Characterization of glycosphingolipid mixtures with up to ten sugars by gas chromatography and gas chromatography-mass spectrometry as permethylated oligosaccharides and ceramides released by ceramide glycanase

A novel, effective method for structural characterization of glycosphingolipids has been devised. It employs ceramide glycanase to release intact oligosaccharides followed by analysis using high-mass gas chromatography-mass spectrometry. The oligosaccharides and ceramides released by the glycanase were permethylated and analyzed. The capillary gas chromatography gave excellent resolution and separated, for example, two isomeric 10-sugar oligosaccharides with a molecular mass of 2150 daltons differing only by a Gal1-3GlcNAc and a Gal1-4GlcNAc linkage. The oligosaccharides released from sialic acid containing glycosphingolipids (gangliosides) were also analyzed for monosialo compounds. This analytical approach is simple, is quick, and can readily allow quantitation of individual glycosphingolipids.     

Analysis of protein-linked glycosylation in a sperm-somatic cell adhesion system

Murine sperm initiate fertilization by binding to the specialized extracellular matrix of their complementary eggs, known as the zona pellucida. On the basis of data reported in this study, mouse sperm also bind to rabbit erythrocytes with higher affinity than they do to murine eggs. This unusual interaction between a germ cell and a somatic cell ("sperm-somatic cell adhesion system") is also carbohydrate dependent based on its sensitivity to mild periodate oxidation. To determine what types of carbohydrate sequences could be involved in this interaction, the protein-linked oligosaccharides of rabbit erythrocytes were sequenced using novel matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry methods that enabled the analysis of individual components up to m/z 9000. The N-glycans are primarily complex biantennary and triantennary types terminated with Galalpha1-3Gal sequences. The majority of these oligosaccharides also possess one antenna consisting of a highly branched polylactosamine-type sequence that is also associated with many glycosphingolipids that coat rabbit erythrocytes. These erythrocytes also express Core 1 and Core 2 O-glycans terminated primarily with Galalpha1-3Gal sequences and to a lesser extent sialic acid. These results confirm that rabbit erythrocytes and mouse eggs present very different types of carbohydrate sequences on their surfaces. However, oligosaccharides terminated with beta1-6-linked N-acetyllactosamine or its alpha1-3 galactosylated analog are expressed on both the mouse zona pellucida and this somatic cell type. The far more abundant presentation of such sequences on rabbit erythrocytes compared with murine eggs could explain why mouse sperm display such exceptional affinity for this somatic cell type.     

The use of gas chromatography and gas chromatography-mass spectrometry for the characterization of permethylated oligosaccharides with molecular mass up to 2300

Gas chromatography and gas chromatography-mass spectrometry were adapted for the analysis of large permethylated oligosaccharides of different types. Permethylated isomaltooligosaccharides with up to 11 sugar residues and a mass of 2291 Da and two branched blood group H-type decasaccharides derived from the corresponding glycosphingolipids with masses of 2150 Da were successfully analyzed. The capillary columns used have extremely good resolution exemplified by the separation of the two decasaccharides which only differed by one internal linkage position and by the separation of four isomeric tetrasaccharides. The combined information of retention times and mass spectra gave detailed information of 22 neutral oligosaccharides from porcine intestinal mucin and the approach thus allow quick screening of O-linked-type glycans. The procedure for permethylation of oligosaccharides using solid NaOH has been investigated and adapted for structures having a glucose alditol as in reduced oligosaccharides derived from milk and glycosphingolipids.     

Novel mannitol-containing oligosaccharides obtained by mild alkaline borohydride treatment of a chondroitin sulfate proteoglycan from brain.

Mannitol-containing oligosaccharides have been isolated from a rat brain proteoglycan after mild alkaline borohydride treatment under conditions which prevent "peeling." Their structural properties were studied by gas-liquid chromatography-mass spectrometry of disaccharides as their trimethylsilylated and permethylated derivatives, methylation, analysis, specific degradations, and CrO3 oxidation. The following components were identified: Gal(beta 1 leads to 4) [Fuc(alpha 1 leads to 3)]GlcNAc(beta 1 leads to 3)Manol,GlcNAc(beta 1 leads to 3)Manol, and Manol. Evidence was also obtained for the occurrence of a sialylated oligosaccharide and another (possibly sulfated) acidic oligosaccharide, both having the sequence GlcNAc(beta 1 leads to 3)Manol at their proximal ends. These mannitol-containing oligosaccharides constitute a novel group of alkali-labile oligosaccharides in mammalian glycoconjugates. The origin of the oligosaccharides and the possible occurrence of a carbohydrate-peptide linkage involving mannose are discussed.     

Conformation analysis of carbohydrate chains of glycoconjugates

A problem of conformations of carbohydrate chains of glycoconjugates-glycoproteins and glycolipids--is reviewed. Experimental data (NMR, X-Ray) and theoretical conformational analysis data are discussed. Spatial structures of O-linked oligosaccharides from blood-group glycoproteins, N-linked oligosaccharides of different types (oligomannosidic, complex, hybrid, bisect) and carbohydrate chains of glycosphingolipids are considered.     

Polyglycosylceramides with branched N-acetyllactosamine sequences are synthesized by the human pancreatic carcinoma cell line PANC-1.

We have metabolically labeled the human pancreatic tumor cell line PANC-1 with high specific activity tritiated sugar precursors to study the expression of glycosphingolipids by this cell type. We have used a combination of detergent solubilization, exhaustive protease digestion, ceramide glycanase digestion, and reverse-phase chromatography to isolate glycosphingolipid-derived oligosaccharides specifically labeled in their component sugars. A significant proportion of the oligosaccharides derived from polar glycosphingolipids were of high molecular mass (greater than 2000 Da). The results of compositional studies, lectin affinity chromatography, and methylation analysis suggested that this high molecular weight fraction consists of lactosaminoglycan type oligosaccharides derived from polyglycosylceramides. There are on average three beta 1-6 linked N-acetyllactosamine branches attached to the polylactosamine backbone in this type of glycosphingolipid-derived oligosaccharide. The majority of the oligosaccharides also contain 1-2 mol of sialic acid that are linked alpha 2-3 to penultimate galactose. The results indicate that PANC-1 cells, like human colorectal tumor cells, express highly extended neolacto type glycosphingolipids. However, the lactosaminoglycan sequences are highly branched, unlike those associated with colorectal tumor cells.     

Transglycosylation and reverse hydrolysis reactions of endoglycoceramidase from the jellyfish, Cyanea nozakii

Endoglycoceramidase (EGCase: EC 3.2.1.123) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids. We report here transglycosylation and reverse hydrolysis reactions of EGCase from the jellyfish Cynaea nozakii. Various alkyl-GM1 oligosaccharides (alkyl-II(3)NeuAcGgOse4) were synthesized when GM1 ganglioside was treated with the EGCase in the presence of 1-alkanols. Among various 1-alkanols tested, methanol was found to be the most preferential acceptor, followed by 1-hexanol and 1-pentanol. GM1 was the best donor, followed by GD1b and GT1b, when methanol was used as an acceptor. However, neither globoside nor glucosylceramide was utilized by the enzyme as a donor substrate. The enzyme transferred oligosaccharides from various glycosphingolipids to NBD-ceramide, a fluorescent ceramide, producing NBD-labeled glycosphingolipids. In addition to the transglycosylation reaction, the enzyme catalyzed the reverse hydrolysis reaction; lactose was condensed to ceramide to generate lactosylceramide in the presence of the enzyme. These results indicate that the jellyfish enzyme will facilitate the synthesis of various neoglycoconjugates and glycosphingolipids.     

High-temperature gas chromatography and gas chromatography-mass spectrometry of glycoprotein and glycosphingolipid oligosaccharides

High-temperature gas chromatography and gas chromatography-inass spectrometry for the analyses of oligosaccharides derived from glycoproteins or glycosphingolipids has been developed. Pcrmethylatcd oligosaccharides with up to about 12 sugar residues and masses up to 2500 Daltons can be analyzed. This approach is discussed and exemplified.     

Galectin-3 Interactions with Glycosphingolipids

Galectins have essential roles in pathological states including cancer, inflammation, angiogenesis and microbial infections. Endogenous receptors include members of the lacto- and neolacto-series glycosphingolipids present on mammalian cells and contain the tetrasaccharides lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) that form their core structural components and also ganglio-series glycosphingolipids. We present crystallographic structures of the carbohydrate recognition domain of human galectin-3, both wild type and a mutant (K176L) that influenced ligand affinity, in complex with LNT, LNnT and acetamido ganglioside a-G_M3 (α2,3-sialyllactose). Key structural features revealed include galectin-3's demonstration of a binding mode towards gangliosides distinct from that to the lacto/neolacto-glycosphingolipids, with its capacity for recognising the core β-galactoside region being challenged when the core oligosaccharide epitope of ganglio-series glycosphingolipids (G_M3) is embedded within particular higher-molecular-weight glycans. The lacto- and neolacto- glycosphingolipids revealed different orientations of their terminal galactose in the galectin-3-bound LNT and LNnT structures that has significant ramifications for the capacity of galectin-3 to interact with higher-order lacto/neolacto-series glycosphingolipids such as ABH blood group antigens and the HNK-1 antigen that is common on leukocytes. LNnT also presents an important model for poly-N-acetyllactosamine-containing glycans and provides insight into galectin-3's accommodation of extended oligosaccharides such as the poly-N-acetyllactosamine-modified N- and O-glycans that, via galectin-3 interaction, facilitate progression of lung and bladder cancers, respectively. These findings provide the first atomic detail of galectin-3's interactions with the core structures of mammalian glycosphingolipids, providing information important in understanding the capacity of galectin-3 to engage with receptors identified as facilitators of major disease.     

Two-dimensional mapping by the high-performance liquid chromatography of oligosaccharides released from glycosphingolipids by endoglycoceramidase

A two-dimensional sugar mapping method has been developed by which sensitive, reproducible, and simple analysis can be carried out on the structures and compositions of oligosaccharides released from glycosphingolipids by endoglycoceramidase. The oligosaccharides were labeled quantitatively with an ultraviolet-absorbing compound, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-oligosaccharides were separated first on an amide-silica column and then on a C4-silica column by high-performance liquid chromatography. The acidic ABEE-oligosaccharides were eluted as a group at the start of the chromatography while the neutral ABEE-oligosaccharides were separated according to size and structure on an amide-silica column using an eluent without salt. The acidic oligosaccharides were separated according to size and structure when rechromatographed on the same column using an eluent containing KH2PO4. NeuAc-containing ABEE-oligosaccharides were extensively separated from the corresponding NeuGc derivatives. The ABEE-oligosaccharides separated on an amide-silica column were then chromatographed on a column of C4-silica on which lactotriose and neolacto-series oligosaccharides were clearly shown to be separated from the others. On the basis of the retention times of the individual ABEE-oligosaccharides on two separate columns, 9 neutral and 15 acidic oligosaccharides derived from glycosphingolipid standards were two-dimensionally mapped without overlapping. The gangliosides of a human chondrosarcoma tissue and glycosphingolipids of tumor tissue of FBJ virus-transformed murine osteosarcoma cells were analyzed by this method in conjunction with exoglycosidase treatment. At least 11 species of glycosphingolipids were identified in both cases.     

Synthesis of fluorescent glycosphingolipids and neoglycoconjugates which contain 6-gala oligosaccharides using the transglycosylation reaction of a novel endoglycoceramidase (EGALC)

Endoglycoceramidase is a glycohydrolase capable of hydrolysing the O-glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. However, no endoglycoceramidase reported so far can hydrolyse 6-gala series glycosphingolipids which possess the common structure R-Gal beta1-6Gal beta1-1'Cer. Recently, we found a novel endoglycoceramidase (endogalactosylceramidase, EGALC) which specifically hydrolyses 6-gala series glycosphingolipids. Here, we report that EGALC catalyses the hydrolysis as well as transglycosylation. An intact sugar chain of neogalatriaosylceramide (Gal beta1-6Gal beta1-6Gal beta1-1'Cer) was found to be transferred by EGALC to a primary hydroxyl group of various alkanols and non-ionic detergents such as Triton X-100 generating corresponding alkyl- and Triton-trigalactooligosaccharides. Furthermore, fluorescent 6-gala series glycosphingolipids were synthesized by transglycosylation in a reaction with EGALC using fluorescent ceramides as acceptors. Because of high efficiency and broad acceptor specificity, EGALC would facilitate the synthesis of fluorescent glycosphingolipids and neoglycoconjugates which contain 6-gala oligosaccharides.     

Glycosphingolipids of human KB cells grown in monolayer, suspension, and synchronized cultures.

Studies have been carried out on the glycosphingolipids of human KB cells grown in monolayer and suspension culture, and by synchronization of the latter with a double thymidine (2mM) block. Glycosphingolipids were identified tentatively by thin layer chromatography, gas-liquid chromatography, and combined gas-liquid chromatography and mass spectrometry. The predominant gangliosides in the these cells were AcNeu-Gal-Glc-Cer and AcNeu-Gal-GalNAc-Gal-(AcNeu). Glc-Cer. Theprincipal neutral glycosphingolipids were Glc-Cer, Gal-Glc-Cer, Gal-Gal-Glc-Cer, and GalNAc-Gal-Gal-Glc-Cer. Incubation of KB cells (grown in monolayer and subsequently in suspension culture) for 48 hours with D-[1-14Clgalactose resulted in appreciable incorporation of radioactivity into all of the principal glycosphingolipids of these cells. These experiments confirmed that KB cells are capable of synthesizing their constituent glycosphingolipids. KB cells grown in suspension culture showed A 2- to 3-fold increase inthe concentration of Glc-Cer, Gal-Glc-Cer, GalNAc-Gal-Gal-Glc-Cer, and AcNeu-Gal1NAc-Gal-Gal-Glc-Cer, and AcNeu-Gal-Ga1NAcGal-(AcNeu)-Glc-Cer. Thus, the occurrence of tissue culture-dependent changes in the level of glycosphingolipids is demonstrated. Perhaps messages governing the synthesis of glycosphingolipids are translated earlier in thecell cycle under certain conditions of growth and are affected by cell-cell contact and cell adhesion.     

A novel glycosphingolipid-degrading enzyme cleaves the linkage between the oligosaccharide and ceramide of neutral and acidic glycosphingolipids

A novel glycosphingolipid-degrading enzyme was found in the cultured supernatant of Rhodococcus sp. G-74-2. It was purified 34.7-fold from the supernatant with 32.2% recovery by ammonium sulfate precipitation followed by Sephadex G-100 chromatography. The enzyme was demonstrated capable of cleaving the linkage between the oligosaccharide and ceramide of various acidic and neutral glycosphingolipids, producing intact oligosaccharides and ceramides. However, it was noted to hardly make any attack on linkages between monosaccharides and ceramides (cerebrosides) or between oligosaccharides and diacylglycerol (glycoglycerolipids). The enzyme preparation was completely free from various exoglycosidases and proteases. Furthermore, it was found to degrade neither N-linked nor O-linked glycoproteins. This enzyme, which is tentatively called endoglycoceramidase, should greatly facilitate the study of glycosphingolipids.     

Purification and characterization of membrane-bound endoglycoceramidase from Corynebacterium sp.

Endoglycoceramidase catalyzes the hydrolysis of the linkage between oligosaccharides and ceramides of various glycosphingolipids. We found that a bacterial strain Corynebacterium sp., isolated from soil, produced endoglycoceramidase both intracellularly and extracellularly. The intracellular enzyme bound to the cell membrane was solubilized with 1% Triton X-100 and purified to homogeneity about 170-fold with 60% recovery. The molecular mass of the enzyme was approximately 65 kDa. The enzyme is most active at pH 5.5-6.5 and stable at pH 3.5-8.0. Various neutral and acidic glycosphingolipids were hydrolyzed by the enzyme in the presence of 0.1% Triton X-100. Ganglio- and lacto-type glycosphingolipids were readily hydrolyzed, but globo-type glycosphingolipids were hydrolyzed slowly.     

Biochemical studies on sphingolipids of Artemia franciscana: novel neutral glycosphingolipids

Neutral glycosphingolipids containing one to six sugars in their oligosaccharide chains have been isolated from cysts of the brine shrimp Artemia franciscana. The structures of these glycolipids were identified by methylation analysis, partial acid hydrolysis, gas-liquid chromatography, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and proton nuclear magnetic resonance spectroscopy to be Glcβ1-Cer, Manβ1-4Glcβ1-Cer, Fucα1-3Manβ1-4Glcβ1-Cer, GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GlcNAcα1-2Fucα1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer (CPS), and GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer (CHS). Two glycosphingolipids, CPS and CHS, were characterized as novel structures. Because Artemia contains a certain series of glycosphingolipids (-Fucα3Manβ4GlcβCer), which differ from the core sugar sequences reported thus far, we tentatively designated the glycosphingolipids characterized as nonarthro-series ones. Furthermore, CHS exhibited a hybrid structure of arthro-series and nonarthro-series sugar chain. Two novel glycosphingolipids were characterized from the brine shrimp Artemia franciscana; one was composed of arthrotetraose and a branching fucose attached to N-acetylglucosamine residue, and the other was composed of CPS with an additional N-acetylglucosamine residue attached to the branching fucose.     

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.     

Study of the fucose-rich oligosaccharides in the urine of healthy and melituric subjects with A, B and O blood groups]

The application of adsorption chromatography on charcoal-Celite leads the authors to characterize in normal urines a class of fucose-rich oligosaccharides which possess blood group activities and are related to the phenotypes ABH, Le and secretor. Most of these oligosaccharides have a glucose residue in reducing terminal positions. Excretion of some oligosaccharides increases in the urine of diabetic and lactosuric subjects. In spontaneous or induced galactosurias, the elimination of oligosaccharides with a glucose residue in reducing terminal position decreases while appears a large amount of new oligosaccharides which all possess a galactose residue in reducing terminal position. These results lead to the conclusion that urinary oligosaccharides do not originate from glycosphingolipids, but from transglycosylation on carbohydrates which exist free in the organism: glucose for normal and diabetic subjects, lactose or galactose for lactosuric and galactosuric subjects, respectively.     

Developmental change and genetic defect in the carbohydrate structure of band 3 glycoprotein of human erythrocyte membrane.

The chemical structure of Band 3 glycopeptide prepared from erythrocytes of normal adult (blood group OI), umbilical cord vessels (Oi), and an i adult variant who fails to develop I antigen (Oi), has been compared. Band 3 glycopeptide of cord erythrocytes gave, on permethylation analysis, predominantly 2,4,6-tri-O-methylgalactose and 3,6-di-O-methyl-2-N-methylacetamido-2-deoxyglucose, whereas the same glycopeptide of normal adult erythrocytes gave much higher amounts of 2,3,4,6-tetra-O-methylgalactose and 2,4-di-O-methylgalactose as compared with that of cord erythrocytes. Band 3 glycopeptide from i adult showed the same methylation pattern as cord-Band 3 glycopeptide. In accordance with these results, Band 3 glycopeptide of cord and i adult erythrocytes were hydrolyzed to mostly small oligosaccharides by endo-beta-galactosidase from Escherichia freundii, whereas that of normal adult produced a number of oligosaccharides with various sizes which was caused by branched structures. Based on these results and structures of released oligosaccharides, the major developmental change of carbohydrate structure in the erythrocyte membrane is the conversion of linear repeating Galbeta1 leads to 4GlcNAcbeta1 leads to 3Gal to a branched Galbeta 1 leads to 4GlcNAcbeta 1 leads to 3 (R leads to 6) Gal structure. i individual may result from the lack of the branching enzyme.     

Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling

Interest in cellular glycosphingolipid (GSL) function has necessitated the development of a rapid and sensitive method to both analyze and characterize the full complement of structures present in various cells and tissues. An optimized method to characterize oligosaccharides released from glycosphingolipids following ceramide glycanase digestion has been developed. The procedure uses the fluorescent compound anthranilic acid (2-aminobenzoic acid; 2-AA) to label oligosaccharides prior to analysis using normal-phase high-performance liquid chromatography. The labeling procedure is rapid, selective, and easy to perform and is based on the published method of Anumula and Dhume [Glycobiology 8 (1998) 685], originally used to analyze N-linked oligosaccharides. It is less time consuming than a previously published 2-aminobenzamide labeling method [Anal. Biochem. 298 (2001) 207] for analyzing GSL-derived oligosaccharides, as the fluorescent labeling is performed on the enzyme reaction mixture. The purification of 2-AA-labeled products has been improved to ensure recovery of oligosaccharides containing one to four monosaccharide units, which was not previously possible using the Anumula and Dhume post-derivatization purification procedure. This new approach may also be used to analyze both N- and O-linked oligosaccharides.     

Immunochemistry of Ii-active glycosphingolipids of erythrocytes.

Fractions of complex glycosphingolipids were prepared from adult, cord, and i phenotype erythrocytes by the method elaborated for the isolation of poly(glycosyl)ceramides. In contrast to poly(glycosyl)ceramides which comprise on the average 30 glycosyl units and about 5 branching points, i.e. 3,6-di-O-substituted galactopyranosyl residues, per mole of glucose, complex glycosphingolipids from cord and i erythrocytes comprise 6 and 15 glycosyl units respectively and only 0.7 branching points. The latter substances exhibited also a high i activity which was not detected in poly(glycosyl)ceramides. Erythrocyte membranes were labeled with radioactive N-acetylgalactosamine (GalNAc) from UDP-GalNAc using a purified A-blood-group gene-specified transfered of GalNAc. It was found that electrophoretic mobilities in dodecylsulfate-gel electrophoresis of all glycoconjugates which accepted GalNAc were increased in i as compared to I membranes. We conclude that the absence of highly branched glycosphingolipids in cord and i erythrocytes as well as the reduction of apparent molecular weights of the glycoconjugates, which are substrates for A-gene-specified transferase of GalNAc, result from a single cause, that is an inadequacy of the biosynthetic process which is responsible for the formation of GlcNAc1 leads to 6Gal structures.