Lipid-bound Sialic Acid Content Research Articles

Storage of Gangliosides GM2 and Fucosyl GM1 in the Kidney of MCC Strain of Mastomys (Praomys coucha)

Previously, we histochemically examined the kidney of the MCC strain of mastomys (Praomys coucha) and found the storage of gangliosides. In the present studies, the lipid-bound sialic acid content of gangliosides in the MCC kidney was about 9- to 14-fold higher than that of the control (MWC strain). In the MCC kidney, sialic acids of male gangliosides were composed of N-acetylneuraminic acid at 91.5%; sialic acids of female gangliosides, however, were composed almost entirely of N-glycolylneuraminic acid. TLC of gangliosides showed that the MCC kidney contained four abundant gangliosides (two gangliosides each in males and females). These gangliosides isolated by HPLC were identified to be GM2(NeuAc) and fucosyl GM1(NeuAc) in the male MCC kidney and GM2(NeuGc) and fucosyl GM1(NeuGc) in the female MCC kidney by secondary ion mass spectrometry, TLC/immunostaining and TLC after enzyme treatments. Although the MCC kidney contained control levels of the activities of beta-N-acetylhexosaminidase, alpha-l-fucosidase, N-acetylgalactosaminyltransferase and fucosyltransferase, the activity of beta-galactosidase in the MCC kidney was increased to 400-500% of that in the MWC kidney. Therefore, we discussed the possibility that in the MCC kidney, GM2 was abundantly produced by the effect of increased beta-galactosidase activity.

Thyroid Sialyltransferase mRNA Level and Activity Are Increased in Graves' Disease

Sialylation of cell components is an important immunomodulating mechanism affecting cell response to hormones and adhesion molecules. To study alterations in sialic acid metabolism in Graves' disease (GD) we measured the following parameters in various human thyroid tissues: lipid-bound sialic acid (LBSA) content, ganglioside profile, total sialyltransferase activity, and the two major sialyltransferase mRNAs for sialyltransferase-1 (ST6Gal I) and for sialyltransferase-4A (ST3Gal I). Fragments of toxic thyroid nodules (TN), nontoxic thyroid nodules (NN) and nontumorous tissue from patients with nodular goiter or thyroid cancer were used as a control (C). The LBSA content and sialyltransferase activity were the highest in the GD group (164 +/- 4.44 versus 120 +/- 2.00 nmoL/g, p = 0.005 and 1625 +/- 283.5 versus 324 +/- 54.2 cpm/mg of protein, p < 0.005 compared to control group C). Ganglioside profile in the GD group was similar to that in control tissues. Sialyltransferase- 1 mRNA and sialyltransferase-4A mRNA levels were significantly higher in the GD group than in the control group (12.52 +/- 6.90 versus 2.54 +/- 1.24 arbitrary units, p < 0.005 and 2,49 +/- 1.16 versus 1.23 +/- 0.46 arbitrary units, p < 0.05, respectively). There was a positive correlation between the increased sialyltransferase-1 mRNA level and the TSH-receptor antibody titer determined by the TRAK test. These results indicate that sialyltransferases expression and activity are increased in GD. Exact mechanism of this upregulation remains unknown, though one of possible explanations is the activation of the thyrotropin (TSH) receptor.

This month in J Lab Clin Med: Issue highlights for August 2002

This month in J Lab Clin Med: Issue highlights for August 2002

Detection of Sialic Acids and Gangliosides with Special Reference to 9-O-Acetylated Species in Basaliomas and Normal Human Skin

Basal cell carcinomas and normal skin were examined in relation to the abnormal expression of gangliosides. The content of gangliosides with 9-O-acetylated sialic acids of 26 sample pairs was analyzed by a microtiter assay using influenza C virus as well as by fluorimetric high-performance liquid chromatography of the sialic acids released. The 9-O-acetylation levels were significantly (up to 56-fold) higher in basal cell carcinoma tissues than in the skin surrounding basal cell carcinomas. Slightly elevated amounts of O-acetylated gangliosides were also seen in the skin marginal to the basaliomas. The ganglioside composition of four sets of pooled samples of basal cell carcinoma and one pool of normal skin were studied by high-performance thin-layer chromatography and immune high-performance thin-layer chromatography using monoclonal antibodies against 9-O-acetyl GD3. The lipid-bound sialic acid content of normal skin was 0.029 microg dry weight, whereas in nodular basal cell carcinomas it was approximately twice as much. Several O-acetylated sialic acids were seen by high-performance liquid chromatography analysis, but N-acetyl-9-O-acetylneuraminic acid prevailed. Only in the tumor ganglioside fraction, a small amount of N-glycolylneuraminic acid was found. The 9-O-acetylated gangliosides with mainly 9-O-acetyl-GD3 can be considered as tumor-associated antigens or markers for basal cell carcinomas. This finding about tumor-associated carbohydrates may contribute to new strategies in current tumor diagnosis and therapy.

Unusual gangliosidosis in emu (Dromaius novaehollandiae).

A previous study has demonstrated an unusual gangliosidosis in emu that is characterized by the accumulation of gangliosides in the brain tissues with GM3 and GM1 predominating. To provide insight into this unique disorder of emu gangliosidosis, the current study focused on analysis of neutral glycosphingolipids and gangliosides from brain and liver tissues of affected birds and healthy controls. We found not only that the total lipid-bound sialic acid content was increased three- and fourfold in the affected brain and liver, respectively, but also that the ganglioside pattern was rather complex as compared with the control. The absolute ganglioside sialic acid content was significantly increased in the diseased tissues, with the highest elevation levels of GM3 (14-fold) and GM1 (ninefold) in the affected brain. Relative increases in content of these monosialogangliosides were also significant. GM2 was only detected in the affected brain, but not in normal controls. The neutral glycosphingolipid fraction showed accumulation of many oligosylceramides, with six- and 5.5-fold increases in lactosylceramide levels for brain and liver, respectively. The level of myelin-associated galactosylceramide (GalCer) in the brain was decreased to only 41% of that in the healthy control, whereas no difference was found in liver tissues from both groups. Besides GalCer, the brain content of sulfatide (cerebroside-sulfate esters), another myelin-associated glycolipid, decreased to only 16% of the control. The loss of myelin-associated GalCer and sulfatide strongly suggests demyelination in the affected emu brain. Our overall data are consistent with the presence of a unique form of sphingolipidosis in the affected emus, perhaps with secondary demyelination, and suggest a metabolic disorder related to total sphingolipid activator deficiency.

Characterization of gangliosides from Ehrlich ascites tumour cells and their variants.

Differences in the nature of the gangliosides present in two types of Ehrlich ascites tumour (EAT) cells, the adherent and non-adherent EAT cells, were studied. Gangliosides were isolated by DEAE Sephadex column chromatography and analysed by high-performance thin-layer chromatography (HPTLC). The non-adherent EAT (na-EAT) cells which grow in the peritoneal cavity of mice were selected for growth on basement membrane and tissue culture plastic to give the adherent EAT (a-EAT) cells. na-EAT cells contained 1.57 nmol lipid-bound sialic acid per mg protein and at least 12 different gangliosides, including major gangliosides such as GM3, GM2, GM1, GD3, GD1a and GT1b. On the other hand, the ganglioside pattern of a-EAT cells differed significantly from that of na-EAT cells, both quantitatively and qualitatively. The content of lipid-bound sialic acid in a-EAT cells was only 0.24 nmol per mg of protein. The gangliosides in a-EAT cells were characterized as GD1a and trisialogangliosides and, significantly, a-EAT cells did not contain monosialogangliosides. Neutral glycolipids were isolated from both cell lines and their patterns were compared. In contrast to the gangliosides pattern, their neutral glycolipid patterns were similar. Glucosylceramide and lactosylceramide were the major components in both types of cells. In addition to na- and a-EAT cells, a-EAT cells were passaged in mice by intraperitoneal injection, giving rise to a third variant (c/m EAT cells). We analysed the gangliosides in c/m EAT cells to determine whether there was a change in the ganglioside pattern found in na-EAT cells. After repeated passage of c/m EAT cells in mice, the pattern of gangliosides shifted to that of na-EAT cells. Alterations of ganglioside composition may be associated with the growth environment of the murine peritoneal cavity; alternatively, a selection process may have occurred.

N-Butyrate Mediation of Ganglioside Expression of Human and Murine Cancer Cells Demonstrates Relative Cell Specificity

1. n-Butyrate, a short chain fatty acid produced by colonic fermentation, induces differentiation in human neoplastic cell lines, and reduces expression in vitro of a sialyltransferase that glycosylates N-linked glycoproteins in hepatoblastoma cells. Gangliosides are amphipathic, sialylated glycosphingolipids that undergo profound changes in many transformed cells and may protect neoplastic cells from host immune surveillance. Colonic mucosal cells are exposed to luminal short-chain fatty acid concentrations of up to 80 mmol/l, and there is some evidence that short-chain fatty acids may alter ganglioside expression in colon cancer cells. 2. Because of the importance of gangliosides in cancer pathogenesis, we investigated the effects of n-butyrate on ganglioside expression of colonic (human and murine) and non-colonic cancer cells. 3. Three separate colon cancer cell lines (LS174T, T84 and MCA-38), when butyrate treated, demonstrated striking amplification of specific individual gangliosides. However, the total lipid-bound sialic acid content of gangliosides of butyrate-treated LS174T cells diminished. In contrast to earlier reports, n-butyrate did not mediate expression of all gangliosides and specifically did not mediate expression of GM3. This effect persisted even after removal of butyrate. 4. In contrast, exposure of extracolonic cells to butyrate, including cervical cancer (HeLa) and laryngeal cancer (HEp-2) cell lines in this study and hepatoblastoma cells (Hep G2) in our previous work, caused no detectable changes in ganglioside expression. 5. In conclusion, our results indicate a relative tissue specificity of butyrate-mediated alterations in ganglioside expression that is not universal but is limited to specific gangliosides.

Rat brain gangliosides during hypoxia

The content and composition of rat brain gangliosides in various forms of hypoxia is studied. The content of lipid-bound sialic acid is found to be considerably elevated in the earliest stages of hypoxia combined with hypercapnia and cooling. The content of gangliosides remains elevated over the hypoxia period and returns to control values 48 hours after its discontinuation. Repeated hypoxia results in the same changes in rat brain gangliosides. Hemic sodium nitrite-induced hypoxia also leads to a reliable elevation of these lipids. Hypoxia also causes changes in the content of individual ganglioside fractions: a decreased content of mono- and an increased content of polysialogangliosides.

Membrane lipids of human peripheral nerve and spinal cord

Major membrane lipids were determined in specimens of human peripheral nerve (cauda equina) and spinal cord of 10 subjects aged 20–70 years. The same lipids were also assayed in myelin from the same tissues isolated with two different procedures and in myelin of cauda equina from 3 subjects aged 17–91 years isolated with a third method. The concentrations (mean and standard deviation) of phospholipids were 90 ± 11 and 96 ± 9 nmol/g fresh weight; of cholesterol 70 ± 15 and 101 ± 16; of cerebroside 19 ± 3 and 41 ± 7; of sulfatide 10 ± 1 and 11 ± 1; and of gangliosides 0.80 ± 0.08 and 0.40 ± 0.05 N in cauda equina and spinal cord, respectively. The proportion of ethanolamine phosphoglyceride was lower and that of sphingomyelin higher in cauda equina than in spinal cord. The myelin of peripheral nerve and spinal cord contained almost the same proportions of lipids as the whole tissue. The protein-bound sialic acid content was 3-fold higher than the lipid-bound sialic acid content in cauda myelin. The fatty acid patterns of choline, ethanolamine, inositol and serine phosphoglycerides of spinal cord and its myelin, were very similar to those of cerebral white matter, while the phosphoglycerides of cauda equina had higher proportions of monoenoic acids and lower proportions of polyunsaturated fatty acids. The fatty acid patterns of sphingomyelin, cerebroside and sulfatide of spinal cord were similar to those of cerebral white matter, while those of cauda equina contained significantly more saturated fatty acids. This suggests that the lipid and fatty acid compositions of peripheral nerve are particularly suitable for the formation of a tightly packed myelin membrane which can be a powerful shield against infections and other injuries.

Gangliosides in normal human serum. Concentration, pattern and transport by lipoproteins.

The total content and pattern of gangliosides were determined in the unfractionated sera of 11 healthy human adults and in isolated lipoproteins. The total content of lipid-bound sialic acid was 10.5 +/- 3.2 nmol/ml serum. The ganglioside profile consisted of more than ten different components. The major ganglioside was GM3, followed by GD3, GD1a, GM2, GT1b, MG-3 (sialosyllactoneotetraosylceramide), GD1b and GQ1b. Traces of four additional gangliosides could not be quantified reliably. Ganglioside patterns did not vary in sera taken from healthy adults of different age and sex. Approximately 98% of human serum gangliosides were transported by serum lipoproteins, predominantly by LDL (66%), followed by HDL (25%) and VLDL (7%). The quantitative distribution of individual gangliosides in VLDL and LDL was almost the same as that in the unfractionated serum; some differences existed with the ganglioside profile in HDL.

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.

Ganglioside Profiles of Metastases and of Metastasizing and Nonmetastasizing Rat Primary Mammary Carcinomas&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="fn3"&gt;3&lt;/xref&gt;&lt;xref ref-type="fn" rid="fn4"&gt;4&lt;/xref&gt;

The possible relationship between ganglioside levels and ganglioside profiles in malignant tumors and the formation of metastasis was investigated by the analysis of gangliosides in metastasizing SMT-2A and nonmetastasizing MT-W9a mammary carcinomas as well as in metastases formed from SMT-2A tumors. The extracted lipid of SMT-2A tumors contained 3.3-fold more lipid-bound sialic acid than did that of MT-W9a tumors. THe differences were also substantial in the ganglioside profiles in these 2 tumors. Plasma membranes isolated from SMT-2A tumors also contained 1.8-fold more lipid-bound sialic acid than did plasma membranes from MT-W9a tumors. Ganglioside profiles in two types of SMT-2A secondary tumors were investigated. The lipid-bound sialic acid content was 1.5-fold higher in tumor nodules in the lung and 1.9-fold higher in axillary lymph node tumors than it was in primary SMT-2A tumors. The ganglioside pattern in these 2 secondary tumors generally reflected that found in SMT-2A: high levels of gangliosides containing three or four sialic acid molecules. The lung nodule retained its specificity with respect to lipid-bound sialic acid content and ganglioside pattern after the lung nodule was sequentially transplanted three times to the site of the original SMT-2A tumor growth.

Possible Relationship Between Glycosphingolipids and the Formation of Metastasis in Certain Human Experimental Tumors&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;

Two tumors, human sarcoma #1 (HS #1) and human epidermoid carcinoma #3 (HEp #3), were cultured on the chorioallantoic membrane of chick embryos. Under experimental conditions, HS #1 does not metastasize, whereas HEp #3 metastasizes extensively to chick embryo lungs and other organs. The glycosphingolipid profiles of these tumors were studied and HEp #3 wad found to contain about 2.5-fold less lipid-bound sialic acid per 100 mg of total lipid extracted than did HS #1, due mainly to smaller levels of monosialoganglioside (3.7-fold) and disialoganglioside (3.8-fold) in HEp #3. The total amount of neutral glycosphingolipids was approximately the same in both tumors, but their profiles differed. Treatment of these tumors with 6,7,8,9-tetrahydro-1-mercapto-1,2,4-triazolo-[4,3-a]quinazolin-5-ol (2.5 mg/egg/tumor) completely inhibited the formation of metastases in HEp #3 and increased the total content of lipid-bound sialic acid in the tumor by 63% (hematoside, monosialoganglioside, and disialoganglioside by 71, 99, and 67%, respectively). No change was seen in the content of lipid-bound sialic acid in HS #1. Treatment of HEp #3 with a smaller dose of te quinazolinol derivative (1.25 mg/egg) caused an average of 88% inhibition of metastasis, with a 37% increase in lipid-bound sialic acid. Another compound, 2,5-diphenylthiazolo-[5,4-d]thiazole (500 microgram/egg), completely inhibited the formation of metastasis and caused a substantial increase in the amount of lipid-bound sialic acid (77%). The data showed the existence of a correlation between the level of gangliosides in HEp #3 and the ability of these tumors to metastasize.

Action of Vibrio cholerae Neuraminidase (Sialidase) upon the Surface of Intact Cells and Their Isolated Sialolipid Components

Abstract The effect of strong electrolyte upon the activity of Vibrio cholerae neuraminidase (VC neuraminidase) toward the surface of intact cells and toward their isolated, purified sialolipid components, was studied in detail. A linear dependence of the logarithm of initial velocity on the square root of ionic strength was observed for purified GM3 over a concentration range of 0 to 0.05 m NaCl in 0.01 m Tris-acetate, pH 6.5. The rate of decrease of the logarithm of the optimum initial velocity, obtained in 0.01 m Tris-acetate alone, with increasing square root of ionic strength was 3 times more rapid with GM1, GD1a, GD1b, and GT1 as substrate mixture than with the smaller sialolipid, GM3, as substrate. These data are consistent with previous results which indicate that ionic interactions upon the enzyme conformationally govern steric availability of the catalytic site. The effect of strong electrolyte upon the activity of neuraminidase toward the surface of intact cells was small in comparison with that observed with purified sialolipid substrates. Analysis of lipid extracts from untreated control and VC neuraminidase-treated samples by thin layer chromatography revealed no detectable difference between the sialolipid content of untreated control samples and samples treated with VC neuraminidase. These findings suggest that the sialolipid components in the intact cell surface are relatively unavailable to the extracellular action of VC-neuraminidase. This is supported by evidence obtained by detailed analysis of the sialyl components in four cell types (Green monkey kidney cells, Morris hepatoma, B-16 melanoma, and human neuroblastoma) before and after VC neuraminidase treatment. The total cellular sialic acid content of these cell types, untreated, varied from 2.3 to 7.1 µg of sialic acid per mg of protein; lipid-bound sialic acid content varied from 0.7 to 1.6 µg of sialic acid per mg of protein. Progress curves for sialic acid release from the surface of intact cells were hyperbolic and had reached a plateau within approximately 30 min. In the plateau region approximately 20% of the total cellular sialic acid had been released consistently, in each case. Analysis of the lipid-bound sialic acid content of the VC neuraminidase-treated intact cells revealed no major changes, all values being greater than 90% of the untreated, control values. The sialolipid composition of each cell type was examined by thin layer chromatography and found nevertheless to consist predominately of VC neuraminidase-labile components. Therefore, the sialolipids of the intact cell surface appear to be more deeply localized so as to be sterically inaccessible to VC neuraminidase, and release of sialic acid from intact cells by VC neuraminidase appears to be from surface sialoglycoproteins.