Characterization of a Novel Rhamnose-Containing Acidic

Characterization of a Novel Rhamnose-containing Acidic Glycosphingolipid from the Ascidian Halocynthia aurantium Hisao Kojima1,† , Ryota Shinohara1, 2,† , Saki Itonori3 and Masahiro Ito1,＊ 1 Department of Bioinformatics, College of Life Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, JAPAN 2 Division of Pharmacology, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, JAPAN 3 Department of Chemistry, Faculty of Liberal Arts and Education, Shiga University, 2-5-1 Hiratsu, Otsu, Shiga 520-0862, JAPAN † These authors contributed equally to this work

Abstract: Halocynthia aurantium, an edible ascidian species belonging to Urochordata, was subjected to structural characterization of acidic glycosphingolipids to investigate these molecules in ascidians: sulfatide from Ciona intestinalis and the glucuronic acid-containing acidic glycosphingolipid from H. roretzi. Acidic glycosphingolipids containing three or five sugars were isolated from soft parts of the ascidian H. aurantium by chloroform–methanol extraction, mild-alkaline hydrolysis, precipitation with cold acetone, and subsequent column chromatography using a DEAE-Sephadex A-25 column, a Florisil column, and an Iatrobead column. The structures of these glycosphingolipids were determined by methylation studies, sugar analysis, fatty acid analysis, sphingoid analysis, mass spectrometry, and proton nuclear magnetic resonance spectroscopy. A novel glucuronic acid-containing glycosphingolipid having a rhamnose residue was identified as Rhaα1-3GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcAβ1-Cer (UGL-2). This novel structure is particularly unusual given that it contains both a rhamnose residue and a reducing terminal glucuronic acid residue within a single molecule. Rhamnose is a characteristic sugar, which is a component of cell wall pectin in plants and exopolysaccharides in bacteria. Ascidians acquired the cellulose synthase gene via lateral gene transfer, and therefore, it can be speculated that they also acquired the rhamnosyltransferase gene in the same manner. We also detected Galβ1-4(Fucα1-3)GlcAβ1-Cer (UGL-1), which was already identified in another ascidian, H. roretzi.

Key words: acidic glycosphingolipid, glucuronic acid, rhamnose, structural characterization, ascidian

1 INTRODUCTION fact, dominant acidic GSLs in flies（Arthropoda）belong to Glycosphingolipids（GSLs）are expressed on the outer the glucuronic acid-containing type; the ones in Nematoda surface of the plasma membrane, forming a microdomain （genus Ascaris）belong to both the inositol phosphate and and playing a role in several types of signal transduction1）. sulfatide types; the ones in a brackish water lugworm in GSLs comprise a sugar chain and an aliphatic portion com- Annelida belong to the inositol phosphate types4）; and the posed of an amide-bonded fatty acid and a sphingoid. They are largely divided into two groups: neutral and acidic Abbreviations: GSL, glycosphingolipid; GC, gas–liquid types. Acidic GSLs are grouped into four types: sialic acid- chromatography; MALDI-TOF MS, matrix-assisted laser containing GSLs（gangliosides）, sulfuric-substituted GSLs desorption/ionization time-of-flight mass spectrometry; 1 （sulfatides）, inositol phosphate-containing GSLs, and H-NMR, proton nuclear magnetic resonance; UGL, uronic acid-containing glycosphingolipid; TLC, thin-layer uronic acid-containing GSLs（UGLs）. chromatography; HPTLC, high-performance thin-layer In phylogenetic analyses of acidic GSLs, gangliosides chromatography; LPS, lipopolysaccharide; Rha, rhamnose; have been found to be the dominant type in vertebrates Man, mannose; Glc, glucose; GlcA, glucuronic acid; GlcN, and echinodermata, but only a few reports on the existence glucosamine; GlcNAc, N-acetylglucosamine; Fuc, fucose; Gal, of gangliosides in protostomes have been published2, 3）. In galactose; Xyl, xylose; Cer, ceramide

＊Correspondence to: Masahiro Ito, Department of Bioinformatics, College of Life Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, JAPAN E-mail: [email protected] Accepted September 29, 2016 (received for review July 28, 2016) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs

285 H. Kojima, R. Shinohara and S. Itonori et al.

ones in the bivalves Hyriopsis schlegelii5） and Meretrix Therefore, sulfated GSLs in C. intestinalis are rare in lusoria6） belong to the methylated glucuronic acid type. In terms of sulfate group-substituted galactose at the C6 posi- the bivalve H. schlegelii, a methylated glucuronic acid- tion. We also investigated acidic GSLs in the ascidian H. type acidic GSL was shown to be present on the surface of roretzi and characterized a novel UGL［Galβ1-4（Fucα1-3） the sperm, indicating its involvement in fertilization7）. GlcAβ1-1Cer; UGL-1］within this species20）. USLs were Rhamnose is a type of methyl-pentose corresponding to characterized from bivalves as GlcA4Meβ1-4（GalNAc- deoxy-mannose. It is widely distributed as a component of 3Meα1-3）Fucα1-4GlcNAcβ1-2Manα1-3（Xylβ1-2）Manβ1- bacterial exopolysaccharides8）. Plants exhibit rhamnose- 4Glcβ1-Cer）, from the fly Calliphora vicina as GlcAβ1- containing pectins composed of a repeated galacturonic 3Galβ1-3GalNAcβ1-4GlcNAcβ1-3Manβ1-4Glcβ1-Cer, and acid-rhamnose（Rha）backbone or a poly-galactose back- from the fly Lucilia caesar as GlcAβ1-3Galβ1-3GalNAcα1- bone attached to Rha, forming a matrix surrounding cellu- 4GalNAcβ1-4GlcNAcβ1-3Manβ1-4Glcβ1-Cer4）. UGLs other lose microfibrils with hemicellulose and proteins on the than H. roretzi contain a glucuronic acid residue at the primary cell wall9）. An inositol phosphate-containing rham- non-reducing terminus, whereas the H. roretzi UGL con- nolipid was identified in the protozoan Leptomonas tains one at the reducing terminus with an attached samueli: Xylβ1-4Xylβ1-4（Rhaα1-3）Manα1-2Manα1-3 branching fucose. Hence, the H. roretzi UGL is also （aminoethylphosphate-6）Manα1-3Manα1-4（aminoeth- unusual in terms of position on the sugar chain and pres- 10） ylphosphate-6）GlcNα1-6Ins（1/2）-PO4-Cer ; moreover, a ence of a branching fucose. In both ascidians, we could not neutral rhamno-GSL was identified in the marine sponge detect any ganglioside despite our expectations. We further Agelas clathrodes: Rhaα1-3GalNAcβ1-6Galα1-2Galα1- conducted genome-wide analyses by phylogenetic profiling Cer11）. These are the only published reports on rhamno- of glycosyltransferases to confirm that ascidians had actu- GSLs. ally lost sialyltransferases21）. Given that genome sequences Ascidians are largely divided into three orders on the were analyzed only for the Ciona species（C. intestinalis basis of their branchial sac in adults: Aplousobranchia, and C. savignyi）, we could not investigate other species. Phlebobranchia, and Stolidobranchia. Their habitats are We confirmed that these ascidians belonging to the genus widely distributed in the sea from the equator to the Arctic Ciona had lost majority sialyltransferases. Based on these and Antarctic12）. Over 3000 ascidian species have been results, we investigated acidic GSLs in the ascidian H. au- identified. Ascidians commonly possess a notochord in rantium for the presence of gangliosides. their larval stage13）, and are therefore classified as chor- In the present study, we extracted and characterized dates, close to vertebrates. Ascidians belong to Urochorda- acidic GSLs from H. aurantium; we found UGL-1 and a ta; therefore, they are closer to mammals than lancelets novel rhamnose-containing UGL, which were speculated to （Cephalochordata）, as determined by comparative genome be elongated via UGL-1. The presence of a rhamnose-con- analyses14）. Notably, in animals, urochordates, including taining GSL in H. aurantium is interesting, given that few Ascidians, have been proven to produce cellulose15）. The examples of such GSLs have been reported10, 11）. gene that enables cellulose production was identified in as- cidians16） and shown to have been acquired from bacteria via lateral gene transfer17）. We conducted a structural characterization of acidic 2 EXPERIMENTAL PROCEDURES GSLs with the expectation of discovering gangliosides con- 2.1 Isolation of acidic GSLs sidering that deuterostomes such as echinoderms and ver- Fifty individuals（wet weight 4.5 kg）of the ascidian H. tebrates contain gangliosides. Among acidic GSLs in Uro- aurantium, purchased from the company Nishikiya Saito

chordata, we reported a sulfated GSL（HSO3-6GlcCer）in （2-19-5 Akaiwa, Otaru, Japan）, were boiled and the encyst- the ascidian Ciona intestinalis18）. The most well-known ment was removed. The remaining materials were dehy- sulfated GSL is sulfatide, a sulfate group-substituted galac- drated using acetone, dried, and pulverized in a blender.

tose at the C3 position（HSO3-3Galβ1-Cer）. Other sulfated Acidic GSLs were isolated and purified as described previ- GSLs are common in sulfate group-substituted sugar resi- ously20）. In brief, they were extracted twice with chloro- dues at the C3 position, except for types of sulfate group- form/methanol（C/M）2:1（v/v）, extracted once with C/M 1:1, substituted N-acetyl/glycolyl neuramic acid at the C8 posi- and evaporated to dryness. To remove glycerolipids, the tion19）. In protostomes, sulfated GSLs have been residues were treated with mild-alkaline hydrolysis and characterized as sulfatides in the nematode Ascaris suum, then neutralized, dialyzed, and evaporated to dryness. The as the sulfate group-substituted N-acetyl/glycolyl neuramic resulting material was suspended in cold acetone and cen- acid type at the C8 position in Echinodermata, as the trifuged, and the precipitate was recovered as crude sphin- sulfate group -substituted N-acetyl neuramic acid type at golipids. Acidic GSLs were separated using a DEAE-Sepha-

the C4 position in sea cucumber, and as HSO3-3Galβ1- dex A-25 column with methanolic 0.05, 0.15, and 0.45 M 4Galβ1-4Glcβ1-Cer in the starfish Luidia maculate4）. ammonium acetate, evaporated to dryness, acetylated, and

286 J. Oleo Sci. 66, (3) 285-295 (2017) An acidic rhamno-glycosphingolipid from the ascidian H. aurantium

then separated using a Florisil column. The resulting frac- extracted with chloroform and analyzed by GC. tions eluted by dichloroethane（DCE）/methanol 1:1 and 3:1 were deacetylated, neutralized, concentrated, and dialyzed. 2.6 Acid hydrolysis The residue was evaporated to dryness and again chroma- Acid hydrolysis was performed as follows: purified UGL tographed on an Iatrobead column, using a gradient system was hydrolyzed in 2 M HCl. The hydrolysate was washed of C/M/W 70:30:3（v/v/v）to 40:60:6. Eluates were collected, with n-hexane to remove fatty acids and deacidified by N2 analyzed by thin-layer chromatography（TLC）, and sepa- flow with toluene. After alkalization with 0.01 M NaOH, the rated into 11 fractions. Fraction 10 was further chromato- hydrolysate was reduced with 1％ NaBH4 in 0.01 M NaOH, graphed in the Iatrobead column, using an isocratic elution neutralized by acetic acid, and subsequently dried by N2 of C/M/W 60:40:10, and separated into six fractions: frac- flow with MeOH. The resulting material was acetylated by tions 10-1 to 10-6. pyridine/acetic anhydride（1:1, v/v）in boiling water. The acetylated material was mixed with chloroform, washed 2.2 TLC three times with water, concentrated to dryness, and then A silicagel 60 plate was developed in a solvent mixture of subjected to GC analysis. For defucosylation, purified GSL

C/M/W 60:40:10 and visualized using the orcinol–H2SO4 was hydrolyzed with 0.1 M HCl and partitioned with C/M reagent22） to detect sugar and by Dittmer-Lester reagent23）（2:1, v/v）. The resulting lower phase was evaporated to to detect phosphate groups. dryness and then subjected to GC.

2.3 Sugar composition and fatty acid analyses 2.7 Reduction of uronic acid Analyses of sugar composition and fatty acids were per- The reduction of uronic acid was conducted by following formed as described previously24）. In brief, acidic GSLs a slightly modified version of the method reported by were methanolyzed with anhydrous methanolic HCl using a Tayler et al.27）. In brief, 1 mL of a 2％ 1-ethyl-3-（3-dimeth- microwave oven. The resulting fatty acid methylester was ylaminopropyl）carbodiimide solution and a few drops of extracted using n-hexane, and the residual methanolic 0.01 M HCl to adjust the pH to 4.7–5.0 were added to the phase was neutralized with silver carbonate and evaporated purified UGL（1 mg）, and the resulting mixture was incu- to dryness under a N2 stream after the removal of silver bated for 2 h at room temperature. After alkalization（pH chloride and subsequently N-acetylated. The resulting ma- 8）with 1 M NH4OH, approximately 10 mg of NaBH4 and a terial was dried, trimethylsilylated, and analyzed by gas– few drops of BuOH as an antifoaming agent were mixed liquid chromatography（GC）. Sugar composition analysis by and stirred at 55℃ for 18 h. Reduction was stopped by the alditol acetate method was conducted omitting the adding a few drops of acetic acid, and subsequently the methylation step in the sugar linkage analysis described reduced GSL was dialyzed, evaporated to dryness, and below. subjected to various analyses.

2.4 Sphingoid analysis 2.8 GC analysis Sphingoid analysis was performed as described previ- GC analysis was performed using a Shimadzu GC-18A ously25）. In brief, purified UGL was hydrolyzed with an and a Shimadzu Hi Cap-CBP5 column（0.22 mm×25 m） aqueous HCl–methanol reagent at 70℃ for 18 h. The hy- under the following conditions: 140℃–230℃ with heating drolysate was washed with n-hexane to remove fatty acids, at 2℃/min for sugar composition analysis, 140℃–230℃ alkalized with a methanol-NaOH solution, extracted with with heating at 4℃/min for the methylation study, 210℃ chloroform, dried, trimethylsilylated, and analyzed by GC. –230℃ with heating at 2℃/min for sphingoid analysis, and 170℃–240℃ with heating at 4℃/min for fatty acid analy- 2.5 Sugar linkage analysis sis. Sugar linkage analysis by the partially methylated alditol acetate method was performed as described previously26）. 2.9 Mass spectrometry In brief, purified UGL was dissolved in dimethylsulfoxide The molecular weight of purified GSL was measured by sonication. GSL was immediately permethylated by with a Voyager DE-STR（Applied Biosystems）in negative- adding sodium hydroxide（NaOH）and methyl iodide and ion mode with 2,5-dihydroxybenzoic acid（Wako Pure acetolyzed with acetic acid–HCl–water（16:1:3, v/v/v）using Chemicals）as a matrix reagent. a microwave oven24）. The acetolysate was reduced by incu- bation with a 0.01 M NaOH solution containing 2％ sodium 2.10 1H-NMR borohydride at room temperature overnight. Reduced and Purified GSL was solubilized in 2％ D2O/DMSO and ana- partially methylated alditols were acetylated by pyridine lyzed using JEOL JNM-ALPHA400 at 60℃. The measured followed by acetic anhydride and subsequently incubated spectrum was integrated 512 times for UGL-1 or 2048 in boiling water. Partially methylated alditol acetates were times for UGL-2.

287 J. Oleo Sci. 66, (3) 285-295 (2017) H. Kojima, R. Shinohara and S. Itonori et al.

2.11 TLC immunostaining Rabbits were immunized with UGL-1 purified from H. roretzi20）; 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）. In brief, semi-purified acidic glycosphingolipid fractions were spotted onto a TLC plate（Polygram Sil G; Macherey-Nagel GmbH, Germany）and developed with C/ M/W 60:40:10（v/v/v）. After development, the plate was sprayed with phosphate-buffered saline（pH 7.2）and blocked by Blocking One（Nacalai Tesque, Inc.）. The anti- bodies used were an antiserum specific for UGL-1 from H. roretzi used as a primary antibody and a peroxidase-con- jugated goat anti-rabbit IgG antibody employed as a sec- ondary antibody. Visualization was performed by spraying immunostaining HRP-1000（Konica Minolta Co. Ltd.）.

3 RESULTS 3.1 Isolation of acidic GSLs Fig. 1 Immunostaining using an anti-UGL-1 antibody. In total, 3.3 g of the alkaline-stable fraction was subject- Purified UGL-1 from H. aurantium（lane 1）, ed to DEAE-Sephadex column chromatography. The eluate antigen UGL-1 from H. roretzi（lane 2）, and total resulting from a treatment with methanolic 0.05 M ammo- acidic GSLs from H. aurantium（lane T）were nium acetate, which was positive on immunostaining with spotted onto a Polygram SilG plate and developed anti-UGL-1 antibody, was subjected to Florisil column with C/M/W（60:40:10, v/v/v）. After cutting, each chromatography to remove phosphosphingolipids, given plate was visualized using the orcinol–H2SO4 that the eluate was positive for both the orcinol–H2SO4 reagent（A）or immunostaining（B）. reagent and the Dittmer-Lester reagent. The acidic GSL fraction（64.4 mg）was again subjected to chromatography 3.3 Sugar linkage analysis using an Iatrobead column and separated into 11 fractions Following the partially methylated alditol acetate （Supplemental Fig. 1）. Immunostaining using an anti- method, non-reduced UGL-1 yielded only two peaks UGL-1 antiserum revealed that fraction 9（8.5 mg）was showing a non-reducing terminus［1,5-di-O-methyl-2,3,4- positive（Fig. 1）; therefore, it was subjected to GC analyses. tri-O-acetylfucitol（1Fuc）and 1,5-di-O-methyl-2,3,4,6-tetra- Fraction 10 comprising two bands was subjected to further O-acetylgalactitol（1Gal）］（ data not shown）. Hence, UGL-1 chromatography analysis using an Iatrobead column and was reduced and subjected to methylation analysis. As a separated into six fractions. In fraction 10-5, the lower result, the peak of 1,3,4,5-tetra-O-methyl-2,6-di-O-acetyl- band component was eluted（＜1 mg）. We refer to the gluctitol（1,3,4Glc）（ peak c）was newly detected in addition eluates in fractions 9 and 10-6 as UGL-1 and UGL-2, re- to the peaks of 1Fuc（peak a）and 1Gal（peak b）（ Fig. 4A）. spectively. To determine the position of the glycosidic substitution of GlcA with a Gal residue, reduced UGL-1 was de-fucosylated 3.2 Sugar composition analysis by hydrolysis with 2 M HCl and subjected to GC analysis. H. aurantium acidic GSLs were analyzed as methylgly- The resulting gas chromatogram showed the presence of coside–TMS derivatives（Fig. 2）. For UGL-1, reduced 1,4,5-tri-O-methyl-2,3,6-tetra-O-acetylgluctitol（1,4Glc） UGL-1 was subjected to analysis, and one mole of Fuc, one （peak d）（ Fig. 4B）. Therefore, we concluded that the mole of Gal, and one mole of Glc were detected. However, branching Fuc residue attaches to the 3-position of the unknown peaks were detected, besides the peaks of Gal GlcA residue to form a UGL-1 sugar chain, Gal1-4（Fuc1-3） and GlcNAc in non-reduced UGL-2. Therefore, sugar com- GlcA. With regard to UGL-2（Fig. 4C）, our methylation position analysis using the alditol acetate method was con- study revealed the presence of 1,5-di-O-methyl-2,3,4-tri-O- ducted to identify such unknown peaks of UGL-2（Fig. 3）. acetylrhamnitol（1Rha）（ peak e）, 1Fuc（peak a）, 1,3,5-tri- One unknown peak was identical in terms of retention time O-methyl-2,4,6-tetra-O-acetylgalactitol（1,3Gal）（ peak f）, to an acetylated alditol peak from standard Rha; therefore, and 1,3,5-tri-O-methyl-4,6-di-O-acetylglucosaminitol UGL-2 was determined to contain Rha in its sugar chain. （1,3GlcNAc）（ peak g）.

288 J. Oleo Sci. 66, (3) 285-295 (2017) An acidic rhamno-glycosphingolipid from the ascidian H. aurantium

Fig. 3 Sugar composition analysis of UGL-2 by the alditol acetate method.（A）Gas chromatogram of reduced UGL-2 showed five peaks: one unknown alditol acetate peak（x）was detected in addition to the hexa-acetyl-derivatized peaks from Fuc（a）, Glc （b）, Gal（c）, and GlcNAc（d）（. B）Gas chromatogram of hexa-acetyl-derivatized Rha（peak e）.

droxyheptadeca（t17:0）-sphinganine.

Fig. 2 Sugar composition analysis by methylglycoside– 3.5 Mass spectrometry TMS derivatization. Gas chromatograms of reduced In combination with the components identified by GC UGL-1（A）and non-reduced UGL-2（B）. Peaks of analysis, the putative UGL structure was confirmed by Fuc（a）, Gal（b）, and Glc（c）were detected in negative-mode MALDI-TOF MS analysis, as shown in Fig. 5. － reduced UGL-1. Although peaks of Gal（b）and Peaks were assigned as［M-H］ ions with different combi- GlcNAc（d）were detected in non-reduced UGL-2, nations of aliphatic components; specifically, peak（A）a at unknown peaks（x）were detected close to the m/z 1083.9（t18:0-h18:0, t17:0-h19:0, t16:0-h20:0）, peak retention time of Fuc. （A）b at m/z 1098.6（t18:0-h19:0, t17:0-h20:0）, peak（A）c at m/z 1111.0（t18:0-C21:0, t17:0-C22:0, t16:0-C23:0）, peak 3.4 Ceramide composition （A）d at m/z 1124.5（t18:0-C22:0, t17:0-C23:0, t16:0-C24:0）, The aliphatic components of fatty acids and sphingoid in peak（A）e at m/z 1138.9（t18:0-C23:0, t17:0-C24:0）, and purified UGLs are given in Table 1. Fatty acids were mainly peak（A）f at m/z 1153.0（t18:0-C24:0）for UGL-1 and peak normal saturated acids ranging from C14 to C24. Among （B）a at m/z 1430.8（t18:0-C19:0, t17:0-C20:0, t16:0-C21:0）, them, the C16 and C18 acids were predominant in both peak（B）b at m/z 1447.3（t18:0-h19:0, t17:0-h20:0）, peak UGL-1 and UGL-2. Odd-numbered saturated acids ranging （B）c at m/z 1461.6（t18:0-h20:0, t16:0-h22:0）, peak（B）d at from C15 to C23 were also detected at low levels except m/z 1474.2（t18:0-C22:0, t16:0-C24:0）, peak（B）e at m/z for C19 and C21. The monoenoic acids of C16 and C18 1487.3（t17:0-C24:0）, and peak（B）f at m/z 1503.7（t18:0- were present in both UGLs. Approximately 10％ 2-hydroxy h23:0）for UGL-2. acids were contained as minor components in both UGLs. The sphingoids of UGL-1 mainly comprised 4-hydroxyocta- 3.6 Anomeric con gurations decasphinganine（phytosphingosine）（ t18:0）, together with Anomeric configurations of the sugar residues in UGLs smaller amounts of 4-hydroxyhexadeca（t16:0）- and 4-hy- were determined using a 1H-NMR spectrometer（Fig. 6,

289 J. Oleo Sci. 66, (3) 285-295 (2017) H. Kojima, R. Shinohara and S. Itonori et al.

Table 1 Ceramide composition of the acidic GSLs purified from H. aurantium.

Fatty acid (%) UGL-1 UGL-2 C14:0 3.2 1.4 C15:0 2.5 1.6 C16:1 2.7 3.9 C16:0 20.7 22.2 C17:0 2.5 3 C18:1 6.1 11.2 C18:0 14.5 21.3 C19:0 9.4 6 C20:0 10.4 8.7 C21:0 9.7 6.9 C22:0 3.6 3.3 C23:0 0.9 － C24:0 2.1 1.6 2h18:0 2.8 1 2h19:0 2.7 1.1 2h20:0 2.6 1.8 2h22:0 2.7 3.4 2h23:0 0.9 1.6 Sphingoid (%) UGL-1 t16:0 20.9 t17:0 11.1 t18:0 68 2 h, 2-hydroxy; t, trihydroxy sphingoid; －, not detected.

Table 2）. Configurations were assigned with reference to data obtained on Fuc and GlcNAc29）, GlcA30）, and Rha31） in terms of chemical shifts and coupling constants. In the anomeric region of the spectrum for each UGL, the following

anomeric proton resonances were observed: 4.34 ppm（J1,2

＝6.8 Hz）and 4.24 ppm（J1,2＝7.3 Hz）for β-GlcA and β-Gal,

and 5.19 ppm（J1,2＝2.9 Hz）for α-Fuc（Fig. 6A, UGL-1）; 4.35

ppm（J1,2＝7.3 Hz）and 4.31 ppm（J1,2＝7.3 Hz）for β-GlcA

and β-Gal, 5.20 ppm（J1,2＝3.9 Hz）for α-Fuc, 4.74 ppm（J1,2

＝7.3 Hz）for β-GlcNAc, and 4.78 ppm（J1,2＝1.5 Hz）for α-Rha（Fig. 6B, UGL-2）.

4 DISCUSSION Our immunostaining results, sugar composition analysis, Fig. 4 Sugar linkage analysis of reduced UGL-1（A）, de- and methylation studies confirmed the sugar chain struc- fucosylated reduced UGL-1（B）, and non-reduced ture of H. aurantium UGL-1 to be identical to that of H. UGL-2（C）. a: 1Fuc, b: 1Gal, c: 1,3,4Glc, d: 1,4Glc, roretzi UGL-120）. Given that rhamnose is a 6-deoxyman- e: 1Rha, f: 1,3Gal, and g: 1,3GlcNAc. nose, protons H1/H2 of rhamnose are in the gauche confor- mation, despite an anomeric configuration, showing small

290 J. Oleo Sci. 66, (3) 285-295 (2017) An acidic rhamno-glycosphingolipid from the ascidian H. aurantium

Fig. 5 MALDI -TOF MS analyses of non-reduced UGL-1（A）and non-reduced UGL-2（B）. Predicted molecular species of their aliphatic components are mentioned in the text.

1–3-Hz coupling constants. Hence, we could not determine bic bacteria Serratia marcescens31）, chemical shift of the anomeric configuration of rhamnose in H. aurantium α-Rha in the Serratia polysaccharide resembled that of UGL-2 on the basis of its coupling constant. However, the signal that was speculated as rhamnose in H. auran- signals at around 5 ppm generally seem to represent an tium UGL-2. Therefore, we speculated that rhamnose in α-anomer. All rhamnoses identified to date as components UGL-2 is also an α-anomer. Both UGL-2 and UGL-1 were of GSLs have been shown to have α-anomeric configura- eluted in an identical fraction using methanolic 0.05 M am- tions10, 11）. A disaccharide residue Rhaα1-3GlcNAcβ, found monium acetate on a DEAE-Sephadex A-25 column. UGL-2 at the non-reducing terminus of H. aurantium UGL-2, has was speculated to be catabolized via UGL-1 as a precursor. been found as a repeating unit in a polysaccharide part of Therefore, we conclude that the sugar chain structure of lipopolysaccharides（LPS）from the Gram-negative anaero- UGL-2 is Rha1-3GlcNAc1-3Gal1-4（Fuc1-3）GlcA. In our

291 J. Oleo Sci. 66, (3) 285-295 (2017) H. Kojima, R. Shinohara and S. Itonori et al.

1 Fig. 6 Anomeric proton regions of H-NMR spectra of separated UGLs.（A）UGL-1;（B）UGL-2; I: GlcAβ and Galβ; II: Fucα; III: GlcNAcβ; IV: Rhaα; *: FucH5.

Table 2 C hemical shifts（ppm）and J1,2 coupling constants（Hz）of the protons of the isolated UGLs in the anomeric regions.

Gal1-4 (Fuc1-3) GlcA1 UGL-1 I II I Chemical shifts 4.34 (6.8) 4.24 (7.3) 5.19 (2.9) (Coupling constants) 4.24 (7.3) 4.34 (6.8) Rha1-3 GlcNAc1-3 Gal1-4 (Fuc1-3) GlcA1 UGL-2 IV III I II I Chemical shifts 4.35 (7.3) 4.31 (7.3) 4.78 (1.5) 4.74 (7.3) 5.20 (3.9) (Coupling constants) 4.31 (7.3) 4.35 (7.3) previous analysis on UGL-1 in the related ascidian species proximately 4.2–4.4 ppm for GlcAβ and Galβ. To precisely H. roretzi20）, signals for GlcAβ and Galβ were determined assign these signals, detailed 2D-NMR analysis is required. from 2D-NMR data obtained from its structural analog, sal- In the present study, we identified UGL-2 as a novel caceramide［Galβ1-4（Fucα1-3）Glcβ1-Cer］, from the ascid- rhamnose-containing acidic GSL（Fig. 7）. An inositol phos- ian Microcosmus sulcatus32）. As a result, a signal at the phate-containing rhamnolipid has been reported from the high-field side was determined as GlcAβ and a signal at the protozoan L. samueli10）, and a neutral rhamno-GSL has downfield side was determined as Galβ in H. roretzi been reported from the marine sponge A. clathrodes11）. UGL-1. However, signals that shifted downfield were iden- There are only two reports about rhamno-GSLs. It is inter- tified upon comparison between UGL-1 and UGL-2 in the esting that such an unusual rhamno-GSL was found in H. present study. Concerning the downfield shift of the glyco- aurantium. Rhamnose is known to be a constituent of cell syl substitution, the non-reducing terminal Gal in UGL-1 wall polysaccharides9） and of bacterial exopolysaccha- was speculated to be elongated with Rha-GlcNAc residues rides8）. The disaccharide residue, Rha-GlcNAc, found at to form UGL-2. Hence, signal assignment of β-Gal in UGL-2 the non-reducing terminus of H. aurantium UGL-2 has could also be interpreted as a downfield-shifted signal by been found as a repeating unit in the polysaccharide part attachment of GlcNAc compared with the data obtained on of LPS from S. marcescens31） and as a linker unit between UGL-1. Therefore, we could not assign two signals at ap- arabinogalactan and peptidoglycan in mycobacterial cell

292 J. Oleo Sci. 66, (3) 285-295 (2017) An acidic rhamno-glycosphingolipid from the ascidian H. aurantium

Fig. 7 Chemical structure of UGL-2 characterized from the ascidian H. aurantium in the present study. R1: hydrocarbon chain in its sphingoid, R2: hydrocarbon chain in its fatty acid. With regard to hydrocarbon chain length, see Table 1. walls33）. Both the anomeric configuration and sugar linkage but we detected two UGLs in the present study. For the found in the former case are completely identical to the di- novel UGL-2, it is particularly unusual to have both a rham- saccharide at the non-reducing terminus of H. aurantium nose residue and a reducing terminal glucuronic acid UGL-2. Ascidians have been shown to acquire a cellulose residue within a single molecule. We could not detect gan- synthase gene from bacteria via lateral gene transfer17）. In glioside in both ascidians: H. roretzi and C. intestinalis. the present study, rhamnose, which is characteristic of Our previous phylogenetic analysis on glycosyltransferases plants and bacteria, was detected as a sugar chain compo- in the Ciona species showed a loss of the majority of sialyl- nent in UGL-2 from H. aurantium. Taking these findings transferases21）. Therefore, we conclude that apparently, in together, H. aurantium might also have acquired rhamno- general, ascidians have lost their sialyltransferases. syltransferase via lateral gene transfer, as in the case of the cellulose synthase gene. We characterized UGL-2 as a novel glucuronic acid-containing GSL as well as UGL-1. Glucuronic acid-containing 5 CONCLUSION GSLs have been found in bivalves such as H. schlegelii and We isolated two UGLs from soft parts of the ascidian H. M. lusoria and shown to be present on the surface of aurantium: UGL-1, which is Galβ1-4（Fucα1-3） sperm7）. In addition, in sea urchin, sperm gangliosides have GlcAβ1-Cer; and UGL-2, which is Rhaα1-3GlcNAcβ1- been shown to bind to sperm-binding proteins localized on 3Galβ1-4（Fucα1-3）GlcAβ1-Cer. The former is identical to the vitelline layer of the egg surface34）. Furthermore, in UGL-1 from the ascidian H. roretzi, whereas the latter is mice, sperm sulfogalactosylglycerolipids have been shown characterized as a novel structure. to bind to the egg extracellular sulfoglycoprotein matrix, the zona pellucida35）. These data suggest that sperm glycolipids are involved in fertilization. The possible involvement of H. aurantium UGLs in fertilization should also be con- Acknowledgements sidered. With respect to the functional analysis of UGL-2, it This work was supported by the JSPS KAKENHI Grant might be interesting to investigate whether sperms from H. Number 15K00412. aurantium could aggregate by the action of anti-UGL-2 antibody in the same manner as that observed in the bivalves. Oral administration of glucuronyl sphingolipid Cer-β-glucuronide was shown to inhibit chemically induced Supplemental figure is available colon cancer in mice36）. Based on the finding that more This material is available free of charge via the Internet than 75％ of orally administered Cer-β-glucuronide was di- at http://dx.doi.org/jos.64.10.5650/jos.ess.16150 gested in colonic segments, Cer-β-glucuronide is expected to be a candidate material for delivering bioactive sphingolipids to the colon. Being one of the glucuronyl sphingolipids, UGL-2 might be useful in research related to colon References cancer. 1） Hakomori, S. Cell adhesion/recognition and signal We never detected any gangliosides from H. aurantium, transduction through glycosphingolipid microdomain.

293 J. Oleo Sci. 66, (3) 285-295 (2017) H. Kojima, R. Shinohara and S. Itonori et al.

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