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PubMed2002

Synthetic studies on glycosphingolipids from the Protostomia phyla: syntheses of arthro-series glycosphingolipids.

Glycosphingolipids isolated from larvae of the green-bottle fly, Lucilia caesar, have quite unique structures containing GlcNAcbeta-(1 --> 3)-Man and GalNAcbeta-(1 --> 4)-GlcNAcbeta-(1 --> 3)-Man. We have synthesized two glycosphingolipids, beta-D-GlcNAcp-(1 --> 3)-beta-D-Manp-(1 --> 4)-beta-D-Glcp-(1 --> 1)-Cer and beta-D-GalNAcp-(1 --> 4)-beta-D-GlcNAcp-(1 --> 3)-beta-D-Manp-(1 --> 4)-beta-D-Glcp-(1 --> 1)-Cer. A key reaction in the synthetic sequence is the application of the intramolecular aglycon delivery (IAD) approach for the synthesis of the beta-mannopyranosidic linkages.

Animals↗

PubMed1998

Glycosphingolipids of skeletal muscle: I. Subcellular distribution of neutral glycosphingolipids and gangliosides in rabbit skeletal muscle.

Membrane vesicles were prepared from rabbit skeletal muscle, separated by sucrose density gradient centrifugation and characterized by their specific marker enzymes, ligand binding, and ion flux activities. The fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmic reticulum (SR1 and SR2) and triads/mitochondria (Tr/M). Their glycosphingolipid compositions were analyzed by biochemical and immunochemical methods with specific antibodies (TLC immunostaining) and characteristic patterns were obtained from respective membrane fractions, expressed on a protein basis. Glucosylceramide, the main neutral glycosphingolipid of rabbit muscle, was found in SL and TT fractions, whereas SR and Tr/M vesicles lack this compound. Lactosylceramide was selectively recovered in the SR1 fraction. GM3(Neu5Ac), the main ganglioside in rabbit muscle, was found to account for 64% in the SL, 13% in the TT, 7% in the SR1, 3% in the SR2 and 13% in the Tr/M fractions. IV3Neu5Ac-nLcOse4Cer was mostly abundant in SL and decreased in the order SL > TT, Tr/M > SR1, SR2. IV6Neu5Ac-nLcOse4Cer was only detected in the SL and Tr/M fractions in noteworthy quantities. Ganglioseries gangliosides GM1, GD1a, GD1b and GT1b displayed homogeneous distribution patterns in each membrane preparation. They were expressed only in small amounts but mainly in SL, TT and Tr/M vesicles and to less extent in SR1 and SR2 fractions. The presence of GM3(Neu5Ac) in the SL as well as on subcellular level was confirmed in transverse muscle cryosections by means of indirect immunofluorescence microscopy. The SL was brightly stained, but considerable intracellular fluorescence was observed as expected from the biochemical analyses. Thus, the neutral GSL and ganglioside expression of the SL and the intracellular membraneous network is different in skeletal muscle both in terms of quantitative and qualitative GSL composition as demonstrated in details by means of biochemical and immunochemical techniques. The modulatory functions of GM3 and gangliosides of the neolacto- and ganglio-series towards the voltage dependent Ca(2+)-channel, largely preponderant in the triads-containing Tr/M fraction, is the subject of the accompanying paper (J. Müthing, U. Maurer, and S. Weber-Schürholz, Carbohydr. Res., 307 (1998) 147-157).

Animals↗

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↗

PubMed2006

Dissecting Ascaris glycosphingolipids for immunomodulatory moieties--the use of synthetic structural glycosphingolipid analogues.

We have previously shown glycosphingolipids of Ascaris suum to have phosphorylcholine (PC) and non-PC immunomodulatory moieties. In the present study we further investigated the nature of the immunomodulatory moieties by employing three synthetic glycosphingolipids each possessing features of the original molecule to examine effects on macrophage and dendritic cell (DC) cytokine production and surface co-stimulatory molecule expression. Compound 2, which lacked PC but contained ceramide, had no effect on either macrophages or DCs. Surprisingly however, Compound 1, which contained PC and hence arguably most resembled the native material, had, with the exception of a small increase in surface antigen expression, no immunomodulatory properties. Conversely, Compound 3, which contained PC but was otherwise least like the native molecule, demonstrated a number of effects on both macrophages and DCs, including induction of Th-1/pro-inflammatory cytokines, inhibition of such cytokines induced by IFN-gamma/LPS and increased expression of co-stimulatory molecules. Taken together these results indicate: (i) that although PC is an immunomodulatory component of the native molecule other structural feature are necessary to allow it to act; (ii) that carbohydrate rather than ceramide is likely to represent a non-PC immunomodulatory moiety; and (iii) that synthetic PC-containing molecules have the potential to act as immunomodulatory drugs.

Animals↗

PubMed2003

Simultaneous quantification of lyso-neutral glycosphingolipids and neutral glycosphingolipids by N-acetylation with [3H]acetic anhydride.

We describe a new method that permits quantification in the pmol to nmol range of three lyso-neutral glycosphingolipids (lyso-n-GSLs), glucosylsphingosine (GlcSph), galactosylsphingosine (GalSph), and lactosylsphingosine, in the same sample as neutral glycosphingolipids (n-GSLs). Lyso-n-GSLs and n-GSLs are initially obtained from a crude lipid extract using Sephadex G25 chromatography, followed by their isolation in one fraction, which is devoid of other contaminating lipids, by aminopropyl solid-phase chromatography. Lyso-n-GSLs and n-GSLs are subsequently separated from one another by weak cation exchange chromatography. N-GSLs are then deacylated by strong alkaline hydrolysis, and the N-deacylated-GSLs and lyso-n-GSLs are subsequently N-acetylated using [3H]acetic anhydride. An optimal concentration of 5 mM acetic anhydride was established, which gave >95% N-acetylation. We demonstrate the usefulness of this technique by showing an approximately 40-fold increase of both GlcSph and glucosylceramide in brain tissue from a glucocerebrosidase-deficient mouse, as well as significant lactosylceramide accumulation. The application and optimization of this technique for lyso-n-GSLs and lyso-GSLs will permit their quantification in small amounts of biological tissues, particularly in the GSL storage diseases, such as Gaucher and Krabbe's disease, in which GlcSph and GalSph, respectively, accumulate.

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↗

PubMed1999

A convenient oxidation of natural glycosphingolipids to their "ceramide acids" for neoglycoconjugation. Bovine serum albumin-glycosylceramide acid conjugates as investigative probes for HIV gp120 coat protein-glycosphingolipid interactions.

A new method to cleave the double bond of sphingolipids has been developed. Using limited concentrations of KMnO4 and an excess of NaIO4, in a neutral aqueous tert-butanol solvent system gave nearly quantitative yields of the oxidized product. A variety of natural glycosphingolipids (GSLs): GlcC, GalC, SGC, LC, Gb3C, Gb4C, Gg4C, Gb5C, and GM1C, gave the corresponding acids: 2-hydroxy-3-(N-acyl)-4-(O-glycosyl)-oxybutyric acids, i.e. "glycosyl ceramide acids" (GSL.CCOOH) in excellent yields (80-90%). Deacyl GSLs (dGSLs) were oxidized to acids containing the oligosaccharides devoid of hydrocarbon chains, i.e. "ceramide oligosaccharides" (dGSL. NRR1CCOOH, where R = R1 = H; R = H, R1 = CH3CO; or R = R1 = Me). The efficacy of this method was demonstrated by transforming natural GSLs: GlcC, GalC, GalS, SGC, LC, Gb3C, and Gb4C into neoglycoproteins via coupling glycosyl ceramide acids (except GalS, which was coupled directly) to bovine serum albumin (BSA). Mass spectroscopic analysis of GalC-BSA conjugates, (GalC.CONH)nBSA and (GalS.NHCO)nBSA gave a value of 9 +/- 1 and 16 +/- 2 for n. Neoglycoconjugates derived from GlcC, GalC (type I and II and the behenic analog), SGC, LC, and Gb3C were recognized by the recombinant human immunodeficiency virus coat protein gp120 (rgp120). The GalS conjugate showed significantly reduced binding, and the Gb4C conjugate showed no binding. Thus, rgp120/GSL-BSA interaction requires a terminal galactose and/or glucose residue. Terminal N-acetylgalactosamine containing GSLs are not bound. The ceramide acid conjugates provide a more effective scaffold for presentation of glycone for rgp120 binding than those derived from dGSLs. The retention of receptor specificity of the glycoconjugates was validated by retention of the expected binding specificity of VT1 and VT2e for Gb3C and Gb4C conjugates, respectively. These studies open a new vista in the generation of glycoconjugates from GSLs and further emphasize the role of aglycone in glycolipid recognition.

Animals↗

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↗

PubMed1981

Studies on glycosphingolipids of fresh-water bivalves. VI. Isolation and chemical characterization of neutral glycosphingolipids from spermatozoa of the fresh-water bivalve, Hyriopsis schlegelii.

The spermatozoa of the fresh-water bivalve, Hyriopsis schlegelii, had a complex spectrum of neutral glycosphingolipids. CDS, CTS, CTeS were isolated as minor components (together they accounted for 3% of the total neutral glycolipid fraction), while CMS and the glycolipids (Lipid I, II, and III) with larger saccharide chains that were previously characterized (1--4) occurred as major ones. The chemical structures were determined by employing exoglycosidases, partial acid hydrolysis and permethylation analysis. The proposed structures are as follows: Man alpha(1-3)Man beta(1-4)Glc-Cer for CTS, and Man alpha(1-3)[Xyl beta(1-2)]Man beta(1-4)Glc-Cer for CTeS. CTeS was the first example of a tetraglycosyl ceramide containing xylose. In another fraction, two types of diglycosyl ceramides were obtained as a mixture, and identified as Man beta(1-4)Glc-Cer and Gal beta(1-4)Glc-Cer, respectively. The long-chain base was predominantly composed of sphingosine, and the acyl groups were mainly of palmitate and stearate.

Animals↗

PubMed1982

Studies on glycosphingolipids of larvae of the green-bottle fly, Lucilia caesar. II. Isolation and structural studies of three glycosphingolipids with novel sugar sequences.

Three novel glycosphingolipids containing five to seven sugars in their saccharide chains have been isolated from larvae of the green-bottle fly, Lucilia caesar. The structures of these glycolipids were identified by sequential enzymatic degradation with exoglycosidases, methylation analysis, and partial acid hydrolysis to be: Gal-NAc alpha (1-4)GalNAc beta (1-4)GlcNAc beta (1-3)Man beta (1-4)Glc-Cer, Gal alpha (1-4)GalNAc alpha (1-4)GalNAc beta (1-4)GlcNAc beta (1-3)Man beta (1-4)Glc-Cer, and GlcNAc beta (1-4)Gal alpha (1-4)Gal-NAc alpha (1-4)GalNAc beta (1-4)GlcNAc beta (1-3)Man beta (1-4)Glc-Cer. It should be noted that one N-acetylgalactosamine in their saccharide chains is alpha-glycosidically linked to another N-acetylgalactosamine by a 1-4 bond and that this is the first example of the presence of an alpha (1-4) linked galactosamine unit in nature. The major fatty acids were normal saturated fatty acids with carbon numbers of 16 to 22. The long-chain bases were dominated by C14 and C16-4-sphingenine homologues.

Animals↗

PubMed1993

Specific hydrolysis of intact erythrocyte cell-surface glycosphingolipids by endoglycoceramidase. Lack of modulation of erythrocyte glucose transporter by endogenous glycosphingolipids.

This study represents the specific hydrolysis of cell-surface glycosphingolipids (GSLs) of intact cells by endoglycoceramidase (EGCase; EC.3.2.1.123) which cleaves the linkage between oligosaccharides and ceramides of various GSLs. After a 2-h incubation of horse intact erythrocytes with 20 mU EGCase II in the presence of activator at 37 degrees C, 68% of the N-glycolylneuraminic-acid-containing ganglioside GM3(NeuGc) and 70% of 4-O-acetyl GM3(NeuGc) were found to be hydrolyzed without hemolysis, accompanied by a corresponding increase in ceramide but not sphingosine or N,N-dimethylsphingosine. No hydrolysis was observed for sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, cholesterol or membrane proteins. The decrease in immunoreactivity with GMR8 antibody, specific to NeuGc alpha 2,3Gal- of GM3(NeuGc), corresponded to that of cell-surface GM3(NeuGc) by the enzyme, and almost no immunoreactivity was found when 70% of the GM3(NeuGc) was hydrolyzed. Besides the cell-surface GM3(NeuGc) of horse erythrocytes, Gg3Cer of guinea pig, GM3(NeuAc) and LcCer of human, and bovine and rabbit erythrocyte IV3Gal alpha-nLc4Cer were found to be efficiently hydrolyzed by EGCase II even when present in intact cells, while human erythrocyte Gb4Cer is quite resistant to hydrolysis by the enzyme on the cell surface as well as in detergent micelles. Glucose incorporation via the glucose transporter in erythrocytes was not affected at all by the specific and exhaustive hydrolysis of cell-surface GSLs by EGCase II. This result strongly suggested that glucose transporter function was not directly modulated by endogenous GSLs. In summary, this paper demonstrates that, together with the assistance of activator protein, EGCase II will become a powerful tool for selectively removing sugar chains from cell-surface GSLs without damaging other cell membrane components, and will be useful for describing the biological functions of endogenous GSLs.

Animals↗

PubMed2006

Soluble adamantyl glycosphingolipid analogs as probes of glycosphingolipid function.

Despite the extensive structural characterization of glycosphingolipids (GSLs), their functions in cell physiology and pathobiology remain elusive. This is largely owing to the fact that they are difficult to handle, being insoluble in aqueous media, and that no one gene alone determines their synthesis. The heterogeneity of the lipid moiety provides a further confounding factor. GSLs are central components within lipid rafts, which are major foci for transmembrane signaling and interactions between eukaryotic cells and microbial pathogens. GSL receptor function often requires the lipid moiety, and lipid-free sugar analogs are ineffective inhibitors. In order to overcome some of these problems, we have synthesized adamantyl GSL analogs which, in part, mimic GSL membrane receptor function in solution. These compounds are made by replacing the endogenous fatty acid with an adamantan frame. This rigid hydrophobic structure surprisingly increases the water solubility of the conjugate and retains receptor function. These GSL mimics provide probes to study GSL receptor function within cells. They compete with native GSLs for ligand binding and are taken up by cells to potentially alter GSL-mediated interaction. We are focused on two derivatives, adamantyl globotriaosyl ceramide and adamantyl sulfogalactosyl ceramide, and have used these analogs to probe GSL function in microbial pathology and hsp70 function. This chapter describes the syntheses and uses of these mimics.

Adamantane↗