Isolation and characterization of a novel uronic acid-containing acidic glycosphingolipid from the ascidian Halocynthia roretzi

Abstract

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...

Characterization of a Novel Rhamnose-containing Acidic Glycosphingolipid from the Ascidian <i>Haloc</i><i>ynthia aurantium</i>

“…Our immunostaining results, sugar composition analysis, and methylation studies confirmed the sugar chain structure of H. aurantium UGL-1 to be identical to that of H. roretzi UGL-1 20 . Given that rhamnose is a 6-deoxymannose, protons H1/H2 of rhamnose are in the gauche conformation, despite an anomeric configuration, showing small 1-3-Hz coupling constants.…”

“…Therefore, we conclude that the sugar chain structure of UGL-2 is Rha1-3GlcNAc1-3Gal1-4 Fuc1-3 GlcA. In our previous analysis on UGL-1 in the related ascidian species H. roretzi 20 , signals for GlcAβ and Galβ were determined from 2D-NMR data obtained from its structural analog, salcaceramide Galβ1-4 Fucα1-3 Glcβ1-Cer , from the ascidian Microcosmus sulcatus 32 . As a result, a signal at the high-field side was determined as GlcAβ and a signal at the downfield side was determined as Galβ in H. roretzi UGL-1.…”

“…We also investigated acidic GSLs in the ascidian H. roretzi and characterized a novel UGL Galβ1-4 Fucα1-3 GlcAβ1-1Cer; UGL-1 within this species 20 . USLs were characterized from bivalves as GlcA4Meβ1-4 GalNAc3Meα1-3 Fucα1-4GlcNAcβ1-2Manα1-3 Xylβ1-2 Manβ1-4Glcβ1-Cer , from the fly Calliphora vicina as GlcAβ1-3Galβ1-3GalNAcβ1-4GlcNAcβ1-3Manβ1-4Glcβ1-Cer, and from the fly Lucilia caesar as GlcAβ1-3Galβ1-3GalNAcα1-4GalNAcβ1-4GlcNAcβ1-3Manβ1-4Glcβ1-Cer 4 .…”

“…Rabbits were immunized with UGL-1 purified from H. roretzi 20 ; the resulting polyclonal antiserum was utilized as an anti-UGL-1 antibody. TLC immunostaining was performed in accordance with the method reported by Itonori et al 28 .…”

“…The remaining materials were dehydrated using acetone, dried, and pulverized in a blender. Acidic GSLs were isolated and purified as described previously 20 . In brief, they were extracted twice with chloroform/methanol C/M 2:1 v/v , extracted once with C/M 1:1, and evaporated to dryness.…”