Cloning and expression of a human choline/ethanolaminephosphotransferase: synthesis of phosphatidylcholine and phosphatidylethanolamine

Abstract

Cholinephosphotransferase catalyses the final step in the synthesis of phosphatidylcholine (PtdCho) via the Kennedy pathway by the transfer of phosphocholine from CDP-choline to diacylglycerol. Ethanolaminephosphotransferase catalyses an analogous reaction with CDP-ethanolamine as the phosphobase donor for the synthesis of phosphatidylethanolamine (PtdEtn). Together these two enzyme activities determine both the site of synthesis and the fatty acyl composition of PtdCho and PtdEtn synthesized de novo. A human choline/ethanolaminephosphotransferase cDNA (hCEPT1) was cloned, expressed and characterized. Northern blot analysis revealed one hCEPT1 2.3 kb transcript that was ubiquitous and not enriched, with respect to actin, in any particular cell type. The open reading frame predicts a protein (hCEPT1p) of 416 amino acid residues with a molecular mass of 46550 Da containing seven membrane-spanning domains. A predicted amphipathic helix resides within the active site of the enzyme with the final two aspartic residues of the CDP-alcohol phosphotransferase motif, DG(X)2AR(X)8G(X)3D(X)3D, positioned within this helix. hCEPT1p was successfully expressed in a full-length, active form in Saccharomyces cerevisiae cells devoid of endogenous cholinephosphotransferase or ethanolaminephosphotransferase activities (HJ091, cpt1::LEU2 ept1-). In vitro, hCEPT1p displayed broad substrate specificity, utilizing both CDP-choline and CDP-ethanolamine as phosphobase donors to a broad range of diacylglycerols, resulting in the synthesis of both PtdCho and PtdEtn. In vivo, S. cerevisiae cells (HJ091, cpt1::LEU2 ept1-) expressing hCEPT1 efficiently incorporated both radiolabelled choline and ethanolamine into phospholipids, demonstrating that hCEPT1p has the ability to synthesize both choline- and ethanolamine- containing phospholipids in vitro and in vivo.

Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells

“…Characterization of the ethanolaminephosphotransferase, and elucidation of its molecular structure, has been hampered by difficulties in purification of this integral membrane protein. However, a yeast gene that encodes a dual specificity ethanolamine/choline phosphotransferase activity was identified [188], and the human ortholog of this gene was subsequently cloned [187]. As is the case for the yeast enzyme, the human enzyme exhibits both choline-and ethanolamine-phosphotransferase activities and was originally proposed to be the sole protein catalyzing the final reaction of the CDP-ethanolamine pathway [187].…”

“…However, a yeast gene that encodes a dual specificity ethanolamine/choline phosphotransferase activity was identified [188], and the human ortholog of this gene was subsequently cloned [187]. As is the case for the yeast enzyme, the human enzyme exhibits both choline-and ethanolamine-phosphotransferase activities and was originally proposed to be the sole protein catalyzing the final reaction of the CDP-ethanolamine pathway [187]. However, another human ethanolaminephosphotransferase, that appears to be specific for CDP-ethanolamine, was more recently identified and is presumed to participate in the CDP-ethanolamine pathway for PE synthesis [189] although its distribution in mouse tissues (mainly in testis and small intestine) is more restricted than that of the dual-specificity enzyme.…”

“…These studies indicate that PE made by the CDP-ethanolamine pathway is required in non-hepatic tissues during mouse development. The final step of the CDP-ethanolamine pathway for PE synthesis is the reaction between diacylglycerol and CDP-ethanolamine, catalyzed by CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, an integral protein that resides primarily in ER membranes [72,74,187]. Characterization of the ethanolaminephosphotransferase, and elucidation of its molecular structure, has been hampered by difficulties in purification of this integral membrane protein.…”

Use of Phosphatide Precursors to Promote Synaptogenesis

“…CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT; EC 2.7.8.2) catalyzes the final reaction in the Kennedy cycle; it transfers the phosphocholine moiety from CDP-choline to DAG (198), thus yielding PC (Figure 3). CPT is an integral membrane protein that is now cloned in humans (75) and is present primarily in the endoplasmic reticulum (41). CPT may be a reversible enzyme, synthesizing CDPcholine from PC and CMP in microsomal preparations from liver (91, 92) and brain (68,69,146).…”

Lipids of mitochondria

“…104 2. Lipid composition of mitochondria 105 Each subcellular compartment of eukaryotes has a distinct set 106 of proteins and lipids with phosphatidylcholine (PC) and phospha- 107 tidylethanolamine (PE) being the most abundant phospholipids 108 found in membranes from yeast to mammalian cells (Tables 1-3; 109 and reviews Refs. [5,14,15]).…”