Characterization of glycosphingolipids from gastrointestinal stromal tumours.
ABSTRACT: Gastrointestinal stromal tumours (GISTs) are the major nonepithelial neoplasms of the human gastrointestinal tract with a worldwide incidence between 11 and 15 per million cases annually. In this study the acid and non-acid glycosphingolipids of three GISTs were characterized using a combination of thin-layer chromatography, chemical staining, binding of carbohydrate recognizing ligands, and mass spectrometry. In the non-acid glycosphingolipid fractions of the tumors globotetraosylceramide, neolactotetraosylceramide, and glycosphingolipids with terminal blood group A, B, H, Lex, Lea, Ley and Leb determinants were found. The relative amounts of these non-acid compounds were different in the three tumour samples. The acid glycosphingolipid fractions had sulfatide, and the gangliosides GM3, GD3, GM1, Neu5Ac?3neolactotetraosylceramide, GD1a, GT1b and GQ1b. In summary, we have characterized the glycosphingolipids of GISTs and found that the pattern differs in tumours from different individuals. This detailed characterization of glycosphingolipid composition of GISTs could contribute to recognition of new molecular targets for GIST treatment and sub-classification.
Project description:As a part of a systematic investigation of the species-specific expression of glycosphingolipids, acid and non-acid glycosphingolipids were isolated from three small intestines and one large intestine of the moose (Alces alces). The glycosphingolipids were characterized by binding of monoclonal antibodies, lectins and bacteria in chromatogram binding assays, and by mass spectrometry. The non-acid fractions were complex mixtures, and all had glycosphingolipids belonging to the lacto- and neolactoseries (lactotriaosylceramide, lactotetraosylceramide, neolactotetraosylceramide, Gal?3-Le(x) hexaosylceramide, and lacto-neolactohexaosylceramide), globo-series (globotriaosylceramide and globotetraosylceramide), and isogloboseries (isoglobotriaosylceramide). Penta- and heptaglycosylceramides with terminal Galili determinants were also characterized. Furthermore, glycosphingolipids with terminal blood group O determinants (H triaosylceramide, H type 2 pentaosylceramide, H type 1 penta- and heptaosylceramide) were characterized in two of the moose small intestines, and in the one large intestine, while the third small intestine had glycosphingolipids with terminal blood group A determinants (A tetraosylceramide, A type 1 hexa- and octaosylceramide, A dodecaosylceramide). The acid glycosphingolipid fractions of moose small and large intestine contained sulfatide, and the gangliosides GM3, GD3, GD1a, GD1b, and also NeuGc and NeuAc variants of the Sd(a) ganglioside and the sialyl-globopenta/SSEA-4 ganglioside. In humans, the NeuAc-globopenta/SSEA-4 ganglioside is a marker of embryonic and adult stem cells, and is also expressed in several human cancers. This is the first time sialyl-globopentaosylceramide/SSEA-4 has been characterized in a fully differentiated normal tissue, and also the first time NeuGc-globopentaosylceramide has been characterized.
Project description:Acinetobacter baumannii is an opportunistic bacterial pathogen associated with hospital-acquired infections, including pneumonia, meningitis, bacteremia, urinary tract infection, and wound infections. Recognition of host cell surface carbohydrates plays a crucial role in adhesion and enables microbes to colonize different host niches. Here the potential glycosphingolipid receptors of A. baumannii were examined by binding of 35S-labeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby a selective interaction with two non-acid glycosphingolipids of human and rabbit small intestine was found. The binding-active glycosphingolipids were isolated and, on the basis of mass spectrometry, identified as neolactotetraosylceramide (Gal?4GlcNAc?3Gal?4Glc?1Cer) and lactotetraosylceramide (Gal?3GlcNAc?3Gal?4Glc?1Cer). Further binding assays using reference glycosphingolipids showed that A. baumannii also bound to lactotriaosylceramide (GlcNAc?3Gal?4Glc?1Cer) demonstrating that GlcNAc was the basic element recognized. In addition, the bacteria occasionally bound to galactosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, isoglobotriaosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide, in analogy with binding patterns that previously have been described for other bacteria classified as "lactosylceramide-binding". Finally, by isolation and characterization of glycosphingolipids from human skin, the presence of neolactotetraosylceramide was demonstrated in this A. baumannii target tissue.
Project description:Bacterial adherence to mucosal surfaces is an important virulence trait of pathogenic bacteria. Adhesion of enterotoxigenic Escherichia coli (ETEC) to the intestine is mediated by a number of antigenically distinct colonization factors (CFs). One of the most common CFs is CFA/I. This has a fimbrial structure composed of a major repeating subunit, CfaB, and a single tip subunit, CfaE. The potential carbohydrate recognition by CFA/I was investigated by binding CFA/I-fimbriated bacteria and purified CFA/I fimbriae to a large number of variant glycosphingolipids separated on thin-layer chromatograms. For both fimbriated bacteria and purified fimbriae, specific interactions could be identified with a number of nonacid glycosphingolipids. These included glucosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, neolactotetraosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, the H5 type 2 pentaglycosylceramide, the Lea-5 glycosphingolipid, the Lex-5 glycosphingolipid, and the Ley-6 glycosphingolipid. These glycosphingolipids were also recognized by recombinant E. coli expressing CFA/I in the absence of tip protein CfaE, as well as by purified fimbriae from the same strain. This demonstrates that the glycosphingolipid-binding capacity of CFA/I resides in the major CfaB subunit.
Project description:Helicobacter pylori has a number of well-characterized carbohydrate-binding adhesins (BabA, SabA, and LabA) that promote adhesion to the gastric mucosa. In contrast, information on the glycoconjugates present in the human stomach remains unavailable. Here, we used MS and binding of carbohydrate-recognizing ligands to characterize the glycosphingolipids of three human stomachs from individuals with different blood group phenotypes (O(Rh-)P, A(Rh+)P, and A(Rh+)p), focusing on compounds recognized by H. pylori We observed a high degree of structural complexity, and the composition of glycosphingolipids differed among individuals with different blood groups. The type 2 chain was the dominating core chain of the complex glycosphingolipids in the human stomach, in contrast to the complex glycosphingolipids in the human small intestine, which have mainly a type 1 core. H. pylori did not bind to the O(Rh-)P stomach glycosphingolipids, whose major complex glycosphingolipids were neolactotetraosylceramide, the Lex, Lea, and H type 2 pentaosylceramides, and the Ley hexaosylceramide. Several H. pylori-binding compounds were present among the A(Rh+)P and A(Rh+)p stomach glycosphingolipids. Ligands for BabA-mediated binding of H. pylori were the Leb hexaosylceramide, the H type 1 pentaosylceramide, and the A type 1/ALeb heptaosylceramide. Additional H. pylori-binding glycosphingolipids recognized by BabA-deficient strains were lactosylceramide, lactotetraosylceramide, the x2 pentaosylceramide, and neolactohexaosylceramide. Our characterization of human gastric receptors required for H. pylori adhesion provides a basis for the development of specific compounds that inhibit the binding of this bacterium to the human gastric mucosa.
Project description:1. The lipid fraction of the plasma membrane of pig mesenteric lymph-node lymphocytes contained primarily (94%) neutral lipids and phospholipids in about equal weights. The remianing lipid comprised glycosphingolipids (1.8%), gangliosides (o.27%)and probably ceramides (1.3%). The major phospholipid was phosphatidylcholine (46% of the total), and mono- and tri-hexosylceramides accounted for 72% of the glycosphingolipids. Haematoside was distributed between the glycosphingolipid and ganglioside fractions. The major ganglioside was monosialoganglioside. About 90% of the sialic acid was N-glycollylated. 2. A comparision of the lipid composition of the plasma-membrane fraction with that of the initial lymph-node homogenate showed that the purified membrane contained increased proportions of phospholipids, especially sphingomyelin, glycosphingolipids and gangliosides. 3. Fatty acid analyses showed that the membrane phosphatidylcholine was rich in palmitic acid, that the sphingomeyelin and phosphatidylethanolamine were high in myristic acid and that the glycosphingolipids were rich in oleic acid.
Project description:The glycosphingolipids of normal human lymphocytes from individual donors were analysed by high-pressure liquid chromatography. In addition, purified T- and B-lymphocytes were examined separately. Lactosylceramide was shown to be the major neutral glycosphingolipid in human lymphocytes, and monohexosylceramide, trihexosylceramide, globoside and paragloboside were all detected in smaller amounts. Analysis of purified B- and T-cell fractions revealed that each of these populations contained a similar qualitative profile for neutral glycosphingolipids, but that quantitatively, B-cells contained several times more of each glycosphingolipid per cell than did T-cells.
Project description:Due to their pluripotency and growth capability, there are great expectations for human embryonic stem cells, both as a resource for functional studies of early human development and as a renewable source of cells for use in regenerative medicine and transplantation. However, to bring human embryonic stem cells into clinical applications, their cell surface antigen expression and its chemical structural complexity have to be defined. In the present study, total non-acid glycosphingolipid fractions were isolated from two human embryonic stem cell lines (SA121 and SA181) originating from leftover in vitro fertilized human embryos, using large amounts of starting material (1 × 10(9) cells/cell line). The total non-acid glycosphingolipid fractions were characterized by antibody and lectin binding, mass spectrometry, and proton NMR. In addition to the globo-series and type 1 core chain glycosphingolipids previously described in human embryonic stem cells, a number of type 2 core chain glycosphingolipids (neo-lactotetraosylceramide, the H type 2 pentaosylceramide, the Le(x) pentaosylceramide, and the Le(y) hexaosylceramide) were identified as well as the blood group A type 1 hexaosylceramide. Finally, the mono-, di-, and triglycosylceramides were characterized as galactosylceramide, glucosylceramide, lactosylceramide, galabiaosylceramide, globotriaosylceramide, and lactotriaosylceramide. Thus, the glycan diversity of human embryonic stem cells, including cell surface immune determinants, is more complex than previously appreciated.
Project description:In order to help determine whether alterations of the profiles of glycosphingolipids occur consistently in human tumours, the neutral glycosphingolipids and gangliosides of nine lung tumours (one adenocarcinoma, four squamous cell, two mixed adeno-squamous cell, one large cell and one oat-cell carcinomata) were analysed. The control tissue consisted of adjacent lung; it contained neutral glycosphingolipids corresponding in properties to glucosyl-, lactosyl-, globotriaosyl- and globotetraosyl-ceramides. All of the tumours also contained these four neutral glycosphingolipids. However, in addition, five of the tumours (two of the squamous, the large cell and the two mixed adeno-squamous cell carcinomata) contained neutral glycosphingolipids corresponding in properties to lactotriaosyl- and neolactotetraosyl-ceramides; these same tumours also exhibited higher amounts of lactosylceramide than the other tumours analysed. Both of the two former neutral glycosphingolipids and very substantial amounts of the latter neutral glycosphingolipid were detected in pneumonic lung and in polymorphonuclear leucocytes; it thus appears possible that these particular compounds were derived from these latter cells rather than from the tumour cells. The ganglioside patterns of the tumours were almost equivalent in complexity to that exhibited by the control lung tissue. This study shows that the profiles of two major classes of glycosphingolipids (neutral glycosphingolipids and gangliosides) occurring in lung tumours are almost as complex as those of the parent tissue, a finding in contrast with the notably simplified patterns of these lipids found in many cancer cells grown in vitro. It also suggests that when lactotriaosyl- and neolactotetraosyl-ceramides and high amounts of lactosylceramide are detected in human tumours, the possibility must be considered that these compounds are derived from polymorphonuclear leucocytes.
Project description:Adhesion of Helicobacter pylori to the gastric mucosa is a prerequisite for the pathogenesis of H. pylori related diseases. In this study, we investigated the ganglioside composition of human stomach as the target for attachment mediated by H. pylori SabA (sialic acid binding adhesin). Acid glycosphingolipids were isolated from human stomach and separated into subfractions, which were characterized by mass spectrometry and by binding of antibodies, bacteria, and Solanum tuberosum lectin. H. pylori SabA binding gangliosides were characterized as Neu5Acα3-neolactohexaosylceramide and Neu5Acα3-neolactooctaosylceramide, while the other acid human stomach glycosphingolipids characterized (sulfatide and the gangliosides GM3, GD3, GM1, Neu5Acα3-neolactotetraosylceramide, GD1a and GD1b) were not recognized by the bacteria. Defining H. pylori binding glycosphingolipids of the human gastric mucosa will be useful to specifically target this microbe-host interaction for therapeutic intervention.
Project description:Glycosphingolipids are a diverse family of biologically important glycolipids. In addition to variations on the lipid component, more than 300 glycosphingolipid glycans have been characterized. These glycans are directly involved in various molecular recognition events. Several naturally occurring sialic acid forms have been found in sialic acid-containing glycosphingolipids, namely gangliosides. However, ganglioside glycans containing less common sialic acid forms are currently not available. Herein, highly effective one-pot multienzyme (OPME) systems are used in sequential for high-yield and cost-effective production of glycosphingolipid glycans, including those containing different sialic acid forms such as N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid (Kdn), and 8-O-methyl-N-acetylneuraminic acid (Neu5Ac8OMe). A library of 64 structurally distinct glycosphingolipid glycans belonging to ganglio-series, lacto-/neolacto-series, and globo-/isoglobo-series glycosphingolipid glycans is constructed. These glycans are essential standards and invaluable probes for bioassays and biomedical studies.