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At least 19 recordsLinked to original sources
PubMed1988

A novel glycosphingolipid hydrolyzing enzyme, glycosphingolipid ceramide deacylase, which cleaves the linkage between the fatty acid and sphingosine base in glycosphingolipids.

It has been demonstrated that the GM2 ganglioside cannot be cleaved by exo-beta-N-acetylhexosaminidases isolated from molds or bacterial sources. Here, a novel GM2 ganglioside-degrading enzyme was found in cells of Nocardia sp. This enzyme releases free fatty acids from the GM2 ganglioside. The chemical structure of the resultant lyso-GM2 ganglioside has been characterized by fast atom bombardment mass spectrometry, gas chromatography, and proton nuclear magnetic resonance spectroscopy. Using 14C-labeled GM2, at the fatty acid moiety, with stearic acid as the substrate, the optimum pH was determined to be 5.8. The enzyme was demonstrated to be capable of releasing fatty acids from GM3, GM2, GM1, and GD1a, and from neutral glycosphingolipids including Gb3-Cer, asialo-GM2, and asialo-GM1, but not from sphingolipids including Cer, Gal-Cer, Glc-Cer, and Lac-Cer. This enzyme, tentatively called glycosphingolipid ceramide deacylase, was found to be a tightly membrane-bound enzyme.

Amidohydrolases↗
PubMed1974

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↗
PubMed1982

Isolation and characterization of glycosphingolipids from human leukocytes. A unique glycosphingolipid pattern in a case of acute myelomonoblastic leukemia.

Neutral glycosphingolipids and gangliosides were isolated from the malignant cells of a patient with acute myelomonoblastic leukemia. Structural analyses were performed by gas-liquid chromatography and by high-performance liquid chromatography combined with enzymatic hydrolysis of glycosphingolipids using glycosidases. We found that, in contrast to normal leukocytes and chronic leukemia cells which have only a single tetraosylceramide species, these acute myelomonoblastic leukemia cells have approximately equal amounts of both globo- and neolactotetraosylceramide. This is the first population of human leukocytes in which we found two families of neutral glycosphingolipids to be present. The ganglioside fraction was composed of appreciable quantities of both NeuAc alpha 2 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer (GM3, hematoside) and NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer (sialoparagloboside). These cells did not have the 'leukocyte-specific' N-acetylneuraminosyllactotriaosylceramide found in normal human lymphocytes and neutrophils. These results are discussed in relation to normal leukocyte differentiation and acute leukemia. The present study also illustrates the usefulness of combining enzymatic degradation with high-performance liquid chromatography for glycosphingolipid structural determination.

Carbohydrates↗
PubMed1991

Activator proteins for glycosphingolipid hydrolysis by endoglycoceramidases. Elucidation of biological functions of cell-surface glycosphingolipids in situ by endoglycoceramidases made possible using these activator proteins.

Endoglycoceramidase (EGCase) cleaves the linkage between oligosaccharides and ceramides of various glycosphingolipids (Ito, M., and Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282). Recently, by extensive purification, it was separated from cell-lytic factor (hemolysin) and found to consist of three molecular species each with its own specificity (EGCases I, II, and III) (Ito, M., and Yamagata, T. (1989) J. Biol. Chem. 264, 9510-9519). A detergent was required for EGCases to express full activity, possibly due to their hydrophobic nature, and thus EGCases cannot be used for research on live cells. This paper presents findings on activator proteins in the culture supernatant of Rhodococcus sp. M-777 regarding the stimulation of EGCase activity in the absence of detergents. The activator protein, exhaustively purified and designated as activator II in this study, showed a single protein band on sodium dodecyl sulfate-, native-, and isoelectrofocussing-polyacrylamide slab gel electrophoresis after being stained with Coomassie Brilliant Blue. Its molecular weight and pI were 69,200 and 4.0, respectively. The activator protein enhanced the hydrolysis of glycosphingolipids in vitro and on the cell-surface by EGCase II in the absence of detergents in a concentration-dependent manner. Interestingly, activator II stimulated the activity of EGCase II much more than that of EGCase I on using asialo-GM1 as the substrate. This activator protein was found nonspecific to substrates susceptible to hydrolysis with EGCase II. Besides activator II, strain M-777 produced a second minor molecular species of activator protein designated as activator I which appeared specific for stimulating the activity of EGCase I in contrast to activator II. Following the addition of activator II, EGCase II hydrolyzed cell-surface glycosphingolipids quite efficiently at neutral pH at which hydrolysis hardly occurred at all in its absence. When using activator II in place of Triton X-100 for stimulating EGCase II activity, it was also noted to cause no damage to intact cells. It is thus possible by activator proteins to elucidate the biological functions of endogenous glycosphingolipids in situ by EGCases.

Amino Acid Sequence↗
PubMed1983

Characterization of glycosphingolipids from cells of various types of human leukemia: occurrence of two glycosphingolipids, one reacting with anti-asialo GM1 antibody and one with anti-Forssman antibody.

Glycosphingolipids of neutrophils, lymphocytes and leukocytes from patients with various types of human leukemia [acute lymphoblastic (ALL), acute unclassified type (AUL), acute myeloblastic (AML), acute monocytic (AMoL), chronic myeloblastic (CML)] and the hypereosinophilic syndrome (HES) were analyzed chemically and immunochemically. No distinct difference was found in the molar ratio of lipid-bound sialic acid to lipid-bound phosphorus in these cells, but a low ratio of cholesterol to lipid-bound phosphorus was found in ALL (3 of 4 cases), AML, CML and AMoL (one of 2 cases). The predominant glycosphingolipid was ceramide dihexoside (CDH) in all cells analyzed, but the amount and the molar ratio of lipid-bound phosphorus to CDH were clearly different in different cell types, indicating that the molar ratio is a useful criterion in the classification of types of leukemia. In addition, molecular diversity of minor glycosphingolipid components was observed in various leukemic cells. Two of the neutral glycosphingolipids in AMoL were tentatively identified as asialo GM1 and Forssman glycolipids by comparing their mobilities on thin-layer chromatography with those of standard glycolipids and by observing the formation of precipitin lines on a double diffusion agar plate with anti-asialo GM1 and anti-Forssman antibodies.

G(M1) Ganglioside↗
PubMed1984

Hydrazinolysis of glycosphingolipids. A new method for preparation of N-deacylated (lyso) glycosphingolipids.

A useful method for N-deacylation of the ceramide moiety of glycosphingolipids has been developed. Galactosylceramide, glucosylceramide, lactosylceramide, and galactosyllactosylceramide were effectively deacylated by heating with anhydrous hydrazine at 150 degrees C for 15-25 h. The lyso-derivative as the deacylated product of the ceramide moiety of each glycosphingolipid was isolated by preparative silica gel thin layer chromatography with a 70-85% yield from the starting glycolipids. Hydrazine sulfate was an effective catalyst for the deacylation of the ceramide moiety. No dissociation of oligosaccharide moieties of the glycolipids on hydrazinolysis was confirmed by gas chromatographic analysis and N-acylation of these lysoderivatives. The free amino groups of the lysoglycosphingolipids can be combined with various kinds of probes giving useful derivatives for biochemical and immunological studies on glycosphingolipids.

Acylation↗
PubMed1992

[The role of glycosphingolipids in the expression of neoplastic phenotype. III. Changes in glycosphingolipids related to the capacity of neoplastic cells for metastasis and tumor growth in vivo].

The role of glycosphingolipids in the expression of neoplastic phenotype. III. Alterations in glycosphingolipids of tumor cells related to their metastatic potential and tumorigenicity. This review describes changes in glycosphingolipids related to metastatic and tumorigenic properties of malignant cells. The possible effects of such alterations on malignant behavior of cells is discussed.

Animals↗
PubMed1986

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.

Carbohydrate Sequence↗
PubMed1975

Mass spectrometric analysis of permethylated glycosphingolipids I. Sequence analysis of two blood-group B active glycosphingolipids from human B erythrocyte membranes.

Two blood group B active glycosphingolipids (B-I and B-II) formerly isolated and purified from human B erythrocytes (16) were investigated by mass spectrometry after permethylation. B-I yielded fragments up to m/e 1266 and B-II up to m/e 1495, showing the sequence of six and seven carbohydrate residues respectively. In combination with additional experimental evidence (18) the glycosphingolipids are demonstrated to be a gal-[ fuc ]-gal-glcNAc-gal-glc-ceramide (B-I) and a gal-[ fuc ]-gal-glcNAc-gal-glcNAc-gal-glc-ceramide (B-II). Mass spectrometric evidence for the ceramide residues are also obtained indicating besides spingosine C24-,C24:1-, and C22-fatty acids as main constituents.

ABO Blood-Group System↗
PubMed1984

Fast atom bombardment mass spectrometry of glycosphingolipids. Glycosphingolipids containing neutral sugars.

Natural and synthetic glycosphingolipids containing neutral sugars have been analyzed by positive and negative ion fast atom bombardment mass spectrometry. Basic structural characterization including saccharide size and sequence and ceramide composition is possible on the basis of the fragment ions observed. The degree of fragmentation could be increased by using higher sample concentrations and lower fast atom beam energies. Commercially available synthetic compounds that had been presumed to be pure were shown to contain homologous fatty acids. Mixtures of glycosphingolipids such as those obtained from Gaucher's spleen and from human erythrocytes can be characterized and quantitated.

Carbohydrates↗
PubMed1982

Studies on glycosphingolipids of larvae of the green-bottle fly, Lucilia caesar. I. Isolation and characterization of glycosphingolipids having novel sugar sequences.

The neutral glycosphingolipids from larvae of the green-bottle fly, Lucilia caesar, were analyzed. Thin-layer chromatograms showed that the larvae contained at least seven major glycolipid components. Of them, the four components with shorter sugar chains consisting of one to four sugar units, were purified by Iatrobeads column chromatography. The structures were identified by partial acid hydrolysis, sequential enzymatic hydrolysis, chromium trioxide oxidation and methylation analysis as: Glc beta (1-1)Cer, Man beta (1-4)Glc beta (1-1)Cer, GlcNAc beta (1-3)Man beta (1-4)Glc beta (1-1)Cer and GalNAc beta (1-4)GlcNAc beta (1-3)Man beta (1-4)Glc(1-1)Cer. These glycolipids altogether comprised 38.3% of the total neutral glycolipid fraction. Unlike common vertebrate glycosphingolipids, the larval ones appear to be quite unique in having the sugar structures, -GlcNAc beta (1-3)Man- and -GalNAc beta (1-4)GlcNAc beta (1-3)Man-, which are novel finding in the natural systems examined so far. The ceramide moieties were composed of normal fatty acids (16:0-22:0) with a small amount of branched acids (16 and 18), and tetradeca- and hexadeca-4-sphingenines as the long-chain bases.

Animals↗
PubMed1981

Glycosphingolipid-high density lipoprotein-3 interactions. I. Transfer of glycosphingolipid from phosphatidylcholine vesicles to high density lipoprotein-3.

Single bilayer vesicles (d less than 1.02 g/ml) of 3H-glycosphingolipids and [14C]phosphatidylcholine in the molar ratio of 1:7 were prepared by ethanolic injection of the lipid mixture into buffer, concentrated, and incubated with human serum high density lipoprotein-3 (HDL3; d = .14 g/ml) at 37 degrees C. Equilibrium ultracentrifugation of the incubation mixtures on a 0-22% NaBr gradient revealed the presence of three discrete lipid-protein complexes of density 1.03, 1.06, and 1.12 g/ml (Peaks I, II, and III, respectively). Each peak was homogeneous upon reultracentrifugation and the protein and radioactivity eluted as a single peak upon Sepharose CL-6B chromatography. Compositional analysis showed peak I to contain 2.6% protein (apo-A-I peptide) and 4.3% cholesterol, peak II to contain 17.6% protein (apo-A-I peptide) and 6.3% cholesterol, and peak III to have a composition similar to HDL3. Electron microscopy of negatively stained samples confirmed the homogeneity of the peaks and the similarity between peak III and HDL3. Peak II particles were larger than HDL3; peak I particles resembled fused or aggregated vesicles which could be removed by ultracentrifugation; disc-shaped particles were not seen in any of the fractions. Direct incubation of HDL3 or human serum with 3H-glycosphingolipid dispersions did not yield a glycolipid . HDL3 complex as judged by density gradient ultracentrifugation and Sepharose CL-6B chromatography. However, incubation of 3H-glycolipid/phosphatidylcholine vesicles with serum did result in transfer of 3H-glycolipid to the HDL fraction. It was concluded that glycolipids incorporated into a lipid membrane structure can interact with, and become incorporated into, high density lipoprotein.

Gangliosidoses↗
PubMed1990

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.

Animals↗
PubMed1987

Predefined gene transfer for expression of a glycosphingolipid antigen by transfection with a cosmid genomic library prepared from a cell line in which the specific glycosphingolipid is highly expressed.

The deliberate transfer of globotriaosylceramide (Gb3) expression in mouse lymphoma L5178 cells was achieved by transfection with a cosmid DNA library prepared from human Burkitt lymphoma Ramos cells in which Gb3 was highly expressed. The recipient mouse lymphoma cells did not contain Gb3 but did contain its direct precursor, lactosylceramide. The transfected cells expressed Gb3, detected both chemically and immunologically, and contained human DNA detected by an Alu sequence probe. This model demonstrates a general method for studying glycosyltransferase genes and other factors necessary for the expression of glycosphingolipid antigens.

Animals↗
PubMed1976

Mass spectrometric analysis of permethylated glycosphingolipids II. Comparative studies on different blood-group active and related erythrocyte membrane glycosphingolipids.

Three isomeric ceramide tetrasaccharides--P blood-group active globoside, lacto-N-neotetraosyl ceramide as ABH blood-group precursor, both isolated from human erythrocytes and "asialo ganglioside" from human brain as reference standard--and two ceramide pentasaccharides--H blood-group active glycosphingolipid, obtained from blood-group B active ceramide hexasaccharide of human B erythrocytes after alpha-galactosidase treatment and ceramide pentasaccharide from rabbit erythrocytes with B-like blood-group activity--were investigated by mass spectrometry after permethylation. The carbohydrate moiety exhibits differences not only concerning the sugar sequence but also with regard to the position of some glycosidic linkages: Oligosaccharides containing N-acetylhexosamine substituted at position 4 produce spectra that are distinctly different from those containing C-3 substituted N-acetylhexosamines, thus allowing the differentation between type 1 and type 2 carbohydrate chains. Moreover, oligosaccharide ions with a hexose at the cleavage site exhibit a fragmentation pattern different from those with a N-acetylhexosamine at the "reducing terminal". The intensity ratio between parent ion and parent ion -32 mass units is Q greater than or equal to 3 in the first case, whereas in the latter case Q is less than 1. The Q-values are given for 14 oligosaccharide ions. Differences in the composition of the ceramide residues can also be deduced from the mass spectra.

ABO Blood-Group System↗
PubMed1991

Synergistic effect of two cell recognition systems: glycosphingolipid-glycosphingolipid interaction and integrin receptor interaction with pericellular matrix protein.

GM3-expressing cells adhere, spread and migrate on plastic plates coated with Gg3, LacCer and Gb4, but not with other glycosphingolipids (GSLs). Thus, cell adhesion, spreading and migration through GSL-GSL interaction occur in an analogous fashion to the interaction of cells with adhesive matrix proteins [AP, e.g. fibronectin (FN), laminin (LN)] through their integrin receptors. In this study, the adhesion of two GM3-expressing cell lines (B16 melanoma and HEL299 fibroblast) on plastic plates co-coated with GSL plus AP is compared with adhesion on plates coated with GSL (Gg3 or LacCer) alone, or coated with AP alone. Results show that: (i) cell adhesion on GSL-coated plates takes place earlier in the incubation period than that on AP-coated plates; (ii) cell adhesion, as well as spreading, was greatly enhanced (in terms of strength and rapidity) on plates co-coated with GSL plus AP; (iii) repulsion (negative adhesion) of cells was observed on plates co-coated with AP plus N-acetyl-GM3 (NAcGM3) and was presumably based on repulsive NAcGM3-NAcGM3 interaction; (iv) GM3-dependent cell adhesion on GSL-coated plates, as well as synergistic promotion of cell adhesion (based on the GSL-GSL and AP-integrin systems), was suppressed by incubation of cells with anti-GM3 monoclonal antibody DH2 or sialidase. Synergistic adhesion of cells on GSL/AP co-coated plates was less inhibited by incubation with peptide sequences RGDS or YIGSR than was adhesion on plates coated with AP alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Polar glycosphingolipids in insect: chemical structures of glycosphingolipid series containing 2'-aminoethylphosphoryl-(----6)-N-acetylglucosamine as a polar group from larvae of the green-bottle fly, Lucilia caesar.

A series of glycosphingolipids containing 2'-aminoethylphosphoryl(----6)-N-acetylglucosamine as a polar group has been demonstrated in larvae of the green-bottle fly, Lucilia caesar. The thin-layer chromatographic pattern of the total polar glycolipid revealed the presence of more than eight components, of which five major components were purified by the use of successive column chromatography on QAE- and DEAE-Sephadex and silicic acid (Iatrobeads). From structural studies including compositional sugar analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy, methylation analysis, and fast atom bombardment mass spectrometry, their structures were deduced to be as follows: 2'-aminoethylphosphoryl----6GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, GalNAc beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, GalNAc alpha 1-4GalNAc beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, Gal beta 1-3GalNAc alpha 1-4GalNAc-beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, and GlcNAc beta 1-3Gal-beta 1-3GalNAc alpha 1-4GalNAc beta 1-4 (2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc-beta 1-Cer. The main molecular species of the ceramide moiety was arachidinyltetradecasphingenine in all of the major glycolipids.

Acetylglucosamine↗
PubMed1981

Purification of anti-glycosphingolipid antibody and topological localization of glycosphingolipid on the cell surface of rat ascites hepatomas.

A simple method for the preparation of oligosaccharide-linked aminohexyl-Sepharose 4B (AH-Sepharose 4B) and its application to the purification of anti-glycosphingolipid antibody which is specific for the oligosaccharide moiety are described. The oligosaccharide, which was obtained from galactosyl(beta 1 leads to 3) N-acetylgalactosaminyl(beta 1 leads to 4)galactosyl(beta 1 leads to 4)glucosylceramide (asialo-GM1) by ozonolysis and subsequent alkali treatment, was covalently linked to the AH-Sepharose 4B by reductamination in the presence of NaBCNH3. Anti-asialo-GM1 antibody was purified by means of an affinity technique with the oligosaccharide-linked AH-Sepharose 4B. The antibody bound to the affinity adsorbent was eluted with 0.5 M NaSCN and 3.0 M NaSCN. Antibody with higher specific activity was recovered in the 3.0 M NaSCN fraction with 50% recovery of the activity of the starting material. The purified antibody was found to be quite specific for asialo-GM1. The presence of asialo-GM1 on the cell surface of free-type rat ascites hepatomas was confirmed by the immunofluorescence technique. The cell aggregates induced by the purified antibody were observed under a scanning electron microscope. The cell connection was found to occur at the tips of microvilli of the surface membrane. The localization of asialo-GM1 on the tips of the surface membrane was confirmed by means of the ferritin-conjugated antibody technique.

Animals↗