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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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Disruption of the plat1 gene in A. limacinum F26-b.(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 plat1-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 plat1 gene in A. limacinum F26-b. The PL fraction was prepared from A. limacinum F26-b before and after the disruption of the plat1 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.
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pone-0102377-g006: Disruption of the plat1 gene in A. limacinum F26-b.(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 plat1-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 plat1 gene in A. limacinum F26-b. The PL fraction was prepared from A. limacinum F26-b before and after the disruption of the plat1 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.

Mentions: A decrease in LPCAT activity was observed in plat1-disrupted mutants when activity was measured using LPC and 15:0-CoA or DHA-CoA as substrates; LPCAT activities were 90% and 80% lower than the wild-type when 15:0-CoA (Figure 6A) and 16:0-CoA (data not shown) were used, respectively, and 50% lower than the wild-type when DHA-CoA was used (Figure 6B). These results suggest that the acylation of LPC with saturated fatty acids was mainly catalyzed by PLAT1, while that with DHA was catalyzed by PLAT1 and other LPCATs in A. limacinum F26-b. The various LPLAT activities of plat1-disrupted mutants were examined using different LPLs and [14C]16:0-CoA. As shown in Figure 6C, the activities of not only LPCAT, but also LPEAT were significantly decreased after the disruption of the plat1 gene; however, those of LPSAT and LPAAT were not, which indicated that PLAT1 is likely to function in vivo as LPCAT and LPEAT, but not other LPLATs. LPIAT activity was found to increase in plat1-knockout mutants under the conditions used (Figure 6C).


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)

Disruption of the plat1 gene in A. limacinum F26-b.(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 plat1-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 plat1 gene in A. limacinum F26-b. The PL fraction was prepared from A. limacinum F26-b before and after the disruption of the plat1 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0102377-g006: Disruption of the plat1 gene in A. limacinum F26-b.(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 plat1-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 plat1 gene in A. limacinum F26-b. The PL fraction was prepared from A. limacinum F26-b before and after the disruption of the plat1 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.
Mentions: A decrease in LPCAT activity was observed in plat1-disrupted mutants when activity was measured using LPC and 15:0-CoA or DHA-CoA as substrates; LPCAT activities were 90% and 80% lower than the wild-type when 15:0-CoA (Figure 6A) and 16:0-CoA (data not shown) were used, respectively, and 50% lower than the wild-type when DHA-CoA was used (Figure 6B). These results suggest that the acylation of LPC with saturated fatty acids was mainly catalyzed by PLAT1, while that with DHA was catalyzed by PLAT1 and other LPCATs in A. limacinum F26-b. The various LPLAT activities of plat1-disrupted mutants were examined using different LPLs and [14C]16:0-CoA. As shown in Figure 6C, the activities of not only LPCAT, but also LPEAT were significantly decreased after the disruption of the plat1 gene; however, those of LPSAT and LPAAT were not, which indicated that PLAT1 is likely to function in vivo as LPCAT and LPEAT, but not other LPLATs. LPIAT activity was found to increase in plat1-knockout mutants under the conditions used (Figure 6C).

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