Molecular Species Of Phosphatidylserine Research Articles

P-243 - Radical induced oxidation of di-oleoyl phosphatidylserine using mass spectrometry revealed preferential oxydation of Sn-2 oleoyl chain

Phosphatidylserine (PS) oxidation products (oxPS) play several roles in cell signaling, in immunity and in cell apoptosis. These different roles seem to be related to different oxPS species that can be generated in vivo. Yet, oxPS formed in biological systems are far from being totally characterized. In this study we evaluated the oxidation pattern of PS bearing two oleic acids (OOPS), one of the most abundant PS molecular species present in mammalian cells, using mass spectrometry (MS) approach. Oxidative modifications of OOPS was induced by hydroxyl radical generated under Fenton reaction. Oxidation products with hydroperoxide, hydroxy and keto groups were identified, similarly to what was previous reported for PS species with one oleic acid (OA). Multiple oxidation products, such as di-hydroperoxides were also observed, and revealed that both hydroperoxy moieties are preferentially linked to the oleoyl fatty acid in sn-2, revealing that radical mediate oxidation is not random. Also, this study shows for the first time that OA can suffer the insertion of multiple oxidative groups, providing further insight on possible oxPS formed in vivo.

Acyl chain dependent product inhibition by phosphatidylserine synthases

Phosphatidylserine (PS), the major anionic phospholipid in eukaryotic cell membranes, is synthesized by PS synthase 1 (PSS1) and PSS2 through the serine‐base exchange reaction from phosphatidylcholine (PC) and phosphatidylethanolamine (PE), respectively. Membrane PS is critically involved in key cell signaling processes such as Akt, PKC and Raf‐1 activation. The membrane PS profile is well maintained although mechanisms for the regulation of the PS profile are not clearly understood. In this study, we investigated the effect of PS acyl chain on the feedback mechanism in which activities of PSSs are inhibited by product PS. We found that PSS1 and PSS2 have significantly different inhibitory response to two PS molecular species, oleic acid (OA, 18:1n‐9) containing (OA‐PS) and docosahexaenoic acid (DHA, 22:6n‐3) containing PS species (DHA‐PS). PSS1 was inhibited more strongly by OA‐PS, while PSS2 was more by DHA‐PS. Together with the differential expression between PSS1 and PSS2 in variety of cells and tissues, these results indicate that the level of individual PS molecular species is regulated through feedback mechanism by which synthetic activity of each enzyme is inhibited differently.Intramural Program of National Institutes of Health

Enzymatic measurement of phosphatidylserine in cultured cells

Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid involved in diverse cellular functions. In this study, we developed a new fluorometric method for measuring PS using combinations of specific enzymes and Amplex Red. The calibration curve for PS measurement was linear and hyperbolic at low (0-50 µM) and high (50-1000 µM) concentrations, respectively, and the detection limit was 5 µM (50 pmol in the reaction mixture). This assay quantified PS regardless of the chain length and the number of double bonds. We applied this new method to the determination of PS content in HEK293 cells, which was validated by a recovery study and comparison with TLC-phosphorus assay. We showed that the PS content was high in sparse cells. The overexpression of PS synthase 1 elevated not only the cellular PS content but also the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents, suggesting the conversion of PS into PE and the enhancement of PC production. This new assay for PS measurement is simple, specific, sensitive, and high throughput, and it will be useful to clarify the metabolism and biological functions of PS.

Triacylglycerol lipolysis is linked to sphingolipid and phospholipid metabolism of the yeast Saccharomyces cerevisiae

Previous work from our laboratory had demonstrated that deletion of TGL3 encoding the major yeast triacylglycerol (TAG) lipase resulted in decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids, especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG fraction [K. Athenstaedt and G. Daum, J. Biol. Chem. 278 (2003) 23317–23323]. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we carried out metabolic labeling experiments with wild type and deletion strains bearing defects in the three major yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Using [ 3H]inositol, [ 32P]orthophosphate, [ 3H]palmitate and [ 14C]acetate as precursors for complex lipids we demonstrated that tgl mutants had a lower level of sphingolipids and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG accumulation in these mutant cells resulted in reduced amounts of phospholipids and sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected the metabolic flux of long chain fatty acids and very long chain fatty acids required for sphingolipid and glycerophospholipid synthesis. Activity and expression level of fatty acid elongases, Elo1p and Elo2p were enhanced as a consequence of reduced TAG lipolysis. Finally, the pattern of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine molecular species was altered in tgl deletion strain underlining the important role of TAG turnover in maintaining the pool size of these compounds and the remodeling of complex membrane lipids.

Oxidative lipidomics of hyperoxic acute lung injury: mass spectrometric characterization of cardiolipin and phosphatidylserine peroxidation

Reactive oxygen species have been shown to play a significant role in hyperoxia-induced acute lung injury, in part, by inducing apoptosis of pulmonary endothelium. However, the signaling roles of phospholipid oxidation products in pulmonary endothelial apoptosis have not been studied. Using an oxidative lipidomics approach, we identified individual molecular species of phospholipids involved in the apoptosis-associated peroxidation process in a hyperoxic lung. C57BL/6 mice were killed 72 h after exposure to hyperoxia (100% oxygen). We found that hyperoxia-induced apoptosis (documented by activation of caspase-3 and -7 and histochemical terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining of pulmonary endothelium) was accompanied by nonrandom oxidation of pulmonary lipids. Two anionic phospholipids, mitochondria-specific cardiolipin (CL) and extramitochondrial phosphatidylserine (PS), were the two major oxidized phospholipids in hyperoxic lung. Using electrospray ionization mass spectrometry, we identified several oxygenation products in CL and PS. Quantitative assessments revealed a significant decrease of CL and PS molecular species containing C(18:2), C(20:4), C(22:5), and C(22:6) fatty acids. Similarly, exposure of mouse pulmonary endothelial cells (MLEC) to hyperoxia (95% oxygen; 72 h) resulted in activation of caspase-3 and -7 and significantly decreased the content of CL molecular species containing C(18:2) and C(20:4) as well as PS molecular species containing C(22:5) and C(22:6). Oxygenated molecular species were found in the same two anionic phospholipids, CL and PS, in MLEC exposed to hyperoxia. Treatment of MLEC with a mitochondria-targeted radical scavenger, a conjugate of hemi-gramicidin S with nitroxide, XJB-5-131, resulted in significantly lower oxidation of both CL and PS and a decrease in hyperoxia-induced changes in caspase-3 and -7 activation. We speculate that cytochrome c driven oxidation of CL and PS is associated with the signaling role of these oxygenated species participating in the execution of apoptosis and clearance of pulmonary endothelial cells, thus contributing to hyperoxic lung injury.

Oxidized phosphatidylserine–CD36 interactions play an essential role in macrophage-dependent phagocytosis of apoptotic cells

The phagocytosis of apoptotic cells within an organism is a critical terminal physiological process in programmed cell death. Evidence suggests that apoptotic cell engulfment and removal by macrophages is facilitated by phosphatidylserine (PS) displayed at the exofacial surface of the plasma membrane; however, neither the macrophage receptors responsible for PS recognition, nor characterization of the PS molecular species potentially involved, have been clearly defined. We show that the class B scavenger receptor CD36 plays an essential role in macrophage clearance of apoptotic cells in vivo. Further, macrophage recognition of apoptotic cells via CD36 is shown to occur via interactions with membrane-associated oxidized PS (oxPS) and, to a lesser extent, oxidized phosphatidylcholine, but not nonoxidized PS molecular species. Mass spectrometry analyses of oxPS species identify structures of candidate ligands for CD36 in apoptotic membranes that may facilitate macrophage recognition. Collectively, these results identify oxPS–CD36 interactions on macrophages as potential participants in a broad range of physiologic processes where macrophage-mediated engulfment of apoptotic cells is involved.

Studies on Phosphatidylserine by Tandem Quadrupole and Multiple Stage Quadrupole Ion-Trap Mass Spectrometry with Electrospray Ionization: Structural Characterization and the Fragmentation Processes

Low-energy CAD product-ion spectra of various molecular species of phosphatidylserine (PS) in the forms of [M-H]- and [M-2H+Alk]- in the negative-ion mode, as well as in the forms of [M+H]+, [M+Alk]+, [M-H+2Alk]+, and [M-2H+3Alk]+ (where Alk=Li, Na) in the positive-ion mode contain rich fragment ions that are applicable for structural determination. Following CAD, the [M-H]- ion of PS undergoes dissociation to eliminate the serine moiety (loss of C3H5NO2) to give a [M-H-87]- ion, which equals to the [M-H]- ion of a phoshatidic acid (PA) and give rise to a MS3-spectrum that is identical to the MS2-spectrum of PA. The major fragmentation process for the [M-2H+Alk]- ion of PS arises from primary loss of 87 to give rise to a [M-2H+Alk-87]- ion, followed by loss of fatty acid substituents as acids (RxCO2H, x=1,2) or as alkali salts (e.g., RxCO2Li, x=1,2). These fragmentations result in a greater abundance of [M-2H+Alk-87-R2CO2H]- than [M-2H+Alk-87-R1CO2H]- and a greater abundance of [M-2H+Alk-87-R2CO2Li]- than [M-2H+Alk-87-R1CO2Li]-; while further dissociation of the [M-2H+Alk-87-R2(or 1)CO2Li]- ions gives a preferential formation of the carboxylate anion at sn-1 (R1CO2-) over that at sn-2 (R2CO2-). Other major fragmentation process arises from differential loss of the fatty acid substituents as ketenes (loss of Rx'CH=CO, x=1,2). This results in a more prominent [M-2H+Alk-R2'CH=CO]- ion than [M-2H+Alk-R1'CH=CO]- ion. Ions informative for structural characterization of PS are of low abundance in the MS2-spectra of both the [M+H]+ and the [M+Alk]+ ions, but are abundant in the MS3-spectra. The MS2-spectrum of the [M+Alk]+ ion contains a unique ion corresponding to internal loss of a phosphate group probably via the fragmentation processes involving rearrangement steps. The [M-H+2Alk]+ ion of PS yields a major [M-H+2Alk-87]+ ion, which is equivalent to an alkali adduct ion of a monoalkali salt of PA and gives rise to a greater abundance of [M-H+2Alk-87-R1CO2H]+ than [M-H+2Alk-87-R2CO2H]+. Similarly, the [M-2H+3Alk]+ ion of PS also yields a prominent [M-2H+3Alk-87]+ ion, which undergoes consecutive dissociation processes that involve differential losses of the two fatty acyl substituents. Because all of the above tandem mass spectra contain several sets of ion pairs involving differential losses of the fatty acid substituents as ketenes or as free fatty acids, the identities of the fatty acyl substituents and their positions on the glycerol backbone can be easily assigned by the drastic differences in the abundances of the ions in each pair.

Structural identification of human blood phospholipids using liquid chromatography/quadrupole-linear ion trap mass spectrometry

A normal-phase liquid chromatography/quadrupole-linear ion trap mass spectrometry method was developed for the separation and mass spectral characterization of the main phospholipid species in human blood. The instrument combines the capabilities of a triple quadrupole mass spectrometer and ion trap technology on a single platform. The optimal separation was achieved by using hexane/1-propanol as mobile phase and 0.6% formic acid, 0.06% ammonia as modifiers. The HPLC/MS technique was able to provide information about the molecular mass of individual homologues by positive and negative turbo ionspray. More complete characterization of fatty acid chains and of the polar head group was obtained by using a quadrupole collisionally activated dissociation (CAD) spectrum with ion trap sensitivity. The mass spectra and molecular species of phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysophosphatidylcholine (lysoPC) and sphingomyelin (SM) are presented.

Molecular Defects in Sarcolemmal Glycerophospholipid Subclasses in Diabetic Cardiomyopathy

A. Vecchini, F. Del Rosso, L. Binaglia, N. S. Dhalla and V. Panagia. Molecular Defects in Sarcolemmal Glycerophospholipid Subclasses in Diabetic Cardiomyopathy. Journal of Molecular and Cellular Cardiology (2000)32, 1061–1074. Although still scarcely studied, the phospholipid component of the cell membrane is of absolute importance for cell function. Experimental evidence indicates that individual molecular species of a given phospholipid can influence specific membrane functions. We have examined the changes in molecular species of diacyl and alkenylacyl choline/ethanolamine glycerophospholipid subclasses and those of phosphatidylserine in purified cardiac sarcolemma of healthy and streptozotocin-induced insulin dependent diabetic rats without or with insulin treatment. The relative content of plasmalogens increased in all the phospholipid classes of diabetic sarcolemma under study. Phosphatidylcholine and phosphatidylethanolamine were mostly enriched with molecular species containing linoleic acid in sn -2 position and deprived of the molecular species containing arachidonic acid. The molecular species of phosphatidylserine containing either arachidonic or docosahexaenoic acid were less abundant in membranes from diabetic rats than in membranes from controls. Insulin treatment of diabetic rats restored the species profile of phosphatidylethanolamine and overcorrected the changes in molecular species of phosphatidylcholine. The results suggest that the high sarcolemmal level of plasmalogens and the abnormal molecular species of glycerophospholipids may be critical for the membrane dysfunction and defective contractility of the diabetic heart.

Preferential Decarboxylation of Hydrophilic Phosphatidylserine Species in Cultured Cells: IMPLICATIONS ON THE MECHANISM OF TRANSPORT TO MITOCHONDRIA AND CELLULAR AMINOPHOSPHOLIPID SPECIES COMPOSITIONS

In baby hamster kidney and other cultured cells the majority of phosphatidylethanolamine (PE) is synthesized from phosphatidylserine (PS) in a process which involves transport of PS from the endoplasmic reticulum to mitochondria and decarboxylation therein by PS decarboxylase. To study the mechanism of this transport process, we first determined the molecular species composition of PE and PS from baby hamster kidney and Chinese hamster ovary cells. Interestingly, the hydrophilic diacyl molecular species were found to be much more abundant in PE than in PS, suggesting that hydrophilic PS species may be more readily transported to mitochondria than the hydrophobic ones. To study this, we compared the rates of decarboxylation of different PS molecular species in these cells. The cells were pulse labeled with [3H]serine whereafter the distribution of the labels among PS and PE molecular species was determined by reverse phase high performance liquid chromatography and liquid scintillation counting. The hydrophilic PE species contained relatively much more 3H label than those of PS, which indicates that they are more readily decarboxylated than the hydrophobic ones. Control experiments showed that differences in [3H]PS and -PE molecular species profiles are not due to (i) incorporation of 3H label to some PE species via alternative pathways, (ii) differences in degradation or remodeling among species, or (iii) selective decarboxylation of PS molecular species by the enzyme. Therefore, hydrophilic PS species are indeed decarboxylated faster than the hydrophobic ones. The rate of decarboxylation decreased systematically with hydrophobicity, strongly suggesting that formation of so called activated monomers, i.e. lipid molecules perpendicularly displaced from the membrane (Jones, J. D., and Thompson, T. E. (1990) Biochemistry 29, 1593-1600), is the rate-limiting step in the transport of PS from the endoplasmic reticulum to mitochondria. The formation of activated monomers and thus the rate of transfer is probably greatly enhanced by frequent collisions between the two membranes which tend to be closely associated. The present data also provides a feasible explanation why hydrophilic molecular species in these cells are much more abundant in PE as compared with PS, its immediate precursor.

High-performance liquid chromatographic method for determination of the metabolism of polyunsaturated molecular species of phosphatidylserine labeled in the polar group.

A reversed-phase HPLC method to monitor the incorporation of radiolabeled precursors into the polar group of individual polyunsaturated molecular species of phosphatidylserine (PS) is presented. PS labeled in the polar group was decarboxylated and subsequently converted to trinitrophenyl-phosphatidylethanolamine (Tnp-PE), which was separated into its molecular species by reversed-phase HPLC within 90 min, using a gradient of acetonitrile-methanol and ammonium acetate. A major feature of the method is the complete resolution of the stearoyl species, 18:0/20:4 and 18:0/22:6, at ambient temperature. By determining the amount of radioactivity incorporated into each fraction, the metabolism of individual molecular species of PS, and also of PE, labeled in the polar group can be investigated.

The selective use of stearoyl-polyunsaturated molecular species of phosphatidylcholine and phosphatidylethanolamine for the synthesis of phosphatidylserine

In rat liver microsomes, [ 3H]serine was incorporated primarily into the two most abundant molecular species of microsomal phosphatidylserine (PS), 18:0 20:4 and 18:0 22:6 , by Ca 2+-dependent base exchange. The pattern of PS molecular species synthesized was very similar to the species composition of PS and markedly different from the species composition of either microsomal precursor, phosphatidylcholine (PC) or phosphatidylethanolamine (PE). The data indicated that the enrichment of rat liver PS in mainly three fatty acids — stearic, arachidonic, and docosahexaenoic acids, occurred by (1) the preference by PS synthases for the stearoyl-polyunsaturated molecular species, 18:0 20:4 and 18:0 22:6 , of PC and PE and (2) a discrimination against the use of the palmitoyl-polyunsaturated species, 16:0 20:4 and 16:0 22:6 , and the stearoyl-diunsaturated species, 18:0 18:2 . The preferential use of the two species of PC and PE, based on their acyl chain content and not on their relative abundance, demonstrates that an individual molecular species can be selected out of the total pool for a defined function.

Separation of Molecular Species of Phosphatidylserine by Reverse-Phase Ion-Pair HPLC

Abstract Molecular species of phosphatidylserines (PS) were separated by reverse-phase ion-pair high-performance liquid chromatography (RP-IP-HPLC). HPLC mobile phases contained either tetraalkyl ammonium phosphates (TAAP) or alkyltriethyl ammonium phosphates (ATAP) in acetonitrile-methanol-water. Separations of the PS components on several different alkylbonded silica stationary phases were compared. The presence of high concentrations of TAAP (or ATAP) in mobile phases invariably enhanced the retention of the analytes on a RP column. Capacity factors (k') of the lipid components increased with increasing hydrophobicity of the ammonium counter ions. The HPLC results are indicative of solute retention via ion-pairing processes. Logarithmic k' values of analytes were linearly related to the total number of carbons in each series of the quaternary ammonium salts. However, linear relationships failed when correlated with counter-ions of different structural series. The counter-ion effects were also influence...

Aminophospholipid molecular species asymmetry in the human erythrocyte plasma membrane

The transbilayer distribution of the molecular species of aminophospholipids in human red blood cell plasma membrane has been investigated using a covalent labelling technique. Separation and quantitative analysis of the molecular species of phosphatidylethanolamine (PE) and phosphatidylserine (PS) was performed using high-performance liquid chromatography with UV detection of the trinitrophenyl derivatives obtained after reaction with trinitrobenzenesulfonic acid (TNBS). When the molecular species distribution obtained with intact cells was compared to that of the whole membrane, a molecular species asymmetry was evident. This phenomenon was most clearly evident when the reaction was performed at low temperatures (0°C) and was obscured by the excessive labelling or probe permeation associated with higher temperatures or longer incubation times. The monoene species were enriched in the outer leaflet, they comprised about 30% of the PE species in this leaflet. The polyunsaturates were preferentially localized in the inner leaflet and this was true of the arachidonyl species in particular as they represented up to 35% of this pool. The w-3 polyunsaturated fatty acids displayed a preferential localization in the plasmalogen subclass in comparison to the diacyl fraction, i.e., they comprised about 58 of the former and 42% of the latter subclass of cellular PE w-3 species. Data concerning the separation, identification and quantification of PS molecular species in human erythrocytes is also presented. The internal localization of the polyunsaturated species as well as the compartmentalization of the w-3 and w-6 pools will have metabolic, structural and physical implications for membrane function.

Interactions of mitochondrial precursor protein apocytochrome c with phosphatidylserine in model membranes. A monolayer study

(1) The interaction of apocytochrome c with different molecular species of phosphatidylserine was studied using monolayers at constant surface area or constant surface pressure. The protein inserted readily into dioleoylphosphatidylserine monolayers up to a limiting pressure of 50 mN/m, whereas the interaction decreased with increasing molecular packing of the phosphatidylserine species, indicating the importance of the hydrophobic core of the lipid layer for the interaction. (2) The high affinity of apocytochrome c for dioleoylphosphatidylserine is indicated by the low K d of 0.017 μM. There is little or no interaction with phosphatidylcholines. The importance of charge interactions is underlined by its ionic strength and pH dependency. (3) Experiments using 14C-labelled apocytochrome c indicate that cholesterol can enhance the protein binding. (4) It was demonstrated that apocytochrome c monomers penetrate the monolayer whereas oligomers can be formed in an adsorbed layer and washed off without changing the surface pressure. Preincubation of apocytochrome c in 3 M guanidine, to obtain the monomeric form, was essential to measure the full effect of interfacial interaction. (5) The molecular area of apocytochrome c changed from 1200–1300 Å 2/molecule in the absence of lipid to 700–900 Å 2/molecule after penetration of dioleoylphosphatidylserine monolayers. (6) Apocytochrome c-dileoylphosphatidylserine interactions are only possible when the monolayer is approached from the subphase. It is concluded that the charge interactions are required for binding and penetration of the protein.