For chemical characterization of glycosphingolipids, it is necessary to determine the chemical compositions of three constituents, i.e., sugars, fatty acids, and sphingoids. A new rapid analytical method is described using a one-pot reaction in a household microwave oven, producing sugars, fatty acids, and especially sphingoids free of byproducts, from a single aliquot of a biological sample. Glycosphingolipids were hydrolyzed by microwave exposure with 0.1 M NaOH/CH 3 OH for 2 min followed by 1 M HCl/ CH 3 OH for 45 s. The alkaline methanolysis step produced intermediate lysoglycosphingolipids virtually free of byproducts such as the O -methyl ethers usually seen. The fatty acid methyl esters were extracted with n-hexane, and other reaction products were dried, taken up in aqueous alkaline methanol, and shaken with chloroform. Sphingoids partitioned into the organic phase under these conditions, whereas the sugar portion that partitioned into the aqueous phase was re-N -acetylated and remethanolyzed for 30 s by microwave exposure.Analysis of the profiles of glycosphingolipid constituents obtained using the microwave oven method showed that they were quantitatively and qualitatively comparable to those obtained by time-consuming conventional methods, which require reaction for several hours. Analysis of the three constituents, including analysis by gas chromatography, may be obtained within 1 day using the method described here.
A novel uronic acid-containing glycosphingolipid (UGL-1) was isolated from the ascidian Halocynthia roretzi. UGL-1 was prepared from chloroform-methanol extracts and purified by the use of successive column chromatography on DEAE-Sephadex, Florisil, and Iatrobeads. Chemical structural analysis was performed using methylation analysis, gas chromatography, gas chromatography-mass spectrometry, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry, and 1 H-NMR spectra. The chemical structure of UGL-1 was determined to be a glucuronic acid-containing glycosphingolipid, Galb1-4(Fuca1-3)GlcAb1-1Cer. The ceramide component was composed of C16:0 and C18:0 acids and C 16 -, C 17 -, and C 18 -phytosphingosines as major components. Glycosphingolipids have been characterized in a range of animal phyla, including arthropods, and have been shown to participate in important functions such as cellular development (1-6). Comparison of protostome and deuterostome glycolipids shows dramatic structural differences even between species with closely related glycolipid expression. In particular, the structures of acidic glycolipids are unique. The sialylated glycosphingolipids, called gangliosides, have been widely distributed within the Echinodermata and in deuterostomes. For example, although gangliosides have not been detected in protostomia, other acidic glycosphingolipids containing uronic acid or inositol phosphate have been characterized (7-9).Ascidians, which belong to the phylum Urochordata, are often referred to as protochordates because during the larval stage they possess chordate characteristics, most notably the tail contains a notochord and a dorsal hollow nerve cord. After a free-swimming stage, the simple tadpolelike larvae attach to a substrate and undergo metamorphosis that includes tail loss and rearrangement of the internal organs. Subsequently, in the adult form, the similarities to chordates are lost. In addition, ascidians are good model organisms for understanding vertebrates (and their development) because their cell lineage can be traced (10, 11) and a large number of genome-related data have been accumulated, including genome sequences, expressed sequence tag sequences, and gene expression patterns (12-15). However, very few studies have been directed to ascidian glycolipids, with the exception of some neutral glycolipids (16)(17)(18).In this article, we describe the structural characterization of a novel acidic glycosphingolipid containing a uronic acid residue (UGL-1) from the ascidian Halocynthia roretzi. UGL-1 exhibits unique structural features, such as Galb1-4(Fuca1-3)GlcAb1-1Cer. The chemical structure of UGL-1, which has a uronic acid with a fucosyl residue bond at the reducing end, is the first example to our knowledge. MATERIALS AND METHODS Isolation and purification of acidic glycolipidsSpecimens of H. roretzi were obtained from Hiroyo Fisheries, Ltd., at Miyagi in Japan, and the cellulose crust was removed, Abbreviations: MALDI-TOF MS, matrix-assisted laser-desorp...
A novel sulfated glycosphingolipid, SGL-1, was isolated from the ascidian Ciona intestinalis, prepared from chloroform/methanol extracts and fractionated successively on DEAE Sephadex-A25, Florisil and Iatrobeads column chromatographies. Chemical structural analysis was performed using methylation analysis, gas-liquid chromatography, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and (1)H-NMR spectroscopy. This chemical structure is presented as GlcCer I(6)-Sulfate. The ceramide moiety was specified by t16:0, t17:0, br,t17:0, t18:0 and br,t18:0 as sphingoids, and 2-hydroxy, saturated fatty acids as represented by docosanoic and tetracosanoic acids.
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