The isolated neutral glycosphingolipid fraction from the pig parasitic nematode, Ascaris suum, was fractionated by silica gel chromatography to yield a neutral and a zwitterionic glycosphingolipid fraction, the latter of which mainly contained two zwitterionic glycosphingolipids termed components A and C. Preliminary chemical characterization with hydrofluoric acid treatment and immunochemical characterization with a phosphocholine-specific monoclonal antibody indicated that both components contained phosphodiester substitutions: phosphocholine for component A, and phosphocholine and phosphoethanolamine for component C. Both components were biologically active in inducing human peripheral blood mononuclear cells to release the inflammatory monokines tumor necrosis factor ␣, interleukin 1, and interleukin 6. Component A was the more bioactive molecule, and its biological activity was abolished on removal of the phosphocholine substituent by hydrofluoric acid. The glycosphingolipid components were structurally analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, liquid secondary ion mass spectrometry, methylation analysis, 1 H NMR spectroscopy, exoglycosidase cleavage, and ceramide analysis. Their chemical structures were elucidated to be (see Structure I below), The carbohydrate moiety oligosaccharide core was characterized as belonging to the arthro series of protostomial glycosphingolipids. The ceramide moiety was distinguished by (R)-2-hydroxytetracosanoic acid as the dominant fatty acid species and by the C17 iso-branched sphingosine and sphinganine bases, 15-methylhexadecasphing-4-enine and 15-methylhexadecasphinganine, respectively.Analyses of the immunoreactivity between neutral fraction glycolipids derived from various species of parasitic nematodes have indicated a high degree of serological cross-reactivity (1, 2). The structural basis for this immunological cross-reactivity between parasitic nematodes at the level of glycolipids is at present unknown. Structural studies on the neutral fraction glycosphingolipids from adults of the porcine parasitic nematode Ascaris suum have revealed that the identified arthro series oligosaccharide chain was not immunogenic, i.e. did not exhibit immunoreactivity toward infection sera from A. suuminfected mice (2, 3). However, a zwitterionic glycosphingolipid fraction was also isolated from A. suum that demonstrated a phosphodiester sidechain as a structural modification of possibly phosphocholine (PC) 1 and phosphoethanolamine (PE). In addition, these zwitterionic glycolipids were immunogenic/antigenic, i.e. exhibited immunoreactivity toward infection sera from A. suum-infected mice (2).PC-containing macromolecules have been regularly detected * This project was supported by the Deutsche Forschungsgemeinschaft (SFB 535 and Graduiertenkolleg Molecular Biology and Pharmacology) and by the Bundesministerium fü r Bildung, Wissenschaft, Forschung und Technologie Grant 01 KI 9471. The costs of publication of this article were defrayed in...
The oligosaccharide structures of glycolipids from cercariae of the human blood fluke, Schistosoma mansoni, were analyzed in the form of their corresponding, pyridylaminated oligosaccharides by methylation analysis, partial hydrolysis, exoglycosidase treatment, on-target exoglyco-sidase cleavage and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The six, dominant chemical structures present have been determined as: GalNAc(beta1-4)Glc1-ceramide; GlcNAc(beta1-3)Gal-NAc(beta1-4)Glc1-ceramide; Gal(beta1-4)GlcNAc(beta1-3)Gal-NAc(beta1-4)Glc1-ceramide; Gal(beta1-4)[Fuc(alpha1-3)]Glc-NAc(beta1-3)Gal-NAc(beta1-4)Glc1-++ +ceram ide (Lewis X pentasaccharide structure); Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Glc-NAc(beta1-3)GalNAc(beta 1-4)Glc1-ceramide (Lewis X hexa-saccharide structure); and, Fuc(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)GalNAc(beta1-4 )Glc1-ceramide (pseudo-Lewis Y hexasaccharide structure). These structures belong to the characterized schisto-series of protostomial glycosphingolipids. The Lewis X and pseudo-Lewis Y glyco-lipids are stage-specifically expressed by the cercarial life-cycle stage, and not by the adult or egg.
The aim of the present study was the characterization of the dominant epitope present on Schistosoma mansoni glycolipids, which causes cross-reactivity of S. mansoni and S. haematobium infection sera with keyhole-limpet haemocyanin (KLH). To this end, the monoclonal antibody M2D3H was chosen for its similar behaviour in high-performance TLC immunostaining and inhibition-ELISA to infection sera. Individual, structurally defined oligosaccharides derived from S. mansoni egg glycolipids were tested for their binding to this monoclonal antibody by immunoaffinity chromatography. A terminal fucose residue linked in the (alpha1-->3) position to N-acetylgalactosamine was found to be the common structural determinant of the four oligosaccharides binding to M2D3H. The Fuc(alpha1-->3)GalNAc-motif also appeared to be the basis for the cross-reactivity with KLH, a phenomenon used in the serodiagnosis of S. mansoni, S. haematobium and S. japonicum infections.
Caenorhabditis elegans displays three neutral glycosphingolipids with structural homology to glycosphingolipids from the porcine nematode parasite, Ascaris suum. The present findings extend the degree of structural conservation between the two nematode species to glycosphingolipids with a phosphodiester substitution. Using a combination of hydrofluoric acid pretreatment, immunochemical characterization and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, three zwitterionic, phosphorylcholine-substituted glycosphingolipids could be identified in the neutral glycolipid fraction of C. elegans. The components were isolated as their zwitterionic, phosphorylcholine-substituted, pyridylaminated oligosaccharides by HPLC. Structural analysis was performed using hydrofluoric acid treatment, partial acid hydrolysis, methylation analysis, gas chromatography±mass spectrometry, cleavage with exoglycosidases and matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. Their chemical structures are proposed as: component Nz1,The oligosaccharide core is characteristic of the biosynthetic arthro-carbohydrate series of protostomial glycosphingolipids. The ceramide moiety was specified by a d17 : 1 sphingoid-base with iso-branching and anteiso-branching, and 2-hydroxy, saturated fatty acids as represented by docosanoic and tetracosanoic acids. Analysis of the spatial and temporal expression of the phosphorylcholine epitope, during embryonic and postembryonic development, showed it to be localized predominantly in seam cells and basement membranes, respectively. In early embryonic ontogenesis the phosphorylcholine epitope was only lipid bound, while in late embryonic and postembryonic development this epitope was both lipid bound and protein bound.Keywords: Caenorhabditis elegans; developmental expression; glycolipid; phosphorylcholine; structural analysis. The logical conclusion from the frequency of the immunological detection of phosphorylcholine (PCho) among parasitic nematodes [1] is that the distribution of this epitope is widespread, if not ubiquitous, within the Nematoda. In fact, the frequent cross-reactivity observed for parasitic nematode antigens is thought to be due to the presence of the PChoantigenic determinant [2]. Its mode of attachment depends on the nature of the acceptor molecule. When the acceptor molecule is a glycoprotein, the PCho may be bound to both N-linked and O-linked glycans [3,4]. The precise structures of these PCho-substituted oligosaccharides have not been elucidated. For an excretory±secretory glycoprotein from Acanthocheilonema viteae it has been demonstrated that the respective carbohydrate moieties represent truncated N-glycans comprising a partially fucosylated, trimannosyl core substituted with between one and four N-acetylglucosamine residues [5]. When glycolipid, the PCho-moiety was found to be C6-linked to the third monosaccharide (N-acetylglucosamine) of a ceramidebound, pentasaccharide chain [6]. The biological significance of such prote...
The presence of glycosphingolipids in the pupae of the blowfly, Calliphora vicina, was established. The thin layer chromatographic pattern of the total neutral glycolipids revealed the presence of more than 13 components, the major one being ceramide monohexoside. By the use of high performance liquid chromatography, the three simplest components were isolated and their chemical structures determined: Glc(b1-1)Cer, Man(B1-4)-Glc(B1-1)Cer [with minor component Gal(fl1-4)Glc(fl1-1)Cerl and GlcNAc(b1-3)Man(~1-4)Glc(/?I-1)-Cer.The ceramide composition of the parent insect glycosphingolipids is dominated by the 20 : 0 fatty acid, arachidic acid, and the sphingoid tetradecasphing-4-enine.The sphingolipids, in general, are to be regarded as plasma membrane components situated at the outer monolayer [I]. The sphingolipids can be structurally divided into the phosphosphingolipids and glycosphingolipids, and together with cholesterol and phosphatidylcholine they comprise the major lipids of the outer monolayer leaflet [2]. The major phosphosphingolipid in vertebrates is ceramide phosphocholine, as well as in the honey bee, Apis mellfera [3]. In contrast, the diptera are characterized by a paucity of choline-containing phospholipids, apparently replaced by ceramide phosphoethanolamine and phosphatidylethanolamine [4, 51. The ceramide moiety from C. erythrocephala (= C. vicina) phosphosphingolipid contains as the major fatty acids : stearic (18:0), arachidic (20:0), and behenic (22:O) acid [4]; whilst the dipteran long-chain bases are tetradecasphing-4-enine and hexadecasphing-4-enine [5, 61. Prior to a comparison of possible parallelism of insect plasma-membrane-located glycolipids with their counterparts in the vertebrata, in the belief that they are involved in the same or similar cell-surface phenomena (recognition, growth, cell cycle control and expression of transformation [7]), it is necessary to isolate and identify the comparable components.Therefore, a systematic structural analysis was begun of the glycolipids of C. vicina pupae and the three simplest neutral fraction glycosphingolipids are described.Abbreviations. GbOse3Cer, globotriaosylceramide or Gal(ctl-4)-Gal(j1-4)Glc(j1-1)Cer; GgOse4Cer, gangliotetraosylceramide or Gal(j1-3)GalNAc(j1-4)Gal(j1-4)Glc(~I -1
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