1-3Gal Alpha Research Articles

Lewis X-containing glycans are specific and potent competitive inhibitors of the binding of ZP3 to complementary sites on capacitated, acrosome-intact mouse sperm.

Mammalian fertilization requires a cascade of interactions between sperm and the egg's zona pellucida (ZP). O-linked glycans on mouse glycoprotein ZP3 have been implicated in mediating one step of the fertilization process, the firm adhesion of acrosome-intact sperm to the ZP. Experiments to identify structural requirements of a sperm-binding glycan have demonstrated that a Lewis X (Le(x))-containing glycan (Gal beta 4[Fuc alpha 3]GlcNAc-R) was a potent, competitive inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273: 1888-1895). However, those experiments did not define the particular step in the fertilization pathway that was blocked. The experiments described herein test the hypothesis that Le(x)-containing glycans are specific, competitive inhibitors of the binding of Alexa Fluor 568 fluorochrome (Alexa(568))-labeled ZP3 to sperm and, thus, bind the same sperm surface sites as ZP3. Dose-response analyses demonstrated that these glycans are potent inhibitors (IC(50) approximately 180 nM), which at saturation, reduced Alexa(568)-ZP3 binding by approximately 70%. A Lewis A (Le(a))-capped glycan (Gal beta 3[Fuc alpha 4]GlcNAc) was also a potent inhibitor (IC(50) approximately 150-200 nM), but at saturation, it reduced Alexa(568)-ZP3 binding by only 30%. In contrast, nonfucosylated glycans with nonreducing GlcNAc beta 4 or Gal beta 4 residues did not compete; neither did sialyl-Le(x) (Neu5Ac alpha 3Gal beta 4[Fuc alpha 3]GlcNAc-Lewis X) nor sulfo-Le(x) (3'-O-SO(3)-Lewis X). However, at saturation, Gal alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc reduced Alexa(568)-ZP3 binding by approximately 70% but with moderate apparent affinity (IC(50) approximately 3000 nM). Fluorescence microscopy revealed that Alexa(568)-labeled Le(x)-Lac-BSA, Le(a)-Lac-BSA, and ZP3 bound to the same sperm surface domains. However, Le(a)-Lac did not inhibit binding of Alexa(568)-Le(x)-Lac-BSA, and Le(x)-Lac did not inhibit binding of Alexa(568)-Le(a)-Lac-BSA. Finally, Le(x)-Lac and Le(a)-Lac had an additive inhibitory effect on Alexa(568)-ZP3 binding. Thus, Le(x) is a ligand for a major class of ZP3 binding sites on mouse sperm, whereas Le(a) binding defines a different but less-abundant class of sites.

Studies on gangliosides with affinity for Helicobacter pylori: binding to natural and chemically modified structures

Helicobacter pylori, like many other microbes, has the ability to bind to carbohydrate epitopes. Several sugar sequences have been reported as active for the bacterium, including some neutral, sulfated, and sialylated structures. We investigated structural requirements for the sialic acid-dependent binding using a number of natural and chemically modified gangliosides. We have chosen for derivatization studies two kinds of binding-active glycolipids, the simple ganglioside S-3PG (Neu5Ac alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, sialylparagloboside) and branched polyglycosylceramides (PGCs) of human origin. The modifications included oxidation of the sialic acid glycerol chain, reduction of the carboxyl group, amidation of the carboxyl group, and lactonization. Binding experiments confirmed a preference of H. pylori for 3-linked sialic acid and penultimate 4-linked galactose. As expected, neolacto gangliosides (with Gal beta 4GlcNAc in the core structure) were active in our assays, whereas gangliosides with lacto (Gal beta 3GlcNAc) and ganglio (Gal beta 3GalNAc) carbohydrate chains were not. Negative binding results were also obtained for disialylparagloboside (with terminal NeuAc alpha 8NeuAc) and NeuAc alpha 6-containing glycolipids. Chemical studies revealed dependence of the binding on Neu5Ac and its glycerol and carboxyl side chains. Most of the derivatizations performed on these groups abolished the binding; however, some of the amide forms turned out to be active, and one of them (octadecylamide) was found to be an excellent binder. The combined data from molecular dynamics simulations indicate that the binding-active configuration of the terminal disaccharide of S-3PG is with the sialic acid in the anticlinal conformation, whereas in branched PGCs the same structural element most likely assumes the synclinal presentation.

Suppression of beta 1,3galactosyltransferase beta 3Gal-T5 in cancer cells reduces sialyl-Lewis a and enhances poly N-acetyllactosamines and sialyl-Lewis x on O-glycans.

We investigated the role of beta 3 Gal-T5, a member of the beta 1,3galactosyltransferase (beta 1,3Gal-T) family, in cancer-associated glycosylation, focusing on the expression of sialyl-Lewis a (sLea, the epitope of CA19.9 antigen), poly N-acetyllactosamines, and sialyl-Lewis x (sLex) antigen. A clone permanently expressing an antisense fragment of beta 3Gal-T5 was obtained from the human pancreas adenocarcinoma cell line BxPC3 and characterized. Both beta 1,3Gal-T activity and sLea expression are dramatically impaired in the clone. Analysis of the oligosaccharides synthesized in cells metabolically labelled with tritiated galactose shows that a relevant amount of radioactivity is associated to large O-glycans. Endo-beta-galactosidase mostly releases NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal and NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal from such O-glycans of BxPC3 membranes, but GlcNAc beta 1-3Gal and type 2 chain oligosaccharides, including NeuAc alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-3Gal, from those of the antisense clone. Furthermore, BxPC3 cells secrete sLea in the culture media but not sLex, while antisense clone secretes mostly sLex, and accumulation of both antigens is prevented by benzyl-alpha-GalNAc. These data indicate that beta 3Gal-T5 suppression turns synthesis of type 1 chain O-glycans to poly N-acetyllactosamine elongation and termination by sLex. In other cell lines and clones, beta 3Gal-T5 transcript, beta 1,3Gal-T activity, and sLea antigen are also correlated, but quantitatively the relative expression ratios are very different from cell type to cell type. We suggest that beta 3Gal-T5 plays a relevant role in gastrointestinal and pancreatic tissues counteracting the glycosylation pattern associated to malignancy, and is necessary for the synthesis and secretion of CA19.9 antigen, whose expression still depends on multiple interacting factors.

α2,3-Sialylation of Terminal GalNAcβ1–3Gal Determinants by ST3Gal II Reveals the Multifunctionality of the Enzyme: THE RESULTING Neu5Acα2–3GalNAc 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.

Blocking of human anti-pig xenoantibodies by soluble GAL alpha 1-3Gal and Gal alpha 1-2Gal disaccharides; studies in a pig kidney in vitro perfusion model.

Depletion of anti-pig xenoantibodies reduces cell cytotoxicity of human serum to pig endothelial cells and lymphocytes. The aim of this study was to test, in a pig kidney xenoperfusion model, the ability of soluble alpha Gal terminated disaccharides to prevent the hyperacute rejection process in an organ. Porcine kidneys were perfused with whole human blood lacking saccharide and blood supplemented with Gal alpha 1-3GAL, Gal alpha 1-2Gal and lactose. Parameters evaluated were, urine production, renal blood flow, vascular resistance, renal clearance, blood cell counts, xenoantibody titers, complement activation and histopathology. The blood flow was higher in the Gal alpha 1-3Gal (155 +/- 31 ml/min x 100 g-1 kidney tissue) group compared to Gal alpha 1-2Gal (138 +/- 16), lactose (92 +/- 78) and controls (69 +/- 16), When calculated as percent of the blood flow value at 1 min, the blood flow at 30 min was 157% for the Gal alpha 1-3Gal and for 187% the Gal alpha 1-2Gal. The corresponding values for the lactose and control groups were 102% and 74%, respectively. Urine production in the lactose/control groups was lower (0.7 ml/min x 100 g-1 kidney tissue) compared to Gal alpha 1-3Gal (3.0) and Gal alpha 1-2Gal (3.7). Urine sodium excretion was reduced in the lactose/control groups, compared to the Gal alpha alpha 1-groups during the perfusions. An increase in urine potassium excretion was found in the Gal alpha alpha 1-groups while a reduction occurred in the lactose/control experiments. An initial 40-50% reduction in platelet count was observed in all groups while the leukocyte count showed a continuous decrease. Immunohistochemistry revealed less deposition of IgM, IgG, C3 and C1 q in the Gal alpha alpha 1-saccharide groups compared to the lactose/control groups. Soluble Gal alpha a1-disaccharides improved both functional and histological parameters. However, significant pathological changes were still present indicating that this approach to inhibit HAR must be used in combination with additional therapeutic approaches such as solid phase xenoantibody immunoadsorption and blocking of complement activation.

Characterization of the sulfated fucose-containing trisaccharides by fast atom bombardment tandem mass spectrometry in the study of the acrosome reaction-inducing substance of the starfish, Asterias amurensis.

Fast atom bombardment mass spectrometry (FABMS) and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) were applied to the investigation of the anomeric isomerism of synthetic trisaccharides consisting of xylose, galactose and sulfated fucose {Xyl1-->3Gal alpha 1-->3(4-OSO3Na)Fuc} and {Xyl1-->3Gal alpha 1-->4(3-OSO3Na)Fuc} and the linkage position of the sulfate group. It was possible to differentiate between various glycosidic linkages in several synthetic trisaccharides. The position of a sulfate group in synthetic methyl O-sulfo-alpha-L-fucopyranoside isomers was elucidated from the fragmentation patterns. Comparing the data from the synthetic sulfated trisaccharides with the spectra from the natural compound derived from glycan chains of the acrosome reaction-inducing substance (ARIS) from starfish, the anomeric structure and the position of the sulfate group in the natural sample were determined without ambiguity as Xyl beta 1-->3Gal alpha 1-->3(4-OSO3-)Fuc, in agreement with the result from an independent study based on nuclear magnetic resonance.

Establishment of a monoclonal antibody directed against Gb3Cer/CD77: a useful immunochemical reagent for a differentiation marker in Burkitt's lymphoma and germinal centre B cells.

A new monoclonal antibody (TU-1) directed against the Gal alpha 1-4Gal beta 1-4Glc residue of the Gb3Cer/CD77 antigen was prepared by the hybridoma technique following immunization of mice with an emulsion composed of monophosphoryl lipid A, trehalose dimycolate, and Gb3Cer isolated from porcine erythrocytes. TU-1 showed reactivity towards Gb3Cer and lyso-Gb3Cer(Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Sph), although the reactivity towards lyso-Gb3Cer was about 10-fold lower than that to Gb3Cer. But it did not react with other structurally-related glycolipids, such as LacCer (Gal beta 1-4Glc beta 1-1'Cer), Gg3Cer, Gg4Cer, Gb4Cer (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer), galactosylparagloboside (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer), sulfatide (HSO3-3Gal beta 1-1'Cer), other gangliosides (GM3, GM2, GM1a, GD1a and GT1b), or P1 antigen (Gal alpha 1-4Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer) among neutral glycolipids prepared from P1 phenotype red blood cells. Furthermore, TU-1 reacted with viable lymphoma cells, such as human Burkitt lymphoma cell line, Daudi, and Epstein-Barr virus (EBV)-transformed B cells by the immunofluorescence method, and also with germinal centre B cells in human tonsil and vessel endothelial cells in human thymus histochemically. These results indicate that TU-1 is a monoclonal antibody directed against Gb3Cer/CD77 antigen and can be utilized as a diagnostic reagent for Burkitt's lymphoma and also for detection of the blood group Pk antigen in glycolipid extracts of erythrocytes.

Extracellular matrix upregulates synthesis of glucosylceramide-based glycosphingolipids in primary Schwann cells.

The formation of basement membrane around Schwann cells that are in contact with axons is necessary for Schwann cell differentiation and myelin formation in the peripheral nervous system. However, primary Schwann cells grown on basement membrane in the absence of neuronal influence show increased proliferation rather than differentiation, which implies that the signals generated by Schwann cell-basement membrane interactions are multipotential. We examined the effect of matrigel, an exogenous basement membrane preparation, and other extracellular matrix growth surfaces on primary Schwann cells to determine if the resulting interactions play a role in the control of glycosphingolipid synthesis. Isolated primary Schwann cells grown on a thin layer of matrigel rapidly adhered to the surface and exhibited a greater degree of cell spreading when compared to cells grown on the nonspecific substrate polylysine. Labeling of the cells with [3H]galactose between 24 and 48 hr after plating revealed that the incorporation of [3H]galactose into glucosylceramide-based glycosphingolipids increased from 1.5-3-fold on matrigel in comparison to cells grown on polylysine. The major labeled glycolipids under both conditions were GM3 ganglioside and two neutral glycolipids that comigrated with GbOse4Cer (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-1Cer) and GbOse5Cer (GalNAc alpha 1-3Gal-NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer) standards. There was little or no increase in the incorporation of [3H]leucine, [3H]galactose, or [3H]glucosamine into proteins or [3H]palmitic acid into phospholipids, free ceramides, or sphingomyelin, suggesting that the matrigel-induced increase in the synthesis of the glycolipids was selective. In the absence of serum, there was little or no difference in the levels of glycolipid labeling between cells grown on the two substrata, demonstrating that serum factors were required for matrigel to have this effect. When cells were grown on surfaces coated with individual extracellular matrix components, those cells grown on laminin and collagen IV showed an increase in glycolipid labeling similar to that produced by matrigel, while labeling increased to a lesser degree for the other components tested. Thus, the signals generated by interactions between Schwann cells and basement membrane, particularly the laminin and collagen IV constituents, contribute to the regulation of glycolipid synthesis which in turn may affect cell morphology and proliferation.

Molecular mimicry in the recognition of glycosphingolipids by Gal alpha 3 Gal beta 4 GlcNAc beta-binding Clostridium difficile toxin A, human natural anti alpha-galactosyl IgG and the monoclonal antibody Gal-13: characterization of a binding-active human glycosphingolipid, non-identical with the animal receptor.

Glycoconjugates with terminal Gal alpha 3Gal beta 4GlcNAc beta sequences have been shown to be recognized by three carbohydrate-binding proteins; toxin A of Clostridium difficile, human natural anti alpha-galactosyl IgG and the monoclonal antibody Gal-13. However, the biological significance of this binding specificity in humans is unclear, since unsubstituted Gal alpha 3Gal beta 4GlcNAc beta sequences are not found in human tissues, due to suppression of the gene coding for the enzyme Gal beta 3-transferase. To explore this inconsistency, the binding of toxin A, human natural anti alpha-galactosyl IgG, and the Gal-13 monoclonal antibody to various glycosphingolipids was examined using the thin-layer chromatogram binding assay. The binding to Gal alpha 3Gal beta 4GlcNAc beta-terminated glycosphingolipids of rabbit erythrocytes was confirmed. A minor binding-active compound was also detected in the non-acid glycosphingolipid fraction of human erythrocytes. This glycosphingolipid was isolated and characterized by EI mass spectrometry, gas chromatography-EI mass spectrometry after degradation, and proton NMR spectroscopy, as GalNAc beta 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, corresponding to the x2 glycosphingolipid isolated before form this source. Two additional binding-active glycosphingolipids were found. One was GalNAc alpha 3Gal beta 4GlcNAc beta 3Gal beta 4 Glc beta 1 Cer, produced from blood group A-active GalNAc alpha 3 (Fuc alpha 2) Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1 Cer by acid-induced defucosylation. The other was GlcNAc beta 3 Gal beta 4 GlcNAc beta 3 Gal beta 4 Glc beta 1 Cer, generated from NeuGc alpha 3Gal beta 4GlcNAc beta 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer by enzymatic hydrolysis. A number of other glycosphingolipid sequences, including the human Le(x), Le(y), and I blood group determinants, suggested to act as receptors for toxin A, were not recognized by the three ligands. Despite the different terminal substituents and anomerity of the binding-active glycosphingolipids, calculated minimum energy conformations demonstrated topographical similarities in the spatial orientation of the terminal trisaccharides, possibly accounting for the cross-reactivity.

Chemical and immunological characterization of galactosyl-beta 1-3-globoside in bovine, human, and rat brain.

Our studies of bovine brain neutral glycosphingolipids (Ngsls) have revealed the presence of several short-chain (containing -CHO 1-4) and previously uncharacterized long-chain (-CHO > 4-5) Ngsls. We reported the structural characterization of brain GgOse4Cer (GA1) and have now purified another brain Ngsl to homogeneity. The purified Ngsl migrated close to standard GgOse4Cer and nLcOse5Cer on a TLC plate employing two different solvent systems. The carbohydrate molar composition indicated the presence of Gal/Glc/GalNAc in a ratio of 2.8:1.0:0.9. Five permethylated alditol acetate peaks were characterized as 2,3,4,6-OMe4Gal, 2,4,6-OMe3Gal, 2,3,6-OMe3Gal, 2,3,6-OMe3Glc, and 4,6-OMe2GalNAcMe by gas chromatography-mass spectrometry. The anomeric carbohydrate sequence has been determined by specific exoglycosidase digestion. Six-hundred megahertz 1H NMR spectroscopy of the oligosaccharide released by ceramide glycanase hydrolysis confirmed the structure of the Ngsl as Gal beta 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer or IV3GalGbOse4Cer. Using the immunooverlay technique with anti-stage-specific embryonic antigen 3 antibody, it was found in bovine, rat, and normal adult human brain and bovine myelin, but not in human or rat myelin.

Galactosylgloboside expression in seminoma. Inverse correlation with metastatic potential

Altered glycosylation is a common phenotype expressed in essentially all types of human cancer and has been found to be correlated closely with the invasive and metastatic properties of a given tumor. Because there was no prognostic information concerning aberrant glycosylation of seminoma, the authors studied this topic. Glycosphingolipid (GSL) composition of orchiectomy samples of seminoma were analyzed systematically. GSL patterns from seminoma samples of the following three groups were compared after a 44-month postoperative period: Stage I disease with no evidence of metastasis during the 44-month postoperative period, Stage I with metastatic relapse during this period, and Stage II with retroperitoneal lymph node metastasis. Unknown GSLs detected were analyzed chemically by 1H-nuclear magnetic resonance spectroscopy and mass spectrometry. All nonmetastatic seminomas (n = 12) contained a GSL band that was identified as galactosylgloboside (Gb5; Gal beta 1-->3GalNAc beta 1-->3Gal alpha 1-->4 Gal beta 1-->4Glc beta 1-->1Cer). All metastatic seminomas (n = 5) lacked this GSL, although the sample sizes were admittedly small. Only the presence or absence of galactosylgloboside (Gb5), but of no other GSL or gangliosides, clearly correlated with metastatic potential in patients with seminoma. This observation is useful in the estimation of prognosis of patients with seminoma, especially those with Stage I disease.

High Affinity Binding of the Entamoeba histolytica Lectin to Polyvalent N-Acetylgalactosaminides

Entamoeba histolytica trophozoites initiate pathogenic colonization by adherence to host glycoconjugates via an amebic surface lectin which binds to galactose (Gal) and N-acetylgalactosamine (GalNAc) residues. Monovalent and multivalent carbohydrate ligands were screened for inhibition of E. histolytica lectin-mediated human red cell hemagglutination, revealing that: (i) the synthetic multivalent neoglycoprotein GalNAc39BSA (having an average of 39 GalNAc residues linked to bovine serum albumin) was 140,000-fold more potent an inhibitor than monovalent GalNAc and 500,000-fold more potent than monovalent Gal; and (ii) small synthetic multivalent ligands which bind with high affinity to the mammalian hepatic Gal/GalNAc lectin do not bind with high affinity to the E. histolytica lectin. Radioligand binding studies revealed saturable binding of 125I-GalNAc39BSA to E. histolytica membranes (KD = 10 +/- 3 nM, Bmax = 0.9 +/- 0.08 pmol/mg membrane protein). Maximal binding required the presence of calcium chloride (300 microM) or sodium chloride (50 mM), and had a broad pH maximum (pH 6-9). GalNAc39BSA was 200,000-fold more potent than monovalent GalNAc in blocking radio-ligand binding. Among synthetic saccharide-derivatized linear polymers, the GalNAc beta and GalNAc alpha 3Gal beta derivatives were the most potent, with GalNAc alpha and GalNAc alpha 3(Fuc alpha 2)Gal beta derivatives much weaker. The data support a model in which a unique pattern of spaced multiple GalNAc residues are the highest affinity targets for the E. histolytica lectin.

Enhanced Binding of Enterotoxigenic Escherichia coli K99 to Amide Derivatives of the Receptor Ganglioside NeuGc-GM3

A natural receptor in pig small intestine [Teneberg, S., Willemsen, P., de Graaf, F. K., & Karlsson, K.-A. (1990) FEBS Lett. 263, 10-14] for the enterotoxigenic bacteria Escherichia coli K99 is the ganglioside NeuGc-GM3 (NeuGc alpha 3Gal beta 4Glc beta Cer) [e.g., H. Smit, W. Gaastra, J. P. Kamerling, J. F. G. Vliegenthart, & F. K. de Graaf (1984) Infect. Immun. 46, 578-584]. Chemical modifications of the carboxyl group of this ganglioside were performed, giving five different amides, the methyl ester, and the primary alcohol. The products were purified, and their structures were investigated by negative FAB mass spectrometry. Binding of E. coli K99 was tested by incubating 35S-labeled bacteria with derivatized compounds separated on thin-layer chromatograms. Modification of the carboxyl group to a primary amide strengthened the binding at least 5-fold, as estimated from autoradiography of dilutions on thin-layer plates. Some strengthening of the binding was also obtained with the methylamide as well as with the carboxyl group reduced to the alcohol. The ethylamide bound equally well as the underivatized NeuGc-GM3. Amide substituents as large as propyl amide and benzyl amide were still recognized by the bacteria, although they bound weaker. The methyl ester was not stable in the chromatogram-binding assay with silica gel and water present, and it reverted to the acid.

Effect of nerve growth factor and forskolin on glycosyltransferase activities and expression of a globo-series glycosphingolipid in PC12D pheochromocytoma cells.

The glycosphingolipid (GSL) composition of cells changes dramatically during cellular differentiation. Nerve growth factor (NGF) or forskolin (FRK) are known to induce cellular differentiation including process formation in PC12 pheochromocytoma cells. In this respect, we present the NGF/FRK-dependent regulation of glycosyltransferase activities and the corresponding GSL expression in PC12D cells. After treatment of PC12D cells with NGF or FRK, the cell processes, including varicoses and growth cones, became strongly immunoreactive with an antibody against a unique globo-series neutral GSL, Gal alpha 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer (GalGb3), and the activity of GalGb3-synthase increased significantly. Other glycosyltransferase activities, including GM1 containing blood group B determinant (BGM1)-, GM3-, GD1a-, and GM2-synthases, also increased significantly upon NGF treatment, but the immunoreactivity against BGM1 did not show any appreciable change. For the parent PC12 cells, NGF/FRK treatment significantly increased the percentage of anti-GalGb3 positive cells and induced some immunoreactive cell processes. Because the parent PC12 cells do not express appreciable amounts of GalGb3, and because PC12D cells are considered to be more differentiated than the parent PC12 cells, the expression of GalGb3 and the increase of GalGb3-synthase activity may be closely related to the cellular differentiation process in this cell line.

Characterization of the specificity of binding of Moluccella laevis lectin to glycosphingolipids.

The specificity of Moluccella laevis lectin was investigated by analysing its binding to glycosphingolipids separated on thin-layer chromatograms or adsorbed on microtitre wells. The binding activity of the lectin was highest for glycosphingolipids with terminal alpha-linked N-acetylgalactosamine, both in linear structures, as the Forssman glycosphingolipid, GalNAc alpha 3GalNAc beta 3Gal alpha 4Glc beta 1Cer, and in branched structures, as glycosphingolipids with the blood group A determinant, GalNAc alpha 3(Fuc alpha 2)Gal beta. In addition, the lectin bound, though considerably more weakly, to linear glycosphingolipids with terminal alpha-linked galactose. When considering the use of the M. laevis lectin for biochemical and medical purposes this cross-reactivity may be of importance.

Isolation and Characterization of a Rat Testis Glycosphingolipid Based on Gangliotetraosylceramide and Having a Blood Group B Determinant Extended with β3-Linked N-Acetylgalactosamine1

Monoclonal antibody 14.2, directed against the GalNAc beta 3Gal alpha 3Gal beta epitope, was used to monitor the isolation of a novel glycosphingolipid from rat testes. Structural analysis was performed by mass spectrometry, proton NMR spectroscopy, gas chromatography-mass spectrometry after degradation, and exoglycosidase digestion. It was concluded that the structure was [formula: see text] This is the first report on an extended blood group B determinant.

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

The carbohydrate-binding specificity of Pseudomonas 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 Gal alpha 3Gal- or Gal alpha 4Gal-structures.

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: Gal beta 1-4GlcCer(LacCer), Gal alpha 1-4GalCer(Ga2Cer), Gal alpha 1-4Gal beta 1-4GlcCer(Gb3Cer), GlcNAc beta 1-3Gal beta 1-4GlcCer(Lc3Cer), GalNAc beta 1-4Gal beta 1-4GlcCer(Gg3Cer), GalNAc beta 1-4[GalNAc beta 1-3] Gal beta 1-4GlcCer(LcGg4Cer), and GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer (Forssman glycolipid) as well as glucosylceramide, galactosylceramide (Nishimura K et al. 1984) Biochim Biophys Acta 796:269-76) and the LeX glycolipid, III3 Fuc alpha-nLc4Cer (Nishimura K et 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 LeX), those rich in the ceramides of N-acyl trihydroxysphinganine with normal fatty acids (Lc3Cer), and glycolipids rich in the ceramides of N-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.

Stage-specific glycosphingolipids from amastigote forms of Leishmania (L.) amazonensis. Immunogenicity and role in parasite binding and invasion of macrophages

Neutral glycosphingolipids (GSLs) from amastigote forms of Leishmania (L.) amazonensis were isolated, and their structures and biological properties were characterized. Based on various immunochemical methods, these GSLs were shown to be expressed at certain stages of amastigote development. GSLs were extracted and purified from amastigotes of hamster foot lesions by established procedures. Three mouse monoclonal antibodies (MoAbs) specific for carbohydrate epitopes of these GSLs were established, and their inhibition of parasite binding and macrophage invasion was analyzed. MoAb ST-3 inhibited 80% of macrophage invasion by amastigotes and 60% of that by promastigotes. Since GSLs reacting with MoAb ST-3 were found in amastigotes but not in promastigotes, ST-3 reactivity with promastigotes presumably depends on an epitope present on an unidentified promastigote glycoconjugate. MoAbs ST-4 and ST-5 inhibited 60-80% of macrophage invasion by amastigotes but were not effective in preventing macrophage invasion by promastigotes. Fab fragments of ST-3 inhibited invasion of cultured mouse macrophages by amastigotes (80%) or promastigotes (60%). The GSL with the simplest structure recognized by these MoAbs was isolated and characterized (by negative ion fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry of the permethylated compound, degradation with exoglycosidases, and 1H NMR) as the novel globoseries structure Gal beta 1-->3Gal alpha 1-->4Gal beta 1-->4Glc beta 1-->Cer, which has beta 1-->3Gal in place of the beta 1-->3GalNAc of globoside. The ceramide contains a 16:0 fatty acid and d18:1 sphingosine as the long chain base. The MoAbs also reacted with a series of GSLs from amastigote forms of L. amazonensis, with longer carbohydrate chains, probably containing identical end groups Gal beta 1-->3Gal alpha 1-->R. Expression of surface GSLs may render amastigote forms more effective than promastigotes in binding and invading host macrophages, thus enhancing the infectious process.

Glycosphingolipid backbone conformation and behavior in cholesterol-containing phospholipid bilayers.

2H NMR spectroscopy was used to consider correspondence between existing single-crystal X-ray data for glycosphingolipids and their ceramide backbone conformation in fluid phospholipid membranes. A monoglycosylated sphingolipid, glucosylceramide (GlcCer), which represents the core structure of many important glycosphingolipids, was derived by partial synthesis through replacement of all native fatty acids with the 18-carbon species, stearic acid, deuterated at C2. N-[2,2-2H2]stearoyl-GlcCer was used to probe glycosphingolipid orientation and motion at low concentration in "fluid" phospholipid bilayers composed of dimyristoylphosphatidylcholine (DMPC), with and without physiological amounts of cholesterol. Spectral analysis, aided by stereoselective monodeuteration of the GlcCer fatty acid at C2, demonstrated that glycosphingolipid average acyl chain backbone conformation in fluid phospholipid membranes, with or without cholesterol, is likely closely related to that predicted from single crystal X-ray studies [Pascher, I. (1976) Biochim. Biophys. Acta 455, 433-451; Pascher, I., & Sundell, S. (1977) Chem. Phys. Lipids 20, 175-191]. To test the generality of this observation, specific comparisons were made involving galactosylceramide (GalCer) and globoside. GalCer provided a glycolipid differing only in monosaccharide stereochemistry (galactose vs glucose). Globoside permitted isolation of the effect of headgroup size, since it is derived from GlcCer via extension of the carbohydrate portion by the oligosaccharide, GalNAc beta 1-->3Gal alpha 1-->4Gal attached in beta 1-->4 linkage to the Glc residue.(ABSTRACT TRUNCATED AT 250 WORDS)