Abstract
Choline phospholipids (PLs) such as phosphatidylcholine (PC) and 1-alkyl-2-acyl-sn-glycerophosphocholine are important components for cell membranes and also serve as a source of several lipid mediators. These lipids are biosynthesized in mammals in the final step of the CDP-choline pathway by the choline phosphotransferases choline phosphotransferase 1 (CPT1) and choline/ethanolamine phosphotransferase 1 (CEPT1). However, the contributions of these enzymes to the de novo biosynthesis of lipids remain unknown. Here, we established and characterized CPT1- and CEPT1-deficient human embryonic kidney 293 cells. Immunohistochemical analyses revealed that CPT1 localizes to the trans-Golgi network and CEPT1 to the endoplasmic reticulum. Enzyme assays and metabolic labeling with radiolabeled choline demonstrated that loss of CEPT1 dramatically decreases choline PL biosynthesis. Quantitative PCR and reintroduction of CPT1 and CEPT1 revealed that the specific activity of CEPT1 was much higher than that of CPT1. LC-MS/MS analysis of newly synthesized lipid molecular species from deuterium-labeled choline also showed that these enzymes have similar preference for the synthesis of PC molecular species, but that CPT1 had higher preference for 1-alkyl-2-acyl-sn-glycerophosphocholine with PUFA than did CEPT1. The endogenous level of PC was not reduced by the loss of these enzymes. However, several 1-alkyl-2-acyl-sn-glycerophosphocholine molecular species were reduced in CPT1-deficient cells and increased in CEPT1-deficient cells when cultured in 0.1% FBS medium. These results suggest that CEPT1 accounts for most choline PL biosynthesis activity, and that both enzymes are responsible for the production of different lipid molecular species in distinct organelles.
Highlights
Choline phospholipids (PLs) such as phosphatidylcholine (PC) and 1-alkyl-2-acyl-sn-glycerophosphocholine are important components for cell membranes and serve as a source of several lipid mediators
choline phosphotransferase 1 (CPT1) is distributed in the trans-Golgi network (TGN), whereas choline/ethanolamine phosphotransferase 1 (CEPT1) is localized in the endoplasmic reticulum (ER)
We compared the intracellular distribution of CPT1 and CEPT1 in the cells by transfecting the expression vectors encoding CPT1 or CEPT1 fused with an HA tag at the C terminus into human embryonic kidney 293 (HEK293) cells, staining with anti-HA-tag antibody followed by the second antibody conjugated with Alexa Fluor 488
Summary
Choline phospholipids (PLs) such as phosphatidylcholine (PC) and 1-alkyl-2-acyl-sn-glycerophosphocholine are important components for cell membranes and serve as a source of several lipid mediators These lipids are biosynthesized in mammals in the final step of the CDP-choline pathway by the choline phosphotransferases choline phosphotransferase 1 (CPT1) and choline/ethanolamine phosphotransferase 1 (CEPT1). Several 1-alkyl-2-acyl-sn-glycerophosphocholine molecular species were reduced in CPT1-deficient cells and increased in CEPT1-deficient cells when cultured in 0.1% FBS medium These results suggest that CEPT1 accounts for most choline PL biosynthesis activity, and that both enzymes are responsible for the production of different lipid molecular species in distinct organelles. Kinetic analysis revealed that CEPT1 prefers CDP-choline over CDPethanolamine as the phosphobase donor [9, 10] Another type of choline PL in mammalian cells is ether-linked lipids such as 1-alkyl-2-acyl-sn-glycerophosphocholine (plasmanyl-PC) and 1-alkenyl-2-acyl-snglycerophosphocholine (plasmenyl-PC). It is widely accepted that plasmanyl-PC is distributed in most tissues, whereas plasmenyl-PC is distributed in limited organs such as the heart and skeletal muscle [14]
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