A Single Enzyme Catalyzes Both Platelet-activating Factor Production and Membrane Biogenesis of Inflammatory Cells

Abstract

Platelet-activating factor (PAF) is a potent proinflammatory lipid mediator eliciting a variety of cellular functions. Lipid mediators, including PAF are produced from membrane phospholipids by enzymatic cascades. Although a G protein-coupled PAF receptor and degradation enzymes have been cloned and characterized, the PAF biosynthetic enzyme, aceyl-CoA:lyso-PAF acetyltransferase, has not been identified. Here, we cloned lyso-PAF acetyltransferase, which is critical in stimulusdependent formation of PAF. The enzyme is a 60-kDa microsomal protein with three putative membrane-spanning domains. The enzyme was induced by bacterial endotoxin (lipopolysaccharide), which was suppressed by dexamethasone treatment. Surprisingly, the enzyme catalyzed not only biosynthesis of PAF from lyso-PAF but also incorporation of arachidonoyl-CoA to produce PAF precursor membrane glycerophospholipids (lysophosphatidylcholine acyltransferase activity). Under resting conditions, the enzyme prefers arachidonoyl-CoA and contributes to membrane biogenesis. Upon acute inflammatory stimulation with lipopolysaccharide, the activated enzyme utilizes acetyl-CoA more efficiently and produces PAF. Thus, our findings provide a novel concept that a single enzyme catalyzes membrane biogenesis of inflammatory cells while producing a prophlogistic mediator in response to external stimuli.Platelet-activating factor (PAF 3 ; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a phospholipid mediator that activates a G protein-coupled receptor (1-3) and results in pleiotropic and potent biological effects, including platelet activation, airway constriction, and hypotension (1). PAF is synthesized in various cells and tissues via two distinct pathways, the de novo and remodeling pathways (2, 4, 5), and the latter is regulated by extracellular signals and plays a critical role in stimulus-coupled PAF biosynthesis (2, 4 -6). PAF synthesis induced by extracellular signals has been reported in murine peritoneal cells stimulated by calcium ionophore (7) or by PAF (8), in human eosinophils stimulated by fMet-Leu-Phe (9), in human neutrophils stimulated by acid stress (10), and in murine peritoneal macrophages stimulated by lipopolysaccharide (LPS) (11). In the remodeling pathway, the precursor of PAF, 1-Oalkyl-sn-glycero-3-phosphocholine (lyso-PAF), is synthesized from 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (1-alkyl-phosphatidylcholine; PC) by the action of phospholipase A 2 (2, 4, 12, 13). Subsequently, lyso-PAF is converted to PAF by acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAF acetyltransferase) (EC 2.3.1.67) (14). PAF is then rapidly degraded to lyso-PAF by PAF acetylhydrolases (15). Alternatively, lyso-PAF is again transformed into PC by the action of lysophosphatidylcholine (LPC) acyltransferase (2.3.1.23) (16).A G protein-coupled PAF receptor was cloned in our laboratory (17), and PAF acetylhydrolases have been cloned and characterized by others (18,19). Lyso-PAF acetyltransferase was initially demonstrated and partially characterized by ...

Signaling Role for Lysophosphatidylcholine Acyltransferase 3 in Receptor-Regulated Arachidonic Acid Reacylation Reactions in Human Monocytes

“…However, no enhanced lysoPC acyltransferase activity was observed in these experiments, suggesting that LPCAT2 participated in platelet-activating factor metabolism rather than in a more general fatty acid remodeling role. On the other hand, endogenous LPCAT activity in murine peritoneal macrophages was found to increase in response to bacterial LPS, although the basal expression of lyso-platelet-activating factor acetyltransferase/LPCAT2 was almost undetectable, suggesting that the LPCAT activity measured was due to other forms (53). In this regard, using siRNA technology, our studies suggest that the LPCAT form involved in AA reacylation in activated cells is LPCAT3 and that LPCAT2 appears to have, if any, only a minor role.…”

“…In human cells, it is LPCAT3 the form that appears to exhibit preference for AA, although also by linoleic acid (50,52). Shindou et al (53) have recently reported the increase of lyso-platelet-activating factor acetyltransferase activity in RAW364.7 cells transfected with the mouse lyso-platelet-activating factor acetyl transferase/LPCAT2 gene and stimulated with TLR agonists. However, no enhanced lysoPC acyltransferase activity was observed in these experiments, suggesting that LPCAT2 participated in platelet-activating factor metabolism rather than in a more general fatty acid remodeling role.…”

“…Four isoforms of LPCAT exist in mammalian cells, termed LPCAT1, LPCAT2, LPCAT3, and LPCAT4 (45-51), but only two of them, LPCAT2 and LPCAT3, have been documented to participate in AA reacylation reactions (49)(50)(51)(52)(53). Human peripheral blood monocytes express both LPCAT2 and LPCAT3 (data not shown).…”

Caenorhabditis elegans mboa-7, a Member of the MBOAT Family, Is Required for Selective Incorporation of Polyunsaturated Fatty Acids into Phosphatidylinositol

“…The rapid turnover of the sn-2 acyl chains of glycerophospholipids was originally described by Lands (Lands, 1958;Lands, 1960;Lands and Merkl, 1963;Merkl and Lands, 1963), who proposed that acyl moieties of membrane phospholipids are rapidly metabolized by the action of phospholipase A 2 s and subsequently by lysophospholipid acyltransferases (Lands' cycle). To date, at least two lysophosphatidylcholine acyltransferases (LPCAT) were cloned from mammals: LPCAT1) catalyzes the incorporation of saturated fatty acids into lysoThis article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-09 -0893) on December 19, 2007. phosphatidylcholine (lysoPC) (Chen et al, 2006;Nakanishi et al, 2006), and LPCAT2 catalyzes the incorporation of acetic acid and AA into lysoPC (Shindou et al, 2007). These enzymes have conserved motifs in common with the enzymes involved in the de novo phospholipid synthesis pathway (Kennedy pathway) such as acylglycerolphosphate acyltransferase and lysophosphatidic acid acyltransferase (LPAAT).…”

“…© 2008 by The American Society for Cell Biology phosphatidylcholine (lysoPC) (Chen et al, 2006;Nakanishi et al, 2006), and LPCAT2 catalyzes the incorporation of acetic acid and AA into lysoPC (Shindou et al, 2007). These enzymes have conserved motifs in common with the enzymes involved in the de novo phospholipid synthesis pathway (Kennedy pathway) such as acylglycerolphosphate acyltransferase and lysophosphatidic acid acyltransferase (LPAAT).…”