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Glycosphingolipids from rabbit aorta, plasma, and red blood cells: effects of high cholesterol-high fat diets on fatty acid distribution and quantity of glycosphingolipids.

Four glycosphingolipids were isolated from rabbit aorta, plasma, and red blood cells. They were identified, by thin-layer chromatography and by quantitative analysis of hexose and fatty acid, as cerebroside, diglycosyl ceramide, triglycosyl ceramide, and globoside. The rabbits had been maintained on a normal diet or on one of three high cholesterol diets for 180 days. The quantities of the glycosphingolipids and their fatty acid distributions were determined, and comparisons were made between the control and experimental animals. Aorta and plasma glycosphingolipids were more affected by the high cholesterol diets than were those from red blood cells. The effects on aorta and plasma glycosphingolipids were similar. The amount of cerebroside was increased in aorta and plasma in all animals in the experimental groups. The amount was also increased in red blood cells in rabbits from two of the experimental groups. The average fatty acid chain length was greater in the lipids from the experimental animals than in those from the control animals for all measured glycosphingolipids from aorta. The average chain length was also greater in cerebrosides from the experimental animals from all three tissues. Probably the most notable differences in the experimental animals were the increased 24:1/24:0 ratios and the increased concentrations of 24:2. These increases occurred in nearly all samples from plasma and aorta, but not in red blood cells. There was also an increase of total unsaturated fatty acids in aorta cerebrosides from the experimental animals. Except for the increase in 24:2, lard generally caused more deviation from normal than did cottonseed oil when the level of cholesterol in the diet was 1%.

Animals↗

On the structure of cytolipin R, a ceramide tetrahexoside hapten from rat lymphosarcoma.

Cytolipin R, a ceramide tetrahexoside isolated from rat lymphosarcoma, was studied by sequential hydrolysis with specific glycosidases which revealed the anomeric configurations of the glycosidic bonds. Sugar linkages were established by combined gas-liquid chromatography and mass spectrometry of the partially methylated alditol acetates prepared after permethylation and hydrolysis of the intact lipid. Results indicated the structure of cytolipin R to be N-acetylgalactosaminyl(beta1-->3)galactosyl(alpha1-->3) galactosyl(beta1-->4)glucosyl ceramide. Cytolipin K (globoside I) differs in having a -galactosyl(alpha1-->4)galactosyl- internal linkage, and this difference must account for the immunological differences between cytolipin K and cytolipin R.

Alkylation↗

[Differential diagnosis of congenital lipidoses by lipid analyses of body fluids, biopsy and autopsy tissue].

1. Presentation of the commomly used procedures for the extraction and separation of total lipids, glycolipids and phosholipids from fresh and formalin-fixed organs tissues (brain, liver, spleen, kidney) as well as from serum, CSF and urine. II. Description of the qualitative and quantitative analysis of individual lipid fractions (glycolipids, gangliosides, phospholipids, neutral lipids) by thin-layer chromatograhy and photodensitometry. III. Results of investigations performed on biopsy material, autopsy material, serum and urine in the following diseases: 1. Infantile, juvenile and adult Gaucher's disease: accumulation of glucocerebroside in liver and spleen. 2. Infantile and adult Niemann-Pick disease: accumulation of sphingomyelin in liver, spleen, kidney and lung. 3. Fabry's disease: increased urinary excretion of trihexosyl-ceramide and dihexosyl-ceramide. 4. Infantile and adult metachromatic leukodystrophy: accumulation of sulfatides in the central and peripheral nervous system and kidney, increased urinary excretion of sulfatides. 5. Austin's variant of metachromatic leukodystrophy: besides an increase of sulfatides in the white matter of brain accumulation of glycolipids in the cerebral cortex. 6. Tay-Sachs disease (GM2-gangliosidosis): cerebral accumulation of GM2-ganglioside and trihexosylceramide (enzyme variant B), additional visceral accumulation (liver, spleen, kidney) of tetrahexosyl-ceramide = globoside (enzyme variant 0). 7. Infantile generalized GM1-gangliosidosis: cerebral (and visceral) accumulation of GM1-ganglioside and tetrahexosyl-ceramide. 8. Late infantile GM1-gangliosidosis: Cerebral accumulation of GM1-ganlioside and tetrahexosylceramide. 9. GM3-gangliosidosis (lactosyl-ceramidosis): neuronal accumulation of lactosyl-ceramide, GM2-ganglioside and GM3-ganglioside. 10. Refsum's disease: demonstration of phytanic acid esters of cholesterol in serum.

Autopsy↗

Concentration and fatty acid composition of cerebrosides and sulfatides in mature and immature human brain.

The fatty acid composition of cerebrosides and sulfatides from frontal lobe gray and white matter was determined for five fresh and four formalinized adult brains and for eight infants. Fatty acid patterns were unaffected by formalinization, but varied considerably from one another in the proportion of saturated to unsaturated fatty acids. The percentages of 24:0 and 24:1 increased with age. Cerebrosides obtained from areas such as the brainstem and cerebellum, where myelination was more advanced, tended to have a larger proportion of long-chain fatty acids than samples extracted from frontal or parietal lobe white matter. Hydroxy fatty acids showed an adult pattern in all instances in which amounts sufficient for accurate quantification could be isolated. Lipid hexose, cerebroside + sulfatide hexose, and methanoleluted hexose were measured in the brains of 12 infants ranging in age from a 4 month fetus to 2 yr. In the most immature, the majority of lipid hexose was in the form of glycolipids more polar than cerebrosides and sulfatides. These have tentatively been identified as hematosides and globosides. With maturation, cerebrosides and sulfatides increased progressively, but the amounts of the more polar glycolipids remained constant in relation to the total lipid content of tissue.

Adult↗

Quantitative determination of the neutral glycosyl ceramides in human blood.

A method is described for the qualitative and quantitative estimation of four neutral glycosyl ceramides from human plasma and erythrocytes. Total lipids extracted from 50 ml of plasma or packed erythrocytes were separated by silicic acid chromatography into neutral lipids, a fraction of mixed glycolipids that was eluted with acetone-methanol 9:1, and phospholipids. After mild alkali-catalyzed methanolysis to remove contaminants from the crude fraction of glycolipids, individual glycosyl ceramides were isolated by preparative thin-layer chromatography. The oligosaccharide portions of these lipids were characterized by cleavage with methanolic hydrogen chloride and gas chromatography of the O-trimethylsilyl methyl glycosides. It was possible to study the composition of the carbohydrate and sphingolipid base fractions in the same gas chromatographic analysis. With mannitol as an internal standard for gas chromatographic estimation of glucose, concentrations of each of the glycosyl ceramides were determined with a precision of about 10%. Recoveries of the lipids from plasma varied with the complexity of the oligosaccharide moiety and ranged from 94% with glucosyl ceramide to 71% with globoside. Concentrations of the four glycosyl ceramides in plasma and in erythrocytes were determined for samples from young, healthy males. Amounts of glycolipid as low as 0.1 micromole can be determined conveniently by this procedure.

Adult↗

Neutral beta-N-acetylhexosaminidases of rat brain. Purification and enzymatic and immunological characterization.

The two neutral beta-N-acetylhexosaminidases of rat brain have been purified by procedures involving extraction, concanavalin A-Sepharose, ammonium sulfate precipitation, DEAE-cellulose, hydroxyapatite, Sepharose 4B, and an affinity chromatography with 2-acetamido-N-(epsilon-aminocaproyl)-2-deoxy-beta-galactopyranosylamine bound to Sepharose. The neutral beta-N-acetylgalactosaminidase was purified to homogeneity at 2000-2500-fold purification over the post-concanavalin A fraction. It was specific to beta-N-acetylgalactosaminide and was inactive to glucosaminide. It appeared to possess extremely small but detectable activity to hydrolyze the terminal N-acetylgalactosamine residue from GM2, asialo-GM2, and globoside. The other enzyme, neutral N-acetylglucosaminidase, was 85-90% pure, judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was predominantly active toward the N-acetylglucosaminide substrate but hydrolyzed N-acetylgalactosaminide at 0.5% of the rate toward glucosaminide. The possibility of this very small galactosaminidase activity being due to contamination by the first enzyme could be excluded by several criteria. Three different criteria, gel filtration, acrylamide gel electrophoresis, and sucrose density gradient centrifugation, gave inconsistent results in the estimated molecular size of either enzyme. The two enzymes were, however, different in molecular size by any of the criteria. Precipitating antibody was produced in rabbits against the neutral beta-N-acetylgalactosaminidase. It specifically precipitated the enzyme but did not cross-react with the neutral beta-N-acetylglucosaminidase or the acid beta-N-acetylhexosaminidase A and B. Physiological substrates for these neutral hexosaminidases are yet to be clarified.

Animals↗

Gas chromatography/mass spectrometry analysis of oligosaccharides from neutral glycosphingolipids of murine B cell hybridomas.

Monoclonal B cell hybridoma cell lines express glycolipids characteristic of both the myeloma and normal lymphocyte parents. The neutral glycolipids from hybridoma cell lines metabolically radiolabeled with [14C]galactose plus [14C]glucosamine separate by high performance thin layer chromatography into patterns that may reflect differences in glycolipid expression among B cell subsets. Gas chromatography/mass spectrometry of oligosaccharides released by trifluoroacetolysis from neutral glycolipids extracted from 10(9) clonally expanded hybridoma cells reveals the carbohydrate composition of the major glycolipid components detected by thin layer chromatography. Glycolipids differentially expressed among six cell lines analyzed include monohexosylceramide, lactosylceramide, globotriaosylceramide, globoside, asialo-GM2, and 2'fucosyllactosylceramide. The latter compound has not been described previously in cells from the mouse.

Animals↗

Purification and characterization of UDP-GalNAc:polypeptide N-acetylgalactosamine transferase from an ascites hepatoma, AH 66.

The membrane-bound UDP-GalNAc:polypeptide N-acetylgalactosamine transferase from an ascites hepatoma, AH 66, has been purified 48,100-fold, mainly by affinity chromatography in aqueous Triton X-100 on apomucin (deglycosylated bovine submaxillary mucin) coupled to Sepharose. The purified preparation behaved homogeneously on gel filtration on Sephadex G-150 in aqueous Triton X-100 and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of about 55,000. The enzyme requires Mn2+, and only UDP-GalNAc served as a sugar donor. Apomucin, A1 protein, kappa-casein, apofetuin, and apoantifreeze glycoproteins served as acceptors, but the rate and amount of the transfer varied considerably from one acceptor to another. The transfer reaction terminated at the level of glycosylation of from only a few to at most about 40% of the serine plus threonine residues from which mucin-type oligosaccharides had been removed. This indicates that the transferase requires a certain conformation surrounding the acceptor site, but suggests also that a special mechanism may be functioning in vivo for frequent glycosylation of the abundant serine plus threonine residues of mucins. Lacto-N-fucopentaose I, ceramide di- and trihexosides, and globoside were not acceptors.

Animals↗

Characterization of mouse helper and suppressor T cell subsets separated by lectins.

Concanavalin A (Con A)-activated cells were fractionated by separating cells that agglutinate with peanut agglutinin (PNA) which binds preferentially to the sugar sequence beta-D-Gal-(1 leads to 3)-D-GalNAc. PNA-agglutinable cells were found to exert a significant suppressor effect on the primary antibody response to sheep red blood cells, whereas PNA-nonagglutinable cells did not affect the antibody response. It was also found that PNA-agglutinable suppressor cells were sensitive to treatment with anti-Thy 1.2 and complement (C), anti-I-J antiserum and complement, and anti-Ly 2.2 antiserum and complement. These results suggest that the PNA-agglutinable suppressor cells, separated from Con A-activated cells, were T cells and that these suppressor T cells possess abundant beta-galactosyl residues exposed on the cell surface, not masked by sialyl residues. Furthermore, it was found that antigen-specific suppressor cells were also separable by use of the agglutinability with PNA. The PNA-agglutinable suppressor T cell activity was eliminated by treatment of the Con A-activated PNA-agglutinable cells with anti-asialo GM1 and complement, anti-Forssman antiserum and complement, and anti-globoside antiserum and complement, but not by treatment with anti-GM1 antiserum and complement. Conversely, the helper T cell activity that was induced by Lens culinalis agglutinin and separated by Limulus polyphemus agglutinin, was eliminated by treatment of the cells with anti-GM1 and complement, but not by the treatment with the other anti-glycolipid antisera and complement.

Animals↗

Abnormalities in cerebral lipids and hepatic cholesterol glucuronide of a patient with GM1-gangliosidosis type 2.

The brain tissue of a patient with GM1-gangliosidosis Type 2 showed an abnormal distribution pattern of lipids, that is, a marked increase in GM1-ganglioside, asialo-GM1 and cholesterol ester, a significant increase of glucosylceramide, lactosylceramide and asialo-GM2, and a decrease in galactocerebroside and sulfatide. On the other hand, the liver tissue showed a normal pattern of glycolipids, that is, the presence of normal amounts of glucosylceramide, lactosylceramide, globotriaosylceramide, globoside, hematoside (GM3) and GD3. However, it was found that the patient's liver contained a larger amount of cholesterol glucuronide than normal liver and that it was present only in the liver and not in the other tissues. This represents the first report of elevated cholesterol glucuronide in liver of GM1-gangliosidosis.

Brain↗

Neutral glycolipid composition in human cancer.

Neutral glycolipid composition of human gastric and colonic cancer was studied in comparison to healthy mucosa. The following compounds were identified: galactosylceramide, glucosylceramide, trihexosylceramide, globoside, small amounts of neotetraosylceramide and fucoglycolipids. The latter comprised both 3-0- and 4-0-substituted residues of N-acetylglucosamine. In cancer tissue the contents of lactosylcermide and of fucoglycolipids were increased. Fatty acid compositions of neutral glycolipids derived from both cancer and control samples were similar. The most abundant fatty acids were palmitic and nervonic acids.

Breast Neoplasms↗

Common tetrasaccharide epitope NeuAc alpha 2-->3Gal beta 1-->3(Neu-Ac alpha 2-->6)GalNAc, presented by different carrier glycosylceramides or O-linked peptides, is recognized by different antibodies and ligands having distinct specificities.

A novel globo-series disialoganglioside, disialosyl galactosyl globoside (Structure 1 below), defined by new monoclonal antibody (mAb) RM2, was isolated and characterized as having terminal structure identical to that of ganglio-series ganglioside GD1 alpha (Structure 2) and a common mucin-type epitope (Structure 3) widely distributed in glycoproteins such as glycophorin A. While these three structures share a common nonreducing tetrasaccharide terminus, mAb RM2 showed strong specific reactivity only with Structure 1, not with Structures 2 or 3. Another mAb, QSH2, reacted strongly with Structure 3 but did not cross-react with Structures 1 or 2. Conformational molecular models based on minimum energy hard sphere exoanomeric calculations suggest that Structure 1 presents a unique surface topology distinct from that of Structures 2 or 3. Our findings suggest the novel concept that reactivity of a common carbohydrate epitope with different antibodies or ligands is highly dependent on the type of carrier glycosylceramide or carrier O-linked peptide. [formula: see text]

Animals↗

Endogenous ganglioside GM1 modulates L-type calcium channel activity in N18 neuroblastoma cells.

Digital imaging fluorescence microscopy was used to investigate the effect of the B subunit of cholera toxin on calcium homeostasis in neuroblastoma N18 cells. The B subunit, which binds specifically to ganglioside GM1 in the outer leaflet of the cell membrane, was found to induce a sustained increase of intracellular calcium concentration ([Ca2+]i). The increase in [Ca2+]i was not observed in the absence of extracellular calcium, or in the presence of the calcium chelator EGTA, and was blocked by nickel. The B subunit was also found to induce an influx of manganese ions, as indicated by a quench of the intracellular fura-2 fluorescence. These data suggest that the B subunit induces an increase in calcium influx in N18 cells. Potassium-induced depolarization also stimulated manganese influx; however, after the onset of depolarization-induced influx, the B subunit had no further effect. This occlusion suggests involvement of voltage-dependent calcium channels. Treatment with BayK8644, a dihydropyridine agonist selective for L-type calcium channels, induced manganese influx that was not altered by the B subunit and apparently blocked the effect of the B subunit itself. Furthermore, the dihydropyridine L-type channel antagonists niguldipine or nicardipine completely inhibited B subunit-induced manganese influx. Thus, the B subunit-induced manganese influx is likely due to activation of an L-type voltage-dependent calcium channel. Spontaneous influx of manganese ions was also inhibited by nicardipine or niguldipine and by exogenous gangliosides. Ganglioside GM1 was more potent than GM3, but globoside had no significant effect. The modulation of L-type calcium channels by endogenous ganglioside GM1 has important implications for its role in neural development, differentiation, and regeneration and also for its potential function in the electrical excitability of neurons.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

Anti-glycolipid antibodies produced by Epstein-Barr virus (EBV)-transformed oligoclonal B cell lines obtained from normal persons and autoimmune disease patients.

Oligoclonal B-lymphoblastoid cell lines (LCL) were blindly established by nonimmunized protocol from natural populations of two normal persons and 8 autoimmune disease patients using Epstein-Barr virus (EBV)-induced transformation. We systematically screened these LCLs on antibodies against a panel of glycolipids using liposome immune lysis assay (LILA). Eventually we found antibodies to 12 out of 15 compounds containing CTH, globoside, Forssman, paragloboside, CPH, sulfatide, NAGM3, NGGM3, i active glycolipid, GM1, GA1 and GA2 in 81 out of 950 LCLs from normal PBL, and antibodies to 2 out of 15 compounds containing sulfatide and CPH were also detected in 11 out of 430 LCLs from autoimmune disease patients. Unexpectedly, the antibody repertoire of LCLs from autoimmune disease patients was impoverished. The possibility of the EBV technique for production of various kinds of human monoclonal antibodies (MAbs) to glycolipid was shown.

Antibodies, Monoclonal↗

Oligosaccharide-receptor interaction of the Gal alpha 1-4Gal binding adhesin of Streptococcus suis. Combining site architecture and characterization of two variant adhesin specificities.

The sugar binding specificities of two groups of Streptococcus suis, a pig pathogen that causes meningitis also in man, were determined. Both the group represented by a recently characterized strain inhibitable by galactose and N-acetylgalactosamine (type PN) and the group inhibitable by galactose (type PO) were found by hemagglutination and solid-phase binding inhibition experiments to recognize the disaccharide Gal alpha 1-4Gal of the P1 and Pk blood group antigens. Both types preferred the disaccharide in terminal position. PN showed some, whereas PO showed almost no, binding to the globoside oligosaccharide containing an additional GalNAc beta 1-3 residue. The complete hydrogen bonding patterns were determined by using deoxy and other synthetic derivatives of the receptor disaccharide, and the constructed models of the interactions were compared with that of Escherichia coli PapG396 adhesin. The essential hydroxyls for binding were the HO-4', HO-6', HO-2, and HO-3 hydroxyls on the beta' alpha-side of the Gal alpha 1-4Gal molecule. Type PO adhesin also formed weak interactions with the hydroxyls HO-6 and HO-3'. The mechanism differed from that of E. coli, which binds to a cluster of five hydroxyls (HO-6, HO-2', HO-3', HO-4', and HO-6') and thus to a different part of the receptor disaccharide. These results represent the first example of the comparison of the saccharide receptor hydrogen bonding patterns of two bacterial organisms of different origin and show that the same saccharide may be recognized by two different binding mechanisms.

Adhesins, Bacterial↗

B.C.P. Jansen Institute, University of Amsterdam, Amsterdam, The Netherlands.

In a patient with Fabry's disease who had undergone kidney transplantation to correct uremia, the neutral glycosphingolipids and alpha-galactosidase activity have been measured in plasma and urine and, 9 months later, after the death of the patient, in autopsy material. After transplantation, there was no significant increase in alpha-galactosidase activity in plasma; the activity found never exceeded 3% of the mean control value. A striking parallelism was found during the follow-up period in the increase and decrease of trihexosylceramide and globoside and also of glucosylceramide and dihexosylceramide. The alpha-galactosidase activity in spleen and liver was as low as that observed in untreated Fabry hemizygotes. These data and those obtained from autopsy material provide evidence that renal transplantation does not lead to a specific enzymic breakdown of trihexosylceramide in Fabry patients. However, no trihexosylceramide accumulation was observed in the transplanted kidney.

Adult↗

Characterization of a novel bacterial adhesion specificity of Streptococcus suis recognizing blood group P receptor oligosaccharides.

Streptococcus suis causes sepsis, meningitis, and other serious infections in piglets, and meningitis in humans. Hemagglutination inhibition experiments with mono- and oligosaccharides and glycoproteins indicated that galactose-binding strains of S. suis recognized the Gal alpha 1-4Gal sequence present in the P1 and Pk blood group antigen structures. In thin-layer chromatography overlay assays the bacteria bound to trihexosylceramide (GbO3) but not to globoside (GbO4) or Forssman glycolipid (GbO5), in contrast to P-fimbriated Escherichia coli, which bound only to the latter two. The S. suis adhesin also differed from that of E. coli in that some of the hydrogen bonds formed with the receptor, as determined with chemically modified receptor analogues, were different. In agreement with the binding specificity, the S. suis bacteria agglutinated best among P blood group erythrocytes those of the P1k and P2k type, and from different animal erythrocytes those from rabbit, which express GbO3 as the predominant glycolipid. Binding to frozen sections of pig pharyngeal tissue was decreased by the free GbO3 oligosaccharide and its protein conjugate, which indicated that the corresponding glycolipid may function as receptor for galactose-binding strains of S. suis in pig pharyngeal epithelium.

Bacterial Adhesion↗

Stage-specific glycosphingolipids from amastigote forms of Leishmania (L.) amazonensis. Immunogenicity and role in parasite binding and invasion of macrophages.

Neutral glycosphingolipids (GSLs) from amastigote forms of Leishmania (L.) amazonensis were isolated, and their structures and biological properties were characterized. Based on various immunochemical methods, these GSLs were shown to be expressed at certain stages of amastigote development. GSLs were extracted and purified from amastigotes of hamster foot lesions by established procedures. Three mouse monoclonal antibodies (MoAbs) specific for carbohydrate epitopes of these GSLs were established, and their inhibition of parasite binding and macrophage invasion was analyzed. MoAb ST-3 inhibited 80% of macrophage invasion by amastigotes and 60% of that by promastigotes. Since GSLs reacting with MoAb ST-3 were found in amastigotes but not in promastigotes, ST-3 reactivity with promastigotes presumably depends on an epitope present on an unidentified promastigote glycoconjugate. MoAbs ST-4 and ST-5 inhibited 60-80% of macrophage invasion by amastigotes but were not effective in preventing macrophage invasion by promastigotes. Fab fragments of ST-3 inhibited invasion of cultured mouse macrophages by amastigotes (80%) or promastigotes (60%). The GSL with the simplest structure recognized by these MoAbs was isolated and characterized (by negative ion fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry of the permethylated compound, degradation with exoglycosidases, and 1H NMR) as the novel globoseries structure Gal beta 1-->3Gal alpha 1-->4Gal beta 1-->4Glc beta 1-->Cer, which has beta 1-->3Gal in place of the beta 1-->3GalNAc of globoside. The ceramide contains a 16:0 fatty acid and d18:1 sphingosine as the long chain base. The MoAbs also reacted with a series of GSLs from amastigote forms of L. amazonensis, with longer carbohydrate chains, probably containing identical end groups Gal beta 1-->3Gal alpha 1-->R. Expression of surface GSLs may render amastigote forms more effective than promastigotes in binding and invading host macrophages, thus enhancing the infectious process.

Animals↗