Oxidatively modified fatty acyl chain determines physicochemical properties of aggregates of oxidized phospholipids

In vivo oxidation of glycerophospholipid generates a variety of products including truncated oxidized phospholipids (tOx-PLs). The fatty acyl chains at the sn-2 position of tOx-PLs are shorter in length than the parent non-oxidized phospholipids and contain a polar functional group(s) at the end. The effect of oxidatively modified sn-2 fatty acyl chain on the physicochemical properties of tOx-PLs aggregates has not been addressed in detail, although there are few reports that modified fatty acyl chain primarily determines the biological activities of tOx-PLs. In this study we have compared the properties of four closely related tOx-PLs which differ only in the type of modified fatty acyl chain present at the sn-2 position: 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC). Aggregates of individual tOx-PL in aqueous solution were characterized by fluorescence spectroscopy, size exclusion chromatography, native polyacrylamide and agarose gel electrophoresis. The data suggest that aggregates of four closely related tOx-PLs form micelle-like particles of considerably different properties. Our result provides first direct evidence that because of the specific chemical composition of the sn-2 fatty acyl chain aggregates of particular tOx-PL possess a distinctive set of physicochemical properties.     

Yeast cells accumulate excess endogenous palmitate in phosphatidylcholine by acyl chain remodeling involving the phospholipase B Plb1p

In the yeast Saccharomyces cerevisiae, the molecular species profile of the major membrane glycerophospholipid phosphatidylcholine (PC) is determined by the molecular species-selectivity of the biosynthesis routes and by acyl chain remodeling. Overexpression of the glycerol-3-phosphate acyltransferase Sct1p was recently shown to induce a strong increase in the cellular content of palmitate (C16:0). Using stable isotope labeling and mass spectrometry, the present study shows that wild type yeast overexpressing Sct1p incorporates excess C16:0 into PC via the methylation of PE, the CDP-choline route, and post-synthetic acyl chain remodeling. Overexpression of Sct1p increased the extent of remodeling of PE-derived PC, providing a novel tool to perform mechanistic studies on PC acyl chain exchange. The exchange of acyl chains occurred at both the sn-1 and sn-2 positions of the glycerol backbone of PC, and required the phospholipase B Plb1p for optimal efficiency. Sct1p-catalyzed acyl chain exchange, the acyl-CoA binding protein Acb1p, the Plb1p homologue Plb2p, and the glycerophospholipid:triacylglycerol transacylase Lro1p were not required for PC remodeling. The results indicate that PC serves as a buffer for excess cellular C16:0.     

Changes in the content and acyl group composition of glycerophospholipids of brain endothelial cells of the developing rat

The glycerophospholipid (GPL) content and acyl group compositions of isolated brain endothelial fractions have been determined in the developing rat. During development there is a marked change in proportions of ethanolamine glycerophospholipids (EGP) to choline glycerophospholipids (CGP), the former rising while CGP falls with age. The acyl group compositions of plasmenylethanolamine (P-GPE) and 1,2-diacyl-sn-glycero-3-phosphocholine (D-GPC) alter significantly during development; both show a decline in saturated fatty acids (SFAs) and a rise in then-6/SFA ratio, in contrast to a constancy in composition of 1,2-diacyl-sn-glycero-3-phosphoethanolamine (D-GPE). The degree of change in the acyl group composition in a particular GPL fraction is related to the rate of its accumulation and to the proportional increase in concentration, fractions accumulating most rapidly or increasing markedly in concentration showing the greatest acyl group compositional change. The possible significance of the high proportion of SFAs in P-GPE and D-GPC fractions in the developing brain endothelial fraction is discussed in relation to the altering blood-brain barrier capacities observed with age.     

Glycerophospholipids in brain: their metabolism, incorporation into membranes, functions, and involvement in neurological disorders

Neural membranes contain several classes of glycerophospholipids which turnover at different rates with respect to their structure and localization in different cells and membranes. The glycerophospholipid composition of neural membranes greatly alters their functional efficacy. The length of glycerophospholipid acyl chain and the degree of saturation are important determinants of many membrane characteristics including the formation of lateral domains that are rich in polyunsaturated fatty acids. Receptor-mediated degradation of glycerophospholipids by phospholipases A(l), A(2), C, and D results in generation of second messengers such as arachidonic acid, eicosanoids, platelet activating factor and diacylglycerol. Thus, neural membrane phospholipids are a reservoir for second messengers. They are also involved in apoptosis, modulation of activities of transporters, and membrane-bound enzymes. Marked alterations in neural membrane glycerophospholipid composition have been reported to occur in neurological disorders. These alterations result in changes in membrane fluidity and permeability. These processes along with the accumulation of lipid peroxides and compromised energy metabolism may be responsible for the neurodegeneration observed in neurological disorders.     

Glycerophospholipid acquisition in Plasmodium - A puzzling assembly of biosynthetic pathways

Throughout the Plasmodium life cycle, malaria parasites repeatedly undergo rapid cellular growth and prolific divisions, necessitating intense membrane neogenesis and, in particular, the acquisition of high amounts of phospholipids. At the intraerythrocytic stage, glycerophospholipids are the main parasite membrane constituents, which mostly originate from the Plasmodium-encoded enzymatic machinery. Several proteins and entire pathways have been characterized and their features reported, thereby generating a global view of glycerophospholipid synthesis across Plasmodium spp. The malaria parasite displays a panoply of pathways that are seldom found together in a single organism. The major glycerophospholipids are synthesized via ancestral prokaryotic CDP-diacylglycerol-dependent pathways and eukaryotic-type de novo pathways. The parasite exhibits additional reactions that bridge some of these routes and are otherwise restricted to some organisms, such as plants, while base-exchange mechanisms are largely unexplored in Plasmodium. Marked differences between Plasmodium spp. have also been reported in phosphatidylcholine and phosphatidylethanolamine synthesis. Little is currently known about glycerophospholipid acquisition at non-erythrocytic stages, but recent data reveal that intrahepatocytic parasites, oocysts and sporozoites import various host lipids, and that de novo fatty acid synthesis is only crucial at the late liver stage. More studies on the different Plasmodium developmental stages are needed, to further assemble the different pieces of this glycerophospholipid synthesis puzzle, which contains highly promising therapeutic targets.     

In Vivo Incorporation of a Polyunsaturated Fatty Acid into the sn-C-1 and sn-C-2 Positions of Tetrahymena Glycerophospholipids1

The glycerophospholipids of the protozoon Tetrahymena pyriformis W are unique in that the polyunsaturated fatty acid γ-linolenate (18:3Δ^6,9,12) is a major component of both the sn-C-1 and sn-C-2 positions. Tetrahymena were incubated with [1-¹⁴C]γ-linolenate. The positional distribution of the radiolabeled fatty acid in the three major glycerophospholipids was determined. [1-¹⁴C]γ-linolenate was found at both carbons of the three lipids, in general agreement with the mass distribution of γ-linolenate, except for markedly greater labeling at the sn-C-2 position of phosphatidylcholine. We hypothesize that an acyltransferase exists in Tetrahymena that can esterify γ-linolenate at both carbons during glycerophospholipid biosynthesis.     

Fatty acid positional distribution in phospholipids of a psychrophilic bacterium during changes in growth temperature

The major phospholipids of the psychrophilic bacteriumMicrococcus cryophilus, phosphatidylethanolamine and phosphatidylglycerol, have similar fatty acid compositions, comprising almost entirely palmitoleic and oleic acids. We show that there is a preference for the longer chains in thesn-1 position of both phosphatidylethanolamine and phosphatidylglycerol, both during isothermal growth and after temperature shifts, despite the fact that the overall phospholipid C18/C16 acyl chain ratio decreases with a lowering of growth temperature. Although it has been shown using model systems that the isomeric configurationsn-1-long,sn-2-short lowers lipid melting temperature, this paper reports the first clear-cut demonstration of such an isomeric preference in a natural system. We discuss how this acyl chain configuration contributes to membrane fluidity inM. cryophilus, in terms of adaptation to its psychrophilic habitat.     

Metabolic interrelations of hydroxy-substituted ether-linked glycerolipids in the pink portion of the rabbit harderian gland.

The pink portion of the rabbit harderian gland is known to contain a preponderance of ether-linked glycerolipids consisting primarily of 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols and smaller amounts of 1-alkyl-2,3-diacyl-sn-glycerols. In the present study, we have used a combination of chemical, enzymatic, and chromatographic techniques to identify two minor lipid components in the gland as 1-hydroxyalkyl-2-acyl-sn-glycerols and 1-hydroxyalkyl-2,3-diacyl-sn-glycerols. The long-chain acyl groups occurring in the 1-hydroxyalkyl-2-acyl-sn-glycerols and 1-hydroxyalkyl-2,3-diacyl-sn-glycerols are almost exclusively hexadecanoic acid, whereas the 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols have a ratio of hexadecanoic acid to octadecanoic acid of $\text{2}\text{1}$. The 1-(O-acyl) hydroxyalkyl-2,3-diacyl-sn-glycerols and the 1-hydroxyalkyl-2,3-diacyl-sn-glycerols also contain a short-chain acyl moiety identified as 3-methylbutanoic acid (isovaleric acid). This acid was found to occupy the 3-position of the glycerol backbone in these lipid classes.Metabolic experiments demonstrate that 3-methylbutanoic acid in the lipids of the gland is derived from the catabolism of l-leucine. Pulse-chase data show a precursor-product relation between the 1-hydroxyalkyl-2,3-diacyl-sn-glycerols and 1-(O-acyl-hydroxyalkyl-2,3-diacyl-sn-glycerols and rule out direct hydroxylation of 1-alkyl-2,3-diacyl-sn-glycerols as a possible biosynthetic route to the 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols.Characterization of the alkyl and acyl groups and the positional distributions of the acyl moieties in combination with the metabolic information indicated the acylation sequence involved in the formation of 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerol is 1-hydroxyalkyl-2-acyl-sn-glycerols → 1-hydroxyalkyl-2,3-diacyl-sn-glycerols → 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols. The data also suggest that hydroxylation of the alkyl side-chain occurs before or at the alkylacylglycerol stage.     

Closely related oxidized phospholipids differentially modulate the physicochemical properties of lipid particles

Oxidation of glycerophospholipids results in the formation of large variety of oxidized phospholipid products that differs significantly in their chemical compositions and molecular structures. Biological activities of these oxidized products also differ considerably. Here we report the comparisons of the physicochemical properties of non-oxidized phospholipid particle containing two closely related tOx-PLs: 1-palmitoyl-2-(5-keto-6-octendioyl)-sn-glycero-3-phosphocholine (KOdiA-PC) and 1-palmitoyl-2-(9-keto-10-dodecendioyl)-sn-glycero-3-phosphocholine (KDdiA-PC). DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) was used as a model membrane non-oxidized phospholipid. Physicochemical properties of the lipid particles were characterized by using fluorescence spectroscopy, native polyacrylamide gel and agarose gel electrophoresis. Our result shows that the presence of closely related tOx-PLs, which differ only in the chemical composition of the oxidized fatty acyl chains at the sn-2 position, exerts considerably different effect on the physicochemical properties of non-oxidized phospholipid particles containing them.     

The glycerophospholipid inventory of Pseudomonas putida is conserved between strains and enables growth condition‐related alterations

Microorganisms, such as Pseudomonas putida, utilize specific physical properties of cellular membrane constituents, mainly glycerophospholipids, to (re‐)adjust the membrane barrier to environmental stresses. Building a basis for membrane composition/function studies, we inventoried the glycerophospholipids of different Pseudomonas and challenged membranes of growing cells with n‐butanol. Using a new high‐resolution liquid chromatography/mass spectrometry (LC/MS) method, 127 glycerophospholipid species [e.g. phosphatidylethanolamine PE(32:1)] with up to five fatty acid combinations were detected. The glycerophospholipid inventory consists of 305 distinct glycerophospholipids [e.g. PE(16:0/16:1)], thereof 14 lyso‐glycerophospholipids, revealing conserved compositions within the four investigated pseudomonads P. putida KT2440, DOT‐T1E, S12 and Pseudomonas sp. strain VLB120. Furthermore, we addressed the influence of environmental conditions on the glycerophospholipid composition of Pseudomonas via long‐time exposure to the sublethal n‐butanol concentration of 1% (v/v), focusing on: (i) relative amounts of glycerophospholipid species, (ii) glycerophospholipid head group composition, (iii) fatty acid chain length, (iv) degree of saturation and (v) cis/trans isomerization of unsaturated fatty acids. Observed alterations consist of changing head group compositions and for the solvent‐sensitive strain KT2440 diminished fatty acid saturation degrees. Minor changes in the glycerophospholipid composition of the solvent‐tolerant strains P. putida S12 and Pseudomonas sp. VLB120 suggest different strategies of the investigated Pseudomonas to maintain the barrier function of cellular membranes.     

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252–6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 μmol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.     

Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C₁₈-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4–6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism.     

Factors regulating the substrate specificity of cytosolic phospholipase A2-alpha in vitro

Cytosolic phospholipase A₂ alpha (cPLA₂α) plays a key role in signaling in mammalian cells by releasing arachidonic acid (AA) from glycerophospholipids (GPLs) but the factors determining the specificity of cPLA₂α for AA-containing GPLs are not well understood. Accordingly, we investigated those factors by determining the activity of human cPLA₂α towards a multitude of GPL species present in micelles or bilayers. Studies on isomeric PC sets containing a saturated acyl chain of 6 to 24 carbons in the sn1 or sn2 position in micelles showed an abrupt decrease in hydrolysis when the length of the sn1 or sn2 chain exceeded 17 carbons suggesting that the acyl binding cavity on the enzyme is of the corresponding length. Notably, the saturated isomer pairs were hydrolyzed identically in micelles as well as in bilayers suggesting promiscuous binding of acyl chains to the active site of cPLA₂α. Such promiscuous binding would explain the previous finding that cPLA₂α has both PLA₁ and PLA₂ activities. Interestingly, increasing the length of either the sn1 or sn2 acyl chain inhibited the hydrolysis in bilayers far more than that in micelles suggesting that with micelles (loosely packed) substrate accommodation at the active site of cPLA₂α is rate-limiting, while with bilayers (tightly packed) upward movement of the substrate from the bilayer (efflux) is the rate-limiting step. With the AA-containing PCs, the length of the saturated acyl chain also had a much stronger effect on hydrolysis in bilayers vs. micelles in agreement with this model. In contrast to saturated PCs, a marked isomer preference was observed for AA-containing PCs both in micelles and bilayers. In conclusion, these data significantly help to understand the mode of action and specificity of cPLA₂α.     

Selective plasmenylcholine oxidation by hypochlorous acid: formation of lysophosphatidylcholine chlorohydrins

The plasmalogen sn-1 vinyl ether bond is targeted by hypochlorous acid (HOCl) produced by activated phagocytes. In the present study, the attack of the plasmalogen sn-1 vinyl ether bond by HOCl is shown to be preferred compared to the attack of double bonds present in the sn-2 position aliphatic chain (sn-2 alkenes) of both plasmenylcholine and phosphatidylcholine. Lysophosphatidylcholine (LPC) is a product from the initial HOCl attack of plasmenylcholine and the sn-2 alkene bonds present in this LPC product are secondary targets of HOCl leading to the production of LPC-chlorohydrins (ClOH). The aliphatic ClOH was demonstrated in both the positive and negative ion mode using collisionally-activated dissociation (CAD) of the molecular ion of LPC-ClOH. Furthermore, HOCl treatment of endothelial cells led to the preferential attack of plasmalogens in comparison to that of diacyl choline glycerophospholipids. Taken together, plasmenylcholine is oxidized preferentially over phosphatidylcholine and leads to the production of LPC-ClOH.     

Selectivity of phospholipid hydrolysis by phospholipase A2 enzymes in activated cells leading to polyunsaturated fatty acid mobilization

Phospholipase A₂s are enzymes that hydrolyze the fatty acid at the sn-2 position of the glycerol backbone of membrane glycerophospholipids. Given the asymmetric distribution of fatty acids within phospholipids, where saturated fatty acids tend to be present at the sn-1 position, and polyunsaturated fatty acids such as those of the omega-3 and omega-6 series overwhelmingly localize in the sn-2 position, the phospholipase A₂ reaction is of utmost importance as a regulatory checkpoint for the mobilization of these fatty acids and the subsequent synthesis of proinflammatory omega-6-derived eicosanoids on one hand, and omega-3-derived specialized pro-resolving mediators on the other. The great variety of phospholipase A₂s, their differential substrate selectivity under a variety of pathophysiological conditions, as well as the different compartmentalization of each enzyme and accessibility to substrate, render this class of enzymes also key to membrane phospholipid remodeling reactions, and the generation of specific lipid mediators not related with canonical metabolites of omega-6 or omega-3 fatty acids. This review highlights novel findings regarding the selective hydrolysis of phospholipids by phospholipase A₂s and the influence this may have on the ability of these enzymes to generate distinct lipid mediators with essential functions in biological processes. This brings a new understanding of the cellular roles of these enzymes depending upon activation conditions.     

Molecular basis of phospholipase A2 activity toward phospholipids with sn-1 substitutions

We studied secretory phospholipase A₂ type IIA (sPLA₂) activity toward phospholipids that are derivatized in the sn-1 position of the glycerol backbone. We explored what type of side group (small versus bulky groups, hydrophobic versus polar groups) can be introduced at the sn-1 position of the glycerol backbone of glycerophospholipids and at the same time be hydrolyzed by sPLA₂. The biophysical characterization revealed that the modified phospholipids can form multilamellar vesicles, and several of the synthesized sn-1 functionalized phospholipids were hydrolyzed by sPLA₂. Molecular dynamics simulations provided detailed insight on an atomic level that can explain the observed sPLA₂ activity toward the different phospholipid analogs. The simulations revealed that, depending on the nature of the side chain located at the sn-1 position, the group may interfere with an incoming water molecule that acts as the nucleophile in the enzymatic reaction. The simulation results are in agreement with the experimentally observed sPLA₂ activity toward the different phospholipid analogs.     

Barotropic and thermotropic bilayer phase behavior of positional isomers of unsaturated mixed-chain phosphatidylcholines

The bilayer phase transitions of six kinds of mixed-chain phosphatidylcholines (PCs) with an unsaturated acyl chain in the sn-1 or sn-2 position, 1-oleoyl-2-stearoyl- (OSPC), 1-stearoyl-2-oleoyl- (SOPC), 1-oleoyl-2-palmitoyl- (OPPC), 1-palmitoyl-2-oleoyl- (POPC), 1-oleoyl-2-myristoyl- (OMPC) and 1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light transmittance measurements. Bilayer membranes of SOPC, POPC and MOPC with an unsaturated acyl chain in the sn-2 position exhibited only one phase transition, which was identified as the main transition between the lamellar gel (L_β) and liquid crystalline (L_α) phases. On the other hand, the bilayer membranes of OSPC, OPPC and OMPC with an unsaturated acyl chain in the sn-1 position exhibited not only the main transition but also a transition from the lamellar crystal (L_c) to the L_β (or L_α) phase. The stability of their gel phases was markedly affected by pressure and chain length of the saturated acyl chain in the sn-2 position. Considering the effective chain lengths of unsaturated mixed-chain PCs, the difference in the effective chain length between the sn-1 and sn-2 acyl chains was proven to be closely related to the temperature difference of the main transition. That is, a mismatch of the effective chain length promotes a temperature difference of the main transition between the positional isomers. Anomalously small volume changes of the L_c/L_α transition for the OPPC and OMPC bilayers were found despite their large enthalpy changes. This behavior is attributable to the existence of a cis double bond and to significant inequivalence between the sn-1 and sn-2 acyl chains, which brings about a small volume change for chain melting due to loose chain packing, corresponding to a large partial molar volume, even in the L_c phase. Further, the bilayer behavior of unsaturated mixed-chain PCs containing an unsaturated acyl chain in the sn-1 or sn-2 position was well explained by the chemical-potential diagram of a lipid in each phase.     

Separation and quantification of 2-acyl-1-lysophospholipids and 1-acyl-2-lysophospholipids in biological samples by LC-MS/MS

Lysophospholipids (LysoGPs) serve as lipid mediators and precursors for synthesis of diacyl phospholipids (GPs). LysoGPs detected in cells have various acyl chains attached at either the sn-1 or sn-2 position of the glycerol backbone. In general, acyl chains at the sn-2 position of 2-acyl-1-LysoGPs readily move to the sn-1 position, generating 1-acyl-2-lyso isomers by a nonenzymatic reaction called intra-molecular acyl migration, which has hampered the detection of 2-acyl-1-LysoGPs in biological samples. In this study, we developed a simple and versatile method to separate and quantify 2-acyl-1- and 1-acyl-2-LysoGPs. The main point of the method was to extract LysoGPs at pH 4 and 4°C, conditions that were found to completely eliminate the intra-molecular acyl migration. Under the present conditions, the relative amounts of 2-acyl-1-LysoGPs and 1-acyl-2-LysoGPs did not change at least for 1 week. Further, in LysoGPs extracted from cells and tissues under the present conditions, most of the saturated fatty acids (16:0 and 18:0) were found in the sn-1 position of LysoGPs, while most of the PUFAs (18:2, 20:4, 22:6) were found in the sn-2 position. Thus the method can be used to elucidate the in vivo role of 2-acyl-1-LysoGPs.     

Plasmenylethanolamine synthesis in Leishmania major

Ethanolamine glycerophospholipids are ubiquitous cell membrane components. Trypanosomatid parasites of the genus Leishmania synthesize the majority of their ethanolamine glycerophospholipids as 1‐O‐alk‐1′‐enyl‐2‐acyl‐sn‐glycero‐3‐phosphoethanolamine or plasmenylethanolamine (PME) through the Kennedy pathway. PME is a subtype of ether phospholipids also known as ethanolamine plasmalogen whose functions are not well characterized. In this study, we investigated the role of PME synthesis in Leishmania major through the characterization of an ethanolamine phosphotransferase (EPT) mutant. EPT‐null parasites are largely devoid of PME and fully viable in regular medium but fail to proliferate in the absence of fetal bovine serum. They exhibit significant abnormalities in the synthesis and localization of GPI‐anchored surface molecules. EPT‐null mutants also show attenuated virulence in BALB/c mice. Furthermore, in addition to PME synthesis, ethanolamine also contributes to the production of phosphatidylcholine, the most abundant class of lipids in Leishmania. Together, these findings suggest that ethanolamine production is likely required for Leishmania promastigotes to generate bulk phospholipids, to handle stress, and to control the expression of membrane bound virulence factors.