Neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii

The neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii were characterized. The most abundant glycolipid was ceramide monosaccharide, followed by ceramide trisaccharide, ceramide tetrasaccharide, and ceramide disaccharide. More complex neutral glycolipids accounted for almost one-third of the total. The total amount of these glycolipids was 0.59 mg/g of dry weight of the ova preparation, a yield which was one-seventh of that of spermatozoa neutral glycolipids. Structural analyses were performed by enzymatic hydrolysis of the glycolipids with exoglycosidases, permethylation experiments, and also immuno-chemical assays. The proposed structures are as follows: ceramide monosaccharides, Gal-Cer and Glc-Cer; ceramide disacharides, Gal(beta 1-4)Gal-Cer, Gal(beta 1-4)Glc-Cer, and Man(beta 1-4)Glc-Cer; ceramide trisaccharide, Man(alpha 1-3)Man(beta 1-4)Glc-Cer; ceramide tetrasaccharides, Man(alpha 1-3)[Xyl(beta 1-2)]Man(beta 1-4)Glc-Cer, GlcNAc(beta 1-2)Man(alpha 1-3)Man(beta 1-4)Glc-Cer, Man(alpha 1-3)[Gal(beta 1-2)]Man(beta 1-4)Glc-Cer, and Man(alpha 1-2?)Man(alpha 1-3)Man(beta 1-4)Glc-Cer. The latter two ceramide tetrasaccharides were new types of glycosphingolipids. The spectrum of ova glycolipids appeared to be more complicated than that of the spermatozoa glycolipids. The ova glycolipids characterized here, with the exception of ceramide tetrasaccharides, contained considerable amounts of 2-hydroxy fatty acids, which were not observed in the spermatozoa glycolipids. The major sphingosine base was C18-sphingenine in all the ova glycolipids as well as in the spermatozoa glycolipids. However, the content of anteiso type of sphingosine base was 2- to 3-fold higher in the ova than in the spermatozoa.     

Proton nuclear magnetic resonance of neutral and acidic glycosphingolipids

A number of intact neutral glycosphingolipids (globo, asialoganglio, neolacto, and gala series), gangliosides, and sulfatide were analyzed by proton nuclear magnetic resonance (NMR) using dimethyl-d6 sulfoxide as a solvent at different conditions of measurement. The chemical shifts of amide proton of ceramide, N-acetylhexosamine and sialic acid moieties were positioned with regularity, thus providing their molar composition. The chemical shifts of amide proton in ceramide moiety differed with respect to constituent fatty acids; delta 7.45 to 7.52 ppm at 25 degrees C for the nonhydroxy acids and 7.32 to 7.42 ppm for the hydroxy acids. The chemical shifts of methyl proton in N-acetyl group were distinguished between N-acetylhexosamine and N-acetylneuraminic acid, and those in N-acetylgalactosamine were discriminated between neutral glycolipids and gangliosides. In the presence or absence of D2O in dimethyl sulfoxide at 110 degrees C, the anomeric protons resonated with regularity characteristic of respective monosaccharide linkages, and the anomeric protons of N-acetylgalactosamine in neutral glycolipids and gangliosides were clearly distinguished. The present study thus demonstrates the general applicability of NMR procedure to glycosphingolipids, providing the determination of chemical composition of both the lipophilic and carbohydrate moieties and the structural elucidation.     

Fast atom bombardment-mass spectrometry of glycosphingolipids extracted from thin-layer chromatography plates

A method is described for analysis of glycosphingolipids extracted from thin-layer chromatography plates. Mixtures of glycolipids and gangliosides were separated by thin-layer chromatography and the individual bands were eluted, permethylated, and, after purification, analyzed by fast atom bombardment-mass spectrometry. The glycosphingolipids could be characterized from their fast atom bombardment mass spectra in terms of partial monosaccharide sequence, ceramide composition, and molecular weight. The sensitivity of the method allows characterization of 1-5 micrograms of glycosphingolipid.     

Characterization of two glucuronic acid-containing glycosphingolipids in larvae of the green-bottle fly, Lucilia caesar

Two glucuronic acid-containing glycosphingolipids were purified from larvae of the green-bottle fly, Lucilia caesar by DEAE-Sephadex and Iatrobeads column chromatography. Structures of these acidic glycolipids, glycolipids X and Y, were elucidated by means of sugar analysis, permethylation, enzymatic hydrolysis, negative-ion fast atom bombardment mass spectrometry, and NMR studies. Glycolipid X was determined to have the following structure: GlcA beta 1-3Gal beta 1-3GalNAc alpha 1-4 GalNAc beta 1-4 GlcNAc beta 1-3Man beta 1-4Glc beta 1-1 ceramide. The other acidic glycolipid, glycolipid Y contains a phosphoethanolamine residue linked through the 6-hydroxy group of the N-acetyl-glucosamine unit of glycolipid X. The ceramide moieties were composed of saturated fatty acids (16:0-22:0) and tetradeca- and hexadeca-4-sphingenines. Based on the structural similarity of the ceramide moieties it appears likely that glycolipid X is an intermediate from which glycolipid Y is synthesized by addition of a phosphoethanolamine residue.     

Surface carbohydrates of hamster fibroblasts. I. Chemical characterization of surface-labeled glycosphingolipids and aspecific ceramide tetrasaccharide for transformants.

1. Neutral glycosphingolipids of hamster fibroblast NIL cells have been characterized as follows: glucosylceramide, lactosylceramide (betaGall yields 4Glc yields Cer), a digalactosylceramide (alphaGall yields 4betaGal yields Cer), a trihexosylceramide (alphaGall yields 4betaGall yields 4Glc yields Cer), two kinds of ceramide tetrasaccharides (A: alphaGa1NAcl yields 3betaGalNAcl yields 3alphaGall yields 4betaGall yields 1Cer, a new type of Forssman active glycolipid; B: globoside, betaGalNAcl yields 3alphaGall yields 4betaGall yields 4betaGlc yields Cer), and a ceramide pentasaccharide having a classical structure for Forssman antigen (alphaGalNAcl yields 3betaGalNAcl yields 3alphaGall yields 4betaGall yields 4Glc yields Cer). 2. Neutral glycosphingolipids of polyoma virus-transformed NIL cells (NILpy) have been characterized as having an additional ceramide tetrasaccharide which was absent in normal NIL cells. The structure of this specific glycolipid was identified as lacto-N-neotetraosylceramide (betaGall yields 4betaGlc-NAcl yields 3betaGall yields 4Glc yields Cer). Chemical quantities of ceramide tetra- and pentasaccharides in NILpy cells were much lower than in NIL cells. 3. All of these glycolipids, except glucosylceramide and lactosylceramide, were labeled externally by galactose oxidase and tritiated borohydride according to the method previously described (GAHMBERG, C. G, and HAKOMORI, S. (1973) J. Biol. Chem. 248, 4311-4317). The specific activities of the label in glycolipid of NIHpy cells were much greater than that in NIL cells, i.e. reactivity of glycolipids with galactose oxidase in NIHpy cells was much higher than for NIL cells. The surface label in glycolipids was cell cycle-dependent in NIL cells, and a remarkable exposure of a galactosyl residue of a ceramide tetrasaccharide was demonstrated only on the surface of NILpy cells, due to the presence of lacto-N-neotetraosylceramide.     

Structural studies on glycolipids of shellfish. V. Gala-6 series glycosphingolipids of the marine snail, Chlorostoma argyrostoma turbinatum

A series of glycosphingolipids and phosphonoglycosphingolipid containing only galactose as the sugar component were isolated from the marine snail, Chlorostoma argyrostoma turbinatum. The structures of these lipids were studied by methylation analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy and fast atom bombardment mass spectrometry, and characterized as follows: the glycosphingolipids galactosyl beta(1-1)ceramide, galactosyl beta(1-6)galactosyl beta(1-1)ceramide, galactosyl beta(1-6)galactosyl beta(1-6)galactosyl beta(1-1)ceramide and galactosyl beta(1-6)galactosyl beta(1-6)galactosyl beta(1-6)galactosyl beta(1-1)ceramide, and phosphonoglycosphingolipid N-methylaminoethylphosphonyl galactosyl(1-1)ceramide. The main molecular species of the ceramide moiety were hexadecanoyl-octadecasphingenine and hydroxyhexadecanoyl-octadecasphingadienine in all of these sphingolipids.     

Retinoic acid alters the metabolic 3H-labelling of glycosphingolipids

Retinoic acid increases the incorporation of radioactivity from a mixture of [3H]-galactose and [3H]-glucosamine into glycosphingolipids of serum-starved quiescent human foreskin fibroblasts with a preferential labelling of ceramide mono- and dihexoside as compared to ceramide tri- and tetrahexoside. Under the conditions used, no similar change in the specific labelling of glycoprotein is observed. Alteration in [3H]-precursor uptake into glycolipids comparable to that seen under the influence of retinoic acid does not occur in the presence of phorbolester, colchicine, butyrate or after infection with cytomegalovirus.     

Blood group A-active glycosphingolipids analysis by the combination of TLC-immunostaining assay and TLC/SIMS mass spectrometry

Blood group A-active glycosphingolipids from human erythrocyte membranes were identified by the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry (TLC/SIMS). Partially purified lipid extracts were chromatographed by TLC and then blood group A-active glycolipids were detected by TLC-immunostaining assay using anti-A antibody. The parts of the plates which contained the same Rf area as anti-A positive spots were cut out and subjected to direct SIMS analysis. The TLC/SIMS spectra were quite similar to those obtained by ordinary SIMS. Detailed information, such as molecular weight, molecular species, ceramide portion, and oligosaccharide sequence, was obtained. Also, peracetylated blood group A-active glycolipids were analyzed in a similar manner. After the position of A-active glycolipids on a TLC plate was confirmed by in situ deacetylation and TLC-immunostaining, acetylated A-active glycolipids were also analyzed by the TLC/SIMS. Enhanced sensitivity was obtained with peracetylated glycolipids. Consequently, small amounts of unpurified bioactive glycolipids can be readily analyzed by TLC/SIMS.     

Synthetic studies on glycosphingolipids from the parasite Echinococcus multilocularis.

Novel neutral glycosphingolipids isolated from the metacestodes of Echinococcus multilocularis by Persat, may be expected to be involved in host-parasite interactions. We have synthesized these glycosphingolipid analogues containing 2-branched fatty alkyl residues in place of ceramide. The glycosylation of galactosyl donors 4 and 5 with each of the acceptors 2 and 11 in the presence of N-iodosuccinimide (NIS)/TfOH, and the glycosylation of fucosyl donor 13 with acceptors 12 and 20 in the presence of dimethyl(methylthio)sulfonium triflate (DMTST) gave the desired oligosaccharide derivatives at good yield. The fully per-O-acylated 2-(trimethylsilyl)ethyl glycosides 6, 15, 21, and 26 were converted to glycosylimidates 7, 16, 22, and 27, which were condensed with 2-(tetradecyl)hexadecanol and subsequently deacylated give four target glycosphingolipid analogues.     

Biosynthesis and degradation of mammalian glycosphingolipids

Glycolipids are a large and heterogeneous family of sphingolipids that form complex patterns on eukaryotic cell surfaces. This molecular diversity is generated by only a few enzymes and is a paradigm of naturally occurring combinatorial synthesis. We report on the biosynthetic principles leading to this large molecular diversity and focus on sialic acid-containing glycolipids of the ganglio-series. These glycolipids are particularly concentrated in the plasma membrane of neuronal cells. Their de novo synthesis starts with the formation of the membrane anchor, ceramide, at the endoplasmic reticulum (ER) and is continued by glycosyltransferases of the Golgi complex. Recent findings from genetically engineered mice are discussed. The constitutive degradation of glycosphingolipids (GSLs) occurs in the acidic compartments, the endosomes and the lysosomes. Here, water-soluble glycosidases sequentially cleave off the terminal carbohydrate residues from glycolipids. For glycolipid substrates with short oligosaccharide chains, the additional presence of membrane-active sphingolipid activator proteins (SAPs) is required. A considerable part of our current knowledge about glycolipid degradation is derived from a class of human diseases, the sphingolipidoses, which are caused by inherited defects within this pathway. A new post-translational modification is the attachment of glycolipids to proteins of the human skin.     

High-performance liquid affinity chromatography and in situ fluorescent labeling on thin-layer chromatography of glycosphingolipids

A method combining high-performance liquid affinity chromatography and in situ fluorescent labeling on thin-layer chromatography is introduced for determination of glycosphingolipids. Glycolipids in crude extract from rat liver were separated quantitatively from neutral lipids and phospholipids with a phenylboronic acid-derivatized silica gel column. Glycolipids were eluted quantitatively with approximately 98% of crude extract recovered. This column is useful for selective cleanup of glycosphingolipids in crude extract from tissue. Simultaneously, a fluorometric determination of glycosphingolipids with 7-amino-4-methylcoumarin after NaIO4 oxidation on a TLC plate was introduced and its condition was optimized. Glycolipids in amounts ranging from 1 to 100 pmol are easily detectable and give linear responses over the respective ranges. The method is fast and useful for the determination of glycolipids from small amounts of biological samples and requires a minimum amount of about 1 mg of biological specimen for determination of glycolipids.     

Studies on glycosphingolipids in larvae of the green-bottle fly, Lucilia caesar: two neutral glycosphingolipids having large straight oligosaccharide chains with eight and nine sugars

Two neutral glycosphingolipids having large straight oligosaccharide chains with eight and nine sugars, provisionally named COS and CNS, were isolated and purified from larvae of the green-bottle fly, Lucilia caesar, as the only two remaining unidentified significant neutral glycolipids in this organism. From the results of sugar analysis, permethylation, negative-ion fast atom bombardment mass spectroscopy (FAB-MS), and 1H-NMR studies, the structures of the two glycolipids are proposed to be: COS, GalNAc beta 1-3GlcNAc beta 1-3Gal beta 1-3GalNAc alpha 1-4GalNAc beta 1-4GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer; and CNS, Gal beta 1-3GalNAc beta 1-3GlcNAc beta 1-3Gal beta 1-3GalNAc alpha 1-4GalNAc beta 1-4GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer. The fatty acid and long-chain base compositions of the above glycolipids were very similar, and were dominated by arachidic acid, and tetradeca- and hexadeca-4-sphingenines. The great similarity between the compositions of their ceramide moieties suggests that COS may be a precursor in the glycosylation reaction yielding CNS.     

Sphingomyelin, glycosphingolipids and ceramide signalling in cells exposed to P-fimbriated Escherichia coli

Uropathogenic Escherichia coli attach to epithelial cells through P fimbriae that bind Galα1-4Galβ-oligosaccharide sequences in cell surface glycosphingolipids. The binding of P-fimbriated E. coli to uroepithelial cells causes the release of ceramide, activation of the ceramide signalling pathway and a cytokine response in the epithelial cells. The present study examined the molecular source of ceramide in human kidney A498 cells exposed to P-fimbriated E. coli . Agonists such as TNF-α and IL-1β released ceramide from sphingomyelin by the activation of endogenous sphingomyelinases and hydrolysis of sphingomyelin, and triggered an IL-6 response. P-fimbriated E. coli caused a slight increase in endogenous sphingomyelinase activity, but there was no associated sphingomyelin hydrolysis. Instead, the concentration of galactose-containing glycolipids decreased. We propose that P-fimbriated E. coli differ from other activators of the ceramide pathway, in that release of ceramide is from receptor glycolipids and not from sphingomyelin. Receptor breakdown may be an efficient host defence strategy, as it reduces the concentration of cell surface receptors, releases soluble receptor analogues and activates an inflammatory response.     

Polar glycosphingolipids in annelida. A novel series of glycosphingolipids containing choline phosphate from the earthworm, Pheretima hilgendorf.

A novel series of glycosphingolipids containing choline phosphate has been demonstrated in whole tissues of the earthworm, Pheretima hilgendorfi. The thin layer chromatographic pattern of the total polar glycolipids revealed the presence of more than three components with positive reactions toward orcinol-sulfuric acid (sugar), molybdate (phosphate), and Dragendorff's (choline) spray reagents. Two of these polar glycolipids (PGL1 and PGL2) were purified by the use of successive column chromatography on QAE-Sephadex A-25 and silicic acid (Iatrobeads) and detected during elution by the presence of galactose-bound choline phosphate. The structural elucidation of the oligosaccharide moieties was performed by compositional sugar analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, and methylation analysis. Thus, the structures of PGL1 and PGL2 were deduced to be as follows: cholinephosphoryl-->6Gal beta 1-1Cer and cholinephosphoryl-->6Gal beta 1-6Gal beta 1-1Cer. Although the oligosaccharide structures of both PGL1 and PGL2 have previously been found in other organisms, the presence of a choline phosphate group as an oligosaccharide substituent is the first finding in nature. The main molecular species of the ceramide moieties were composed of beheninyl- and lignocerinyloctadecasphingenines and their nonadecasphingenine homologues.     

Quantitative isolation of total glycosphingolipids from animal cells

The quantitative isolation of total glycosphingolipids from crude lipid extracts without contamination from other lipid classes is described. The method consists of (a) acetylation of total lipids with pyridine and acetic anhydride, (b) separation of acetylated glycolipids from nonglycolipids on a magnesia-silica gel (Florisil) column, and (c) deacetylation of glycolipid in chloroform-methanol-sodium methoxide. This method is useful for determination of microgram quantities of glycolipids derived from less than 1 ml of packed cells.     

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.     

Isolation and structural analysis of three neutral glycosphingolipids from a mixed population of Caenorhabditis elegans (Nematoda: Rhabditida)

The free-living nematode, Caenorhabditis elegans, has been proposedand analyzed as a prototypic model for parasitic nematodes.In order to study whether there is a structural basis for theproposed analogy with respect to nematode glycoconjugates, wehave analyzed Caenorhabditis elegans glycosphingolipids. Three,simple neutral glycosphingo-lipid components of the neutralglycolipid fraction were isolated by high-performance liquidchromatography. Structural analysis was performed by methylationanalysis, exoglycosidase cleavage, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and ceramide analysis.The chemical structures have been determined as Glcß1Cer,Manß4Glcß1Cer and GlcNAcß3Manp4Glcß1Cer;that are characterized as belonging to the arthro-series ofprotostomial glycosphingolipids. The ceramide moiety of theparent glycosphingolipid-ceramide mono-hexoside was dominatedby 2-hydroxy fatty acids, and a d17:1 sphingoid-base with aniso- or anteiso-branched chain. The chemical composition ofthe three glycosphingolipids from Caenorhabditis elegans displayedclose structural coincidence with the equivalent structuresfrom the porcine parasitic nematode, Ascaris suum (G.Lochnit,R.D. Dennis, U.Zähringer, and R.Geyer, Glycoconjugate J.,1997), in support of this organism as a prototypic glyco-sphingolipidmodel for parasitic nematodes. Arthro-series glycosphingolipids Caenorhabditis elegans d17:1 branched sphingoid-bases MALDI-TOF-MS nematode neutral glycosphingolipids     

Acidic glycosphingolipids of human amnion

Six major acidic glycosphingolipids were isolated from human amnion using DEAE Sephadex A-25 and silica beads column chromatography. The structures of these glycosphingolipids were determined by methylation analysis, TLC immunostaining and/or negative ion FAB-MS, and were concluded to be II3 alpha NeuAcLacCer(GM3), IV3 alpha NeuAcnLc4-Cer (sialyl[alpha 2-3]paragloboside), IV6 alpha NeuAcnLc4Cer (sialyl[alpha 2-6]paragloboside), IV3 alpha NeuAcIII4 alpha FucLc4Cer (sialyl Lea), VI3 alpha NeuAcnLc6Cer (i-ganglioside) and II3 alpha (NeuAc alpha 2----8NeuAc)LacCer (GD3). In addition, several minor glycosphingolipids were detected with specific monoclonal antibodies, including glycolipids with NeuAc alpha 2-3Gal beta 1-4GlcNAc-beta 1- or NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1- determinant. Our results show that the glycosphingolipids of human amnion are characterized by having mainly type II chain analogues and onco-fetal antigens.     

Immunochemistry of the Lewis-blood-group system: Proton nuclear magnetic resonance study of plasmatic Lewis-blood-group-active glycosphingolipids and related substances

The Le^a-, Le^b-, and H-type 1 (Le^dH)-blood-group-active glycosphingolipids, as well as H-I-type 2 glycolipid, lactotetraosyl ceramide, and neo-lactotetraosyl ceramide were examined by ¹H nuclear magnetic resonance at 360 MHz in dimethyl-d₆ sulfoxide as solvent. The resonances of almost all protons of the sugar rings were assigned with the aid of spin decoupling and nuclear Overhauser difference spectroscopy. The latter technique was also applied to establish the sequences and sites of glycosidic linkage. This information, combined with the chemical shift-structure correlations established in our previous work, led to an independent identification of those six glycolipids. Type 1 (Galβ1 → 3GlcNAc) and type 2 (Galβ1 → 4GlcNAc) saccharide chains can be distinguished by this approach. Some deviations from additivity in chemical shifts, calculated for oligosaccharides from the data on their constituent sugar residues, furnished information on the conformational changes in crowded glycolipid molecules.     

Fatty acids of glycosphingolipids from pig blood fractions

Four glycolipids have been isolated from three fractions of pig blood. The glycolipids were presumably cerebroside, diglycosyl ceramide, triglycosyl ceramide, and globoside. The blood fractions were erythrocytes and plasma high and low density lipoproteins. Fatty acid distributions were determined for each glycolipid as a means to assist in identifying relationships among the several glycolipids. Normal fatty acids predominated in all glycolipids except the globosides from erythrocytes in which the amount of hydroxy acids was slightly greater than the amount of normal acids. Hydroxy acids appeared to be present in all the glycolipids, but the concentration was very low in cerebrosides isolated from high density lipoproteins and erythrocytes, and in diglycosyl ceramide and globoside of the low density lipoproteins. In general, the average fatty acid chain length increased from cerebroside to globoside. This was most apparent in erythrocytes and also greater for normal acids than for hydroxy acids. Fatty acid distributions of erythrocyte glycolipids had sufficient variation to make a metabolic relationship by simple addition of a hexose appear doubtful. While the fatty acid distributions found in plasma lipoproteins were more similar, some means of acyl group selection is probably present for either the synthesis or degradation of these glycolipids.