Glycosphingolipid expression in human skeletal and heart muscle assessed by immunostaining thin-layer chromatography

In this study the comparative TLC immunostaining investigation of neutral GSLs and gangliosides from human skeletal and heart muscle is described. A panel of specific polyclonal and monoclonal antibodies as well as the GM1-specific choleragenoid were used for the overlay assays, combined with preceding neuraminidase treatment of gangliosides on TLC plates. This approach proved homologies but also quantitative and qualitative differences in the expression of ganglio-, globo- and neolacto-series neutral GSLs and gangliosides in these two types of striated muscle tissue within the same species. The main neutral GSL in skeletal muscle was LacCer, followed by GbOse3Cer, GbOse4Cer, nLcOse4Cer and monohexosylceramide, whereas in heart muscle GbOse3Cer and GbOse4Cer were the predominant neutral GSLs beside small quantities of LacCer, nLcOse4Cer and monohexosylceramide. No ganglio-series neutral GSLs and no Forssman GSL were found in either muscle tissue. GM3(Neu5Ac) was the major ganglioside, comprising almost 70% in skeletal and about 50% in cardiac muscle total gangliosides. GM2 was found in skeletal muscle only, while GD3 and GM1b-type gangliosides (GM1b and GD1) were undetectable in both tissues. GM1a-core gangliosides (GM1, GD1a, GD1b and GT1b) showed somewhat quantitative differences in each muscle; lactosamine-containing IV3Neu5Ac-nLcOse4Cer was detected in both specimens. Neutral GSLs were identified in TLC runs corresponding to e.g. 0.1 g muscle wet weight (GbOse3Cer, GbOse4Cer), and gangliosides GM3 and GM2 were elucidated in runs which corresponded to 0.2 g muscle tissue. Only 0.02 g and 0.004 g wet weight aliquots were necessary for unequivocal identification of neolacto-type and GM1-core gangliosides, respectively. Muscle is known for the lowest GSL concentration from all vertebrate tissues studied so far. Using the overlay technique, reliable GSL composition could be revealed, even from small muscle probes on a sub-orcinol and sub-resorcinol detection level. Abbreviations: ATCC, American Type Culture Collection; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid [78]; PBS, phosphate buffered saline. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [79] and the ganglioside nomenclature system of Svennerholm [80]. Lactosylceramide or LacCer, Gal1-4Glc1-1Cer; gangliotriaosylceramide or GgOse3Cer, GalNAc1-4Gal1-4Glc1-1Cer; gangliotetraosylceramide or GgOse4Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; globotriaosylceramide or GbOse3Cer, Gal1-4Gal1-4Glc1-1Cer; globoside or globotetraosylceramide or GbOse4Cer, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; Fo or Forssman GSL, GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; paragloboside or lacto-N-neotetraosylceramide or nLcOse4Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; lacto-N-norhexaosylceramide or nLcOse6Cer, Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; GM3, II3Neu5Ac-LacCer; GM2, II3Neu5Ac-GgOse3Cer; GM1 or GM1a, II3Neu5Ac-GgOse4Cer; GM1b, IV3Neu5Ac-GgOse4Cer; GD3, II3(Neu5Ac)2-LacCer; GD1a, IV3Neu5Ac,II3Neu5Ac-GgOse4Cer; GD1b, (II3Neu5Ac)2-GgOse4Cer; GD1, IV3Neu5Ac,III6Neu5Ac-GgOse4Cer; GT1b, IV3Neu5Ac,II3(Neu5Ac)2-GgOse4Cer; GQ1b, IV3(Neu5Ac)2, II3(Neu5Ac)2-GgOse4Cer.     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

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).     

The glycolipid specificity ofErythrina cristagalli agglutinin

The interaction of¹²⁵I-labeledErythrina cristagalli agglutinin (ECA) with neutral glycosphingolipids on thin layer chromatograms was examined by the overlay technique followed by radioautography. The lectin bound topara-globoside with a sensitivity about 10 times higher than to lactosylceramide or globoside, in agreement with the specificity of the lectin forN-acetyllactosamine. The lower limit of detection ofpara-globoside was about 0.66 nmol. The specific binding of ECA to this glycolipid was confirmed by a highly sensitive enzyme-linked lectin assay (ELLA), utilizing the horseradish peroxidase-avidin-biotin system for detection of bound lectin. Overlays of neutral glycosphingolipid extracts from human erythrocyte membranes and from human granulocytes with ECA demonstrated that the lectin can be employed for the detection of small amounts ofpara-globoside in biological materials also in the presence of excess globoside. No staining was obtained when thin layer chromatograms of neutral glycosphingolipid extracts from rabbit erythrocyte membranes were overlayed with¹²⁵I-ECA. Afterin situ treatment of the chromatograms with -galactosidase, the lectin bound to several components, one of which had a mobility corresponding to that of the pentahexosylceramide Gal3Gal4GlcNAc3Gal4Glc1Cer, the major neutral glycosphingolipid of rabbit erythrocytes, thus providing further evidence for the specificity of ECA forpara-globoside.Abbreviations GSL glycosphingolipid(s) - CDH lactosylceramide, Gal4Glc1Cer - CTH trihexosylceramide, Gal4Gal4Glc1Cer - GLOB globoside, GalNac3Gal4Gal4Glc1Cer - PG para-globoside, Gal4GlcNAc3Gal4Glc1Cer - AsG_M1 asialo-G_M1, Gal3GalNAc4Gal4Glc1Cer - FORS Forsmann antigen, GalNAc3GalNAc3Gal4Gal4Glc1Cer - CPH pentahexosylceramide, Gal3Gal4GlcNAc3Gal4Glc1Cer - ECA Erythrina cristagalli agglutinin - SBA soybean agglutinin - PBS phosphate-buffered saline - PVP-40 polyvinylpyrrolidone M.W. 40000 - BSA bovine serum albumin - HRP-avidin horseradish peroxidase conjugated to avidin - ELLA enzyme-linked lectin assay - ELISA enzyme-linked immunosorbent assay - PMNL polymorphonuclear leukocytes - HPTLC high performance thin layer chromatography     

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     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

Further studies on cell adhesion based on Lex-Lex interaction,with new approaches: embryoglycan aggregation of F9 teratocarcinoma cells,and adhesion of various tumour cells based on Lex expression

We previously proposed specific interaction of Le^x (Gal1 4[Fuc1 3]-GlcNAc1 3Gal) with Le^x as a basis of cell adhesion in pre-implantation embryos and in aggregation of F9 teratocarcinoma cells, based on several lines of evidence (Eggenset al., J Biol Chem (1989)264:9476–9484). We now present additional evidence for this concept, based on autoaggregation studies of plastic beads coated with glycosphingolipids (GSLs) bearing Le^x or other epitopes, and affinity chromatography on Le^x-columns of multivalent lactofucopentaose III (Le^x oligosaccharide) conjugated with lysyllysine. Comparative adhesion studies of Le^x-expressing tumour cellsvs their Le^x-non-expressing variants showed that only Le^x-expressing cells adhere to Le^x-coated plates and are involved in tumour cell aggregation, in analogy to F9 cell aggregation. The major carrier of Le^x determinant in F9 cells is not GSL but rather polylactosaminoglycan (embryoglycan), and we demonstrated autoaggregation of purified embryoglycan in the presence of Ca²⁺, and reversible dissociation in the absence of Ca²⁺ (addition of EDTA). Defucosylated embryoglycan did not show autoaggregation under the same conditions. Thus, Le^x-Le^x interaction has been demonstrated on a lactosaminoglycan basis as well as a GSL basis. A molecular model of Le^x-Le^x interaction based on minimum energy conformation with involvement of Ca²⁺ is presented.Abbreviations BSA bovine serum albumin - CHO carbohydrate - DMEM Dulbecco's modified Eagle's medium - EDTA ethylenediaminetetraacetic acid - GP glycopeptide - GSL glycosphingolipid - LAG lactosaminoglycan - Le^x Gal1 4[Fuc-1 3]GlcNAc1 R - LFP lacto-N-fucopentaose - LysLys-OH lysyllysinol - Mr relative molecular weight - PBS phosphate-buffered saline - PG paragloboside (Gal1 4GlcNAc1 3Gal1 4Glc1 1Cer) - TBS Tris-buffered saline (10mM Tris-HCl, pH 7.4, containing 0.15M NaCl) - TC tumour cell     

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).     

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     

Developmental changes in neutral glycosphingolipids of mouse placenta

The mammalian placenta is a unique organ for the study of developmental changes. Placentas of laboratory animals such as the mouse allow for the determination of the exact stage of pregnancy, which cannot be achieved with human placenta. In this study, neutral glycosphingolipids were isolated from mouse (inbred strain C57BL/6) placentas, from day 10 to day 18 of gestation, and were separated by high performance thin layer chromatography. Densitometric measurements after orcinol staining showed, at day 10 of gestation, the presence of mono-, tetra-, tri- and dihexosylceramide in decreasing quantities, as well as four unidentified spots. On day 12, the glycosphingolipid composition changed with the disappearance of the unidentified spots and the appearance of an orcinol positive spot migrating similarly to the Forssman antigen; no further changes occurred between days 12 and 18 of gestation. The identity of the Forssman-like glycosphingolipid with the Forssman antigen was established by binding of¹²⁵I labelledHelix pomatia agglutinin (-GalNAc specific) to glycosphingolipids separated on high performance thin layer chromatography plates, and by the reaction of the isolated glycosphingolipid with a monoclonal anti-Forssman antibody. The appearance of the Forssman antigen at day 12 of gestation coincided with the day of final maturation of the mouse placenta and subsequent cessation of growth, suggesting a possible role of the glycosphingolipid during embryonic development.Abbreviations asialo-GM₁ Gal 3GalNAc4Gal4Glc1Cer - BCIP 5-bromo-4-chloro-3-indolylphosphate - DHC lactosylceramide, Gal4Glc1Cer - Forssman antigen GalNAc3GalNAc3Gal4Gal4Glc1Cer - globoside GalNAc3Gal4Gal4Glc1Cer - GSL glycosphingolipids - HPA Helix pomatia agglutinin - HPTLC high performance thin layer chromatography - MHC galactosylceramide, Gal1Cer - MHC glucosylceramide, Glc1Cer - PBS phosphate-buffered saline - PNA peanut agglutinin - PVP poly(vinylpyrrolidone), mol. wt 40 000 - SBA soybean agglutinin - THC trihexosylceramide, Gal4Gal4Glc1Cer. To whom correspondence should be addressed.     

Single mid-chain GlcNAcβ1-6Galβ1-4R sequences of linear oligosaccharides are resistant to endo-β-galactosidase ofBacteroides fragilis

Endo--galactosidase (EC 3.2.1.103) ofBacteroides fragilis, at 250 mU ml^–1, did not cleave the internal galactosidic linkage of the linear radiolabelled trisaccharide GlcNAc1-6Gal1-4GlcNAc, or those of the tetrasaccharides Gal1-4GlcNAc1-6Gal1-4GlcNAc and Gal1-4GlcNAc1-6Gal1-4Glc. The isomeric glycans which contained the GlcNAc1-3Gal1-4GlcNAc/Glc sequence were readily cleaved.Abbreviations GlcNAc 2-acetamido-2-deoxy-d-glucose - Lact lactose - MT maltotriose - MTet maltotetraose - R _MTet chromatographic migration rate in relation to that of maltotetraose     

Characterization of gangliosides from Ehrlich ascites tumour cells and their variants

Differences in the nature of the gangliosides present in two types of Ehrlich ascites tumour (EAT) cells, the adherent and non-adherent EAT cells, were studied. Gangliosides were isolated by DEAE Sephadex column chromatography and analysed by high-performance thin-layer chromatography (HPTLC). The non-adherent EAT (na-EAT) cells which grow in the peritoneal cavity of mice were selected for growth on basement membrane and tissue culture plastic to give the adherent EAT (a-EAT) cells. na-EAT cells contained 1.57 nmol lipid-bound sialic acid per mg protein and at least 12 different gangliosides, including major gangliosides such as GM3, GM2, GM1, GD3, GD1a and GT1b. On the other hand, the ganglioside pattern of a-EAT cells differed significantly from that of na-EAT cells, both quantitatively and qualitatively. The content of lipid-bound sialic acid in a-EAT cells was only 0.24 nmol per mg of protein. The gangliosides in a-EAT cells were characterized as GD1a and trisialogangliosides and, significantly, a-EAT cells did not contain monosialogangliosides. Neutral glycolipids were isolated from both cell lines and their patterns were compared. In contrast to the gangliosides pattern, their neutral glycolipid patterns were similar. Glucosylceramide and lactosylceramide were the major components in both types of cells. In addition to na- and a-EAT cells, a-EAT cells were passaged in mice by intraperitoneal injection, giving rise to a third variant (c/m EAT cells). We analysed the gangliosides in c/m EAT cells to determine whether there was a change in the ganglioside pattern found in na-EAT cells. After repeated passage of c/m EAT cells in mice, the pattern of gangliosides shifted to that of na-EAT cells. Alterations of ganglioside composition may be associated with the growth environment of the murine peritoneal cavity; alternatively, a selection process may have occurred.Abbreviations EAT cells Ehrlich ascites tumour cells - na-EAT cells non-adherent EAT cells - a-EAT cells adherent EAT cells - c/m EAT cells cultured a-EAT cells passaged in mice - HPTLC high-performance thin-layer chromatography - PBS 10 mM phosphate-buffered saline, pH 7.2, containing 0.15 M NaCl - EDTA ethylene-diaminetetraacetic acid - TFA trifluoroacetic acid - TG thioglycollate - Cer ceramide (N-fatty acyl sphingosine) - GM3 NeuAc2-3Gal1-4Glc-Cer - GM2 GalNAc1-4(NeuAc2-3)Gal1-4Glc-Cer - GM1a Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-Cer - GD3 NeuAc2-8NeuAc2-3Gal1-4Glc-Cer - GD1a NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-Cer - GT1b NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-8NeuAc2-3)Gal1-4Glc-Cer - LacCer Gal1-4Glc-Cer - Gb3 Gal1-4Gal1-4Glc-Cer - Gb4 GalNAc1-4Gal1-4Gal1-4Glc-Cer This paper is dedicated to my esteemed colleague, Sen-itiroh Hakomori on the occasion of his 65th birthday.     

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.     

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)     

Ganglioside lactones:1H-NMR determination of the inner ester position of GD1b-ganglioside lactone naturally occurring in human brain or produced by chemical synthesis

The complete definition of the chemical structure of G_D1b-ganglioside (G_D1b) lactone isolated from human brain has been given by means of spectrometric and spectroscopic analyses. G_D1h lactone contains a single ester linkage involving the external sialic acid carboxyl group and the C-9 hydroxyl group of the internal sialic acid unit. A synthetic lactone of G_D1b prepared treating G_D1b with glacial acetic acid characterized in the same way showed an identical chemical structure.Abbreviations: Ganglioside nomenclature is according to Svennerholm [16] and the IUPAC-IUB Recommendations [17] G_M1 G_M1-ganglioside, II³NeuAc-GgOse₄Cer, Gal1-3GalNac1-4[NeuAc2-3]Gal1-4Glc1-1Cer - G_D1b G_D1b-ganglioside, II³(NeuAc)₂GgOse₄Cer, Gal1-3GalNAc1-4[NeuAc2-8NeuAc2-3]Gal1-4Glc1-1Cer - G_D1b lactone G_D1b-L, Gal1-3GalNAc1-4[NeuAc(1-9)2-8NeuAc2-3]Gal1-4Glc1-1Cer - Cer ceramide - FAB-MS fast atom bombardment-mass spectrometry - ¹H-NMR proteon nuclear magnetic resonance - 1D-NMR one dimensional NMR - 2D-COSY two dimensional correlated spectroscopy - DMSO-d₆ deuterated dimethylsulfoxide     

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     

Carbohydrate antigen expression in murine embryonic stem cells and embryos. II. Sialylated antigens and glycolipid analysis

Summary A mouse embryonic stem (ES) cell line E14 and early mouse embryos were stained with a panel of 15 monoclonal antibodies recognizing sialylated or potentially sialylated carbohydrate determinants, Sialyl Le-x and sialyl Le-a were detected on the pre-implantation embryo from the 8-cell stage, and sialyl Le-a weakly on undifferentiated ES cells. Changes in cell surface carbohydrates occurred after induction of ES cell differentiation with retinoic acid (RA) and dibutyryl cAMP. Qualitative analysis of the neutral glycolipids of untreated and RA-treated ES cells using high-performance thin-layer chromatography (HPTLC) revealed few differences between the two types of culture. The major gangliosides in both cultures were indicative of an active a ganglioside synthesis pathway. GD₃, a precursor of the b synthesis pathway, previously reported to be characteristic of embryonal carcinoma (EC) cells, was absent. RA-induced differentiation caused a shift in the spectrum to more complex gangliosides. Application of fast atom bombardment mass spectrometry (FAB-MS) to permethylated derivatives of individual bands permitted partial characterization of an unusual sialylated glycolipid and a rare ganglioside with the suggested structure of GalNAc-GD1a.Abbreviations NeuAc N-acetylneuraminic acid - Cer ceramide - CMH monohexosylceramide - CDH lactosylceramide (Gal1-4Glc1-Cer) - CTH ceramide trihexoside (Gal1-4Gal1-4Glc1-Cer) - globoside (GalNAc1-3 Gal1-4Gal1-4Glc1-Cer) - Forssman antigen (GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-Cer) - GM₃ (NeuAc2-3Gal1-4Glc1-Cer) - GD₃ (NeuAc2-8NeuAc2-3Gal1-4Glc1-Cer) - GM1 (Gal1-3GalNAc1-4[NeuAca2-3]Gal1-4Glc1-Cer) - GD1a (NeuAc2-3Gal1-3GalNAc1-4[NeuAc2-3]Gal1-4Glc1-Cer) - GT1b (Neu5Ac2-3Gal1-3GalNAc1-4[Neu5Ac2-8Neu5Ac2-3]Gal1-4Glc1-Cer) The glycolipids are named according to Svennerholm (1963) and the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (1978).     

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.     

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.     

Studies of the action of ceramide-like substances (d- andl-PDMP) on sphingolipid glycosyltransferases and purified lactosylceramide synthase

We have studied the effects ofD-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and itsL-enantiomer on glycosphingolipids in cultured normal human kidney proximal tubular cells. We found thatD-PDMP exerted a concentration-dependent reduction in the metabolic labelling and cellular levels of glucosylceramide (GlcCer), lactosylceramide (LacCer), and the globo-series glycosphingolipids, GbOse₃Cer and GbOse₄Cer. It also directly inhibited the activity of UDP-glucose:ceramide 1 4-glucosyltransferase (GlcT-1) and UDP-galactose: GlcCer 1 4 galactosyltransferase (GalT-2). In contrast,L-PDMP had opposite effects on the metabolic labelling of GlcCer, LacCer, and GbOse₃Cer. The levels of GlcCer and LacCer were increased, while the labelling and level of GbOse₄Cer were strongly reduced. Purified GalT-2 from human kidney was inhibited byD-PDMP and stimulated byL-PDMP. It appears likely that the different glycosphingolipid glycosyltransferases possess similar binding sites for the ceramide moiety, which are blocked by binding toD-PDMP and, in the case of GbOse₄Cer synthase, byL-PDMP as well. The stimulatory effects ofL-PDMP on GlcCer and LacCer synthases may be the result of binding to a modulatory site on the glycosyltransferases; in intact cells, the enzyme-analog complex may afford protection against the normal catabolic inactivation of the enzymes.Abbreviations GalT-2 UDP-galactose:GlcCer -galactosyltransferase - GbOse₃Cer Gal1 4Gal1 GlcCer - GbOse₄Cer GalNAc1 3Gal1 4Gal1 GlcCer - GlcCer glucosylceramide - GlcT-1 UDP-glucose:ceramide -glucosyltransferase - GSLs glycosphingolipids - LacCer lactosylceramide - PDMP threo-1-phenyl-2-decanolyamino-3-morpholino-1-propanol