Coenzyme-A-Independent Transacylation System; Possible Involvement of Phospholipase A2 in Transacylation

The coenzyme A (CoA)-independent transacylation system catalyzes fatty acid transfer from phospholipids to lysophospholipids in the absence of cofactors such as CoA. It prefers to use C20 and C22 polyunsaturated fatty acids such as arachidonic acid, which are esterified in the glycerophospholipid at the sn-2 position. This system can also acylate alkyl ether-linked lysophospholipids, is involved in the enrichment of arachidonic acid in alkyl ether-linked glycerophospholipids, and is critical for the metabolism of eicosanoids and platelet-activating factor. Despite their importance, the enzymes responsible for these reactions have yet to be identified. In this review, we describe the features of the Ca2+-independent, membrane-bound CoA-independent transacylation system and its selectivity for arachidonic acid. We also speculate on the involvement of phospholipase A2 in the CoA-independent transacylation reaction.


Fatty Acid Remodeling System of Glycerophospholipids
Various types of fatty acids are present in each glycerophospholipid at the sn-1 or -2 position of the glycerol backbone. Observation of differences in turnover rates of fatty acyl and glycerol moieties of phospholipids led to the identification of acyl-CoA:lysophospholipid acyltransferase in rat liver and the subsequent discovery of the fatty acid remodeling system (Table 2 and Figure 2) [14,15], which increases the diversity of fatty acids in phospholipids [1][2][3][4][5][6]. Fatty acids are first incorporated into glycerophospholipids during de novo synthesis; some polyunsaturated fatty acids (PUFAs) such as arachidonic acid (5,8,11,14-eicosatetraenoic acid, 20:4 n-6) are then incorporated by the remodeling system. Stearic acid (octadecanoic acid, 18:0) is also incorporated at the sn-1 position during the remodeling process.
The remodeling system includes acyltransferases and transacylation of lysophospholipids that catalyze fatty acid transfer between an acyl donor and acceptor lysophospholipid ( Table 2) [4][5][6][7][8][9][10][11][12][13]. The acyl donors for acyl-CoA:lysophospholipid acyltransferases are fatty acyl-CoAs, the activated forms of fatty acids that facilitate fatty acid transfer to acceptor molecules. The activation of free fatty acids consumes ATP. In contrast, the transacylation system employs the esterified fatty acids in phospholipids as acyl donors that are transferred to acceptor lysophospholipids. This transacylation reaction is essentially ATP-independent.

Fatty Acid Remodeling System of Glycerophospholipids
Various types of fatty acids are present in each glycerophospholipid at the sn-1 or -2 position of the glycerol backbone. Observation of differences in turnover rates of fatty acyl and glycerol moieties of phospholipids led to the identification of acyl-CoA:lysophospholipid acyltransferase in rat liver and the subsequent discovery of the fatty acid remodeling system (Table 2 and Figure 2) [14,15], which increases the diversity of fatty acids in phospholipids [1][2][3][4][5][6]. Fatty acids are first incorporated into glycerophospholipids during de novo synthesis; some polyunsaturated fatty acids (PUFAs) such as arachidonic acid (5,8,11,14-eicosatetraenoic acid, 20:4 n-6) are then incorporated by the remodeling system. Stearic acid (octadecanoic acid, 18:0) is also incorporated at the sn-1 position during the remodeling process.
The remodeling system includes acyltransferases and transacylation of lysophospholipids that catalyze fatty acid transfer between an acyl donor and acceptor lysophospholipid ( Table 2) [4][5][6][7][8][9][10][11][12][13]. The acyl donors for acyl-CoA:lysophospholipid acyltransferases are fatty acyl-CoAs, the activated forms of fatty acids that facilitate fatty acid transfer to acceptor molecules. The activation of free fatty acids consumes ATP. In contrast, the transacylation system employs the esterified fatty acids in phospholipids as acyl donors that are transferred to acceptor lysophospholipids. This transacylation reaction is essentially ATP-independent. Exogenous arachidonic acid is not typically introduced into phospholipids via de novo synthesis but is instead incorporated during fatty acid remodeling [3][4][5][6][7][8][9][10][11][12][13]. This is important for the biosynthesis of lipid messengers since arachidonic acid is the precursor of eicosanoids such as prostaglandins and leukotrienes. In de novo synthesis of phospholipids, fatty acids that are saturated or exhibit a low degree of unsaturation, such as oleic acid (9-octadecenoic acid, 18:1 n-9), are often incorporated at the sn-2 positions of phospholipids. Their hydrolysis by phospholipase A2 (PLA2) releases the fatty acid, and the resultant lysophospholipids are acylated by acyl-CoA:lysophospholipid acyltransferases. Since acyltransferases prefer PUFA-CoAs such as arachidonoyl-CoA, PUFAs accumulate in the sn-2 positions of phospholipids during the deacylation-reacylation cycle (Lands cycle) ( Figure 2).  Exogenous arachidonic acid is not typically introduced into phospholipids via de novo synthesis but is instead incorporated during fatty acid remodeling [3][4][5][6][7][8][9][10][11][12][13]. This is important for the biosynthesis of lipid messengers since arachidonic acid is the precursor of eicosanoids such as prostaglandins and leukotrienes. In de novo synthesis of phospholipids, fatty acids that are saturated or exhibit a low degree of unsaturation, such as oleic acid (9-octadecenoic acid, 18:1 n-9), are often incorporated at the sn-2 positions of phospholipids. Their hydrolysis by phospholipase A2 (PLA2) releases the fatty acid, and the resultant lysophospholipids are acylated by acyl-CoA:lysophospholipid acyltransferases. Since acyltransferases prefer PUFA-CoAs such as arachidonoyl-CoA, PUFAs accumulate in the sn-2 positions of phospholipids during the deacylation-reacylation cycle (Lands cycle) ( Figure 2). The Lands cycle begins with PLA2, which releases a fatty acid from a phospholipid, and then another fatty acid is incorporated into the phospholipid by an acyl-CoA:1-acyl lysophospholipid acyltransferase. Exogenous PUFAs, such as arachidonic acid, are incorporated into phospholipids during the cycle. Exogenous stearic acid is also concentrated at the sn-1 positions of phospholipids by a similar deacylation-reacylation cycle consisting of phospholipase A1 (PLA1) and acyl-CoA:2-acyl lysophospholipid acyltransferases. ATP is required for acyl-CoA synthesis in the reacylation step. The Lands cycle begins with PLA2, which releases a fatty acid from a phospholipid, and then another fatty acid is incorporated into the phospholipid by an acyl-CoA:1-acyl lysophospholipid acyltransferase. Exogenous PUFAs, such as arachidonic acid, are incorporated into phospholipids during the cycle. Exogenous stearic acid is also concentrated at the sn-1 positions of phospholipids by a similar deacylation-reacylation cycle consisting of phospholipase A1 (PLA1) and acyl-CoA:2-acyl lysophospholipid acyltransferases. ATP is required for acyl-CoA synthesis in the reacylation step. Two different transacylation systems (CoA-dependent and CoA/cofactor-independent) are also involved in fatty acid remodeling ( Figure 3). CoA-dependent transacylation activity was first reported in rat liver microsomes [16]. In this reaction, esterified fatty acids in phospholipids are transferred to lysophospholipids to form phospholipids in the presence of CoA without generating free fatty acids [7][8][9][10][11][16][17][18][19][20][21][22][23]. CoA-dependent transacylation uses a variety of glycerophospholipids, such as phosphatidylcholine (PC) and phosphatidylinositol (PI), as acyl donors and lyso-type glycerophospholipids, such as lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI), as acyl acceptors (Table 1). This activity is distributed in the membrane fractions of mammalian tissues and cells, including in microsomes. The K m for CoA in the CoA-dependent transacylation reaction is very low (1-4 µM), suggesting that the reaction proceeds in the presence of physiological levels of CoA. We have proposed that the CoA-dependent transacylation reaction is mediated by a combination of reverse and forward reactions of acyl-CoA:lysophospholipid acyltransferases [7,8,[23][24][25][26][27]. Two different transacylation systems (CoA-dependent and CoA/cofactor-independent) are also involved in fatty acid remodeling ( Figure 3). CoA-dependent transacylation activity was first reported in rat liver microsomes [16]. In this reaction, esterified fatty acids in phospholipids are transferred to lysophospholipids to form phospholipids in the presence of CoA without generating free fatty acids [7][8][9][10][11][16][17][18][19][20][21][22][23]. CoA-dependent transacylation uses a variety of glycerophospholipids, such as phosphatidylcholine (PC) and phosphatidylinositol (PI), as acyl donors and lyso-type glycerophospholipids, such as lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI), as acyl acceptors (Table 1). This activity is distributed in the membrane fractions of mammalian tissues and cells, including in microsomes. The Km for CoA in the CoA-dependent transacylation reaction is very low (1-4 μM), suggesting that the reaction proceeds in the presence of physiological levels of CoA. We have proposed that the CoA-dependent transacylation reaction is mediated by a combination of reverse and forward reactions of acyl-CoA:lysophospholipid acyltransferases [7,8,[23][24][25][26][27].   In contrast to CoA-dependent transacylation, the mechanisms of CoA-independent transacylation are not fully understood. In this review, we describe the properties and possible mechanisms of the CoA/cofactor-independent transacylation system. We hypothesize that this reaction is catalyzed by PLA2, as described in detail below.
Biology 2017, 6, 23 6 of 26 lysophospholipids are effective acyl acceptors. The fatty chain at sn-1 and fatty acid at sn-2 are shown in blue and red, respectively; the transferred fatty acid is shown on a yellow background.
In contrast to CoA-dependent transacylation, the mechanisms of CoA-independent transacylation are not fully understood. In this review, we describe the properties and possible mechanisms of the CoA/cofactor-independent transacylation system. We hypothesize that this reaction is catalyzed by PLA2, as described in detail below.
Plasmalogens are present in various human tissues of the nervous and cardiovascular systems and are especially enriched in the myelin sheath, where they may contribute to membrane insulation. The vinyl ether bonds of plasmalogens can protect mammalian cells against the damaging effects of reactive oxygen species; reduced levels of brain tissue plasmalogens have been associated with Alzheimer's disease [32], X-linked adrenoleukodystrophy [33], and defects in central nervous system myelination [34].
An important function of ether-linked membrane phospholipids is arachidonic acid storage. As mentioned earlier, arachidonic acid is highly enriched in ether-linked phospholipids such as 1-O-alkyl-2-acyl-GPC and 1-O-alkenyl-2-acyl-GPE ( Figure 4C). The former is the precursor of platelet-activating factor (PAF), a potent bioactive mediator. The synergistic synthesis of PAF and eicosanoids is discussed in Section 6.
The substrate specificity and tissue distribution of CoA-independent transacylation explain the complement of alkyl ether-linked phospholipids in mammalian cells and tissues. Large amounts of ether-linked phospholipids, including 1-O-alkyl-2-acyl-GPC and 1-O-alkenyl-2-acyl-GPE, are detected in tissues such as the brain and heart and in inflammatory cells such as neutrophils, macrophages, platelets, and lymphocytes. Although a high proportion of arachidonic acid (20:4 n-6) is found at the sn-2 positions of ether-linked glycerophospholipids [6], arachidonoyl-CoA acyltransferase activity for 1-O-alkyl-GPC and 1-O-alkenyl-GPC is very low [47][48][49]. In contrast, CoA-independent transacylation was observed in the membrane fractions of various mammalian tissues excluding the liver [17,18], in which ether-containing phospholipids are almost completely absent [3]. The tissue distribution of CoA-independent transacylation activity appears to be closely related to the amounts of ether-linked glycerophospholipids in these tissues. The fatty acid specificity of this activity in vitro reflects the fatty acid pattern at the sn-2 positions of ether-linked glycerophospholipids [3,11,18]. CoA-independent transacylation is assumed to play an important role in the acylation of ether-linked lysophospholipids to generate PUFA-containing ether-linked glycerophospholipids.
There are few studies on the upregulation of CoA-independent transacylation activity. Treatment of platelets with phorbol ester or diacylglycerol was shown to enhance CoA-independent transacylation [50], which was also increased by treatment of human neutrophils with tumor necrosis factor-α [51].

Incorporation and Mobilization of Arachidonic Acid in Lipid Subclasses via Acyl-CoA Acyltransferases and CoA-Independent Transacylation
The behavior of arachidonic acid in cells, including its incorporation into each lipid class, highlights the distinct roles and substrate specificities of acyl-CoA:lysophospholipid acyltransferases and the CoA-independent transacylation system. Exogenously added arachidonic acid is primarily incorporated into phospholipids in various cell types. Radiolabeled arachidonic acid was mainly incorporated into the diacyl-GPC subclass in intact rabbit alveolar macrophages ( Figure 5A, red circles after a brief incubation (7.5 min) [54,55]. However, very little was incorporated into ether-linked phospholipids ( Figure 5A,B, blue squares and green diamonds). Incorporation of arachidonic acid into diacyl-GPC during short-term incubation is thought to occur via sequential reactions of acyl-CoA synthetase and acyl-CoA:1-acyl-GPC acyltransferase (Lands pathway; Figure 2). The fact that ATP, CoA, and Mg 2+ are required for the formation of acyl-CoA from free fatty acids by acyl-CoA synthetase indicates that energy is needed for fatty acid incorporation via the Lands pathway.
The transfer of arachidonic acid or DHA from diacyl-GPC to 1-O-alkyl-2-acyl-GPC and 1-O-alkenyl-2-acyl-GPE can be explained by CoA-independent activity, since the fatty acid and acceptor specificities resemble those of the CoA-independent transacylation reaction in in vitro cell-free assays. Such gradual transfers may account for the accumulation of arachidonic acid or DHA in ether-linked glycerophospholipids, such as 1-O-alkyl-2-acyl-GPC and 1-O-alkenyl-2-acyl-GPE, in inflammatory cells [18,54].
The incorporation of exogenous arachidonic acid into PI was also observed during short-term incubation [54,55]. However, the arachidonic acid in PI did not serve as an acyl donor in the CoA-independent transacylation system. 1-O-alkenyl-2-acyl-GPE was not observed [54]. The transfer of arachidonic acid or DHA from diacyl-GPC to 1-O-alkyl-2-acyl-GPC and 1-O-alkenyl-2-acyl-GPE can be explained by CoA-independent activity, since the fatty acid and acceptor specificities resemble those of the CoA-independent transacylation reaction in in vitro cell-free assays. Such gradual transfers may account for the accumulation of arachidonic acid or DHA in ether-linked glycerophospholipids, such as 1-O-alkyl-2-acyl-GPC and 1-O-alkenyl-2-acyl-GPE, in inflammatory cells [18,54]. The incorporation of exogenous arachidonic acid into PI was also observed during short-term incubation [54,55]. However, the arachidonic acid in PI did not serve as an acyl donor in the CoA-independent transacylation system.

Involvement of the CoA-Independent Transacylation System in PAF Metabolism
PAF was originally identified as a potent bioactive phospholipid mediator released from IgE-stimulated basophils that can induce aggregation of rabbit platelets [60]. Its chemical structure was identified as 1-O-alkyl-2-acetyl-sn-GPC [60][61][62][63]. PAF is a biologically active glycerophospholipid that is presumed to mediate inflammatory responses, including anaphylaxis

Involvement of the CoA-Independent Transacylation System in PAF Metabolism
PAF was originally identified as a potent bioactive phospholipid mediator released from IgE-stimulated basophils that can induce aggregation of rabbit platelets [60]. Its chemical structure was identified as 1-O-alkyl-2-acetyl-sn-GPC [60][61][62][63]. PAF is a biologically active glycerophospholipid that is presumed to mediate inflammatory responses, including anaphylaxis and septic shock, through the seven transmembrane-type G protein-coupled PAF receptor ( Figure 6) [64].
CoA-independent transacylation also triggers PAF biosynthesis through the formation of lysoPAF (Figure 3, deacylation). The addition of 1-O-alkenyl-GPE (lysoplasmalogen) has been shown to increase the amount of PAF produced by opsonized zymosan-stimulated polymorphonuclear leukocytes, although 1-O-alkenyl-GPE is not a direct precursor of PAF synthesis [71]. The addition of 1-O-alkenyl-GPE to cells induces the formation of lysoPAF from Figure 6. Biosynthesis and degradation of PAF. The PAF precursor 1-O-alkyl-2-acyl-GPC is hydrolyzed by a PLA2 protein such as cPLA2α to form 1-O-alkyl-GPC (lysoPAF) (Reaction 1). LysoPAF is also formed by the CoA-independent transacylation system through fatty acid transfer from 1-O-alkyl-2-acyl-GPC to a lysophospholipid (Reaction 1'). The lysophospholipid acceptor for the CoA-independent transacylation system is formed by hydrolysis mediated by a PLA2 protein such as cPLA2α (Reaction 0). The bioactive phospholipid PAF is formed by lysoPAF acetyltransferase (LPCAT1 and LPCAT2) through the reacetylation of lysoPAF. PAF activates the PAF receptor to trigger intracellular signaling. Because arachidonic acid is formed by PLA2 in the deacylation step (Reactions 1 and 0), eicosanoids, prostaglandins (PGs), and leukotrienes (LTs) are synthesized simultaneously by the COX and LOX pathways. PGs and LTs activate PG and LT receptors. PAF and PGs/LTs simultaneously activate the cells. In contrast, PAF is degraded through the deacetylation process by PAF acetylhydrolase (PAF-AH, Reaction 3). The resultant lysoPAF is further converted to 1-O-alkyl-2-acyl-GPC through the CoA-independent transacylation system (Reaction 4). The reactions involved in the synthesis of PAF are indicated in red, and those involved in the degradation of PAF are indicated in blue.
PAF and eicosanoids are simultaneously formed in inflammatory cells [3][4][5][6][7][8][9][10][11]. A portion of the arachidonic acid (20:4n-6) released from the PAF precursor 1-O-alkyl-2-arachidonoyl-GPC by cPLA2α may be metabolized to prostaglandins and leukotrienes by the cyclooxygenase (COX) and lipoxygenase (LOX) pathways, respectively ( Figure 6). Accumulation of arachidonic acid in 1-O-alkylacyl-GPC is favorable for the simultaneous synthesis of PAF and various eicosanoids in inflammatory cells. As stated earlier, supplementation of eosinophilic leukemia cells with DHA decreased the arachidonic acid content in 1-O-alkyl-2-acyl-GPC via competition with the CoA-independent transacylation reaction [77] and resulted in a reduction in PAF formation. These results suggest that phospholipid remodeling by CoA-independent transacylation is important for the synthesis of not only eicosanoids but also PAF in accelerated inflammation.

Possible Molecular Mechanisms of CoA-Independent Transacylation Reactions
Little is known about the molecular mechanisms of CoA-independent transacylation, such as the enzymes responsible for the reaction, which have yet to be purified; these enzymes are difficult to solubilize due to the high sensitivity of their enzymatic activity to various detergents [18]. Additionally, the genes encoding these enzymes have not yet been identified.
It was previously reported that several forms of extracellular secreted PLA2 (sPLA2), including pancreatic and venom PLA2s, operated in a reverse reaction in low-polarity solvents [78]-that is, free fatty acids were transferred to LPC (1-acyl-GPC) to form PC ( Figure 7A, red arrows). Results indicated that PLA2 could catalyze the acyltransferase reaction under specific conditions. This prompted us to examine the potential contribution of PLA2 to acyltransferase and transacylation activities in membranes, which resemble the environments of low-polarity solvents that exclude water molecules, although a transacylation reaction was not reported in the earlier study [78].
We propose that CoA-independent transacylation reactions are catalyzed by enzymes belonging to the PLA2 family. Based on the characteristics of the CoA-independent transacylation system (Sections 4 and 5), we speculate that Ca 2+ -independent and membrane-bound PLA2 proteins that selectively transfer C20-22 PUFAs including arachidonic acid are involved in this process (Figure 7).
The CoA-independent transacylation reaction comprises two steps: the first is the formation of the fatty acyl-PLA2 complex by the half-reaction of PLA2 (step 1a in Figure 7B), and the second is the transfer of fatty acids from this complex to lysophospholipids (step 2b in Figure 7B), which is the reverse reaction of step 1a. The fatty acyl-PLA2 complex can also react with a water molecule to complete the hydrolytic (PLA2) reaction (step 2a). It is critical that CoA-independent transacylation activity be present in the membrane fraction, including the microsomes, and nearly absent from the cytosolic fraction [18]. The catalytic site should be in a hydrophobic environment within the membrane to limit access to water molecules, thereby inhibiting hydrolysis and promoting transacylation.
It has been reported that CoA-independent transacylation involves enzyme(s) that are biochemically and pharmacologically distinct from cPLA2α and the 14-kDa sPLA2 [79]. This is consistent with our hypothesis that the enzyme in question is a membrane-bound and non-soluble PLA2 ( Figure 7B). Acyltransferase activity of venom PLA2 in a low-polarity organic solvent. We hypothesize that an intermediate fatty acyl-enzyme complex mediates the PLA2 reaction, which includes formation of a fatty acyl-enzyme complex by the half-reaction of PLA2 (step 1a) and fatty acid transfer from the complex to a water molecule (step 2a). Acyltransferase activity is demonstrated in the reverse PLA2 reaction and consists of steps −2a and −1a (red arrows). (B) Proposed model of the CoA-independent transacylation system. We hypothesize that a Ca 2+ -independent and membrane-bound PLA2 catalyzes the CoA-independent transacylation reaction, which consists of two steps: formation of a fatty acyl-enzyme complex as an intermediate by the half-reaction of PLA2 (step 1a) and transfer of a fatty acid from the acyl-enzyme complex to a lysophospholipid (step 2b), which is the reverse of step 1a. (C) Lysophospholipase/transacylation and CoA-independent transacylation reactions catalyzed by cPLA2γ. cPLA2γ can catalyze CoA-independent transacylation (described in panel B) as well as the lysophospholipase/transacylation reaction. Lysophospholipase/transacylation involves two steps: formation of a fatty acyl-enzyme complex by the half-reaction of lysophospholipase (or PLA1) (step 1b), and fatty acid transfer from the acyl-enzyme complex to lysophospholipid (step 2b). The transferred fatty acid is shown in red. X and Y represent parts of polar headgroups of phospholipids.

Intracellular PLA2
We propose that CoA-independent transacylation is catalyzed by enzymes belonging to the PLA2 family-specifically, intracellular PLA2s. Before discussing the candidate enzymes, we first describe intracellular PLA2, which hydrolyzes glycerophospholipids at the sn-2 position to release (A) Acyltransferase activity of venom PLA2 in a low-polarity organic solvent. We hypothesize that an intermediate fatty acyl-enzyme complex mediates the PLA2 reaction, which includes formation of a fatty acyl-enzyme complex by the half-reaction of PLA2 (step 1a) and fatty acid transfer from the complex to a water molecule (step 2a). Acyltransferase activity is demonstrated in the reverse PLA2 reaction and consists of steps −2a and −1a (red arrows). (B) Proposed model of the CoA-independent transacylation system. We hypothesize that a Ca 2+ -independent and membrane-bound PLA2 catalyzes the CoA-independent transacylation reaction, which consists of two steps: formation of a fatty acyl-enzyme complex as an intermediate by the half-reaction of PLA2 (step 1a) and transfer of a fatty acid from the acyl-enzyme complex to a lysophospholipid (step 2b), which is the reverse of step 1a. (C) Lysophospholipase/transacylation and CoA-independent transacylation reactions catalyzed by cPLA2γ. cPLA2γ can catalyze CoA-independent transacylation (described in panel B) as well as the lysophospholipase/transacylation reaction. Lysophospholipase/transacylation involves two steps: formation of a fatty acyl-enzyme complex by the half-reaction of lysophospholipase (or PLA1) (step 1b), and fatty acid transfer from the acyl-enzyme complex to lysophospholipid (step 2b). The transferred fatty acid is shown in red. X and Y represent parts of polar headgroups of phospholipids.

Intracellular PLA2
We propose that CoA-independent transacylation is catalyzed by enzymes belonging to the PLA2 family-specifically, intracellular PLA2s. Before discussing the candidate enzymes, we first describe intracellular PLA2, which hydrolyzes glycerophospholipids at the sn-2 position to release fatty acids and lysophospholipids. As stated earlier (Figure 2), PLA2 is involved in the first step of the deacylation-reacylation cycle (Lands cycle) as well as in the synthesis of lipid mediators such as eicosanoids or PAF ( Figure 6).
The PLA2 enzyme superfamily consists of over 30 enzymes, with members such as sPLA2, cPLA2, Ca 2+ -independent PLA2 (iPLA2), PAF-AH, and lysosomal PLA2 classified according to localization (extracellular vs. intracellular), sequence homology, or biochemical characteristics. A systematic group numbering system has been proposed for these enzymes [80]. Figure 8 shows a phylogenetic tree of intracellular and related PLA2s.
cPLA2α was identified as a key enzyme in eicosanoid synthesis [4,[65][66][67][81][82][83] that selectively hydrolyzes arachidonic acid-containing phospholipids when cells are activated by extracellular stimuli, thereby stimulating eicosanoid synthesis. It is also involved in PAF synthesis ( Figure 6). cPLA2α (PLA2G4A) harbors the conserved GXSXG sequence for lipase activity as well as a calcium-dependent lipid-binding domain that resembles the C2 domain of calcium-dependent protein kinase C enzymes ( Figure 9). cPLA2α also has several serine residues that are phosphorylated by protein kinases, and it is activated by intracellular Ca 2+ via C2 domain-mediated membrane binding and phosphorylation in response to extracellular stimuli. A study using transgenic mice lacking cPLA2α demonstrated the importance of prostaglandin and leukotriene synthesis in the allergic reaction and in parturition [84]. cPLA2 (PLA2G4) family enzymes were identified based on their sequence similarity to cPLA2α [85][86][87]. cPLA2β (PLA2G4B), cPLA2δ (PLA2G4D), cPLA2ε (PLA2G4E), and cPLA2ζ (PLA2G4F) were found to form the cPLA2β gene cluster, and all have both the lipase consensus sequence (GXSXG) and the C2 domain [87] (Figure 8).
Another isoform of iPLA2, iPLA2γ (PLA2G6B, PNPLA8), is a membrane-bound enzyme that may also be involved in the deacylation step of phospholipid remodeling. Recombinant iPLA2γ exhibits PLA1 and PLA2 activities depending on substrate type (Figure 8) [103]. Purified iPLA2γ was shown to hydrolyze oleic acid at the sn-2 position of 1-stearoyl-2-oleoyl-GPC, suggesting that it possesses PLA2 activity. Mass spectrometric analyses demonstrated that purified iPLA2γ readily hydrolyzed saturated or monounsaturated aliphatic groups at either the sn-1 or -2 positions of phospholipids.

cPLA2γ Catalyzes CoA-Independent Transacylation Reactions that Transfer Arachidonic Acid to Ether-Linked Lysophospholipids
We previously suggested that cPLA2γ (PLA2G4C) may be responsible for CoA-independent transacylation [92,93]. cPLA2γ (PLA2G4C) was identified as an ortholog of cPLA2α (PLA2G4A) [85,86], whose activity was shown to be Ca 2+ independent due to the absence of a C2 domain that is conserved in other cPLA2 (PLA2G4) family enzymes and is involved in Ca 2+ -dependent lipid binding. cPLA2γ is membrane bound owing to the presence of lipidation motifs, including a C-terminal CAAX farnesylation motif ( Figure 9A,B). The membrane-bound and Ca 2+ -independent properties of cPLA2γ are similar to those of the CoA-independent transacylation system.
When purified cPLA2γ was incubated with [ 14 C]LPC (1-[ 14 C]palmitoyl-GPC), [ 14 C]palmitic acid was released, suggesting that cPLA2γ catalyzes the lysophospholipase reaction ( Figure 7C, reactions 1b + 2a) [92,93]. In addition, [ 14  1-O-Alkyl-GPC acts only as an acyl acceptor due to the resistance of the alkyl ether bond to cleavage. The addition of LPC increased the acylation activity of 1-O-alkyl-GPC rather than that of PC ( Figure 9B), suggesting that cPLA2γ prefers lysophospholipids (lysophospholipase/transacylation) to phospholipids (CoA-dependent transacylation reaction) as acyl donors ( Figure 9B) [92,93]. CoA-independent transacylation and lysophospholipase/transacylation differ in terms of the acyl donor: the former uses phospholipids such as PC and PE, whereas the latter uses LPC. The lysophospholipase/transacylation activity of cPLA2γ was markedly higher than its CoA-independent transacylation activity. cPLA2γ also exhibited higher lysophospholipase activity (sequential reactions, steps 1b and 2a in Figure 7C) than PLA2 activity (sequential reactions, steps 1a and 2a in Figure 7C) [92,93], which may explain the ratio of each transacylation reaction (step 1a vs. 1b). These results indicate that cPLA2γ is not the enzyme responsible for CoA-independent transacylation-mediated accumulation of arachidonic acid in ether-linked phospholipids.
The physiological roles of cPLA2γ remain unclear. We also predict that cPLA2γ has important functions in the heart because it is highly expressed in this tissue [85,86]. In addition, lysophospholipids including lysoplasmalogen have been shown to accumulate in ischemic heart tissue [112][113][114]. Under hypoxic conditions, Ca 2+ -independent PLA2 (iPLA2β) is thought to be involved in lysophospholipid accumulation, since iPLA2β and its substrate plasmalogen are abundant in the myocardium [100][101][102]. Lysoplasmalogen as well as LPC are also known to trigger cardiac arrhythmia [112][113][114]. In iPLA2β transgenic mice, cardiac ischemia enhances iPLA2β activity, leading to increased lysophospholipid accumulation and aggravation of arrhythmia [102]. Given that the lysophospholipase and transacylation reactions of cPLA2γ can reduce the levels of toxic lysophospholipids, we hypothesize that cPLA2γ has a protective role against arrhythmia [92,93]. tissue [112][113][114]. Under hypoxic conditions, Ca 2+ -independent PLA2 (iPLA2β) is thought to be involved in lysophospholipid accumulation, since iPLA2β and its substrate plasmalogen are abundant in the myocardium [100][101][102]. Lysoplasmalogen as well as LPC are also known to trigger cardiac arrhythmia [112][113][114]. In iPLA2β transgenic mice, cardiac ischemia enhances iPLA2β activity, leading to increased lysophospholipid accumulation and aggravation of arrhythmia [102]. Given that the lysophospholipase and transacylation reactions of cPLA2γ can reduce the levels of toxic lysophospholipids, we hypothesize that cPLA2γ has a protective role against arrhythmia [92,93].  that is involved in Ca 2+ -dependent phospholipid binding. Human cPLA2γ has a CAAX box and putative N-myristoylation site at the C-and N-termini, respectively. cPLA2α can be phosphorylated at Ser505 by mitogen-activated protein kinases (MAPKs), at Ser515 by Ca 2+ /calmodulin-dependent protein kinase II, and at Ser727 by MAPK-interacting kinase 1 or a closely related isoform. Post-translational modifications such as C-terminal farnesylation and N-terminal N-myristoylation are depicted. (B) Transacylation activity of cPLA2γ. Purified cPLA2γC-FLAG was incubated with [ 3 H]alkyl-GPC in the absence or presence of PC or LPC as acyl donors; the products were analyzed by thin-layer chromatography and the radioactivity of [ 3 H]alkylacyl-GPC was measured. cPLA2γ exhibits CoA-independent transacylation activity for 1-O-alkyl-GPC and uses both phospholipids (upper, CoA-independent transacylation) and lysophospholipids (lower, lysophospholipase/transacylation) as the acyl donor, with preference for the latter. The transferred fatty acyl moiety is shown on a yellow background.

Other Enzymes that Catalyze Transacylation Reactions
Our group has investigated the enzymes responsible for CoA-independent transacylation [92,93]. Although cPLA2γ can catalyze this reaction, it also exhibits high lysophospholipase/transacylation activity ( Figure 9B). Below, we discuss the possibility that other PLA2s participate in CoA-independent transacylation (Table 3). cPLA2α (PLA2G4A), another cPLA2 family enzyme, was also shown to exhibit transacylation activity [115]. However, cPLA2α preferentially uses lysophospholipids as substrates in the transacylation reaction, transferring the fatty acids in LPC to another LPC to yield PC in the lysophospholipase/transacylation reaction (Table 3). Ca 2+ is essential for cPLA2α-catalyzed PLA2 activity and for the binding of PLA2 to the membrane or micelles where the substrate is located. However, cPLA2α-catalyzed lysophospholipase activity towards micelles was not stimulated by Ca 2+ , and cPLA2α-catalyzed lysophospholipase/transacylase activity was low.
Another PLA2 isoform may participate in the CoA-independent transacylation system. The 30-kDa PLA2 belonging to the 14-3-3 protein family catalyzes the cleavage of the sn-2 arachidonic acid in phospholipids though the formation of a stable acyl-enzyme complex as an intermediate [116]. The occurrence of this arachidonic acid-enzyme complex may suggest that arachidonic acid in the complex to specifically transfer to putative nucleophilic acceptors including water (PLA2 activity) and lipids (transacylation activity).
The PLA/AT family enzymes [HRASLS3 (H-rev107), HRASLS4 (TIG3), and HRASLS2] were also shown to exhibit CoA-independent transacylation activity [108][109][110][111]. The fatty acids at the sn-1 or sn-2 positions of donor phospholipids are transferred to LPC in a Ca 2+ -independent manner, with preference for the transfer of sn-1 rather than sn-2 fatty acids. These enzymes also exhibit PLA1 or PLA2 activities, releasing free fatty acids from glycerophospholipids with a preference for hydrolysis at the sn-1 position [108][109][110][111]. Although these enzymes could catalyze CoA-independent transacylation, they are not thought to be involved in the acylation of ether-linked lysophospholipids with arachidonic acid based on their positional preference ( Figure 10A) ( Table 3).
Lysosomal PLA2 (LPLA2, PLA2G15) was also reported to catalyze transacylation reactions ( Figure 8) (Table 3). Abe et al. identified this enzyme as having novel activity for the synthesis of 1-O-acyl-ceramide [127]. Activity was characterized by transfer of the sn-2 fatty acid of PC to the 1-hydroxyl group of N-acetylsphingosine. The product of this reaction was 1-O-acylceramide (1-O-acyl-N-acetylsphingosine). Further characterization employing the recombinant protein revealed that the enzyme predominantly exhibited PLA2 activity rather than ACS activity. Thus, this enzyme is now known as lysosomal PLA2 (LPLA2) [128,129].

Concluding Remarks and Future Directions
In this review, we summarized the characteristics and physiological significance of the CoA-independent transacylation system, which is involved in fatty acid remodeling of glycerophospholipids and in the accumulation and enrichment of arachidonic acid in alkyl ether-linked phospholipids in the brain, heart, and various inflammatory cells. This system is also important for the metabolism of eicosanoids and PAF. Despite its importance, the component enzyme(s) have not yet been identified. We described here the reaction mechanism of CoA-independent transacylation and discussed the involvement of Ca 2+ -independent and membrane-bound PLA2. In Section 11, we discussed how many lipases, including PLA2s, can catalyze transacylation, even although their substrate specificities differ. This lends support to the rationale behind our proposed model (Figure 7) for the involvement of PLA2 in CoA-independent transacylation.
A detailed analysis of enzymological data revealed that cPLA2γ is not the enzyme responsible for CoA-independent transacylation, since it prefers to use lysophospholipids rather than phospholipids as acyl donors. However, this in vitro substrate specificity may be due to the solubilities of lyso and diradyl phospholipids as acyl donors. Further detailed characteristics of cPLA2γ will be needed, including analyses using cPLA2γ-knockdown or -knockout macrophages or neural cells, to investigate the involvement of cPLA2γ in the accumulation of arachidonic acid in ether-linked phospholipids.
Identifying the enzyme(s) involved in the CoA-dependent transacylation system will require a combination of highly specific probes-including substrates/inhibitors with covalent modifications such as photoaffinity labeling-and sensitive methodologies such as liquid chromatography-tandem mass spectrometry. This information will improve our understanding of the physiological significance of alkyl ether phospholipids and the biosynthesis of PAF and eicosanoids.