ラビリンチュラ類のDHA含有グリセロ脂質生合成機構に関する研究

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The glycerol-3-phosphate acyltransferase PLAT2 functions in the generation of DHA-rich glycerolipids in Aurantiochytrium limacinum F26-b.

Thraustochytrids possess docosahexaenoic acid (DHA, 22:6n-3) as acyl chain(s) of triacylglycerol (TG) and phosphatidylcholine (PC), some of which contain multiple DHAs. However, little is known about how these DHA-rich glycerolipids are produced in thraustochytrids. In this study, we identified PLAT2 in Aurantiochytrium limacinum F26-b as a glycerol-3-phosphate (G3P) acyltransferase (GPAT) by heterologous expression of the gene in budding yeast. Subsequently, we found that GPAT activity was reduced by disruption of the PLAT2 gene in A. limacinum, resulting in a decrease in DHA-containing lysophosphatidic acid (LPA 22:6). Conversely, overexpression of PLAT2 increased both GPAT activity and LPA 22:6. These results indicate that PLAT2 is a GPAT that transfers DHA to G3P in vivo as well as in vitro. Overexpression of the PLAT2 gene increased the production of a two DHA-containing diacylglycerol (DG 44:12), followed by an increase in the three DHA-containing TG (TG 66:18), two-DHA-containing TG (TG 60:12), and two DHA-containing PC (PC 44:12). However, overexpression of PLAT2 did not increase DHA-free DG (DG32:0), which was preferentially converted to three 16:0-containing TG (TG 48:0) but not two 16:0-containing PC (PC 32:0). Collectively, we revealed that DHA-rich glycerolipids are produced from a precursor, LPA 22:6, which is generated by incorporating DHA to G3P by PLAT2 in the A. limacinum

Correction: Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate-Docosahexaenoate-Phosphatidylcholine and Phosphatidylethanolamine.

[This corrects the article DOI: 10.1371/journal.pone.0102377.]

Novel Lysophospholipid Acyltransferase PLAT1 of <i>Aurantiochytrium limacinum</i> F26-b Responsible for Generation of Palmitate-Docosahexaenoate-Phosphatidylcholine and Phosphatidylethanolamine

<div><p>N-3 polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA, 22:6n-3), have been reported to play roles in preventing cardiovascular diseases. The major source of DHA is fish oils but a recent increase in the global demand of DHA and decrease in fish stocks require a substitute. Thraustochytrids, unicellular marine protists belonging to the Chromista kingdom, can synthesize large amounts of DHA, and, thus, are expected to be an alternative to fish oils. DHA is found in the acyl chain(s) of phospholipids as well as triacylglycerols in thraustochytrids; however, how thraustochytrids incorporate DHA into phospholipids remains unknown. We report here a novel lysophospholipid acyltransferase (PLAT1), which is responsible for the generation of DHA-containing phosphatidylcholine and phosphatidylethanolamine in thraustochytrids. The PLAT1 gene, which was isolated from the genomic DNA of <i>Aurantiochytrium limacinum</i> F26-b, was expressed in <i>Saccharomyces cerevisiae</i>, and the FLAG-tagged recombinant enzyme was characterized after purification with anti-FLAG affinity gel. PLAT1 shows wide specificity for donor substrates as well as acceptor substrates <i>in vitro</i>, <i>i.e</i>, the enzyme can adopt lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophosphatidylinositol as acceptor substrates, and 15:0/16:0-CoA and DHA-CoA as donor substrates. In contrast to the <i>in vitro</i> experiment, only lysophosphatidylcholine acyltransferase and lysophosphatidylethanolamine acyltransferase activities were decreased in <i>plat1</i>-knockout mutants, resulting in a decrease of 16:0-DHA-phosphatidylcholine (PC) [PC(38∶6)] and 16:0-DHA-phosphatidylethanolamine (PE) [PE(38∶6)], which are two major DHA-containing phospholipids in <i>A. limacinum</i> F26-b. However, the amounts of other phospholipid species including DHA-DHA-PC [PC(44∶12)] and DHA-DHA-PE [PE(44∶12)] were almost the same in plat-knockout mutants and the wild-type. These results indicate that PLAT1 is the enzyme responsible for the generation of 16:0-DHA-PC and 16:0-DHA-PE in the thraustochytrid.</p></div

Phylogenetic tree of LPLAT family members.

<p>The phylogenetic tree was drawn using CLUSTALW, DDBJ (<a href="http://clustalw.ddbj.nig.ac.jp/top-j.html" target="_blank">http://clustalw.ddbj.nig.ac.jp/top-j.html</a>). LPLAT sequences are available on the NCBI database. The accession numbers are as follows: mGPAT1 (NP_032175), mGPAT2 (XP_130488), mGPAT3/LPAATθ (NP_766303), mLPAATα (NP_061350), mLPAATβ (NP_080488), mLPAATγ (NP_443747), mLPAATδ (NP_080920), mLPAATε (NP_081068), mGPAT4/LPAATζ (NP_061213), mAT Like 1B (NP_081875), mLPGAT1 (NP_758470), mALCAT (Q3UN02), mLPCAT1 (BAE94687), mLysoPAFAT/LPCAT2 (BAF47695), mTafazzin (NP_852657), mMGAT1 (NP_080989), mMGAT2 (NP_803231), mDGAT2 (NP_080660), mDGAT2Like3 (NP_001074605), mDGAT2Like4 (NP_808414), mDGAT2Like6 (CAM19588), mLPCAT3/MBOAT5 (NP_660112), mLPCAT4/MBOAT2 (NP_080313), mLPEAT1/MBOAT1 (NP_705774), mMBOAT4 (XP_134120), mDGAT1 (NP_034176), mACAT1 (NP_033256), mACAT2 (NP_666176), mPorcupine-a (NP_058609), mLRC4 (NP_084210), sLpt1 (BAF93897), and sAle1 (EWH15997); s, <i>Saccharomyces cerevisiae</i>, m, <i>Mus musculus</i>.</p

Specificity of PLAT1 for acyl-CoAs (A) and LPLs (B, C).

<p>(A) FLAG-PLAT1 was purified using the ANTI-FLAG M2 Affinity Gel and 3X FLAG peptide to remove the endogenous LPLs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102377#s2" target="_blank">Materials and Methods</a>. Each assay contained 25 µM acyl-CoA, 1 µM [1-¹⁴C]palmitoyl-LPC, and purified enzyme (10 µl of gel suspension) in 100 µl of 100 mM Tris-HCl, pH 7.5, containing 1 mM EDTA and 0.01% sodium cholate. The reaction was conducted at 30°C for 20 min and terminated by the addition of 500 µl of CHCl₃/CH₃OH (2∶1, v/v). The reaction mixture was loaded on the TLC plate and developed with CHCl₃/CH₃OH/H₂O (65/25/4, v/v/v). The radioactivity of the band corresponding to PC on the TLC plate was quantified by a FLA 5100 Bio-imaging analyzer. Data represent the mean ± SD (n = 3). (B) Each assay contained 1 µM of LPLs, 1 µM [1-¹⁴C]palmitoyl-CoA, and the purified enzyme (10 µl of gel suspension) in 100 µl of 100 mM Tris-HCl, pH 7.5, containing 1 mM EDTA and 0.01% sodium cholate. Mock represents the experiment using the enzyme prepared from the lysate of yeast cells harboring the empty vector. The assay was conducted at 30°C for 20 min and terminated by the addition of 500 µl of CHCl₃/CH₃OH (2∶1, v/v). The reaction mixture was loaded on the TLC plate, developed with CHCl₃/CH₃OH/H₂O (65/25/4, v/v/v), and analyzed with a FLA 5100 Bio-imaging analyzer. The typical TLC is presented here. (C) The enzyme reaction was performed by the same procedure as shown in (B). The reaction products were applied on TLC plates and the radioactivity of the band corresponding to each PL was quantified by a FLA 5100 Bio-imaging analyzer (n = 3). Data represent the mean ± SD.</p

Metabolic map of PLs (A) and comparison of the specificity of PLAT1 with those of other LPCATs (B, C).

<p>Panel A represents the LPLATs shown in this study along with the metabolic map of PLs in mammals. The specificity of PLAT1 for acyl-CoAs (B) and LPLs (C) was compared with those of the LPCATs reported so far. The darker color represents higher activity toward the substrates indicated. White (blank) and ND show the absence of activity and not determined, respectively. The specificities of LPCATs are mainly referred from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102377#pone.0102377-Hishikawa1" target="_blank">[27]</a>.</p

Western blot (A), intracellular localization (B), activity (C), and Ca²⁺-dependency (D) of FLAG-PLAT1 expressed in budding yeasts.

<p>(A) Western blot of FLAG-tagged PLAT1 at the N-terminal (FLAG-PLAT1). FLAG-PLAT1, expressed in yeast, was detected on 10% SDS-PAGE using the anti-FLAG mouse monoclonal antibody as the primary antibody, followed by HRP-conjugated anti-mouse IgG antibody as the secondary antibody. (B) The localization of PLAT1 was estimated by Western blot using the microsomal and cytosolic fractions of FLAG-PLAT1-expressing yeast cells. (C) LPCAT activity was measured based on the incorporation of 16∶0 from 16:0-CoA into [1-¹⁴C]palmitoyl-LPC. The reaction mixture contained 25 µM 16:0-CoA and 1 µM [1-¹⁴C]palmitoyl-LPC, and 10 µg of cell protein (cell lysate) in 100 µl of reaction buffer (100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.01% sodium cholate). Mock represents the experiment using the enzyme prepared from the lysate of yeast cells harboring the empty vector. The reaction was conducted at 30°C for 20 min and terminated by the addition of 500 µl of CHCl₃/CH₃OH (2∶1, v/v). The reaction mixture was loaded on the TLC plate, developed with CHCl₃/CH₃OH/H₂O (65/25/4, v/v/v), and analyzed with a FLA 5100 Bio-imaging analyzer. (D) LPCAT activity (left column) was measured by the method described in the legend (C), while the reaction mixture contained 1 mM EGTA (center column) or 1 mM CaCl₂ (right column) instead of EDTA. The radioactivity of the band corresponding to PC on the TLC plate was quantified by a FLA 5100 Bio-imaging analyzer. Data represent the mean ± SD (n = 3). Blue and red bars represent the FLAG-PLAT1 and mock, respectively.</p

Disruption of the <i>plat1</i> gene in <i>A. limacinum</i> F26-b.

<p>(A, B) LPCAT activity was measured using 25 µM of 15:0-CoA (A) or DHA-CoA (B), 1 µM [1-¹⁴C]palmitoyl-LPC and 10 µg of the cell protein (cell lysate) from the <i>plat1</i>-knockout mutant (KO; red column) or wild-type (WT; blue column). (C) Various LPLAT activities were measured using 25 µM of the corresponding LPLs, 1 µM [1-¹⁴C]palmitoyl-CoA, 10 µg of the cell protein from KO (red column) or WT (blue column). Data represent the mean ± SD (n = 3). * and ** represent p<0.05 and not significant (p>0.10), respectively. (D) Change in the molecular species of PC and PE after the disruption of the <i>plat1</i> gene in <i>A. limacinum</i> F26-b. The PL fraction was prepared from <i>A. limacinum</i> F26-b before and after the disruption of the <i>plat1</i> gene. PLs were analyzed by Chip-based nanoESI-MS using a 4000Q TRAP with chip-based ionization source, TriVersa NanoMate (Advion BioSystems, Ithaca, NY, USA). The intensities at m/z 850 for 16:0-22:6-PC [PC(38∶6)], m/z 764 for 16:0-22:6-PE [PE(38∶6)], m/z 922 for 22:6-22:6-PC [PC(44∶12)], and m/z 836 for 22:6-22:6-PE [PE(44∶12)] were extracted and analyzed. The intensity of PL species of KO (red column) was expressed as a percentage of that of WT (blue column). Data represent the mean ± SD for 3 independent experiments (n = 1). * and ** represent p<0.05 and not significant (p>0.10), respectively.</p

Identification of strain F26-b by 18S r RNA gene.

<p>A neighbor-joining tree was drawn based on 18S rRNA gene sequences of the genus <i>Aurantiochytrium</i> with five strains of other thraustochytrid genera as outgroups. Bootstrap values (>50%, 1,000 replicates) are shown on each internal branch. GenBank/DDBJ accession numbers of each sequence are indicated in parentheses.</p