Lipidomic profiling reveals biosynthetic relationships between phospholipids and diacylglycerol ethers in the deep-sea soft coral Paragorgia arborea

The cold-water gorgonian coral Paragorgia arborea is considered as a foundation species of deep-sea ecosystems in the northern Atlantic and Pacific oceans. To advance lipidomic studies of deep-sea corals, molecular species compositions of diacylglycerol ethers (DAGE), which are specific storage lipids of corals, and structural glycerophospholipids (GPL) including ethanolamine, choline, inositol and serine GPL (PE, PC, PI, and PS, respectively) were analyzed in P. arborea by HPLC and tandem mass spectrometry. In DAGE molecules, alkyl groups (16:0, 14:0, and 18:1), polyunsaturated fatty acids (PUFA), and monounsaturated FA are mainly substituted the glycerol moiety at position sn-1, sn-2, and sn-3, respectively. The ether form (1-O-alkyl-2-acyl) predominates in PE and PC, while PI is comprised of the 1,2-diacyl form. Both ether and diacyl forms were observed in PS. At position sn-2, C20 PUFA are mainly attached to PC, but C24 PUFA, soft coral chemotaxonomic markers, concentrate in PS, PI, and PE. A comparison of non-polar parts of molecules has shown that DAGE, ether PE, and ether PC can originate from one set of 1-O-alkyl-2-acyl-sn-glycerols. Ether PE may be converted to ether PS by the base-exchange reaction. A diacylglycerol unit generated from phosphatidic acid can be a precursor for diacyl PS, PC, and PI. Thus, a lipidomic approach has confirmed the difference in biosynthetic origins between ether and diacyl lipids of deep-sea gorgonians.

by analyzing the lipidome of reef-building corals [32][33][34]43 . Different patterns of distribution of polyunsaturated FA (PUFA) among structural lipid classes were demonstrated for different taxa of soft corals and hydrocorals from tropical and cold-water regions 30 . A lipidome analysis allowed simultaneous monitoring of lipid dynamics of both symbiotic dinoflagellates and host tissues during coral bleaching 44,45 . An in situ lipidome analysis of symbiotic dinoflagellates revealed possible mechanisms of heat stress tolerance in reef-building corals 46 . The accumulation of dietary very-long-chain FA (VLCFA) in nudibranch mollusks preying on soft corals was explained by comparing the lipidomes of soft corals and predators 28 .
In corals, a lipidomic comparison between different phospholipid classes has been applied for resolving biosynthetic relationships of these classes 30,47 . The major storage lipid classes of corals are wax esters, triacylglycerols, and specific ether lipids such as 1-O-alkyl-2,3-diacyl-sn-glycerols or diacylglycerol ethers (DAGE) 48 . The structural lipids classes of corals include ceramide aminoethylphosphonate (CAEP) and four glycerophospholipid (GPL) classes: ethanolamine, choline, inositol and serine GPL (abbreviated here as PE, PC, PI, and PS, respectively) 30 . Besides 1,2-diacyl GPL, corals contain also large amounts of ether phospholipids, which are divided into two types, plasmanyl (alkyl moiety at position sn-1, 1-O-alkyl-2-acyl GPL), and plasmenyl (alkenyl moiety with vinyl ether linkage at position sn-1, 1-O-(alk-1´-enyl)-2-acyl GPL) 30 . According to general biosynthetic pathways, 1-O-alkyl-2-acyl-sn-glycerol is converted into PE and PC by enzyme systems used to produce diacyl forms 49,50 . Presumably, 1-O-alkyl-2-acyl-sn-glycerols are the main source of DAGE in animal cells 49 . Hence, a comparison of non-polar parts of lipid molecules (alkyl/acyl composition) can confirm a hypothesis that DAGE and alkyl/acyl PE and PC in coral cells originated from a single precursor, 1-O-alkyl-2-acyl-sn-glycerol. Chemical structures of DAGE and triacylglycerols are similar and, therefore, DAGE may be a form of coral storage lipids. However, data on DAGE molecular species composition of corals still remains very limited 37,44 .
The cold-water gorgonian coral Paragorgia arborea (Paragorgiidae) mainly inhabits a depth range between 50 and 1300 m. This coral is widely distributed in the northern Atlantic and northern Pacific oceans, and is considered as a foundation species of deep-sea ecosystems 51 . The FA and lipid class compositions of P. arborea were described earlier 52,53 . In the present study, the profile of the lipid molecular species of phospholipid classes and DAGE was analyzed by a combination of high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). The lipidomics strategy was applied and individual molecular species were manually identified. A comparison of the lipidomes of the gorgonian coral P. arborea and other corals was carried out. Similarities of non-polar parts of molecules of each lipid class of P. arborea were tested to confirm possible biosynthetic relationships between the coral lipid classes.
The composition of DAGE molecular species is shown in Fig. 1A and Supplementary Table S2. The letter "e" after the first fatty acids in DAGE means 1-O-alkyl group. The distribution of alkyl and acyl groups at positions sn-1/sn-2/sn-3 of a glycerol ether backbone of each DAGE molecular species was determined ( Supplementary  Fig. S5). Three O-alkyl groups such as hexadecyl (16:0), tetradecyl (14:0), and octadecenyl (18:1) mainly attached to glycerol at position sn-1. The proportions of DAGE molecules with these major O-alkyl groups showed no significant (ANOVA, p > 0.01) differences from the proportions of the corresponding 1-O-alkyl-sn-glycerols obtained by hydrolysis of DAGE. Only polyunsaturated FA (PUFA) were found at position sn-2 of the glycerol backbone. At position sn-3, 60.2 ± 3.5% of DAGE molecules attached monounsaturated FA (MUFA), and 11.9 ± 3.6% of DAGE molecules attached saturated FA (SFA). VLCFA were found at both sn-2 and sn-3 of the glycerol backbone.  Table S3, Supplementary Fig. S7). The letter "a" after the first fatty acids in GPL means 1-O-alkyl group (plasmanyl form), the letter "p"-plasmenyl form, 1-O-alk-1´-enyl group. The contents of 22 major molecular species of five polar lipid classes (more than 5% of each lipid class) are shown in Fig. 1B. These major molecular species made up 67% of total polar lipids. CAEP molecular species differed in sphingoid bases, but only palmitic acid (16:0) was detected as N-acyl groups. The letter "d" means a sphingoid base in the names of CAEP molecules. The sphingoid bases had a 2-amino-1,3-dihydroxy long-chain core structure with 18-22 carbon atoms and 1-4 double bonds. Both ether and diacyl forms of GPL were identified. The ether form dominated PE, PC, and PS. The content of the ether molecular species in PE, PC, PS, and PI was 99.3, 85.4, 64.1, and 2.0%, respectively. The noticeable level of the alkenylacyl (plasmalogen) form was found only in PE and PS (29.7 and 5.9% of lipid class, respectively).
Saturated and monounsaturated C 16 and C 18 alkyl/acyl groups were most abundant at position sn-1 in GPL molecules. Clear differences in the PUFA composition between GPL classes were observed. At position sn-2, C 20 PUFA such as 20:4n-6 and 20:5n-3 dominated acyl groups in PC molecular species, while C 24 VLCFA dominated acyl groups in PS and PI. Approximately half of PE molecules contained C 20 PUFA at position sn-2, but 47% of PE molecules were esterified by C 24 VLCFA such as 24:5n-6 and 24:6n-3.  Table S4). A cluster analysis (Fig. 2B) revealed a similarity in the sn-1/sn-2 composition between DAGE, PC and PE. The molecular species 16:0a/20:4 (sn-1/sn-2) was most abundant in these three lipid classes. The dendrogram also shows that the group of DAGE, PC, and PE differed from PS and PI that contained high proportions of VLCFA at position sn-2 ( Fig. 2A). The same diacyl forms (18:0/24:5 and 18:0/24:6) were observed in both PI and PS molecular species. Due to the trace amounts of PI ether forms, we do not consider the biosynthetic relationship between DAGE and PI below.

Discussion
In addition to triacylglycerols, the common storage lipid class, corals contain diacylglycerol ethers (DAGE). Up to 50% of coral total lipids may be comprised of DAGE 48 . These two storage lipid classes have similar chemical structures but different biosynthetic pathways 54,55 . Coral colonies exposed to thermal stress demonstrate different dynamics of the triacylglycerol and DAGE catabolism 44 . The molecular species profiles of triacylglycerols and DAGE were earlier described from two alcyonarian species of the genus Sinularia 37,44 , the reef-building coral Acropora cerealis 45 , and the zoanthid Palithoa tuberculosa 38 . In these coral polyps, the PUFA acyl groups were mainly attached at positions sn-1 (3) in triacylglycerols but at position sn-2 in DAGE molecules. This observation thereby confirms the different biosynthetic origins of these lipid classes in corals. A comparison of coral polyps showed order-specific differences in the FA composition at position sn-2 between DAGE profiles. SFA were mainly attached to the glycerol moiety in DAGE of tropical alcyonarians 37,45 , whereas PUFA dominated acyl groups at position sn-2 of DAGE in the tropical zoanthid P. tuberculosa and the deep-sea gorgonian P. arborea.
Most of these PUFA are represented by long-chain acids (20:4n-6 and 20:5n-3). Furthermore, the markers of symbiotic zooxanthellae (18:3n-6, 18:4n-3, and C 16 PUFA) were presented in DAGE of the zoanthid, while the chemotaxonomic markers of octocorals (24:5n-6 and 24:6n-3) were found in DAGE of P. arborea. Thus, mediumchain PUFA, which can be transferred from photosynthetic symbionts 56,57 or possibly synthesized in the coral The distribution of PUFA among structural lipid classes in P. arborea was typical for the soft corals. It was found that C 20 PUFA mainly located in PE and PC, while VLCFA concentrated in PS and, partly, in PI 30,35 . In contrast to PS of tropical alcyonarians, which contain only an ether form, both diacyl and ether forms of PS were observed in deep-sea P. arborea, similarly to the cold-water soft corals Gersemia rubiformis and G. fruticosa 30,36,47 . We suppose that the presence of diacyl PS is necessary to maintain the membrane fluidity of coral cells under lower water temperatures.
The high level of PE with VLCFA (46.6% of total PE) in P. arborea distinguishes this species from other corals studied. The PC molecular species with VLCFA were also found in P. arborea. In our earlier studies, PE and PC with VLCFA were not detected in corals 60 . Subsequently, PE 18:0ap/24:5 (the sum of plasmanyl and plasmenyl forms) was found in the tropical alcyonarians Capnella sp., Sinularia siaesensis, and S. heterospiculata (2.1, 5.9, and 10.8% of total PE, respectively) 35,37 . Thus, C 24 PUFA can be incorporated in all structural lipid classes (except for CAEP) of soft corals. To recognize the high level of PE with VLCFA as a chemotaxonomic trait of the genus Paragorgia, further analyses of lipidomes of other tropical and cold-water gorgonian species are required.
The plasmanyl PE (alkyl PE) can further be transformed into their corresponding plasmenyl form, 1-O-(alk-1′-enyl)-2-acyl-sn-glycero-3-phosphoethanolamine (plasm PE) 49 . In P. arborea, this transformation is confirmed by the high level of plasm PE. No enzymes converting plasmanyl PS (alkyl PS) into plasmenyl PS (plasm PS) are known to date, but PS can be synthesized by the base-exchange reaction between PE and serine 50 . We suppose that this reaction can lead to the conversion of a part of ether PE to ether PS in the soft coral P. arborea. It should be noted that this base-exchange reaction mainly involves the molecular species of PE with VLCFA at position sn-2. Possible pathways of synthesis of ether lipids in P. arborea are summarized in Fig. 3.
The synthesis of mammalian diacyl GPL requires either 1,2-diacylglycerol or CDP-diacylglycerol generated from phosphatidic acid 50 . A diacylglycerol unit can be a precursor in the synthesis of diacyl PI in the soft coral P. arborea. The same synthetic way or the base-exchange reaction between diacyl PI and serine may lead to the formation of diacyl PS in P. arborea. Similarly to ether PS, diacyl PS molecular species are esterified by VLCFA at position sn-2. Recently, a biosynthetic relationship between PS and PI in hydrocoral species has been reported www.nature.com/scientificreports/ on the basis of lipidomic data 30 . The reaction between diacylglycerol and CDP-choline may produce diacyl PC. Possible pathways of synthesis of diacyl GPL in P. arborea are shown in Fig. 3. Thus, a lipidomic approach has shown the biosynthetic origins of ether and diacyl lipids, confirmed the general pathways of lipid biosynthesis, and revealed relationships between DAGE and GPL, as well as among GPL classes, in gorgonian corals.

Methods
Chemicals. The use of chemicals was described previously 28,47 .  www.nature.com/scientificreports/ Analyses of fatty acid, alkylglycerol, and molecular species compositions of DAGE. Fatty acid methyl esters (FAME) were obtained by acidic methanolysis of pure DAGE as described previously 28 . 4,4-Dimethyloxazoline (DMOX) derivatives of FA were prepared according to Svetashev 62 . A GC analysis of FAME and GC−MS analysis of FAME and DMOX derivatives were conducted according to Imbs et al. 28 The methods described by Rybin et al. 59  Quantitative analyses of molecular species and their identification using fragmentation pattern were performed on a Shimadzu LCMS-8060 triple-quadrupole mass spectrometer (Kyoto, Japan) operated in electrospray ionization (ESI) conditions. The temperature of the interface, heat block, and desolvation line was 300, 400, and 250 °C, respectively. The drying gas (N 2 ) flow was 10 L min −1 . The nebulizer gas (N 2 ) flow rate was 3 L min −1 . The heating gas (zero air) flow rate was 10 L min −1 . The negative ion mode was applied for analyzing PI, and the positive mode was applied for analyzing others polar lipid classes. CAEP, PE, and PS were detected by scanning for a neutral loss of 125, 141, and 185 Da, respectively, for their precursor ions [63][64][65]  To determine exact molecular masses of polar lipids, we carried out high-resolution tandem ion trap-time of flight mass spectrometry on a Shimadzu LCMS-IT-TOF instrument (Kyoto, Japan) operated in the positive and negative ion modes during each analysis in ESI conditions. The ion source temperature was 200 °C; the range of detection was m/z 600-1100; the potential in the ion source was − 3.5 for the negative mode and 4.5 kV for the positive mode. The drying gas (N 2 ) pressure was 100 kPa. The nebulizer gas (N 2 ) flow rate was 1.5 L min −1 . Precise molecular masses were used to calculate the brutto-formulas and distinguish diacyl lipids and ether lipids. The chemical structure of the polar lipid molecular species was identified as described earlier (Supplementary Fig. S7) 65,66 .
To confirm the presence of molecular species with the plasmalogen structure, the lipidomic profiles of samples were compared before and after mild acid hydrolysis. In brief, about 50 µg of the lipid sample was evaporated to dryness under argon stream in a glass vial (1 mL). The vial was placed bottom up on the drop of concentrated HCl for 3 min. Then HCl was blown off with argon stream, lipids dissolved in 50 µL of chloroform and analysed again by LC-MS. The disappearance of peaks of certain lipid molecular species on the chromatogram confirms the plasmalogen structure of these molecules. Statistical analysis. The analysis approach was recently described 28 . In brief, significance of differences between mean contents of DAGE and alkylglycerols was tested by one-way analysis of variance (ANOVA). Raw data were used following evaluation of the homogeneity of variances (Levene's test) and the normality of data distribution (Shapiro-Wilk test). For the cluster analysis, the unweighted pair-group method with arithmetic mean and the Euclidean distance as dissimilarity metric were applied to the percentage content of molecular species of DAGE and four GPL classes according to the structure of their non-polar parts. All statistical analyses were performed using STATISTICA 5.1 (StatSoft, Inc., USA). A statistical probability of p < 0.01 was considered significant. Values are represented as mean ± standard deviation.