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(The FASEB Journal. 2007;21:1164-1176.)
© 2007 FASEB

Molecular basis for susceptibility of plasma platelet-activating factor acetylhydrolase to oxidative inactivation

Amy N. MacRitchie1, Alison A. Gardner, Stephen M. Prescott2 and Diana M. Stafforini3

Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah, USA

3Correspondence: Huntsman Cancer Institute, 2000 Cir. of Hope, University of Utah, Salt Lake City, UT 84112-5550, USA. E-mail: diana.stafforini{at}hci.utah.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Platelet-activating factor acetylhydrolase (PAF-AH) is a phospholipase A2 that inactivates potent lipid messengers, such as PAF and modified phospholipids generated in settings of oxidant stress. The catalytic activity of PAF-AH is sensitive to oxidants, a feature that may have pathological consequences. We report that peroxynitrite, an oxidant species generated after cellular activation, mediates oxidative inactivation of PAF-AH. We found that peroxynitrite inactivated and derivatized the recombinant protein and obtained evidence supporting a role for a methionine and two tyrosine residues in this process. We employed interspecies comparisons and site-directed mutagenesis and identified a role for M-117, and a smaller contribution of Y-307 and Y-335 as targets of oxidant attack using free and lipoprotein-associated recombinant proteins. M-117 is adjacent to W-115 and L-116, which are essential for association of PAF-AH with LDL. Oxidation of LDL-associated PAF-AH partially dissociated the enzyme from the particles. Similarly, oxidation of the purified enzyme in the absence of lipoproteins prevented subsequent association with LDL. These results provide new insights into the molecular mechanisms that mediate inactivation of PAF-AH in settings of oxidant stress and the consequences of oxidation on the ability of this enzyme to associate with LDL.— MacRitchie, A. N., Gardner, A. A., Prescott, S. M., Stafforini, D. M. Molecular basis for susceptibility of plasma platelet-activating factor acetylhydrolase to oxidative inactivation.


Key Words: PAF acetylhydrolase • phospholipase A2 • oxidant stress • peroxynitrite • reactive oxygen species • protein oxidation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE PLATELET-ACTIVATING FACTOR (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) (2) acetylhydrolase (PAF-AH) activity that circulates in mammalian plasma is a phospholipase A2 secreted primarily by macrophages (1) . This enzyme is also known as lipoprotein-associated phospholipase A2 (Lp-PLA2), and it catalyzes the hydrolysis of short and/or oxidized acyl groups present in phospholipids, such as PAF, oxidatively fragmented phospholipids, and F2-isoprostanes esterified in phospholipids (2 , 3) . A related enzymatic activity that shares homology to the plasma PAF-AH exists in the cytosolic fraction of mammalian tissues (4) . The substrates of PAF-AH have potent and varied biological effects, and some of them, e.g., PAF and oxidatively-fragmented phospholipids, elicit their functions after interaction with the PAF receptor expressed on the surface of target cells (5 , 6) . The biological activities of the substrates of this enzyme have made PAF-AH the focus of many investigations aimed at establishing its pathophysiological role using biochemical, molecular, genetic, and in vivo approaches (7) .

There is evidence in a variety of human syndromes and in animal models of disease that exposure to oxidants and subsequent increases in PAF and/or PAF-like activity contribute to tissue injury (8 9 10) . Reperfusion injury is mediated, at least in part, by oxidants, and PAF-like bioactivity has been identified in injured tissues after ischemia and reperfusion (10 , 11) . Oxidants and oxidant injury are also important in the development of atherosclerosis as cell-mediated oxidative modification of LDL occurs, owing to cellular generation of toxic oxygen metabolites (12) . The known mitogenic effect of modified LDL on vascular smooth muscle has been shown to derive, at least in part, from lipids with PAF-like activity that can be inactivated by PAF-AH (13) . Elevated PAF-like bioactivity has been observed in lung lavage fluid from adult respiratory distress syndrome (ARDS) patients (14) and infants with bronchopulmonary dysplasia (15) and necrotizing enterocolitis (16) . Animal models have shown that combined exposure to PAF and endotoxin results in lung injury that recapitulates many features characteristic of ARDS (17) . These data suggest that a component of the pathological response to oxidant injury is related to the accumulation of PAF and oxidatively fragmented phospholipids. Thus, the mechanisms that control the abundance of these mediators are essential in regulating the extent of oxidant-associated injury.

While PAF concentrations are controlled at the levels of both synthesis and degradation (18) , PAF-AHs provide a mechanism that limits accumulation of oxidatively fragmented phospholipids and esterified F2-isoprostanes, in addition to inactivating PAF. Paradoxically, even though PAF-AHs function to inactivate PAF and phospholipid analogs generated in settings of oxidant stress, both the plasma enzyme (19 , 20) and the intracellular PAF-AH II (D. M. Stafforini, unpublished observations) are inhibited by oxidants. The inhibition seems to involve irreversible modification of key functional groups of the enzymes, and it can result in the accumulation of substrates that may worsen syndromes in which inactivation of these molecules is beneficial.

NO and superoxide (O2–) are potent oxidants released in vivo by activated endothelial cells, polymorphonuclear leukocytes, and macrophages (21 22 23) . NO and O2– can act alone or combine to form peroxynitrite (OONO–), which is also produced during oxidation in vivo (24) . OONO– can further react to form peroxynitrous acid, hydroxyl radical, and nitrite. It has recently been reported that myeloperoxidase oxidatively modifies tyrosine residues within apolipoprotein A-I and that this modification changes the properties of HDL particles from anti- to proinflammatory (25) . This illustrates the functional changes that can take place when certain proteins become oxidatively modified.

The role of the plasma form of PAF-AH in physiological and pathological settings is the subject of much controversy, in particular, as it relates to its participation in vascular disease (26 27 28) . This enzyme circulates in plasma as a complex with low- and high-density lipoproteins (LDL and HDL) (29) . Elevated expression of PAF-AH activity has been reported in the plasma of patients with vascular disease by a large number of groups (30 , 31) . In addition, PAF-AH protein has been detected in atherosclerotic plaques of both humans and experimental animals (32) . However, the functional state of the enzyme present in these structures has not been investigated in detail. An important consideration is that macrophage activation within vascular lesions results in elevated local levels of ROS (33 , 34) , a process that could lead to oxidative inactivation of PAF-AH. Here, we used approaches aimed at characterizing the oxidant species likely to target PAF-AH in settings of oxidant stress and then utilized this information to investigate the molecular basis for PAF-AH oxidative inactivation. Our work provides new insights into the molecular mechanisms that mediate inactivation of PAF-AH by oxidants, including the contribution of the lipoprotein environment to this process.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
We obtained OONO–, antinitrotyrosine and anti-PAF-AH antibodies from Cayman Chemical (Ann Arbor, MI, USA). Secondary antibodies were purchased from BioSource International (Camarillo, CA, USA). Horseradish peroxidase-labeled biotin, PEO-Maleimide Activated Biotin (PMAB) and the BCA protein assay were purchased from Pierce (Rockford, IL, USA). [3H-acetyl]PAF was from Amersham Biosciences (Piscataway, NJ, USA), and unlabeled PAF was obtained from Avanti Polar Lipids (Alabaster, AL, USA). Pfu was obtained from Stratagene (La Jolla, CA, USA), and dNTPs were purchased from Fermentas Inc. (Hanover, MD, USA). Pefabloc was purchased from Calbiochem (San Diego, CA, USA). Three-morpholino-sydnonimine (SIN-1), tetranitromethane (TNM), and all other reagents were obtained from Sigma (St. Louis, MO, USA). Lipofectamine and the mammalian expression vector plasmid construct DNA (pcDNA) 3.1/Zeo– were obtained from Invitrogen (Carlsbad, CA, USA). In some experiments, we utilized recombinant human PAF-AH (Pafase®), which was a generous gift from ICOS (Bothell, WA, USA). The preparation had a specific activity of 135 µmol/min/mg. PNU101033E was a generous gift from Pharmacia & Upjohn (Kalamazoo, MI, USA). LDL particles were isolated, as described previously (29) , and were treated with Pefabloc according to the instructions provided by the manufacturer. The Pefabloc-treated LDL fraction was subjected to exhaustive dialysis against PBS, at 4°C.

PAF-AH activity and protein determinations
PAF-AH activity was determined by our previously described radiometric assay, using [3H-acetyl]PAF as the substrate (35) . Briefly, we incubated an appropriately diluted source of enzyme (10–20 µl) with 40–30 µl of 0.1 mM [3H-acetyl]PAF (10 nCi/nmol) in a total volume of 50 µl. The samples were incubated for 30 min at 37°C, and the reactions were quenched using 50 µl of 10 M acetic acid. Released [3H]acetate was separated from excess substrate by transferring the entire reaction product to individual C-18 reversed-phase cartridges equilibrated with water, using two 1.5-ml-aliquots of 0.1 M sodium acetate, as described (35) . The amount of product released was quantitated in the effluents by scintillation counting; the data were corrected for a small amount of radioactivity resulting from nonenzymatic hydrolysis of PAF. Protein content was determined using the BCA protein assay.

Mutant generation and vectors
Site-directed mutagenesis was performed by a two-step amplification protocol using Pfu as the polymerase (36) . A FLAG tag was inserted at the amino terminal end for immunoblot detection and purification purposes. The products were inserted into a pUC cloning vector under the control of the tryptophan promoter, as described previously (36) . Plasmid DNA was purified using a miniprep purification kit (Qiagen, Valencia, CA, USA). For mammalian expression, we introduced the wild-type PAF-AH cDNA into the mammalian expression vector pcDNA 3.1/Zeo–.

Expression and purification of mutant and chimeric proteins
We expressed wild-type murine and human PAF-AHs in the E. coli strain BL-21. Protein extracts were obtained from pelleted bacterial cells after 30 min incubation at room temperature in 50 mM Tris-HCl (pH 8) containing 0.1% Triton X-100, 0.5 M NaCl, and 0.02% lysozyme. The lysates were briefly sonicated and then centrifuged at 21,000 g for 15 min at 4°C. The supernatants then were purified using anti-FLAG affinity beads, following the instructions provided by the manufacturer (Sigma). We determined the enzymatic activity and protein content of the mutant preparations recovered after purification and determined the level of expression by immunoblot analysis using a monoclonal anti-FLAG (M2) antibody (Sigma), as described previously (3) . Unless otherwise stated, the purified mutants expressed high levels of PAF-AH activity, suggesting that the folding of the recombinant proteins was comparable to that of the wild-type enzyme.

Transfections
COS-7 cells were plated at a density of 250,000–300,000 cells/35 mm well the day before transfection. Each well was transfected with 1 µg of plasmid DNA using lipofectamine, according to the instructions provided by the manufacturer (Invitrogen). Five hours after DNA addition, the culture medium was removed and fresh serum-containing medium was supplemented for an additional 43–44 h. The wells were washed twice with PBS and the protein was harvested using 2 x 200 µl of reporter lysis buffer (Promega Corp., Madison, WI, USA) following the instructions provided by the manufacturer.

Oxidations
Peroxynitrite treatment
Working solutions of OONO– were prepared by diluting stocks in 100 mM NaOH prior to use. OONO– was added at the final indicated concentrations to recombinant human PAF-AH in 0.1 M KPi, pH 7.4, while vortexing. The incubations took place at room temperature. Derivatization of PAF-AH was assessed by immunoblot analysis using an antinitrotyrosine antibody.

SIN-1 treatment
Working solutions of SIN-1 were prepared freshly in PBS. The pH was adjusted to 7.4 with sodium phosphate buffer and the incubations took place at 37°C for 4 h. Where indicated, NaHCO3 was supplemented at a final concentration of 20 mM. In these cases, the buffers were subjected to prolonged bubbling with a stream of N2.

TNM treatment
Working solutions of TNM were prepared freshly in ethanol. The incubations took place at room temperature for 10 min. Appropriate controls were performed using ethanol as the vehicle.

Analysis of sulfhydryl status after OONO– treatment
We determined the effect of OONO– or SIN-1 on reduced cysteines in PAF-AH using PMAB, a thiol-reactive biotinylation reagent, as described (37) . PAF-AH was labeled with PMAB essentially as recommended by the manufacturer (Pierce) for 2 h at room temperature in the dark. The samples then were subjected to electrophoresis on SDS-PAGE followed by transfer and immunoblotting using horseradish peroxidase-labeled avidin (1:1000). Immunoreactive proteins were visualized using chemiluminescence detection reagents (PerkinElmer Life Sciences).

Statistical analyses
Enzymatic activity assays were conducted in duplicate and the results typically varied by no more than 5%. The data reported represent the mean ± the SD of representative experiments. The number of times each experiment was conducted (n) is indicated in the legend of each figure. Student’s t test was used for evaluation of statistical significance of differences among independent experiments; *P < 0.05; **P < 0.001.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Peroxynitrite inhibits PAF-AH and derivatizes tyrosine residues
To investigate the mechanism that underlies PAF-AH inactivation by oxidants, we asked whether NO and superoxide (O2–), two biologically relevant oxidant species produced in vivo by a number of activated cells, affected PAF-AH activity. We used two NO donors [S-nitroso-N-acetylpenicillamine and (DETA) NONOate)] and a superoxide radical generating system (xanthine/xanthine oxidase). We found either moderate or no significant inhibition of PAF-AH activity after a 4-hour incubation period in response to NO or O2– alone (not shown). However, in systems that generate both of these species, NO and O2– combine to form ONOO–, which is frequently the active oxidant species and nitrating mediator formed in various pathological situations (38) . ONOO– is a highly reactive molecule that can oxidize cysteines and methionines and is known to nitrate tryptophan and tyrosine residues (39) . We found that SIN–1, a compound that undergoes oxygen–dependent release of ONOO–, inhibited purified recombinant PAF-AH activity and derivatized tyrosine residues present in the enzyme (Fig. 1 A). The possibility that protein degradation accounted for the loss of enzymatic activity was ruled out as most of the PAF-AH protein remained intact after SIN-1 treatment (Fig. 1A ). We found that exposure to synthetic OONO– also inhibited and nitrated PAF-AH (Fig. 1B ). From these observations, we concluded that the active oxidant species was either OONO– or one of its degradation products. We tested responses to SIN-1 using various levels of PAF-AH over the same concentration range of SIN-1 (Fig. 1C ). We found that inactivation was independent of protein concentration up to a level of PAF-AH of ~200 ng per oxidation assay. At higher levels of enzyme, the degree of inactivation depended on the concentration of both enzyme and oxidant. To investigate whether metals contributed to SIN-1-mediated oxidative inactivation, we tested the effect of diethylenetriaminepentaacetate (DTPA, 100 µM) and deferoxamine (50 µM) and found that these chelating agents had minor effects (data not shown). These results ruled out potentially confounding effects of redox active adventitial metals. We next considered the possibility that the oxidative susceptibility of the full-length protein might differ from that observed in the truncated form utilized in the studies shown in Fig. 1A-C . To address this issue, we tested the effect of SIN-1 on both the full-length and truncated protein and observed minor differences in oxidative susceptibility of these constructs, with the full-length construct being somewhat more resistant (not shown). This ruled out major contributions related to tertiary structure differences that could alter access of oxidants to target residues. Thus, subsequent studies were conducted with truncated wild-type and mutant proteins closely representing the processed enzyme that circulates in human plasma (40) . Finally, we found that the inhibition mediated by SIN-1 was recapitulated when we used preparations rich in PAF-AH, such as COS-7 cells transfected with the PAF-AH cDNA (Fig. 1D ), LDL (see below), and extracts from cultured human monocyte-derived macrophages that release the extracellular, plasma form of PAF-AH (data not shown) (1) .


Figure 1
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  		Figure 1. PAF-AH is nitrated and inactivated by SIN-1 and OONO–. A) Aliquots of Pafase® (160 ng) were treated with SIN-1 (0–20 mM) at pH 7.4 for 2 h at 37°C and then assayed for PAF-AH activity, PAF-AH mass, and nitrated tyrosine content. B) Aliquots of human recombinant PAF-AH (960 ng) were treated with OONO– (0–250 µM) at pH 7.4 for 10 min at room temperature and then assayed for PAF-AH activity and for nitrotyrosine (NT) content by immunoblot (IB) analysis. C) Various amounts of recombinant PAF-AH were subjected to SIN-1 (0–500 µM) at pH 7.4 for 4 h at 37°C, and then assayed for PAF-AH activity. D) COS-7 cells transfected with vector or a cDNA encoding PAF-AH were harvested, and the extracts then were exposed to SIN-1. The data represent the difference between PAF-AH-transfected and vector-transfected cells.

A tyrosine residue(s) confers susceptibility of PAF-AH to oxidative inactivation
The results depicted in Fig. 1 showed that both tyrosine nitration and loss of catalytic function occur on exposure of PAF-AH to SIN-1 and ONOO– but did not establish a causal relationship between the two parameters. To investigate whether derivatization of one or more tyrosine residues by nitration-mediated inactivation of PAF-AH, we tested the effect of PNU-101033E, a pyrrolopyrimidine antioxidant previously shown to specifically block peroxynitrite-mediated tyrosine nitration (41) . PNU-101033E (100 µM) completely prevented tyrosine nitration of PAF-AH (Fig. 2 A), had no significant effect on enzymatic activity in the absence of SIN-1, and prevented oxidative inactivation at concentrations of SIN-1 up to 1 mM (Fig. 2B ). These results indicated that a tyrosine residue(s) was a target of oxidative inactivation at relatively low oxidant levels and suggested that mechanisms other than tyrosine nitration contributed to enzymatic inactivation at higher oxidant concentrations. This issue was addressed in experiments described in the next sections. A second line of evidence suggesting that tyrosine nitration participated in PAF-AH inactivation was obtained in experiments that tested the effect of TNM, an oxidizing agent that nitrates tyrosine residues. TNM can form radical species and can damage proteins independently of its ability to nitrate tyrosine residues, but the latter reaction occurs with relative selectivity at pH 8. In contrast, TNM has predominantly oxidative effects at pH 6 (42 , 43) . We used this pH-dependent reactivity as a tool to differentiate between nitration and nonspecific oxidation reactions. We found that TNM treatment of PAF-AH at pH 8 resulted in higher inhibition and tyrosine nitration of the protein compared to pH 6 (Fig. 3 A, B). These combined results suggested that nitration of a tyrosine residue(s) in PAF-AH contributes to enzymatic inactivation, particularly at the concentrations of oxidants encountered in biological systems.


Figure 2
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  		Figure 2. Inactivation of PAF-AH and nitration of tyrosine residues is prevented by PNU-101033E. A) Aliquots of Pafase (1.6 µg containing 0.22 µmol/min of PAF-AH activity) were treated with SIN-1 (0, 0.3, 1, 3.0, 10 mM) and either DMSO or PNU-101033E in DMSO (1–100 µM) at pH 7.4 for 4 h at 37°C, in a total volume of 50 µl. Ten-microliter aliquots were subjected to immunoblot analysis using an antinitrotyrosine antibody. B) Appropriate aliquots from (A) were assayed for PAF-AH activity.


Figure 3
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  		Figure 3. Tetranitromethane selectively nitrates and inactivates PAF-AH at pH 8.0. Aliquots of Pafase (900 ng) were treated with tetranitromethane (TNM, 0–500 µM, dissolved in ethanol) in a total volume of 25 µl. The pH was adjusted to 6 or 8 with phosphate buffer (final concentration=50 mM), and the mixtures were incubated at room temperature for 15 min. Individual aliquots were subjected to immunoblot analysis using an antinitrotyrosine antibody (A) and assayed for PAF-AH activity (B).

Additional mechanisms participate in the oxidative inactivation of PAF-AH
The results from our analyses using PNU-101033E suggested that, in addition to tyrosine, other amino acids contribute to oxidative inactivation of PAF-AH. To investigate the involvement of methionine and/or cysteine residues, we made use of the observation that in the presence of physiological concentrations of HCO3–, ONOO– forms the nitrosoperoxycarbonate (ONOOCO2–) intermediate, which is a more effective tyrosine nitrating agent and a less effective cysteinyl and methionyl oxidant than ONOO– (39) . We tested the effect of HCO3– on tyrosine nitration and found that this treatment robustly increased nitration of PAF-AH by ONOO– (Fig. 4 ), as reported for actin (44) and other proteins. The fact that HCO3– strongly affected tyrosine nitration established that our precautions to exclude CO2 and HCO3– from all buffers were successful. Surprisingly, HCO3– partially protected PAF-AH from enzymatic inactivation (Fig. 4) . The immunoblot analysis shown in Fig. 4 reflects HCO3–-mediated effects on tyrosine nitration, while the enzymatic activity measurements represent the balance between opposite effects of HCO3– on tyrosine nitration and methionine/cysteine oxidation. The dual effects of HCO3– on ONOO–-mediated tyrosine nitration and PAF-AH enzymatic inactivation suggested that residues additional to tyrosine(s) constituted targets for oxidation. This possibility was explored in the experiments described in the next sections.


Figure 4
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  		Figure 4. Bicarbonate effects on nitration and inactivation of PAF-AH. Aliquots of Pafase (800 ng) were treated with ONOO- (0–250 µM, dissolved in 50 mM NaOH) in a total volume of 30 µl. We supplemented NaHCO3- (10 mM) where indicated. Individual aliquots were assayed for PAF-AH activity and subjected to immunoblot analysis using an antinitrotyrosine antibody.

The murine form of PAF-AH is more resistant to inactivation than the human ortholog: studies on chimeric constructs
To simplify the task of identifying the precise residues whose oxidation inactivated PAF-AH, we compared the ability of SIN-1 to inhibit the purified murine and human orthologs and found that the human enzyme was much more sensitive to inhibition than the murine protein (Fig. 5 B). We next investigated which domain was responsible for this difference by generating chimeric proteins in which discrete regions spanning ~20–30% of the human ortholog were individually replaced by the equivalent sequences derived from the murine enzyme (Fig. 5A ). We then tested the susceptibility of the affinity-purified chimeric proteins to SIN-1-mediated inhibition. Replacing the 20% amino-terminal end of the human form of PAF-AH (residues I-42 to N-119) with the equivalent sequences from the murine enzyme (residues L-41 to N-118) resulted in a chimeric protein that was more resistant to oxidation than the human wild-type form (Fig. 5B ). Sequential replacement of the remaining sequences of the human ortholog with the equivalent murine sequences generated chimeric proteins (constructs II, III, IV, and V) that had similar susceptibility to inactivation compared to wild-type human PAF-AH (not shown).


Figure 5
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  		Figure 5. The human and murine PAF-AHs differ in their susceptibilities to oxidative inactivation owing to a residue(s) located at the amino-terminal end. A) Diagrammatic representation of chimeric constructs expressing various portions of the murine form of PAF-AH (shown in white bars) replacing the corresponding human sequences (shown in gray bars). B) Aliquots (25–100 ng) of purified FLAG-tagged human, murine or chimeric PAF-AHs were treated with SIN-1 (0–500 µM) for 4 h at 37°C in a total volume of 10 µl and then assayed for PAF-AH activity.

Our next goal was to identify the amino acids in the amino-terminal end of human PAF-AH that conferred higher susceptibility to oxidation compared to the murine ortholog. Alignment of the sequences corresponding to domain I (Fig. 6 A) revealed that two segments comprising residues 73–81 and 114–117 in the human ortholog are divergent between the two species. However, only the second segment contained the types of residues known to constitute targets for ONOO- attack. We generated constructs within human PAF-AH that progressively changed the human HWLM domain to mimic the murine sequence and then tested the purified recombinant proteins for susceptibility to SIN-1. We found that constructs in which the HWLM motif present in the wild-type human protein was replaced with PWLM, PSLM, or PSIM behaved similarly to the wild-type protein (not shown). However, when we also replaced M-117 with valine to mimic the corresponding residue in murine PAF-AH, the resulting purified PSIV mutant was more resistant to oxidation than the wild-type form (Fig. 6B ). Single replacement of M-117 with valine, isoleucine, or alanine (Fig. 6B and not shown) increased resistance to oxidation, confirming a role for M-117 as a target for attack. The PSIV mutant was more susceptible to oxidation compared to the single M117V mutation, perhaps owing to a modest protective effect of residues W-115 and L-116, which were absent in the PSIV construct (not shown). We also utilized the converse approach by replacing the PSIV domain in murine wild-type PAF-AH with HWLM. We observed increased susceptibility to oxidative inactivation of the HWLM murine form compared to that of the wild-type murine enzyme (Fig. 6C ). These studies pointed at M-117 as a target whose oxidation likely inhibits human PAF-AH activity. We speculate that W-115 and L-116, owing to their proximity to M-117, may limit M-117 oxidation.


Figure 6
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  		Figure 6. Identification of M-117 as a target of oxidative inactivation of PAF-AH. A) Amino acid alignment of human and murine PAF-AHs in the regions comprised between residues 42 and 119 and 41 and 118, respectively. B) Aliquots of FLAG-tagged human wild-type PAF-AH, and mutants PSIV and M117V (25–100 ng) were treated with SIN-1 (0–500 µM) for 4 h at 37°C in a total volume of 10 µl, and then assayed for PAF-AH activity. C) Aliquots of FLAG-tagged murine wild-type PAF-AH and the HWLM mutant (25–100 ng) were treated with SIN-1 (0–500 µM) for 4 h at 37°C in a total volume of 10 µl and then assayed for PAF-AH activity.

Role of additional methionine residues in the oxidative inactivation of PAF-AH
Our next goal was to identify features that accounted for oxidative inactivation of PAF-AH from both human and murine sources. There are a total of 12 methionine residues in full-length human PAF-AH, 10 of which are present in the enzyme form utilized in these studies (see Materials and Methods). A comparison of the human and murine orthologs revealed that only M-46, -71, -255, -299, -331, and -343 are conserved between these proteins. Replacement of M-46 in the human PSIV construct with valine did not significantly affect oxidative susceptibility (not shown). We were unable to study the contribution of M-71 because the M71V mutant lacked enzymatic activity. Contributions of M-255 were ruled out when we found that the canine ortholog, which contains a valine residue at this position, had equal susceptibility to inactivation compared to the human ortholog (not shown). In contrast, we found that replacement of M-299, -331, and -343 with valine substantially increased susceptibility to oxidation (Supplemental Fig. 1S). These mutants were generated within the PSIV human construct to rule out contributions from this domain. We speculate that replacement of M-299, -331, and -343 results in conformational changes that facilitate access of oxidants to target residues or that these residues function as scavengers of reactive oxidant species in the wild-type protein.

Identification of target tyrosine residues common to human and murine PAF-AHs
There are 16 tyrosine residues in human PAF-AH, 14 of which are present in the enzyme form utilized in these studies (see Materials and Methods) and 10 of which (located at positions 63, 84, 85, 103, 144, 160, 188, 205, 307, and 335) are conserved between the murine and human orthologs. We generated constructs harboring individual tyrosine to phenylalanine mutations that also contained the PSIV sequence at positions 114–117 to rule out contributions from this domain. We found that Y-63, -144, and -188 did not alter oxidative susceptibility when replaced with phenylalanines (not shown). In contrast, replacement of Y-307 and -335 in the human PSIV construct with phenylalanine decreased oxidative susceptibility (Fig. 7 A). PNU101033E had almost no protective effect on the tyrosine mutants, a result that is consistent with participation of these residues as targets of oxidative attack and enzymatic inactivation (Fig. 7A ). A triple mutant in which M-117, Y-307, and Y-335 were replaced with valine, phenylalanine, and phenylalanine, respectively, displayed significant resistance even to very high oxidant levels (Fig. 7B ). It is unlikely that the observed effects occurred owing to folding differences among the mutants because the specific activities of the key recombinant proteins were comparable (Fig. 7C ). We also observed attenuation of tyrosine nitration following mutation of Y-307 and Y-335 (Fig. 7D ), although the effect was moderate possibly owing to nitration of additional tyrosine residues in the protein. Replacement of Y-84, -85, -103, -160, and -205 in the PSIV construct substantially increased susceptibility to oxidative inactivation (Supplemental Fig. 2S). The folding of these proteins may facilitate access of oxidants to target sites. Alternatively, these residues could have protective scavenging functions in the wild-type protein.


Figure 7
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  		Figure 7. Identification of Y-307 and Y-335 as targets of nitration and oxidative inactivation of PAF-AH. A) Aliquots of the purified FLAG-tagged mutants indicated in each case (25–100 ng) were treated with vehicle, SIN-1 (500 µM) and/or PNU-101033E (100 µM) for 4 h at 37°C in a total volume of 10 µl. The samples then were assayed for PAF-AH activity. B) Aliquots of FLAG-tagged human wild-type PAF-AH and mutants M117V and M117V:Y307F:Y335F (25–100 ng) were treated with the indicated concentrations of SIN-1 for 4 h at 37°C in a total volume of 10 µl, and then assayed for PAF-AH activity. C) Comparison of specific activities of purified FLAG-tagged wild-type, M117V, and M117V:Y307F:Y335F recombinant human PAF-AHs. D) Aliquots of FLAG-tagged mutants containing 66 nmol/min of enzymatic activity were incubated at pH 7.4 with 50 µM SIN-1 for 4 h at 37°C in a total volume of 200 µl and then subjected to immunoblot analysis using antinitrotyrosine and anti-FLAG antibodies. Densitometric scanning was conducted using NIH image software.

Identification of target tryptophan residues common to human and murine PAF-AHs
Tryptophan residues can be nitrated by ONOO– on the benzene ring, a reaction that is moderately increased by the addition of HCO3— (39) . There are nine tryptophan residues in human PAF-AH, but only seven of them are present in the enzyme form utilized in these studies (see Materials and Methods) and only W-97, -134, -203, -298, and -405 are conserved between the two orthologs. We generated constructs harboring individual tryptophan to phenylalanine mutations that also contained the PSIV sequence at positions 114–117, to rule out contributions from this domain. We found that replacement of tryptophan residues located at positions 134 and 405 with phenylalanine generated recombinant proteins that behaved in manners similar to that of the PSIV control mutant (not shown). In contrast, replacement of W-97, -203, and -298 with phenylalanine increased susceptibility to oxidative inactivation compared to that of the parent PSIV mutant (Supplemental Fig. 3S). We speculate that W-97, -203, and -298 may function as scavengers of reactive oxidant species or that replacement of these residues facilitates access of oxidants to target residues in the mutant proteins.

Cysteine residues do not participate in the oxidative inactivation of PAF-AH
Cysteine residues are well-recognized targets of oxidative attack. The circulating form of PAF-AH has five cysteine residues, which indicates that at least one free sulfhydryl group exists in the mature form of the protein. To investigate whether a cysteine residue(s) was a target of oxidation, we treated the recombinant protein with PMAB, a sulfhydryl-reactive biotinylation reagent that derivatized the enzyme (Supplemental Fig. 4SA). Next, we asked whether derivatization of free cysteines with the sulfhydryl reagent DTNB altered responses to SIN-1. We found that DTNB effectively derivatized free cysteines (Supplemental Fig. 4SA), did not affect enzymatic activity, and did not protect the enzyme from SIN-1 inhibition (Supplemental Fig. 4SB). Second, substitution of three conserved cysteine residues (C-67, -229, and -291) with alanine did not affect susceptibility to oxidation (Supplemental Fig. 4SC). These combined observations suggested that cysteine residues in PAF-AH are not likely targets of oxidative inactivation.

Studies using lipoprotein-associated wild-type and mutant enzymes
Our next goal was to investigate whether association with HDL and LDL influenced susceptibility to oxidant attack and whether our findings in purified systems were recapitulated using lipoprotein-associated enzymes. To investigate this, we first established that the key recombinant proteins studied (M117V and M117V:Y307F:Y335F) associated with HDL and LDL particles in a manner identical to that of the wild-type protein (Fig. 8 A, C). Next, we tested the effect of SIN-1 on the lipoprotein-associated enzymes and found somewhat higher susceptibility to oxidation of the wild-type protein compared to the free enzyme (compare Figs. 1A , 8B, D) . This effect could occur owing to increased stability and/or solubility of ROS in the lipid environment provided by the lipoprotein particles or to a role for lipoprotein-associated lipids in oxidative inactivation. In addition, the qualitative behavior of the lipoprotein-associated proteins was comparable to that of the free purified proteins. Replacement of M-117, Y-307 and Y-335 increased resistance to oxidation both in HDL and in LDL (Fig. 8B, D ), providing additional support for a role of these residues as targets of oxidation in physiological settings.


Figure 8
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  		Figure 8. Studies using lipoprotein-associated wild-type and mutant enzymes. Aliquots of the FLAG-tagged proteins indicated containing 9 nmol/min of PAF-AH activity were incubated with Pefabloc-treated LDL (A; 245 µg) or Pefabloc-treated HDL (C; 1.77 mg) for 30 min at 37°C in a total volume of 500 µl. The mixtures then were adjusted to a density of 1.3 g/ml with KBr and to a volume of 10 ml with PBS and were centrifuged for 3 h at 50,000 rpm in a Beckman VTi50 rotor. Fractions (~1.3 ml) were collected and assayed for PAF-AH activity. B, D) Aliquots of LDL- and HDL-associated activities (fractions 13 and 5, respectively) were treated with the indicated concentrations of SIN-1 for 4 h at 37°C in a total volume of 20 µl and then assayed for PAF-AH activity.

Oxidation of PAF-AH affects the ability of the enzyme to associate with LDL
We previously demonstrated that W-115 and L-116, which neighbor M-117, participate in the interaction between PAF-AH and LDL (36) . The observation that a residue next to the LDL binding domain is a target of oxidation led us to hypothesize that oxidation of wild-type PAF-AH might interfere with its ability to remain associated with LDL. To test this, we incorporated PAF-AH into LDL particles previously treated with Pefabloc to inactivate the endogenous activity, then oxidized the particles, separated the soluble and LDL fractions by ultracentrifugation, and subjected these extracts to enzymatic activity and immunoblot analyses using anti-PAF-AH antibodies. A control incubation conducted in the absence of oxidants showed that most of the PAF-AH protein remained associated with LDL throughout the procedure (Fig. 9 , lanes 1 and 2 showing the soluble and LDL fractions, respectively). Treatment with SIN-1 attenuated association with LDL (Fig. 9 , compare LDL fractions depicted in lanes 2, 4, and 6) and increased the amount of immunoreactive protein in the soluble fraction (Fig. 9 , compare soluble fractions depicted in lanes 1, 3, and 5). These results indicated that oxidation led to partial dissociation of PAF-AH from LDL. In complementary studies, we investigated whether oxidation of the free enzyme affected subsequent association with LDL. A control incubation conducted in the absence of oxidants provided a measure of the amount of free PAF-AH that retained the ability to bind to LDL (Fig. 9 , lanes 7 and 8). Treatment of the free enzyme with oxidants inhibited enzymatic activity, attenuated association with LDL (Fig. 9D , compare LDL fractions depicted in lanes 8, 10, and 12), and increased the amount of immunoreactive protein in the soluble fraction (Fig. 9D , compare soluble fractions depicted in lanes 7, 9, and 11). These results indicated that oxidation of the free enzyme partially prevented subsequent association with LDL. In summary, our combined results indicated that oxidation interfered with the ability of PAF-AH to bind to, and remain associated with, LDL. These findings are consistent with a model wherein oxidation of M-117 results in local structural alterations that prevent adjacent residues W-115 and L-116 to effectively mediate binding to LDL.


Figure 9
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  		Figure 9. Oxidation affects association of PAF-AH with LDL. Aliquots of Pafase (16.4 µg containing 2.2 µmol/min of PAF-AH activity) were incubated for 60 min at 37°C with Pefabloc-treated LDL (680 µg, left) or PBS (right) in a total volume of 2 ml. SIN-1 was supplemented at relatively high levels owing to the high concentration of Pafase in this experiment. The incubations were continued for 4 additional hours at 37°C. We then added PBS (left) or Pefabloc-treated LDL (680 µg, right) and incubated the samples for 60 additional min at 37°C in a total volume of 4 ml. The mixtures were subjected to ultracentrifugation, fractionated, and assayed for PAF-AH activity (top). Fractions 1–8 (soluble, S) and 9–16 (LDL, L) were pooled and dialyzed overnight against PBS at 4°C. We then subjected aliquots of the dialyzed fractions to electrophoresis and immunoblotting using an anti-PAF-AH antibody (bottom).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we showed that PAF-AH is inhibited by oxidants known to be generated during cellular oxidation in vivo. Concomitant exposure to NO and O2– inhibited PAF-AH activity, suggesting that ONOO– or a degradation product was responsible for the observed effects. Other physiological [i.e., heavy metals (35 , 45) ], and nonphysiological oxidants [i.e., TNM and cigarette smoke (20 , 46) ] also inactivate PAF-AH, suggesting that the enzyme is sensitive to a variety of oxidants. In the present study, we identified ONOO– as one of the species that inactivates PAF-AH using purified recombinant proteins. In biological settings, ROS that can lead to ONOO– generation are likely involved as well. For example, Ambrosio and coworkers first reported that O2–-mediated, iron catalyzed formation of hydroxyl radicals constitutes a key mechanism for enzymatic inactivation of PAF-AH in whole plasma and in intact LDL (19) . LDL is known to contain an array of nitrogen species that may generate ONOO– by interaction with oxygen radicals such as O2– (47 48 49) . Thus, our results combined with those of Ambrosio and coworkers are consistent with participation of both ONOO– and O2– in the inactivation of PAF-AH in biological settings.

Peroxynitrite has been shown to affect proteins by modifying free tyrosine, methionine, tryptophan, and cysteine residues (39) . Nitrosylation of thiols, S-nitrosylation, can regulate protein function by several mechanisms (50 , 51) . Here, we found no evidence that the inactivation of PAF-AH by ONOO– involved cysteine residues. Ambrosio and coworkers and Bielicki et al. reached similar conclusions using other experimental approaches and different oxidant sources (19 , 46) .

ROS can modify proteins by derivatizing tyrosine residues, resulting in changes in protein function. In our studies, we found evidence for overall modification of PAF-AH protein by tyrosine nitration and for participation of Y-307 and Y-335 in the oxidative inactivation of PAF-AH. Interestingly, these residues are the only tyrosines that are in close proximity to two acidic residues and a bending residue, a topology reported to favor tyrosine nitration (51) . The stimulation of ONOO–-mediated tyrosine nitration by HCO3– without parallel effects on enzymatic activity suggested that multiple tyrosines may become nitrated without affecting function. Biologically significant oxidation of tyrosines and methionines is usually associated with an impairment of functional activity, as is the case for several enzymes (52 , 53) , some of which are involved in defense against oxidative stress (54) . In other cases, however, oxidants can be stimulatory. For example, ONOO– increased glutathione S-transferase activity by nitration of Y-92 (55) . Our functional analyses suggested that tyrosine nitration other than that of residues 307 and 335 may have no functional consequences or even be protective of PAF-AH, potentially owing to an oxidant scavenging effect. However, our studies do not rule out the possibility that altered folding of the recombinant mutant proteins resulted in increased access of oxidants to susceptible sites.

Our studies generated evidence for participation of M-117 as a target of oxidation. The role of methionine oxidation in the inactivation of PAF-AH is clearly more important than that of tyrosine nitration, as mutation of M-117 resulted in higher protection than that afforded by replacement of Y-307 and Y-335. Oxidation of M-117 not only resulted in enzymatic inactivation but also affected association with LDL, possibly owing to the fact that this residue is immediately adjacent to the LDL binding domain. Dissociation of the LDL-associated enzyme following oxidation may have functional consequences additional to inhibition, as we previously demonstrated that lipoprotein association profoundly affects the function of the enzyme in vitro (56) . Our studies also showed that oxidation of the free enzyme prevented subsequent association with LDL. These findings, combined with our observation that PAF-AH can transfer between HDL and LDL in a pH-dependent manner (29) , leads us to speculate that dissociation of PAF-AH following oxidation and during transit between lipoproteins may impair subsequent association with the particles and may have pathological consequences.

There are additional interesting features that highlight the complexity of oxidant-mediated effects on PAF-AH expression. It has been reported that plasma from cigarette smokers has increased levels of PAF-AH activity compared to plasma from nonsmokers (57) . This suggests that cigarette smoke components activate the enzyme or induce enzyme synthesis. However, cigarette smoke extracts, or products derived from exposure to these extracts, inhibit PAF-AH activity (20 , 46) . These combined observations indicate that oxidants directly inactivate the enzyme and that a compensatory mechanism(s) mediates de novo synthesis of the protein. The mechanism of oxidant-mediated modulation of PAF-AH expression has not been investigated in detail, but our work suggests that oxidants transcriptionally regulate expression of PAF-AH (S. Green and D. M. Stafforini, unpublished observations). These findings have implications for pathological conditions, in which oxidant stress is thought to play an important role, such as pathological inflammation, cancer, and atherosclerosis. Our findings that oxidative modification of LDL is accompanied by inactivation of PAF-AH (58) , combined with studies that report elevated expression of the protein in settings of oxidant stress such as atherosclerosis (32) , also illustrate the existence of dual effects of oxidants on the expression and activity of this enzyme. These results underscore the importance of pursuing further investigations aimed at dissecting the compensatory effects of oxidants on the regulation of expression and physiological function of this enzyme.


   ACKNOWLEDGMENTS
 
This work was supported in part by a grant from the National Institutes of Health HL35828 to D.M.S. We thank L. Jeff Johnson and Damian Dayton for providing excellent technical assistance.


   FOOTNOTES
 
1 Current address: University of Colorado Health Sciences Center, Section of Neonatology, 4200 East Ninth Ave., B195 Denver, CO 80262, USA.

2 Current address: Oklahoma Medical Research Foundation, 825 NE 13th St., Oklahoma City, OK 73104, USA.

Received for publication June 29, 2006. Accepted for publication November 9, 2006.


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