Estrogen-induced alterations of hematoside of rat small intestinal mucosa

Prior studies have suggested that sex hormones could influence the ganglioside and/or neutral glycosphingolipid composition of various organs. To date, the effects of sex hormones on the glycosphingolipid composition of the rat small intestinal mucosa, however, have not been examined. In the present studies, male albino rats of the Sherman strain were subcutaneously administered the synthetic estrogen, ethinylestradiol (5 mg/kg body wt. per day), or diluent for 5 days, and the ganglioside, neutral glycosphingolipid and ceramide composition of the small intestinal mucosa of these animals were analyzed and compared. The results of these experiments demonstrate that estrogen administration: increased the ganglioside concentration of this tissue, including hematoside (Gm3); increased the percentage of the long-chain base phytosphingosine of hematoside; and did not appear to significantly influence the concentration or composition of the neutral glycosphingolipids or ceramide in this tissue. These data, therefore, indicate that estrogen administration induces quantitative and qualitative alterations in the gangliosides but not in the neutral glycosphingolipids or ceramide of rat small intestinal mucosa.     

Dexamethasone-induced alterations in the glycosphingolipids of rat proximal small-intestinal mucosa

Prior studies have demonstrated that glucocorticoids can influence the structure and function of several different organs, including the small intestine. However, to date, the effects of glucocorticoids on the glycosphingolipids of the rat small intestinal mucosa have not been examined. In the present experiments, male albino rats of the Sherman strain were subcutaneously administered dexamethasone (100 micrograms/100 g body wt. per day) or diluent for 4 days, and the ceramide, acidic and neutral glycosphingolipid compositions of the proximal small intestine of these animals were examined and compared. The results of these studies demonstrate that dexamethasone administration: (1) increased the content and relative percentage of hematoside (GM3) in this tissue; (2) increased the percentage of N-glycoylneuraminic acid of hematoside; (3) decreased the percentage of the long-chain base phytosphingosine of hematoside, glucosyl- and globotriaosylceramide; and (4) did not appear to influence significantly the concentration of the neutral glycosphingolipids or ceramide in this tissue. These data, therefore, indicate that dexamethasone administration induces alterations in the glycosphingolipids, particularly hematoside, of rat small-intestinal mucosa.     

Estrogen-induced alterations of the acidic and neutral glycosphingolipids of rat kidney

In order to determine whether female sex hormones could influence the glycosphingolipid composition of the rat kidney, male albino rats of the Sherman strain were subcutaneously administered the synthetic estrogen, ethinylestradiol (5 mg/kg body wt. per day) or vehicle for 5 days, and the ganglioside, ceramide and neutral glycosphingolipid compositions of the kidneys of these animals were analyzed and compared. The results of these experiments demonstrate that estrogen treatment: (1) increased the ceramide, acidic and neutral glycosphingolipid contents of this tissue; (2) decreased the relative percentages of glucosyl- and globotetraosylceramide and hematoside (GM3), but increased the relative percentage of globotriaosylceramide and 'other' gangliosides; (3) increased the relative percentage of N-acetyl- to N-glycolylneuraminic acid in GM3; and (4) altered the long-chain bases of GM3, glucosyl- and globotetraosylceramide in this organ. These data, therefore, demonstrate that estrogen administration induces quantitative and qualitative alterations in the gangliosides, neutral glycosphingolipids and ceramide of the rat kidney. This data as well as a discussion of the possible physiological consequences of these estrogen-induced alterations in kidney glycosphingolipids serve as the basis for this report.     

Sphingolipid composition of human platelets

Total lipid extracts from washed trypsinized human platelets were fractionated into neutral lipids, glycosphingolipids, and phospholipids by silicic acid chromatography. The concentrations and chemical structures of the neutral and acidic glycosphingolipids were then studied in detail. On the basis of sugar molar ratios, studies of permethylation products, and the action of stereospecific glycosidases on the lipids, identifications were made of four neutral glycosphingolipids. Lactosylceramide was the most abundant type and accounted for 64% of the total neutral glycolipid mixture. The major fatty acids of the lactosylceramide were 20:0, 22:0, 24:0, and 24:1; the major long-chain base was 4-sphingenine. The platelets were surprisingly rich in a ceramide fraction, which represented 1.3% of the total platelet lipids. It had a different fatty acid composition than the neutral glycosphingolipid and ganglioside fractions. Hematoside was also isolated from the total lipid fraction of platelets; the neuraminic acid component was N-acetylneuraminic acid. Treatment of platelets with trypsin, chymotrypsin, or thrombin increased the yield of hematoside as compared with a control, while the level of ceramides was not changed. It was concluded that the platelets are similar to leukocytes, liver, and spleen in that lactosylceramide and hematoside are the principal neutral and acidic glycosphingolipids. The presence of a relatively high proportion of ceramide in platelets may be a unique characteristic of this cellular fraction of blood.     

Neutral glycolipids of atherosclerotic plaques and unaffected human aorta tissue

The composition, structure and localization of neutral glycosphingolipids of human aorta taken from subjects who had died after myocardial infarction were studied. Individual glycosphingolipids were purified by high-performance liquid chromatography and were characterized on the basis of their chromatographic mobility, carbohydrate composition, methylation analysis and by 1H-NMR spectroscopy. The main aortic glycosphingolipids were identified as glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. Significant differences in the neutral glycosphingolipid composition of intima and media were detected. The neutral glycosphingolipid profile of medial plaques resembled that of unaffected media; however, significant differences were detected between intimal plaques and unaffected intima. Whereas the latter contained trihexosylceramide and globoside as the only neutral glycolipids, the intimal plaque glycolipids consisted mainly of glucosylceramide and also contained appreciable amounts of lactosylceramide which were completely absent in the unaffected intima. In comparison to intimal plaques, unaffected intima is characterized by a much higher content of cerebrosides terminating by beta-galactosyl residues which are known to interact with growth factors and other external stimuli. It thus seems possible that the proliferative activity of smooth muscle cells in atherosclerotic diseases is to some extent associated with their neutral glycolipid profile.     

Glycosphingolipid patterns of the gastrointestinal tract and feces of germ-free and conventional rats

Acid and non-acid glycosphingolipids of stomach, small and large intestine, and stimulated feces of germ-free and conventional rats of the same stain have been isolated and characterized. The glycosphingolipid patterns of the intestinal organs were chemically and immunologically very similar between the two groups of rats and relatively unaffected by the presence of an intestinal microbial flora. The major exception was the presence of hematoside with N-glycoloylneuraminic acid (NeuGc) (NeuGc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) in the stomach of conventional rats not found in the stomach of germ-free animals. Glycosphingolipids of stimulated feces of germ-free animals were derived from epithelial cells mainly of the small intestine and showed no signs of degradation. Glycosphingolipids of feces of conventional rats completely retained the pattern of blood group A-, B-, and H-active glycolipids as found in sterile feces but contained less of hematoside and more of lactosylceramide. This effect was probably due to degradation by bacteria, as demonstrated in vitro with the production of lactosylceramide after treatment of the isolated acid glycolipids of sterile feces with neuraminidase from Clostridium perfringens. The amount of total non-acid glycosphingolipids per dry weight was similar for stomach, was 50% higher for small intestine, and 300% higher for large intestine of germ-free animals compared to conventional animals. Due to the presence of large amounts of mucins the dry sterile feces contained 12% less non-acid glycolipids than conventional feces. However, calculated per rat per day the germ-free animal excreted more of non-acid glycosphingolipids (1.8 and 1.2 mg, respectively).     

Influence of monovalent cation transport on anabolism of glycosphingolipids in cultured human fibroblasts

We have reported [Saito, M., Saito, M., & Rosenberg, A. (1984) Biochemistry 23, 1043-1046] that the monovalent cationic ionophore monensin reduced the incorporation of labeled galactose into oligosaccharidyl glycosphingolipids (globotriaosylceramide, globotetraosylceramide, and gangliosides) and induced a cellular accumulation of glucosyl- and lactosylceramide in cultured diploid human fibroblasts. We have undertaken further studies on the effects of monensin and made comparison with the effects of related monovalent cation transporters on plasma membrane glycosphingolipid anabolism in human fibroblasts. Our results demonstrate that ionic flux can markedly influence glycosphingolipid synthesis, and they indicate that, like glycoprotein, the sites of glycosylation of the initial, precursor glycosphingolipids are different from the sites of higher glycosylation. At a concentration of 10(-7) M, monensin induced the maximum inhibition of incorporation of labeled galactose into polyglycosyl sphingolipids: globotriaosylceramide, globotetraosylceramide, and gangliosides; increased incorporation of labeled galactose into glucosyl- and lactosylceramide was clearly evident, and their content rose measurably in the cell at concentrations of monensin as low as 10(-8) M. These effects of monensin were reversible. Incorporation of labeled galactose into higher glycosylated neutral glycosphingolipids and gangliosides slowly resumed, and the accumulated glycosylceramide diminished after removal of monensin from the culture medium. Ouabain (plasma membrane Na+,K+-ATPase inhibitor) and A23187 (Ca2+ ionophore) also caused a rapid increase in incorporation of labeled hexose into glucosylceramide and decreased its incorporation into higher neutral glycosphingolipids and into gangliosides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization, distribution and biosynthesis of the major ganglioside of rat intestinal mucosa.

The major sialic acid containing glycolipid has been isolated from rat intestinal mucosa. Characterization of this ganglioside by thin layer and gas chromatographic analysis indicates that it is an hematoside (GM3) with the major portion of the sialic acid in the N-glycolyl form. The distribution of this ganglioside was determined in villus and crypt cells isolated from rat intestine. The hematoside content of crypt cells was found to be significantly decreased when compared to villus cells. CMP-sialic acid:lactosylceramide sialyltransferase, responsible for the sialylation of lactosylceramide, was measured in differentiated villus and undifferentiated crypt cells and found to be greatly reduced in the crypt cell fraction. The present study demonstrates that marked differences in ganglioside content and biosynthesis occur in contiguous populations of cells in varying states of differentiation when isolated from normal rat intestine.     

Neutral Glycosphingolipids and Ceramide Composition of Ethylnitrosourea-Induced Rat Neural Tumors: Accumulation of Ceramide in Tumors

Abstract: Experimental rat neural tumors in offspring were induced transplacentally by a single injection of a chemical carcinogen, ethylnitrosourea, 20 mg/kg body weight, in the tail vein of the mother. The neutral glycosphingolipid, sulfatide, and ceramide composition of the tumors and the normal tissues from which the tumors originated is described. The content of nonhydroxy fatty acid (NFA) and hydroxy fatty acid (HFA) containing ceramide in all the neural tumors so far examined was significantly increased compared with the corresponding normal neural tissue. Some 8 to 18 mol% of total neutral glycolipids was as ceramide in neurinomas, oligodendrogliomas, and menin-giomas. Lactosylceramide in normal neural tissues was about 1 mol% of the total neutral glycosphingolipids. In various neural tumors lactosylceramide increased up to 8 mol%. NFA- and HFA-containing cerebrosides constitute 94–100% of the neutral glycosphingolipids in normal neural tissues. In various neural tumors the mol percent of cerebrosides was significantly reduced. A high performance liquid chromatographic method was modified to analyze simultaneously ceramides, cerebrosides, and higher neutral glycosphingolipids.     

Glycosphingolipids from bovine milk and milk fat globule membranes: a comparative study. Adhesion to enterotoxigenic Escherichia coli strains

Several components of milk fat globule membranes (MFGMs) have been reported to display beneficial health properties and some of them have been implicated in the defense of newborns against pathogens. These observations prompted us to determine the glycosphingolipid content of MFGMs and their interaction with pathogens. A comparative study with whole milk components was also carried out. Milk fat globules and MFGMs were isolated from milk. Gangliosides and neutral glycosphingolipids were obtained from MFGMs and whole milk and their fatty acid contents were determined by gas chromatography-mass spectrometry (GC-MS). MFGMs and whole milk showed similar ganglioside and neutral glycosphingolipid contents, with whole milk having more GM3 and glucosylceramide and less GD3, O-acetyl GD3, O-acetyl GT3, and lactosylceramide. The fatty acid content of gangliosides from both sources showed a similar composition. However, the neutral glycosphingolipid fatty acid content seemed to be quite different. Whole milk had fewer very-long-chain fatty acids (18.1% vs. 46.4% in MFGMs) and more medium-chain and unsaturated C18:1 and C18:2 fatty acids. Milk fat globules, MFGMs, lactosylceramide, and gangliosides GM3 and GD3 were observed to bind enterotoxigenic Escherichia coli strains. Furthermore, bacterial hemagglutination was inhibited by MFGMs and glycosphingolipids.     

P blood group regulation of glycosphingolipid levels in human erythrocytes.

The neutral glycosphingolipid content of normal human erythrocytes was analyzed by a new method which utilizes high performance liquid chromatography. This rapid and accurate technique permits the quantitation of each of the major neutral glycolipids from individual blood samples. A correlation between the P blood group and the relative quantities of neutral glycosphingolipids is demonstrated. Erythrocytes from P1 individuals are shown to contain more globotriaosylceramide and less lactosylceramide than do erythrocytes from P2 individuals. The results of these experiments suggest the existence of a new phenotype in the P blood group system, and have further implications regarding the biosynthesis of the P blood group glycosphingolipids.     

Glycosphingolipid patterns of fetal and adult small intestinal mucosa in the sheep

The ganglioside and neutral glycosphingolipid composition of fetal and adult sheep small intestinal mucosa were characterized and compared. Mono- and tetrahexosylceramide were the major neutral glycolipids of both fetal and adult tissue. Fetal mucosa, however, possessed a higher content of monohexosylceramide than its adult counterpart. Similarly, G_D1a, G_M3 and G_M2 were found to be the principal gangliosides in fetal and adult tissue. Adult intestinal mucosa possessed significant amounts of G_T1a not present in fetal tissue. Analysis of the hydroxy and nonhydroxy fatty acids as well as the long-chain bases of the major glycosphingolipids revealed differences between these lipophilic components of glycolipids in fetal and adult intestinal mucosa. The results, therefore, indicate that both quantitative and qualitative differences in glycosphingolipid composition exist between fetal and adult sheep small intestinal mucosa.     

Glycosphingolipids of human aorta

The structures of the main gangliosides of human aorta (intima and media) were elucidated. The main component (67%) was identified as N-acetylneuraminosyl-lactosylceramide (ganglioside GM3). The aorta tissue contained also gangliosides GM1, GD3, GD1a, and GT1. All sialic acid residues in gangliosides were present as N-acetyl-neuraminosyl derivatives. Among neutral glycosphingolipids of human aorta, the main components were identified as glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. The preliminary data suggest that the composition of the investigated glycosphingolipids in tissue might vary upon atherosclerosis lesions of aorta.     

The effect of thrombin induced aggregation on human platelet glycosphingolipids

Analyses were made of the glycosphingolipid composition of human platelets before and after treatment with thrombin. Within 10 min after incubation was initiated, there was approximately a two-fold increase in the concentration of hematoside. Concomitantly, the level of lactosylceramide was decreased about three-fold, and there was also a slight diminution in the ceramide levels of these cells. Platelets treated with phenylmethyl-sulfonyl fluoride and thrombin did not exhibit such an effect. These results suggest that lactosylceramide and ceramide are utilized as precursors in the biosynthesis of hematoside and that this conversion accompanies the aggregation of human platelets.     

An improved technique for separation of neutral glycosphingolipids by high-performance liquid chromatography

We have developed a high-performance liquid chromatographic (HPLC) procedure for separation of O-acetyl-N-p-nitrobenzoyl derivatives of six neutral glycosphingolipids: glucosylceramide, lactosylceramide, globotriaosylceramide, lactotriaosylceramide, globotetraosylceramide, and neolactotetraosylceramide. The recoveries of glucosylceramide and globotetraosylceramide for the derivatization procedure and HPLC analysis were approximately 75%, and one nanomole of glycolipid could be detected. The procedure was used for analysis of human erythrocyte neutral glycolipids.     

Mast cell heterogeneity. Differential synthesis and expression of glycosphingolipids by mouse serosal mast cells as compared to IL-3-dependent bone marrow culture-derived mast cells before or after coculture with 3T3 fibroblasts

The synthesis and intracellular expression of glycosphingolipids by mouse serosal mast cells (SMC) have been characterized by radiolabeling and TLC and by immunodetection in situ. Chromatographic analysis of purified glycosphingolipids from SMC intrinsically labeled with [14C]galactose and [14C]glucosamine hydrochloride revealed the predominant synthesis of only the simplest neutral glycosphingolipid and ganglioside, glucosylceramide and ganglioside GM3, respectively. Intracellular indirect immunofluorescence staining of permeabilized SMC demonstrated the absence of the more complex neutral glycosphingolipids lactosylceramide, globotriosylceramide, globotetraosylceramide, and globopentaosylceramide, the absence of ganglioside GM1, and the presence of ganglioside GM3. By contrast, permeabilized mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) and mast cells recovered after 21 days of coculture of BMMC with mouse 3T3 fibroblasts expressed lactosylceramide, globotriosylceramide, globotetraosylceramide, ganglioside GM1, and ganglioside GM3, but not globopentaosylceramide intracellularly as determined by immunofluorescence. The findings indicate a loss of biosynthetic capacity and epitope maintenance for glycosphingolipids with in vivo differentiation of SMC from IL-3-dependent BMMC progenitors. Thus, although mast cells derived after coculture of these progenitors for 21 days with fibroblasts assume multiple SMC-like properties in terms of their histochemical staining and their secretory granule proteoglycan and neutral protease constituents, they do not lose the ability to express complex glycosphingolipids. The finding that glycosphingolipid composition does not change coordinately with other secretory granule markers defines a new stage of mouse mast cell development between the BMMC and SMC and provides evidence that mast cell development is more complex than previously appreciated.     

Human gastric glycosphingolipids recognized by Helicobacter pylori vacuolating cytotoxin VacA

Many bacterial toxins utilize cell surface glycoconjugate receptors for attachment to target cells. In the present study the potential carbohydrate binding of Helicobacter pylori vacuolating cytotoxin VacA was investigated by binding to human gastric glycosphingolipids on thin-layer chromatograms. Thereby a distinct binding of the toxin to two compounds in the non-acid glycosphingolipid fraction was detected. The VacA-binding glycosphingolipids were isolated and characterized by mass spectrometry and proton NMR as galactosylceramide (Galbeta1Cer) and galabiosylceramide (Galalpha4Galbeta1Cer). Comparison of the binding preferences of the protein to reference glycosphingolipids from other sources showed an additional recognition of glucosylceramide (Glcbeta1Cer), lactosylceramide (Galbeta4Glcbeta1Cer) and globotriaosylceramide (Galalpha4Galbeta4Glcbeta1Cer). No binding to the glycosphingolipids recognized by the VacA holotoxin was obtained with a mutant toxin with deletion of the 37 kDa fragment of VacA (P58 molecule). Collectively our data show that the VacA cytotoxin is a glycosphingolipid binding protein, where the 37 kDa moiety is required for carbohydrate recognition. The ability to bind to short chain glycosphingolipids will position the toxin close to the cell membrane, which may facilitate toxin internalization.     

Expression of glycosphingolipids in serum-free primary cultures of mouse kidney cells: male-female differences and androgen sensitivity

The expression of neutral glycosphingolipids was examined in primary kidney cell cultures derived from adult male and female beige mutant mice (C57BL/6J;bg ^j/bg ^j) with enrichment for proximal tubule cells during preparation of explants and using defined serum-free medium for the culture conditions. Cell proliferated for 7 daysin vitro to provide confluent or nearly confluent monolayers of epithelial-type growth indicative of proximal tubule cells. The malevs female differences in neutral glycosphingolipids seen in the kidneyin vivo were retained in these 7 day cultures. Cultures derived from males contained galacto- and digalactosylceramides whereas those from females did not express these types of glycolipids. Also, male cells had higher ratios of sphingosine: phytosphingosine containing species in Nfa (non-hydroxy fatty acid) globotriaosylceramide and in glucosylceramide than females. The shift in sphingosine: phytosphingosine to male ratios in Nfa globotriaosylceramide and in glucosylceramide could be stimulated in female kidney cells by treatment with 10^–5 M testosterone or 5-dihydrotestosterone. The male-specific expression of neutral glycosphingolipids, then, appears to be stable character of male-type differentiation in mouse kidney that is passed on during proliferation in culture. Female kidney cells retain an ability to respond to androgens with specific changes in neutral glycosphingolipid expression during 7 days of growthin vitro in serum-free conditions, but do not respond with the induction of the male-specific glycolipids galacto-and digalactosylceramides as seenin vivo.     

Sialoglycoconjugate changes during 2-acetylaminofluorene-induced hepatocarcinogenesis in the rat

Previous studies indicated a reproducible pattern of altered glycosphingolipid biosynthesis accompanying late stages of liver tumorigenesis in the rat induced by the carcinogen 2-acetylaminofluorene. The sequence began with a dramatic elevation in CMP-sialic acid:lactosylceramide sialyltransferase and was followed by sequential elevations and eventual depressions in other enzymes catalyzing sugar transfers to glycolipid acceptors. The present study focused on the early events of glycolipid biosynthesis during the first 11 weeks of 2-acetylaminofluorene administration according to the same feeding schedule as used previously. Transient elevations in CMP-sialic acid synthetase and elevations in neutral glycosphingolipid precursors to gangliosides were found to precede the major elevations in CMP-sialic acid:lactosylceramide sialyltransferase (GM3 synthetase) noted earlier. Two cycles of response were observed prior to the initiation of the sustained enhancement of biosynthesis of precursor ganglioside, GM3, and/or a significant increase in total or lipid-soluble sialic acid. In vitro rates of sialyl transfer from CMP-sialic acid to endogenous protein acceptors were not altered. The results suggest that the previous observations of altered ganglioside biosynthesis following 2-acetylaminofluorene administration are not an isolated occurrence but may represent late events in a sequence or 'cascade' of biochemical change involving, as well, biosynthesis of ganglioside precursors, CMP-sialic acid and neutral glycosphingolipids.     

Convenient structural analysis of glycosphingolipids using MALDI-QIT-TOF mass spectrometry with increased laser power and cooling gas flow

Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) was applied to the structural characterization of neutral glycosphingolipids. Lithium adduct ions of glycosphingolipids were analyzed using MALDI-QIT-TOF MS under strong conditions of increased laser power and cooling gas flow. The relative intensities of fragment ions were increased under the strong conditions, and the resulting spectra revealed the presence of oligosaccharide ions fragmented from the glycosphingolipids. Consequently, the oligosaccharide sequences of the glycosphingolipids were readily obtained. To obtain more detailed structural information, MS/MS (MS2) and MS/MS/MS (MS3) analyses were performed with selection of the lactosylceramide and ceramide ions, respectively. The resulting data were sufficient to determine the structures of both the oligosaccharide and the ceramide moiety of each glycosphingolipid. The fragmentation patterns of MS2 and MS3 for Forssman glycolipid under the strong conditions were comparable to those of MS3 and MS4 obtained under standard conditions, respectively. Thus, MALDI-QIT-TOF MS with increased laser power and cooling gas flow is a convenient method for glycosphingolipid analysis.