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Locations and contributions of the phosphotransferases EPT1 and CEPT1 to the biosynthesis of ethanolamine phospholipids.

ABSTRACT: The final step of the CDP-ethanolamine pathway is catalyzed by ethanolamine phosphotransferase 1 (EPT1) and choline/EPT1 (CEPT1). These enzymes are likely involved in the transfer of ethanolamine phosphate from CDP-ethanolamine to lipid acceptors such as 1,2-diacylglycerol (DAG) for PE production and 1-alkyl-2-acyl-glycerol (AAG) for the generation of 1-alkyl-2-acyl-glycerophosphoethanolamine. Here, we investigated the intracellular location and contribution to ethanolamine phospholipid (EP) biosynthesis of EPT1 and CEPT1 in HEK293 cells. Immunohistochemical analyses revealed that EPT1 localizes to the Golgi apparatus and CEPT1 to the ER. We created EPT1-, CEPT1-, and EPTI-CEPT1-deficient cells, and labeling of these cells with radio- or deuterium-labeled ethanolamine disclosed that EPT1 is more important for the de novo biosynthesis of 1-alkenyl-2-acyl-glycerophosphoethanolamine than is CEPT1. EPT1 also contributed to the synthesis of PE species containing the fatty acids 36:1, 36:4, 38:5, 38:4, 38:3, 40:6, 40:5, and 40:4. In contrast, CEPT1 was important for PE formation from shorter fatty acids such as 32:2, 32:1, 34:2, and 34:1. Brefeldin A treatment did not significantly affect the levels of the different PE species, indicating that the subcellular localization of the two enzymes is not responsible for their substrate preferences. In vitro enzymatic analysis revealed that EPT1 prefers AAG 16-20:4 > DAG 18:0-20:4 > DAG 16:0-18:1 = AAG 16-18:1 as lipid acceptors and that CEPT1 greatly prefers DAG 16:0-18:1 to other acceptors. These results suggest that EPT1 and CEPT1 differ in organelle location and are responsible for the biosynthesis of distinct EP species.

SUBMITTER: Horibata Y

PROVIDER: S-EPMC7397746 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The Saccharomyces cerevisiae CPT1 and EPT1 genes encode for a cholinephosphotransferase (CPT) and choline/ethanolaminephosphotransferase, respectively. Both Cpt1p and Ept1p activities display an absolute requirement for cations and phospholipids. A mixed-micelle assay was employed to determine cation and lipid activators of parental and chimaeric Cpt1p/Ept1p enzymes to gain insight into their mechanism(s) of activation. Mg2+, Mn2+ and Co2+ were the only cations capable of activating Cpt1p and Ept1p in vitro. Kinetic data revealed that only Mg2+ is present in appropriate amounts to activate CPT activity in vivo. Kinetic data revealed that only Mg2+ is present in appropriate amounts to activate CPT activity in vivo. The two enzymes displayed distinct activation profiles on the basis of their relative affinities for Mg2+, and Mn2+ and Co2+. This allowed the use of chimaeric enzymes to determine the mechanism of cation activation. Cations do not activate Cpt1p or Ept1p by complexing with the substrate, CDP-choline, but instead bind to disparate regions within the enzymes themselves. Cpt1p and Ept1p also displayed distinct phospholipid activation profiles. Phospholipid activation required a phosphate and/or glycero-phosphoester linkage, with the phospho-head group moiety positioned at the surface of the micelle. Assays with parental and chimaeric Cpt1p/Ept1p constructs revealed that the phospholipid binding/activation domains are not located within linear segments of the protein, but instead are contained within distinct and separate regions of the proteins that require an intact tertiary structure for formation. Phosphatidylcholine (and its structural analogue sphingomyelin) were the best lipid activators of Cpt1p, the main biologically relevant CPT activity in S. cerevisiae. Hence CPT displays product activation. Because phosphatidylcholine is an efficient activator of CPT activity (and hence Cpt1p is not subject to feedback inhibition by its product), Cpt1p is incapable of functioning as a direct monitor of membrane phosphatidylcholine composition.

Project description:?-Hydroxy-?,?-unsaturated aldehydes, generated by oxidative damage of polyunsaturated phospholipids, form pyrrole derivatives that incorporate the ethanolamine phospholipid (EP) amino group such as 2-pentylpyrrole (PP)-EP and 2-(?-carboxyalkyl)pyrrole (CAP)-EP derivatives: 2-(?-carboxyethyl)pyrrole (CEP)-EP, 2-(?-carboxypropyl)pyrrole (CPP)-EP, and 2-(?-carboxyheptyl)pyrrole (CHP)-EP. Because EPs occur in vivo in various forms, a complex mixture of pyrrole-modified EPs with different molecular weights is expected to be generated. To provide a sensitive index of oxidative stress, all of the differences in mass related to the glycerophospholipid moieties were removed by releasing a single CAP-ethanolamine (ETN) or PP-ETN from each mixture by treatment with phospholipase D. Accurate quantization was achieved using the corresponding ethanolamine-d4 pyrroles as internal standards. The product mixture obtained by phospholipolysis of total blood phospholipids from sickle cell disease (SCD) patients was analyzed by LC-MS/MS. The method was applied to measure CAP-EP and PP-EP levels in blood plasma from clinical monitoring of SCD patients. We found uniformly elevated blood levels of CEP-EP (63.9 ± 9.7 nM) similar to mean levels in blood from age-related macular degeneration (AMD) patients (56.3 ± 37.1 nM), and 2-fold lower levels (27.6 ± 3.6 nM, n = 5) were detected in plasma from SCD patients hospitalized to treat a sickle cell crisis, although mean levels remain higher than those (12.1 ± 10.5 nM) detected in blood from healthy controls. Plasma levels of CPP-EPs from SCD clinic patients were 4-fold higher than those of SCD patients hospitalized to treat a sickle cell crisis (45.1 ± 10.9 nM, n = 5 versus 10.9 ± 3.4 nM, n = 6; p < 0.002). PP-EP concentration in plasma from SCD clinic patients is nearly 4.8-fold higher than its level in plasma samples from SCD patients hospitalized to treat a sickle cell crisis (7.06 ± 4.05 vs 1.48 ± 0.92 nM; p < 0.05). Because CAP-EPs promote angiogenesis and platelet activation, the elevated levels present in SCD blood can contribute to the hypercoaguability and vaso-occlusive events that are critical pathophysiologic features of SCD.

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Locations and contributions of the phosphotransferases EPT1 and CEPT1 to the biosynthesis of ethanolamine phospholipids.

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