Glycosphingolipids of leukocytes are unbranched at the galactopyranosyl residue and contain fucosylα-1-3-N-acetylglucosamine structures

Glycolipids of peripheral leukocytes which had been used for the production of interferon were separated into oligoglycosylceramides, polyglycosylceramides and polyglycosylpeptides (erythroglycan). Neutral oligoglycosylceramides comprised glucosylceramide, galactosylceramide, lactosylceramide, lactotriaosylceramide, globotriaosylceramide andneolactotetraosylceramide. Globotetraosylceramide was not detected. Glycolipids which were more complex thanneolactotetraosylceramide belonged exclusively to theneolacto series of compounds and were essentially unbranched at galactopyranosyl residues. The polyglycosylceramide fraction contained a glycolipid with a probable structure Gal1-4(Fuc1-3) GlcNAc1-3Gal1-4GlcNAc1-3 Gal1-4GlcNAc1-3Gal1-4Glc1-1ceramide. Polyglycosylpeptides were found only in trace amounts and were also unbranched at galactopyranosyl residues. All glycoconjugates studies did not contain significant amounts of carbohydrate structures derived from ABH immunodominant groups.Nomenclature Gal1-4Gal1-4GlcCer Lactotrioasylcermide (LcOse₃Cer) - Gal1-4Gal1-4GlcCer globotriaosylceramide, (GbOse₄Cer) - GalNAc1-3Gal1-4 Gal1-4GlcCer globoside (globotetraosylceramide, GbOse₄Cer) - Gal1-4GlcNAc1-3Gal1-4GlcCer paragloboside (lacto-N-neo tetraosylceramide,nLcOse₄Cer)     

Stage-specific expression of fuco-neolacto- (Lewis X) and ganglio-series neutral glycosphingolipids during brain development: characterization of Lewis X and related glycosphingolipids in bovine,human and rat brain

We have purified and characterized a bovine brain pentaglycosylceramide as Lewis X and identified it in human and rat brain using anti-Lewis X (anti-SSEA 1) monoclonal antibody. Neutral glycosphingolipid expression in developing rat brain has been examined by digoxigenin immunostaining and TLC-immunostaining using anti-SSEA 1 and anti-GgOse₄Cer (GA1) monoclonal antibodies. Five transient Lewis X-series bands were identified in brain at embryonic day 15 that disappear by postnatal day 5 (one disappears at embryonic day 18). Gangliotetraosylceramide (GA1) first appears at embryonic day 21 and increases in concentration with age until postnatal day 21. In addition, we have purified another minor brain neutral glycosphingolipid and tentatively identified it as a Lewis X-series glycolipid by gas chromatography-mass spectrometry analysis followed by TLC-immunostaining with anti-SSEA 1 antibody.Abbreviations Cer Ceramide, GlcCer, Glc1-1Cer - LacCer Gal1-4GlcCer - CTH Gal1-4LacCer - nLcOse₄Cer Gal1-4GlcNAc1-3LacCer - nLcOse₅Cer Gal1-3nLcOse₄Cer - GgOse₄Cer Gal1-3GalNAc1-4LacCer - DPA diphenylamine-aniline-phosphoric acid - SSEA stage-specific embryonic antigen - NGSL neutral glycosphingolipid - TLC thin-layer tomography - HPTLC high performance thin-layer chromatography - GA1 gangliotetraosyleramide - SAT-2 sialytransferase-2 - GalNAcT-1 galactosaminyltransferase-1 - DIG-IS digoxigenin-immunostaining - PMAAS partially methylated alditol acetates - DCE dichloroethane - TLC-IS TLC-immunostaining - (Le^x) Lewis X - NK murine natural killer     

Isolation and characterization of a Forssman antigen-binding lectin from velvet bean (Mucuna derringiana) seeds

A Forssman antigen (GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer)-binding lectin has been purified from velvet bean (Mucuna derringiana) seeds by a combination of affinity chromatography and reversed phase HPLC. This lectin agglutinates both native and trypsin-treated sheep erythrocytes as well as trypsinized rabbit erythrocytes, but neither native rabbit nor human erythrocytes, irrespective of blood group type. SDS-PAGE and gel filtration chromatography reveal the lectin to be a homodimer consisting of two 54 kDa subunits linked by non-covalent bonds. The results obtained by quantitative precipitation, haemagglutination inhibition and TLC overlay assays indicate that theMucuna lectin specifically recognizes Forssman antigen and Forssman disaccharide (GalNAc1-3GalNAc)-related structures. Abbreviations: The abbreviations and the trivial names used are: AH, 6-aminohexyl; BSA, bovine serum albumin; Cer, ceramide; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PBS, 10mm phosphate-buffered saline, pH 7,2, containing 0.15m NaCl; PMSF, phenyl methyl sulfonyl fluoride; SDS, sodium dodecyl sulphate; TFA, trifluoroacetic acid; TBS, 20mm tris-buffered saline, pH 7.2; TLC, thin-layer chromatography; A disaccharide, GalNAc1-3Gal; A trisaccharide, GalNAc1-3[Fuc1-2]Gal; Forssman disaccharide, GalNAc1-3GalNAc; CDH (ceramide dihexoside or lactosyl ceramide) Gal1-4Glc1-1Cer (LacCer); CTH (ceramide trihexoside or globotriosyl ceramide), Gal1-4Gal1-4Glc1-1Cer (GbOse₃Cer or Gb₃); globoside (globotetraosyl ceramide), GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₄Cer or Gb₄); Forssman antigen (globopentaosyl ceramide), GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₅Cer).     

Molecular species analysis of glycosphingolipids from small intestine of Japanese quail,Coturnix coturnix Japonica by HPLC/FAB/MS

Neutral glycosphingolipids were isolated from quail small intestine and their structures were analysed. They contained: Gal1-4GlcCer(LacCer), Gal1-4GalCer(Ga₂Cer), Gal1-4Gal1-4GlcCer(Gb₃Cer), GlcNAc1-3Gal1-4GlcCer(Le₃Cer), GalNAc1-4Gal1-4GlcCer(Gg₃Cer), GalNAc1-4[GalNAc1-3]Gal1-4GlcCer(LcGg₄Cer), and GalNAc1-3GalNAc1-3Gal1-4Gal1-4GlcCer (Forssman glycolipid) as well as glucosylceramide, galactosylceramide (Nishimura Ket al. 1984)Biochim Biophys Acta 796:269–76) and the Le^x glycolipid, III³ Fuc-nLc₄Cer (Nishimura Ket al. (1989)J. Biochem (Tokyo) 101:1315–18). The molecular species compositions of these glycosphingolipids were examined using fast atom bombardment-mass spectrometry linked with reversed-phase high-performance liquid chromatography. By such analysis, we could classify the quail glycosphingolipids into at least three classes: glycolipids rich in species having four hydroxyl groups in the ceramides (GalCer, Gg₃Cer, LcGg₄Cer and Le^x), those rich in the ceramides ofN-acyl trihydroxysphinganine with normal fatty acids (Lc₃Cer), and glycolipids rich in the ceramides ofN-acyl sphingenine with normal fatty acids (LacCer, Gb₃Cer and Forssman glycolipid). Immunohistochemical observation implies that the differences in the hydrophobic moieties specified the localization of glycosphingolipids in the tissue.     

Developmental control of the expression of two ten-sugar branched chain fucolipids in rat small intestine

Two branched decaglycosylceramides, apparently identical to those identified in the small intestine of adult rats [Breimer ME, Falk K-E, Hansson GC, Karlsson K-A (1982) J Biol Chem 257:50–59], were absent during the three weeks following birth. They appeared abruptly at around 21 days. After their appearance, their tissue concentration and their base composition did not change during development. Their fatty acids were non-hydroxylated and the percentage of C₂₂–C₂₄ fatty acids, which was low at 24 days, increased and reached 48.6% by 27 days.Nomenclature Gal1-4Gal1-4GlcCer Globotriaosylceramide (GbOse₃Cer) - Il³NeuAc-LacCer M_M3-ganglioside - GalNAc1-3Gal1-4Gal1-4GlcCer globoside (globotetraosylceramide, GbOse₄Cer)     

A comparative assessment of TLC overlay technique and microwell adsorption assay in the examination of influenza A and Sendai virus specificities towards oligosaccharides and sialic acid linkages of gangliosides

Influenza A and Sendai viruses bind toneolacto-series gangliosides isolated from human granulocytes. Differences in receptor specificity of influenza viruses A/PR/8/34 (H1N1), A/X-31 (H3N2), and parainfluenza Sendai virus (HNF1, Z-strain) were determined by two direct solid phase binding assays: the overlay technique, which combines high-resolution in the separation of gangliosides on thin-layer chromatograms with direct binding; and the microwell adsorption assay as a convenient binding assay which is performed in microtitre wells to estimate the avidity of binding to an isolated ganglioside. Both methods were applied for comparative binding studies. Viruses were found to exhibit specificity for oligosaccharides and sialic acids as well as for chain length of the neutral carbohydrate backbone, whereas differing fatty acids (C_24:1 and C_16:0) in the ceramide portion had no impact on virus adsorption. Terminal sialyloligosaccharides Neu5Ac2-3Gal1-4Glc-R of G_M3, and Neu5Ac2-3Gal1-4GlcNAc-R as well as Neu5Ac2-6Gal1-4GlcNAc-R ofneolacto-series gangliosides with nLcOse₄Cer and nLcOse₆Cer backbone, exhibited significant specific receptor activity towards the different viruses. To compare the data revealed from both test systems, values of virus binding were ascertained by a non-parametric statistical approach based on rank correlation. The rank correlation coefficientr _s was calculated according to Spearman from each virus binding towards G_M3, IV³Neu5Ac-nLcOse₄Cer, IV⁶Neu5Ac-nLcOse₄Cer and VI³Neu5Ac-nLcOse₆SCer. The rank correlation coefficients 0.74, 0.95 and 0.92, which were determined for A/PR/8/34 (H1N1), A/X-31 (H3N2) and Sendai virus (HNF1, Z-strain), respectively, indicated that both assays generate highly correlated experimental data. Based on these results, analyses of virus binding on thin-layer chromatograms as well as in microwells were found equivalent tools for ganglioside receptor studies. Abbreviations: BSA, bovine serum albumin; GSL(s), glycosphingolipids; HPTLC, high performance thin-layer chromatography; PBS, phosphate buffered saline; Neu5Ac,N-acetylneuraminic acid [35];r _s = rank correlation coefficient according to Spearman. The designation of the glycosphingolipids follows the IUPAC-IUB recommendations [36]. LacCer or lactosylceramide, Gal1-4Glc1-1Cer; lacto-N-neotetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; lacto-N-norhexaosylceramide or nLcOse₆Cer, Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; G_M3 (according to Svennerholm [37]) or II³Neu5AcLacCer.     

Immunohistological analyses of neutral glycosphingolipids and gangliosides in normal mouse skeletal muscle and in mice with neuromuscular diseases

The expression of neutral glycosphingolipids (GSLs) and gangliosides was investigated in cryosections of normal mouse skeletal muscle and in muscle of mice with neuromuscular diseases using indirect immunofluorescence microscopy. Transversal and longitudinal sections were immunostained with specific polyclonal antibodies against lactosylceramide, lacto-N-neotetraosylceramide, globoside, G_M3(Neu5Ac), G_M3(Neu5Gc) and G_M1(Neu5Ac) as well as monoclonal anti-Forssman GSL antibody. In normal CBA/J mouse muscle (control) the main immunohistochemically detected ganglioside was G_M3(Neu5Ac) followed by moderately expressed G_M3(Neu5Gc) and G_M1. The neutral GSLs lactosylceramide and globoside were stained with almost identical, high fluorescence intensity. Low amounts of lacto-N-neotetraosylceramide and trace quantities of Forssman GSL were immunostained. All GSLs were detected in the sarcolemma, but also in considerable amounts at the intracellular level. Mice with neuromuscular diseases were the A2G-adr mouse mutant (a model for human recessive myotonia of Becker type), the BL6-wr mutant (a model for motor neuron disease) and the BL10-mdx mouse mutant (a model for human Duchenne muscular dystrophy). No changes in GSL expression were found in the A2G-adr mouse, while muscle of the BL6-wr mouse showed increased intensity of immunofluorescence in stainings with anti-lactosylceramide and anti-G_M3(Neu5Ac) antibodies. Muscle of BL10-mdx mice showed the most prominent changes in GSL expression with reduced fluorescence intensity for all antibodies. Major differences were not observed in the intensities of GSLs, but there were significant differences in the patterns of distribution on plasma membrane and at the subcellular level. The exact nature and pathogenesis of these changes should be elucidated since such investigations could furnish advances in understanding the functional role of neutral GSLs and gangliosides in normal as well as in diseased muscle. Abbreviations: BSA, bovine serum albumin; DAPI, 4, 6-diamidine-2-phenylindole-dihydrochloride; DTAF, dichlorotriazinylamino-fluorescein; GSL(s), glycosphingolipid(s); Neu5Ac,N-acetylneuraminic acid; Neu5Gc,N-glycolylneuraminic acid [53]; PBS, phosphate buffered saline. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [54] and the nomenclature of Svennerholm [55]. Lactosylceramide or LacCer, Gal1-4Glc1-1Cer; gangliotriaosylceramide or GgOse₃Cer, GalNAc1-4Gal1-4Glc1-1Cer; globotriaosylceramide or GbOse₃Cer, Gal1-4Gal1-4Glc1-1Cer; gangliotetraosylceramide or GgOse₃Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; globotetraosylceramide or GbOse₄Cer, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; lacto-N-neotetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; Forssman GSL or GbOse₅Cer, GalNAc1-3GalNAc1-3GAl1-4Gal1-4Glc1-1Cer; G_M3, II³Neu5Ac-LacCer; G_M1, II³Neu5Ac-GgOse₄Cer.     

N-andO-alkylation of glycoconjugates and polysaccharides by solid base in dimethyl sulphoxide/alkyl lodide

O-Methylation of simple neutral oligosaccharides is readily accomplished in dimethyl sulphoxide containing solid sodium hydroxide and methyl iodide [Cincanu I, Kerek F (1984) Carbohydr Res 131209-17]. This procedure has been extended to 2-acetamido-2-deoxy sugars and sialic acid-containing oligosaccharides. CompleteO-andN-methylation was in most cases achieved in 15 min. Esterification of carboxylic groups in uronic acids was fast and resulted in concomitant -elimination. The method is also suitable for methylation of glycoproteins and glycosphingolipids. Polysaccharides can also be methylated by this technique. Analysis of the products by gas-liquid chromatography and mass spectrometry showed no degradation products.Abbreviations lacto-N-tetraose LcOse₄, Gal3GlcNAc3Gal4Glc - lacto-N-fucopentaose III III³Fuc-nLcOse₄, Gal4[Fuc3]GlcNAc3Gal4Glc - trihexosylceramide GbOse₃Cer, Gal4Gal4Glc1-1Cer - globoside GbOse₄Cer, GalNAc3Gal4Glc1-1Cer - FAB-MS fas atom bombardment mass spectrometry     

Structural characterization of a novel mono-sulfated gangliotriaosylceramide containing a 3-O-sulfatedN-acetylgalactosamine from rat kidney

A novel mono-sulfated glycosphingolipid based on the gangliotriaose core structure was isolated from rat kidney. The isolation procedure involved extraction of lipids with chloroform/methanol, mild alkaline methanolysis, column chromatographies with anion exchangers and silica beads. The structure was characterized by compositional analysis, FTIR spectroscopy, methylation analysis,¹H-NMR spectroscopy and negative-ion liquid secondary ion mass spectrometry (LSIMS) using the intact glycolipid and its desulfation product. The two dimensional chemical shift correlated spectroscopy provided information on the sugar sequence as well as anomeric configurations, and indicated the presence of a 3-O-sulfatedN-acetylgalactosamine within the molecule. Negative-ion LSIMS with high- and low-energy collision-induced dissociation defined the sugar sequence and ceramide composition, confirming the presence of a sulfatedN-acetylgalactosamine at the non-reducing terminus. From these results, the complete structure was proposed to be HSO₃-3GalNAc1-4Gal1-4Glc1-1Cer (Gg₃Cer III³-sulfate, SM2b). Abbreviations: Abbreviations for sulfated glycolipids [17] follow the modifications of the nomenclature system of Svennerholm for gangliosides [37], and the designation of the other glycosphingolipids follows the IUPAC-IUB recommendations [38]. Cer, ceramide; LacCer, lactosylceramide, Gal1-4Glc1-1Cer; Gg₃Cer, gangliotriaosylceramide, GalNAc1-4Gal1-4Glc1-1Cer; Gg₄Cer, gangliotetraosylceramide, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; iGb₄Cer, isoglobotetraosylceramide, GalNAc1-3Gal1-3Gal1-4Glc1-1Cer; Gb₄Cer, globotetraosylceramide, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; SM4s, galactosylceramide sulfate, GalCer I³-sulfate; SM3, lactosylceramide sulfate, LacCer II³-sulfate; SM2a, Gg₃Cer II³-sulfate; SM2b, Gg₃Cer III³-sulfate; SB2, Gg₃Cer II³,III³-bis-sulfate; SM1a, Gg₄Cer II³-sulfate; SM1b, Gg₄Cer IV³-sulfate; SB1a, Gg₄Cer II³,IV³-bissulfate; GLC, gas-liquid chromatography; GC-MS, gas chromatography-mass spectrometry; DQF, double quantum filtered; COSY, chemical-shift-correlated spectroscopy; LSIMS, liquid secondary ion mass spectrometry; CID, collision-induced dissociation; MS/MS, tandem mass spectrometry.     

UDP-galactose:lactosylceramide α-galactosyltransferase activity in human placenta

The activity of UDP-Gal: LacCer galactosyltransferase in human placenta was studied by using crude homogenate and Triton CF-54 extract as the source of enzyme. Transfer of galactose to lactosylceramide was optimal in the presence of 0.1% Triton CF-54 and Mn²⁺ at pH 6.3, and the reaction product was susceptible to -galactosidase.Abbreviations LacCer lactosylceramide (Gal1-4Glc1-1Cer) - Gb₃ globotriaosylceramide (Gal1-4Gal1-4Glc1-1Cer) - Gb₄ globoside (GalNAc1-3Gal1-4Gal1-4Glc1-1Cer) - TLC thin-layer chromatography - GC/MS gas chromatography/mass spectrometry - NMR nuclear magnetic resonance - EDTA ethylenediamine tetraacetic acid - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate     

The effect of fumonisin B1 on developing chick embryos: correlation between de novo sphingolipid biosynthesis and gross morphological changes

Fumonisins, mycotoxins produced byFusarium moniliforme and a number of other fungi, are potent inhibitors of the sphinganine-N-acyltransferase, a key enzyme of sphingolipid biosynthesis, and cause neuronal degeneration, liver and renal toxicity, cancer and other injury to animals.In this study we investigated the effect of fumonisin B₁ on the sphingolipids of developing chick embryos. After yolk sac injection of fumonisin B₁ a concentration and time dependent increase of the sphinganine-over-sphingosine ratio of the embryos could be demonstrated. Studies were done to evaluate the effect of fumonisin B₁ on the glycosphingolipid pattern of the chick embryos. In the presence of 72 µg fumonisin B₁ per egg the incorporation of [¹⁴C]galactose and of [¹⁴C]serine into embryonic glycosphingolipids was reduced by about 70%, although the mass of glycosphingolipids was not affected by the toxin. However, a reduction of the wet weight of the treated embryos was observed. Additionally, histological examinations of whole embryo sections of control and fumonisin B₁ treated embryos are presented. Fumonisin B₁ caused haemorrhages under the skin as well as in the liver of treated embyros. A close correlation between disruption of sphingoid metabolism and light microscopic detectable tissue lesions could be observed.Abbreviations Cer ceramide (N-acylsphingosine) - FB₁ fumonisin B₁ - GM3 NeuAc23Gal14Glc11Cer - GD3 NeuAc28NeuAc23Gal14Glc11Cer - GD1a NeuAc23Gal13GalNAc14(NeuAc23)Gal14Glc11Cer - GT1b NeuAc23Gal13GalNAc14(NeuAc28NeuAc23) Gal14Glc11Cer - HPLC high pressure liquid chromatography - PBS phosphate buffered saline - PDMP 1-phenyl-2-dodecanoylamino-3-morpholino-1-propanol - Sa sphinganine - So sphingosine - Sa/So sphinganine-over-sphingosine - TLC thin layer chromatography - Tris Tris(hydroxymethyl)aminomethan Dedicated to Dr Sen-itiroh Hakomori in celebration of his 65th birthday.     

Biosynthesisin vitro of neolactotetraosylceramide by a galactosyltransferase from mouse T-lymphoma: purification and kinetic studies; synthesis of neolacto and polylactosamine core

The galactosyltransferase, GalT-4, which catalyses the biosynthesisin vitro of neolactotetraosylceramide, nLcOse4Cer (Gal1-4GleNAc1-3Gal1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (GlcNAc1-3Gal1-4Glc-Cer), and UDP-galactose has been purified 107 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified enzyme is partially stabilized in the presence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indicating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine lactose synthetase (UDP-Gal:Glc 4-galactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all other galactosyltransferases, GalT-4 has absolute requirements for divalent cation (Mn²⁺). TheK _m values for the substrate LcOse3Cer and donor UDP-galactose are 110 and 250 µm, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-1-acid glycoprotein orN-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which showed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAc1-1-3Gal1-4Lc3), the precursor for polylactosamine antigens. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental values were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an absolute requirement for anN-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH₂1-3Gal1-4Glc-sphingosine) of the acceptor glycolipid LcOse3Cer is completely inactive as substrate while theK _m andV _max of the reacetylated substrate (GlcNac1-3Gal1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic glycolipid. The monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified enzyme from mouse P-1798 T-lymphoma.Abbreviations EDTA ethylenediamine tetraacetate - ME -mercaptoethanol - PEG polyethylene glycol - PBS phosphate buffered saline - Suc sucrose - Mn²⁺ manganese - Gal galactose - GlcNAc N-acetylglucosamine - UDP-Gal Uridine diphosphate galactose - Ab antibody - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ECB embryonic chicken brain - Cer ceramide - nLc4 or NlcOse4Cer Gal1-4GleNac1-3Gal1-4Glc-Cer, neoLactotetraosylceramide - Lc3 or LcOse3Cer GlcNac1-3Gal1-4Glc-Cer, lactotriaosylceramide - iLc5 iLcOse5Cer, GlcNAc1-3nLcOse4Cer - nLc6 nLcOse6Cer, Gal1-4iLcOse5Cer - SA^–Gal^–₁AGP asialo-agalacto1-acid glycoprotein - TLC thin layer chromatography     

Detection of the gangliosideN-glycolyl-neuraminyl-lactosyl-ceramide by biotinylatedEscherichia coli K99 lectin

K99 lectin fromEscherichia coli was purified and biotinylated via its carboxyl groups using biocytin hydrazide and a water soluble carbodiimide. Biotinylation of two out of the nine carboxyl groups was sufficient to permit detection of the lectin by avidin and did not cause any loss of the haemagglutinating activity. It was demonstrated that the biotinylated K99 lectin retained other important properties of native K99 and that it will probably become a very sensitive detecting reagent. Indeed, it was able to bind to HeLa cells, as do intact bacteria carrying K99 fimbriae, and also to recognizeN-glycolyl-neuraminyl-lactosyl-ceramide in an overlay binding assay. Abbreviations: NeuAc,N-acetylneuraminic acid; NeuGc,N-glycolylneuraminic acid; PBS, phosphate buffered saline (0.9% NaCl containing 150mm sodium phosphate, pH 7.2); LPS, lipopolysaccharide; BCHZ, biocytin hydrazide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle medium. For the gangliosides, trivial names and structures are given according to the recommendations in [43]. NeuAc2-3Gal1-4Glc1-1Cer (NeuAc-GM₃); NeuGc2-3Gal1-4Glc1-1Cer (NeuGc-GM₃); GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer (GM₂); NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer (GD3); Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer (GM₁); NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer (GD_1a); Gal1-3GalNAc1-4(NeuAc2-8NeuAc2-3)Gal1-4Gle1-1Cer (GD_1b); NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-8NeuAc2-3) Gal1.-4Glc1-1Cer (GT_1b). NeuGc2-3Gal1-4GleNAc1-4Gal1-4Glc1-1Cer (NeuGc-SPG).     

Cloned β1,4N-acetylgalactosaminyltransferase: subcellular localization and formation of disulfide bonded species

Cloned human 1,4N-acetylgalactosaminyltransferase (GalNAcT) catalyses the synthesis of the glycosphingolipids GM2, GD2, and gangliotriosylceramide. To determine the subcellular location of this enzyme and whether it exists in intermolecular disulfide bonded species, we stably transfected Chinese hamster ovary (CHO) cells with three myc epitope-tagged forms of the GalNAcT gene: the native enzyme; the lumenal domain of GalNAcT fused to the cytoplasmic and transmembrane domains ofN-acetylglucosaminyltransferase I (GNT); and the transmembrane and lumenal domains of GalNAcT fused to the cytoplasmic domain of the Iip33 form of human invariant chain in order to retain the enzyme in the endoplasmic reticulum (ER). Immunoelectron microscopic analysis with anti-myc revealed that GalNAcT/myc was present throughout the Golgi stack, the GNT/GalNAcT/myc form was restricted primarily to the medial Golgi cisternae, and the Iip33/GalNAcT/myc form was restricted to the ER. Cells transfected with each of the three constructs contained high levels of GM2 synthase activityin vitro, but only the GalNAcT/myc form and the GNT/GalNAcT/myc forms were able to synthesize the GM2 productin vivo. The enzyme produced by all three constructs was present in the transfected cells in a disulfide bonded form having a molecular size consistent with that of a homodimer or higher aggregate.Abbreviations GSL glycosphingolipid(s) - CHO Chinese hamster ovary - GSL structures: GM2 GalNAc1,4(NeuAc2,3)Gal1,4GlcCer - GD2 GalNac1,4(NeuAc2,8NeuAc2,3)Gal1,4GlcCer - GM3 NeuAc2,3Gal1,4GlcCer - Gg₃ GalNAc1,4Gal1,4GlcCer - LacCer Gal1,4GlcCer - GlcCer glucosylceramide - PBS-BSA phosphate buffered saline pH 7.4 containing 1% bovine serum albumin - GalNAcT N-acetylgalactosaminyltransferase - GNT N-acetylglucosaminyltransferase I - Iip33 p33 form of human invariant chain - HPTLC high performance thin layer chromatography - PCR polymerase chain reaction - BFA Brefeldin A This paper is dedicated to Professor Sen-itiroh Hakomori on the occasion of his 65th birthday.     

A monoclonal antibody specific for non-reducing terminal Galα1-4Gal residues

A mouse monoclonal antibody (87.5) against Gal1-4Gal has been obtained after immunization with the disaccharide glycosidically coupled to a protein. The specificity was determined by studying its binding to a number of glycoconjugates and oligosaccharides.The antibody which was found to be highly specific for terminal Gal1-4Gal residues is a powerful tool for the detection of this structure in glycoproteins and glycolipids by immunochemicalin vitro methods. It is also useful forin vitro quantification of the free disaccharide.A thin layer chromatographic overlay assay using glycolipids and an immunoperoxidase technique is also described. The antibody 87.5 is used in this assay to identify human uroepithelium glycolipids with terminal Gal1-4Gal residues.Abbreviations Lactosylceramide Gal1-4GlcCer - globotriaosylceramide GbOse₃-ceramide, Gal1-4Gal1-4GlcCer - globotetraosylceramide globoside, GbOse₄-ceramide, GalNAc1-3Gal1-4Gal1-4GlcCer     

Expression of neutral glycosphingolipids and gangliosides in human skeletal and heart muscle determined by indirect immunofluorescence staining

The expression of neutral glycosphingolipids and gangliosides has been studied in human skeletal and heart muscle using indirect immunofluorescence microscopy. Transversal and longitudinal cryosections were immunostained with specific monoclonal and polyclonal antibodies against the neutral glycosphingolipids lactosylceramide, globoside, Forssman glycosphingolipid, gangliotetraosylceramide, lacto-N-neotetraosylceramide and against the gangliosides G_M3(Neu5Ac) and G_M1(Neu5Ac). To confirm the lipid nature of positive staining, control sections were treated with methanol and chloroform:methanol (1:1) before immunostaining. These controls were found to be either negative or strongly reduced in fluorescence intensity, suggesting that lipid bound oligosaccharides were detected. In human skeletal muscle, lactosylceramide was found to be the main neutral glycosphinogolipid. Globoside was moderately expressed, lacto-N-neotetraosylceramide and gangliotetraosylceramide were minimally expressed and Forssman glycosphingolipid was not detected in human skeletal muscle. The intensities of the immunohistological stains of G_M3 and G_M1 correlated to the fact that G_M3 is the major ganglioside in skeletal muscle whereas G_M1 is expressed only weakly. In human heart muscle globoside was the major neutral glycosphingolipid. Lactosylceramide and lacto-N-neotetraosylceramide were moderately expressed, gangliotetraosylceramide was weakly expressed and the Forssman glycosphingolipid was not expressed at all in cardiac muscle. G_M3 and G_M1 were detected with almost identical intensity. All glycosphingolipids were present in plasma membranes as well as at the intracellular level. Abbreviations used: BSA, bovine serum albumin; DAPI, 4,6-diamidine-2-phenylindole-dihydrochloride; DTAF, fluorescein isothiocyanate derivative; GSL(s), glycosphingolipid(s); Neu5Ac,N-acetylneuraminic acid [50]; PBS, phosphate buffered saline. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [51] and the nomenclature of Svennerholm [52]. Lactosylceramide or LacCer, Gal1-4Glc1-1Cer; gangliotriaosylceramide or GgOse₃Cer, GalNAc1-4Gal1-4Glc1-1Cer; globotriaosylceramide or GbOse₃Cer, Gall-4Gall-4Glcl-1Cer; gangliotetraosylceramide or GgOse₄Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; globotetraosylceramide or GbOse₄Cer, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; lacto-N-neotetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; Forssman GSL or GbOse₃Cer, GalNAc1-3GalNAc1-3Gal1-4Gal1-4Gle1-1Cer; G_M3, II³Neu5Ac-LacCer; G_M2, II³Neu5Ac-GgOse₃Cer; G_M1, II³Neu5Ac-GgOse₄Cer; G_D3 II³(Neu5Ac)₂-LacCer; G_D2, II³(Neu5Ac)₂-GgOse₃Cer; G_D1a, IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer; G_D1b, II³(Neu5Ac)₂-GgOse₄Cer.     

Über die Eignung von Naphthyl-α-l-fucosiden zur Untersuchung der α-l-Fucosidasen

Zusammenfassung Verglichen mit 1- und 2-Naphthyl--d-glucosid,--d-galactosid,--d-glucuronid,--d-N-acetylglucosaminid,--d-glucosid,--d-galactosid und--d-mannosid werden 1- und 2-Naphthyl--l-fucosid schneller oder im gleichen Ausmaß von Homogenaten verschiedener Rattenorgane hydrolysiert. Trotzdem fällt der histochemische Nachweis der -l-Fucosidasen methodenunabhängig im Gegensatz zu dem der anderen Glykosidasen überwiegend negativ aus. Ursache dafür ist die massive Hemmung der -l-Fucosidase durch Aldehydfixation und Diazoniumsalze; die Inhibitionsrate liegt bei 90% bzw. zwischen 85 und 98%; die - und -d-Glucosidase, - und -d-Galactosidase, -d-Mannosidase, -d-Glucuronidase sowie -d-N-Acetylglucosaminidase werden durch Aldehydfixation oder Kuppler höchstens zu 70% gehemmt. Daher können 1- und 2-Naphthyl--l-fucosid für die histochemische Darstellung der -l-Fucosidase nicht einschränkungslos empfohlen werden. Kleine Mengen Dimethylformamid hemmen die meisten Glykosidasen nicht.Für biochemische Messungen der -l-Fucosidase eignet sich speziell 1-Naphthyl--l-fucosid und läßt sich an Stelle von p-Nitrophenyl--l-fucosid werwenden. Bei der fluorometrischen Untersuchung der -l-Fucosidase in Rattenorganen mit dem 2-Naphthylderivat ergeben sich bemerkenswerte Aktivitätsunterschiede.

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Suitability of naphthyl--l-fucosides for the investigation of -l-fucosidases

Summary In comparison with 1- and 2-naphthyl -d-glucoside, -d-galactoside, -d-glucuronide, -d-N-acetylglucosaminide, -d-glucoside, -d-galactoside and -d-mannoside 1- and 2-naphthyl -l-fucoside are hydrolyzed more quickly or to the same extent by homogenates prepared from freezedried cryostate sections of various rat organs. Nevertheless, when the fucosides are employed for the histochemical demonstration of -l-fucosidase mostly negative data were obtained independent on the method used, whereas all other naphthyl glycosides deliver positive results. The reasons for these discrepancies are the marked inhibition of -l-fucosidase by aldehyde fixation and diazonium salts. Then, -l-fucosidase activity is suppressed to 90% and between 85 and 98% respectively; the inhibition of - and -d-glucosidase, - and -d-galactosidase, -d-mannosidase, -d-glucuronidase and -d-N-acetylglucosaminidase by the fixative or coupling reagent does not exceed 70%. Therefore 1- and 2-naphthyl -l-fucoside cannot be recommended in general for histochemical purposes. Small amounts of dimethylformamide do not influence the activity of most of the glycosidases investigated.For biochemical measurements, however, especially 1-naphthyl -l-fucoside represents a suitable alternative in a fluorometric procedure instead of p-nitrophenyl -l-fucoside used for the photometric evaluation of -l-fucosidase. With the fluorometric method the enzyme was measured in rat organs, which posses remarkably different activities of -l-fucosidase.

     

Purification, properties, and immunological characterization of GalT-3 (UDP-galactose: GM2 ganglioside, β1-3 galactosyltransferase) from embryonic chicken brain

A 1-3 galactosyltransferase (GalT-3; UDP-Gal; GM2 1-3galactosyltransferase) was purified over 5100-fold from 19-day-old embryonic chicken brain homogenate employing detergent solubilization, -lactalbumin Sepharose, Q-Sepharose, UDP-hexanolamine Sepharose, and GalNAc1-4Gal-Synsorb column chromatography. The purified enzyme was resolved into two bands on reducing gels with apparent molecular weights of 62 kDa and 65 kDa, respectively. GalT-3 activity was also localized in the same regions by activity gel analysis and sucrose-density gradient centrifugation of a detergent-solubilized extract of 19-day-old embryonic chicken brain. Purified GalT-3 exhibited apparentK _mS of 33 µm, 22 µm and 14.4mM with respect to the substrates GM2, UDP-galactose, and MnCl₂, respectively. Substrate specificity studies with the purified enzyme and a variety of glycosphingolipids, glycoproteins, and synthetic substrates revealed that the enzyme was highly specific only for the glycosphingolipid acceptors, GM2 and GgOse3Cer (asialo-GM2). Ovine-asialo-agalacto submaxillary mucin inhibited the transfer of galactose to GM2 but did not act as an acceptor in the range of concentrations tested. Polyclonal antibodies raised against purified GalT-3 inhibited GalT-3 activityin vitro and Western-immunoblot analysis of purified GalT-3 showed immunopositive bands at 62 and 65 kDa.Abbreviations CNS central nervous system - GM1 monosialotetraosylganglioside, Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer - GM2 monosialotriaosylganglioside, GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer - DSS detergent solubilized supernatant - ECB embryonic chicken brain - TBS Tris-buffered saline     

Microscale analysis of glycosphingolipids by TLC blotting/secondary ion mass spectrometry: a novel blood group A-active glycosphingolipid and changes in glycosphingolipid expression in rat mammary tumour cells with different metastatic potentials

The glycosphingolipid compositions of rat mammary tumour cell lines with different metastatic potentials for the lung [a parental tumour cell line (MTC) and its subclones MTLn2 (a non metastatic subclone) and MTLn3 (a subclone with high metastatic potential to the lung)] were studied using a newly developed TLC blotting/secondary ion mass spectrometry system and crude glycosphingolipids obtained from 0.5–1×10⁷ cells of each cell line. G_M3 and G_M2 were the major components of the MTC cell line, but they were very minor components in the MTLn2 and MTLn3 cell lines, G_Dla being the major ganglioside. HexNAc-fucosyl-G_Mla was found in the MTLn2 cells by the TLC blotting/SIMS method, and the terminal sugar linkage was shown to be a blood group A-type structure by immunostaining. These findings suggest that the ganglioside is a novel type of blood group A-active ganglioside, GalNAc1-3(Fuc1-2)G_Mla. No blood group A-active lipid was present in MTLn3 cells, whereas Hex-G_Mla and neutral glycosphingolipids with more than 5 sugar residues were. Abbreviations: TLC, thin-layer chromatography; HPTLC plate, high performance thin-layer chromatography-plate; PVDF, polyvinylidene difluoride; SIMS, secondary ion mass spectrometry; GC-MS, gas chromatography-mass spectrometry; C16:0, hexadecanoic acid; C18:0, octadecanoic acid; C22:0, docosanoic acid; C24:0, tetracosanoic acid; d18:1, 2-amino-4-octadecene-1,3-diol; Hex, hexose; HexNAc,N-acetylhexosamine; Gal, galactose; Glc, glucose; GalNAc,N-acetylgalactosamine; Lac, lactose; NeuAc,N-acetylneuraminic acid; Cer, ceramide; Glob, globoside; iGlob, isogloboside; GlcCer, glucosylceramide; LacCer, lactosylceramide; Gb₃Cer, Gal1-4Gal1-4Glc1-1Cer; Gb₄Cer (Glob), GalNAc1-3Gal1-4Glc1-1Cer; iGb₃Cer, Gal1-3Gal1-4Glc1-1Cer; iGb₄Cer (iBlob), GalNAc1-3Gal1-4Glc1-1Cer; Ganglio-series gangliosides are named according to Svennerholm [1].     

Ganglioside alterations in YAC-1 cells cultivated in serum-supplemented and serum-free growth medium

Gangliosides of the G_M1b-pathway (G_M1b and GalNAc-G_M1b) have been found to be highly expressed by the mouse T lymphoma YAC-1 grown in serum-supplemented medium, whereas G_M2 and G_M1 (G_M1a-pathway) occurred only in low amounts [Müthing, J., Peter-Katalini, J., Hanisch, F.-G., Neumann, U. (1991)Glycoconjugate J 8:414–23]. Considerable differences in the ganglioside composition of YAC-1 cells grown in serum-supplemented and in well defined serum-free medium were observed. After transfer of the cells from serum-supplemented medium (RPMI 1640 with 10% fetal calf serum) to serum-free medium (RPMI 1640 with well defined supplements), G_M1b and GalNAc-G_M1b decreased and only low amounts of these gangliosides could be detected in serum-free growing cells. The expression of G_M1a was also diminished but not as strongly as that of G_M1b and GalNAc-G_M1b. These growth medium mediated ganglioside alterations were reversible, and the original ganglioside expression was achieved by readaptation of serum-free growing cells to the initial serum-supplemented medium. On the other hand, a new ganglioside, supposed to represent GalNAc-G_D1a and not expressed by serum-supplemented growing cells, was induced during serum-free cultivation, and increased strongly after readaptation. These observations reveal that the ganglioside composition ofin vitro cultivated cells can be modified by the extracellular environment due to different supplementation of the basal growth medium. Abbreviations: BSA, bovine serum albumin GSL(s), glycosphingolipid(s); HPTLC, high-performance thin-layer chromatography; LDL, low density lipoprotein; NeuAc,N-acetylneuraminic acid; NeuGc,N-glycoloylneuraminic acid. The designation of the following glycosphingolipids follows IUPAC-IUB recommendations. GgOse₃Cer or gangliotriaosylceramide, GalNAc1-4Gal1-4GlcCer; GgOse₄Cer or gangliotetraosylceramide, Gal1-3GalNAc1-4Gla1-4GlcCer; GgOse₅Cer or gangliopentaosylceramide, GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer; GgOse₆Cer or gangliohexaosylceramide, Gal1-3GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer or GgOse₆Cer; II³NeuAc-GgOse₃Cer or G_M2; II³NeuAc-GgOse₄Cer or G_M1 or G_M1a; IV³NeuAc-GgOse₄Cer or G_M1b; IV³NeuAc-GgOse₅Cer or GalNAc-G_M1b; IV³NeuAc-GgOse₆Cer or Gal-GalNAc-G_M1b; IV³NeuAc, II³NeuAc-GgOse₄Cer or G_D1a; II³(NeuAc)₂-GgOse₄Cer or G_D1b; IV³NeuAc, III⁶NeuAc-GgOse₄Cer or G_D1a; IV³NeuAc, II³NeuAc-GgOse₅Cer or GalNAc-G_D1a. Enzymes: Vibrio cholerae andArthrobacter ureafaciens neuraminidase (EC 3.2.1.18).