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Publication
Journal: Journal of Biological Chemistry
December/28/2008
Abstract
The cycle of deacylation and reacylation of phospholipids plays a critical role in regulating availability of arachidonic acid for eicosanoid production. The major yeast lysophospholipid acyltransferase, Ale1p, is related to mammalian membrane-bound O-acyltransferase (MBOAT) proteins. We expressed four human MBOATs in yeast strains lacking Ale1p and studied their acyl-CoA and lysophospholipid specificities using novel mass spectrometry-based enzyme assays. MBOAT1 is a lysophosphatidylserine (lyso-PS) acyltransferase with preference for oleoyl-CoA. MBOAT2 also prefers oleoyl-CoA, using lysophosphatidic acid and lysophosphatidylethanolamine as acyl acceptors. MBOAT5 prefers lysophosphatidylcholine and lyso-PS to incorporate linoleoyl and arachidonoyl chains. MBOAT7 is a lysophosphatidylinositol acyltransferase with remarkable specificity for arachidonoyl-CoA. MBOAT5 and MBOAT7 are particularly susceptible to inhibition by thimerosal. Human neutrophils express mRNA for these four enzymes, and neutrophil microsomes incorporate arachidonoyl chains into phosphatidylinositol, phosphatidylcholine, PS, and phosphatidylethanolamine in a thimerosal-sensitive manner. These results strongly implicate MBOAT5 and MBOAT7 in arachidonate recycling, thus regulating free arachidonic acid levels and leukotriene synthesis in neutrophils.
Publication
Journal: Genes to Cells
October/22/2008
Abstract
Glycerophospholipids in biological membranes are metabolically active and participate in a series of deacylation-reacylation reactions, which may lead to accumulation of polyunsaturated fatty acids (PUFAs) at the sn-2 position of the glycerol backbone. The reacylation reaction is believed to be catalyzed by acyl-coenzyme A (acyl-CoA):lysophospholipid acyltransferase. Very recently, we have shown that Caenorhabditis elegans mboa-7, which belongs to the membrane-bound O-acyltransferase (MBOAT) family, encodes lysophosphatidylinositol (LPI)-specific acyltransferase (LPIAT). In this study, we found that knockdown of another member of the MBOAT family in C. elegans, named mboa-6, reduced incorporation of exogenous PUFAs into phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE) in C. elegans. Knockdown of a human mboa-6 homologue, referred to as MBOAT5, also impaired the incorporation of PUFAs into PC, PS and PE in HeLa cells. In in vitro assays, lysoPC (LPC), lysoPS (LPS) and lysoPE (LPE) acyltransferase activities using [(14)C]arachidonoyl-CoA were significantly reduced in the microsomes of MBOAT5 knockdown cells. Conversely, over-expression of MBOAT5 in human embryonic kidney (HEK) 293 cells resulted in great increases in LPC, LPS and LPE acyltransferase activities but not in LPIAT or lysophosphatidic acid (LPA) acyltransferase (LPAAT) activities. These results indicate that human MBOAT5 is a lysophospholipid acyltransferase acting preferentially on LPC, LPS and LPE.
Publication
Journal: Prostaglandins and Other Lipid Mediators
August/16/2015
Abstract
Eicosanoids derived from the enzymatic oxidation of arachidonic acid play important roles in a large number of physiological and pathological processes in humans. Many animal and cellular models have been used to investigate the intricate mechanisms regulating their biosynthesis and actions. Zebrafish is a widely used model to study the embryonic development of vertebrates. It expresses homologs of the key enzymes involved in eicosanoid production, and eicosanoids have been detected in extracts from adult or embryonic fish. In this study we prepared cell suspensions from kidney marrow, the main hematopoietic organ in fish. Upon stimulation with calcium ionophore, these cells produced eicosanoids including PGE2, LTB4, 5-HETE and, most abundantly, 12-HETE. They also produced small amounts of LTB5 derived from eicosapentaenoic acid. These eicosanoids were also produced in kidney marrow cells stimulated with ATP, and this production was greatly enhanced by preincubation with thimerosal, an inhibitor of arachidonate reacylation into phospholipids. Microsomes from these cells exhibited acyltransferase activities consistent with expression of MBOAT5/LPCAT3 and MBOAT7/LPIAT1, the main arachidonoyl-CoA:lysophospholipid acyltransferases. In summary, this work introduces a new cellular model to study the regulation of eicosanoid production through a phospholipid deacylation-reacylation cycle from a well-established, versatile vertebrate model species.