Structural Elucidation and Monokine-inducing Activity of Two Biologically Active Zwitterionic Glycosphingolipids Derived from the Porcine Parasitic Nematode Ascaris suum

Abstract

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...

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