Oxidatively modified LDL contains phospholipids with platelet-activating factor-like activity and stimulates the growth of smooth muscle cells.

Oxidative modification of lipoproteins is believed to be important in the genesis of atherosclerosis. We established cultures of smooth muscle cells (SMC) and exposed them to native LDL or oxidized LDL. Oxidized LDL, but not native LDL, was mitogenic as measured by incorporation of [3H]-thymidine into DNA. This effect was concentration dependent, averaged 288% of control, and was blocked by a platelet-activating factor (PAF) receptor antagonist. We hypothesized that phospholipids with PAF-like activity were generated during the oxidation of LDL. To test this hypothesis we extracted phospholipids from copper-oxidized LDL and assayed for PAF-like activity. Phospholipids extracted from oxidized LDL and purified by HPLC induced neutrophil adhesion equivalent to PAF (10 nM) and were mitogenic for smooth muscle cells. These effects were not seen with phospholipids extracted from native LDL and were blocked by two structurally different, competitive antagonists of the PAF receptor. The effects of these lipids were also abolished by pretreating them with PAF acetylhydrolase. Finally, we used Chinese hamster ovary cells that had seen stably transfected with a cDNA for the PAF receptor to confirm that phospholipids from oxidized LDL act via this receptor. We found that PAF (control) and the oxidized phospholipids each induced release of arachidonic acid from the transfected cells, but had no effect on wildtype Chinese hamster ovary cells, which lack the PAF receptor. This effect was also blocked by a PAF receptor antagonist. Thus, phospholipids generated during oxidative modification of LDL may participate in atherosclerosis by stimulating SMC proliferation and leukocyte activation.

Effect of platelet activating factor-acetylhydrolase on the formation and action of minimally oxidized low density lipoprotein.

Mildly oxidized low density lipoprotein (MM-LDL) produced by oxidative enzymes or cocultures of human artery wall cells induces endothelial cells to produce monocyte chemotactic protein-1 and to bind monocytes. HDL prevents the formation of MM-LDL by cocultures of artery wall cells. Using albumin treatment and HPLC we have isolated and partially characterized bioactive oxidized phospholipids in MM-LDL. Platelet activating factor-acetylhydrolase (PAF-AH), a serine esterase, hydrolyzes short chain acyl groups esterified to the sn-2 position of phospholipids such as PAF and particular oxidatively fragmented phospholipids. Treatment of MM-LDL with PAF-AH (2-4 x 10(-2) U/ml) eliminated the ability of MM-LDL to induce endothelial cells to bind monocytes. When HDL protected against the formation of MM-LDL by cocultures, lysophosphatidylcholine was detected in HDL; whereas when HDL was pretreated with diisopropyl fluorophosphate, HDL was no longer protective and lysophosphatidylcholine was undetectable. HPLC analysis also revealed that the active oxidized phospholipid species in MM-LDL had been destroyed after PAF-AH treatment. In addition, treatment of MM-LDL with albumin removed polar phospholipids that, when reisolated, induced monocyte binding to endothelial cells. These polar phospholipids, when treated with PAF-AH, lost biological activity and were no longer detected by HPLC. These results suggest that PAF-AH in HDL protects against the production and activity of MM-LDL by facilitating hydrolysis of active oxidized phospholipids to lysolipids, thereby destroying the biologically active lipids in MM-LDL.

Deficiency of platelet-activating factor acetylhydrolase is a severity factor for asthma.

Asthma, a family of airway disorders characterized by airway inflammation, has an increasing incidence worldwide. Platelet-activating factor (PAF) may play a role in the pathophysiology of asthma. Its proinflammatory actions are antagonized by PAF acetylhydrolase. A missense mutation (V279F) in the PAF acetylhydrolase gene results in the complete loss of activity, which occurs in 4% of the Japanese population. We asked if PAF acetylhydrolase deficiency correlates with the incidence and severity of asthma in Japan. We found that the prevalence of PAF acetylhydrolase deficiency is higher in Japanese asthmatics than healthy subjects and that the severity of this syndrome is highest in homozygous-deficient subjects. We conclude that the PAF acetylhydrolase gene is a modulating locus for the severity of asthma.

Anti-inflammatory HDL becomes pro-inflammatory during the acute phase response. Loss of protective effect of HDL against LDL oxidation in aortic wall cell cocultures.

We previously reported that high density lipoprotein (HDL) protects against the oxidative modification of low density lipoprotein (LDL) induced by artery wall cells causing these cells to produce pro-inflammatory molecules. We also reported that enzyme systems associated with HDL were responsible for this anti-inflammatory property of HDL. We now report studies comparing HDL before and during an acute phase response (APR) in both humans and a croton oil rabbit model. In rabbits, from the onset of APR the protective effect of HDL progressively decreased and was completely lost by day three. As serum amyloid A (SAA) levels in acute phase HDL (AP-HDL) increased, apo A-I levels decreased 73%. Concomitantly, paraoxonase (PON) and platelet activating factor acetylhydrolase (PAF-AH) levels in HDL declined 71 and 90%, respectively, from days one to three. After day three, there was some recovery of the protective effect of HDL. AP-HDL from human patients and rabbits but not normal or control HDL (C-HDL) exhibited increases in ceruloplasmin (CP). This increase in CP was not seen in acute phase VLDL or LDL. C-HDL incubated with purified CP and re-isolated (CP-HDL), lost its ability to inhibit LDL oxidation. Northern blot analyses demonstrated enhanced expression of MCP-1 in coculture cells treated with AP-HDL and CP-HDL compared to C-HDL. Enrichment of human AP-HDL with purified PON or PAF-AH rendered AP-HDL protective against LDL modification. We conclude that under basal conditions HDL serves an anti-inflammatory role but during APR displacement and/or exchange of proteins associated with HDL results in a pro-inflammatory molecule.

Platelet-activating factor acetylhydrolase deficiency. A missense mutation near the active site of an anti-inflammatory phospholipase.

Deficiency of plasma platelet-activating factor (PAF) acetylhydrolase is an autosomal recessive syndrome that has been associated with severe asthma in Japanese children. Acquired deficiency has been described in several human diseases usually associated with severe inflammation. PAF acetylhydrolase catalyzes the degradation of PAF and related phospholipids, which have proinflammatory, allergic, and prothrombotic properties. Thus, a deficiency in the degradation of these lipids should increase the susceptibility to inflammatory and allergic disorders. Miwa et al. reported that PAF acetylhydrolase activity is absent in 4% of the Japanese population, which suggests that it could be a common factor in such disorders, but the molecular basis of the defect is unknown. We show that inherited deficiency of PAF acetylhydrolase is the result of a point mutation in exon 9 and that this mutation completely abolishes enzymatic activity. This mutation is the cause of the lack of enzymatic activity as expression in E. coli of a construct harboring the mutation results in an inactive protein. This mutation as a heterozygous trait is present in 27% in the Japanese population. This finding will allow rapid identification of subjects predisposed to severe asthma and other PAF-mediated disorders.

Platelet-activating factor acetylhydrolases in health and disease.

The platelet-activating factor (PAF) acetylhydrolases catalyze hydrolysis of the sn-2 ester bond of PAF and related pro-inflammatory phospholipids and thus attenuate their bioactivity. One secreted (plasma) and four intracellular isozymes have been described. The intracellular isozymes are distinguished by differences in primary sequence, tissue localization, subunit composition, and substrate preferences. The most thoroughly characterized intracellular isoform, Ib, is a G-protein-like complex with two catalytic subunits (alpha1 and alpha2) and a regulatory beta subunit. The beta subunit is a product of the LIS1 gene, mutations of which cause Miller-Dieker lissencephaly. Isoform II is a single polypeptide that is homologous to the plasma PAF acetylhydrolase and has antioxidant activity in several systems. Plasma PAF acetylhydrolase is also a single polypeptide with a catalytic triad of amino acids that is characteristic of the alpha/beta hydrolases. Deficiency of this enzyme has been associated with a number of pathologies. The most common inactivating mutation, V279F, is found in >30% of randomly surveyed Japanese subjects (4% homozygous, 27% heterozygous). The prevalence of the mutant allele is significantly greater in patients with asthma, stroke, myocardial infarction, brain hemorrhage, and nonfamilial cardiomyopathy. Preclinical studies have demonstrated that recombinant plasma PAF acetylhydrolase can prevent or attenuate pathologic inflammation in a number of animal models. In addition, preliminary clinical results suggest that the recombinant enzyme may have pharmacologic potential in human inflammatory disease as well. These observations underscore the physiological importance of the PAF acetylhydrolases and point toward new approaches for controlling pathologic inflammation.

The effect of glyoxalase I on the metabolism of 4,5-dioxovaleric acid.

Biosynthesis of 5-aminolevulinic acid in mammalian cells is catalyzed by aminolevulinic acid synthase in a condensation reaction utilizing glycine and succinyl X coenzyme A. An alternate pathway in mammalian cells may involve the biosynthesis of aminolevulinic acid via a transamination reaction in which L-alanine is the amino donor and 4,5-dioxovaleric acid is the acceptor. This transamination reaction, or one very similar, is employed by plants for the biosynthesis of aminolevulinic acid which is ultimately converted to chlorophyll. The effect of glyoxalase I on the diversion of dioxovaleric acid to other products was tested using both purified glyoxalase I and crude tissue homogenates. Glyoxalase I is a metalloenzyme and glutathione is a co-substrate. Purified glyoxalase I reduced the amount of aminolevulinic acid formed in the presence of dioxovaleric acid, L-alanine, glutathione, and purified L-alanine: 4,5-dioxovaleric acid aminotransferase (dioxovalerate transaminase). The conversion of dioxovaleric acid to aminolevulinic acid was inhibited by the addition of glutathione when a dialyzed bovine liver homogenate served as the source of both glyoxalase I and dioxovalerate transaminase. Removal of metals from bovine liver homogenates produced an 85% decrease in glyoxalase I activity. These 'metal-free' homogenates still affected the conversion of dioxovaleric acid to aminolevulinic acid after preincubation with MgSO4. The effect of glyoxalase I on the metabolism of dioxovaleric acid was also studied using a fluorometric enzyme assay for the quantification of dioxovaleric acid via a coupled enzyme reaction converting it to uroporphyrin. Homogenates of both liver and barley diminished the amount of dioxovaleric acid detected by the coupled assay, but this effect could be prevented by dialysis of the homogenates. Addition of glutathione to dialyzed homogenates markedly reduced the amount of uroporphyrin generated from dioxovaleric acid. Metal-free homogenates supplemented with glutathione reduced the conversion of dioxovaleric acid to uroporphyrin in the coupled assay, but preincubation with MgSO4 greatly augmented this effect. These studies point out the difficulty in evaluating dioxovaleric acid as a heme precursor using whole cell homogenates.

PAF acetylhydrolase gene polymorphisms and asthma severity.

This review describes the current understanding of the contributions of genetic alterations in platelet-activating factor (PAF) acetylhydrolase to the pathogenesis of asthma. A variety of in vitro and in vivo studies, performed by multiple laboratories, suggest that the lipid substrates of this enzyme, PAF and oxidised derivatives of phosphatidylcholines, play important roles as causative factors in many diseases including asthma. PAF acetylhydrolase inactivates PAF and oxidatively-fragmented lipids thus providing a mechanism to prevent their pro-inflammatory effects. Since it is a most unusual protein, the biochemical, structural and functional characteristics of PAF acetylhydrolase continue to be unravelled. First, the ability of this enzyme to inactivate pro-inflammatory lipid mediators is modulated by its association with lipoproteins and by its susceptibility to oxidative inactivation. Second, mediators of inflammation, such as the substrates for PAF acetylhydrolase, alter expression of the protein at the transcriptional level. Third, naturally-occurring variants of PAF acetylhydrolase have catalytic properties different from those exhibited by the most common form of this protein. Thus, a variety of factors, including genetics, contribute to determine the biological level of lipid substrates known to act as mediators of asthma and other diseases. Here, I summarise key studies that implicate PAF and related molecules as important mediators in the pathogenesis of asthma. Next, I describe clinical findings that are consistent with a role of PAF acetylhydrolase as a modulator of asthma. Third, I focus on the biochemical effects associated with naturally-occurring mutations and polymorphisms in the PAF acetylhydrolase gene and the incidence of these genetic variations in populations of asthmatic subjects. Finally, I present my views on the future of this emerging field and the potential utility of performing additional studies aimed at further characterising the contribution of PAF acetylhydrolase to the pathogenesis of a complex syndrome generally recognised as a multifactorial and heterogeneous disease.

Characterization of human UMP-CMP kinase enzymatic activity and 5' untranslated region.

We have cloned a cDNA for human UMP-CMP kinase from a macrophage cDNA library. Sequence analysis showed that this cDNA is derived from the same gene as a previously reported EST-derived cDNA. Here we show that a conspicuous difference between these two clones, 73 additional 5' nucleotides in the EST clone, including a putative translational start site, is not functionally significant. This work shows that the additional 5'sequence in the EST clone was unnecessary for enzymatic activity and nonfunctional in the initiation of translation. Specifically, we found that protein expressed by both the macrophage-derived cDNA and the extended cDNA had the same relative molecular mass, consistent with use of an ATG internal to the macrophage-derived clone as the functional start site. In addition, this work more precisely defines the catalytic activity of UMP-CMP kinase. Here, we show a 3-fold greater substrate preference for CMP relative to UMP, identify ATP and UTP as the preferred phosphate donors for the reaction, and demonstrate that the reaction is Mg2+-dependent. In addition, investigation of UMP-CMP-kinase expression revealed two mRNA products in immune tissues and cancer cell lines. The smaller RNA product was previously undescribed.

Platelet-activating factor acetylhydrolase activity in human tissues and blood cells.

Human tissues, blood cells, and plasma have enzymes that catalyze the hydrolysis of PAF (1-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine). The activities are not due to phospholipases A2 that hydrolyze long chain acyl groups at the sn-2 position of glycerophospholipids, since they are calcium-independent and are specific for hydrolysis of short chain acyl groups. We examined the biochemical properties of these PAF acetylhydrolase activities (EC 3.1.1.47) in homogenates of human liver and spleen, in white blood cells (neutrophils and monocytes), and in erythrocytes. The data suggest that the plasma and intracellular PAF acetylhydrolase activities are likely due to different proteins. Second, the intracellular PAF acetylhydrolase activities in liver and spleen share several biochemical features that differentiate them from the activities in blood cells. Third, the activities in monocytes and neutrophils have properties that differentiate them from the activity present in human erythrocytes. Finally, the erythrocyte activity has unique properties that place it in a separate category of short chain acylhydrolases. In conclusion, there is a family of distinct enzymes that can be identified as PAF acetylhydrolases based on their calcium-independence and specificity for a short residue at the sn-2 position of phospholipids.

Diacylglycerol kinase η modulates oncogenic properties of lung cancer cells.

PURPOSE: Lung cancer is a leading cause of cancer deaths and efforts are underway to identify novel therapies to treat these tumors. Diacylglycerol kinase η (DGKη), an enzyme that phosphorylates diacylglycerol to form phosphatidic acid, has been shown to modulate MAPK signaling downstream of EGFR, which is an oncogenic driver in some lung cancers. Since mutations in EGFR and K-Ras are common in lung cancer, we hypothesized that limiting the function of DGKη would attenuate oncogenic properties of lung cancer cells. METHODS: We determined the expression levels of DGKη in a mouse models of mutant EGFR and K-Ras lung cancer and in human lung cancer cell lines with activating mutations in either EGFR or K-Ras. We also tested the effects of shRNA-mediated depletion of DGKη in lung cancer cells and tested if DGKη depletion augmented the effects of afatinib, a new generation EGFR inhibitor. RESULTS: DGKη was expressed in malignant epithelium from mice with mutant EGFR or K-Ras lung cancer. It was also expressed in human lung cancer cell lines with EGFR or K-Ras mutations. Depleting DGKη in lung cancer cell lines, harboring mutant EGFR, reduced their growth on plastic and in soft agar and also augmented the effects of afatinib, an EGFR inhibitor. DGKη depletion also reduced growth of one of two lung cancer cell lines that harbored mutant K-Ras. CONCLUSIONS: Our data indicate that DGKη is a potential therapeutic target in lung cancers, especially those harboring EGFR mutations. Our findings warrant further studies to examine the effects of limiting its function in vivo.

Human plasma platelet-activating factor acetylhydrolase. Purification and properties.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid synthesized by a variety of cell types upon appropriate stimulation. PAF is a potent hypotensive factor and it activates platelets and inflammatory cells at concentrations as low as 10(-10) M. Removal of the acetyl moiety at the sn-2 position abolishes the biological activity and this reaction is catalyzed by a specific acetylhydrolase present in plasma and animal tissues. Ultracentrifugation in density gradients showed that 30% of the activity is associated with high density lipoproteins and 70% with low density lipoproteins. We have purified the plasma low density lipoprotein-associated activity to near homogeneity using a rapid assay based on the separation of [3H]acetate from 1-O-alkyl-2-[3H]acetyl-sn-glycerol-3-phosphocholine on disposable reversed-phase columns. The enzyme was purified by 25,000-fold and approximately 10% of the starting activity was recovered. Plasma PAF-acetylhydrolase has an apparent molecular weight of 43,000, does not require calcium, has preference for micellar versus monomeric substrate, and exhibits surface dilution kinetics. The purified protein has an apparent Km of 13.7 microM and a Vmax of 568 mumol/h/mg with micellar PAF. It can act both on 1-O-alkyl and 1-acyl substrates and on ethanolamine analogs of PAF. However, the enzyme has a marked preference for the sn-2 acetyl residue and therefore can be considered as a specific PAF-acetylhydrolase.

The platelet-activating factor acetylhydrolase from human erythrocytes. Purification and properties.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine) is a biologically active phospholipid. Tissues, blood cells, and plasma contain PAF acetylhydrolases (calcium independent phospholipase A2 activities) that catalyze the hydrolysis of phospholipids containing short chain sn-2 acyl groups. They inactivate PAF and thereby determine PAF accumulation. We purified the PAF acetylhydrolase from human erythrocytes 15,600-fold. The enzyme has a molecular weight of 25,000, it behaves as a dimer during gel filtration, and it is a previously uncharacterized cytosolic esterase, as it has a unique amino-terminal sequence. The erythrocyte PAF acetylhydrolase requires the addition of sulfhydryl agents for maximal activity, is inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), NaF, diisopropyl fluorophosphate, diethylpyrocarbonate, p-bromophenacylbromide, and a number of proteases. Antibodies against the purified protein precipitate all PAF hydrolase activity from erythrocyte lysates. The erythrocyte PAF acetylhydrolase is specific for short or oxidized sn-2 acyl residues. It exhibits surface dilution kinetics, suggesting that hydrolysis occurs at lipid interfaces. This suggests that this enzyme acts in vivo as a scavenger of oxidatively fragmented phospholipids that are toxic to the cell.

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.