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Journal of Lipid Research, Vol. 41, 1222-1230, August 2000
Copyright © 2000 by Lipid Research, Inc.

Original Article

Purification, characterization, and cDNA sequencing of cytosolic phospholipase A2 from equine neutrophils1

Pontus K. A. Larsson Forsella, Åsa Lindberga, Sture Karlssona, Jan Åke Lindgrena, and Hans-Erik Claessona
a Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden

Correspondence to: Pontus K. A. Larsson Forsell


  ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

It has been demonstrated that equine neutrophils, but not eosinophils, require exogenous arachidonic acid for calcium ionophore A23187-induced leukotriene synthesis. Because cytosolic phospholipase A2 (cPLA2) plays an essential role in leukotriene formation in leukocytes, we investigated the presence of a functional cPLA2 in equine neutrophils. To determine whether cPLA2 from neutrophils was catalytically active, we purified the enzyme >6,500 fold with 3% recovery from equine neutrophils. The full-length cDNA sequence encoded a 749-amino acid protein. The deduced amino acid sequence demonstrated 95% identity with human and mouse cPLA2, as well as 83 and 73% identity with chicken and zebra fish cPLA2 protein, respectively. The equine cPLA2 possessed some properties that distinguished the equine enzyme from the human enzyme. First, the enzyme activity of the equine cPLA2 was differently influenced by cations as compared with the human cPLA2. Second, the equine neutrophil cPLA2 migrated as an approximately 105-kDa protein, in comparison with human cPLA2 which migrated as a 110-kDa protein. A difference between equine neutrophils and eosinophils in the degree of phosphorylation of the cPLA2 protein was observed. Thus, the cPLA2 protein from eosinophils was constitutively phosphorylated, while the cPLA2 protein from neutrophils was unphosphorylated.

In summary, these results demonstrate that equine neutrophils indeed express an active cPLA2 protein but that there is a difference in the degree of phosphorylation of the cPLA2 protein between equine neutrophils and eosinophils. This difference might explain the difference between the two cell types in the capacity to produce leukotrienes from endogenous substrate.—Larsson Forsell, P. K. A., Å. Lindberg, S. Karlsson, J. Å. Lindgren, and H-E. Claesson. Purification, characterization, and cDNA sequencing of cytosolic phospholipase A2 from equine neutrophils. J. Lipid Res. 2000. 41: 1222;–1230.

Supplementary key words: phospholipase A2, arachidonic acid, leukotriene, equine, neutrophils, eosinophils, phosphorylation


  INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

The phospholipase A2 (PLA2) family of enzymes hydrolyzes the fatty acid ester bond at the sn-2 position in phospholipids (1). PLA2 enzymes play an important role in the release of lysophospholipids and the eicosanoid precursor arachidonic acid. Two major types of intracellular PLA2 have been recognized, that is, calcium independent and calcium dependent. A group VI calcium-independent PLA2 (iPLA2) was originally purified from P388D1 cells and was later cloned from several species (2) (3) (4) (5) (6). In addition, a group IV cytosolic calcium-dependent PLA2 (cPLA2) was originally discovered and characterized biochemically from the macrophage cell line RAW 264.7 (7). Subsequently, it was isolated, cloned, and sequenced from the human monocytic cell line U937 (8) (9) (10). The cDNA sequence of cPLA2 has also been determined from rat (11), mouse (9), chicken, and zebra fish (12). The cPLA2 enzyme is expressed in a variety of cells including human neutrophils and B lymphoblasts (13) (14). Hormonally induced phosphorylation of cPLA2 occurs in several tissues and cell types and this modification appears to increase the activity of the enzyme in vitro and to decrease the electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (15) (16) (17) (18). Several investigators have tried to define the role of cPLA2 in leukotriene formation and gene-targeting experiments indicated that cPLA2 is essential for leukotriene synthesis in mice (19) (20).

Arachidonic acid plays a crucial role as a precursor for the synthesis of eicosanoid, such as prostaglandins and leukotrienes. Leukotrienes are mediators of inflammation and are thought to be involved in human diseases such as asthma (21). Leukotrienes have also been suggested to be of importance in several pathophysiological conditions of the horse such as inflammation (22) and chronic obstructive pulmonary disease (23) (24).

Equine eosinophils and neutrophils display differential capacity to produce leukotrienes from endogenous substrate after calcium ionophore A23187 stimulation because neutrophils are dependent on exogenous arachidonic acid for leukotriene formation (25). Therefore, we explored the presence of a functional cPLA2 enzyme in equine neutrophils. This article demonstrates the purification, characterization, and cDNA sequence of cPLA2 from these cells. Furthermore, we also present data suggesting a difference in enzyme phosphorylation in equine neutrophils as compared with eosinophils from the same species.


  MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES

Materials
High-performance liquid chromatography (HPLC) solvents were purchased from Rathburn Chemicals (Walkerburn, UK) and arachidonic acid from Biomol (Plymouth Meeting, PA). MonoflowTM 2 scintillation liquid was obtained from National Diagnostics (Manville, NJ). 1-palmitoyl 2-[1-14C]arachidonyl phosphatidylcholine (PC), (57 mCi/mmol), 1-palmitoyl 2-[1-14C]palmitoyl PC (55.5 mCi/mmol), 1-palmitoyl 2-[1-14C]arachidonyl phosphatidylethanolamine (PE) (57 mCi/mmol), 1-palmitoyl 2-[1-14C] palmitoyl phosphatidic acid (PA) (144 mCi/mmol), and [1-14C] arachidonic acid (57 mCi/mmol) were obtained from New England Nuclear (Boston, MA); 1-stearoyl 2-[1-14C]arachidonyl PC (56 mCi/mmol) and 1-stearoyl 2-[1-14C]arachidonyl diacylglycerol (DAG) (53 mCi/mmol) were purchased from Amersham International (Amersham, UK). The FPLC SystemTM and all protein purification columns were from Pharmacia Biotech (Uppsala, Sweden) if not stated otherwise. The rabbit anti-human cPLA2 polyclonal antibody (amino acids 731;–749 as immunogen) and the corresponding synthetic peptide were kindly provided by N. Tremblay (Merck Frosst, Pointe-Claire-Dorval, PC, Canada).

Preparation of equine neutrophils
Horse blood from healthy, adult Swedish Warmblood horses was collected by jugular vein puncture into EDTA-containing Vacutainer blood collection tubes (Becton Dickinson, Rutherford, NJ), or into blood bags (Teruflex B-450; Terumo, Tokyo, Japan) containing EDTA (final concentration, 4.1 mM). Equine neutrophils suspensions were prepared as follows: after centrifugation of the equine blood at 140 g for 15 min, the platelet-rich plasma was removed and the neutrophils were isolated from the remaining lower phase by dextran sedimentation, hypotonic ammonium chloride lysis, and LymphoprepTM centrifugation at 400 g for 40 min. The isolated cells contained >96% neutrophils, with 1;–4% contaminating eosinophils as judged by light microscopy. Highly enriched preparations of equine neutrophils and eosinophils were obtained as described (25). These preparations had less than 1% contaminating cells (data not shown). Cells were resuspended, to a final concentration of 5 x 107 cells/mL, in homogenization buffer (20 mM Tris-HCl [pH 7.5], 2 mM EGTA, 1 mM EDTA, 2 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], soybean trypsin inhibitor [20 µg/mL], bacitracin [0.1 mg/mL], 0.5 mM benzamidine, 0.02 mM leupeptin, and 10% glycerol).

PLA2 assay
Cells were homogenized twice by nitrogen cavitation at 800 psi for 10 min at 4°C. The supernatant was obtained after centrifugation at 100,000 g for 60 min. PLA2 activity was assayed with 1-palmitoyl 2-[1-14C]arachidonyl phosphatidylcholine (PC) as a standard substrate. Phospholipids were dried under nitrogen and resuspended in assay buffer (final concentration, 80 mM glycine [pH 9.0], 5 mM CaCl2, 5 mM DTT, albumin [1 mg/mL], and 10% glycerol) to yield a final concentration of 2 µM PC. Subsequently this preparation was sonicated in a water bath for 10 min at 4°C. The reaction was initiated by adding either cell homogenate, 100,000 g supernatant, or purified PLA2 and the reaction mixture was incubated for the indicated times at 37°C in a shaking water bath. The reaction was terminated by the addition of 2 volumes of methanol containing 0.5% acetic acid and 40 µM stearic acid, as carrier, followed by vigorous vortexing. Precipitated proteins and cell debris were removed by centrifugation at 800 g for 10 min. Thereafter, the supernatants were applied to a octadecyl reversed-phase column (Chromabond, C18, 100 mg; Macherey-Nagel, Duren, Germany). After washing, the fatty acids were eluted with 500 µL of methanol. The samples were analyzed in a reversed-phase (RP) HPLC system equipped with a Radial-Pak cartridge (5 x 100 mm) packed with 4-µm Novapak C18 material, guarded by a Novapak C18 column (Waters, Milford, MA). The mobile phase was methanol;–water;–trifluoroacetic acid 85:15:0.007 (v/v/v) and the flow rate was 1.2 mL/min. Analysis was performed by comparing retention times with authentic standards. Radioactivity was detected with a ß-RAM HPLC flowthrough monitoring system (Inus System, Tampa, FL) coupled online to a Waters 991 diode array spectrophotometer. Quantitative determination was performed by peak area integration.

Purification of cytosolic PLA2
Liquid chromatography was performed on a Pharmacia FPLC SystemTM. All buffers were made fresh daily, passed through a 0.22-µm pore size filter, and degassed before use. The 100,000 g supernatant was applied to a 10-mL heparin-Sepharose column equilibrated with buffer A (20 mM Tris-HCl [pH 7.5], 1 mM EGTA, 1 mM EDTA, 0.5 mM DTT). Proteins were eluted by a two-step gradient from 0 to 0.5 M NaCl and from 0.5 to 1 M NaCl, respectively. Fractions of 3 mL were collected and 30-µL aliquots of these were assayed for PLA2 activity. The flowthrough fractions from the heparin-Sepharose step were pooled and applied to a Resource-Q (6-mL) column preequilibrated with buffer A. After washing, a gradient was developed from 0.15 to 0.55 M NaCl in buffer A at a flow rate of 2 mL/min. Fractions of 1.5 mL were collected and 10-µL aliquots of these were then assayed for PLA2 activity. Sodium chloride (1 M final concentration) was added to pooled, active fractions from the Resource-Q column and in turn applied to a phenyl-Superose HR 5/5 column preequilibrated with 1 M NaCl in buffer A. Proteins were eluted in a stepwise gradient at a flow rate of 0.3 mL/min. Fractions of 0.5 mL were collected and 10-µL aliquots were then assayed for PLA2 activity. Fractions with enzyme activity were analyzed further on a Resource-Q (1-mL) column equilibrated with buffer A. After washing, proteins were eluted in a linear gradient from 0.15 to 0.5 M NaCl. Aliquots (20 µL) of resulting 0.5-mL fractions were assayed for PLA2 activity. Active fractions from this purification step were concentrated with a Centricon 10 (Amicon, Danvers, MA) at 4,700 g to a final volume of approximately 250 µL and then applied to a Superdex 200 column equilibrated with buffer A supplemented with 0.15 M NaCl. Proteins were eluted at a flow rate of 0.35 mL/min and 0.4-mL fractions were collected. Aliquots (25 µL) of these fractions were analyzed for PLA2 activity. PLA2 active fractions were further analyzed by SDS-PAGE. Protein concentrations were measured by Coomassie assay as described by the manufacturer (Bio-Rad, Hercules, CA) against bovine serum albumin as the standard protein. Protein content after the last purification step was estimated from the absorbance at 280 nm.

Polyacrylamide gel electrophoresis
SDS-PAGE was performed as described previously (26). Samples were mixed with loading buffer (50 mM Tris [pH 6.8], 10% glycerol, 0.1% SDS, 3 mM 2-mercaptoethanol, and 0.005% bromophenol blue), heated for 3 min at 95°C, and loaded onto a 5;–16% SDS-PAGE (16 x 18 or 8 x 10 cm). After the run, proteins were visualized by silver staining (Bio-Rad silver stain kit), as described by the manufacturer, or subjected to Western blot. Molecular weight markers, SeeBlue and Mark 12, were obtained from Novex (San Diego, CA).

Immunoblotting
Western blots were performed according to Towbin et al. (27). Briefly, after SDS-PAGE the samples were transferred to HybondTM-C nitrocellulose sheets (Amersham, Buckinghamshire, UK). Afterward, membranes were blocked with 1% milk powder and 0.5% bovine albumin in Tris-buffered saline with 0.1% Tween (TBS-T) for 60 min. After blocking, the membranes were incubated overnight at 4°C with a sequence-specific rabbit anti-human cPLA2 polyclonal antibody (1:1,000 dilution). In one set of control experiments the corresponding synthetic peptide was added to the polyclonal antibody before immunodetection. The peptide/antibody ratio exceeded a 100:1 molar ratio in these experiments. The second antibody was anti-rabbit (1:10,000) coupled to horseradish peroxidase. Enhanced chemiluminescence (ECLTM) was used for detection as described by the manufacturer (Amersham).

Isolation of total cellular RNA, reverse transcription, and PCR
Total cellular RNA was isolated with Trizol reagent (Life Technologies, Gaithersburg, MD) according to the instructions of the manufacturer. The amount and purity of isolated total RNA were determined spectrophotometrically by analyzing the absorbance at 260 nm. Isolated total RNA (2 µg) was subjected to reverse transcription in order to obtain cDNA. The reverse transcription mixture contained 2 µg of total RNA, 1x reverse transcriptase buffer (50 mM KCl, 10 mM Tris-HCl [pH 8.3], 2 mM MgCl2, and 0.01% gelatin), 5 mM DTT, 0.5 mM dNTPs (SDS-Promega, Falkenberg, Sweden), 200 ng of oligo(dT)12;–18 (Pharmacia, Uppsala, Sweden), 39 U of RNasin (SDS-Promega), and 500 U of Moloney murine leukemia virus reverse transcriptase (Mo-MuLV RT; United States Biochemical, Cleveland, OH) in a total volume of 40 µL. The reverse transcription reaction was carried out at 37°C for 60 min and then terminated by heating for 5 min at 95°C. To determine whether a successful cDNA was obtained, polymerase chain reaction (PCR) amplification of a 784-bp ß-actin fragment was performed (data not shown). PCR was performed with the following cycle parameters. First cycle: denaturation for 4.0 min at 94°C, annealing for 1.0 min at 65°C, and extension for 1.5 min at 72°C. Subsequent cycles: denaturation for 1.0 min at 94°C, annealing for 1.0 min at 65°C, and extension for 1.5 min at 72°C. Last cycle: denaturation for 1.0 min at 94°C, annealing for 1.0 min at 65°C, and a final extension of 10.0 min at 72°C. Altogether, 33 cycles were carried out for each fragment to be amplified. The following cPLA2 primer pairs were used:

1. 5'-CTCGCTCGCCCACTCTAGTCCTCCGTTCAAGGAAC-3'

5'-CTGGTTCGGCCCATTGCAAACTGCCTCAGCATCAG-3'

2. 5'-CTCGCTCGCCCAGGCTCCACAATGGAGGAAGAAT-3'

5'-CTGGTTCGGCCCAGTTCCTTGAACGGAGGACTAGA-3'

3. 5'-AGCCACAATCCCCTTTTACTT-3'

5'-GCTATCATTACTCACAATATGCT-3'

4. 5'-AGTGCCAAAGAAGACTTTGAAGT-3'

5'-TCTCTTAACTTTCTGTGGCGTGA-3'

The primer pairs used were designed on the basis of the human cPLA2 cDNA sequence. After the PCR, aliquots of the amplified product was analyzed on a 1% low melting point agarose gel. The PCR-derived fragment was excised and extracted with a Qiaex II agarose gel extraction kit (Qiagen, Chatsworth, CA).

Cloning and sequencing of the PCR products
The PCR products were cloned with the PCR-DIRECT cloning system (Clontech, Palo Alto, CA) or with the pCR-Script Amp SK(+) cloning kit (Stratagene, La Jolla, CA). Plasmid DNA was purified and the insert was sequenced with M13 forward and reverse primers as well as internal sequencing primers, using the T7 sequencing kit (Pharmacia). An aliquot (2 µL) of the sequencing reaction was subjected to electrophoresis with a Macrophore sequencing system (Pharmacia LKB Biotechnology, Uppsala, Sweden). After electrophoresis the gels were fixed, dried, and subjected to autoradiography.


  RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES

Purification of cPLA2 from equine neutrophils
Because equine neutrophils are unable to produce leukotrienes from endogenous arachidonic acid after calcium ionophore A23187 stimulation (25), we investigated whether these cells contained active cPLA2 enzyme. When 100,000 g supernatants obtained from neutrophil homogenates were incubated with PC and PE containing 14C-labeled arachidonic acid at the sn-2 position, a phospholipase activity corresponding to 39 pmol mg-1 min-1 was observed. For comparison, 100,000 g supernatants from a corresponding number of U937 cells, possessing a phospholipase activity of 46 pmol mg-1 min-1, were used.

To investigate whether the phospholipase activity observed in the 100,000 g supernatants from equine neutrophils could be ascribed to cPLA2, the PLA2 enzyme was isolated and purified by sequential heparin-Sepharose chromatography, anion-exchange chromatography, hydrophobic interaction chromatography, and a second anion-exchange chromatography followed by size-exclusion chromatography (Superdex 200) ( Table 1). After the last purification step, PLA2 activity declined rapidly, either because of inactivation or aggregation. Aliquots of PLA2 active fractions eluting from the Superdex 200 column were subjected to gel electrophoresis. One major band with the apparent molecular mass of 105 kDa was detected in the PLA2 active fractions by silver staining ( Fig 1A). The intensity of the 105-kDa protein, eluting in fractions 21;–23, correlated with PLA2 activity (Fig 1C). Furthermore, Western blot analysis of the PLA2 active fractions demonstrated correlation between immunodetected proteins, PLA2 activity, and the silver-stained 105-kDa protein (Fig 1B). One contaminating protein was present in all examined fractions. The contaminating protein, with a molecular mass of approximately 45 kDa, is likely to be the equine form of a protein disulfide isomerase, as previously reported by Gronich et al. (28). The identity of this contaminating protein was not further analyzed. In summary, these results demonstrate that active cPLA2 from equine neutrophils has been purified to near homogeneity. This purification scheme resulted in >6,500-fold purification above levels found in total cellular homogenate, with a recovery of 2.9% (Table 1). The apparent increase in total activity after the first chromatographic steps might be due to the removal of either endogenous substrate or inhibiting factors.




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  		Figure 1. Analysis of PLA2 active fractions after gel filtration. (A) Silver staining of PLA2 active fractions after gel electrophoresis. An aliquot of each fraction from Superdex 200 chromatography was subjected to SDS-PAGE on a 12% gel. The fraction numbers are indicated above the lanes. Molecular mass markers are indicated on the left. (B) Western blot analysis of cPLA2. An aliquot of each fraction from the Superdex 200 column was subjected to SDS-PAGE followed by immunoblotting. Recombinant cPLA2 (15 ng) was run in parallel as indicated. Immunoblot analysis was performed with polyclonal antibodies raised against a C-terminal peptide of cPLA2. Arrow on the left indicates the position of the 98-kDa marker. (C) PLA2 activity profile of fractions from the Superdex 200 column. PLA2 activity was determined with 1 µM 1-palmitoyl 2-[14C]arachidonyl PC as substrate. Aliquots from each fraction were incubated for 30 min. The activity represents the activity found in each analyzed aliquot.


 
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  		Table 1. Scheme for purification of cytosolic phospholipase A2 from equine neutrophils

Biochemical characterization of equine neutrophil cPLA2
The substrate specificity of partially purified cPLA2 (Table 1, step V) was investigated with various radiolabeled substrates as outlined in Table 2. The enzyme selectively hydrolyzed phospholipids containing arachidonic acid, as compared with palmitic acid, at the sn-2 position. As shown in Table 2, the PLA2 activity toward PC or PA, containing palmitic acid at the sn-2 position, and 1-stearoyl 2-[1-14C]arachidonoyl DAG was below detection limits in a vesicle-based assay. As shown in Fig 2, the equine neutrophil cPLA2 had an alkaline pH optimum, with maximal enzyme activity at pH 9.0. Furthermore, the cPLA2 active site inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), dose dependently inhibited equine neutrophil cPLA2 activity ( Fig 3). The inhibition displayed an apparent median inhibitory concentration (IC50) of less than 0.1 µM on partially purified equine cPLA2. The enzyme was calcium dependent because no release of arachidonic acid from PC:PE was observed in the presence of EGTA ( Table 3). No other divalent metal cations such as Sr2+, Ba2+, Mn2+, Mg2+, Cd2+, and Zn2+ could replace Ca2+ and restore activity completely, but Sr2+, Ba2+, and Mn2+ were capable of partial restoration (Table 3). For comparison, cPLA2 from U937 cells, purified 380-fold in a similar manner as described for equine neutrophil cPLA2 (steps I;–IV), was analyzed for cation dependency under identical conditions (Table 3). In contrast to equine neutrophil cPLA2, Mn2+ could replace Ca2+ to restore full activity of U937 cPLA2, as reported by others (29) (30). On the other hand, Ba2+ restored more activity in equine neutrophil cPLA2 than in U937 cPLA2 preparations. The other divalent cations were equipotent in restoring the activity of equine neutrophil cPLA2 as compared with U937 cPLA2. Thus, these results might demonstrate a species difference in cationic ability to restore PLA2 activity.



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  		Figure 2. pH dependency of the release of arachidonic acid. Partially purified PLA2 (step V) was added to the assay mixture, containing 80 mM HEPES (pH 6.5;–8.0) or 80 mM glycine (pH 8.5;–10), with pH as indicated, and incubated for 30 min with a mixture (1:1) of 2 µM 1-palmitoyl 2-[14C]arachidonyl PC and 1-palmitoyl 2-[14C]arachidonyl PE. The results represent the means ± range of duplicate samples from one of two representative experiments.


 
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  		Table 2. Acyl-chain specificity of partially purified equine cPLA2


 
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  		Table 3. Effects of divalent cations on the activities of equine and human cPLA2

Preliminary Western blot results demonstrated that the equine cPLA2 had higher electrophoretic mobility than human recombinant cPLA2 protein when analyzed by SDS-PAGE (data not shown). To explore further the observed size differences, cPLA2 partially purified from equine as well as human neutrophils and from unstimulated U937 cells, and recombinant cPLA2, were loaded on a 5;–10% polyacrylamide gel ( Fig 4). The gel was run for an additional 30 min after the front had eluted from the gel in order to obtain sufficient separation. Subsequently, Western blotting was performed and immunoreactive proteins were detected with a polyclonal anti-cPLA2 antibody. Fig 4 demonstrates that equine neutrophil cPLA2 had a higher electrophoretic mobility than either cPLA2 derived from human neutrophils or U937 cells, or recombinant cPLA2, and that equine neutrophil cPLA2 migrated as a 105-kDa protein. This observed apparent size difference is unlikely to be due to phosphorylation differences between the equine and human cPLA2 proteins because both partially purified and recombinant human cPLA2 had a lower electrophoretic mobility than the equine cPLA2 protein (Fig 4).

cDNA sequencing of equine neutrophil cPLA2
In an attempt to explain the observed differences in migration on SDS-polyacrylamide gels between human and equine cPLA2, the equine cPLA2 cDNA sequence was determined. Fig 5 depicts the deduced amino acid sequence of the equine neutrophil cPLA2 aligned with the human sequence. The equine protein demonstrated 95% identity with the human protein. As indicated in Fig 5, two charged amino acids were different in the vicinity of Ser-505, which is the target for phosphorylation by mitogen-activated protein (MAP) kinase. This phosphorylation, and hence the introduction of a negatively charged phosphate group, is suggested to result in a slower migrating protein cPLA2 (31). Ser-508 and Asp-509 in the human sequence were found to be Arg-508 and Asn-509 in the equine sequence, and thus a net charge corresponding to -1 in the human sequence is replaced by a net charge of +1 in the equine sequence, in this specified region of the protein. We suggest that this charge difference is the cause for the different mobility observed between the two species (Fig 4). Furthermore, the equine protein demonstrated 95% identity with human and mouse cPLA2 as well as 83 and 73% identity with chicken and zebra fish cPLA2 protein, determined by using the Genetics Computer Group (GCG, Madison, WI) program GAP.

Expression of cPLA2 protein in equine eosinophils and neutrophils
Having established that equine neutrophils indeed express an active cPLA2 enzyme, we continued to explore the discrepancy between equine neutrophils and eosinophils in their capacity to produce leukotrienes from endogenous substrate by analyzing phosphorylation of cPLA2 in highly enriched preparations of these cells. It is well documented that the phosphorylated cPLA2 enzyme migrates slower than the unphosphorylated enzyme (15) (32). Interestingly, cPLA2 from both resting and calcium ionophore A23187-stimulated eosinophils had slower electrophoretic mobility than cPLA2 from neutrophils ( Fig 6), suggesting that eosinophilic cPLA2 was phosphorylated to a major extent because no slower migrating band could be observed in these samples. On the other hand, cPLA2 protein from resting neutrophils seemed to be unphosphorylated and did not change its electrophoretic mobility after calcium ionophore A23187 stimulation (Fig 6). Thus, the phosphorylation pattern of cPLA2 in equine neutrophils and eosinophils correlates with the capacity of these cells to produce leukotrienes after calcium ionophore stimulation (Fig 6 and ref. 25). However, incubation of total homogenate from eosinophils with alkaline phosphatase did not affect the mobility of cPLA2 from equine eosinophils (data not shown).


  DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES

Human neutrophils produce leukotriene B4 (LTB4) after stimulation with calcium ionophore A23187. A species difference, between human and horse, in leukotriene production after A23187 stimulation has been demonstrated (25). Thus, equine neutrophils did not produce leukotrienes after calcium ionophore A23187 stimulation, unless low concentrations of exogenous arachidonic acid were added. However, calcium ionophore A23187 was a sufficient stimulus to induce leukotriene synthesis in equine eosinophils (25). To elucidate why equine neutrophils failed to produce leukotrienes after calcium ionophore A23187 stimulation, we investigated whether the cPLA2 enzyme from neutrophils was catalytically active. The equine form of the cPLA2 protein was purified >6,500 fold with a recovery of 3% from equine neutrophils. The equine cytosolic PLA2 activity could be ascribed to a protein migrating as a 105-kDa protein on SDS-PAGE (Fig 1). This protein was also recognized by cPLA2 polyclonal antibody, thus establishing that the purified protein was the equine cPLA2. The partially purified cPLA2 was insensitive to reducing conditions and displayed an alkaline pH optimum. The enzyme was dependent on Ca2+ for its activity and showed a marked preference for phospholipids containing arachidonic acid at the sn-2 position, as compared with palmitic acid, when sonicated phospholipid vesicles were used as substrates (Table 2).

The equine neutrophil cPLA2 possessed some biochemical features that distinguished it from the human cPLA2 and hence could represent species-specific differences. First, the dependence on divalent metal cations was not similar for the equine neutrophil cPLA2 as compared with U937 cPLA2 (Table 3), THP-1 cPLA2 (33), or recombinant cPLA2 (30). The observed differences in cation dependence for the equine neutrophil cPLA2, as compared with previous studies, could simply be due to different assay conditions. However, cPLA2 from U937 cells, isolated in a similar manner as cPLA2 from equine neutrophils, showed a dependency on divalent cations different from that of equine neutrophil cPLA2 (Table 3). A possible explanation for this finding might be differences in the calcium-binding domain between the two species. The crystal structure of the C2 domain of cPLA2 was resolved and it was demonstrated that two calcium ions were ligated to this domain (34). The amino acid residue Asn-65 participates in ligation of one of the calcium ions via its side-chain oxygen. Interestingly, one neighboring amino acid residue to Asn-65 was found to be different in the equine cPLA2 protein as compared with the human protein. The exchange of Asp-66 in the human protein to Asn-66 in the equine protein might affect the binding of divalent cations and thereby explain the observed differences in the dependency on cations for activity (Table 3). Interestingly, mutation of Asn-65 to alanine had a larger effect on cPLA2 activity than on membrane binding, suggesting that Asn-65 is important for activity (35). Thus, comparative studies using cPLA2 from human and horse might be useful for further exploration of cation-mediated mechanisms.

Second, a significant difference in migration on SDS-PAGE could be observed between equine neutrophil cPLA2 as compared with cPLA2 derived from human neutrophils or U937 cells or with recombinant cPLA2, with the equine neutrophil cPLA2 having a higher electrophoretic mobility (Fig 4). Phosphorylation of cPLA2 results in a gel mobility shift, with the phosphorylated protein migrating slower than the unphosphorylated protein (15) (17) (18). However, in this study both the phosphorylated form of equine cPLA2 from equine eosinophils, and the unphosphorylated form of cPLA2, from equine neutrophils, had higher electrophoretic mobility than the human forms (Fig 4 and Fig 6). It is therefore highly unlikely that the observed difference in electrophoretic mobility of cPLA2 between human and equine cells could be due to phosphorylation differences. A more likely explanation for the differences in electrophoretic mobility might involve differences in the primary structure of the enzymes. The equine neutrophil cPLA2 protein demonstrated two changes in charged amino acids in the vicinity of Ser-505 (Fig 5), which when phosphorylated is known to cause a slower migrating protein on SDS-PAGE (15) (17) (18). Furthermore, mutation of Ser-505 to glutamate resulted in a protein with gel mobility properties similar to those of phosphorylated cPLA2 (31). This demonstrates that reduced gel mobility is likely to be dependent on the presence of a negatively charged residue in this region. Taken together, the changes in charged amino acid residues between equine and human cPLA2 might explain the observed gel mobility differences (Fig 4).

Having established that equine neutrophils indeed express an active cPLA2 enzyme, we tried to elucidate the role of phosphorylation of cPLA2 in leukotriene synthesis in the horse. Our results indicate that cPLA2 in equine eosinophils is constitutively phosphorylated, whereas the cPLA2 in neutrophils is unphosphorylated, even after calcium ionophore A23187 stimulation (Fig 6). There was a clear difference in electrophoretic mobility between the cPLA2 from eosinophils as compared with neutrophils and it is well known that the phosphorylated enzyme migrates slower than the unphosphorylated enzyme. No other posttranslational modification of the cPLA2 protein is known to cause a gel shift similar to that caused by a phosphorylation event (36).

Phosphorylation of Ser-505 by MAP kinase has been demonstrated to increase the specific activity of the enzyme in vitro. However, the increase in activity is only modest and it has been suggested that phosphorylation of cPLA2 facilitates the calcium-dependent association between enzyme and membrane, thereby leading to increased release of fatty acid (18) (37). In this study, a majority of cPLA2 from equine eosinophils seemed to be in the phosphorylated state, because no faster migrating form could be observed, whereas a majority of cPLA2 from equine neutrophils seemed to be nonphosphorylated. This coincided with the greater capacity of the eosinophils to produce leukotrienes from endogenous arachidonic acid, as compared with equine neutrophils (25). Phosphorylation of cPLA2 might be one of several mechanisms necessary for the formation of leukotrienes from endogenous substrate in horse neutrophils. However, the precise mechanism governing leukotriene formation in equine neutrophils needs further elucidation. In conclusion, comparative studies of cPLA2 from different species could contribute to a better mechanistic understanding of the enzyme.


  FOOTNOTES

1 The sequence reported herein has been submitted to GenBank under the accession number AF092539. Back


  ACKNOWLEDGMENTS

The authors are greatly indebted to Ms. Hélène Ax:son-Johnson for technical assistance. This work was financially supported by grants from the Swedish Foundation for Health Care Sciences and Allergy Research, the research funds of Karolinska Institutet, Bayer AG, the Swedish Rheumatism Association, the Swedish Council for Forestry and Agricultural Research, and the Swedish Medical Research Council.

Manuscript received December 14, 1999; and in revised form March 23, 2000

Abbreviations: AACOCF3, arachidonoyl trifluoromethyl ketone; DAG, diacylglycerol; DTT, dithiothreitol; LTB4, leukotriene B4; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PLA2, phospholipase A2; RP-HPLC, reversed-phase high-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis


  REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

  1. Dennis, E. A. 1997. The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem. Sci. 22:1-2[Medline].

  2. Ackermann, E. J., Kempner, E. S., Dennis, E. A. 1994. Ca2+-independent cytosolic phospholipase A2 from macrophage-like P388D1 cells. J. Biol. Chem. 269:9227-9233[Abstract/Free Full Text].

  3. Tang, J., Kriz, R. W., Wolfman, N., Shaffer, M., Seehra, J., Jones, S. S. 1997. A novel cytosolic calcium-independent phospholipase A2 contains eight ankyrin motifs. J. Biol. Chem. 272:8567-8575[Abstract/Free Full Text].

  4. Ma, Z., Ramanadham, S., Kempe, K., Sherry Chi, X., Ladenson, J., Turk, J. 1997. Pancreatic islet express a Ca2+-independent phospholipase A2 enzyme that contains a repeated structural motif homologous to the integral membrane protein binding domain of ankyrin. J. Biol. Chem. 272:11118-11127[Abstract/Free Full Text].

  5. Balboa, M. A., Balsinde, J., Jones, S. S., Dennis, E. A. 1997. Identity between the Ca2+-independent phospholipase A2 enzymes from P388D1 macrophages and Chinese hamster ovary cells. J. Biol. Chem. 272:8576-8580[Abstract/Free Full Text].

  6. Larsson, P. K. A., Claesson, H-E., Kennedy, B. P. 1998. Multiple splice variants of the human calcium-independent phospholipase A2 and their effect on enzyme activity. J. Biol. Chem. 273:207-214[Abstract/Free Full Text].

  7. Leslie, C. C., Voelker, D. R., Channon, J. Y., Wall, M. M., Zelarney, P. T. 1988. Properties and purification of an arachidonoyl-hydrolyzing phospholipase A2 from a macrophage cell line, RAW 264.7. Biochim. Biophys. Acta. 963:476-492[Medline].

  8. Clark, J. D., Milona, N., Knopf, J. L. 1990. Purification of a 110-kilodalton cytosolic phospholipase A2 from the human monocytic cell line U937. Proc. Natl. Acad. Sci. USA. 87:7708-7712[Abstract/Free Full Text].

  9. Clark, J. D., Lin, L. L., Kriz, R. W., Ramesha, C. S., Sultzman, L. A., Lin, A. Y., Milona, N., Knopf, J. L. 1991. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP. Cell. 65:1043-1051[Medline].

  10. Kramer, R. M., Roberts, E. F., Manetta, J., Putnam, J. E. 1991. The Ca2+-sensitive cytosolic phospholipase A2 is a 100-kDa protein in human monoblast U937 cells. J. Biol. Chem. 266:5268-5272[Abstract/Free Full Text].

  11. Owada, Y., Tominaga, T., Yoshimoto, T., Kondo, H. 1994. Molecular cloning of rat cDNA for cytosolic phospholipase A2 and the increased gene expression in the dentate gyrus following transient forebrain ischemia. Brain Res. Mol. Brain Res. 25:364-368[Medline].

  12. Nalefski, E. A., Sultzman, L. A., Martin, D. M., Kriz, R. W., Towler, P. S., Knopf, J. L., Clark, J. D. 1994. Delineation of two functionally distinct domains of cytosolic phospholipase A2, a regulatory Ca2+-dependent lipid-binding domain and a Ca2+-independent catalytic domain. J. Biol. Chem. 269:18239-18249[Abstract/Free Full Text].

  13. Ramesha, C. S., Ives, D. L. 1993. Detection of arachidonoyl-selective phospholipase A2 in human neutrophil cytosol. Biochim. Biophys. Acta. 1168:37-44[Medline].

  14. Feltenmark, S., Runarsson, G., Larsson, P., Jakobsson, P. J., Bjorkholm, M., Claesson, H-E. 1995. Diverse expression of cytosolic phospholipase A2, 5-lipoxygenase and prostaglandin H synthase 2 in acute pre-B-lymphocytic leukaemia cells. Br. J. Haematol. 90:585-594[Medline].

  15. Lin, L. L., Lin, A. Y., Knopf, J. L. 1992. Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid. Proc. Natl. Acad. Sci. USA. 89:6147-6151[Abstract/Free Full Text].

  16. Durstin, M., Durstin, S., Molski, T. F., Becker, E. L., Sha'afi, R. I. 1994. Cytoplasmic phospholipase A2 translocates to membrane fraction in human neutrophils activated by stimuli that phosphorylate mitogen-activated protein kinase. Proc. Natl. Acad. Sci. USA. 91:3142-3146[Abstract/Free Full Text].

  17. Sa, G., Murugesan, G., Jaye, M., Ivashchenko, Y., Fox, P. L. 1995. Activation of cytosolic phospholipase A2 by basic fibroblast growth factor via a p42 mitogen-activated protein kinase-dependent phosphorylation pathway in endothelial cells. J. Biol. Chem. 270:2360-2366[Abstract/Free Full Text].

  18. De Carvalho, M., McCormack, A. L., Olson, E., Ghomashchi, F., Gelb, M. H., Yates, J. R., Leslie, C. C. 1996. Identification of phosphorylation sites of human 85-kDa cytosolic phospholipase A2 expressed in insect cells and present in human monocytes. J. Biol. Chem. 271:6987-6997[Abstract/Free Full Text].

  19. Bonventre, J. V., Huang, Z., Taheri, M. R., O'Leary, E., Li, E., Moskowitz, M. A., Sapirstein, A. 1997. Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2. Nature. 390:622-625[Medline].

  20. Uozumi, N., Kume, K., Nagase, T., Nakatani, N., Ishii, S., Tashiro, F., Komagata, Y., Maki, K., Ikuta, K., Ouchi, Y., Miyazaki, J., Shimizu, T. 1997. Role of cytosolic phospholipase A2 in allergic response and parturition. Nature. 390:618-622[Medline].

  21. Claesson, H-E., Dahlen, S. E. 1999. Asthma and leukotrienes: antileukotrienes as novel anti-asthmatic drugs. J. Intern. Med. 245:205-227[Medline].

  22. Higgins, A. J., Lees, P. 1984. Detection of leukotriene B4 in equine inflammatory exudate. Vet. Rec. 115:275[Medline].

  23. Doucet, M. Y., Jones, T. R., Ford-Hutchinson, A. W. 1990. Responses of equine trachealis and lung parenchyma to methacholine, histamine, serotonin, prostanoids and leukotrienes in vitro. Can. J. Physiol. Pharmacol. 68:379-383[Medline].

  24. Watson, E. D., Sweeney, C. R., Steensma, K. A. 1992. Arachidonate metabolites in bronchoalveolar lavage fluid from horses with and without COPD. Equine Vet. J. 24:379-381[Medline].

  25. Lindberg, A., Tornhamre, S., Mugnai, S., Lindgren, J. 1998. Ionophore A23187-induced leukotriene biosynthesis in equine granulocytes-neutrophils, but not eosinophils, requires exogenous arachidonic acid. Biochim. Biophys. Acta. 1391:247-255[Medline].

  26. Laemmli, U. 1970. Most commonly used discontinuous buffer system for SDS electrophoresis. Ann. N.Y. Acad. Sci. 209:477-496[Medline].

  27. Towbin, H., Staehelin, T., Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA. 76:4350-4354[Abstract/Free Full Text].

  28. Gronich, J. H., Bonventre, J. V., Nemenoff, R. A. 1990. Purification of a high-molecular-mass form of phospholipase A2 from rat kidney activated at physiological calcium concentrations. Biochem. J. 271:37-43[Medline].

  29. Rehfeldt, W., Hass, R., Goppelt-Struebe, M. 1991. Characterization of phospholipase A2 in monocytic cell lines. Functional and biochemical aspects of membrane association. Biochem. J. 276:631-636.

  30. Reynolds, L. J., Hughes, L. L., Louis, A. I., Kramer, R. M., Dennis, E. A. 1993. Metal ion and salt effects on the phospholipase A2, lysophospholipase, and transacylase activities of human cytosolic phospholipase A2. Biochim. Biophys. Acta. 1167:272-280[Medline].

  31. Huang, Z., Payette, P., Abdullah, K., Cromlish, W. A., Kennedy, B. P. 1996. Functional identification of the active-site nucleophile of the human 85-kDa cytosolic phospholipase A2. Biochemistry. 35:3712-3721[Medline].

  32. Kramer, R. M., Roberts, E. F., Manetta, J. V., Hyslop, P. A., Jakubowski, J. A. 1993. Thrombin-induced phosphorylation and activation of Ca2+-sensitive sensitive cytosolic phospholipase A2 in human platelets. J. Biol. Chem. 268:26796-26804[Abstract/Free Full Text].

  33. Rehfeldt, W., Resch, K., Goppelt-Struebe, M. 1993. Cytosolic phospholipase A2 from human monocytic cells: characterization of substrate specificity and Ca2+-sensitive dependent membrane association. Biochem. J. 293:255-261.

  34. Perisic, O., Fong, S., Lynch, D. E., Bycroft, M., Williams, R. L. 1998. Crystal structure of a calcium-phospholipid binding domain from cytosolic phospholipase A2. J. Biol. Chem. 273:1596-1604[Abstract/Free Full Text].

  35. Bittova, L., Sumandea, M., Cho, W. 1999. A structure-function study of the C2 domain of cytosolic phospholipase A2. J. Biol. Chem. 274:9665-9672[Abstract/Free Full Text].

  36. Clark, J. D., Schievella, A. R., Nalefski, E. A., Lin, L. L. 1995. Cytosolic phospholipase A2. J. Lipid Med. Cell Signal. 12:83-117[Medline].

  37. Abdullah, K., Cromlish, W. A., Payette, P., Laliberte, F., Huang, Z., Street, I., Kennedy, B. P. 1995. Human cytosolic phospholipase A2 expressed in insect cells is extensively phosphorylated on Ser-505. Biochim. Biophys. Acta. 1244:157-164[Medline].


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