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

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Horibata Y, Ando H, and Sugimoto H. (2020) Locations and contributions of the phosphotransferases EPT1 and CEPT1 to the biosynthesis of ethanolamine phospholipids. Journal of Lipid Research.

One of the most abundant phospholipids in mammals is phosphatidylethanolamine (PE) contributing 20-40% of all cellular phospholipids. To produce these molecules, ethanolamine is phosphorylated to produce ethanolamine phospholipid, which is then transferred from CDP-ethanolamine to 1,2-diacylglycerol (DAG), producing PE. Another type of ethanolamine phospholipid in mammalian cells are ether lipids, including 1-alkenyl-1-acyl-glycerophosphoethanolamine (plasmenyl-PE), also called plasmalogens. Ether lipids make up around 20% of phospholipids, but within the brain they are the major contributor and are found as high as 80% of total ethanolamine phospholipids. As these classes make up such a large portion of the lipids in the human body, the ability to synthesize them is essential. Two phosphotransferases shown to be involved in the final step of PE biosynthesis and in the later stages of plasmalogen synthesis are choline/ethanolamine phosphotransferase1 (CEPT1) and ethanolamine phosphotransferase 1 (EPT1). Horibata et al determined the intercellular location of EPT1 and CEPT1 and whether their roles differed by making EPT1- and CEPT1-deficient human HEK293 cell lines and performed immunocytochemistry, enzymatic assays, and lipid analyses.

It has been well-established that CEPT1 is found in the endoplasmic reticulum (ER) and along the nuclear envelope, however the location of EPT1 in mammalian cells had not been previously identified. To determine the location of EPT1 and CEPT1, each gene was fused with a HA-tag at the C-terminus and then incubated with antibodies for the HA-tag, ER or the Golgi apparatus. The green colour associated with the HA-tagged CEPT1 co-localized with the ER antibody confirming its presence in this organelle. The fluorescence associated with the HA-tagged EPT1 localized with the Golgi apparatus antibody, indicating its presence in this organelle and, interestingly, that these two similar enzymes have different locations in the cell, seen in the figure below.

To determine the effect of knocking out the EPT1 and CEPT1 genes on their enzymatic activity, radiolabeled CDP-ethanolamine was used as a phosphoethanolamine donor and DAG 18:1-18:1 was used as a lipid acceptor. If any PE were produced, it would be radioactive and could be quantified to determine what was synthesized during the experiment time using thin layer chromatography (TLC). The phosphotransferase activities of the EPT1-deficient cell line were reduced by 90% and CEPT1-deficient cell line decreased by 25% compared to the wild-type cells. This suggests that EPT1 has a larger role in the biosynthesis of PE compared to CEPT1.

Biosynthesis of various ethanolamine phospholipid species was studied using the EPT1- and CEPT1-deficient cell lines to determine their respective roles. Radiolabeled ethanolamine allowed the de novo biosynthesis of any PE species to be analyzed. Based on the different banding profiles found when separating the lipids with TLC, it could be determined which species require either enzyme to be produced. The banding profiles were different between the deficient cell lines, indicating that EPT1 and CEPT1 contribute to the synthesis of different PE species. Through this analysis, Horibata et al also found that EPT1 is essential in the formation of plasmalogens, but not CEPT1. To provide more detail about the specific PE species produced by either enzyme using liquid chromatography-tandem mass spectrometry (LC-MS/MS). These results suggested that EPT1 is involved in the synthesis of fatty acid species including 32:2, 32:1, 34:2, and 34:1, while CEPT1 produced more longer fatty acids such as 36:1, 38:5, 38:4, 40:6, 40:5, and 40:4.

Horibata et al have confirmed the location of CEPT1 and EPT1 in mammalian cells and identified the roles that they have in the biosynthesis of PE, plasmalogens, and fatty acids. It was shown that CEPT1 is found within the ER and, for the first time, it has been documented that EPT1 localizes to the Golgi apparatus. EPT1 and CEPT1 being found in different organelles could be due to both needing to produce different lipid species or preventing redundancy by having two enzymes in different locations producing different essential lipid molecules. This was supported by the finding that each have larger roles in the production of different ethanolamine phospholipids: CEPT1 has a greater role in the synthesis of larger fatty acids and, interestingly, EPT1 is important for producing smaller fatty acids and plasmalogen biosynthesis. However, Horibata et al also state that the enzyme responsible for the final step of plasmalogen biosynthesis is within the ER, indicating that the plasmanyl-PE, the plasmalogen precursor produced by EPT1, would need to be trafficked to the ER before synthesis can be completed. Learning more about the biosynthetic steps for these lipids could elucidate further ambiguities about disorders caused by plasmalogen deficiency, especially through studying EPT1.

Figure 2: Immunocytochemistry of EPT1 and CEPT1 in HEK293 cells. HEK293 cells were transfected with the expression vector for CEPT1 (A) or EPT1 (B) tagged with HA at the C-terminus. After fixation, the cells were permeabilized with 0.1% Triton X-100 (w/v), then immunostained with anti-HA antibody, followed by anti-mouse IgG Alexa488 (green). The ER and Golgi apparatus were stained with anti-ERp72 and GM130 antibodies, respectively, followed by anti-rabbit IgG Alexa594 (red). Nuclei were stained with DAPI (blue). Bars indicate 10uM