Complex Gangliosides Research Articles

Gangliosides of cultured cells of a rat mammary carcinoma cell line.

Ganglioside content of rat mammary carcinoma-derived cells grown in layers in vitro was nearly as high as that of apical membrane-derived milk fat globule membrane and nearly four time higher than the content of normal, lactating mammary tissue on a protein basis. The major ganglioside of these carcinoma-derived cells was identified as GDla (sialic acid-Gal-GalNAc-sialic acid)-Gal-Glc-Cer. Relative to carcinoma-derived cells, rat mammary tissue and milk fat globule membrane had more complex ganglioside patterns but appeared to lack substantial quantities of GDla.

Gangliosides and their cell density-dependent changes in control and chemically transformed C3H/10T1/2 cells

The cloned C3H/10T1/2 mouse embryo cells contained a complex pattern of gangliosides. Two cloned chemical transformants obtained from the C3H/10T1/2 cell line by treatment with 7,12-dimethylbenz(a) anthracene (DMBA-TCL1) and 3-methylcholanthrene (MCA-TCL15) also had complex ganglioside patterns; but the transformants had increased levels of the simplest ganglioside, N-acetylneuraminylgalactosylglucosylceramide (GM 3), and reduced levels of more complex gangliosides. Incorporation of [ 14C]glucosamine into gangliosides, as cell-to-cell contact increased in C3H/10T1/2 cells, showed that GM 3 synthesis was decreased and that the synthesis of the more complex ganglioside N-acetylneuraminylgalactosyl- N-acetylgalactosaminyl-( N-acetylneuraminyl)-galactosylglucosylceramide (GD 1a) was increased. In the two transformants the percentage each individual ganglioside was of total labeled gangliosides was only slightly altered with changing cell density. Turnover of [ 14C]glucosamine-labeled gangliosides, as cell density increased, was approximately equal in C3H/10T1/2 cells and MCA-TCL15 cells, but more rapid in the DMBA-TCL1 cells. Most individual gangliosides turned over at about the same rate in the respective cell lines. However, GD 1a increased slightly as a percentage of total labeled gangliosides with increasing cell density in both C3H/10T1/2 cells and transformed cells. The labeling data indicated that the majority of GD 1a synthesis was de novo and only a small part occurred by transfer of sialyl or glycosyl residues to simpler gangliosides or catabolism of more complex gangliosides already present in the outer membrane. Exogenous complex gangliosides added to the medium were more effective inhibitors of DMBA-TCL1 cell growth than of C3H/10T1/2 cell growth. Furthermore, gangliosides added to exponentially growing C3H/10T1/2 and DMBA-TCL1 cells caused both cell lines to incorporate a greater percentage of [ 14C]glucosamine into gangliosides more complex than GM 3.

Similar content of phospholipids and gangliosides in normal and homozygous familial hypercholesterolemia fibroblasts.

The cellular content of total and individual phospholipids and gangliosides was measured in fibroblasts cultured from four normal subjects, three patients with lysosomal lipid storage diseases, and two subjects with homozygous familial hypercholesterolemia. Measurements were made on cells grown in medium containing fetal calf serum under conditions in which normal cells derive cholesterol for cell growth from low density lipoprotein present in the fetal calf serum, whereas familial hypercholesterolemia homozygote cells, which lack cell surface low density lipoprotein receptors, derive cholesterol from endogenous synthesis. No difference was observed in the cellular content of total or individual phospholipids and gangliosides in the normal and familial hypercholesterolemia homozygote cells. In contrast, cells from a patient with Niemann-Pick disease and a patient with Sandhoff disease showed elevations in the content of sphingomyelin and complex gangliosides, respectively.

Interaction of human chorionic gonadotropin with membrane components of rat testes.

Previous studies demonstrating that gangliosides interacted with thyrotropin and human chorionic gonadotropin (hCG) suggested that gangliosides participate in the transduction of the hormonal message across the target cell membrane. As a continuation of these investigations, we examined the effects of down-regulation of hCG receptors on the interaction of hCG with rat testis membrane components. Rat testes contained a complex ganglioside pattern that did not appear to change qualitatively or quantitatively after the injection of hCG into the animals, although testis membranes from hCG-treated rats lost their capacity to bind (125)I-labeled hCG ((125)I-hCG). Gangliosides extracted from the testes of control and treated animals were equally effective inhibitors of (125)I-hCG binding to testis membranes. However, inhibition of binding was observed only under conditions (pH 6.0, low ionic strength) such that unlabeled hCG (>2500-fold excess) did not block (125)I-hCG binding, and (125)I-hCG bound similarly to testis membranes from control and treated rats. Under conditions such that hCG binding was specific (blocked by 250-fold excess of unlabeled hCG), testis gangliosides were noninhibitory. Liposomes containing gangliosides from the testes of control or hCG-treated rats bound similar small amounts of (125)I-hCG. These same liposomes bound 50 and 1000 times more thyrotropin and cholera toxin, respectively, than hCG. Oligosaccharides derived from gangliosides did not inhibit (125)I-hCG binding to testis membranes nor did they alter the fluorescence of hCG conjugated with fluorescent probes, whereas the gangliosides themselves were inhibitory and enhanced the fluorescence intensity of the hCG derivatives. Exposure of testis membranes from hCG-treated rats to 4 M MgCl(2), which displaces bound hCG [Chen, Y.-D. I. & Payne, A. H. (1977) Biochem. Biophys. Res. Commun. 74, 1589-1596], did not restore their ability to bind (125)I-hCG. When membranes were solubilized with Triton X-100, a solubilized receptor was detected from testis membranes of control but not hCG-treated rats. These findings and the absence of demonstrable changes in the composition or quantity of rat testis gangliosides when hCG receptors are down-regulated suggest that gangliosides do not represent the primary binding determinants of hCG receptors.

GANGLIOSIDE COMPOSITION AND BIOSYNTHESIS IN CULTURED CELLS DERIVED FROM CNS

Abstract— Cultured mouse neuroblastoma cells (clone N18) contained a homologous series of gangliosides, GM3, GM2, GM1 and GD1a; the total lipid bound sialic acid (LBSA) was 3.3 nmol per mg of protein, of which GD1a comprised two‐thirds. In contrast, neonatal hamster astrocytes (clone NN) and human glioblastoma cells (Cox clone) contained mainly GM3, which represented 95% of the 2 nmol of LBSA per mg protein in these cells. When the cells were grown in the presence of [14C]galactose, label was incorporated into all of the gangliosides isolated from the cells. The labeling pattern corresponded to the ganglioside composition of the cell lines; GD1a was more extensively labeled in N18 cells and GM3 was the major labeled ganglioside extracted from glial cells. In addition to in rivo biosynthesis, in vitro synthesis of gangliosides was also determined. The activities of five glycosyltransferases of the ganglioside biosynthetic pathway were measured in homogenates of the three cell lines. The neuroblastoma cells contained all five enzyme activities whereas the two glial cell lines were deficient in UDP‐N‐acetylgalactosamine: GM3N‐acetylgalactosaminyltransferase activity, which catalyzes the synthesis of GM2 from GM3. The results indicated that cells of neuronal origin contain the more complex gangliosides associated with CNS and the requisite biosynthetic enzymes and that cells of glial origin are missing these complex gangliosides and the key glycosyltransferase required for their synthesis.

Synthesis and uptake of gangliosides by choleragen-responsive human fibroblasts

Human fibroblasts, cultured in medium containing 10% fetal calf serum, responded dramatically to choleragen with an increase in cyclic adenosine monophosphate content to greater than 48 times basal levels. Analysis of these cells for gangliosides indicated that the major ganglioside was N-acetylneuraminylgalactosylglucosylceramide (GM3) with trace amounts (less than or equal to 100 pmol/mg of protein) of other gangliosides including GM1, the putative choleragen receptor. Although the cells contained three glycosyltransferases required for ganglioside synthesis, the N-acetylgalactosaminyltransferase activity necessary for the conversion of GM3 to more complex gangliosides was not detected. When the cells were grown in medium containing [14C]galactose or N-acety[3H]mannosamine, however, all of the gangliosides became labeled, indicating that the cells can synthesize complex gangliosides. Although fetal calf serum contains gangliosides including GM1, [3H]GM1 was taken up poorly from the growth medium and uptake at the rate observed could have accounted for less than 2% of the GM1 content of the cells. When the cells were incubated in chemically defined medium containing [3H]GM1 at the concentrations present in fetal calf serum, rapid uptake of the ganglioside occurred and the total GM1 content of the cells increased threefold in less than 3 h. Thus, although the cells are capable of binding exogenous gangliosides, the gangliosides in fetal calf serum are in a form not readily available to the cells.

Ganglioside content and pattern in human gliomas in culture

The ganglioside level and pattern of human gliomas in monolayer cultures were examined. These gliomas revealed morphological variations that correlated with several features of ganglioside analysis. Glioblastoma lines TC 178 and TC 501 that morphologically had changed during extended subculture revealed reduced amounts and a simplified pattern of gangliosides with almost total loss of the characteristic brain complex gangliosides. In contrast, two glioblastoma lines TC 526 and TC 593, as well as the oligodendroglioma line TC 620 showed brain-like gangliosides and the cells in these cultures had maintained their characteristic morphology observed during early subcultures. The possibility that altered ganglioside levels occur in conjunction with morphological changes after propagation in vitro is discussed.

Relationship of gangliosides to the structure and function of thyrotropin receptors: their absence on plasma membranes of a thyroid tumor defective in thyrotropin receptor activity.

Plasma membranes derived from rat thyroid tumor (1-8R) which is unresponsive to thyrotropin but is responsive to dibutyryl adenosine 3':5'-cyclic monophosphate bind less than 20% of the [125I] thyrotropin which can be bound to plasma membranes from normal rat thyroids under conditions which optimize tumor membrane binding relative to normal thyroid membranes. In addition, the binding is different from thyrotropin binding to normal thyroid membranes both in its altered sensitivity to changes in hydrogen ion concentration and in a decreased sensitivity to competition by unlabeled thyrotropin. This reduced capacity to bind [125I] thyrotropin cannot be attributed to degradation of the hormone by membrane-associated proteases. Although the supernatant phase of the thyroid tumor homogenates contains a soluble component which inhibits [125I] thyrotropin binding to thyrotropin receptors on plasma membranes, its level is the same as in homogenates of normal thyroid tissue. Trypsin digestion does not expose thyrotropin receptors in a manner analogous to that seen in normal thyroid tissue. The major ganglioside in the tumor membranes is N-acetylneuraminylgalactosylglucosylceramide and the membranes lack the N-acetylgalactosaminyltransferase required for the synthesis of more complex gangliosides. In contrast, the normal rat thyroid membranes contain more complex gangliosides such as galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide and N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide as well as the glycosyltransferase activities required for their syntheses. Galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide can also be detected in normal membranes, but not in tumor membranes, by selective labeling with galactose oxidase (D-galactose: oxygen 6-oxidoreductase, EC 1.1.3.9) and [3H] sodium borohydride. These results support the hypothesis that gangliosides are important structural or functional components of thyrotropin receptors on thyroid plasma membranes.

Abnormalities in the glycosphingolipid content of human Pk and p erythrocytes.

Erythrocytes of the rare Pk phenotype lack the blood group P antigen, and p erythrocytes lack both P and Pk antigens. On the basis of immunological data we suggested previously that the P and Pk antigens are the glycosphingolipids globoside and trihexosyl ceramide, respectively, and we have now confirmed these designations by chemical analysis of erythrocytes lacking these antigens. The Pk erythrocytes contain only traces of globoside and have a marked excess of trihexosyl ceramide in comparison with normal erythrocytes. The p erythrocytes lack globoside and trihexosyl ceramide and contain an excess of lactosyl ceramide and other complex glycolipids. Our analyses of normal erythrocytes also revealed complex gangliosides with the approximate chromatographic mobilities of GD1b and GT1, and several gangliosides containing N-acetylglucosamine.

Gangliosides of the human gastrointestinal mucosa

Gangliosides of the human alimentary mucosa were purified and analysed with thin-layer chromatography and gas chromatography. The content of ganglioside neuraminic acid was 0.16 μmol/g dry weight in the stomach, 0.07 μmol/g dry weight in the small intestine and 0.11 μmol/g dry weight in the large intestine. Mono- and disialosylhemosides were the major gangliosides, on a molar basis 68% of the total found in the stomach and 44% of the total in the small and large intestine. Considerable amounts of more complex gangliosides were found, especially in the small and large intestine, in which the molar content of tri- and tetraglycosylgangliosides containing galactosamine made up 38% of the total. Two glucosamine-containing gangliosides were also found, the probable structures of which were mono- and disialotetraglycosylceramide. The presence of the latter is reported for the first time.

Synthesis of surface glycoproteins and glycosphingolipids in db-cAMP-treated normal and virus-transformed 3T3 cells

Addition of dibutyryl cyclic AMP (db-cAMP) plus theophylline to normal or virus-transformed 3T3 mouse fibroblasts, in concentrations sufficient to alter the growth characteristics, produced no qualitative changes in the pattern of incorporation of glucosamine (Glc.NH 2) into glycoproteins at their extreme periphery. Nor did these drugs qualitatively alter the patterns of incorporation of 1- 14C-galactose (Gal) into the principal glycosphingolipids (GSL) of these cells. In particular, virus-transformed 3T3 cells, which do not synthesize certain complex gangliosides made by normal cells, were not caused to do so. It may, however, be significant that drug treatment did result in increased incorporation of precursor galactose into complex heteroglycans of both normal and virus-transformed cells. The implications of these observations are discussed.

Circular dichroism of gangliosides from normal and Tay-Sachs tissues

The circular dichroism (CD) of mammalian gangliosides and their derivatives has been studied in the ultraviolet region from 185 to 300 mμ. Four major CD bands contribute in varying proportions to the resultant curve, two from the amino sugar (GalNAc) chromophore [(−) at ∼ 210 mμ and (+) at ∼ 187 mμ)] and two from the sialic acid (NANA or NGNA) components [(−) at ∼ 220 mμ and (−) at 196 to 202 mμ]. Comparison of optical parameters derived from the CD curves reveals characteristic differences between G M3 and the more complex ganglioside structures, and between the two disialogangliosides, GD1a and GD1b. Ganglioside G M2, which accumulates in the brain of children with Tay-Sachs disease, has conformations similar to the G M2 prepared from a mixture of normal rat brain gangliosides on the basis of their optical properties. Furthermore, the conformation of the ketosidic bond in CMP-NANA, the intermediate in sialyl transferase reactions, is α as judged by the sign of its 196 mμ sialyl-CD band.

Gangliosides of Cultured Mouse Cells: PARTIAL CHARACTERIZATION AND DEMONSTRATION OF 14C-GLUCOSAMINE INCORPORATION

Abstract The gangliosides of two types of mouse cells in tissue culture, L-929 cells and secondary cultures of mouse embryo cells, have been investigated. Gangliosides were identified tentatively by comparison of the chromatographic behavior of the intact gangliosides and their products of hydrolysis in mild acid with standards of known composition. Monosialogangliosides predominated in L cells whereas the secondary cultures also contained more complex gangliosides. The calf serum used for culturing the cells contained gangliosides but the pattern differed from those of the two types of cells. Both L cell and secondary cultures incorporated significant amounts of 14C-glucosamine into all of their gangliosides. The 14C-glucosamine was found to be principally distributed in the galactosamine, N-acetyl-, and N-glycolylneuraminic acid moieties of the gangliosides. These latter results suggest that the gangliosides are synthesized—at least in part—by the cells de novo. In addition, mono-, di-, and triglycosylceramides were detected in the L cell cultures, whereas the secondary cultures contained mono-, di-, and tetraglycosylceramides.