Human TMEM30a promotes uptake of antitumor and bioactive choline phospholipids into mammalian cells

Antitumor alkylphospholipids initiate apoptosis in transformed HL-60 and Jurkat cells while sparing their progenitors. 1-O-Alkyl-2-carboxymethyl-sn-glycero-3-phosphocholine (Edelfosine) like other short-chained phospholipids--inflammatory platelet-activating factor (PAF) and apoptotic oxidatively truncated phospholipids--are proposed to have intracellular sites of action, yet a conduit for these choline phospholipids into mammalian cells is undefined. Edelfosine is also accumulated by Saccharomyces cerevisiae in a process requiring the membrane protein Lem3p, and the human genome contains a Lem3p homolog TMEM30a. We show that import of choline phospholipids into S. cerevisiae ΔLem3 is partially reconstituted by human TMEM30a and by Lem3p-TMEM30a chimeras, showing the proteins are orthologous. TMEM30a-GFP chimeras expressed in mammalian cells localized in plasma membranes, as well as internal organelles, and ectopic TMEM30a expression promoted uptake of exogenous choline and ethanolamine phospholipids. Short hairpin RNA knockdown of TMEM30a reduced fluorescent choline phospholipid and [(3)H]PAF import. This knockdown also reduced mitochondrial depolarization from exogenous Edelfosine or the mitotoxic oxidatively truncated phospholipid azelaoyl phosphatidylcholine, and the knockdown reduced apoptosis in response to these two phospholipids. These results show that extracellular choline phospholipids with short sn-2 residues can have intracellular roles and sites of metabolism because they are transport substrates for a TMEM30a phospholipid import system. Variation in this mechanism could limit sensitivity to short chain choline phospholipids such as Edelfosine, PAF, and proapoptotic phospholipids.     

Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) activates neutrophils (polymorphonuclear leukocytes, PMN) through a receptor that specifically recognizes short sn-2 residues. We oxidized synthetic [2-arachidonoyl]phosphatidylcholine to fragment and shorten the sn-2 residue, and then examined the phospholipid products for the ability to stimulate PMN. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was fragmented by ozonolysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. This phospholipid activated human neutrophils at submicromolar concentrations, and is effects were inhibited by specific PAF receptor antagonists WEB2086, L659,989, and CV3988. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine next was fragmented by an uncontrolled free radical-catalyzed reaction: it was treated with soybean lipoxygenase to form its sn-2 15-hydroperoxy derivative (which did not activate neutrophils) and then allowed to oxidize under air. The secondary oxidation resulted in the formation of numerous fragmented phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103), some of which activated PMN. Hydrolysis of sn-2 residues with phospholipase A2 destroyed biologic activity, as did hydrolysis with PAF acetylhydrolase. PAF acetylhydrolase is specific for short or intermediate length sn-2 residues and does not hydrolyze the starting material (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103). Neutrophil activation was completely blocked by L659,989, a specific PAF receptor antagonist. We conclude that diacylphosphatidylcholines containing an sn-2 polyunsaturated fatty acyl residue can be oxidatively fragmented to species with sn-2 residues short enough to activate the PAF receptor of neutrophils. This suggests a new mechanism for the appearance of biologically active phospholipids, and shows that PAF receptor antagonists block the action of both PAF and these PAF-like lipids.     

Novel leukocyte agonists are released by endothelial cells exposed to peroxide.

Reactive oxygen species do not activate isolated neutrophils, yet in vivo, such oxidants promote their adhesion to, and subsequent migration through, the vascular wall. We show human endothelial cells exposed to t-butylhydroperoxide shed large, sealed membrane vesicles that contained potent neutrophil agonists. This activity migrated on TLC like platelet-activating factor (PAF). Since neutrophils have a receptor for this phospholipid, which recognizes its unique characteristics including the short sn-2 acetyl residue, we examined the effect of PAF receptor antagonists and PAF acetylhydrolase on this activity. Structurally unrelated PAF receptor antagonists blocked neutrophil stimulation by vesicular phospholipids, and digestion with PAF acetylhydrolase, which is specific for short sn-2 residues, destroyed this activity. However, metabolic labeling, inhibition of synthesis, phospholipase A1 digestion, and high performance liquid chromatographic studies demonstrated that the vesicles did not contain PAF. Instead, the bioactivity migrated on high performance liquid chromatography like the phospholipids generated by oxidative fragmentation of synthetic arachidonoyl phosphatidylcholine that we have shown previously (Smiley, P. L., Stremler, K. E., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11104-11110) to stimulate neutrophils through their receptor for PAF. Thus, peroxide treatment of endothelial cells fragments cellular phosphatidylcholines, forming novel PAF-like phospholipids, and induces the shedding of membrane vesicles that contain these bioactive phospholipids.     

Membrane-bound plasma platelet activating factor acetylhydrolase acts on substrate in the aqueous phase.

Human plasma platelet activating factor acetylhydrolase (pPAF-AH) is a phospholipase A(2) that specifically hydrolyzes the sn-2 ester of platelet activating factor (PAF) and of phospholipids with oxidatively truncated sn-2 fatty acyl chains. pPAF-AH is bound to lipoproteins in vivo, and it binds essentially irreversibly to anionic and zwitterionic phospholipid vesicles in vitro and hydrolyzes PAF and PAF analogues. Substrate hydrolysis also occurs in the absence of vesicles, with a maximum rate reached at the critical micelle concentration. A novel pre-steady-state kinetic analysis with enzyme tightly bound to vesicles and with a substrate that undergoes slow intervesicle exchange establishes that pPAF-AH accesses its substrate from the aqueous phase and thus is not an interfacial enzyme. Such a mechanism readily explains why this enzyme displays dramatic specificity for phospholipids with short sn-2 chains or with medium-length, oxidatively truncated sn-2 chains since a common feature of these lipids is their relatively high water solubility. It also explains why the enzymatic rate drops as the length of the sn-1 chain is increased. pPAF-AH shows broad specificity toward phospholipids with different polar headgroups. Additional results are that PAF undergoes intervesicle exchange on the subminute time scale and it does not undergo transbilayer movement over tens of minutes.     

Human plasma platelet-activating factor acetylhydrolase. Oxidatively fragmented phospholipids as substrates

Human plasma platelet-activating factor (PAF) acetylhydrolase hydrolyzes the sn-2 acetyl residue of PAF, but not phospholipids with long chain sn-2 residues. It is associated with low density lipoprotein (LDL) particles, and is the LDL-associated phospholipase A2 activity that specifically degrades oxidatively damaged phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1989) J. Biol. Chem. 264, 5331-5334). To identify potential substrates, we synthesized phosphatidylcholines with sn-2 residues from two to nine carbon atoms long, and found the V/k ratio decreased as the sn-2 residue was lengthened: the C5 homolog was 50%, the C6 20%, while the C9 homolog was only 2% as efficient as PAF. However, the presence of an omega-oxo function radically affected hydrolysis: the half-life of the sn-2 9-aldehydic homolog was identical to that of PAF. We oxidized [2-arachidonoyl]phosphatidylcholine and isolated a number of more polar phosphatidylcholines. We treated these with phospholipase C, derivatized the resulting diglycerides for gas chromatographic/mass spectroscopic analysis, and found a number of diglycerides where the m/z ratio was consistent with a series of short to medium length sn-2 residues. We treated the polar phosphatidylcholines with acetylhydrolase and derivatized the products for analysis by gas chromatography/mass spectroscopy. The liberated residues were more polar than straight chain standards and had m/z ratios from 129 to 296, consistent with short to medium chain residues. Therefore, oxidation fragments the sn-2 residue of phospholipids, and the acetylhydrolase specifically degrades such oxidatively fragmented phospholipids.     

Cytotoxic phospholipid oxidation products. Cell death from mitochondrial damage and the intrinsic caspase cascade

Phospholipid oxidation products accumulate in the necrotic core of atherosclerotic lesions, in apoptotic cells, and circulate in oxidized low density lipoprotein. Phospholipid oxidation generates toxic products, but little is known about which specific products are cytotoxic, their receptors, or the mechanism(s) that induces cell death. We find the most common phospholipid oxidation product of oxidized low density lipoprotein, phosphatidylcholine with esterified sn-2-azelaic acid, induced apoptosis at low micromolar concentrations. The synthetic ether phospholipid hexadecyl azelaoyl phosphatidylcholine (HAzPC) was rapidly internalized, and overexpression of PLA2g7 (PAF acetylhydrolase) that specifically hydrolyzes such oxidized phospholipids suppressed apoptosis. Internalized HAzPC associated with mitochondria, and cytochrome c, and apoptosis-inducing factor escaped from mitochondria to the cytoplasm and nucleus, respectively, in cells exposed to HAzPC. Isolated mitochondria exposed to HAzPC rapidly swelled and released cytochrome c and apoptosis-inducing factor. Other phospholipid oxidation products induced swelling, but HAzPC was the most effective and was twice as effective as its diacyl homolog. Cytoplasmic cytochrome c completes the apoptosome, and activated caspase 9 and 3 were present in cells exposed to HAzPC. Irreversible inhibition of caspase 9 blocked downstream caspase 3 activation and prevented apoptosis. Mitochondrial damage initiated this apoptotic cascade, because overexpression of Bcl-X(L), an anti-apoptotic protein localized to mitochondria, blocked cytochrome c escape and apoptosis. Thus, exogenous phospholipid oxidation products target intracellular mitochondria to activate the intrinsic apoptotic cascade.     

Oxidatively truncated phospholipids are required agents of tumor necrosis factor α (TNFα)-induced apoptosis

TNFα generates reactive oxygen species (ROS) at the cell surface that induce cell death, but how ROS communicate to mitochondria and their specific apoptotic action(s) are both undefined. ROS oxidize phospholipids to hydroperoxides that are friable and fragment adjacent to the (hydro)peroxide function, forming truncated phospholipids, such as azelaoyl phosphatidylcholine (Az-PC). Az-PC is relatively soluble, and exogenous Az-PC rapidly enters cells to damage mitochondrial integrity and initiate intrinsic apoptosis. We determined whether this toxic phospholipid is formed within cells during TNFα stimulation in sufficient quantities to induce apoptosis and if they are essential in TNFα-induced cytotoxicity. We found that TNFα induced ROS formation and phospholipid peroxidation in Jurkat cells, and either chemical interference with NADPH oxidase activity or siRNA suppression of the NADPH oxidase-4 subunit blocked ROS accumulation and phospholipid peroxidation. Mass spectrometry showed that phospholipid peroxides and then Az-PC increased after TNFα exposure, whereas ROS inhibition abolished Az-PC accumulation and TNFα-induced cell death. Glutathione peroxidase-4 (GPx4), which specifically metabolizes lipid hydroperoxides, fell in TNFα-stimulated cells prior to death. Ectopic GPx4 overcame this, reduced peroxidized phospholipid accumulation, blocked Az-PC accumulation, and prevented death. Conversely, GPx4 siRNA knockdown enhanced phospholipid peroxidation, increasing TNFα-stimulated Az-PC formation and apoptosis. Truncated phospholipids were essential elements of TNFα-induced apoptosis because overexpression of PAFAH2 (a phospholipase A(2) that selectively hydrolyzes truncated phospholipids) blocked TNFα-induced Az-PC accumulation without affecting phospholipid peroxidation. PAFAH2 also abolished apoptosis. Thus, phospholipid oxidation and truncation to apoptotic phospholipids comprise an essential element connecting TNFα receptor signaling to mitochondrial damage and apoptotic death.     

A yeast PAF acetylhydrolase ortholog suppresses oxidative death

Phospholipids containing sn-2 polyunsaturated fatty acyl residues are primary targets of oxidizing radicals, producing proapoptotic and membrane perturbing fragmented phospholipids. The only known phospholipases that specifically select these oxidized and/or short-chained phospholipids as substrates are mammalian group VII phospholipases A2s that were purified and cloned as PAF acetylhydrolases. Platelet-activating factor (PAF) is a short-chained phospholipid, and whether these enzymes actually are PAF hydrolases or evolved as oxidized phospholipid phospholipases is unknown. The fission yeast Schizosaccharomyces pombe, which does not form or use PAF as a signaling molecule, contains an open-reading frame potentially homologous to mammalian group VII phospholipase A2s. We cloned this SPBC106.11c locus and expressed it in distantly related Saccharomyces cerevisiae that lack homologous sequences. The S. pombe locus encoded a functional phospholipase A2, now renamed plg7+, that hydrolyzed PAF and a synthetic oxidized phospholipid. Expression of human type II PAF acetylhydrolase or S. pombe Plg7p enhanced the viability of S. cerevisiae subjected to oxidative stress. We conclude that a single-celled organism with an exceedingly spare genome still expresses an unusually discriminating phospholipase A2, and that selective hydrolysis of phospholipid oxidation products is an early, and critical, way to overcome oxidative membrane damage and oxidant-induced cell death.     

Correlation of antiphospholipid antibody recognition with the structure of synthetic oxidized phospholipids. Importance of Schiff base formation and aldol condensation.

The oxidation of low density lipoproteins (LDL) has been correlated with atherogenesis through a variety of pathways. The process involves nonspecific fragmentation, oxidative breakdown, and modification of the lipids and protein of LDL. The process yields a variety of bioactive products, including aldehyde-containing phospholipids, which can cross-react with primary amines (i.e. peptides or phospholipid head groups) to yield Schiff base products. We also demonstrate that such oxidized phospholipid products may further react through a post-oxidation chemical pathway involving aldol condensation. EO6, an IgM monoclonal autoantibody to oxidized phospholipids, blocks the uptake of oxidized LDL (OxLDL) by macrophages. Because the epitope(s) of EO6 also blocks the uptake of OxLDL, a series of oxidized phospholipids, their peptide complexes, and their aldol condensates have been synthesized and characterized, and their antigenicity has been determined. This study defines structural motifs of oxidized phospholipids responsible for antigenicity for EO6. Certain monomeric phospholipids containing short chain fatty acids were antigenic whether oxidized or not in the sn-2 position. However, oxidized phospholipids containing sn-1 long chain fatty acids were not antigenic unless the sn-2 oxidized fatty acid contained an aldehyde that first reacted with a peptide yielding a Schiff base or the sn-2 oxidized fatty acid underwent an aldol type self-condensation. Our data indicate that the phosphorylcholine head group is essential for antigenicity, but its availability depends on the oxidized phospholipid conformation. We suggest that upon oxidation, similar reactions occur in phospholipids on the surface of LDL, generating ligands for macrophage recognition. Synthetic imine adducts of oxidized phospholipids of this type are capable of blocking the uptake of OxLDL.     

Synthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine by an enriched preparation of the human lung mast cell

Our study has examined the synthesis of platelet activating factor (PAF; 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and of structurally related molecules by an enriched preparation (greater than 70%) of the human lung mast cell (HLMC) in response to immunologic stimulation. Upon activation with anti-IgE, HLMC incorporated exogenously provided acetate into a phospholipid that migrated with authentic PAF on TLC. The formation of this product in HLMC occurred concomitantly with histamine and leukotriene C4 release. Further analysis of this phospholipid revealed that 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (GPC) and not 1-alkyl-2-acetyl-GPC was the major 1-radyl-2-acetyl-GPC subclass formed during cell activation. The presence of 1-alkyl-2-acetyl-GPC was confirmed by negative ion chemical ionization mass spectrometry. In addition to this product, anti-IgE-stimulated HLMC synthesized relatively small quantities of another 2-acetylated phospholipid migrating on TLC between phosphatidylcholine and phosphatidylinositol. The chromatographic characteristics of this product suggested that it is a subclass of 1-radyl-2-acetyl-sn-glycero-3-phosphoethanolamine. The catabolism of both 1-acyl-2-acetyl-GPC and 1-alkyl-2-acetyl-GPC was next examined to determine if the predominant formation of 1-acyl-2-acetyl-GPC over 1-alkyl-2-acetyl-GPC were metabolized by the HLMC at similar rates. There was, however, a qualitative difference in the metabolic products derived from the two phospholipids. 1-Alkyl-2-acetyl-GPC was rapidly inactivated by removal of the acetate moiety at the sn-2 position followed by rapid reacylation with arachidonate. By contrast, 1-acyl-2-acetyl-GPC was catabolized mainly by removal of the fatty acyl moiety at the sn-1 position. These data demonstrate the natural occurrence of PAF and at least two structurally similar molecules in anti-IgE stimulated HLMC. Furthermore, an analog containing an ester linkage at the sn-1 position, 1-acyl-2-acetyl-GPC, appears to be the major acetylated product synthesized under these conditions.     

Molecular and mechanistic characterization of platelet-activating factor-like bioactivity produced upon LDL oxidation

Oxidation of LDL is thought to be involved in both initiating and sustaining atherogenesis through the formation of proinflammatory lipids and the covalent modification of LDL particles. Platelet-activating factor (PAF; 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent phospholipid mediator involved in inflammation. Upon oxidation of LDL, oxidized phospholipids with PAF-like structure are generated, and some of them may act via the PAF receptor. We evaluated the contribution of 1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) and of other PAF analogs on the PAF-like bioactivity formed upon Cu2+-initiated oxidation of LDL. Reverse-phase HPLC purification and electrospray ionization-MS analyses showed that upon oxidation of LDL with inactivated PAF-acetylhydrolase (PAF-AH), C16:0 PAF accounted for >30% of PAF-like biological activity and its sn-2 butenoyl analog accounted for >50%. However, upon LDL oxidation in the presence of exogenous 1-0-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) without PAF-AH inactivation, C16:0 PAF formation accounted for >90% of the biological activity recovered. We suggest that the C16:0 PAF, despite being a minor constituent of the LDL peroxidation products, may contribute substantially to the bioactivity formed in oxidized LDL. The higher bioactivity of C16:0 PAF, and the higher selectivity of the LDL-attached lyso-PAF transacetylase toward very short acyl chains [acetate (C2) vs. butanate (C4)], may explain the contribution described above.     

Ultraviolet B radiation generates platelet-activating factor-like phospholipids underlying cutaneous damage

Ultraviolet B light (UVB) causes cutaneous inflammation and cell death, but the agents responsible are not defined. These studies examined the role of the platelet-activating factor (PAF) signaling system in UVB-mediated effects. Expression of the PAF receptor in the PAF receptor-negative epidermoid cell line KB augmented apoptosis in response to UVB irradiation. Overexpression of the PAF receptor in primary human keratinocytes also enhanced UVB-mediated apoptosis in vitro, and it enhanced apoptosis in an in vivo model of human keratinocytes grafted onto severe combined immune-deficient (SCID) mice. To define the mechanism by which UVB activates the PAF receptor, we used mass spectrometry to demonstrate significant amounts of the C4 PAF analogs 1-alkyl-2-(butanoyl and butenoyl)-sn-glycero-3-phosphocholine, as well as native PAF in an epidermal cell line after UVB irradiation. Supplementing the cells with the precursor phospholipid 1-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine (HAPC) increased the amount of C4 PAF analogs recovered after UVB exposure. We irradiated HAPC directly and found, even in the absence of a photosensitizer, fragmentation to C4-PAF receptor ligands. We conclude UVB photo-oxidizes cellular phospholipids, creating PAF analogs that stimulate the PAF receptor to induce further PAF synthesis and apoptosis. PAF signaling may participate in the cutaneous inflammation that occurs during photo-aggravated dermatoses.     

Platelet activating factor activity in the phospholipids of bovine spermatozoa

Platelet activating factor (PAF) has been detected in sperm from several mammalian species and can affect sperm motility and fertilization. Because bovine sperm contain a high percentage of ether-linked phospholipid precursors required for PAF synthesis, a study was undertaken to determine the PAF activity of bovine sperm phospholipids. Total lipids of washed, ejaculated bull sperm were extracted, and phospholipids were fractionated by thin-layer chromatography. Individual phospholipid fractions were assayed for PAF activity on the basis of [3H]serotonin release from equine platelets. PAF activity was detected in the PAF fraction (1.84 pmol/mumol total phospholipid) and in serine/inositol (PS/PI), choline (CP), and ethanolamine phosphoglyceride (EP) and cardiolipin (CA) fractions. Activity was highest in the CP fraction (8.05 pmol/mumol total phospholipid). Incomplete resolution of PAF and neutral lipids may have contributed to the activity in the PS/PI and CA fractions, respectively. Phospholipids from nonsperm sources did not stimulate serotonin release. Platelet activation by purified PAF and by sperm phospholipid fractions was inhibited by the receptor antagonist SRI 63-675. These results indicate that bovine sperm contain PAF and that other sperm phospholipids, especially CP and EP, which are high in glycerylether components, are capable of receptor-mediated platelet activation.     

Phospholipid hydroxyalkenals, a subset of recently discovered endogenous CD36 ligands, spontaneously generate novel furan-containing phospholipids lacking CD36 binding activity in vivo

We recently identified a novel family of oxidized choline glycerophospholipid (oxPC) molecular species enriched in atheroma that serve as endogenous ligands for the scavenger receptor CD36 (oxPC(CD36)), facilitating macrophage cholesterol accumulation and foam cell formation (Podrez, E. A., Poliakov, E., Shen, Z., et al. (2002) J. Biol. Chem. 277, 38517-38523). A high affinity CD36 recognition motif was defined within oxPC(CD36), an oxidatively truncated sn-2 acyl group with a terminal gamma-hydroxy (or oxo)-alpha,beta-unsaturated carbonyl. The fate of these species once formed in vivo is unknown. Here we show that a subset of oxPC(CD36), a phosphatidylcholine molecular species possessing sn-2 esterified fatty acyl hydroxyalkenal groups, can undergo a slow intramolecular cyclization and dehydration reaction to form novel oxPC species possessing a sn-2 acyl group that incorporates a terminal furyl moiety (oxPC-furan). Using high performance liquid chromatography with on-line tandem mass spectrometry in combination with unambiguous organic synthesis, we confirm that oxPC-furans, ultimately derived from phospholipids with sn-2 esterified docosahexaenoic, arachidonic, or linoleic acids, are formed during exposure of model membranes and isolated lipoproteins to physiological oxidant systems. In vivo generation of oxPC-furans at sites of enhanced oxidant stress is also demonstrated, such as within brain tissues following cerebral ischemia. Cell binding studies reveal that in contrast to their oxPC(CD36) precursors, oxPC-furans lack CD36 binding activity. Taken together, the present studies identify oxPC-furans as a novel family of oxidized phospholipids that are formed in vivo from phospholipid hydroxyalkenals but that lack CD36 binding activity.     

1-O-alkyl-2-N-methylcarbamyl-glycerophosphocholine: a biologically potent, non-metabolizable analog of platelet-activating factor

We prepared unlabeled and 3H-labeled analogs of platelet-activating factor (PAF) containing a N-methylcarbamyl residue at the sn-2 position. PAF and its methylcarbamyl analog competed for binding to high affinity receptors on human polymorphonuclear neutrophils; their respective dissociation constants for these receptors were 0.2 and 1.1 nM. The binding affinities of the two analogs correlated precisely with their capacities to stimulate neutrophil degranulation responses. Unlike PAF, however, the methylcarbamyl analog completely resisted metabolic inactivation by neutrophils and by human sera. Thus, these compounds' biological potencies are determined predominantly by receptor binding: cellular metabolism of the ligands neither contributes to nor appreciably limits their stimulating actions.     

Substrate specificities and properties of human phospholipases A2 in a mixed vesicle model.

Studies of the specificity of phospholipases A2 (PLA2s) for different substrates have usually been carried out in vesicles or mixed micelles, where differences in shape, size, or charge of vesicles formed with different phospholipids may give misleading results. Another factor is binding of the enzyme to the phospholipid surface, which has recently been addressed using vesicles of an anionic phospholipid, dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) to which some extracellular PLA2s were shown to bind with a very high affinity (Jain, M. K., and Berg, O. G. (1989) Biochem. Biophys. Acta 1002, 127-156). In the present report we have used a similar system to study the substrate preferences of two human PLA2s that are thought to be physiologically relevant in the metabolism of arachidonic acid: a recombinant form of the human synovial fluid (14 kDa) PLA2 and the cytosolic (85 kDa) PLA2 found in monocytic cells. It is shown that both human enzymes bind tightly to DMPM vesicles and follow the basic characteristics of processive hydrolysis in this model using analysis of progress curves and substrate competition experiments. Mixed vesicles containing DMPM with small amounts (3-5 mol%) of other phospholipids have been used to study the substrate selectivity of the two human isoenzymes. The synovial fluid PLA2 shows a clear preference (approximately 7-fold) for sn-glycero-3-phosphoethanolamine over sn-glycero-3-phosphocholine. Within glycerophosphocholines, this enzyme displays little preference for the sn-2 fatty acyl group, and a slight preference for phospholipids with sn-1-acyl versus sn-1-alkyl substituents. In contrast, the cytosolic PLA2 shows a marked selectivity for arachidonoyl in the sn-2 position and only minor differences in selectivity for the polar head group in the sn-3 position. This enzyme does not distinguish between sn-1-acyl and sn-1-alkyl subclasses of glycerophosphocholines.     

Properties and purification of an arachidonoyl-hydrolyzing phospholipase A2 from a macrophage cell line, RAW 264.7

The lipid mediators, platelet activating factor (PAF) and the eicosanoids, can be coordinately produced from the common phospholipid precursor, 1-O-alkyl-2-arachidonoylglycerophosphocholine (1-O-alkyl-2-arachidonoyl-GPC), through the initial action of a phospholipase A2 that cleaves arachidonic acid from the sn-2 position. The mouse macrophage cell line RAW 264.7, which was used as a model macrophage system to study the arachidonoyl-hydrolyzing phospholipase A2 enzyme(s), could be induced to release arachidonic acid in response to inflammatory stimuli. A phospholipase A2 that hydrolyzed 1-O-hexadecyl-2-[3H]arachidonoyl-GPC was identified in the cytosolic fraction of these macrophages. This phospholipase activity was optimal at pH 8 and dependent on calcium. Enzyme activity could be stimulated 3-fold by heparin, suggesting the presence of phospholipase inhibitory proteins in the macrophage cytosol. Compared to 1-alkyl-2-arachidonoyl-GPC, the enzyme hydrolyzed 1-acyl-2-arachidonoylglycerophosphoethanolamine (1-acyl-2-arachidonoyl-GPE) with similar activity but showed slightly greater activity against 1-acyl-2-arachidonoyl-GPC, suggesting no specificity for the sn-1 linkage or the phospholipid base group. Although comparable activity against 1-acyl-2-arachidonoylglycerophosphoinositol (1-acyl-2-arachidonoyl-GPI) could be achieved, the enzyme exhibited much lower affinity for the inositol-containing substrate. The enzyme did, however, show apparent specificity for arachidonic acid at the sn-2 position, since much lower activity was observed against choline-containing substrates with either linoleic or oleic acids at the sn-2 position. The cytosolic phospholipase A2 was purified by first precipitating the enzyme with ammonium sulfate followed by chromatography over Sephadex G150, where the phospholipase A2 eluted between molecular weight markers of 67,000 and 150,000. The active peak was then chromatographed over DEAE-cellulose, phenyl-Sepharose, Q-Sepharose, Sephadex G150 and finally hydroxylapatite. The purification scheme has resulted in over a 1000-fold increase in specific activity (2 mumol/min per mg protein). Under non-reducing conditions, a major band on SDS-polyacrylamide gels at 70 kDa was observed, which shifted to a lower molecular weight, 60,000, under reducing conditions. The properties of the purified enzyme including the specificity for sn-2-arachidonoyl-containing phospholipids was similar to that observed for the crude enzyme. The results demonstrate the presence of a phospholipase A2 in the macrophage cell line. RAW 264.7, that preferentially hydrolyzes arachidonoyl-containing phospholipid substrates.     

Suppression of Mitochondrial Function by Oxidatively Truncated Phospholipids Is Reversible, Aided by Bid, and Suppressed by Bcl-XL

Oxidatively truncated phospholipids are present in atherosclerotic lesions, apoptotic cells, and oxidized low density lipoproteins. Some of these lipids rapidly enter cells to induce apoptosis by the intrinsic pathway, but how such lipids initiate this process is unknown. We show the truncated phospholipid hexadecyl azelaoyl glycerophosphocholine (Az-LPAF), derived from the fragmentation of abundant sn-2 linoleoyl residues, depolarized mitochondria of intact cells. Az-LPAF also depolarized isolated mitochondria and allowed NADH loss, but did not directly interfere with complex I function. Cyclosporin A blockade of the mitochondrial permeability transition pore partially prevented the loss of electrochemical potential. Depolarization of isolated mitochondria by the truncated phospholipid was readily reversed by the addition of albumin that sequestered this lipid. Ectopic expression of the anti-apoptotic protein Bcl-X_L in HL-60 cells reduced apoptosis by the truncated phospholipid by protecting their mitochondria. Mitochondria isolated from these cells were also protected from Az-LPAF-induced depolarization. Conversely mitochondria isolated from Bid^−/− animals that lack this pro-apoptotic Bcl-2 family member were resistant to Az-LPAF depolarization. Addition of recombinant full-length Bid, which has phospholipid transfer activity, restored this sensitivity. Thus, phospholipid oxidation products physically interact with mitochondria to continually depolarize this organelle without permanent harm, and Bcl-2 family members modulate this interaction with full-length Bid acting as a co-factor for pro-apoptotic, oxidatively truncated phospholipids.Vascular cells are exposed to oxidizing radicals during normal metabolism, but especially so during physiologic and pathologic inflammatory processes. The double bonds of polyunsaturated fatty acyl residues are particularly prone to attack by radicals because the C-H bond situated between two double bonds is relatively weak, allowing a more facile abstraction of hydrogen to produce a radical (1). Because polyunsaturated fatty acyl residues are abundant and are generally esterified in the sn-2 position of the glycerol backbone, common products of oxidative attack on cells and circulating lipoproteins are phospholipids that have been peroxidized at their sn-2 position. These peroxy radicals abstract hydrogen to form hydroperoxy phospholipids, may be reduced to the corresponding alcohol, rearrange (2, 3), or fragment to generate a host of oxidatively truncated phospholipids (4–7).The shortened sn-2 residue of truncated phospholipids, which may also contain a newly introduced polar oxygen function, does not intercalate into the membrane well and is energetically favored to protrude into the aqueous phase, a conformation that disorders phospholipid packing into a bilayer (8–10). Oxidatively truncated phospholipids are more water soluble than their phospholipid precursors and readily associate with plasma albumin (11), plasma membranes (12), and even traffic into cells to lysosomes (12) or mitochondria (13) depending on the structure of the truncated phospholipid.Phospholipid oxidation products can be cytotoxic (14, 15), and at least some of these are toxic because they initiate the apoptotic process of regulated cell death (13). The manner by which oxidatively truncated phospholipids alter cell viability has been ascribed to solubilization of the plasma membrane (14), adduction of mitochondrial proteins (17), temporary physical distortion of the plasma membrane (18), or activation of acid sphingomyelinase activity that alters plasma membrane microdomains by generating ceramide (15, 19). We found that a common oxidatively truncated phospholipid, containing a 9-carbon azelaoyl fragment derived from fragmentation of sn-2 linoleoyl residues, induces apoptosis by the intrinsic caspase cascade with loss of mitochondrial function and not, apparently, from damage of the plasma membrane (13).Members of the Bcl-2 family modulate mitochondria-dependent apoptosis either by promoting apoptosis (Bid, Bad, and Bax) or obstructing this event (Bcl-2 and Bcl-X_L). Aggregation of Bax on the mitochondrial outer membrane forms ion conducting pores and Bcl-X_L associates with mitochondrial outer membranes to suppress this Bax activity (20). In contrast, Bid promotes apoptosis after cleavage to truncated Bid, a regulatory event catalyzed by activated caspase 8 (21). Bid, alone among Bcl-2 family members, displays homology to plant lipid transfer proteins and both truncated and full-length Bid will incorporate fluorescent phospholipids, and not the cognate fluorescent fatty acid, into mitochondrial membranes (22).We determined whether mitochondrial integrity or function were directly affected by oxidatively truncated phospholipids, and then whether Bcl-2 family members alter these effects as they do in other, established apoptotic signaling pathways. We find that truncated phospholipids accumulated from the extracellular environment depolarize intracellular mitochondria, that these bilayer challenged phospholipids reversibly interact with mitochondria to continually reduce their transmembrane potential, and that Bcl-2 family members modulate this interaction.     

An oxidized derivative of phosphatidylcholine is a substrate for the platelet-activating factor acetylhydrolase from human plasma

Platelet-activating factor (PAF) is a glycerophospholipid that has diverse potent biological actions. A plasma enzyme catalyzes the hydrolysis of the sn-2 acetoyl group of PAF and thereby abolishes its bioactivity. This PAF acetylhydrolase is specific for phospholipids, such as PAF, with a short acyl group at the sn-2 position. The majority of it (60-70%) is associated with low density lipoprotein (LDL), and the remainder is with high density lipoprotein (HDL). LDL also has a phospholipase A2 activity that is specific for oxidized polyunsaturated fatty acids, which may be important in determining how LDL is recognized by cellular receptors. We previously have purified and characterized the PAF acetylhydrolase from human plasma. We now have found that the purified PAF acetylhydrolase catalyzes the hydrolysis of the oxidized fragments of arachidonic acid from the sn-2 position of phosphatidylcholine. One of the preferred substrates appeared by mass spectrometry to have 5-oxovalerate at the sn-2 position. We synthesized 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine and found that the PAF acetylhydrolase had the same apparent Km for it (11.3 microM) as for PAF (12.5 microM), with Vmax values of 100 and 167 mumol/h/mg of protein, respectively. We also conclude that the PAF acetylhydrolase is the sole activity in LDL that degrades oxidized phospholipids since we found co-localization of the activity against both substrates to LDL and HDL, and precipitation of enzyme activity with an antibody to the PAF acetylhydrolase. Thus, the PAF acetylhydrolase in human plasma degrades oxidized phospholipids, which may be involved in the modification of apolipoprotein B100 and other pathological processes.     

A facile synthesis of an aldehydic analog of platelet activating factor and its use in the production of specific antibodies

The multistep synthesis of a platelet activating factor (PAF) analog having a reactive aldehyde group at the omega-end of the sn-1 position is described. A novel ozonolysis of a double bond was employed to generate the aldehyde group in high yield under mild conditions. The aldehyde group was generated at the last step of the synthesis to avoid any reactions of protection and deprotection. The natural chiral center at the sn-2 position was introduced at the first step so that no steric resolution of the final product was needed. This analog of PAF was conjugated to thyroglobulin via reductive amination and then used to immunize rabbits for production of specific antibodies. The purified antibodies bind stereospecifically to tritiated PAF and crossreact minimally with lyso-PAF, plasmalogens and other phospholipids. The solid-phase radioimmunoassay thus developed detects as low as 20 pg of PAF per assay tube and should be applicable to the quantitation of PAF in biological systems.