Structures of blood group glycosphingolipids of human small intestine. A relation between the expression of fucolipids of epithelial cells and the ABO, Le and Se phenotype of the donor

Small intestinal epithelial cells (enterocytes) were isolated from specimens obtained at operation from four human individuals with different blood group ABO, Lewis, and secretor phenotypes. The non-acid glycolipids were isolated and characterized by thin-layer chromatography, mass spectrometry, and proton NMR spectroscopy and for reactivity with monoclonal antibodies on thin-layer chromatograms. Monohexosylceramides and blood group ABH (type 1 chain) and Lewis glycolipids with 5-7 sugar residues were the major compounds present in all cases, and the expression of the major blood group glycolipids was in agreement with the ABO, Lewis, and secretor phenotype of the individual donors. Small amounts of more complex glycolipids with up to 10 sugar residues were identified by mass spectrometry in all cases. In addition, small amounts of lactotetraosylceramide, a blood group H-active triglycosylceramide with the structure of Fuc alpha 1-2Gal-Hex-Cer (where Fuc is fucose, Hex is hexose, and Cer is ceramide), and dihexosylceramides were identified in some cases. Globotriaosyl- and globotetraosylceramides were absent from the epithelial cells. Small amounts of Leb-active glycolipids in blood group OLe(a+b-), non-secretor and OLe(a-b-), secretor individuals as well as trace amounts of type 2 carbohydrate chain compounds in all individuals were detected by specific monoclonal antibodies.     

Chemical and immunological identification of glycolipid-based blood group ABH and Lewis antigens in human kidney

A polar non-acid glycolipid fraction has been isolated from human kidney. It was shown by thin-layer chromatography to be a mixture of glycolipids having more than four carbohydrate residues. Immunological testing revealed strong blood group Lea and A activity together with weak Leb, P1 and B activity. Mass spectrometry of the permethylated and permethylated-reduced (LiAlH4) glycolipid fraction showed that the two major components were a five sugar fucolipid (isomer of Lea) and a glycolipid having four hexoses and one N-acetylhexosamine. In addition, blood group Leb, B and A type hexaglycosylceramides were present. Evidence for small amounts of more complex glycolipids was also found. Acid degradation and gas chromatography of the native fraction revealed fucose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. This is the first chemical isolation and characterization of complex blood group active glycolipids in human kidney. The existence of these molecules is discussed in view of their possible role as transplantation antigens.     

Blood group A glycolipid antigen expression in kidney, ureter, kidney artery, and kidney vein from a blood group A1Le(a-b+) human individual. Evidence for a novel blood group A heptaglycosylceramide based on a type 3 carbohydrate chain

Kidney, ureter, kidney artery, and kidney vein tissue were obtained from a single human transplant specimen. The donors erythrocyte blood group phenotype was A1Le(a-b+). Total non-acid glycolipid fractions were isolated and individual glycolipid components were identified by immunostaining thin layer plates with a panel of monoclonal antibodies and by mass spectrometry of the permethylated and permethylated-reduced total glycolipid fractions. The dominating glycolipids in all tissues were mono- to tetraglycosylceramides. In the kidney, ureter, and artery tissue less than 1% of the glycolipids were of blood group type, having more than 4 sugar residues. In contrast, 14% of the vein glycolipids were of blood group type, and the dominating components were type 1 chain blood group H pentaglycosylceramides and A hexaglycosylceramides. Trace amounts of structurally different blood group A glycolipids (type 1 to 4 core saccharide chains) with up to 10 sugar residues were found in the kidney, ureter, and vein tissues, including evidence for a novel blood group A heptaglycosylceramide based on the type 3 chain in the vein. The only detected A glycolipid antigen in the artery tissue was the blood group A difucosyl type 1 chain heptaglycosylceramide (ALeb) structure. Blood group Lewis and related antigens (Lea, Leb, and ALeb) were expressed in the kidney, ureter, and artery, but were completely lacking in the vein, indicating that the Le gene-coded alpha 1-4-fucosyltransferase was not expressed in this tissue. The X and Y antigens (type 2 chain isomers of the Lea and Leb antigens) were detected only in the kidney tissue.     

Comparison of the glycolipid-binding specificities of cholera toxin and porcineEscherichia coli heat-labile enterotoxin: identification of a receptor-active non-ganglioside glycolipid for the heat-labile toxin in infant rabbit small intestine

The binding specificities of cholera toxin andEscherichia coli heat-labile enterotoxin were investigated by binding of¹²⁵I-labelled toxins to reference glycosphingolipids separated on thin-layer chromatograms and coated in microtitre wells. The binding of cholera toxin was restricted to the GM1 ganglioside. The heat-labile toxin showed the highest affinity for GM1 but also bound, though less strongly, to the GM2, GD2 and GD1b gangliosides and to the non-acid glycosphingolipids gangliotetraosylceramide and lactoneotetraosylceramide. The infant rabbit small intestine, a model system for diarrhoea induced by the toxins, was shown to contain two receptor-active glycosphingolipids for the heat-labile toxin, GM1 ganglioside and lactoneotetraosylceramide, whereas only the GM1 ganglioside was receptor-active for cholera toxin. Preliminary evidence was obtained, indicating that epithelial cells of human small intestine also contain lactoneotetraosylceramide and similar sequences. By computer-based molecular modelling, lactoneotetraosylceramide was docked into the active site of the heat-labile toxin, using the known crystal structure of the toxin in complex with lactose. Interactions which may explain the relatively high toxin affinity for this receptor were found.Abbreviations CT cholera toxin - CT-B B-subunits of cholera toxin - LT Escherichia coli heat-labile enterotoxin - hLT humanEscherichia coli heat-labile enterotoxin - pLT porcineEscherichia coli heat-labile enterotoxin - EI electron ionization     

Histochemical localization and analysis of blood group-related antigens in human pancreas using immunostaining with monoclonal antibodies and exoglycosidase digestion

We examined the distribution of blood group-related antigens using an indirect immunoperoxidase method with monoclonal antibodies (MAb) directed to A, B, H, Lewis a (Lea), Lewis b (Leb), Lewis x (Lex), and Lewis y (Ley) antigens and Type 1 precursor chain in human pancreas. Effects of prior digestion with exoglycosidases on MAb stainings were simultaneously investigated. A, B, H, Leb, and Ley antigens were detected in acinar cells and interlobular duct cells but not in centroacinar cells, intercalated duct cells, and islet of Langerhans cells. The expression of these antigens in acinar cells was not dependent on Lewis type and secretor status of the tissue donors, whereas that in interlobular duct cells was strictly dependent on secretor status. The distribution pattern of these antigens in acinar cells was not homogeneous, i.e., cells producing H antigens expressed both Leb and Ley antigens but not A or B antigens, whereas those producing A or B antigens did not secrete Leb and Ley as well as H antigens. Digestion with alpha-N-acetylgalactosaminidase or alpha-galactosidase resulted in the appearance of Leb and Ley antigens as well as H antigen in acinar cells producing A and/or B antigens. Type 1 precursor chain was not detected in pancreatic tissues from secretors but appeared in acinar cells producing H antigen after alpha-L-fucosidase digestion, which also disclosed Lex but not Lea antigen in acinar cells expressing both Leb and Ley. In some non-secretors, MAb against Type 1 precursor chain reacted with acinar cells without enzyme digestion. Although Lea antigen was not detected in acinar cells, it was found in centroacinar cells, intercalated duct cells, and interlobular duct cells from all individuals examined except two Le(a-b-) secretors. After sialidase digestion, Lex antigen appeared in centroacinar and intercalated duct cells from some individuals. Sialidase digestion also elicited reactivity with MAb against Type 1 precursor chain in islet of Langerhans cells from some individuals. These results demonstrate the complexity in the pattern of expression and regulation of blood group-related antigens in different cell types of human pancreas. Such complexity may largely be ascribed to differences in individual genotypes and in gene expression patterns of different cell types.     

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.     

Incorporation and metabolism of ganglioside GM2 in skin fibroblasts from normal and GM2 gangliosidosis subjects

Ganglioside GM2, 3H-labeled in the sphingoid base, was added to the culture medium of normal and GM2 gangliosidosis fibroblasts. Ganglioside was found to adsorb rapidly to the cell surface, most of it could however be removed by trypsination. The trypsin-resistant incorporation was about 10 nmol/mg cell protein, after 48 h. The rates of adsorption and incorporation depended strongly on the concentration of fetal calf serum in the medium, higher serum concentrations being inhibitory. After various incubation times, the lipids were extracted, separated by thin-layer chromatography and visualized by fluorography. In normal cells a variety of degradation products as well as sphingomyelin was found whereas in GM2 gangliosidosis cells, only trace amounts of such products (mainly GA2) were found. In contrast, the higher gangliosides GM1 and GD1a were formed in comparable amounts (2.2-3.6% of total radioactivity after 92 h) in normal and pathologic cell lines. Supplementation of cells from GM2 gangliosidosis, variant AB, with purified GM2-activator protein restored ganglioside GM2 degradation to almost normal rates but had no effect on its glycosylation to gangliosides GM1 and GD1a. From these results we conclude that the synthesis of higher gangliosides from incorporated GM2 can occur by direct glycosylation and not only via lysosomal degradation and resynthesis from [3H]sphinganine-containing degradation products. Preliminary studies with subcellular fractionation after various times of [3H]ganglioside incorporation indicated biphasic kinetics for the net transport of membrane-inserted ganglioside to lysosomes, compatible with the notion that a portion of the glycolipids can also escape from secondary lysosomes and migrate to Golgi compartment or cell surface.     

Asymmetric distribution of gangliosides in rat renal brush-border and basolateral membranes

Highly enriched brush-border and basolateral membranes isolated from rat renal cortex were used to study the distribution of endogenous gangliosides in the two distinct plasma membrane domains of epithelial cells. These two membrane domains differed in their glycolipid composition. The basolateral membranes contained more of both neutral and acidic glycolipids, expressed on a protein basis. In both membranes, the neutral glycolipids corresponding to mono-, di-, tri- and tetraglycosylceramides were present. The basolateral membranes contained more diglycosylceramide than the brush-border membranes. The major gangliosides found were GM4, GM3, and GD3 with minor amounts of GM1 and GD1a. The latter were identified and quantified by sensitive iodinated cholera toxin binding assays. When the distribution of individual gangliosides was calculated as a percent of total gangliosides, the brush-border membranes were enriched with GM3, GM1 and GD1a compared to the basolateral membranes, which were enriched with GD3 and GM4. The observation of a distinct distribution of glycolipids between brush-border and basolateral membranes of the same epithelial cell suggests that there may be a specific sorting and insertion process for epithelial plasma membrane glycolipids. In turn, asymmetric glycolipid biogenesis may reflect differences in glycolipid function between the two domains of the epithelial plasma membrane.     

Tissue-specific expression of GM3(NeuGc) and GD3(NeuGc) in epithelial cells of the small intestine of strains of inbred rats. Absence of NeuGc in intestine and presence in kidney gangliosides of brown Norway and spontaneously hypertensive rats

The ganglioside composition of the epithelial cells of the small intestine was investigated in 15 strains of inbred rats. Most of these strains had GM3 as the only detectable ganglioside. In addition to GM3, small amounts of GD3 were found in four strains, AVN, BN, DA, and LE. The fatty acid content of the ceramide portion was composed of a large, although variable, percentage of 2-hydroxy fatty acids. The sphingoid base was always C18-4D-hydroxysphinganine. The highly prominent sialic acid was N-glycolylneuraminic acid (NeuGc) in most strains. However in two strains, Brown Norway (BN) and spontaneously hypertensive rats (SHR), NeuAc was the only sialic acid of the gangliosides of the intestinal epithelium. The analysis of the ganglioside composition of the epithelium of the small intestine of the first generation hybrids of SHR with DA and BN, respectively, demonstrated that the expressions of GM3 (NeuGc) and GD3 were genetically transmitted as dominant traits and that BN and SHR were likely to carry the same deficient gene that led to the expression of GM3(NeuAc) instead of GM3(NeuGc) in the small intestine. For comparison, the sialic acid composition of kidney gangliosides was analyzed in some strains. 21-23% of the kidney gangliosides was GM3(NeuGc) in all tested strains, including BN and SHR. Therefore, the ganglioside composition of the intestinal epithelium could vary in the rat species, and the defect of N-glycolylneuraminic acid was not only strain-specific but also occurred in a tissue-specific way among strains of inbred rats.     

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.     

Specific ganglioside changes in extraneural tissues of adult rats with hypothyroidism

Adults rats with hypothyroidism were prepared by administration of 6-propyl-2-thiouracil (PTU) or methimazole, and the tissues were examined for their gangliosides through methods including glycolipid-overlay techniques. Normal thyroid tissue contained GM3, GD3, and GD1a as the major gangliosides, with GM1, GD1b, GT1b, and GQ1b in lesser amounts. The goitrous tissue of PTU-induced hypothyroid rats had higher concentrations of GM1 and GD1a with a concomitant decrease of GM3. The amount of GT3 in thyroid tissue was increased in hypothyroid animals. While normal liver tissue had a complex ganglioside pattern with a- and b-series gangliosides, the PTU-induced hypothyroid tissue showed a simpler ganglioside profile that consisted mainly of a-series gangliosides with almost undetectable amounts of b-series gangliosides. The expression of c-series gangliosides was suppressed in the hypothyroid liver tissue. Heart tissue had higher contents of GM3 and GT3 than control. No apparent change was observed in the compositions of major and c-series gangliosides in other extraneural tissues (i.e., kidney, lung, spleen, thymus, pancreas, testis, skeletal muscle, and eye lenses), and neural tissues (i.e., cerebrum and cerebellum) from PTU-induced hypothyroid rats. The ganglioside changes of thyroid, liver, and heart tissues were reproduced in corresponding tissues of methimazole-induced hypothyroid rats. These results suggest that hypothyroid conditions affect the biosynthesis and expression of gangliosides in specific tissue and cell types.     

Ganglioside patterns and cholera toxin--peroxidase labeling of aggregating cells from the chick optic tectum.

2The ganglioside compositions of the chick optic tectum and aggregating tectal cell cultures were examined. Both showed similar trends in changes in ganglioside patterns during development. GD3 and GD1b were the predominant gangliosides early in development, while GD1a and several other multisialogangliosides increased in relative amounts with increasing age in vivo and in vitro. Four gangliosides were present early in development which have not previously been reported. These gangliosides are not present at later developmental times suggesting a possible role for them during the critical early stages of nervous tissue differentiation. Some differences were noted when comparing in vivo versus in vitro ganglioside patterns; these differences may possibly be due to the lack of normal retinotectal connections in the cultures. Cytochemical studies on the localization of the presumed cholera toxin--peroxidase binding site GM1 showed conjugate binding correlates with increasing levels of GM1 in the cultures. In older cultures, the conjugate was uniformly localized on all cells and processes in the aggregates. The conjugate also bound to synaptic membranes and intensely stained the synaptic cleft. This latter observation suggests an enrichment of GM1 in the synaptic cleft region.     

Subcellular distribution and biosynthesis of rat liver gangliosides

Gangliosides have generally been assumed to be localized primarily in the plasma membrane. Analysis of gangliosides from isolated subcellular membrane fractions of rat liver indicated that 76% of the total ganglioside sialic acid was present in the plasma membrane. Mitochondria and endoplasmic reticulum fractions, while containing only low levels of gangliosides on a protein basis, each contained approx. 10% of total ganglioside sialic acid. Gangliosides also were present in the Golgi apparatus and nuclear membrane fractions, and soluble gangliosides were in the supernatant. Individual gangliosides were non-homogeneously distributed and each membrane fraction was characterized by a unique ganglioside composition. Plasma membrane contained only 14 and 28% of the total GD1a and GD3, respectively, but 80-90% of the GM1, GD1b, GT1b and GQ1b. Endoplasmic reticulum, when corrected for plasma membrane contamination, contained only trace amounts of GM1, GD1b, GT1b and GQ1b, but 11 and 5% of the total GD1a and GD3, respectively. The ganglioside composition of highly purified endoplasmic reticulum was similar. Ganglioside biosynthetic enzymes were concentrated in the Golgi apparatus. However, low levels of these enzymes were present in the highly purified endoplasmic reticulum fractions. Pulse-chase experiments with [3H]galactose revealed that total gangliosides were labeled first in the Golgi apparatus, mitochondria and supernatant within 10 min. Labeled gangliosides were next observed at 30 min in the endoplasmic reticulum, plasma membrane and nuclear membrane fractions. Analysis of the individual gangliosides also revealed that GM3, GM1, GD1a and GD1b were labeled first in the Golgi apparatus at 10 min. These studies indicate that gangliosides synthesized in the Golgi apparatus may be transported not only to the plasma membrane, but to the endoplasmic reticulum and to other internal endomembranes as well.     

Receptor ganglioside content of three hosts for Sendai virus. MDBK, HeLa, and MDCK cells

Specific gangliosides GD1a, GT1b and GQ1b isolated from brain have been shown to function as receptors for Sendai virus by conferring susceptibility to infection when they are incorporated into receptor-deficient cells (Markwell, M.A.K., Svennerholm, L. and Paulson, J.C. (1981) Proc. Natl. Acad. Sci. USA 78, 5406-5410). The endogenous gangliosides of three commonly used hosts for Sendai virus: MDBK, HeLa, and MDCK cells were analyzed to determine the amount and type of receptor gangliosides present. In all three cell lines, GM3 was the major ganglioside component. The presence of GM1, GD1a and the more complex homologs of the gangliotetraose series was also established. In cell lines derived from normal tissue, MDBK and MDCK cells, gangliosides contributed 47-65% of the total sialic acid. In HeLa cells, gangliosides contributed substantially less (17% of the total sialic acid). The ganglioside content of each cell line was shown not to be immutable but instead to depend on the state of differentiation, passage number, and surface the cells were grown on. Thus, the ganglioside concentration of undifferentiated MDCK cells was found to be substantially greater than that of MDBK or HeLa cells, but decreased as the MDCK cells underwent differentiation. Changes in culture conditions that were shown to decrease the receptor ganglioside content of the cells resulted in a corresponding decrease in susceptibility to infection. The endogenous oligosialogangliosides present in susceptible host cells were shown to function as receptors for Sendai virus.     

The Lewis blood group system among Chinese in Taiwan.

The nonsecretor gene se is absent (or very rare) among Chinese in Taiwan and the previously reported Le(a+b-) phenotype in this population is in fact Le(a+b+) as proven by the presence of small amounts of Leb antigen on red blood cells. Salivary ABH substances in this phenotype are usually (although not always) markedly reduced. The Chinese Le(a+b+) phenotype is postulated to be the result of a weak secretor gene Se omega. Although the Le(a+b+) phenotype is very rare in Caucasians, it has a frequency of 25% in Chinese. All Le(a-b-) Chinese are ABH secretors and have varying amounts of Lea and/or Leb substances in saliva.     

Selective changes in gangliosides of human milk during lactation: a molecular indicator for the period of lactation

Gangliosides of human milk from women at various periods of lactation were analyzed. GD3 in colostrum, particularly in the early period of lactation, was the major ganglioside, and the molar ratio of GM3 to GD3 was 0.2-0.3 in the milk at 2-6 days postpartum. In contrast, milk from women at 60-390 days postpartum contained GM3 as the major ganglioside and the molar ratio of GM3 to GD3 was more than 3. Milk at 8-40 days postpartum represented an intermediate stage in terms of the ratio of GM3 to GD3. The selective change in the molar ratio of gangliosides was observed as a phenomenon common to all human milk from different individuals at different periods of lactation, indicating that the periods of lactation can be defined on the basis of the ratio. Since glycolipids in human milk are preferentially localized in the milk fat globule membrane, which is derived from the plasma membrane of epithelial cells in the mammary gland, the changes in the ganglioside composition reported in this communication may reflect a qualitative change of the cells in the mammary gland.     

Glycosphingolipids of human large intestine: detailed structural characterization with special reference to blood group compounds and bacterial receptor structures

Non-acid glycosphingolipid expression was studied in the large intestines from four individuals with the A1Le(a-b+), BLe(a-b+), and OLe(a-b+) blood group phenotypes. In the A1Le(a-b+) case, specimens were taken from the ascending and sigmoid parts of the large intestine in order to compare the expression of glycolipids in the proximal and distal regions of the intestine. In one blood group OLe(a-b+) individual, epithelial cells were isolated from the residual stroma to compare the glycolipid compositions in these two tissue compartments. GlcCer, GalCer, LacCer, Gb3Cer, and Gb4Cer were the major compounds in all three individuals, as shown by mass spectrometry, proton NMR spectroscopy, and degradation studies. The Lea-5 glycolipid was the major complex blood group glycolipid in all individuals, except in the proximal ascending part of the large intestine of the A1Le(a-b+) case, in which the Leb-6 glycolipid was predominant. There were trace amounts of blood group ABH glycolipids, in agreement with the ABO blood group phenotypes of the donors, Lewis antigens with more than six sugar residues in the carbohydrate chain, and blood group X and Y glycolipid antigens. The epithelial cells were dominated by monoglycosylceramides and the Lea-5 glycolipid, while only trace amounts of di-, tri-, and tetraglycosylceramide structures were present. No reactivity was seen in the epithelial cell fraction with Gal alpha 1-4Gal specific Escherichia coli, anti-Pk, or anti-P antibodies, indicating the absence of the glycolipid-borne Gal alpha 1-4Gal sequence in human large intestinal epithelial cells.     

Neutral glycolipids and gangliosides of concanavalin A-selected SV3T3 revertant cells and of normal and SV40-transformed Balb/c 3T3 cells.

The structural analysis of neutral glycolipids and gangliosides of the SV40 transformed Balb/c3T3 cells (SV3T3 cells) and concanavalin A-selected SV3T3 revertant cells, both compared with untransformed Balb/c3T3 cells, has shown: (i) a content of neutral glycolipids in revertant cells near to that found in the untransformed parental cells; (ii) a similar decrease of the higher gangliosides in transformed and revertant cells; (iii) a content of ganglioside GM3 in revertant cells much higher than that found in both SV3T3 and untransformed Balb/3T3 cells. The possible role of ganglioside GM3 in growth control is discussed.     

Gangliosides modulate sodium transport in cultured toad kidney epithelia

Cultured A6 epithelial cells from toad kidney form confluent monolayers with tight junctions separating the apical and basolateral membranes. These two membrane domains have distinct compositions and functions. Thus, sodium is actively transported across the epithelia from the apical to basolateral surface via amiloride-inhibitable sodium channels located in the apical membrane. Sodium transport is stimulated by vasopressin, cholera toxin, and 8-bromo-cAMP applied to the basolateral surface where the receptors, adenylate cyclase, and Na+/K+-ATPase are located. In a previous study (Spiegel, S., Blumenthal, R., Fishman, P.H., and Handler, J.S. (1985) Biochim. Biophys. Acta 821, 310-318), we demonstrated that exogenous gangliosides inserted into the apical membrane of A6 epithelia do not redistribute to the basolateral membrane. With the ability to vary selectively the ganglioside composition of the apical membrane, we examined the effects of gangliosides on sodium transport in A6 epithelia. When the apical surface of A6 epithelia were exposed to exogenous gangliosides, sodium transport in response to vasopressin, cholera toxin, and 8-bromo-cAMP was enhanced compared to epithelia not exposed to gangliosides. The effect was observed with bovine brain gangliosides, NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer (GD1a) and Gal beta-1----3GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer (GM1), but not with the less complex ganglioside, Neu-Ac alpha 2----3Gal beta 1----4Glc beta 1----Cer (GM3). We examined A6 cells for endogenous gangliosides and found that, whereas GM3 was a major ganglioside, only trace amounts of GM1 and GD1a were present. Based on cell surface and metabolic labeling studies, these gangliosides were synthesized by the cells and were present on the apical as well as the basolateral surface. Bacterial sialidase, which hydrolyzes more complex gangliosides to GM1, was used to modify the endogenous gangliosides on the apical surface; after sialidase treatment, the epithelia were more responsive to vasopressin, cholera toxin, and 8-bromo-cAMP. Thus, gangliosides may be modulators of sodium channels present in the apical membrane of epithelial cells.     

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.