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Novel lysophospholipid acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b responsible for generation of palmitate-docosahexaenoate-phosphatidylcholine and phosphatidylethanolamine.

Abe E, Ikeda K, Nutahara E, Hayashi M, Yamashita A, Taguchi R, Doi K, Honda D, Okino N, Ito M - PLoS ONE (2014)

Bottom Line: 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.PLAT1 shows wide specificity for donor substrates as well as acceptor substrates in vitro, i.e, the enzyme can adopt lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophosphatidylinositol as acceptor substrates, and 15:0/16:0-CoA and DHA-CoA as donor substrates.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan.

ABSTRACT
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 Aurantiochytrium limacinum F26-b, was expressed in Saccharomyces cerevisiae, 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 in vitro, i.e, 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 in vitro experiment, only lysophosphatidylcholine acyltransferase and lysophosphatidylethanolamine acyltransferase activities were decreased in plat1-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 A. limacinum 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.

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Specificity of PLAT1 for acyl-CoAs (A) and LPLs (B, C).(A) FLAG-PLAT1 was purified using the ANTI-FLAG M2 Affinity Gel and 3X FLAG peptide to remove the endogenous LPLs as described in Materials and Methods. 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.
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pone-0102377-g005: Specificity of PLAT1 for acyl-CoAs (A) and LPLs (B, C).(A) FLAG-PLAT1 was purified using the ANTI-FLAG M2 Affinity Gel and 3X FLAG peptide to remove the endogenous LPLs as described in Materials and Methods. 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.

Mentions: The specificity of PLAT1 toward acyl-CoA donors was examined using [14C]LPC and various fatty acyl-CoAs. To remove the endogenous LPLAT activity of S. cerevisiae, recombinant FLAG-PLAT1 was purified by immunoprecipitation using the anti-FLAG antibody immobilized on the agarose gel, as shown in the ‘Materials and Methods’. PLAT1 showed wide specificity and adopted various acyl-CoAs such as CoAs conjugated with 15∶0, 16∶0 (palmitic acid), 18∶0 (stearic acid), 18:3n-3 (α-linolenic acid), 20:4n-6 (arachidonic acid, AA), and 22:6n-3 (DHA). Although PLAT1 appeared to prefer 15:0-CoA and 16:0-CoA, it also adopted PUFA-CoAs such as 18:3-CoA, 20:4-CoA, and 22:6-CoA (Figure 5A). The specificity of PLAT1 toward acceptor substrates was examined using [14C]16:0-CoA and various LPLs. PLAT1 adopted LPC, LPE, LPA, LPI, and LPS as acceptor substrates, which indicated that the specificity of PLAT1 toward acceptor substrates was quite broad in vitro (Figure 5B, C). PLAT1 showed the relatively strong activity toward LPC among the acceptor substrates examined under the conditions used (Figure 5C).


Novel lysophospholipid acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b responsible for generation of palmitate-docosahexaenoate-phosphatidylcholine and phosphatidylethanolamine.

Abe E, Ikeda K, Nutahara E, Hayashi M, Yamashita A, Taguchi R, Doi K, Honda D, Okino N, Ito M - PLoS ONE (2014)

Specificity of PLAT1 for acyl-CoAs (A) and LPLs (B, C).(A) FLAG-PLAT1 was purified using the ANTI-FLAG M2 Affinity Gel and 3X FLAG peptide to remove the endogenous LPLs as described in Materials and Methods. 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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4121067&req=5

pone-0102377-g005: Specificity of PLAT1 for acyl-CoAs (A) and LPLs (B, C).(A) FLAG-PLAT1 was purified using the ANTI-FLAG M2 Affinity Gel and 3X FLAG peptide to remove the endogenous LPLs as described in Materials and Methods. 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.
Mentions: The specificity of PLAT1 toward acyl-CoA donors was examined using [14C]LPC and various fatty acyl-CoAs. To remove the endogenous LPLAT activity of S. cerevisiae, recombinant FLAG-PLAT1 was purified by immunoprecipitation using the anti-FLAG antibody immobilized on the agarose gel, as shown in the ‘Materials and Methods’. PLAT1 showed wide specificity and adopted various acyl-CoAs such as CoAs conjugated with 15∶0, 16∶0 (palmitic acid), 18∶0 (stearic acid), 18:3n-3 (α-linolenic acid), 20:4n-6 (arachidonic acid, AA), and 22:6n-3 (DHA). Although PLAT1 appeared to prefer 15:0-CoA and 16:0-CoA, it also adopted PUFA-CoAs such as 18:3-CoA, 20:4-CoA, and 22:6-CoA (Figure 5A). The specificity of PLAT1 toward acceptor substrates was examined using [14C]16:0-CoA and various LPLs. PLAT1 adopted LPC, LPE, LPA, LPI, and LPS as acceptor substrates, which indicated that the specificity of PLAT1 toward acceptor substrates was quite broad in vitro (Figure 5B, C). PLAT1 showed the relatively strong activity toward LPC among the acceptor substrates examined under the conditions used (Figure 5C).

Bottom Line: 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.PLAT1 shows wide specificity for donor substrates as well as acceptor substrates in vitro, i.e, the enzyme can adopt lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophosphatidylinositol as acceptor substrates, and 15:0/16:0-CoA and DHA-CoA as donor substrates.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan.

ABSTRACT
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 Aurantiochytrium limacinum F26-b, was expressed in Saccharomyces cerevisiae, 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 in vitro, i.e, 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 in vitro experiment, only lysophosphatidylcholine acyltransferase and lysophosphatidylethanolamine acyltransferase activities were decreased in plat1-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 A. limacinum 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.

Show MeSH
Related in: MedlinePlus