Keywords

1 Introduction

Glycerophospholipids (phospholipids) are important not only as structural and functional components of cellular membranes, but also as the precursors of various lipid mediators, such as platelet-activating factor (PAF) and eicosanoids [1, 2]. Phospholipids are also the main components of pulmonary surfactant: they are composed of two fatty acids and a polar head on a glycerol backbone. Tissues and cells contain several phospholipids such as phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin (CL) [3, 4]. Phospholipids are biosynthesized by two pathways that were proposed in the 1950s: the first is the de novo pathway (Kennedy pathway) [5], and the second is the remodeling pathway (Lands’ cycle) [6]. Saturated and monounsaturated fatty acids are usually esterified at the sn-1 position, whereas polyunsaturated acyl groups are esterified at the sn-2 position. Using acyl-CoAs as donors, phospholipids are first produced from glycerol-3-phosphate (G3P) by the de novo pathway. Next, in the remodeling pathway, the fatty acids of phospholipids are maturated by the coordinated actions of phospholipase A2s (PLA2s) and lysophospholipid acyltransferases (LPLATs) (Fig. 1.1) [4, 7, 8].

Fig. 1.1
figure 1

Phospholipid biosynthetic pathways. Acyltransferase steps are indicated in red, phospholipase (PLA) steps to release fatty acids in blue, and phospholipids in yellow

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Recently, several LPLATs were identified in the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) and membrane-bound O-acyltransferase (MBOAT) families. Both these families have motifs that are essential for LPLAT activities. Site-directed mutagenesis revealed four AGPAT motifs ([1] HxxxxD, [2] GxxFxxR, [3] xxEGxx, and [4] xxxxPxx), and four MBOAT motifs ([A] WD, [B] WHGxxxGYxxxF, [C] YxxxxF, and [D] YxxxYFxxH) [7, 9, 10]. AGPAT motif 4 consists of a conserved proline surrounded by hydrophobic amino acids. Although the AGPAT and MBOAT motifs are known to differ completely, more detailed structural information will be provided by future protein crystallization analyses.

Many of the LPLATs have been assigned multiple names because several groups identified them independently or registered different names for the same enzyme based on activity or sequence homology [8]. To eliminate confusion regarding the nomenclature, the proposed names based on their substrate specificities and in order of their publication are summarized in Table 1.1.

Table 1.1 Summary of lysophospholipid acyltransferases (LPLATs) from the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) and membrane-bound O-acyltransferases (MBOAT) families. Other names were registered in HomoloGene of the NCBI database
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2 Lysophosphatidic Acid Acyltransferase (LPAAT) Enzymes

Lysophosphatidic acid (LPA) is the substrate for LPAAT enzymes and is biosynthesized as part of the acyltransferase reaction. Four mammalian GPATs, which synthesize LPA from glycerophospholipids (GP), have been cloned from the AGPAT family [2, 4]. GPAT1 and GPAT2 function in the outer mitochondrial membrane, whereas GPAT3 and GPAT4 are localized to the endoplasmic reticulum (ER) [4]. GPAT4 is also found in lipid droplets [11]. The four GPATs preferentially use saturated and monounsaturated fatty acyl-CoAs to produce LPA, which is then converted to PA by LPAAT. To date, four LPAATs (LPAAT1, -2, -3, and -4) have been cloned and characterized. The representative LPAAT-catalyzed reaction is shown in Fig. 1.2.

Fig. 1.2
figure 2

Representative LPAAT-catalyzed reactions: PA (18:0/18:1) (a); PA (16:0/22:6) (b)

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2.1 LPAAT1 and LPAAT2

Human LPAAT1 and LPAAT2 were cloned based on their homology with yeast, Escherichia coli, and coconut AGPATs. LPAAT1 has a higher activity toward 14:0-, 16:0-, and 18:2-CoAs, whereas LPAAT2 exhibited higher activity toward 20:4-CoA compared with 16:0- or 18:0-CoA. Both mRNAs are found in a broad range of tissues [4]. LPAAT2 mutations have been linked to congenital generalized lipodystrophy (also known as Berardinelli–Seip syndrome) [12], suggesting that LPAAT2 might be involved in triacylglycerol (TAG) synthesis and storage in adipocytes.

2.2 LPAAT3

LPAAT3 is mainly expressed in the testis and is upregulated in an age-dependent manner [13, 14]. In a biochemical assay, LPAAT3 exhibited LPAAT activity with a preference for polyunsaturated fatty acyl-CoAs (PUFA-CoAs) such as 22:6-CoA [15]. Thus, docosahexaenoic acid (DHA, 22:6) could be inserted into phospholipid via the Kennedy pathway. The induction of LPAAT3 during germ cell development might contribute to the accumulation of PUFAs in testicular phospholipids, suggesting that it has a potential role in proper sperm cell differentiation and maturation. LPAAT3 is also reported to affect Golgi structure and function because Golgi membrane tubule formation and trafficking were inhibited by overexpression of LPAAT3 [16]. In addition, LPAAT3 was detected in lipid droplets [11].

2.3 LPAAT4

Recently, LPAAT4 was also reported to possess LPAAT activity with 22:6-CoA [17]. LPAAT4 mRNA is expressed predominantly in the brain. The brain contains an abundant amount of DHA-containing phospholipids. Therefore, LPAAT4 might have important roles in brain function.

2.4 Putative LPAAT

AGPAT5 (also called LPAATε) was reported to be a LPAAT and lyso-PE (LPE) AT, but it has not yet been analyzed in detail [18, 19]. AGPAT5 also contains the AGPAT motifs. Further studies are needed to identify the AGPAT5 roles.

3 Lysophosphatidylcholine Acyltransferase (LPCAT) Enzymes

Phosphatidylcholine (PC) is biosynthesized from LPC by LPCATs in the remodeling pathway. Four LPCATs have been reported to date: LPCAT1 and LPCAT2 from the AGPAT family, and LPCAT3 and LPCAT4 from the MBOAT family. The representative LPCAT-catalyzed reaction is shown in Fig. 1.3.

Fig. 1.3
figure 3

Representative LPCAT-catalyzed reaction: PC (16:0/16:0, DPPC) (a); PC (18:0/20:4) (b)

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3.1 LPCAT1

LPCAT1 was the first enzyme identified as having LPCAT activity [20, 21]. LPCAT1 preferentially uses 16:0-CoA to produce dipalmitoyl-PC (DPPC), which is the main component of the pulmonary surfactant that prevents alveolar collapse, small airway closure, and alveolar flooding by decreasing surface tension. Alveolar type II cells produce the pulmonary surfactant that is essential for respiration. Consistent with this, LPCAT1 is expressed mainly in the lung, particularly in alveolar type II cells, and its mRNA expression is upregulated during the perinatal period [21].

Pulmonary surfactant deficiency is an important contributing factor during the pathogenesis of infant respiratory distress syndrome (IRDS), acute respiratory distress syndrome (ARDS), bronchial asthma, and bronchiolitis because pulmonary surfactant plays a critical role in respiratory physiology [22]. Recently, several groups reported the function of LPCAT1 in the lung. LPCAT1 gene trap mice exhibited a decreased level of saturated PC and increased perinatal mortality because of respiratory failure [23]. In addition, LPCAT1-knockout mice had low levels of DPPC, as well as higher sensitivity for acute lung injury than control mice [24]. These reports suggest that the saturated PC generated by LPCAT1 is important for lung surfactant production and function.

Retinal degeneration and visual dysfunction were also found in a mouse strain with a LPCAT1 mutation (rd11) [25]. LPCAT1 mRNA levels decreased in the retina and the brain in response to the onset of diabetes in Ins2(Akita) and db/db mice, mouse models of type 1 and type 2 diabetes, respectively [26]. LPCAT1 expression was reported in colorectal cancer and was also correlated with the progression of prostate cancer [27, 28].

LPCAT1 also has lyso-PAF acetyltransferase activity to produce PAF. PAF production by LPCAT1 is described as “lyso-PAF acetyltransferase activity.” However, additional studies are required to discover the biological roles of LPCAT1-generated PAF.

3.2 LPCAT2

The main product of the LPCAT2-mediated reaction is thought to be PAF, although LPCAT2 also possesses LPCAT (lyso-PAF acyltransferase) activity with a preference for 20:4-CoA [29]. Therefore, LPCAT2 is not only important during the biosynthesis of PAF, but also for membrane homeostasis in inflammatory cells. However, it is important to identify and characterize the binding sites for each substrate (acetyl-CoA and arachidonoyl-CoA). Both LPCAT1 and LPCAT2 are localized in the endoplasmic reticulum (ER) and are also found on the surface of lipid droplets [20, 29, 30]. PAF biosynthetic activity of LPCAT2 is described as “lyso-PAF acetyltransferase activity.”

3.3 LPCAT3

LPCAT3 mRNA is expressed ubiquitously, and the protein exhibits LPCAT, LPEAT, and LPSAT activities with PUFA-CoAs such as 20:4-CoA and 18:2-CoA [31, 32]. LPCAT3 knockdown in HEK293 cells induced apoptosis and altered cellular morphology [33]. Liver-specific LPCAT3 knockdown in mice showed increased levels of LPC, which led to decreased hepatic triglyceride levels and increased triglyceride-rich lipoprotein production and apolipoprotein-B secretion [34]. LPCAT3 knockdown in mammalian cells also enhanced the palmitic acid-induced unfolded protein response [35]. LPCAT3 is induced by agonists for peroxisome proliferator-activator receptor-α and liver X receptor, as well as during the differentiation of C3H10T1/2 cells into adipocyte-like cells [32, 36, 37]. LPCAT1, LPCAT2, LPCAT3, and LPCAT4 were induced in a model of nonalcoholic steatohepatitis [38]. In addition, hepatic LPCAT3 and LPCAT4 were induced by treatment with fibrates [39]. Interestingly, the Drosophila orthologue of LPCAT3, nessy, is controlled by ultrabithorax (Ubx), homeobox (Hox), and other Hox proteins during Drosophila embryogenesis [40].

3.4 LPCAT4

LPCAT4 has both LPCAT and LPEAT activities [31, 41]. LPCAT4 preferentially uses 18:1-CoA as a donor. Mouse LPCAT4 mRNA is highly expressed in the epididymis, brain, testis, and ovary. However, biological roles of LPCAT4 remain unclear.

4 Lyso-PAF Acetyltransferase Enzymes

The lyso-PAF acetyltransferase-catalyzed reaction is shown in Fig. 1.4.

Fig. 1.4
figure 4

Representative lyso-PAF acetyltransferase-catalyzed reaction: PAF

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4.1 LPCAT1 and LPCAT2

In response to extracellular stimuli, PAF is synthesized rapidly and has important roles as a potent pro-inflammatory lipid mediator [42]. Specifically, it triggers various cellular functions via its G protein-coupled receptor, PAF receptor [43]. PAF is biosynthesized via the activation of acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAFAT). To date, two lyso-PAFAT enzymes have been reported: LPCAT1 and LPCAT2 [9, 29]. LPCAT1 is mainly expressed in the lung, whereas LPCAT2 is mainly observed in inflammatory cells such as peritoneal macrophages. Before the identification of LPCAT1 and LPCAT2, it was reported that endogenous lyso-PAFAT activity is enhanced in three distinct ways in mouse macrophages: (i) rapid activation (30 s) after PAF stimulation and (ii, iii) minutes to hours after lipopolysaccharide stimulation [44]. However, three distinct mechanisms have been clarified. (1) After G protein-coupled receptor (PAF receptor and ATP receptor) stimulation for 30 s, LPCAT2 phosphorylation at Ser34 in the conventional protein kinase C (cPKC) pathway enhances lyso-PAF acyltransferase activity [45]. (2) After lipopolysaccharide stimulation for 30 min, LPCAT2 is phosphorylated at Ser34 and activated via the p38 MAP kinase (MAPK)/MAPK-activated protein kinase 2 (MK2) pathway [46]. (3) LPCAT2 mRNA is upregulated within 16–24 h of lipopolysaccharide stimulation [29].

In contrast, LPCAT1 is unaffected by lipopolysaccharide, PAF, or ATP stimulation. Therefore, LPCAT1 is a constitutively expressed lyso-PAF acetyltransferase, whereas LPCAT2 is an inducible lyso-PAF acetyltransferase. This relationship is similar to cyclooxygenase 1 and 2, which are constitutively expressed and inducible enzymes, respectively [47]. Recently, the LPCAT2-specific inhibitor TSI-01 was identified from 174,131 compounds. TSI-01 might form a better basis for antiinflammatory drugs in PAF-related diseases [48].

LPCAT2 expression is elevated in several conditions. LPCAT2 mRNA was also reported to be upregulated in the ipsilateral spinal cord in a rat model of peripheral nerve injury [49]. In addition, LPCAT2 was increased in the spinal cords of mice with experimental allergic encephalomyelitis (EAE), which is a model of multiple sclerosis [50]. During this condition, inflammatory cells infiltrate into the central nervous system (CNS).

5 Lyso-PE Acyltransferase (LPEAT) Enzymes

A representative LPEAT-catalyzed reaction is shown in Fig. 1.5.

Fig. 1.5
figure 5

Representative LPEAT-catalyzed reaction: PE (18:0/22:6)

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5.1 LPEAT1

LPEAT1 exhibits both LPEAT and LPSAT activities and has a preference for 18:1-CoA as a donor [31, 41]. LPEAT1 mRNA is highly expressed in the stomach, epididymis, and colon. Furthermore, the human LPEAT1 gene, which is located on chromosome 6, was disrupted in a brachydactyly-syndactyly syndrome patient [51], suggesting that LPEAT1 contributes to the turnover of phospholipids during normal development and organogenesis.

5.2 LPEAT2

LPEAT2 was identified from the AGPAT family. Although LPEAT2 exhibits LPEAT, LPGAT, LPSAT, and LPCAT activities using 18:1-CoA or 20:4-CoA as the acyl donor in vitro, only LPEAT activity was decreased by its siRNA transfection in HEK293T cells [52]. LPEAT2 is expressed at high levels in the brain, suggesting that it might be important for the biogenesis of brain PE. However, the reported biochemical activities of LPEAT2 are inconsistent with the brain PE composition [53]. It has been reported that hepatic LPEAT2 expression increases on exposure to lithocholic acid exposure [54]. Confusingly, LPEAT2 is also called LPCAT4 (see Table 1.1).

5.3 LPCAT3 and LPCAT4

LPCAT3 and LPCAT4 both exhibit LPEAT activity [31, 41]; however, their biological roles as LPEAT are yet to be clarified. The regulation of these enzymes is described earlier in the “LPCAT enzymes” section.

6 Lyso-PS Acyltransferase (LPSAT) Enzymes

The representative LPSAT-catalyzed reaction is shown in Fig. 1.6.

Fig. 1.6
figure 6

Representative LPSAT-catalyzed reaction: PS (16:0/22:6)

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6.1 LPCAT3 and LPCAT4

LPCAT3 and LPCAT4 both exhibit LPSAT activity [31, 41]; however, their biological roles as LPSAT are yet to be clarified. The regulation of these enzymes is described earlier in the “LPCAT enzymes” section.

7 Lyso-PI Acyltransferase (LPIAT) Enzymes

The representative LPIAT-catalyzed reaction is shown in Fig. 1.7.

Fig. 1.7
figure 7

Representative LPIAT-catalyzed reaction: PI (18:0/20:4)

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7.1 LPIAT1

LPIAT1 is the first reported LPIAT and was identified from the MBOAT family [55]. LPIAT1 prefers 20:4-CoA and 20:5-CoA as donors. In LPIAT1-KO mice, PI and PI phosphates containing arachidonic acid decreased, and the mice exhibited abnormal brain morphology, delayed neural migration, and reduced neurite outgrowth. LPIAT1-KO mice were significantly smaller than their littermates and were born at a frequency less than would be expected from the Mendelian ratio [56, 57]. Arachidonic acid containing PI produced by LPIAT1 is converted to phosphoinositides. They play an important role in brain development [56]. A Caenorhabditis elegans LPIAT1 (mboa-7) mutant showed a “bag of worms” phenotype, whereby the embryos hatched within the mother, leaving a cuticle sack containing multiple wriggling larvae [55].

7.2 Lyso-CL Acyltransferase (LCLAT)1

LCLAT1, a member of the AGPAT family, has LPIAT activity and uses 18:1-CoA as the donor [58]. LCLAT1 can also incorporate 18:0-CoA into the sn-1 position of LPI [59], as is described in detail in the “LCLAT enzyme” section.

8 Lyso-PG Acyltransferase (LPGAT) Enzyme

The representative LPGAT-catalyzed reaction is shown in Fig. 1.8.

Fig. 1.8
figure 8

Representative LPGAT-catalyzed reaction: PG (16:0/18:1)

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8.1 LPGAT1

PG is synthesized from LPG by LPGAT enzyme during the Lands’ cycle. LPGAT1 was cloned as an LPGAT enzyme from the AGPAT family [60]. Human LPGAT1 has a preference for 16:0-, 18:0-, and 18:1-CoAs as donors and is widely expressed in several tissues. PG is a precursor for the synthesis of CL.

9 LCLAT Enzyme

Representative LCLAT-catalyzed reaction is shown in Fig. 1.9.

Fig. 1.9
figure 9

Representative LCLAT-catalyzed reaction: CL (18:2/18:2/18:2/18:2)

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9.1 LCLAT1

CL, a dimeric glycerophospholipid, is remodeled from dilyso-CL and monolyso-CL by LCLAT. Normal CL contains four linoleoyl group (C18:2) fatty acyl chains. Mouse LCLAT1 was first identified from the AGPAT family as a LCLAT enzyme [58]. Subsequently, it was also reported to possess LPIAT and LPGAT activities [61]. A previous study used site-directed mutagenesis to identify a putative LPI recognition site. Transfection with LCLAT1 siRNA decreased LPIAT and LPGAT activity but not LCLAT activity. It is possible that LCLAT1 functions as both LPIAT and LPGAT enzymes in vivo. An additional report showed that LCLAT1 was localized in the mitochondria-associated membrane (MAM) [62]. LCLAT1 overexpression in cells increased the amount of polyunsaturated fatty acid that contained CL and also induced oxidative stress and mitochondrial dysfunction. LCLAT1-KO mice are protected from diet-induced obesity, insulin resistance, and hypertrophic cardiomyopathy [34, 62, 63].

10 Conclusion

The Kennedy pathway and the Lands’ cycle were first proposed in the 1950s. More than ten LPLATs have been identified during the past decade, resulting in significant advancement of the LPLAT field. However, the nomenclature should be standardized in the international conferences to bring about progress in phospholipid research because most enzymes have several confusing names. It is possible that additional LPLATs with preferences for different substrates might contribute to the generation of membrane diversity and will be identified in future studies. The redundant and pleiotropic substrate preferences of LPLATs might help regulate membrane diversity in tissues, which could be changed in response to external stimuli (Fig. 1.10). Further in vivo studies are needed to elucidate the biological roles of LPLATs and to understand the biological significance of membrane diversity and asymmetry.

Fig. 1.10
figure 10

Generation of tissue-specific membrane diversity. Several LPLATs biosynthesize phospholipids using acyl-CoAs and lysophospholipids. Expression pattern of LPLATs, acyl-CoA synthetases, and phospholipase As determines phospholipid composition in individual cells

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