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Glycosylceramides from marine green microalga Tetraselmis sp

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Page 1: Glycosylceramides from marine green microalga Tetraselmis sp

Phytochemistry 85 (2013) 107–114

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Phytochemistry

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Glycosylceramides from marine green microalga Tetraselmis sp.

Atsushi Arakaki a,⇑, Daisuke Iwama b, Yue Liang c, Nagisa Murakami d, Masaharu Ishikura d,Tsuyoshi Tanaka a,c, Tadashi Matsunaga a

a Department of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japanb Graduate School of Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japanc Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japand Yamaha Motor Co., Ltd., Tokyo, Japan

a r t i c l e i n f o

Article history:Received 22 February 2012Received in revised form 8 August 2012Available online 19 October 2012

Keywords:Tetraselmis sp.PrasinophyceaeGlucosylceramidesSphingolipidsD3-unsaturatation

0031-9422/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.phytochem.2012.09.006

⇑ Corresponding author. Address: Department of BiFaculty of Engineering, Tokyo University of AgricultuNaka-cho, Koganei, Tokyo 184-8588, Japan. Tel.: +81 47713.

E-mail address: [email protected] (A. Arakaki)

a b s t r a c t

Glycosylceramides are ubiquitous and important components of the plasma membrane in most eukary-otic cells and a few bacteria. They play significant roles in a variety of cellular functions. Their molecularstructures are well recognized in animals, higher plants, and fungi, but are poorly characterized in lowerplants. In this study, a high glycosylceramide-producing microalgal strain Tetraselmis sp. NKG 400013was found. TLC and MS analyses established the presence of glycosylceramides, GT1 and GT2, in thisstrain. Their chemical structures were determined by NMR spectroscopy and GC/MS, and were identifiedas glycosylceramides consisting of the typical botanical sphingoid base ([4E, 8E]-sphinga-4, 8-dienine)and 2-hydroxy-D3-unsaturated fatty acyl chains, respectively. To our knowledge, the occurrence of gly-cosylceramides in microalga of the class Prasinophyceae was previously unknown.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Glycosylceramides (a family of glycosphingolipids) are animportant class of membrane lipids with high structural diversityand various biological functions; they are ubiquitous in almostall eukaryotes and some prokaryotes (Olsen, 2001; Warnecke andHeinz, 2003). Glycosylceramides have been implicated in manyfundamental cellular processes of signal transduction, membraneformation, and intercellular reorganization, and most of their func-tions are related to their varied chemical structures (Pata et al.,2010; Sperling and Heinz, 2003; Warnecke and Heinz, 2003).

Glycosylceramides are composed of one or several glycosyl res-idues and a ceramide backbone. The hydrophobic ceramide moietyconsists of a sphingoid long chain base (LCB) to which a fatty acidis attached via an amide bond. Structural variations are found inthe glycosyl moiety, LCB and fatty acyl chain length, alkyl and hy-droxyl branching positions, and degree of unsaturation in bothLCBs and fatty acyl chains (Olsen, 2001; Warnecke and Heinz,2003; Zheng et al., 2006). The structural diversity of glycosylcera-mides has been extensively discussed in mammals, higher plants,and yeasts (Sperling and Heinz, 2003; Warnecke and Heinz,2003). The chemical structures of glycosylceramides in these

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organisms differ from each other in their ceramide backbones.However, compared to the tremendous research on glycosylcera-mides in higher plants, our knowledge of glycosylceramides inlower plants such as marine microalgae is poor.

Marine photosynthetic microalgae compose a large part of theplant kingdom and are the largest primary producers of biomassin the marine environment. They produce various useful sub-stances, such as essential fatty acids (Brossard et al., 1994; Miuraet al., 1993), vitamins (Carballo-Cardenas et al., 2003; Takeyamaet al., 1997), polysaccharides (Sudo et al., 1996), pigments(Matsunaga et al., 1993), and triglycerides (Matsumoto et al.,2010; Matsunaga et al., 2009) through photosynthesis. Moreover,the structures of natural products are usually more diverse inmarine microalgae than in higher plants. However, very limitedinformation is available on glycosphingolipid biosynthesis inmicroalgae. Only a few examples of microalgal glycosphingolipidproduction have been reported and their structures were not fullycharacterized (Iomini et al., 2006; Vardi et al., 2009; Wenzl andSumper, 1986).

In this study, glycosylceramide-producing microalgae from rep-resentative phytoplanktons were screened including Botryococcusbraunii, Chlamydomonas reinhardtii, and Chlorella sp., which havebeen widely used in biofuel studies. The chemical structures ofthe glycosylceramides were characterized by TLC, ESI-MS, MAL-DI-TOF MS, GC/MS, and NMR spectroscopic analyses. Structuralanalysis identified 2 novel glycosylceramides in the marine greenmicroalgae Tetraselmis sp. containing D3-unsaturated fatty acylchains that had not been previously identified in plants (Warnecke

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108 A. Arakaki et al. / Phytochemistry 85 (2013) 107–114

and Heinz, 2003; Zauner et al., 2008). To the best of our knowledge,this is the first report of the full structures of microalgalglycosylceramides.

2. Results

2.1. Identification of glycosylceramide-producing microalgae

Screening of glycosylceramide-producing microalgae was con-ducted by TLC and ESI-MS analyses. Investigated were phylogenet-ically distinct eukaryotic microalgae in the phyla Chlorophyceae (4strains), Prasinophyceae (2 strains), Bacillariophyceae (2 strains),Rhodophyceae (1 strain), and Euglenophyceae (1 strain), respec-tively. Two strains of prokaryotic microalgae (Cyanobacteria) werealso examined. The yeast strain K. lactis NBRC1267, whose glyco-sylceramide production has been elucidated (Takakuwa et al.,2002), was used as a positive control. As a first examination, glyco-staining was used on a TLC plate to identify cells producing prom-inent amounts of glycolipids. Total lipids were extracted from drycells and an aliquot (0.8 lg) of this extract was analyzed by TLC.After staining with orcinol-H2SO4 reagent, bands of glycolipidswere specifically visualized on TLC plates (SupplementaryFig. S1). The Rf values of standard glycosylceramides were detectedbetween 0.4 and 0.6. Bands observed before orcinol H2SO4 stainingwere considered to be pigments from the cells and were not inves-tigated by MS. Ten glycolipids were identified from the lipid ex-tracts of 6 eukaryotic microalgal strains, B. braunii NIES-836,Tetraselmis sp. NKG400013, T. pseudonana CCMP1335, S. costatumNIES-324, P. sordidum NBRC102772, and E. gracilis NIES-48 (Ta-ble 1). The target lipids were determined by mobility and glyco-staining, whereas the detection limit of the staining is ratherlimited (approximately 0.1 lg in this study).

ESI-MS analysis was conducted to characterize the structuralproperties of the analytes with high sensitivity (approximately1 ng in this study). The bands of these 10 glycolipids were scrapedfrom the TLC plates and analyzed by ESI-MS. The TLC of the lipidextract from Tetraselmis sp. NKG400013 showed three discrete gly-colipid bands. Major peaks in the mass spectrum of GT 1 were at m/z 473, 710, 747, 756, 800, 966, and 994, whereas major peaks in themass spectrum of GT 2 were at m/z 473, 748, 762, 800, 822, 859,

Table 1Summary of the screen for glycosylceramide-producing microalgae identified by TLC and

Class Microalgae

Cyanophyceae Spirulina platensis NIES-39Synechocystis sp. PCC6803

Chlorophyceae Botryococcus braunii NIES-836Chlamydomonas reinhardtii NIES-2235Chlorella sp. NKG400014Haematococcus pluvialis NIES-144

Prasinophyceae Micromonas pusilla NBRC102743Tetraselmis sp. NKG400013

Bacillariophyceae Skeletonema costatum NIES-324

Thalassiosira pseudonana CCMP1335

Rhodophyceae Porphyridium sordidum NBRC102772

Euglenophyceae Euglena gracilis NIES-48

–: not detected.a Glycolipids were detected by TLC with orcinol-H2SO4 staining.b Rf values were determined from images of TLC plates stained with orcinol-H2SO4.c Mass values were obtained from predicted m/z values of [M�H]� ions in ESI-MS.

and 911 (Fig 1). The exact mass of a natural hexosylceramide withthe shortest fatty acyl chain (d18:1/C16:0) is 699.5648 (calculatedvalue). Therefore, the major peaks in both MS spectra with m/zover 600 were subjected to MSn analyses. Two bands designatedGTs (1–2) showed peaks of [M+Cl]� ions at m/z 747 and 859,respectively. MS2 spectra of m/z 747 from GT 1 and m/z 859 fromGT 2 generated fragment [M�H]� ions at m/z 710, and 822, respec-tively. MS3 spectra of both m/z 710 and 822 fragments also gener-ated [M�H-162]� and [M�H-180]� ions, corresponding tofragmentation of a monohexose moiety from the parent ions. Thefragments at m/z 269 from GT 1 and m/z 381 from GT 2 were iden-tical to fatty acid fragments 2-hydroxy 16:1 and 2-hydroxy 24:1,respectively. In contrast, ESI-MS spectra of GT 3 did not showany fragments corresponding to glucose or fatty acids, suggestingthat the glycolipid in this band has a low ionization frequency inESI mode. Substance from this band was analyzed by GC/MS and1H NMR, and it was identified as a steryl glycoside. Similarly, B.braunii (GB 1), E. gracilis (GEs (1–2)), T. pseudonana CCMP1335(GThs 1), and S. costatum NIES-324 (GSs (1–3)) showed no defini-tive MS fragments. They were considered to be due to steryl glyco-side or other glycolipids.

Prominent amounts of glycolipids (GPs (1–2)) were also de-tected in the extract from P. sordidum NBRC102772 by orcinol-H2SO4 staining. MS2 analysis of these glycolipids showed [M�H]�

ions at m/z 886 for GP 1 and m/z 870 for GP 2. In the MS3 spectrumof GP 1, the fragments generated m/z 724 [M�H-162]� and 706[M�H-180]� peaks. GP 2 generated m/z 708 [M�H-162]� and m/z 690 [M�H-180]� peaks. These fragmentation patterns suggestedthe possibility of the existence of a sugar constituent in these mol-ecules. However, other fragments were not assigned to any knownlipid components.

Sphingoid bases in GTs (1–2) were further elucidated by detect-ing amine groups from the sphingoid moieties. Methanolysis prod-ucts from GTs (1–2) were loaded onto a TLC plate and stained withninhydrin, which specifically reacts with free terminal aminesfrom sphingoid bases. The staining yielded positive bands for GTs(1–2), but was negative for GPs (1-2) from P. sordidumNBRC102772 (Supplementary Fig. S2).

With this combinatorial strategy, the microalgae producing gly-cosylceramides were successfully screened. Based on the results of

ESI-MS.

Glycolipida Rf valueb Mass value (m/z)c

––

GB 1 0.61 ––––

–GT 1 0.43 711GT 2 0.48 823GT 3 0.45 –

GS 1 0.50 -GS 2 0.20 –GS 3 0.18 –GTh 1 0.36 –

GP 1 0.44 887GP 2 0.36 871

GE 1 0.41 –GE 2 0.49 –

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Fig. 1. ESI-MSn spectra of GTs (1–2) from Tetraselmis sp. strain NKG400013. The separated samples on TLC plates were scraped and resolved in chloroform:methanol (2:1, v/v)solution. Filtered samples were subjected to infusion-ESI-MSn in negative-ion mode using an ion trap mass spectrometer.

A. Arakaki et al. / Phytochemistry 85 (2013) 107–114 109

our TLC and ESI-MS analyses, it was concluded that GTs (1–2) fromTetraselmis sp. NKG400013 are glycosylceramides, and the studythus focused on these compounds.

2.2. Structural characterization of GTs (1–2)

2.2.1. PurificationApproximately 42.5 g of dry cells was obtained from a 40 L cul-

ture of strain NKG400013. The glycosylceramides were separatedfrom crude lipid extracts by ion-exchange, silica acid and reversephase chromatography. Purity and separation were checked byTLC in each separation process. 1 and 2 were successfully separatedfrom other lipid constituents. Approximately 5 mg of 1 and 1 mg of2 from 16 g dry cells of strain NKG400013 were purified. The con-tents of 1 and 2 were estimated to be approximately 0.31 and0.06 mg/g dry cells, respectively. As a comparison, glucosylcera-mide was also purified from K. lactis NBRC1267 using the same

procedure. Approximately 0.16 mg of glucosylceramide was ex-tracted from 1 g of dry yeast cells. Purified 1 and 2 were then usedfor the following structural characterization.

2.2.2. MALDI-TOF MSPositive-ion MALDI-TOF MS of 1 and 2 indicated pseudomolec-

ular ions ([M+Na]+) at m/z 734.5 and m/z 846.6 (Fig. 2). The com-parative experiment with glycosylceramide standards alsoshowed pseudomolecular ions ([M+Na]+). The molecular weightsof 1 and 2 were therefore 711.5 and 823.6. Compounds 1 and 2have been determined by TLC and ESI-MS/MS to contain a sphing-oid long-chain base, a monohexosyl moiety, and a fatty acyl chain.Thus, molecular formulas of 1 and 2 were identified as C40H73NO9

and C48H89NO9, respectively. These results have thus been con-firmed by HR-ESI-MS.

Page 4: Glycosylceramides from marine green microalga Tetraselmis sp

Fig. 2. MALDI-TOF MS spectra of purified glycosylceramides GT 1 (A) and GT 2 (B)from Tetraselmis sp. strain NKG400013. Analyses were performed in positive-ionmode.

Table 21H and 13C NMR spectroscopic data for glycosylceramide GT 1 extracted from strainNKG400013.

Proton (signal form) dH (ppm)/J (Hz) Carbon dC (ppm)

SphingoidH-1a (dd) 4.14/10.3, 5.7 C-1 69.7H-1b (dd) 3.70/10.3, 3.4H-2 (m) 3.99–3.94 C-2 54.6H-3(d) 4.11/7.5 C-3 72.8H-4 (dd) 5.44/7.5, 14.9 C-4 130.8H-5 (m) 5.71/14.3 C-5 134.4H-6 (m) 2.05 C-6 33.5H-7 (m) 2.05 C-7 33.1H-8 (m) 5.43 C-8 131.1H-9(m) 5.43 C-9 132.0H-10 (m) 1.98 C-10 33.7H-11 (d) 1.38/7.5 C-11 33.4H-12–H-17 1.32–1.28 C-12–C-17 30.9–23.8H-18 (t) 0.90/6.9 C-18 14.5H-NH (d) 7.72

Fatty acidC-10 175.5

H-20 (d) 4.43/6.3 C-20 74.1H-30 (dd) 5.49/15.5, 6.3 C-30 129.0H-40 (m) 5.87–5.80/1.2, 6.9 C-40 134.8H-50 (d) 2.03/7.5 C-50 33.5H-60 (d) 1.38/7.5 C-60 30.9H-70–H-150 1.32–1.28 C-70–C-150 30.9–23.8H-160 (t) 0.90/6.9 C-160 14.5

b-D-GlucoseH-100 (d) 4.27/8.0 C-100 104.7H-200 (dd) 3.12/9.2, 8.0 C-200 75.0H-300 (m) 3.37/9.2 C-300 77.9H-400 (m) 3.28 C-400 71.4H-500 (m) 3.28 C-500 77.9H-6a00 (d) 3.87/11.5 C-600 62.6H-6b00 (dd) 3.67/11.5, 4.0

110 A. Arakaki et al. / Phytochemistry 85 (2013) 107–114

2.2.3. GC-MSFatty acid chains were determined by GC/MS. Single peaks were

observed at 17.0 min and 38.5 min for 1 and 2, respectively (Sup-plementary Fig. S3). MS spectra indicated that the fatty acid moie-ties of 1 and 2 are 2-hydroxy C16:1 and 2-hydroxy C24:1,respectively.

2.2.4. 1H and 13C NMR spectroscopyIn order to determine the anomeric configuration, the linkage

sequence of carbohydrates, and the structure of sugar moiety,Compounds 1 and 2 were subjected to NMR spectroscopic analysis.The NMR data for 1 are summarized in Table 2. The b-D-glucosemoiety in 1 was determined by the anomeric proton signal atdH4.27 (1H, d, J = 8.0 Hz, H-100) in the 1H NMR spectra and fromthe carbon resonances at dC104.7 (C-100), 75.0 (C-200), 77.9 (C-300),71.4 (C-400), 77.9 (C-500), and 62.6 (C-600) in the 13C NMR spectra.The signals of two primary methyl groups (dH0.90 [3H, m, J = 6.9,H-18], dH0.90 [3H, m, J = 6.9, H-160], dC14.5 [C-18], and dC14.5 [C-160]), the resonances of three olefinic groups (dH5.44 [1H, dd,1J = 7.5, 2J = 14.9, H-4], dH5.71 [1H, m, J = 14.3, H-5], dH5.43 [2H,m, H-8, H-9], dH5.49 [1H, dd, 1J = 15.5, 2J = 6.3, H-30], dH5.87-5.80[1H, m, 1J = 1.2, 2J = 6.9, H-40], dC130.8 [C-4], dC134.4 [C-5],dC131.1 [C-8], dC132.0 [C-9], dC129.0 [C-30], and dC134.8 [C-40]),the two hetero bearing-methine resonances (dH3.99-3.94 [1H, m,H-2], dH4.11 [1H, d, J = 7.5, H-3], dC54.6 [C-2] and dC72.8 [C-3]),the oxygenated methylene resonances (dH4.14 [1H, dd, 1J = 10.3,2J = 5.7, H-1a], dH3.70 [1H, dd, 1J = 10.3, 2J = 3.4, H-1b], dC69.7 [C-1]), the signals of a hydroxylated methylene (dH4.43 [1H, d,J = 6.3, H-20], and dC74.1 [C-20]), the downfield doublet NH reso-nance at dH7.72 (1H, d, NH), the carbonyl group at dC175.5 (C-10),and the remaining long CH2 chain signals from dH1.28 to 2.05

and dC23.8 to 33.7 established two long chain bases of sphingo-lipid. The resonances at dC33.5 (C-6), dC33.1 (C-7), dC33.7 (C-10),and dC33.5 (C-50) indicate that all three olefinic groups are in thetrans conformation (Noda et al., 1994; Shibuya et al., 1990).

Comparing the 13C NMR spectrum of 1 with those of glucosyl-erythro-ceramide and glucosyl-threo-ceramide, the sphingoid basewas deduced as (4E, 8E)-D-erythro-sphinga-4, 8-dienine (Sarmien-tos et al., 1985).

GT2 showed similar NMR spectrum as 1 (Table 3), therefore, thestructures of 1 and 2 were determined to be glucosylceramidesconsisting of (4E, 8E)-D-erythro-sphinga-4, 8-dienine and (E)-2-hydroxyhexadec-3-enoic acid and (E)-2-hydroxytetracos-3-enoicacid, respectively. These results were confirmed by 2D-NMR spec-troscopic analysis (Supplementary Fig. S4), and the key HMBCinteractions are shown in Fig. 3.

2.3. Quantification of GTs (1-2) by LC/ESI-MS

In order to quantify compounds 1 and 2 in strain NKG400013,these were analyzed by LC/ESI-MS in the MRM mode. They weremonitored at m/z 712.6/262.2 and 824.6/262.2 for Q1/Q3, becauseboth 1 and 2 generate a sphingoid fragment ion (m/z 262.2) in MS2.The calibration curves were constructed by plotting the peak areaversus glucosylceramide concentration standards from soybean(monitored at m/z 714.6/262.2). They were linear in the range of0-2 ng (Supplementary Fig. S5). Both compounds were successfullyanalyzed in a single operation.

The contents of 1 and 2 in were quantified by the same method.Experiments were conducted with stationary phase cultures.Approximately 100 mg of dry cells was obtained from three 500-ml cultures. Under optimized LC conditions, 1 and 2 were eluted

Page 5: Glycosylceramides from marine green microalga Tetraselmis sp

Table 31H and 13C NMR spectroscopic data for glycosylceramide GT 2 extracted from strainNKG400013.

Proton (signal form) dH (ppm)/J (Hz) Carbon dC (ppm)

SphingoidH-1a (m) 4.14/5.7 C-1 69.7H-1b (m) 3.70H-2 (m) 3.99-3.94 C-2 54.6H-3 (d) 4.11/7.5, 4.0 C-3 72.8H-4 (t) 5.46/7.5 C-4 130.8H-5 (m) 5.71/15.5 C-5 134.4H-6(m) 2.05 C-6 33.5H-7 (m) 2.05 C-7 33.1H-8 (m) 5.43 C-8 131.1H-9 (m) 5.43 C-9 132.0H-10 (broad) 2.01-1.96 C-10 33.7H-11(broad) 1.43-1.35 C-11 33.4H-12–H-17 1.33-1.28 C-12–C-17 30.9–23.8H-18 (t) 0.90/6.3 C-18 14.5H-NH (d) 7.72

Fatty acidC-1’ 175.5

H-2’ (d) 4.43/5.7 C-2’ 74.1H-3’ (dd) 5.49/15.5, 6.3 C-3’ 129.0H-4’ (m) 5.83/15.5, 6.9 C-4’ 134.8H-5’ (broad) 2.07–2.01 C-5’ 33.5H-6’ (broad) 1.43–1.35 C-6’ 30.9H-7’–H-23’ 1.33–1.28 C-7’–C-23’ 30.9–23.8H-24’ (t) 0.90/6.3 C-24’ 14.5

b-D-glucoseH-100 (d) 4.27/8.0 C-100 104.7H-200 (dd) 3.19/9.2, 8.0 C-200 75.0H-300 (m) 3.34 C-300 77.9H-400 (m) 3.28 C-400 71.4H-500 (m) 3.28 C-500 77.9H-6a00 (d) 3.86/11.5 C-600 62.6H-6b00 (m) 3.68/4.5

A. Arakaki et al. / Phytochemistry 85 (2013) 107–114 111

at 7.8 and 10.4 min, respectively. The amounts of 1 and 2 wereapproximately 0.54 ± 0.05 and 0.13 ± 0.01 mg/g dry cells. There-fore, the total content of 1 and 2 in strain NKG400013 was esti-mated to be approximately 0.67 mg/g dry cells. This result wasconsistent with the results of the weight measurements of purifiedsamples (0.38 mg/g dry cells). The difference is due to loss duringpurification. The content is relatively higher than in yeast(0.43 mg/g dry cells) (Saito et al., 2006), soy bean (0.39 mg/g drycells), and wheat (0.21 mg/g dry cells) (Takakuwa et al., 2005).

Fig. 3. Key HMBC interacti

3. Discussion

A lipidomics strategy was used with a combination of TLC andESI-MS/MS to screen glycosylceramide-producing microalgalstrains. However, the screening can also be performed by detectinggenes that catalyze glycosphingolipid biosynthesis. Expression of aglucosylceramide synthase gene in a blue-green alga Synechocystissp. has been previously reported (Leipelt et al., 2001), and genes in-volved in glycosphingolipid biosynthesis in the genomes of severalmicroalgae were investigated in this study. A gene of a glucosylcer-amide synthase homolog was found in the genome of Micromonassp. RCC299 (Worden et al., 2009). Serine palmitoyltransferase wasalso identified in the genome of C. reinhardtii (Merchant et al.,2007). However, no glycosphingolipid was detected by TLC andMS screening. Although these cells may be able to synthesizeglycosphingolipids, the product did not reach detectable levels inculture and analytical conditions used here.

Glycosylceramides exhibit high structural diversity betweenorganisms, species, and tissues, while the D3-(E)-unsaturation of2-hydroxy fatty acids appears to be restricted to some fungal gly-cosylceramides (Warnecke and Heinz, 2003; Zauner et al., 2008).These discoveries suggest the possibility of either fungal contami-nation or symbiotic fungi in our cultures. To resolve this, the purityof the culture was confirmed by microscopy (Figure S6A). Cell nu-clei were stained with Hoechst 33342 (Invitrogen, Tokyo, Japan) todetect symbiotic fungi. The cells contained a single nucleus unlessthey were dividing, in which cases 2 nuclei were observed (Fig-ure S6B-E). 18S rDNA sequences from 177 randomly selectedclones were also analyzed. Most clone sequences (173, 98%) withhigh a sequence similarity were affiliated with Tetraselmis sp..These results demonstrated the purity of our strain from fungalcontamination and symbionts.

The glucosylceramide structures from our strain were thencompared with fungal glycosylceramides containing a D3-(E)-unsaturated 2-hydroxy fatty acid moiety. The structural diversityof fungal glycosylceramides was discussed by Warnecke and Heinz(Warnecke and Heinz, 2003). The glycosylceramides from Aspergil-lus fumigatus, Aspergillus niger, Sporothrix schenckii, Fusarium solani,Fusicoccum amygdali, Magnaporthe grisea, Pachybasium sp., and Par-acoccidioides brasiliensis maintain the D3-(E)-unsaturated 2-hydro-xy fatty acid. However, all of the glycosylceramides in theseorganisms contain a characteristic C9-methyl-branched fungal

ons in GT 1 and GT 2.

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112 A. Arakaki et al. / Phytochemistry 85 (2013) 107–114

sphingoid long-chain base that differs from the compounds foundin this study. In addition, the fatty acyl chain length of fungal gly-cosylceramides are predominantly C16 or C18; very long-chainfatty acids of C24-C26 are rarely identified in fungal glycosylcera-mides (Aoki et al., 2004; Wells et al., 1996). D9-methylation is ab-sent in mammalian and botanical sphingoid long-chain bases.Unlike mammalian sphingoid bases, due to additional desaturationat C-8, the dominant unsaturated plant LCBs are (E/Z)-sphing-8-en-ine (d18:18), (4E, 8E/Z)-sphinga-4, 8-dienine (d18:24,8), and (8E/Z)-4-hydroxy-8-sphingenine (t18:18) (Sperling and Heinz, 2003).Therefore, the 2 novel glucosylceramides (GTs (1-2)) in strainNKG400013 were characterized as having a typical botanicalsphingoid base and fungal fatty acyl chains with a glucose moiety.

Further structural analyses of glycosylceramides from a closelyrelated microalga were also performed with Tetraselmis sp.NBRC103003. Based on the TLC, ESI-MS, and GC/MS analyses, Thesame glucosylceramides as GTs (1-2) in the extracts of this strainwere identified. This suggested that the 2 glucosylceramides in thisstudy might be conserved in the Tetraselmis sp. and closely relatedmicroalgal groups. In addition, the new structures suggested pres-ence of unidentified synthetic routes of glucosylceramides as wellas enzymes catalyzing desaturation of fatty acid chains in thisorganism. Further systematic studies of microalgal ceramide pro-duction will elucidate the structural varieties of glycosylceramidesand their cellular functions.

Glycosylceramides in plant cells have been implicated indrought tolerance, in addition to their role in membrane stabilityduring cold acclimation (Pata et al., 2010; Sperling and Heinz,2003; Warnecke and Heinz, 2003). Because Tetraselmis sp. requiressalt for growth, the function of GTs (1–2) may be related to saltand drought tolerances. High motility in both Tetraselmis sp.NKG400013 and NBRC103003 were also observed, while othermicroalgae showed little or no motility. A sphingolipid-enrichedmembrane microdomain was identified in the flagellar membraneof Chlamydomonas and proposed to serve an organizational func-tion for interpreting light signals (Iomini et al., 2006). The glycosyl-ceramides in strain NKG400013 might be involved in cell motilityby forming lipid microdomains.

4. Conclusion

Two new glucosylceramides from marine microalga Tetraselmissp. strain NKG 400013 have been isolated and structurally charac-terized. The composition of the typical botanical sphingoid baseand fungal fatty acyl chains of these compounds may indicatethe intermediate evolutionary state of microalgae, especially Pra-sinophyceae, between higher plants and fungi. This is the first dis-covery of D3-unsaturated 2-hydroxyl fatty acids in plant cells. Toour knowledge, this is also the first identification of glycosylcera-mides in Tetraselmis sp.

5. Experimental

5.1. Strains and culture conditions

Microalgal strains Spirulina platensis (NIES-39), Botryococcusbraunii (NIES-836), Chlamydomonas reinhardtii (NIES-2235),Haematococcus pluvialis (NIES-144), Skeletonema costatum (NIES-324), and Euglena gracilis (NIES-48) were obtained from theNational Institute for Environmental Studies (Ibaraki, Japan).Micromonas pusilla (NBRC102743), Porphyridium sordidum(NBRC102772), Tetraselmis sp. (NBRC103003), and Kluyveromyceslactis (NBRC1267) were acquired from the National Institute ofTechnology and Evaluation Biological Resource Center (Chiba,Japan). Synechocystis sp. (PCC6803) and Thalassiosira pseudonana

(CCMP1335) were obtained from the Institut Pasteur and Provasol-i-Guillard National Center for the Culture of Marine Phytoplankton(Paris, France), respectively. Tetraselmis sp. (NKG400013) and Chlo-rella sp. (NKG400014) were isolated and identified in our labora-tory (Matsunaga et al., 2009). Microalgae were cultured in SOT(S. platensis), BG11 (Synechocystis sp.), AF6 (B. braunii), IMK (M. pu-silla, P. sordidum, Chlorella sp. and Tetraselmis sp.), Proteose (C. rein-hardtii and H. pluvialis), f/2 (S. costatum and T. pseudonana), or HUT(E. gracilis) medium for 2 weeks in Erlenmeyer flasks (40 ml) or flatflasks (500 ml) under continuous illumination with a cool-fluores-cent lamp at 60-70 lmol/m2/s with aeration. K. lactis (NBRC1267)was cultured in a flask containing YPD medium for 2 days. Mediumcomposition was derived from the National Institute for Environ-mental Studies (http://mcc.nies.go.jp/) database.

5.2. Materials

DEAE Sephadex A-25 was purchased from GE Healthcare UK,Ltd. (Buckinghamshire, England). Iatrobeads 6RS-8060 was ob-tained from Mitsubishi Chemical Medience (Tokyo, Japan) and sil-ica gel 60-coated plates were from Merck (Darmstadt, Germany).Glycosylceramide standards were from Avanti Polar Lipids, Inc.(Alabaster, AL, USA). All other reagents were of the highest com-mercial grade available.

5.3. Lipid extraction

Cells were harvested by centrifugation and lyophilized. Lipidextraction was performed as described previously with very littlemodification (Takakuwa et al., 2005). Briefly, lyophilized cells(50 mg) were mixed with CHCl3:MeOH (1 ml, 1:1, v/v) and 0.8 MKOH–MeOH (1.0 ml). The mixture was homogenized with an ultra-sonic homogenizer (W-170-ST, HONDA, Aichi, Japan) for 5 min.The suspension was incubated at 42 �C for 1 h. After mixing withCHCl3 (2 ml) and distilled H2O (1.12 ml) water, the suspensionwas centrifuged at 1000g for 1 min. The organic phase was sepa-rated and dried using a rotary evaporator. The dried powder wasre-suspended in of CHCl3:MeOH (200 ll, 2:1, v/v) and stored at�20 �C.

5.4. TLC and electrospray ionization-mass spectrometry (ESI-MS)

Extracted lipids were analyzed by TLC and ESI-MS. TLC was per-formed on silica gel 60-coated plates with a neutral solvent systemof CHCl3:MeOH:H2O (65:16:2, v/v/v). Glycolipids were visualizedwith orcinol-H2SO4 reagent. The separated lipids on silica gelplates were scraped and resolved in CHCl3:MeOH (200 ll, 2:1, v/v). The recovered samples were filtered through 0.5-lm polytetra-fluoroethylene membrane filters. After filtration, samples weresubjected to infusion-ESI-MSn in negative ion mode on an LCQDECA XP ion trap mass spectrometer (Thermo Fisher ScientificInc., Waltham, MA, USA) with the Xcalibur operating system. Thetemperature of the ion transfer tube was 300 �C, the spray voltagewas 5 kV, and the nitrogen sheath gas pressure was 10 U. The col-lision energy for MSn analysis was fixed at 30-50%.

Sphingoid bases prepared from glycosylceramides (4 lg) bymethanolysis with 1 M methanolic NaOH at 100 �C for 1 h were de-tected by TLC with ninhydrin reagent (Itonori et al., 2004).

5.5. Purification of GTs from microalgae

The crude lipid extracts were fractionated on a DEAE-SephadexA-25 (OAc-form) column (30 � 300 mm) using a solvent systemcontaining CHCl3:MeOH:H2O (70 ml, 5:10:1 v/v/v) at a flow rateof 2 ml/min. The neutral lipid fractions were detected by TLC anddried by Ar gas. The dried neutral lipid fraction was dissolved in

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A. Arakaki et al. / Phytochemistry 85 (2013) 107–114 113

CHCl3 (8 ml) and subjected to silica gel cc (Iatrobeads)(20 � 500 mm), using a linear gradient elution system containinga chloroform-acetone mixture (Momoi et al., 1976) at a flow rateof 2 ml/min. The glycosylceramide fractions were detected byTLC. GTs (1-2) were separated by isocratic reversed-phase HPLCon a Spherisorb ODS2 column (4.6 � 150 mm, Waters, Milford,MI, USA) in MeOH, at a flow rate of 0.5 ml/min for 20 min.

5.6. MALDI-TOF MS and HR-ESI-MS

MALDI-TOF MS analysis of purified glycosylceramides was per-formed on an AXIMA Performance (Shimadzu, Kyoto, Japan) with anitrogen laser (337 nm) and an acceleration voltage of 20 kV. Gly-cosylceramide samples (200 ng/ll in MeOH) were mixed with ma-trix solution (10 mg of 2, 5-dihydroxybenzoic acid in 1 ml H2O)(1:1, v/v) and the suspensions were loaded on a sample plate. Po-sitive mass spectra were measured in reflector mode.

High-resolution ESI mass (HR-ESI-MS) spectra were obtained onan LTQ Orbitrap XL mass spectrometer operating in the positive ionmode using a solvent system of 9: 1 (v/v) CH3CN and H2O with 0.1%(v/v) HCO2H by continuous infusion.

5.7. Fatty acid composition analyses

To determine the fatty acid compositions of the purified glyco-sylceramides, glycosylceramides (50–100 lg) were methanolyzedwith 1 ml freshly prepared 1 M anhydrous methanolic HCl at90 �C for 1 h. After methanolysis, the fatty acid methyl esters wereextracted 3 times with n-hexane (1 ml) and converted to their O-trimethylsilyl derivatives with an equivalent amount of N, O-bis(trimethylsilyl) trifluoroacetamide.

A Shimadzu GCMS QP2010 with a capillary column (0.22 mm �25 m) Shimadzu HiCap-CBP 5 (Shimadzu, Kyoto, Japan) was usedto determine the sugar and aliphatic compositions under the fol-lowing conditions: ionizing voltage, 70 eV; ionizing current,60 lA; interface temperature, 250 �C; injection-port temperature,240 �C; and helium gas pressure, 100 kPa. Oven temperatures wereprogrammed at 80 �C for 2 min, then to 240 �C at 4 �C/min (Sudoet al., 1996). Fatty acid methyl esters were identified by comparingthe peak retention times and mass spectra of samples with those ofa standard fatty acid mixture.

5.8. NMR spectroscopy

1H, 13C, DEPT, 1H/1H correlated spectroscopy (COSY), 1H/13C het-eronuclear multiple quantum coherence (HMQC), and 1H/13C het-eronuclear multiple bond connectivity (HMBC) experiments wererecorded on a JNM ECA-500 NMR spectrometer (JEOL, Tokyo, Ja-pan). Each purified sphingolipid was dissolved in methanol-d4(0.6 ml) and the chemical shift was referenced to internal 0.05%tetramethylsilane (dH 0.00 ppm).

5.9. Quantification of glycosylceramides by liquid chromatography/electrospray ionization-mass spectroscopy (LC/ESI-MS)

Individual glycosylceramides in cells were quantified on an LC/ESI-MS system (Prominence UFLC, Shimadzu, Kyoto, Japan;API4000 QTRAP, AB SCIEX, Tokyo, Japan) as reported previously(Shaner et al., 2009). The glycosylceramides were separated by re-verse phase LC using a TSKgel 2.1 (i.d.) � 50 mm C18 column (TOS-OH, Tokyo, Japan) and a binary solvent system at a flow rate of0.2 ml/min. Prior to injection, the column was equilibrated for0.8 min with a solvent mixture of 60% mobile phase A (CH3OH:H2-

O:HCOOH, 58:41:1, v/v/v, with 5 mM ammonium formate) and40% mobile phase B (CH3OH:HCOOH, 99:1, v/v, with 5 mM ammo-nium formate); after sample injection (1 ll), the A/B ratio was

maintained at 60:40 for 1.0 min, followed by a linear gradient to100% B over 3.6 min, held at 100% B for 10.6 min, followed by a1.0-min wash with 60:40 before the next run. Simultaneous anal-ysis of individual glycosylceramide molecular species was per-formed by multiple reaction monitoring (MRM) in positiveionization mode. Q1 was set to pass the precursor ion of GT 1and GT 2, whereas Q3 was set to pass the fragment ion for sphing-oid base. The declustering potential (DP) and collision energies (CE)were optimized at 70 and 50 eV respectively.

5.10. GT 1

White amorphous powder; TLC: Rf-values (100�) 43; LCMS Rt

(min) 7.8; for 1H and 13C NMR spectroscopic data, see Table 2;HR-ESI-MS calc. for C40H74NO9: 712.5364. Found: 712.5360. GCMSRt (min) for fatty acyl chain: 17.

5.11. GT 2

White amorphous powder; TLC: Rf-values (100�) 48; Rt (min)10.4; for 1H and 13C NMR spectroscopic data, see Table 3; HR-ESI-MS calc. for C48H90NO9: 824.6616. Found: 824.6612. GCMS Rt

(min) for fatty acyl chain: 38.5.

Acknowledgements

This work was partially supported by a Grant-in-Aid for Scien-tific Research on Innovative Areas (no. 2206) from the Ministry ofEducation, Culture, Sports, Science and Technology, Japan (MEXT)and Japan Science, and Technology (JST), Core Research for Evolu-tional Science and Technology (CREST). We thank Dr. Keiichi Nogu-chi for NMR technical support.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.phytochem.2012.09.006.

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