Abstract
Acyl-CoA-dependent lysophospholipid acyltransferases play an important role in attaining the appropriate molecular species of phospholipids. A number of genes encoding these activities were recently identified. It has become clear that multiple genes can encode one enzymatic activity and that a given gene may encode multiple activities. Here we report the identification of a gene encoding a mammalian acyl-CoA-dependent lysophospholipid acyltransferase with prominent activity toward ethanolamine-containing lysophospholipids, which we termed acyl-CoA:lysophosphatidylethanolamine acyltransferase 2, LPEAT2 (previously annotated as AYTL3 or AGPAT7). LPEAT2 is predominantly expressed in brain, coinciding with an enrichment of phosphatidylethanolamine in this tissue. Ectopic expression of LPEAT2 in mammalian HEK293T cells led to a dramatic increase (up to 9-fold) in LPEAT activity when compared with cells transfected with empty vector or an unrelated acyltransferase. LPEAT2 also exhibited significant acyl-CoA-dependent acyltransferase activity toward 1-O-alkenyl-lysophosphatidylethanolamine, lysophosphatidylglycerol, 1-O-alkyl-lysophosphatidylcholine, lysophosphatidylserine, and lysophosphatidylcholine but lacked appreciable acylating activity toward glycerol 3-phosphate, lysophosphatidic acid, lysophosphatidylinositol, and diacylglycerol, demonstrating multiple but selective functions of LPEAT2 as an enzyme involved in phospholipid remodeling. LPEAT2 recognizes a broad range of medium and long chain fatty acyl-CoA, and its activity was not affected by Ca(2+). When overexpressed in mammalian cells, LPEAT2 is localized to the endoplasmic reticulum. siRNA-mediated knockdown of LPEAT2 in HEK293T cells significantly decreased LPEAT and 1-alkenyl-LPEAT activities but did not affect other lysophospholipid acylating activities. These findings identify LPEAT2 as an important enzyme in the biosynthesis of ethanolamine-containing phospholipids, especially in brain.
Highlights
Phospholipids are the major constituents of biological membranes, playing important roles in multiple cellular processes including maintenance of the cellular permeability barrier, regulation of the activities of proteins associated with the membrane, and regulation of intracellular signaling by serving as precursors of signaling molecules [1, 2]
We show that one of these orphan genes, previously named 1-acylglycerol 3-phosphate acyltransferase 7 (AGPAT7) or lysophosphatidic acid acyltransferase (LPAAT) [17], encodes a new lysophospholipid acyltransferase that most prominently utilizes 1-acyllysophosphatidylethanolamine (LysoPtdEtn) and 1-alkenylLysoPtdEtn as substrates
Identification and Cloning of the Human lysophosphatidylethanolamine acyltransferase 2 (LPEAT2) Gene—Using a seed alignment sequence derived from the previously described glycerolipid acyltransferase motif (PF01553) [19], we have previously generated a comprehensive list of human, mouse, and rat genes containing the glycerolipid acyltransferase motif (PF01553) [16]
Summary
Materials—Unless stated otherwise, all lipids and acyl-CoAs were purchased from Avanti Polar Lipids (Alabaster, AL) or Sigma. A mammalian expression vector for full-length human LPEAT2 was engineered by subcloning the insert into EcoRV/NotI sites of the pcDNA3.1/V5-His vector (Invitrogen). HEK293T cells were transiently transfected with the LPEAT2 cDNA-containing vector or empty vector by using FuGENE 6 (Roche Applied Science). To express LPEAT2 in a bacterial system, N-terminally FLAG-tagged LPEAT2 cDNA in pcDNA3.1/V5-His was subcloned into EcoRI and NotI sites of the pET21a vector (Novagen) to ensure an in-frame translation of full-length protein. Immunofluorescence—Indirect immunofluorescence was performed in HEK293T cells or Neural 2A cells transfected with FLAG-tagged LPEAT essentially as described [16]. 48 h after transfection, LPEAT2 mRNA was measured by quantitative PCR, and lysophospholipid acyltransferase activities were determined as described above. Statistical Analysis—Statistical significance was determined by Student’s t test
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