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The proinflammatory mediator Platelet Activating Factor is an effective substrate for human group X secreted phospholipase A2
Biochimica et Biophysica Acta 1761 (2006) 1093 – 1099 www.elsevier.com/locate/bbalip
The proinflammatory mediator Platelet Activating Factor is an effective substrate for human group X secreted phospholipase A2 Sarah Gora a , Gerard Lambeau b , James G. Bollinger c , Michael Gelb c , Ewa Ninio a , Sonia-Athina Karabina a,⁎ a
INSERM U525, Université Pierre et Marie Curie-Paris6, Faculté de Médecine Pierre et Marie Curie, Paris, France b IPMC, CNRSUMR 6097, Sophia Antipolis, Valbonne, France c Department of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195, USA Received 12 April 2006; received in revised form 19 June 2006; accepted 2 August 2006 Available online 5 August 2006
Abstract Platelet Activating Factor (PAF) is a potent mediator of inflammation whose biological activity depends on the acetyl group esterified at the sn2 position of the molecule. PAF-acetylhydrolase (PAF-AH), a secreted calcium-independent phospholipase A2, is known to inactivate PAF by formation of lyso-PAF and acetate. However, PAF-AH deficient patients are not susceptible to the biological effects of inhaled PAF in airway inflammation, suggesting that other enzymes may regulate extracellular levels of PAF. We therefore examined the hydrolytic activity of the recently described human group X secreted phospholipase A2 (hGX sPLA2) towards PAF. Among different sPLA2s, hGX sPLA2 has the highest affinity towards phosphatidylcholine (PC), the major phospholipid of cellular membranes and plasma lipoproteins. Our results show that unlike group IIA, group V, and the pancreatic group IB sPLA2, recombinant hGX sPLA2 can efficiently hydrolyze PAF. The hydrolysis of PAF by hGX sPLA2 rises abruptly when the concentration of PAF passes through its critical micelle concentration suggesting that the enzyme undergoes interfacial binding and activation to PAF. In conclusion, our study shows that hGX sPLA2 may be a novel player in PAF regulation during inflammatory processes. © 2006 Elsevier B.V. All rights reserved. Keywords: PAF; Inflammation; PAF-AH; Phospholipase A2 group X; Lp-PLA2; LDL
1. Introduction Platelet-Activating Factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent lipid mediator which is produced by various inflammatory cells and is involved in a number of inflammatory and allergic processes including atherosclerosis (reviewed in [1]). Its biological activity depends on the acetyl group esterified at the sn-2 position of the glycerol backbone. Inactivation of PAF to form the biologically inactive metabolite lyso-PAF (1-O-alkyl-sn-glycero-3-phosphocholine) and acetate is catalyzed by PAF-acetylhydrolase (PAF-AH), a calcium-independent phospholipase A2 (PLA2) [2,3] also known as lipoprotein associated PLA2 (Lp-PLA2) or GVIIA phospholipase A2 (reviewed in [4]). A loss of function mutation (Val279Phe) has been described for plasma PAF-AH [5] that ⁎ Corresponding author. Tel.: +33 1 40 77 81 39; fax: +33 1 40 77 97 68. E-mail address: [email protected] (S.-A. Karabina). 1388-1981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2006.08.004
results in the inability of the protein to be secreted by mammalian cells [6]. In addition the recombinant bacterial protein is inactive [5]. The mutation Val279Phe occurs in 30% (both homozygotes and heterozygotes) of Japanese [5] and other Eastern populations [7], leading to PAF-AH deficiency and absence of hydrolysis of exogenously added substrate in homozygotes. Although the initial genetic studies showed an association of 279Phe allele with an increased risk for cardiovascular disease, stroke [8] and myocardial infraction [9], as compared to 279Val carriers, a more recent larger study did not confirm such an association [10]. Furthermore, as PAF-AH deficient patients are not susceptible to the biological effects of inhaled PAF in airway inflammation [11], the central role of PAF-AH as a major player in PAF hydrolysis in vivo is being challenged. Secreted PLA2s (sPLA2s) form a group comprising up to 12 mammalian enzymes that have distinct primary structures, specific tissue distributions and different enzymatic properties, suggesting that each enzyme has a distinct physiological role.
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Among sPLA2s, the human group X sPLA2 (hGX sPLA2) has been detected in thymus, spleen and leucocytes, indicating a possible role of this sPLA2 in immunity and/or inflammation [12]. Furthermore, in marked contrast to other Ca2+-dependent sPLA2, hGX sPLA2 is by far the most active enzyme towards phosphatidylcholine (PC), the major phospholipid of mammalian cell membranes and plasma low and high density lipoproteins (LDL and HDL respectively) [13–18]. On the basis of the structural similarities between PAF and PC and the results of clinical trials using recombinant human PAF-AH that have raised doubts about the central role of this enzyme in hydrolyzing physiological concentrations of PAF [19,20], we examined whether hGX sPLA2 could hydrolyze PAF in vitro. Here we demonstrate that unlike hGV, hGIIA sPLA2s and porcine GIB, hGX sPLA2 can efficiently hydrolyze PAF when incorporated into large unilamellar PC phospholipid vesicles and when present in PC-rich lipoproteins indicating that hGX sPLA2 may be a novel player in PAF regulation during inflammatory processes. 2. Materials and methods 2.1. Reagents PAF (1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine), obtained as a powder from Sigma Aldrich (Saint Louis, MO), was dissolved at a final concentration of 50 mM in ethanol. This solution was mixed with 1-O-hexadecyl-2[3H-acetyl]-sn-glycero-3-phosphocholine (21.5 Ci/mmol; DuPont-New England Nuclear) in various proportions, dried under a stream of nitrogen, and dissolved in a solution containing fatty acid-free BSA/saline (0.25%) to obtain [3H-acetyl] PAF final concentrations from 2.5 to 1000 μM solutions. 1-palmitoyl-2arachidonoyl-sn-glycero-3-phosphocholine was from Sigma Aldrich (St Louis, MO) and was kept as a 10 mg/ml solution in 0.25% fatty acid-free BSA saline. Recombinant PAF-AH was prepared in our laboratory as previously described [21,22]. Porcine group IB sPLA2 was obtained from Sigma. Recombinant hGIIA, hGV and wild type and H48Q mutant of hGX sPLA2 were prepared in E. coli as described [13,23]. The hGX-sPLA2 inhibitor, LY 329722 (3-(3-Aminooxalyl-1-benzyl-2-ethyl-6-methyl-1H-indol-4-yl)-propionic acid), was synthesized as described [24].
2.2. hGX sPLA2 activity towards [3H-acetyl]PAF The activity of hGX sPLA2 (50 nM) was measured using a modified PAF-AH assay [25] in Tris–HCl buffer pH 8.0, supplemented with 1 mM CaCl2, containing 50 μM [3H-acetyl]PAF as substrate (prepared as described above), except for the dose–response studies which were performed with final concentrations of [3H-acetyl]PAF ranging from 0.25 to 100 μM. In competition experiments, various concentrations of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine were added to the reaction mixture. Several experiments were performed in the presence of 1–10 mM CaCl2. In selected experiments either 10 μM LY 329722, a potent hGX sPLA2 inhibitor, was added to the reaction mixture or hGX sPLA2 was substituted by its catalytically-inactive mutant H48Q at a concentration of 50 nM. Comparative activity studies were performed using similar amounts of hGX, hGV, hGIIA, porcine pancreas GIB, and human recombinant PAF-AH in the presence of 50 μM [3H-acetyl]PAF final concentration. All enzymatic reactions were performed at 37 °C for 10 min, except for kinetic experiments. The reactions were stopped in an ice bath for 10 min using an excess of BSA (final concentration, 16.6 mg/mL), to bind unreacted [3H-acetyl]PAF, prior to addition of trichloroacetic acid. The samples were then centrifuged for 15 min at 1800 × g, and the [3H]acetate recovered in the aqueous phase was measured by liquid scintillation counting in Optiphase Hi-Safe 3. The results, after correction for the blank (150–200 dpm, which did not exceed 10% of the
experimental values), were expressed as nmol of PAF degraded per min per microgram of protein.
2.3. LDL isolation and treatment with hGX sPLA2 LDL was isolated from frozen plasma by density gradient ultracentrifugation, as described previously [26]. The protein content of LDL was determined by the bicinchoninic acid (BCA) method (Pierce). Freshly prepared, filtered (0.45 μm) sterile LDL, 1 mg protein/mL in buffer containing 1 mM CaCl2, 12.5 mM Tris–HCl (pH 8.0), 0.25 M NaCl and 0.0125% BSA was first incubated for 30 min at 37 °C in the presence of the serine esterase inhibitor Pefabloc (0.1 mM) in order to inactivate the endogenous PAF-AH activity [27], prior to being dialyzed for 24 h against PBS. LDL (with inactivated PAF-AH) was mixed with 50 μM final concentration [3H acetyl]PAF and subsequently incubated with 50 nM hGX, hGV, hGIIA, and porcine group IB sPLA2s at 37 °C for 10 and 60 min. The reaction was stopped on ice with an excess of BSA and the amount of [3H] acetate released was measured as described above. In parallel, the amount of free fatty acids liberated during the treatment of LDL by the different sPLA2s was quantified using the NEFA C kit (Wako Chemicals, GmbH) following the manufacturer's instructions.
2.4. Hydrolysis of PC vesicles containing PAF To study hydrolysis of phosphatidylcholine vesicles containing PAF, we prepared large unilamellar vesicles (0.1 μm) of 1-palmitoyl-2-oleoyl-snglycerol-3-phosphocholine (Avanti Polar Lipids Inc., Alabaster, AL) containing 10% 1-hexadecyl-2-acetyl-sn-glycerol-3-phosphocholine (Avanti Polar Lipids Inc.) by extrusion as described [13]. Reaction mixtures using rat liver fatty acid binding protein were carried out with Hank's balanced salt solution containing calcium and magnesium and 30 μM total phospholipid (as vesicles) as described [13]. Enzyme (30 ng human group V sPLA2 or 40 ng human group X sPLA2) was added to initiate the reaction. The reaction mixture was quenched by adding EGTA to give 2 mM when ∼ 10% of the 1-palmitoyl-2-oleoyl-sn-glycerol-3phosphocholine was hydrolyzed (the assay was calibrated with oleic acid as described) [13]. Samples were processed by solid-phase extraction and analyzed by combined liquid chromatography electrospray ionization mass spectrometry as described [13]. A blank sample was prepared and processed as above except that the sPLA2 was omitted. Prior to processing, 5 nmol of the internal standard, 1-palmitoyl-sn-glycero-3-phosphocholine containing a per-deuterated fatty acyl chain was added. To obtain the relative ionization efficiency of the two reaction products, equal amounts of 1-palmitoyl-sn-glycero-3-phosphocholine and 1hexadecyl-sn-glycero-3-phosphocholine were co-injected onto the liquid chromatography column. The absolute amount of the reaction products was obtained from the relative peak areas (parent ion trace) of enzyme-produced lyso-phospholipid to internal standard with correction for the relative ionization efficiency. The amount of product formed in the time needed to hydrolyze ∼10% of the vesicles together with the amount of added sPLA2 were used to obtain the specific activities reported in Results.
2.5. Statistical analysis Results are expressed as mean ± SE of 3–9 experiments in duplicate or triplicate. Mean values were compared by Student's t test, with significance defined at a value of P ≤ 0.05.
3. Results 3.1. Recombinant hGX sPLA2 efficiently catalyses the hydrolysis of [3H-acetyl]PAF We first compared the enzymatic activities of different purified or recombinant sPLA2s with that of PAF-AH towards [3 H-acetyl]PAF. Recombinant hGX sPLA2, hGV sPLA2, hGIIA sPLA2, purified porcine group IB and human PAFAH were incubated with [3H-acetyl]PAF for 10 min at 37 °C.
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Fig. 1. [3H-acetyl]PAF hydrolysis by recombinant PLA2s. hGX sPLA2, human recombinant PAF-AH, hGV, porcine GIB, hGIIA and the active site mutant of hGX sPLA2, H48Q were incubated at 37 °C for 10 min with [3H-acetyl]PAF. The amount of the [3H]acetate liberated was measured as described in Materials and methods. Results are expressed as nmol of [3H-acetyl]PAF hydrolyzed per minute per μg of each recombinant protein and represent the mean ± SE of three separate experiments performed in duplicate. *P < 0.05 when compared to PAFAH or hGX sPLA2.
In agreement with former studies [22], recombinant PAF-AH efficiently hydrolyzed [3H-acetyl]PAF (Fig. 1), either in the presence or absence of Ca2+ (data not shown). At similar enzyme concentrations (50 nM), hGX sPLA2 showed almost 50% of the PAF-AH hydrolytic activity. In contrast to hGX sPLA2, hGV, hGIIA sPLA2s and porcine group IB, showed very low if any catalytic activity towards [3H-acetyl]PAF (Fig. 1) even after longer incubation periods (data not shown). The activity of hGX sPLA2 on [3H-acetyl]PAF hydrolysis was not influenced by Ca2+ concentrations above 1 mM (data not shown) as 1 mM Ca2+ is sufficient to saturate the enzyme [28]. However, in the presence of 5 mM EDTA, the enzyme activity was completely abolished (95% ± 2% inhibition), a result consistent with the strict Ca2+ requirement of this sPLA2 [12]. The H48Q catalytic site mutant of hGX sPLA2, which is enzymatically-inactive when assayed on PG vesicles or radiolabelled E. coli membranes, did not degrade [3Hacetyl]PAF (Fig. 1). Finally, no enzymatic activity (99% ± 2% of inhibition) was detected in the presence of 10 μM LY 329722, a potent hGX sPLA2 inhibitor [24].
Fig. 2. Concentration dependent hydrolysis of [3H-acetyl]PAF by hGX sPLA2. 50 nM of hGX sPLA2 were incubated with 0.25–100 μM final concentration of [3H-acetyl]PAF for 10 min at 37 °C. The [3H]acetate liberated was measured as described in Materials and methods. Results are expressed as nmol of [3H-acetyl] PAF hydrolyzed per minute per μg of hGX sPLA2 and represent the mean ± SE of 4 separate experiments performed in duplicate or triplicate.
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Fig. 3. Time dependent hydrolysis of [3H-acetyl]PAF by hGX sPLA2. 50 nM of hGX sPLA2 were incubated with 50 μM final concentration of [3H-acetyl]PAF for various times. The amount of the [3H]acetate liberated was measured as described in Materials and methods. Results are expressed as nmol of [3Hacetyl]PAF hydrolyzed per μg of hGX sPLA2 and represent the mean ± SE of 3 separate experiments performed in duplicate or triplicate.
3.2. Interfacial property of hGX sPLA2 on PAF The enzymatic property of hGX sPLA2 was evaluated at 37 °C for 10 min with [3H-acetyl]PAF at concentrations ranging from 0.25 to 100 μM. Consistent with its interfacial binding properties [14], hGX sPLA2 (50 nM) exhibited a maximal enzymatic activity when PAF concentration rose above its critical micellar concentration (∼5 μM) (Fig. 2). Time-course experiments showed that about 40% of [3H-acetyl]PAF was hydrolyzed by hGX sPLA2 in the first 10 min of incubation (Fig. 3). 3.3. Effect of PC on hGX sPLA2 PAF hydrolysis Since arachidonoyl-PC is a very good substrate for hGX sPLA2 [18], we next studied the [3H-acetyl]PAF hydrolysis in the presence of different concentrations (0 to 1278 μM) of this competitor. As shown in Fig. 4 the presence of arachidonoyl-PC in the assay mixture containing hGX sPLA2 decreased the hydrolysis of [3H-acetyl]PAF in a concentration dependent manner.
Fig. 4. Hydrolysis of [3H-acetyl]PAF in the presence of arachidonoyl-PC. Increasing quantities of arachidonoyl PC were added to the assay mixture containing 50 μM [3H-acetyl]PAF. Subsequently 50 nM of hGX sPLA2 was added and the incubation took place for 10 min at 37 °C. The amount of the [3H] acetate liberated was measured as described in Materials and methods. Results are expressed as nmol of hydrolyzed [3H-acetyl]PAF per minute per μg of hGX sPLA2 and represent the mean ± SE of 3 separate experiments performed in duplicate.
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Fig. 5. Hydrolysis of [3H-acetyl]PAF in the presence of LDL treated with Pefabloc. (A) 0.1, 0.4, and 0.8 mg/mL LDL protein were incubated in the presence of 50 nM hGX sPLA2 for 10 and 60 min at 37 °C with 50 μM [3H-acetyl]PAF. The amount of the [3H]acetate liberated was measured as described in Materials and methods. Results are expressed as nmol of hydrolyzed [3H-acetyl]PAF per μg of hGX sPLA2 and represent the mean ± SE of 3 separate experiments performed in duplicate. *P < 0.05 when compared to 0.1 mg/mL LDL. (B) 0.1, 0.4, and 0.8 mg/mL LDL protein were incubated in the presence of 50 nM hGX sPLA2 for 10 and 60 min at 37 °C with 50 μM [3H-acetyl]PAF. In control experiments, LDL and [3H-acetyl]PAF were incubated without hGX sPLA2. The amount of fatty acids liberated was quantified by the NEFA C kit. The result is representative of two experiments.
3.4. Hydrolysis of [3H-acetyl]PAF by hGX sPLA2 on LDL As oxidation of LDL generates PAF [29] and a vast array of phospholipids with truncated, short fatty acids at the sn-2 position of PC, which structurally mimic PAF [30,31], we examined whether hGX sPLA2 could hydrolyze [3H-acetyl] PAF when the latter was incorporated into LDL particles. Since purified LDL particles contain an associated-PAFAH activity, we first treated LDL preparations with Pefabloc to inhibit PAF-AH activity [27] prior to addition of [3Hacetyl]PAF and enzymatic assays with different sPLA2s. Various concentrations of LDL (0.1 to 0.8 mg of protein/ml)
were incubated with 50 μM final concentration of [3H-acetyl] PAF in the presence of 50 nM hGX sPLA2. hGX sPLA2 efficiently hydrolyzed [3H-acetyl]PAF even in the presence of LDL associated PC (Fig. 5A); the reaction was, as expected, inhibited in a dose-dependent manner by LDL. The latter observation is presumably due to two factors: the first is that the surface concentration of PAF in LDL will go down as the amount of LDL is increased in the presence of a fixed amount of PAF. This surface dilution effect would lower the rate of PAF hydrolysis by hGX sPLA2 because the enzyme bound to LDL would encounter a lower concentration of PAF substrate. Secondly, PC is hydrolyzed by hGX sPLA2 (as free fatty
Fig. 6. Hydrolysis of [3H-acetyl]PAF by different recombinant sPLA2 in the presence of LDL treated with Pefabloc. 0.1 and 0.4 mg/mL LDL protein were incubated in the presence of 50 nM hGX, hGV, hGIIA, porcine GIB sPLA2s and PAF-AH for 10 at 37 °C with 50 μM [3H-acetyl]PAF. The amount of the [3H]acetate liberated was measured as described in Materials and methods. Results are expressed as nmol of hydrolyzed [3H-acetyl]PAF per mL of sPLA2 and represent the mean ± SE of 3 separate experiments performed in duplicate.
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acids were concomitantly released in the reaction mixture (Fig. 5B)) and so increased amounts of LDL relative to PAF would increase the concentration of competing PC substrate relative to PAF substrate with a corresponding decrease in the rate of enzymatic PAF hydrolysis. As compared to hGX sPLA2, hGV, hGIIA and porcine group IB showed little, if any, activity towards [3H-acetyl]PAF in the presence of LDL (Fig. 6). 3.5. Hydrolysis of PAF by hGX and hGV sPLA2 in mixed PAF: PC co-vesicles Next, the ability of hGX and hGV sPLA2s to hydrolyze large unilamellar vesicles (0.1 μm) of 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) containing 10 mol% PAF (1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) was studied by measuring the amount of each product formed in this competitive substrate analysis [32]. It was expected that most of the PAF would partition into the vesicles since the phospholipid concentration in the assay (30 μM) is well above the critical micelle concentration of PAF (∼ 2 μM) [33]. The relevant equation for this competitive substrate specificity analysis is [32]: vPOPC =vPAF ¼ ðkcat * =KM * ÞPOPC =ðkcat * =KM * ÞPAF ðXPOPC =XPAF Þ here, vPOPC and vPAF are the initial velocities for the hydrolysis of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and PAF, respectively. (kcat * /KM * )POPC and (kcat * /KM * )PAF are the ratio of interfacial turnover number to interfacial Michaelis constant for each of the two substrates, and XPOPC and XPAF are the mole fractions of each substrate in the vesicle. This equation is analogous to the standard steady-state enzyme kinetic equation for the action of an enzyme on two substrates simultaneously (competitive substrate analysis). Since only 10% of the substrate is hydrolyzed, it is reasonable to assume that the ratio of products formed equals the ratio of initial enzymatic reaction velocities. In the present study, (XPOPC/XPAF) = 9, and thus (kcat * /KM * )POPC/(kcat * /KM * )PAF = 6.8 for hGX sPLA2 (Table 1). Thus, it can be said that hGX sPLA2 intrinsically prefers 1palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine as a substrate over PAF by 6.8-fold. For hGV sPLA2, no PAF hydrolysis could be detected, and thus the preference of hGV sPLA2 for PC over PAF is at least 50-fold (Table 1). Table 1 Hydrolysis of mixed PAF:PC co-vesicles by hGX and hGV sPLA2 POPC specific activity μmol/(min mg)
PAF specific activity μmol/(min mg)
(kcat * /KM * )POPC/ (kcat * /KM * )PAF
hGV hGX
0a 0.14
>50 a, b 6.8
9.97 8.56
Large unilamellar vesicles of PC and PAF prepared by extrusion were incubated with 40 ng of hGX and 30 ng of hGV. The reaction mixture was quenched by adding EGTA and samples were processed as described in Materials and methods. a No PAF hydrolysis was observed. The value of 50 is a lower limit based on the estimated sensitivity of detection limit for lyso-PAF. b Calculated according to the equation in the main text.
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4. Discussion Although the role of plasma PAF-AH in hydrolyzing PAF and controlling inflammation has been well established (reviewed in [34,35]), clinical trials showed that administration of recombinant human PAF-AH failed to improve the clinical outcome of patients [19,20,36]. All these investigations have questioned the role of PAF-AH as the sole enzyme responsible for PAF hydrolysis in vivo, highlighting the possibility that other enzymes may regulate extracellular PAF levels [11]. We show here for the first time that hGX sPLA2, which has high affinity for PC [12–14,18], can efficiently hydrolyze PAF in vitro or ex-vivo on plasma lipoproteins. Interestingly, hGIIA sPLA2 which is highly expressed during inflammation [37] and reviewed in [38] does not hydrolyze PAF as shown in Fig. 1 probably due to its poor binding to PC-rich membranes [14]. The same observation was made for the pancreatic PLA2 and for hGV sPLA2, which were shown to be expressed in certain human pathological conditions [39–41]. Even after prolonged incubation with labelled PAF, both hGIIA and porcine GIB had only minor hydrolytic effect on this substrate. Additionally, as PAF accumulates in oxidized LDL [29], we show here that hGX sPLA2 can also efficiently hydrolyze radiolabelled PAF when it is incorporated into LDL, in a range of concentrations corresponding to either normolipidemic (100 μg/ml) or hypercholesterolemic subjects (800 μg/mL). We observed that hGX sPLA2 was more efficient at hydrolyzing PAF when LDL concentrations were in the normolipidemic range. The more LDL is present, the lower is the surface concentration of PAF in the LDL and the less hGX sPLA2 will have PAF bound to its active site and thus the rate of PAF hydrolysis will go down. In addition one could expect that increasing the amount of LDL should increase the LDLassociated PC concentration and create an increased competition between radiolabelled PAF and PC for hydrolysis by hGX sPLA2. The fact that the amount of LDL bound-PC hydrolyzed by hGX sPLA2 is independent of the amount of LDL added to the assay indicates that all of the hGX-sPLA2 is bound to the LDL particles at all concentrations of LDL studied. The bound enzyme encounters the same surface concentration of PC in LDL as the amount of LDL is increased. Inactivation of the endogenous PAF-AH activity was a prerequisite step in order to measure hydrolysis of PAF incorporated onto LDL particles. Since PAF-AH activity is dramatically decreased during LDL oxidation [42–44], it is possible that circulating or lipoprotein-associated hGX sPLA2 hydrolyzes the newly synthesized PAF during LDL oxidation, thereby eliminating its biological activity. It would be interesting to know whether hGX sPLA2 is able to hydrolyze PAF-like molecules and/or other short chain oxidized phospholipids which are produced upon inflammation and atherosclerosis. In addition to LDL, hGX sPLA2 was able to hydrolyze PAF co-dispersed in PC as mixed vesicles. When data is normalized for the different concentrations of PAF and PC present in the vesicles, it was found that hGX sPLA2 displays an intrinsic preference for PC over PAF by 6.8-fold (Table 1).
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In contrast, the other sPLA2s studied did not yield detectable PAF hydrolysis in this assay, showing that the PC-to-PAF preference for these other sPLA2s is much larger (> 50-fold) than for hGX sPLA2. As plasma PAF-AH deficient individuals do not have PAFrelated clinical symptoms, it is likely that other extracellular and/or intracellular enzymes with PAF degrading activity compensate for the deficient plasma PAF-AH. Two other enzymes were described to exhibit PAF-hydrolyzing activity, namely the cholesterol esterification enzyme Lecithin:cholesterol acyltransferase [45], which inactivates PAF via transacetylation reaction and the HDL-associated esterase, paraoxonase-1 [46]. More recently, it was however shown that the PAF hydrolyzing activity of paraoxonase-1 was due to PAF-AH contamination during its purification, as serum from a donor with an inactivating mutation in the PAF-AH gene (279Phe), did not hydrolyze PAF, and yet displayed a full paraoxonase-1 activity [47]. In summary we report here for the first time that recombinant hGX sPLA2 efficiently hydrolyzes PAF in vitro, in the presence or absence of lipoproteins and in mixed PC:PAF co-vesicles. The physiological meaning of this activity remains unknown, but points to a potential role for hGX sPLA2 not only in producing potent lipid mediators [23,48,49] but also in regulating the degradation of the potent lipid mediator PAF in various inflammatory processes including atherosclerosis. Acknowledgments This study was supported by the Institut National de la Santé et de la Recherche Médicale and a scholarship from the Fondation pour la Recherche Médicale (FRM: ACE20050703776) to S-A K. S.G is a recipient of a doctoral fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche. E.N is a Director of Research at the Centre National de la Recherche Scientifique. The study was also supported in part by the Centre National de Recherche Scientifique and the Association pour la Recherche sur le Cancer (ARC) to G.L and the National Institute of Health Grant HL36235 to M.G. References [1] E. Ninio, Phospholipid mediators in the vessel wall: involvement in atherosclerosis, Curr. Opin. Clin. Nutr. Metab. Care 8 (2005) 123–131. [2] R.S. Farr, C.P. Cox, M.L. Wardlow, R. Jorgensen, Preliminary studies of an acid-labile factor (ALF) in human sera that inactivates platelet-activating factor (PAF), Clin. Immunol. Immunopathol. 15 (1980) 318–330. [3] M.L. Blank, T. Lee, V. Fitzgerald, F. Snyder, A specific acetylhydrolase for 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a hypotensive and plateletactivating lipid), J. Biol. Chem. 256 (1981) 175–178. [4] M.J. Caslake, C.J. Packard, Lipoprotein-associated phospholipase A2 as a biomarker for coronary disease and stroke, Nat. Clin. Pract. Cardiovasc. Med. 2 (2005) 529–535. [5] D.M. Stafforini, K. Satoh, D.L. Atkinson, L.W. Tjoelker, C. Eberhardt, H. Yoshida, T. Imaizumi, S. Takamatsu, G.A. Zimmerman, T.M. McIntyre, P.W. Gray, S.M. Prescott, Platelet-activating factor acetylhydrolase deficiency. A missense mutation near the active site of an antiinflammatory phospholipase, J. Clin. Invest. 97 (1996) 2784–2791. [6] M. Ishihara, T. Iwasaki, M. Nagano, J. Ishii, M. Takano, T. Kujiraoka, M. Tsuji, H. Hattori, M. Emi, Functional impairment of two novel mutations
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The proinflammatory mediator Platelet Activating Factor is an effective substrate for human group X secreted phospholipase A2 Sarah Gora a , Gerard Lambeau b , James G. Bollinger c , Michael Gelb c , Ewa Ninio a , Sonia-Athina Karabina a,⁎ a
INSERM U525, Université Pierre et Marie Curie-Paris6, Faculté de Médecine Pierre et Marie Curie, Paris, France b IPMC, CNRSUMR 6097, Sophia Antipolis, Valbonne, France c Department of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195, USA Received 12 April 2006; received in revised form 19 June 2006; accepted 2 August 2006 Available online 5 August 2006
Abstract Platelet Activating Factor (PAF) is a potent mediator of inflammation whose biological activity depends on the acetyl group esterified at the sn2 position of the molecule. PAF-acetylhydrolase (PAF-AH), a secreted calcium-independent phospholipase A2, is known to inactivate PAF by formation of lyso-PAF and acetate. However, PAF-AH deficient patients are not susceptible to the biological effects of inhaled PAF in airway inflammation, suggesting that other enzymes may regulate extracellular levels of PAF. We therefore examined the hydrolytic activity of the recently described human group X secreted phospholipase A2 (hGX sPLA2) towards PAF. Among different sPLA2s, hGX sPLA2 has the highest affinity towards phosphatidylcholine (PC), the major phospholipid of cellular membranes and plasma lipoproteins. Our results show that unlike group IIA, group V, and the pancreatic group IB sPLA2, recombinant hGX sPLA2 can efficiently hydrolyze PAF. The hydrolysis of PAF by hGX sPLA2 rises abruptly when the concentration of PAF passes through its critical micelle concentration suggesting that the enzyme undergoes interfacial binding and activation to PAF. In conclusion, our study shows that hGX sPLA2 may be a novel player in PAF regulation during inflammatory processes. © 2006 Elsevier B.V. All rights reserved. Keywords: PAF; Inflammation; PAF-AH; Phospholipase A2 group X; Lp-PLA2; LDL
1. Introduction Platelet-Activating Factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent lipid mediator which is produced by various inflammatory cells and is involved in a number of inflammatory and allergic processes including atherosclerosis (reviewed in [1]). Its biological activity depends on the acetyl group esterified at the sn-2 position of the glycerol backbone. Inactivation of PAF to form the biologically inactive metabolite lyso-PAF (1-O-alkyl-sn-glycero-3-phosphocholine) and acetate is catalyzed by PAF-acetylhydrolase (PAF-AH), a calcium-independent phospholipase A2 (PLA2) [2,3] also known as lipoprotein associated PLA2 (Lp-PLA2) or GVIIA phospholipase A2 (reviewed in [4]). A loss of function mutation (Val279Phe) has been described for plasma PAF-AH [5] that ⁎ Corresponding author. Tel.: +33 1 40 77 81 39; fax: +33 1 40 77 97 68. E-mail address: [email protected] (S.-A. Karabina). 1388-1981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2006.08.004
results in the inability of the protein to be secreted by mammalian cells [6]. In addition the recombinant bacterial protein is inactive [5]. The mutation Val279Phe occurs in 30% (both homozygotes and heterozygotes) of Japanese [5] and other Eastern populations [7], leading to PAF-AH deficiency and absence of hydrolysis of exogenously added substrate in homozygotes. Although the initial genetic studies showed an association of 279Phe allele with an increased risk for cardiovascular disease, stroke [8] and myocardial infraction [9], as compared to 279Val carriers, a more recent larger study did not confirm such an association [10]. Furthermore, as PAF-AH deficient patients are not susceptible to the biological effects of inhaled PAF in airway inflammation [11], the central role of PAF-AH as a major player in PAF hydrolysis in vivo is being challenged. Secreted PLA2s (sPLA2s) form a group comprising up to 12 mammalian enzymes that have distinct primary structures, specific tissue distributions and different enzymatic properties, suggesting that each enzyme has a distinct physiological role.
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Among sPLA2s, the human group X sPLA2 (hGX sPLA2) has been detected in thymus, spleen and leucocytes, indicating a possible role of this sPLA2 in immunity and/or inflammation [12]. Furthermore, in marked contrast to other Ca2+-dependent sPLA2, hGX sPLA2 is by far the most active enzyme towards phosphatidylcholine (PC), the major phospholipid of mammalian cell membranes and plasma low and high density lipoproteins (LDL and HDL respectively) [13–18]. On the basis of the structural similarities between PAF and PC and the results of clinical trials using recombinant human PAF-AH that have raised doubts about the central role of this enzyme in hydrolyzing physiological concentrations of PAF [19,20], we examined whether hGX sPLA2 could hydrolyze PAF in vitro. Here we demonstrate that unlike hGV, hGIIA sPLA2s and porcine GIB, hGX sPLA2 can efficiently hydrolyze PAF when incorporated into large unilamellar PC phospholipid vesicles and when present in PC-rich lipoproteins indicating that hGX sPLA2 may be a novel player in PAF regulation during inflammatory processes. 2. Materials and methods 2.1. Reagents PAF (1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine), obtained as a powder from Sigma Aldrich (Saint Louis, MO), was dissolved at a final concentration of 50 mM in ethanol. This solution was mixed with 1-O-hexadecyl-2[3H-acetyl]-sn-glycero-3-phosphocholine (21.5 Ci/mmol; DuPont-New England Nuclear) in various proportions, dried under a stream of nitrogen, and dissolved in a solution containing fatty acid-free BSA/saline (0.25%) to obtain [3H-acetyl] PAF final concentrations from 2.5 to 1000 μM solutions. 1-palmitoyl-2arachidonoyl-sn-glycero-3-phosphocholine was from Sigma Aldrich (St Louis, MO) and was kept as a 10 mg/ml solution in 0.25% fatty acid-free BSA saline. Recombinant PAF-AH was prepared in our laboratory as previously described [21,22]. Porcine group IB sPLA2 was obtained from Sigma. Recombinant hGIIA, hGV and wild type and H48Q mutant of hGX sPLA2 were prepared in E. coli as described [13,23]. The hGX-sPLA2 inhibitor, LY 329722 (3-(3-Aminooxalyl-1-benzyl-2-ethyl-6-methyl-1H-indol-4-yl)-propionic acid), was synthesized as described [24].
2.2. hGX sPLA2 activity towards [3H-acetyl]PAF The activity of hGX sPLA2 (50 nM) was measured using a modified PAF-AH assay [25] in Tris–HCl buffer pH 8.0, supplemented with 1 mM CaCl2, containing 50 μM [3H-acetyl]PAF as substrate (prepared as described above), except for the dose–response studies which were performed with final concentrations of [3H-acetyl]PAF ranging from 0.25 to 100 μM. In competition experiments, various concentrations of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine were added to the reaction mixture. Several experiments were performed in the presence of 1–10 mM CaCl2. In selected experiments either 10 μM LY 329722, a potent hGX sPLA2 inhibitor, was added to the reaction mixture or hGX sPLA2 was substituted by its catalytically-inactive mutant H48Q at a concentration of 50 nM. Comparative activity studies were performed using similar amounts of hGX, hGV, hGIIA, porcine pancreas GIB, and human recombinant PAF-AH in the presence of 50 μM [3H-acetyl]PAF final concentration. All enzymatic reactions were performed at 37 °C for 10 min, except for kinetic experiments. The reactions were stopped in an ice bath for 10 min using an excess of BSA (final concentration, 16.6 mg/mL), to bind unreacted [3H-acetyl]PAF, prior to addition of trichloroacetic acid. The samples were then centrifuged for 15 min at 1800 × g, and the [3H]acetate recovered in the aqueous phase was measured by liquid scintillation counting in Optiphase Hi-Safe 3. The results, after correction for the blank (150–200 dpm, which did not exceed 10% of the
experimental values), were expressed as nmol of PAF degraded per min per microgram of protein.
2.3. LDL isolation and treatment with hGX sPLA2 LDL was isolated from frozen plasma by density gradient ultracentrifugation, as described previously [26]. The protein content of LDL was determined by the bicinchoninic acid (BCA) method (Pierce). Freshly prepared, filtered (0.45 μm) sterile LDL, 1 mg protein/mL in buffer containing 1 mM CaCl2, 12.5 mM Tris–HCl (pH 8.0), 0.25 M NaCl and 0.0125% BSA was first incubated for 30 min at 37 °C in the presence of the serine esterase inhibitor Pefabloc (0.1 mM) in order to inactivate the endogenous PAF-AH activity [27], prior to being dialyzed for 24 h against PBS. LDL (with inactivated PAF-AH) was mixed with 50 μM final concentration [3H acetyl]PAF and subsequently incubated with 50 nM hGX, hGV, hGIIA, and porcine group IB sPLA2s at 37 °C for 10 and 60 min. The reaction was stopped on ice with an excess of BSA and the amount of [3H] acetate released was measured as described above. In parallel, the amount of free fatty acids liberated during the treatment of LDL by the different sPLA2s was quantified using the NEFA C kit (Wako Chemicals, GmbH) following the manufacturer's instructions.
2.4. Hydrolysis of PC vesicles containing PAF To study hydrolysis of phosphatidylcholine vesicles containing PAF, we prepared large unilamellar vesicles (0.1 μm) of 1-palmitoyl-2-oleoyl-snglycerol-3-phosphocholine (Avanti Polar Lipids Inc., Alabaster, AL) containing 10% 1-hexadecyl-2-acetyl-sn-glycerol-3-phosphocholine (Avanti Polar Lipids Inc.) by extrusion as described [13]. Reaction mixtures using rat liver fatty acid binding protein were carried out with Hank's balanced salt solution containing calcium and magnesium and 30 μM total phospholipid (as vesicles) as described [13]. Enzyme (30 ng human group V sPLA2 or 40 ng human group X sPLA2) was added to initiate the reaction. The reaction mixture was quenched by adding EGTA to give 2 mM when ∼ 10% of the 1-palmitoyl-2-oleoyl-sn-glycerol-3phosphocholine was hydrolyzed (the assay was calibrated with oleic acid as described) [13]. Samples were processed by solid-phase extraction and analyzed by combined liquid chromatography electrospray ionization mass spectrometry as described [13]. A blank sample was prepared and processed as above except that the sPLA2 was omitted. Prior to processing, 5 nmol of the internal standard, 1-palmitoyl-sn-glycero-3-phosphocholine containing a per-deuterated fatty acyl chain was added. To obtain the relative ionization efficiency of the two reaction products, equal amounts of 1-palmitoyl-sn-glycero-3-phosphocholine and 1hexadecyl-sn-glycero-3-phosphocholine were co-injected onto the liquid chromatography column. The absolute amount of the reaction products was obtained from the relative peak areas (parent ion trace) of enzyme-produced lyso-phospholipid to internal standard with correction for the relative ionization efficiency. The amount of product formed in the time needed to hydrolyze ∼10% of the vesicles together with the amount of added sPLA2 were used to obtain the specific activities reported in Results.
2.5. Statistical analysis Results are expressed as mean ± SE of 3–9 experiments in duplicate or triplicate. Mean values were compared by Student's t test, with significance defined at a value of P ≤ 0.05.
3. Results 3.1. Recombinant hGX sPLA2 efficiently catalyses the hydrolysis of [3H-acetyl]PAF We first compared the enzymatic activities of different purified or recombinant sPLA2s with that of PAF-AH towards [3 H-acetyl]PAF. Recombinant hGX sPLA2, hGV sPLA2, hGIIA sPLA2, purified porcine group IB and human PAFAH were incubated with [3H-acetyl]PAF for 10 min at 37 °C.
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Fig. 1. [3H-acetyl]PAF hydrolysis by recombinant PLA2s. hGX sPLA2, human recombinant PAF-AH, hGV, porcine GIB, hGIIA and the active site mutant of hGX sPLA2, H48Q were incubated at 37 °C for 10 min with [3H-acetyl]PAF. The amount of the [3H]acetate liberated was measured as described in Materials and methods. Results are expressed as nmol of [3H-acetyl]PAF hydrolyzed per minute per μg of each recombinant protein and represent the mean ± SE of three separate experiments performed in duplicate. *P < 0.05 when compared to PAFAH or hGX sPLA2.
In agreement with former studies [22], recombinant PAF-AH efficiently hydrolyzed [3H-acetyl]PAF (Fig. 1), either in the presence or absence of Ca2+ (data not shown). At similar enzyme concentrations (50 nM), hGX sPLA2 showed almost 50% of the PAF-AH hydrolytic activity. In contrast to hGX sPLA2, hGV, hGIIA sPLA2s and porcine group IB, showed very low if any catalytic activity towards [3H-acetyl]PAF (Fig. 1) even after longer incubation periods (data not shown). The activity of hGX sPLA2 on [3H-acetyl]PAF hydrolysis was not influenced by Ca2+ concentrations above 1 mM (data not shown) as 1 mM Ca2+ is sufficient to saturate the enzyme [28]. However, in the presence of 5 mM EDTA, the enzyme activity was completely abolished (95% ± 2% inhibition), a result consistent with the strict Ca2+ requirement of this sPLA2 [12]. The H48Q catalytic site mutant of hGX sPLA2, which is enzymatically-inactive when assayed on PG vesicles or radiolabelled E. coli membranes, did not degrade [3Hacetyl]PAF (Fig. 1). Finally, no enzymatic activity (99% ± 2% of inhibition) was detected in the presence of 10 μM LY 329722, a potent hGX sPLA2 inhibitor [24].
Fig. 2. Concentration dependent hydrolysis of [3H-acetyl]PAF by hGX sPLA2. 50 nM of hGX sPLA2 were incubated with 0.25–100 μM final concentration of [3H-acetyl]PAF for 10 min at 37 °C. The [3H]acetate liberated was measured as described in Materials and methods. Results are expressed as nmol of [3H-acetyl] PAF hydrolyzed per minute per μg of hGX sPLA2 and represent the mean ± SE of 4 separate experiments performed in duplicate or triplicate.
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Fig. 3. Time dependent hydrolysis of [3H-acetyl]PAF by hGX sPLA2. 50 nM of hGX sPLA2 were incubated with 50 μM final concentration of [3H-acetyl]PAF for various times. The amount of the [3H]acetate liberated was measured as described in Materials and methods. Results are expressed as nmol of [3Hacetyl]PAF hydrolyzed per μg of hGX sPLA2 and represent the mean ± SE of 3 separate experiments performed in duplicate or triplicate.
3.2. Interfacial property of hGX sPLA2 on PAF The enzymatic property of hGX sPLA2 was evaluated at 37 °C for 10 min with [3H-acetyl]PAF at concentrations ranging from 0.25 to 100 μM. Consistent with its interfacial binding properties [14], hGX sPLA2 (50 nM) exhibited a maximal enzymatic activity when PAF concentration rose above its critical micellar concentration (∼5 μM) (Fig. 2). Time-course experiments showed that about 40% of [3H-acetyl]PAF was hydrolyzed by hGX sPLA2 in the first 10 min of incubation (Fig. 3). 3.3. Effect of PC on hGX sPLA2 PAF hydrolysis Since arachidonoyl-PC is a very good substrate for hGX sPLA2 [18], we next studied the [3H-acetyl]PAF hydrolysis in the presence of different concentrations (0 to 1278 μM) of this competitor. As shown in Fig. 4 the presence of arachidonoyl-PC in the assay mixture containing hGX sPLA2 decreased the hydrolysis of [3H-acetyl]PAF in a concentration dependent manner.
Fig. 4. Hydrolysis of [3H-acetyl]PAF in the presence of arachidonoyl-PC. Increasing quantities of arachidonoyl PC were added to the assay mixture containing 50 μM [3H-acetyl]PAF. Subsequently 50 nM of hGX sPLA2 was added and the incubation took place for 10 min at 37 °C. The amount of the [3H] acetate liberated was measured as described in Materials and methods. Results are expressed as nmol of hydrolyzed [3H-acetyl]PAF per minute per μg of hGX sPLA2 and represent the mean ± SE of 3 separate experiments performed in duplicate.
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Fig. 5. Hydrolysis of [3H-acetyl]PAF in the presence of LDL treated with Pefabloc. (A) 0.1, 0.4, and 0.8 mg/mL LDL protein were incubated in the presence of 50 nM hGX sPLA2 for 10 and 60 min at 37 °C with 50 μM [3H-acetyl]PAF. The amount of the [3H]acetate liberated was measured as described in Materials and methods. Results are expressed as nmol of hydrolyzed [3H-acetyl]PAF per μg of hGX sPLA2 and represent the mean ± SE of 3 separate experiments performed in duplicate. *P < 0.05 when compared to 0.1 mg/mL LDL. (B) 0.1, 0.4, and 0.8 mg/mL LDL protein were incubated in the presence of 50 nM hGX sPLA2 for 10 and 60 min at 37 °C with 50 μM [3H-acetyl]PAF. In control experiments, LDL and [3H-acetyl]PAF were incubated without hGX sPLA2. The amount of fatty acids liberated was quantified by the NEFA C kit. The result is representative of two experiments.
3.4. Hydrolysis of [3H-acetyl]PAF by hGX sPLA2 on LDL As oxidation of LDL generates PAF [29] and a vast array of phospholipids with truncated, short fatty acids at the sn-2 position of PC, which structurally mimic PAF [30,31], we examined whether hGX sPLA2 could hydrolyze [3H-acetyl] PAF when the latter was incorporated into LDL particles. Since purified LDL particles contain an associated-PAFAH activity, we first treated LDL preparations with Pefabloc to inhibit PAF-AH activity [27] prior to addition of [3Hacetyl]PAF and enzymatic assays with different sPLA2s. Various concentrations of LDL (0.1 to 0.8 mg of protein/ml)
were incubated with 50 μM final concentration of [3H-acetyl] PAF in the presence of 50 nM hGX sPLA2. hGX sPLA2 efficiently hydrolyzed [3H-acetyl]PAF even in the presence of LDL associated PC (Fig. 5A); the reaction was, as expected, inhibited in a dose-dependent manner by LDL. The latter observation is presumably due to two factors: the first is that the surface concentration of PAF in LDL will go down as the amount of LDL is increased in the presence of a fixed amount of PAF. This surface dilution effect would lower the rate of PAF hydrolysis by hGX sPLA2 because the enzyme bound to LDL would encounter a lower concentration of PAF substrate. Secondly, PC is hydrolyzed by hGX sPLA2 (as free fatty
Fig. 6. Hydrolysis of [3H-acetyl]PAF by different recombinant sPLA2 in the presence of LDL treated with Pefabloc. 0.1 and 0.4 mg/mL LDL protein were incubated in the presence of 50 nM hGX, hGV, hGIIA, porcine GIB sPLA2s and PAF-AH for 10 at 37 °C with 50 μM [3H-acetyl]PAF. The amount of the [3H]acetate liberated was measured as described in Materials and methods. Results are expressed as nmol of hydrolyzed [3H-acetyl]PAF per mL of sPLA2 and represent the mean ± SE of 3 separate experiments performed in duplicate.
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acids were concomitantly released in the reaction mixture (Fig. 5B)) and so increased amounts of LDL relative to PAF would increase the concentration of competing PC substrate relative to PAF substrate with a corresponding decrease in the rate of enzymatic PAF hydrolysis. As compared to hGX sPLA2, hGV, hGIIA and porcine group IB showed little, if any, activity towards [3H-acetyl]PAF in the presence of LDL (Fig. 6). 3.5. Hydrolysis of PAF by hGX and hGV sPLA2 in mixed PAF: PC co-vesicles Next, the ability of hGX and hGV sPLA2s to hydrolyze large unilamellar vesicles (0.1 μm) of 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) containing 10 mol% PAF (1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) was studied by measuring the amount of each product formed in this competitive substrate analysis [32]. It was expected that most of the PAF would partition into the vesicles since the phospholipid concentration in the assay (30 μM) is well above the critical micelle concentration of PAF (∼ 2 μM) [33]. The relevant equation for this competitive substrate specificity analysis is [32]: vPOPC =vPAF ¼ ðkcat * =KM * ÞPOPC =ðkcat * =KM * ÞPAF ðXPOPC =XPAF Þ here, vPOPC and vPAF are the initial velocities for the hydrolysis of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and PAF, respectively. (kcat * /KM * )POPC and (kcat * /KM * )PAF are the ratio of interfacial turnover number to interfacial Michaelis constant for each of the two substrates, and XPOPC and XPAF are the mole fractions of each substrate in the vesicle. This equation is analogous to the standard steady-state enzyme kinetic equation for the action of an enzyme on two substrates simultaneously (competitive substrate analysis). Since only 10% of the substrate is hydrolyzed, it is reasonable to assume that the ratio of products formed equals the ratio of initial enzymatic reaction velocities. In the present study, (XPOPC/XPAF) = 9, and thus (kcat * /KM * )POPC/(kcat * /KM * )PAF = 6.8 for hGX sPLA2 (Table 1). Thus, it can be said that hGX sPLA2 intrinsically prefers 1palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine as a substrate over PAF by 6.8-fold. For hGV sPLA2, no PAF hydrolysis could be detected, and thus the preference of hGV sPLA2 for PC over PAF is at least 50-fold (Table 1). Table 1 Hydrolysis of mixed PAF:PC co-vesicles by hGX and hGV sPLA2 POPC specific activity μmol/(min mg)
PAF specific activity μmol/(min mg)
(kcat * /KM * )POPC/ (kcat * /KM * )PAF
hGV hGX
0a 0.14
>50 a, b 6.8
9.97 8.56
Large unilamellar vesicles of PC and PAF prepared by extrusion were incubated with 40 ng of hGX and 30 ng of hGV. The reaction mixture was quenched by adding EGTA and samples were processed as described in Materials and methods. a No PAF hydrolysis was observed. The value of 50 is a lower limit based on the estimated sensitivity of detection limit for lyso-PAF. b Calculated according to the equation in the main text.
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4. Discussion Although the role of plasma PAF-AH in hydrolyzing PAF and controlling inflammation has been well established (reviewed in [34,35]), clinical trials showed that administration of recombinant human PAF-AH failed to improve the clinical outcome of patients [19,20,36]. All these investigations have questioned the role of PAF-AH as the sole enzyme responsible for PAF hydrolysis in vivo, highlighting the possibility that other enzymes may regulate extracellular PAF levels [11]. We show here for the first time that hGX sPLA2, which has high affinity for PC [12–14,18], can efficiently hydrolyze PAF in vitro or ex-vivo on plasma lipoproteins. Interestingly, hGIIA sPLA2 which is highly expressed during inflammation [37] and reviewed in [38] does not hydrolyze PAF as shown in Fig. 1 probably due to its poor binding to PC-rich membranes [14]. The same observation was made for the pancreatic PLA2 and for hGV sPLA2, which were shown to be expressed in certain human pathological conditions [39–41]. Even after prolonged incubation with labelled PAF, both hGIIA and porcine GIB had only minor hydrolytic effect on this substrate. Additionally, as PAF accumulates in oxidized LDL [29], we show here that hGX sPLA2 can also efficiently hydrolyze radiolabelled PAF when it is incorporated into LDL, in a range of concentrations corresponding to either normolipidemic (100 μg/ml) or hypercholesterolemic subjects (800 μg/mL). We observed that hGX sPLA2 was more efficient at hydrolyzing PAF when LDL concentrations were in the normolipidemic range. The more LDL is present, the lower is the surface concentration of PAF in the LDL and the less hGX sPLA2 will have PAF bound to its active site and thus the rate of PAF hydrolysis will go down. In addition one could expect that increasing the amount of LDL should increase the LDLassociated PC concentration and create an increased competition between radiolabelled PAF and PC for hydrolysis by hGX sPLA2. The fact that the amount of LDL bound-PC hydrolyzed by hGX sPLA2 is independent of the amount of LDL added to the assay indicates that all of the hGX-sPLA2 is bound to the LDL particles at all concentrations of LDL studied. The bound enzyme encounters the same surface concentration of PC in LDL as the amount of LDL is increased. Inactivation of the endogenous PAF-AH activity was a prerequisite step in order to measure hydrolysis of PAF incorporated onto LDL particles. Since PAF-AH activity is dramatically decreased during LDL oxidation [42–44], it is possible that circulating or lipoprotein-associated hGX sPLA2 hydrolyzes the newly synthesized PAF during LDL oxidation, thereby eliminating its biological activity. It would be interesting to know whether hGX sPLA2 is able to hydrolyze PAF-like molecules and/or other short chain oxidized phospholipids which are produced upon inflammation and atherosclerosis. In addition to LDL, hGX sPLA2 was able to hydrolyze PAF co-dispersed in PC as mixed vesicles. When data is normalized for the different concentrations of PAF and PC present in the vesicles, it was found that hGX sPLA2 displays an intrinsic preference for PC over PAF by 6.8-fold (Table 1).
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In contrast, the other sPLA2s studied did not yield detectable PAF hydrolysis in this assay, showing that the PC-to-PAF preference for these other sPLA2s is much larger (> 50-fold) than for hGX sPLA2. As plasma PAF-AH deficient individuals do not have PAFrelated clinical symptoms, it is likely that other extracellular and/or intracellular enzymes with PAF degrading activity compensate for the deficient plasma PAF-AH. Two other enzymes were described to exhibit PAF-hydrolyzing activity, namely the cholesterol esterification enzyme Lecithin:cholesterol acyltransferase [45], which inactivates PAF via transacetylation reaction and the HDL-associated esterase, paraoxonase-1 [46]. More recently, it was however shown that the PAF hydrolyzing activity of paraoxonase-1 was due to PAF-AH contamination during its purification, as serum from a donor with an inactivating mutation in the PAF-AH gene (279Phe), did not hydrolyze PAF, and yet displayed a full paraoxonase-1 activity [47]. In summary we report here for the first time that recombinant hGX sPLA2 efficiently hydrolyzes PAF in vitro, in the presence or absence of lipoproteins and in mixed PC:PAF co-vesicles. The physiological meaning of this activity remains unknown, but points to a potential role for hGX sPLA2 not only in producing potent lipid mediators [23,48,49] but also in regulating the degradation of the potent lipid mediator PAF in various inflammatory processes including atherosclerosis. Acknowledgments This study was supported by the Institut National de la Santé et de la Recherche Médicale and a scholarship from the Fondation pour la Recherche Médicale (FRM: ACE20050703776) to S-A K. S.G is a recipient of a doctoral fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche. E.N is a Director of Research at the Centre National de la Recherche Scientifique. The study was also supported in part by the Centre National de Recherche Scientifique and the Association pour la Recherche sur le Cancer (ARC) to G.L and the National Institute of Health Grant HL36235 to M.G. References [1] E. Ninio, Phospholipid mediators in the vessel wall: involvement in atherosclerosis, Curr. Opin. Clin. Nutr. Metab. Care 8 (2005) 123–131. [2] R.S. Farr, C.P. Cox, M.L. Wardlow, R. Jorgensen, Preliminary studies of an acid-labile factor (ALF) in human sera that inactivates platelet-activating factor (PAF), Clin. Immunol. Immunopathol. 15 (1980) 318–330. [3] M.L. Blank, T. Lee, V. Fitzgerald, F. Snyder, A specific acetylhydrolase for 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a hypotensive and plateletactivating lipid), J. Biol. Chem. 256 (1981) 175–178. [4] M.J. Caslake, C.J. Packard, Lipoprotein-associated phospholipase A2 as a biomarker for coronary disease and stroke, Nat. Clin. Pract. Cardiovasc. Med. 2 (2005) 529–535. [5] D.M. Stafforini, K. Satoh, D.L. Atkinson, L.W. Tjoelker, C. Eberhardt, H. Yoshida, T. Imaizumi, S. Takamatsu, G.A. Zimmerman, T.M. McIntyre, P.W. Gray, S.M. Prescott, Platelet-activating factor acetylhydrolase deficiency. A missense mutation near the active site of an antiinflammatory phospholipase, J. Clin. Invest. 97 (1996) 2784–2791. [6] M. Ishihara, T. Iwasaki, M. Nagano, J. Ishii, M. Takano, T. Kujiraoka, M. Tsuji, H. Hattori, M. Emi, Functional impairment of two novel mutations
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