Carbohydrate analysis of chicken heart glycolipids

Neutral and acidic glycolipids were extracted from chicken hearts. The neutral and acidic compounds were separated by preparative thin-layer chromatography into eight and two fractions, respectively. Total hydrolysis by mineral acid, permethylation analysis, and sequential cleavage with exoglycosidases showed the presence of glycolipids that belong to the globo- and gala-oligosaccharide series, i.e., the monohexosylceramides Glc-Cer and Gal-Cer, the dihexosylceramides Gal beta 1-4Glc-Cer and Gal alpha 1-4Gal-Cer, the tetrahexosylceramides GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer and GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal-Cer (III3GalNAc alpha-Ga3Cer) and four subfractions of the Forssman glycolipid GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer. With the notable exception of III3GalNAc alpha 1-Ga3Cer, all glycolipids with terminal GalNAc alpha 1-3GalNAc1 reacted on thin-layer chromatograms with a monoclonal anti-Forssman antibody. The major components of the acidic fraction glycolipids were characterized as the lactose-based gangliosides Glac1 (GM3) and Glac2 (GD3).     

Globotetraosylceramide is recognized by the pig edema disease toxin

The pig edema disease toxin has been shown by a tlc glycolipid binding assay to bind specifically to globotetraosylceramide (Gb4, GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer.). Binding was reduced for globotriosylceramide (Gb3, Gal alpha 1-4Gal beta 1-4GlcCer) and more markedly for the Forssman antigen (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer). Paragloboside, blood group A glycolipids, glycolipids terminating in Gal NAc beta 1-4Gal-, and glycolipids in which globoside was present as an internal sequence did not bind the toxin. Isogloboside (GalNAc beta 1-3Gal alpha 1-3Gal beta 1-4GlcCer) was efficiently recognized. This toxin is genetically related to the verotoxin (or Shiga-like) family of toxins for which Gb3 has been shown to be the receptor. The difference in susceptibility of cell lines to the cytotoxicity of the pig edema disease toxin and the Shiga and Shiga-like toxins is consistent with the difference in receptor glycolipid binding.     

The parasitic trematode Fasciola hepatica exhibits mammalian-type glycolipids as well as Gal(beta1-6)Gal-terminating glycolipids that account for cestode serological cross-reactivity

Neutral glycosphingolipids from sheep-derived Fasciola hepatica liver flukes were isolated and characterized both structurally and serologically. After HPLC fractionation, glycolipids were analyzed by linkage analysis, enzymatic cleavage, and MALDI-TOF as well as electrospray ionization mass spectrometry. Obtained results revealed the presence of two types of neutral glycolipids. The first group represented mammalian-type species comprising globo- and isoglobotriaosylceramides (Gal(alpha1-4)Gal(beta1-4)Glc(1-1)ceramide and Gal(alpha1-3)Gal(beta1-4)Glc(1-1)ceramide, respectively) as well as Forssman antigen (GalNAc(alpha1-3)GalNAc(beta1-3/4)Gal(alpha1-4/3)Gal(beta1-4)Glc(1-1)ceramide). Applying Helix pomatia agglutinin, recognizing terminal alpha-linked GalNAc, to cryosections of adult flukes, the latter glycolipid could be localized to the F. hepatica gut. As Forssman antigen from the parasite and sheep host led to identical MALDI-TOF MS profiles, this glycolipid might be acquired from the definitive host. As a second group, highly antigenic glycolipids were structurally characterized as Gal(beta1-6)Gal(beta1-4)Glc(1-1)ceramide, Gal(beta1-6)Gal(alpha1-3/4)Gal(beta1-4)Glc(1-1)ceramide and Gal(beta1-6)Gal(beta1-6)Gal(alpha1-3/4)Gal(beta1-4)Glc(1-1)ceramide, the latter two structures of which exhibited both isoglobo- or globo-series core structures. Terminal Gal(beta1-6)Gal1-motifs have previously been shown to represent antigenic epitopes of neogala-series glycosphingolipids from tape worms. Using human Echinococcus granulosus infection sera, Gal(beta1-6)Gal-terminating glycolipids could be allocated to the gut in adult liver fluke cryosections. Corresponding neogala-reactive antibodies in F. hepatica infection serum were detected by their binding to E. granulosus and Taenia crassiceps neogala-glycosphingolipids. These antibodies might contribute to the known serological cross-reactivity between F. hepatica and parasitic cestode infections.     

Characterization of a glycosphingolipid antigen defined by the monoclonal antibody MBr1 expressed in normal and neoplastic epithelial cells of human mammary gland

The antigen defined by a monoclonal antibody, MBr1, was found to be expressed in normal human mammary gland epithelia and human mammary carcinoma cells (Ménard, S., Tagliabue, E., Canevari, S., Fossati, G., and Colnaghi, M. I. (1983) Cancer Res. 43, 1295-1300). The antigen has been isolated from breast cancer cell line MCF-7, which was used as immunogen, and its structure was determined by methylation analysis, NMR spectroscopy, direct probe mass spectrometry, and enzymatic degradation as identified below. Fuc alpha 1----2Gal beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer The antibody cross-reacted weakly with fucosylasialo-GM1 (IV2FucGg4), which shares the same terminal sequence, Fuc alpha 1----2Gal beta 1----3GalNAc, with this antigen. However, various other structures, including lacto-series H structure (Fuc alpha 1----2 Gal beta 1----4/or 3GlcNAc beta 1----3Gal), did not show any reactivity with this antibody. Therefore, this antigen represents a blood group H antigen with a globo-series structure which is abundant in human teratocarcinoma (Kannagi, R., Levery, S. B., Ishigami, F., Hakomori, S., Shevinsky, L. H., Knowles, B. B., and Solter, D. (1983) J. Biol. Chem. 258, 8934-8942), although its presence must be limited in normal adult human tissue.     

The reactivities of human erythrocyte autoantibodies anti-Pr2, anti-Gd, Fl and Sa with gangliosides in a chromatogram binding assay.

The thin layer chromatogram binding assay was used to study the reaction of several natural-monoclonal autoantibodies which recognize sialic acid-dependent antigens of human erythrocytes. Immunostaining of gangliosides derived from human and bovine erythrocytes was achieved with four autoantibodies designated anti-Pr2, anti-Gd, Sa and Fl, each of which has a different haemagglutination pattern with untreated and proteinase-treated erythrocytes and with cells of I and i antigen types. From the chromatogram binding patterns of anti-Pr2 with gangliosides of the neolacto and the ganglio series, it is deduced that this antibody reacts best with N-acetylneuraminic acid when it is alpha 2-3- or alpha 2-6-linked to a terminal Gal(beta 1-4)Glc/GlcNAc GlcNAc sequence and to a lesser extent when it is alpha 2-3-linked to a terminal Gal(beta 1-3)GalNAc sequence or to an internal galactose and when it is alpha 2-8-linked to another, internal N-acetylneuraminic acid residue. The other three antibodies differ from anti-Pr2 in their lack of reaction with glycolipids of the ganglio series. They react with the NeuAc(alpha 2-3)Gal(beta 1-4)Glc/GlcNAc sequence as found in GM3 and in glycolipids of the neolacto series, but show a preference for the latter, longer sequences. Thus all four antibodies react with sialylated oligosaccharides containing i type (linear) and I type (branched) neolacto backbones. Fl antibody differs from the other three in its stronger reaction with branched neolacto sequences in accordance with its stronger agglutination of erythrocytes of I rather than i type. The four antibodies show a specificity for N-acetyl- rather than N-glycolyl-neuraminic acid.     

Gangliosides GM2, IV4GalNAcGM1b, and IV4GalNAcGC1a as antigens for monoclonal immunoglobulin M in neuropathy associated with gammopathy

It was previously reported that monoclonal IgM from two patients with gammopathy and neuropathy showed similar specificity by reacting with the same group of unidentified minor components in the ganglioside fractions of human nervous tissues (Ilyas, A. A., Quarles, R. H., Dalakas, M. C., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6697-6700). Enzymatic degradation, ion-exchange chromatography, and immunostaining of purified ganglioside standards on thin-layer chromatograms have now revealed that the antigenic glycolipids recognized by the IgM from these patients are gangliosides GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1Cer(GM2), GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer (IV4GalNAcGM1b), and GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4 beta Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1-Cer (IV4GalNAcGD1a). The monoclonal IgM appears to be reacting with the terminal [GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-] moiety shared by these three gangliosides and is a useful probe for detecting small amounts of GM2, IV4GalNAcGM1b, IV4GalNAcGD1a, and other gangliosides with the same terminal sugar configuration in tissues. Species distribution studies using the antibody revealed that GM2 is present in the brains and nerves of all species examined, while IV4GalNAcGM1b and IV4GalNAcGD1a exhibit some striking species specificity. GM2, but not IV4GalNAcGD1a, is enriched in purified myelin from human brain.     

Solution conformation of Forssman antigen probed by NOE and exchange interactions

The conformation of Forssman glycolipid, GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1ceramide, was analysed with the aid of the rotating frame NOE and Hartmann-Hahn spectroscopy. NOE contacts between C-, O-, and N-linked protons were used for distance mapping. The glycosidic bonds that are common to globotriaosylceramide and globoside showed a similar flexibility as found for these compounds [Poppe et al., (1990) Eur. J. Biochem. 189, 313-325; J. Am. Chem. Soc. 112, 7762-7771]. In contrast, the conformational mobility of the terminal GalNAc alpha 1-3GalNAc beta linkage appears to be restrained. A new approach, based on 2D exchange spectroscopy, was proposed for revealing of spatial proximities between exchangeable protons in Me2SO solution.     

Two kinds of P-fimbrial variants of uropathogenic Escherichia coli recognizing forssman glycosphingolipid.

Various types of fimbriae on pathogenic Escherichia coli strains have been classified by their antigenicities and recognition specificities for receptors. However, the antigenicity of fimbrial proteins does not always correlate with the fimbrial recognition specificity. In this communication, the exact carbohydrate structures recognized by the fimbriae of two human uropathogenic E. coli strains, KS71 (O4) and IH11024 (O6), that have P-fimbrial antigen, were examined. Strain KS71 showed mannose-resistant (MR) hemagglutination (HA) of human blood group OP1 phenotype erythrocytes, and its HA was inhibited by blood group Pk antigen, Gal(alpha,1-4)Gal(beta,1-4)Glc-ceramide and P antigen, GalNAc(beta,1-3)Gal (alpha,1-4)Gal(beta,1-4)Glc-ceramide but not by Forssman antigen, GalNAc(alpha,1-3)GalNAc(beta,1-3)Gal(alpha,1-4)Gal (beta,1-4)Glc-ceramide, as previously described in many papers. The cells also showed MR HA of sheep erythrocytes, which was potently inhibited by Forssman, and weakly by P and Pk antigens. These phenomena could not be explained by the above P adhesin specificity. This adhesin was called Forssman-like adhesin. Strain IH11024 also caused MR HA of sheep erythrocytes but not of human erythrocytes. The HA was inhibited specifically by Forssman but neither by Pk nor P antigen. This adhesin was completely different from P adhesin and Forssman-like adhesin in recognition of the carbohydrate epitope. This adhesin, until now called a pseudotype of P fimbriae, was renamed Forssman adhesin.     

Biosynthesis in vitro of a globoside containing a 2-acetamido-2-deoxy-beta-D-galactopyranosyl group (1----3)-linked and Forssman glycolipid by two N-acetylgalactosaminyltransferases from chemically transformed guinea pig cells

Two N-acetylgalactosaminyltransferase activities (GalNAcT-2 and GalNAcT-3) have been characterized in chemically transformed, cultured guinea-pig cell lines (104C1 and 106B). Line 104C1 is a benz[a]pyrene-transformed tumorigenic variant, whereas line 106B is a 7,12-dimethylbenz[a]anthracene-transformed nontumorigenic variant obtained from fetal guinea-pig cells at 43 days of gestation. The GalNAcT-2 (UDP-GalNAc:GbOse3Cer beta-N-acetylgalactosaminyltransferase) isolated from both 104C1 and 106B cells catalyzed the transfer of Gal-NAc from UDP-GalNAc to the 3H-labeled terminal galactose group of Gb3 [( 6-3H]Gal alpha 1----4Gal beta 1----4Glc----Cer). The 3H-labeled globoside was purified and then subjected to exhaustive methylation. After acetolysis, the partially methylated sugars were separated by two-dimensional, thin-layer chromatography. 3H-Label was detected in two major areas, 2,4,6-tri-O-Me-Gal (40%) and 2,3,4,6-tetra-O-Me-Gal (46%). In a separate experiment, 80% of the GalNAc was released when labeled GbOse4Cer [( 3H]GalNAc----Gal alpha 1----4Gal beta 1----4Glc----Cer) was treated with purified clam beta-hexosaminidase. The present results establish the formation of a beta-D-GalpNAc-(1----3) linkage in the terminal region of the biosynthesized globoside. GalNAcT-3 activity (UDP-GalNAc:GbOse4Cer alpha-GalNAc-transferase), which catalyzes the transfer of GalNAc from UDP-[14C]- or -[3H]GalNAc to GbOse4Cer (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc----Cer), was three times higher in 106B cells than in 104C1 cells. The isolated, purified radioactive product formed an immunoprecipitin line against rabbit anti-Forssman antibody.     

A new ganglioside of the lactotetraose series, GalNAc-3'-isoLM1, detected in human meconium

A monoclonal antibody produced by immunization with cells of the human glioma cell line D-54 MG reacted with ganglioside GM2. The binding epitope of the antibody was found to be GalNAc beta 1-4(NeuAc alpha 2-3)Gal. Immunological detection of glycolipid antigens on thin-layer plates with this monoclonal antibody, DMAb-1, revealed the presence of a new ganglioside. This ganglioside, co-migrating with NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer(6'-LM1) and GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-3GalNAc beta 1-4Gla beta 1-4Glc beta 1-1Cer (GalNAc-isoGM1) at chromatographic separation was isolated from human meconium. Its structure was determined by permethylation and fast atom bombardment-mass spectometry analyses. The new ganglioside was found to be a combination of the lacto and ganglio series gangliosides, and the structure found to be GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-3GlcNAc alpha 1-3Gal beta 1-4Glc beta 1-1Cer(GalNAc-3'-isoLM1).     

Pseudomonas aeruginosa and Pseudomonas cepacia isolated from cystic fibrosis patients bind specifically to gangliotetraosylceramide (asialo GM1) and gangliotriaosylceramide (asialo GM2)

Pseudomonas aeruginosa infection in the lungs is a leading cause of death of patients with cystic fibrosis, yet a specific receptor that mediates adhesion of the bacteria to host tissue has not been identified. To examine the possible role of carbohydrates for bacterial adhesion, two species of Pseudomonas isolated from patients with cystic fibrosis were studied for binding to glycolipids. P. aeruginosa and P. cepacia labeled with 125I were layered on thin-layer chromatograms of separated glycolipids and bound bacteria were detected by autoradiography. Both isolates bound specifically to asialo GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) and asialo GM2 (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) but not to lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), globoside (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer), paragloboside (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), or several other glycolipids that were tested. Asialo GM1 and asialo GM2 bound the bacteria equally well, exhibiting similar binding curves in solid-phase binding assays with a detection limit of 200 ng of either glycolipid. Both isolates also did not bind to GM1, GM2, or GDla suggesting that substitution of the glycolipids with sialosyl residues prevents binding. As the Pseudomonas do not bind to lactosylceramide, the beta-N-acetylgalactosamine residue, positioned internally in asialo GM1 and terminally in asialo GM2, is probably required for binding. beta-N-Acetylgalactosamine itself, however, is not sufficient as the bacteria do not bind to globoside or to the Forssman glycolipid. These data suggest that P. aeruginosa and P. cepacia recognize at least terminal or internal GalNAc beta 1-4Gal sequences in glycolipids which may be receptors for these pathogenic bacteria.     

Histochemical study of glycoconjugates in the toadfish Halobatrachus didactylus oesophagus epithelium

The carbohydrate expression in the epithelium lining the oesophagus of the toadfish Halobatrachus didactylus was studied by means of conventional and lectin histochemistry. The stratified epithelium was constituted by basal cells, polymorphous cells in the intermediate layer, pyramidal and flattened cells in the outer layer and contained two types of large secretory cells: goblet cells and sacciform cells. PAS, Alcian blue pH 2.5 and pH 1.0 stained very strongly the goblet cells, weakly the surface of the other epithelial cells but did not stain the sacciform cells. The goblet cells cytoplasm contained oligosaccharides with terminal Galbeta1,3GalNAc, alpha/betaGalNAc, Galbeta1,4GlcNAc, alphaL-Fuc and internal betaGlcNAc residues (PNA, SBA, RCA120, UEA I, LTA and KOH-sialidase-WGA affinity). Galbeta1,4GlcNAc, alphaL-Fuc and internal betaGlcNAc were also found in the glycocalyx. The sacciform cells expressed sialyloligosaccharides terminating with Neu5Acalpha2,3Galbeta1,4GlcNac, Neu5Acbeta2,6Gal/GalNAc, Neu5AcForssman pentasaccharide (MAL II, SNA, KOH-sialidase-DBA staining) as well as asialo-glycoconjugates with terminal/internal alphaMan (Con A affinity) and with terminal Galbeta1,3GalNAc, Forssman pentasaccharide, Galbeta1,4GlcNAc, GalNAc (HPA and SBA reactivity), alphaGal (GSA I-B4 reactivity), D-GlcNAc (GSA II labelling), alphaL-Fuc. The basal cells cytoplasm exhibited terminal/internal alphaMan and terminal Neu5Acalpha2,6Gal/GalNAc, Galbeta1,4GlcNAc, alpha/betaGalNAc, alphaGal, GlcNAc, alphaL-Fuc. Intermediate cells showed oligosaccharides with terminal/internal alphaMan and/or terminating with Neu5Acalpha2,6Gal/GalNAc, Galbeta1,4GlcNAc in the cytoplasm and with Neu5Acalpha2,3Galbeta1,4GlcNac, alpha/betaGalNAc, alphaGal, GlcNAc, alphaL-Fuc in the glycocalyx. The pyramidal cells expressed terminal/internal alphaMan and terminal Neu5Acalpha2,6Gal/GalNAc, alpha/betaGalbeta1,4NAc, alphaGal, alphaL-Fuc in the entire cytoplasm, terminal Neu5Acalpha2,3Galbeta1,4GlcNac and Forssman pentasaccharide in the apical extension, internal betaGlcNAc and/or terminal alphaL-Fuc in the luminal surface, Neu5Acalpha2,3Galbeta1,4GlcNac, Neu5Acalpha2,6Gal/GalNAc, Galbeta1,4GlcNAc, alphaGal in the basolateral surface. The flattened cells displayed glycans with terminal/internal alphaMan and terminal Neu5Acalpha2,6Gal/GalNAc, alpha/betaGalNAc, alphaGal, D-GlcNAc in the entire cytoplasm, glycans terminating with Galbeta1,3GalNAc and/or internal betaGlcNAc in the sub-nuclear cytoplasm.     

Novel Leb-like Helicobacter pylori-binding glycosphingolipid created by the expression of human alpha-1,3/4-fucosyltransferase in FVB/N mouse stomach

The "Le(b) mouse" was established as a model for investigations of the molecular events following Le(b)-mediated adhesion of Helicobacter pylori to the gastric epithelium. By the expression of a human alpha-1,3/4-fucosyltransferase in the gastric pit cell lineage of FVB/N transgenic mice, a production of Le(b) glycoproteins in gastric pit and surface mucous cells was obtained in this "Le(b) mouse," as demonstrated by binding of monoclonal anti-Le(b) antibodies. To explore the effects of the human alpha-1,3/4-fucosyltransferase on glycosphingolipid structures, neutral glycosphingolipids were isolated from stomachs of transgenic alpha-1,3/4-fucosyltransferase-expressing mice. A glycosphingolipid recognized by BabA-expressing H. pylori was isolated and characterized by mass spectrometry and proton NMR as Fuc alpha 2Gal beta 3(Fuc alpha 4)GalNAc beta 4 Gal beta 4 Glc beta 1Cer, i.e., a novel Le(b)-like glycosphingolipid on a ganglio core. In addition, two other novel glycosphingolipids were isolated from the mouse stomach epithelium that were found to be nonbinding with regard to H. pylori. The first was a pentaglycosylceramide, GalNAc beta 3 Gal alpha 3(Fuc alpha 2)Gal beta 4 Glc beta 1Cer, in which the isoglobotetrasaccharide has been combined with Fuc alpha 2 to yield an isoglobotetraosylceramide with an internal blood group B determinant. The second one was an elongated fucosyl-gangliotetraosylceramide, GalNAc beta 3(Fuc alpha 2)Gal beta 3GalNAc beta 4Gal beta 4 Glc beta 1Cer.     

Novel isoglobo-neolacto-series hybrid glycolipid detected by a monoclonal antibody is a rat colon tumor-associated antigen

Isoglobotetraosylceramide (GalNAc(beta 1-3)Gal(alpha 1-3)Gal(beta 1-4)Glc (beta 1-1)Cer), the major glycolipid species in dimethylhydrazine-induced rat tumors of colorectal origin, was not detected in epithelial cells of normal colon but was present in the non-epithelial stroma and could be extracted from each of nine tumors studied. Monoclonal antibodies produced against isoglobotetraosylceramide detected this and another novel rat tumor-associated glycolipid not present in epithelial cells nor in non-epithelial stroma of normal rat colon (Brodin, T., Thurin, J., Str?mberg, N., Karlsson, K.-A. and Sj?gren, H.O. (1985) Eur. J. Immunol. 16, 951-956). This novel glycolipid was present in 8/9 of the studied tumors and was also present in two in vitro cell clones. These were originally obtained from a W49/T4 colon tumor isograft. The novel glycolipid was characterized by mass spectrometry, 1H-NMR, and methylation analysis as a hybrid between the isoglobo- and neolacto-series, with the structure GalNAc(beta 1-3)Gal(alpha 1-3)Gal(beta 1-4)GlcNA(beta 1-3)Gal (beta 1-4)Glc(beta 1-1)Cer.     

Histochemical analysis of glycoconjugates in the domestic cat testis

The localization and characterization of oligosaccharide sequences in the cat testis was investigated using 12 lectins in combination with the beta-elimination reaction, N-Glycosidase F and sialidase digestion. Leydig cells expressed O-linked glycans with terminal alphaGalNAc (HPA reactivity) and N-glycans with terminal/internal alphaMan (Con A affinity). The basement membrane showed terminal Neu5Acalpha2,6Gal/GalNAc, Galbeta1,3GalNAc, alpha/betaGalNAc, and GlcNAc (SNA, PNA, HPA, SBA, GSA II reactivity) in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc (RCA120 staining) and alphaMan in N-linked oligosaccharides; in addition, terminal Neu5acalpha2,3Galbeta1,4GlcNac, Forssman pentasaccharide, alphaGal, alphaL-Fuc and internal GlcNAc (MAL II, DBA, GSA I-B4, UEA I, KOH-sialidase-WGA affinity) formed both O- and N-linked oligosaccharides. The Sertoli cells cytoplasm contained terminal Neu5Ac-Galbeta1,4GlcNAc, Neu5Ac-betaGalNAc as well as internal GlcNAc in O-linked glycans, alphaMan in N-linked glycoproteins and terminal Neu5Acalpha2,6Gal/ GalNAc in both O- and N-linked oligosaccharides. Spermatogonia exhibited cytoplasmic N-linked glycoproteins with alphaMan residues. The spermatocytes cytoplasm expressed terminal Neu5Acalpha2,3Galbeta1,4 GlcNAc and Galbeta1,3GalNAc in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-linked glycoconjugates. The Golgi region showed terminal Neu5Acalpha2,3Galbeta1,4GlcNac, Galbeta1,4GlcNAc, Forssman pentasaccharide, and alphaGalNAc in O-linked oligosaccharides, alphaMan and terminal betaGal in N-linked oligosaccharides. The acrosomes of Golgi-phase spermatids expressed terminal Galbeta1,3GalNAc, Galbeta1,4GlcNAc, Forssmann pentasaccharide, alpha/betaGalNAc, alphaGal and internal GlcNAc in O-linked oligosaccharides, terminal alpha/betaGalNAc, alphaGal and terminal/internal alphaMan in N-linked glycoproteins. The acrosomes of cap-phase spermatids lacked internal Forssman pentasaccharide and alphaGal, while having increased alpha/betaGalNAc. The acrosomes of elongated spermatids did not show terminal Galbeta1,3GalNAc, displayed terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-glycans and Neu5Ac-Galbeta1,3GalNAc in O-linked oligosaccharides.     

Production of monoclonal antibodies specific for ganglioside GD3.

Four kinds of anti-GD3 monoclonal antibodies, DSG-1, -2, -3, and -4, of the IgM class were obtained by the immunization of BALB/c mice with enzootic bovine leukosis tumor tissue-derived ganglioside GD3 inserted into liposomes with Salmonella minnesota R595 lipopolysaccharides. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay and by enzyme immunostaining on thin-layer chromatography. The reactivities of the monoclonal antibodies obtained to four ganglioside GD3 variants [GD3(NeuAc-NeuAc), GD3(NeuAc-NeuGc), GD3(NeuGc-NeuAc), and GD3(NeuGc-NeuGc)] were tested. All of the monoclonal antibodies were found to react with GD3(NeuAc-NeuAc) and GD3(NeuAc-NeuGc) but not with GD3(NeuGc-NeuAc) or GD3(NeuGc-NeuGc). Furthermore, various purified glycosphingolipids were used to determine the specificity of these monoclonal antibodies. All 4 antibodies reacted only with ganglioside GD3 [GD3(NeuAc-NeuAc) and GD3(NeuAc-NeuGc)], but not with several gangliosides linking the GalNAc, Gal beta 1-3GalNAc, NeuAc alpha 2-3Gal beta 1-3GalNAc, or NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-3GalNAc residue to the Gal moiety of ganglioside GD3 (GD2, GD1b, GT1b, or GQ1b, respectively), ganglioside GT1a having the same terminal NeuAc alpha 2-8NeuAc alpha 2-3Gal residue as ganglioside GD3, other gangliosides, and neutral glycosphingolipids. These findings suggest that the 4 monoclonal antibodies obtained may be specific for the epitope of NeuAc-alpha 2-8Sia alpha 2-3Gal beta 1-4Glc residue of ganglioside GD3.     

Isolation and characterization of gangliosides from Trypanosoma brucei

Gangliosides were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC) and TLC immunostaining test. Four species of gangliosides, designated as G-1, G-2, G-3, and G-4, were separated by TLC. G-1 ganglioside had the same TLC migration rate as GM3. In contrast, G-2, G-3, and G-4 gangliosides migrated a little slower than GM1, GD1a, and GD1b, respectively. To characterize the molecular species of gangliosides from T. brucei, G-1, G-2, G-3, and G-4 gangliosides were purified and analyzed by TLC immunostaining test with monoclonal antibodies against gangliosides. G-1 ganglioside showed the reactivity to the monoclonal antibody against ganglioside GM3. G-2 was recognized by the anti-GM1 monoclonal antibody. G-3 showed reaction with the monoclonal antibody to GD1a. G-4 had the reactivity to anti-GD1b monoclonal antibody. Using 4 kinds of monoclonal antibodies, we also studied the expression of GM3, GM1, GD1a, and GD1b in T. brucei parasites. GM3, GM1, GD1a, and GD1b were detected on the cell surface of T. brucei. These results suggest that G-1, G-2, G-3, and G-4 gangliosides are GM3 (NeuAc alpha2-3Gal beta1-4Glc beta1-1Cer), GM1 (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), GD1a (NeuAc alpha2-3Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), and GD1b (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-8NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), respectively, and also that they are expressed on the cell surface of T. brucei.     

alpha 2,3-Sialylation of terminal GalNAc beta 1-3Gal determinants by ST3Gal II reveals the multifunctionality of the enzyme. The resulting Neu5Ac alpha 2-3GalNAc linkage is resistant to sialidases from Newcastle disease virus and Streptococcus pneumoniae

Enzymatic alpha 2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing alpha 2,3-sialylated GalNAc residues have been reported. In the present experiments, recombinant soluble alpha 2,3-sialyltransferase ST3Gal II efficiently sialylated the X(2) pentasaccharide GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, globo-N-tetraose GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and the disaccharide GalNAc beta 1-3Gal in vitro. The purified products were identified as Neu5Ac alpha 2-3GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, Neu5Ac alpha 2-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and Neu5Ac alpha 2-3GalNAc beta 1-3Gal, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enzymatic degradations, and one- and two-dimensional NMR-spectroscopy. In particular, the presence of the Neu5Ac alpha 2-3GalNAc linkage was firmly established in all three products by a long range correlation between Neu5Ac C2 and GalNAc H3 in heteronuclear multiple bond correlation spectra. Collectively, the data describe the first successful sialyltransfer reactions to the 3-position of GalNAc in any acceptor. Previously, ST3Gal II has been shown to transfer to the Gal beta 1-3GalNAc determinant. Consequently, the present data show that the enzyme is multifunctional, and could be renamed ST3Gal(NAc) II. In contrast to ST3Gal II, ST3Gal III did not transfer to the X(2) pentasaccharide. The Neu5Ac alpha 2-3GalNAc linkage of sialyl X(2) was cleaved by sialidases from Arthrobacter ureafaciens and Clostridium perfringens, but resisted the action of sialidases from Newcastle disease virus and Streptococcus pneumoniae. Therefore, the latter two enzymes cannot be used to differentiate between Neu5Ac alpha 2-3GalNAc and Neu5Ac alpha 2-6GalNAc linkages, as has been assumed previously.     

Selective expression of different fucosylated epitopes on two distinct sets of Schistosoma mansoni cercarial O-glycans: identification of a novel core type and Lewis X structure.

The glycobiology of Schistosoma mansoni is dominated by developmentally regulated expression of various fucosylated structures, most notably the Lewis X epitope and a multifucosylated sequence, Fuc alpha1-->2Fuc alpha1-->, in its various forms. For the infective cercarial stage, Lewis X has been structurally identified on glycosphingolipids and N-glycans of total glycoprotein extracts, and a population of multifucosylated glycoproteins were found to carry a unique terminal sequence, +/-Fuc alpha1-->2Fuc alpha1-->[3GalNAc beta1-->4(Fuc alpha1-->2Fuc alpha1--> 2Fuc alpha1-->3) GlcNAc beta1-->3Gal alpha1-->](n), on their O-glycans. Using a mass spectrometry approach coupled with chromatographic separation, sequential exoglycosidase digestion, periodate oxidation, and other chemical derivatization, we demonstrate that Lewis X could also be carried on the cercarial O-glycans, but the two distinctive sets of fucosylated epitopes were conjugated to two different core structures. Lewis X, lacNAc, or single GlcNAc was found to attach directly to the -->3Gal beta1-->3GalNAc core and indirectly via another beta-Gal residue branching off from C6 of the reducing end GalNAc to give a biantennary-like structure. The -->3(+/-Gal beta1-->6)Gal beta1-->3(-->3Gal beta1-->6)GalNAc core thus characterized represents a novel core type for O-glycans. In contrast, the previously characterized multifucosylated terminal sequences were carried on conventional type 1 and 2 cores. The smallest structures of the reductively released O-glycans were defined as GalNAc beta1-->4GlcNAc beta1-->3Gal beta1-->3GalNAcitol with a total of two to four fucoses attached to the terminal lacdiNAc. alpha-Galactosylation of the nonreducing terminal beta-GalNAc instead of fucose capping leads to further elongation with another lacdiNAc unit that could also extend directly from C6 of the reducing end GalNAc and similarly elongated or terminated.     

The glycosphingolipid composition of the placenta of a blood group P fetus delivered by a blood group Pk1 woman and analysis of the anti-globoside antibodies found in maternal serum

To further define the molecules that may mediate spontaneous abortion due to maternal-fetal blood group incompatibility within the P blood group system, we have examined the fine specificities of maternal antibodies and the glycolipid antigens from the placenta of a P infant born to a Pk1 mother. Maternal antibodies obtained during therapeutic plasmapheresis were analyzed to determine their reactivities with placental glycolipid extracts on thin-layer plates. Second antibodies specific for IgM, IgG, and IgA revealed immunoglobulins of all of these classes strongly reactive with one major placental glycolipid that comigrates with globoside. GC/MS analysis confirmed that the major P-active pentaglycosylceramide of placenta has the same structure as that previously shown for the P antigen of red blood cells: GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer. Serum antibodies partially purified by affinity chromatography on globoside-octyl-Sepharose specifically recognize glycolipids that contain terminal GalNAc beta 1-3Gal . . . residues and also recognize the same sequence as an internal determinant in some, but not all, glycolipids with extended globoside core regions. Thus, in the blood group P incompatible fetus, the major P antigen present in placenta has the same carbohydrate structure as the P antigen present in fetal and adult erythrocytes and might be a target for the maternal immune system.