为12500倍,产率0.4％。提纯的GD_3合成酶在醋酸纤维膜上经等电聚焦电泳鉴定示单一条带,其pI值为5.25左右。关于糖脂唾液酸转移酶的纯化工作目前还未见报道。     

Changes of the ganglioside pattern and content in human fibroblasts by high density cell population subculture progression

In this study we show that the ganglioside content and pattern of human skin fibroblasts change along the process of cell subculture progression by varying the cell density.GM3, GD3 and GD1a were components of the total cell ganglioside mixtures extracted from cells, but GD1a was in all the extracts a minor component or very scant. Other gangliosides present in traces were not characterised. The fibroblast ganglioside content of 52 pools of cells obtained from 5 different cell lines cultured at variable cell density ranged from 2.0 to 13.1 nmoles per mg of cell protein. The molar ratio between GM3 and GD3 varied from 418 to 0.6 in the ganglioside mixtures, as determined by densitometric quantitative analysis after thin layer chromatographic separation.Both the ganglioside content and the GM3/GD3 molar ratio were constant along several passages of subculture progression performed by plating cells collected at confluence. Instead, when the subculture progression was performed by plating cells collected at a few days after reaching confluence, a progressive increase of the ganglioside content was observed. GD3 increased proportionally more than GM3 so that a progressive decrease of the ratio between GM3 and GD3 was observed. In some experiments, GD3 was very scant at the beginning of the progression, while it was near 30% after 5 passages under these conditions. The progressive increase of GD3 along the high density cell population subculture progression was associated to a moderate increase of the mRNA GD3 synthase. Published in 2003.     

Ganglioside biosynthesis in rat liver: different distribution of ganglioside synthases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells

The activities of five glycolipid-glycosyltransferases, GL2, GM3, GM2, GM1, and GD1a synthase, were determined in a cell-free system with homogenate protein of total rat liver, isolated hepatocytes, Kupffer cells, and sinusoidal endothelial cells. In rat liver parenchymal and nonparenchymal cells ganglioside synthases were distributed differently. Compared to hepatocytes, Kupffer cells expressed a nearly sevenfold greater activity of GM3 synthase, but only 14% of GM2, 19% of GM1, and 67% of GD1a synthase activity. Sinusoidal endothelial cells expressed a pattern of enzyme activities quite similar to that of Kupffer cells with the exception of higher GM2 synthase activity. Activity of GL2 synthase was distributed unifromly in parenchymal and nonparenchymal cells of rat liver, but differed by sex. It was 1 to 2 orders of magnitude below that of all the other ganglioside synthases investigated. The results indicate GL2 synthase regulates the total hepatic ganglioside content, and hepatocytes but not nonparenchymal liver cells have high enzymatic capacities to form a-series gangliosides more complex than GM3.     

Oncogene transgenic mice: an useful model to study in vivo the relationships between gangliosides and oncogenes

Several studies have demonstrated that transfer of oncogenes in cultured cells reproducibly induces transmissible alterations in their ganglioside profile; the transfection of the same oncogene into different cell lines and the different localization of the oncogene product result in a different ganglioside expression. In the present study the modifications of the ganglioside pattern in mammary carcinomas induced in transgenic mice by the activated form of the rat neu oncogene have been investigated. Whereas control mammary tissues contain quite exclusively GM3, all neoplastic samples show a substantial decrease of this ganglioside, an accumulation in variable amount of GM3-derived species (GM1, GD3, GD1a, GD1b, GT and GQ) and the appearance of new, not yet identified, sialic acid containing molecules. Interestingly, three out of 10 tumors analyzed, even if histologically comparable to the others but with a larger dimension, show a significative difference as regard to the GM1, GD3 and GD1a content. Our data suggest that an activated oncogene may induce also in vivo a specific and transmissible alteration in the ganglioside pattern, but this distribution could be susceptible to further modifications during the tumor progression.     

Age- and hormone-dependent ganglioside patterns of rat hepatocytes in primary culture

Using an improved procedure for the quantitative extraction of all glycolipids from small tissue samples the hepatic ganglioside pattern of rats was analysed during development. While this parameter remained fairly constant in adult animals, hepatocytes in primary culture showed drastic changes both in content and relative distribution among the various ganglioside species. The content of lipid-bound sialic acid increased several-fold during 6 days in monolayer and the pattern changed in favour of the higher sialylated forms. Dexamethasone delayed this transition and enhanced the content of GD1a and GM1 relative to GM3. The ganglioside content was also dependent on the density of hepatocytes in the primary culture. If the cell density was insufficient for formation of a confluent monolayer, higher ganglioside-sialic acid contents were found and the relative amount of GD3 increased after 3-4 days. These results support the notion that gangliosides are involved in cellular differentiation and cell-cell contact.     

Biosynthesis of gangliosides in primary cultures of rat hepatocytes. Determination of the net synthesis of individual gangliosides by incorporation of labeled N-acetylmannosamine

The ganglioside content of rat hepatocytes increases several-fold during the first 6 days in monolayer culture. To correlate increased levels with rates of de novo synthesis, the incorporation of N-acetyl-[6-3H]D-mannosamine into individual gangliosides was determined. The calculation of synthetic rates was made possible by the simultaneous measurement of the specific radioactivity of the immediate sialic-acid donor, CMP-Neu5Ac. The CMP-Neu5Ac content of hepatocytes was found by HPLC analysis to be 30.5 nmol/g of plated cells. The specific radioactivity of this precursor pool reached a constant plateau 5 h after addition of the labeled N-acetyl-mannosamine and remained constant for at least 70 h. The incorporation into individual gangliosides was measured in primary cultures of rat hepatocytes between 72 and 144 h after seeding. During this period, the increase in ganglioside levels was greatest. The highest rates of incorporation were seen in GD1a followed by GM3, GM1, GD3 and the polysialylated compounds. The following rates of synthesis (nmol per 60 h and mg of protein) were calculated: GD1a 0.68, GM3 0.59, GM1 0.36, GD3 0.13 and GT1 0.02. These values are compared with the net increase of the gangliosides as measured by the resorcinol reaction.     

The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-to-cell interactions

We describe herein the enzyme behavior of MmNEU3, the plasma membrane-associated sialidase from mouse (Mus musculus). MmNEU3 is localized at the plasma membrane as demonstrated directly by confocal microscopy analysis. In addition, administration of the radiolabeled ganglioside GD1a to MmNEU3-transfected cells, under conditions that prevent lysosomal activity, led to its hydrolysis into ganglioside GM1, further indicating the plasma membrane topology of MmNEU3. Metabolic labeling with [1-(3)H]sphingosine allowed the characterization of the ganglioside patterns of COS-7 cells. MmNEU3 expression in COS-7 cells led to an extensive modification of the cell ganglioside pattern, i.e. GM3 and GD1a content was decreased to about one-third compared with mock-transfected cells. At the same time, a 35% increase in ganglioside GM1 content was observed. Mixed culture of MmNEU3-transfected cells with [1-(3)H]sphingosine-labeled cells demonstrates that the enzyme present at the cell surface is able to recognize gangliosides exposed on the membrane of nearby cells. Under these experimental conditions, the extent of ganglioside pattern changes was a function of MmNEU3 transient expression. Overall, the variations in GM3, GD1a, and GM1 content were very similar to those observed in the case of [1-(3)H]sphingosine-labeled MmNEU3-transfected cells, indicating that the enzyme mainly exerted its activity toward ganglioside substrates present at the surface of neighboring cells. These results indicate that the plasma membrane-associated sialidase MmNEU3 is able to hydrolyze ganglioside substrates in intact living cells at a neutral pH, mainly through cell-to-cell interactions.     

Identity of GA1, GM1a and GD1b synthase in Golgi vesicles from rat liver

Synthesis of ganglioside GD1b from ganglioside GD2 was demonstrated using Golgi membranes isolated from rat liver. Competition experiments using gangliosides GA2, GM2 and GD2 as substrates, and as mutual inhibitors for ganglioside galactosyltransferase activity in preparations of Golgi vesicles derived from rat liver, suggested that galactosyl transfer to these three compounds, leading to gangliosides GA1, GM1a and GD1b respectively, is catalyzed by one enzyme. These results strengthen the hypothesis that the main site for the regulation of ganglioside biosynthesis occurs within the reaction sequence LacCer----GA3----GD3----GT3.     

Coordinate Regulation of Ganglioside Glycosyltransferases in Differentiating NG108-15 Neuroblastoma X Glioma Cells

The enzymatic basis for ganglioside regulation during differentiation of NG108-15 mouse neuroblastoma x rat glioma hybrid cells was studied. This cell line contains four gangliosides that lie along the same biosynthetic pathway: GM3, GM2, GM1, and GD1a. Chemically induced neuronal differentiation of NG108-15 cells led to an 80% drop in the steady-state level of their major ganglioside, GM3, a sixfold increase in the level of a minor ganglioside, GM2 (which became the predominant ganglioside of differentiated cells); and relatively little change in the levels of GM1 and GD1a, which lie further along the same biosynthetic pathway. The enzymatic basis for this selective change in ganglioside expression was investigated by measuring the activity of two glycosyltransferases involved in ganglioside biosynthesis. UDP-N-acetylgalactosamine: GM3 N-acetylgalactosaminyltransferase (GM2-synthetase) activity increased fivefold during butyrate-induced differentiation, whereas UDP-galactose: GM2 galactosyltransferase (GM1-synthetase) activity decreased to 10% of its control level. Coordinate regulation of these two glycosyltransferases appears to be primarily responsible for the selective increase of GM2 expression during NG108-15 differentiation.     

Content, pattern and metabolic processing of rat-liver gangliosides during liver regeneration

During rat liver regeneration, the ganglioside content and distribution undergo significant changes after partial hepatectomy; total liver gangliosides increase remarkably till the 4th day after surgery, thereafter progressively decreasing to reach the values of sham-operated controls at the 12th day. The qualitative pattern is characterized by the 95% relative increase of GD1a at the 4th day and the 40% relative decrease of GD1b. In order to investigate the processes of ganglioside penetration into cells, degradation and biosynthesis, radiolabelled GM1 ([Sph-3H] GM1) was administered. One day after hepatectomy the liver uptake and metabolism of exogenous ganglioside were significantly reduced. Three days post-surgery these parameters were restored to control values; however an increased radioactivity incorporation was found in GD1a, thus suggesting an enhancement of its biosynthesis around the 4th day. The data reported here suggest that in the first two days after partial hepatectomy, the ganglioside degradation is reduced with a consequent increase of ganglioside content; later on the catabolic routes normalize and some biosynthetic processes leading to GD1a are enhanced. GD1a seems to be a marker of a peculiar transition phase of liver regeneration.     

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.     

Genetic polymorphism of rat liver gangliosides

Liver ganglioside patterns of eight rat strains were classified according to two phenotypes: SHR type, characterized by predominance of b-series gangliosides (GD1b, GT1b, GQ1b), and DA type, characterized by predominance of a-series gangliosides (GM1, GD1a). Comparison of ganglioside pattern expressed in the liver of F1 hybrids and backcross F2 hybrids indicated that SHR type is controlled by a single autosomal-dominant gene which probably determines the expression of sialytransferase 2 activity for synthesis of GD3 from GM3.     

A GM1b-derived disialoganglioside GD1c is the predominant ganglioside of rat thymocytes.

The thin-layer chromatographic (TLC) pattern of gangliosides of rat thymocytes showed a profile characterized by the occurrence of a predominant ganglioside which did not correspond to any reference gangliosides of rat brain. The ganglioside was isolated from rat thymus, and characterized by compositional analysis, methylation analysis, sialidase treatment, negative-ion fast atom bombardment (FAB) mass spectrometry, and proton NMR spectroscopy. The structure was elucidated to be NeuGc alpha 2-8NeuGc alpha 2-3Gal beta 1-3GalNac beta 1-4Gal beta 1-4Glc beta 1-1Cer. This is the major ganglioside of rat thymus lymphoid cells and is one of the GM1b-derived gangliosides, GD1c, having two N-glycolylneuraminic acids. This is the first report on the occurrence of GD1c in normal animal cells.     

Differential expression of fucosyl GM1 and a disialoganglioside with a NeuAc alpha 2-6GalNAc linkage (GD1e) in various rat ascites hepatoma cells

A distinct difference in ganglioside composition among various rat ascites hepatomas and Yoshida sarcoma was observed on TLC-immunostaining with anti-fucosyl GM1 antibody, and chemical and enzymatic analyses. Yoshida sarcoma and ascites hepatomas, AH13, AH66F and AH66, but not the other 9 tumor cell lines investigated, specifically contained a disialoganglioside, NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc beta 1-4Gal beta 1-4Glc beta 1-1ceramide (GD1e), whereas the 9 ascites hepatoma cells without GD1e contained fucosyl GM1. The differential expression of fucosyl GM1 and GD1e in various tumor cell lines indicates that different cell lineages express distinct metabolic pathways for gangliosides, and that the gangliosides are useful markers for distinguishing tumor cell lines.     

Ganglioside composition of the rat choriocarcinoma cell line,Rcho-1

The Rcho-1 cell line, originally established from a rat choriocarcinoma, shows differentiation into placental trophoblastic giant cell-like cells and has been used to study the mechanism of placental function control. In the present study, we analysed the ganglioside composition of Rcho-1 cells by HPTLC orcinol/H₂SO₄, TLC/immunostaining and immunohistochemistry. Rcho-1 cells expressed GM3 and GD3 as the major gangliosides and CTH as major neutral glycolipid when they were cultured in growth medium (20% FCS) or transplanted beneath the kidney capsule. The expression of these gangliosides was strong in the undifferentiated small cells, whereas the completely differentiated giant cells showed poor staining with antibodies against the gangliosides. Under culture conditions to induce cell differentiation using horse serum (1–20% HS), the expression of GD3 was suppressed and re-expressed when the medium was changed to growth medium, suggesting that a change of ganglioside components may trigger and define the direction of terminal differentiation. Thus the composition of glycolipids is conserved in Rcho-1 cells and is similar to that of the rat placenta, where GM3 is dominant in mid-pregnancy and decreased in late pregnancy, whereas GD3 is low in mid-pregnancy and increased in late pregnancy.     

Gangliosides of hepatoma 27, normal and regenerating rat liver.

The highly malignant rat hepatoma 27 was found to have increased amounts of lipid-bound sialic acid as compared with normal liver whereas in regenerating liver the lipid-bound sialic acid level was reduced. In contrast to the liver the hepatoma contained higher amounts of disialogangliosides and no trisialogangliosides, which are abundant in the liver. The main disialoganglioside of the hepatoma had no analogue among the liver gangliosides and was identified as Gal-GalNAc(AcNeu-AcNeu)-Glc-Cer (GD1b), which in other tissues is known to be a precursor of trisialogangliosides. These findings may be explained by a reduced activity of glycosyltransferases in the hepatoma and apparently do not simply reflect differences in growth rate since the ganglioside pattern of regenerating rat liver was not altered significantly in comparison with the liver. Liver and hepatoma microsomes were found to be enriched in gangliosides as compared with whole cells, liver mitochondria were slightly poorer, while the ganglioside level of hepatoma mitochondria was much higher than that of the hepatoma cells. It thus appears that the existing image of the plasma membranes as the only sites of high ganglioside concentration may not hold true for weakly differentiated hepatomas of high malignancy.     

Ganglioside composition of growth cone-deficient nerve cell cultures

The ganglioside concentration and composition in growth cone-deficient nerve cells, induced by inclusion of cytochalasin B (CB) are compared with those of 2-day-old control cells from primary cultures of embryonic rat cerebral cortex. Ganglioside GM1 and GD1a are the major gangliosides in the growth cone. Ganglioside GM1 may be one of the membrane components of growth cones that function in neural recognition during development.     

Distribution of gangliosides in parenchymal and non-parenchymal cells of rat liver

Parenchymal and non-parenchymal cells were isolated from rat liver with purities of more than 90%. Total and ganglioside sialic acid contents were higher in non-parenchymal cells than in parenchymal cells. Thin-layer chromatography of gangliosides showed that the main component in rat liver was ganglioside GM3 and that this was abundant in non-parenchymal cells. Parenchymal cells had ganglioside GD1b as the main component and less GM3 than non-parenchymal cells. These results suggested that the main ganglioside of rat liver, GM3, arises mainly from non-parenchymal cells.     

Sub-Golgi distribution in rat liver of CMP-NeuAc GM3- and CMP-NeuAc:GT1b alpha 2----8sialyltransferases and comparison with the distribution of the other glycosyltransferase activities involved in ganglioside biosynthesis

Using a sucrose density gradient fractionation of a highly purified Golgi apparatus from rat liver, we determined the sub-Golgi distribution of CMP-NeuAc:GM3 ganglioside alpha 2----8sialyltransferase (GM3-SAT) and CMP-NeuAc:GT1b ganglioside alpha 2----8sialyltransferase (GT1b-SAT), in comparison with that of the other glycosyltransferase activities involved in ganglioside biosynthesis. While GM3-SAT was recovered in several density fractions, GT1b-SAT was mainly found on less dense sub-Golgi membranes; this indicates that these two activities are physically separate. Moreover, with regard to the monosialo pathway, CMP-NeuAc:lactosylceramide alpha 2----3sialyltransferase, UDP-GalNAc:GM3 ganglioside beta 1----4N-acetylgalactosaminyltransferase, UDP-Gal:GM2 ganglioside beta 1----3galactosyltransferase, and CMP-NeuAc:GM1 ganglioside alpha 2----3sialyltransferase were resolved from more dense to less dense fractions, respectively. In the disialo pathway, UDP-GalNAc:GD3 ganglioside beta 1----4N-acetylgalactosaminyltransferase, UDP-Gal:GD2 ganglioside beta 1----3galactosyltransferase and CMP-NeuAc:GD1b ganglioside alpha 2----3sialyltransferase co-distributed with the corresponding activities of the monosialo pathway. These last results indicate that many Golgi glycosyltransferases involved in ganglioside biosynthesis are localized in the order in which they act.     

Gangliosides in rat kidney: composition, distribution, and developmental changes

Gangliosides in rat kidney were analyzed for their composition, regional distribution, and developmental changes. Renal tissue from 7-week-old rats showed a GM3-dominant pattern with GD3 and several minor ganglioside components including GM4, GM2, GD1a, and an unknown ganglioside (ganglioside X). The tissue also contained c-series gangliosides that included GT3 as the main component with GT2 in a lesser amount. Ganglioside analysis of cortical and medullary regions of renal tissue suggested the restricted localization of some gangliosides. While GM4 and GD3 were enriched in the cortical region, GM2 was distributed mainly in the medullary area. Renal gangliosides showed unique developmental profiles during a period from Embryonic Day 20 (E20) to 7 weeks postnatal. The content of renal gangliosides increased from E20, reached the highest around Postnatal Day 1, and thereafter, decreased rapidly to the adult level. The ratio of N-glycolylneuraminic acid to total sialic acids in gangliosides tended to change in inverse proportion to the amount of total sialic acids. The composition of major gangliosides in renal tissues shifted from GD3-dominant to GM3-dominant patterns with advancing ages. While GM1 was expressed only at early stages of the development, GM4, GM2, and ganglioside X appeared after Postnatal Day 3. The expression of c-series gangliosides was less affected through the period examined. These results suggest that gangliosides may be implicated with development and function of rat kidney.