Structures Of Glycosphingolipids Research Articles

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.

14] Glycosphingolipids: structure, biological source, and properties

Publisher Summary This chapter focuses on the structure, biological source, and properties of glycosphingolipids. Several reasons can be cited for the explosive increase in the number of newly characterized glycosphingolipids. Technical developments (TLC immunostaining and solid-phase microtiter plate binding assays) have provided a means of demonstrating that monoclonal antibodies are immunoreactive with carbohydrate sequences carded by glycosphingolipids. This has led to a greater interest in evaluating the glycosphingolipid composition of a variety of biological tissues and tumors. Improvements in chromatographic procedures, especially high-performance liquid chromatography, have simplified isolation and separation methods. Use of nuclear magnetic resonance (NMR) has improved structural characterization of glycosphingolipids. Development and application of FAB mass spectrometry for glycosphingolipid characterization have been especially important for the characterization of high molecular weight compounds. Isolation of endoglycosidase and new exoglycosidases has provided additional tools for characterization.

Isolation and characterization of four major neutral glycosphingolipids from the liver of the English sole (Parophrys vetulus). Presence of a novel branched lacto-ganglio-iso-globo hybrid structure.

We have previously reported on carbohydrate structures of the major acidic glycosphingolipids from the liver of the English sole, Parophrys vetulus (Ostrander, G. K., Levery, S. B., Hakomori, S., and Holmes, E. H. (1988) J. Biol. Chem. 263, 3103-3110). We have now isolated four major neutral glycosphingolipids from English sole liver. They have been characterized by 1H nuclear magnetic resonance spectroscopy, methylation analysis, and by fast atom bombardment-mass spectrometry. In addition to neutral glycosphingolipids with known structures (CMH, lactosylceramide, and Gg3), a major polar neutral glycosphingolipid was isolated and characterized as having the following novel structure: (formula; see text) The compound represents a novel hybrid of neolacto-, ganglio-, and iso-globo-series glycosphingolipid structures, a combination not previously encountered. Furthermore, the linkage Fuc alpha 1----3GalNac has not been previously reported for a glycosphingolipid. The relationship of these structural elements to known glycoconjugates is discussed.

Thermodynamic-geometric correlations for the morphology of self-assembled structures of glycosphingolipids and their mixtures with dipalmitoylphosphatidylcholine

The morphology of aqueous dispersions of five neutral glycosphingolipids (GalCer, GlcCer, LacCer, asialo-G M2, asialo-G M1), sulfatide, and five gangliosides (G M3, G M2, G M1, G D1a and G t1b) and their mixtures with dipalmitoylphosphatidylcholine was studied by negative staining electron microscopy. The morphological features are interpreted on the basis of thermodynamic and geometric constraints previously studied in these systems (Maggio, B (1985) Biochim. Biophys. Acta 815, 245–258). The correlation between the theoretical predictions and the experimental findings are in reasonable agreement. Small changes in tge molecular parameters of the individual glycosphingolipids or in their proportion in mixtures with dipalmitoylphosphatidylcholine bring about remarkable variations on the type of structure formed, its radius of curvature and thermodynamic stability.

10 オニヒトデより得られる新スフィンゴ糖脂質Acanthaganglioside類の単離と構造(ポスター発表の部)

10 オニヒトデより得られる新スフィンゴ糖脂質Acanthaganglioside類の単離と構造(ポスター発表の部)

Glycosphingolipids of human umbilical vein endothelial cells and smooth muscle cells

Glycosphingolipids (GSLs) represent an important class of immunogens and receptors. Although cell surface antigens and receptors of endothelial cells (ECs) have been the subject of extensive biochemical investigation, no information is available about their GSLs. We report here the characterization by chromatographic and immunological techniques of GSLs of cultured human umbilical vein ECs and, for comparison, umbilical vein smooth muscle cells (SMCs). The most abundant neutral GSLs of both cell types were lactosylceramide, Gb 3, and Gb 4, and both cells contained complex lacto and globo series compounds. Immunostaining revealed that ECs, but not SMCs, contained long chain GSLs bearing a type 2 blood group H determinant. ECs also contained more long chain GSLs bearing an unsubstituted terminal lactosamine structure than SMCs. Labeling with galactose oxidase/NaB 3H 4 demonstrated that neutral glycolipids that contained three or more sugars were accessible on the cell surface. The major gangliosides of both cell types were G M3 and IV 3NeuAcnLc 4. Immunostaining following neuraminidase treatment revealed that most of the long chain gangliosides in both types of cells contained a lacto core structure, and that ganglio series compounds were more abundant in SMCs than ECs. Gangliosides that contain a polyfucosyllactosamine core and a globo core were also present in both cell types. These results demonstrate that endothelial and smooth muscle cells contain a large diversity of GSL structures, and provide the basis for investigation of the role of these GSLs as cell surface antigens and receptors for blood components.

Immunochemistry of the Lewis blood-group system: Isolation and structures of Lewis-c active and related glycosphingolipids from the plasma of blood-group O Le(a-b-) nonsecretors

Five different glycosphingolipid fractions (GL-3, 285 μg; GL-5,1090 μg; GL-6, 615 μg; GL-7, 555 μg; and GL-8, 155 μg) have been isolated from 25 liters of plasma of O Le(a-b-) nonsecretors by means of ethanol extraction, several steps of Folch distribution, and reversed-phase, silicic acid, and ion-exchange column chromatography of native or peracetylated substances. Final purification, accomplished by preparative silica gel highperformance thin-layer chromatography, led to chromatographic homogeneity of GL-3 and GL-6. In the hemagglutination inhibition as well as quantitative passive hemagglutination techniques two of these substances (GL-3, GL-5) exhibited distinct, and the other three (GL-6-GL-8) very strong, Le c blood-group activities when tested against two different Le c antisera of human or goat origin. The fragments' structures were elucidated by fast atom bombardment and electron impact mass spectrometry of permethylated derivatives in order to determine molecular weight, sugar sequence, position of branching points, and type of oligosaccharide chains, as well as fatty acid and sphingosine patterns of the ceramide residue. Combined gas-liquid chromatography and mass spectrometry of partially methylated alditol acetates identified sugar composition and glycosidic linkages. Thus, the following structures could be established: ▪ In contrast to the structurally homogeneous GL-3, minor amounts of 4- O-substituted GlcNAc pointed to a small contamination of GL-6 by branched type 2 ceramide nonasaccharide analogs. Glycolipids containing hepta- or nonasaccharides as in GL-3 or GL-6 could also be identified in fractions GL-5 (ceramide heptasaccharide) and GL-7 and GL-8 (ceramide nonasaccharide). These latter fractions revealed, however, distinct heterogeneity due to the presence of a small amount of either a type 2 analog of GL-3 (GL-5) or linear, mainly type 2, ceramide hexa- (GL-5, GL-7) or octasaccharides (GL-8). In addition to previous immunochemical communications the presented Le c active structures of GL-3 and GL-6 provide evidence that 3-fucosyl- N-acetyllactosamine in combination with a type 1 based oligosaccharide sequence and a 3,6-galactosyl branching point are essential parts of the Le c antigenic determinant (as marked in the formula of GL-6).

Structures of glycosphingolipids isolated from human embryonal carcinoma cells. The presence of mono- and disialosyl glycolipids with blood group type 1 sequence.

Structures of glycolipids present in the human embryonal carcinoma cell PA1, were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase digestion. PA1 cells contain globotriaosylceramide, sialosylgangliotriaosylceramide, sialylated and nonsialylated lacto-N-neotetraosylceramide, and the following glycolipids with a blood group type 1 sequence: (formula; see text) The two former glycolipids, lacto-N-tetraosylceramide and sialosyllacto-N-tetraosylceramide, reacted with monoclonal antibodies, K21 and K4, respectively. K21 and K4 antigens are present in many of the human embryonal carcinoma cells but not in a variety of other cell lines, suggesting that sialylated but not fucosylated blood group type 1 sequences are characteristic markers for human embryonal carcinoma cells and malignant teratocarcinomas.

A description of human placental syncytiotrophoblast membrane glycosphingolipids

The glycosphingolipid (GSL) structure of isolated human term placental syncytiotrophoblast microvillous plasma membrane (StMPM) has been investigated by Folch solvent extraction and thin-layer chromatography. StMPM preparations were shown to contain unique hydrophilic GSL species, including two highly glycosylated (greater than 15 saccharide residues/molecule) components. This fraction consistently included four major gangliosides (sialylated GSL), as well as two highly-sialylated (greater than or equal to 4 sialic acid residues/molecule) minor gangliosides.

Structures of glycosphingolipids isolated from human granulocytes. The presence of a series of linear poly-N-acetyllactosaminylceramide and its significance in glycolipids of whole blood cells.

Structures of glycolipids isolated from human granulocytes were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase treatment. All neutral glycolipids, with saccharide residues ranging from 2 to 10, were found to have linear N-acetyllactosaminyl backbones. The majority of neutral glycolipids contain one or two fucosyl residues attached to N-acetylglucosamine residues through the Fuc alpha 1----3 linkage and were reactive with the monoclonal antibody specific to Gal beta 1----4(Fuc alpha 1----3)GlcNAc, the Lex structure. Their general structure can be expressed as follows: (formula; see text) where n = 0-3. Glycolipids containing sialic acid (gangliosides) were also found to have linear N-acetyllactosaminyl backbones with sialic acid joined to this backbone by either alpha 2----3 or alpha 2----6 linkage. The gangliosides have the following general structure: (formula; see text) where n = 0-3. The ceramide was composed of sphingosine with d18:1 as the long-chain base and C16:0 (as a major component) or C24:1 (as a minor component) fatty acid. Analysis of glycolipids isolated from granulocytes, erythrocytes, and whole blood cells revealed that, among the glycolipids prepared from the whole blood cells, dihexaosylceramide, lactoneotetraosylceramide, and the above described linear lactoneo series neutral glycolipids are present in granulocytes but barely present in erythrocytes.

Recent advances in structural analysis of gangliosides: primary and secondary structures.

High-field (500 MHz) proton NMR has been used to elucidate the primary and secondary structures of glycosphingolipids (GSLs). Using 2-D J-correlated spectroscopy (2-D SECSY) which establishes scalar couplings of protons, the monosaccharide composition, anomeric configuration and aglycon structures of a GSL can be established. 2-D nuclear Overhauser effect spectroscopy (2-D NOE) then establishes through-space intra- and inter-residue couplings of cross-relaxing protons. We have found that each anomeric proton is involved in NOE couplings with inter- and intra-residue protons. The inter-residue coupling, resulting from interaction of protons across the glycosidic linkage, establishes the n-1 sugar residue and specific glycosidation site to which the n-residue is linked. When such information is known for each residue and is combined, the sequence of the core oligosaccharide is obtained. The sialylation-induced glycosidation shift is then used to establish the site of sialic acid residue attachment in a ganglioside molecule. We have also observed that the anomeric proton inter-residue NOE couplings can be used to suggest the preferred conformation of an oligosaccharide. We have found that the oligosaccharide residue of globoside exists in a unique and rather rigid conformation which could be stabilized by hydrogen bonds and van der Waals interactions. Since GSLs are known to have a receptor role and are implicated in cell-cell recognition, enzyme-substrate interaction and antigen-antibody interaction, the determination of their conformation should be useful in understanding their biological functions.

Novel pentahexosyl ganglioside of the globo series purified from chicken muscle.

A monosialosyl ganglioside, sialosylpentaglycosyl-ceramide, has been isolated from chicken pectoral muscle. This ganglioside is the first to be found which contains the glycosphingolipid structure of the globo series. It was extracted from tissue with a mixture of tetrahydrofuran and aqueous KCl and purified by anion exchange and silicic acid chromatography. Enzymic hydrolysis utilizing specific glycosidases and methylation technique were employed to establish the structure which is proposed to be: NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc beta 1 leads to 3Gal alpha 1 leads to 4Gal beta 1 leads to 4GlcCer. The chain base was predominantly sphingosine and the fatty acids present were mainly stearate and oleate.

Antibodies to total brain gangliosides: titer and specificity of antisera.

Our sustained studies of the immunological activity of lipidsl,2 led to the important generalization that such activity was intimately related to glycosphingolipid structures. We therefore had an early interest in the immunological activity of gangliosides and an early appreciation that they exhibited only weak immunogenic properties, much weaker than those of neutral glycosphingolipids.

Proton nuclear magnetic resonance analysis of anomeric structure of glycosphingolipids: Lewis-active and Lewis-like substances

High resolution nuclear magnetic resonance spectra were recorded in a chloroform solution of six Lewis-active or Lewis-like glycosphingolipids in permethylated and permethylated-reduced (LiAlH 4) form. The samples were selected to cover the presently known structural variants of α-fucose linked to galactose and N-acetylglucosamine. Fucα1 → 2Gal, Fucα1 → 3GlcNAc, and Fucα1 → 4GlcNAc gave characteristic and well-separated anomeric resonances. Furthermore, upon reduction there was a strong deshielding effect on Fucα1 → 3GlcNAc and Galβ1 → 3GlcNAc (linkage vicinal to reduced amide), which makes it possible to differentiate type 1 (Galβ1 → 3GlcNAc) and type 2 (Galβ1 → 4GlcNAc) saccharide chains. This improved method of nuclear magnetic resonance spectroscopy is discussed in relation to sequence analysis by mass spectrometry, two microscale structural methods using the same type of derivatives and needing no degradations before analysis.

Proton nuclear magnetic resonance analysis of anomeric structure of glycosphingolipids: Blood group ABH-active substances

High resolution nuclear magnetic resonance spectra of permethylated and permethylated-reduced (LiAlH 4) derivatives were recorded in chloroform solution for the following glycosphingolipids with known structure: lactotriaosylceramide, neolactotetraosylceramide (paragloboside), two blood group H-active pentaglycosylceramides (type 1 and type 2 saccharide chains, respectively), a B-active hexaglycosylceramide, an A-active hexaglycosylceramide, and an A-active octaglycosylceramide. Good quality and resolution allow a clear-cut diagnosis of α-anomeric protons of Fuc, Gal, and GalNAc, and in most cases of all β protons. Upon reduction there is a strong deshielding effect on H-1 of Gal of Galβ1 → 3GlcNAc but not on Gal of Galβ1 → 4GlcNAc. It is therefore possible to differentiate type 1 and type 2 chains by this method, a structural difference of importance for serological specificity. Nuclear magnetic resonance spectroscopy may therefore provide conclusive information on the anomeric structure of the immunodeterminant of blood group-active glycolipids using the same derivatives as for sequence analysis by mass spectrometry.

Proton nuclear magnetic resonance analysis of anomeric structure of glycosphingolipids: The globo-series (one to five sugars)

Proton nuclear magnetic resonance spectroscopy has been reevaluated concerning the assignment of anomeric structure of glycosphingolipids. Solubility problems due to a varying number of sugars are avoided by permethylation, allowing a wide range of glycolipids to be compared. High resolution spectra were recorded in chloroform solution for the following substances with known structure, most of them representing a successive building up of members of the globo-series: ceramide, Galβ1 → 1Cer, a mixture of Glcα1 → 1Cer and Glcβ1 → 1Cer, lactosylceramide, globotriaosylceramide, globotetraosylceramide (globoside), and GalNAcα1 → 3globotetraosylceramide (Forssman hapten). Resonances originating in anomeric protons were identified and possible interference from other signals was defined. A complex set of resonances from H-1 of hexosamines was probably due to two separate conformers of the acetamido group caused by N-methylation. The complexity disappeared upon reduction with LiAlH 4. The chemical shifts and coupling constants were characteristic for the configuration of the glycosidic bond, the type of monomer, and in part for its location in the chain. At present, spectra may be recorded from 200-μg samples. It is concluded that the good quality and resolution obtained make this technique an alternative method to the presently used enzymatic degradation for establishing anomeric structure of glycosphingolipids.

Glycosphingolipids of human plasma lipoproteins

AbstractThe content and structure of glycosphingolipids (GSL) in human plasma lipoproteins were studies. The quantitative distribution of the neutral GSL(Glc‐Cer, Gal‐Glc‐Cer, Gal‐Gal‐Glc‐Cer, and GalNAc‐Gal‐Gal‐Glc‐Cer) and the principal ganglioside (AcNeu‐Gal‐Glc‐Cer) within the different lipoprotein classes was similar to that of whole plasma. The total amounts (μmol glucose/100 ml plasma) of GSL in the plasma lipoproteins of three normal subjects were VLDL (very low density lipoproteins) (trace to 0.46), LDL (low density lipoproteins) (1.08–1.48), HDL2 (high density lipoproteins2) (0.62–0.85), and HDL3 (high density lipoproteins3) (trace to 0.28). In subjects with Lp(a) lipoproteins, HDL2 rather than HDL3 contained most of the GSL in HDL. When the data were corrected for differences in the plasma concentrations of the lipoproteins, the total amounts of GSL(nmol glucose/mg lipoprotein cholesterol) were VLDL(trace to 21.20), LDL(11.70–15.36), HDL2(8.50–9.10), and HDL3(3.12). No GSL were detected in lipoprotein deficient plasma. Mass spectrometry of the trimethylsilyl derivatives of the GSL in LDL showed major fragment ions characteristic of their individual structural components. The elevated plasma levels of the GSL(2–18 fold), in a homozygote for familial hypercholesterolemia, resided in LDL which contained an absolute increase (per mg lipoprotein cholesterol) of GSL. Most, if not all, of the plasma GSL are associated with plasma lipoproteins and may have an important role in their biological functions.

Schistosoma mansoni: Adsorption of Human Blood Group A and B Antigens by Schistosomula

Immediately following skin penetration, schistosomula of Schistosoma mansoni were found capable of passively adsorbing human A and B blood group antigens onto their surfaces. The antigens were adsorbed from intact erythrocytes, boiled human saliva, or alcohol extracts of erythrocyte membranes. Evidence is presented suggesting that a glycosphingolipid structure may be important in the binding of erythrocyte antigens to schistosomes. No uptake of various other blood group antigens could be demonstrated. Mouse erythrocyte antigens have been demonstrated on the surface of adult Schistosoma mansoni reared in mice (Smithers, Terry, and Hockley, 1969; Clegg, Smithers, and Terry, 1971a), and it has been shown that at least one such antigen is passively adsorbed by schistosomula (Dean, 1971; Sell and Dean, 1972; Dean and Sell, 1972). Clegg, Smithers, and Terry (1971b) reported that young schistosomes reared in a culture medium containing human erythrocytes were destroyed following surgical transfer into monkeys which had been immunized against human erythrocytes, indicating that human antigens are also acquired by schistosomes. In one experiment, the erythrocytes used to immunize recipient monkeys differed from the erythrocytes in the worm cultures with respect to five major blood group antigens. Fewer worms were killed upon transfer to monkeys so immunized than was the case with homologous transfers, and this led the authors to conclude that blood group antigens might be acquired by schistosomula. The present study was carried out to deReceived for publication 13 September 1973. * Supported by the Bureau of Medicine and Surgery Work Unit No. MR041.05.01.0023A6GI. The opinions or assertions contained herein are the private ones of the author and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles outlined in the Animal Welfare Act (PL 89-544 as amended) and followed the guidelines prescribed in DHEW Publication No. (NIH) 72-23, formerly PHS Publication No. 1024, Guide for Laboratory Animal Facilities and Care. termine which, if any, human blood group antigens are adsorbed onto the surfaces of schistosomula in vitro. MATERIALS AND METHODS Schistosomula were prepared from Puerto Ricanstrain cercariae of S. mansoni by allowing them to penetrate excised rat skins into Hanks' Balanced Salt Solution (HBSS), by methods described previously (Stirewalt and Uy, 1969; Clegg and Smithers, 1972). Alcohol extracts of erythrocyte membranes were prepared by the method of Koscielak (1963). Wet membranes prepared by hypotonic lysis (Dodge, Mitchell, and Hanahan, 1963) were mixed with ethanol to a final alcohol concentration of 85%, mixed on a shaker at room temperature for 3 days, centrifuged, and the superatant fluid stored at -15 C for 3 days. The resulting precipitate was collected by centrifugation and stored at -70 C. Koscielak showed that the blood group active molecules in these preparations are glycosphingolipids. Saliva of a known secretor of soluble blood group A substance was boiled and centrifuged, and the supernatant fluid employed in appropriate experiments. Neutr AB is a commercial preparation (Dade Reagents, Miami, Florida) containing both A and B blood group activities. The source of this preparation (equine gastric mucin) and the method of preparation (Dade Reagents, pers. comm.) indicate that the active molecules are probably glycoproteins rather than glycosphingolipids (Baer, Kabat, and Knaub, 1950; Kabat, 1956). Specific activities of alcohol-extracted antigens and Neutr AB were measured by means of the hemagglutination inhibition (HI) assay. Serial double dilutions of antigen were prepared in Vbottom wells of Microtiter plates (Cooke Engineering Company, Alexandria, Virginia). Dilutions were made in pH 7.4 phosphate-buffered saline (PBS) at a final volume of 0.05 ml per well. An equal volume of blood group typing antiserum

Determination of glycosphingolipid structures by mass spectrometry

Mass spectrometry is useful for the determination of oligosaccharide structures in complex glycosphingolipids. A partial mass spectrum (scanned from m/e 60 to m/e 800) of fully trimethylsilylated intact glycosphingolipid yields information regarding the positions of substitution of monosaccharide units, the nature of sphingolipid bases, and the relative amounts of constituent fatty acids.