A Novel Cytosolic Calcium-independent Phospholipase A2 Contains Eight Ankyrin Motifs*

(Received for publication, October 16, 1996, and in revised form, December 30, 1996)

Jin Tang , Ronald W. Kriz , Neil Wolfman , Mary Shaffer , Jasbir Seehra and Simon S. Jones Dagger

From the Genetics Institute, Cambridge, Massachusetts 02140

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
Note Added in Proof
REFERENCES


ABSTRACT

We report the purification, molecular cloning, and expression of a novel cytosolic calcium-independent phospholipase A2 (iPLA2) from Chinese hamster ovary cells, which lacks extended homology to other phospholipases. iPLA2 is an 85-kDa protein that exists as a multimeric complex of 270-350 kDa with a specific activity of 1 µmol/min/mg. The full-length cDNA clone encodes a 752-amino acid cytoplasmic protein with one lipase motif (GXS465XG) and eight ankyrin repeats. Expression of the cDNA in mammalian cells generates an active 85-kDa protein. Mutagenesis studies show that Ser465 and the ankyrin repeats are required for activity. We demonstrate that iPLA2 selectively hydrolyzes the sn-2 over sn-1 fatty acid by 5-fold for 1,2-dipalmitoyl phosphatidylcholine in a mixed micelle. Moreover, we found the fatty acid preference at the sn-2 position to be highly dependent upon substrate presentation. However, iPLA2 does have a marked preference for 1,2-dipalmitoyl phosphatidic acid presented in a vesicle, generating the lipid second messenger lysophosphatidic acid. Finally the enzyme is able to hydrolyze the acetyl moiety at the sn-2 position of platelet-activating factor.


INTRODUCTION

Phospholipase A2 enzymes are a broad group of proteins that have been intensively studied due to their potential involvement in the production of proinflammatory mediators, such as prostaglandins and leukotrienes through the release of arachidonic acid from membrane phospholipid (1). Concurrently, with fatty acid hydrolysis, lysophos-pholipid is released, the precursor for the proinflammatory molecule platelet-activating factor (PAF)1 as well as a potent mitogen in the case of lysophosphatidic acid (2, 3).

Phospholipase A2 enzymes can be divided into several groups based on sequence similarity and subcellular localization. Groups I-III consist of low molecular weight, 13-18-kDa, extracellular (sPLA2) enzymes found in snake venoms, mammalian pancreas, and synovial fluid. These enzymes require millimolar concentrations of Ca2+ for maximal activity and fail to exhibit any preference for the fatty acid substituent at the sn-2 position of phospholipids (4, 5). There are numerous reports that have causally linked sPLA2 to various inflammatory conditions, including arthritis. However, in several mouse strains the sPLA2 gene is naturally disrupted, yet these strains have been frequently used as antigen-induced arthritic models (6). Therefore, the role of sPLA2 in eicosanoid production and inflammation has become less clear. Conversely, the widely expressed 85-kDa cytosolic calcium-dependent phospholipase (cPLA2) of Group IV has the expected characteristics of an enzyme mediating hormonally induced eicosanoid and PAF production (7). cPLA2 has a 20-fold preference for arachidonic acid over unsaturated fatty acids in phospholipid substrates (8) and consists of two functional domains: a calcium-dependent lipid binding domain (CaLB) and a calcium-independent catalytic domain (8, 9). The former domain mediates the translocation of cPLA2 to the nuclear envelope and endoplasmic reticulum upon treatment with extracellular factors that raise intracellular Ca2+ concentrations to submicromolar levels (10, 11). In the catalytic domain, Ser228 has been shown to be essential for enzymatic activity (12, 13) and is contained within a partial lipase consensus sequence, GXSG, common to lipases and esterases (14, 15). Finally, cPLA2 is activated through phosphorylation of Ser505 (16) by mitogen-activated protein kinase (17). Other phosphorylation sites have been recently mapped, but their role in the activation of cPLA2 is unclear (18, 19).

Compared with cPLA2, there is a paucity of information on the structure and regulation of intracellular calcium-independent phospholipases (iPLA2) as well as the role of these enzymes in the inflammatory cascade, lipid metabolism, and other signaling pathways. A wide variety of poorly defined iPLA2 activities have been found in different tissues (Ref. 20 and references therein), but only three groups have attributed cytosolic iPLA2 activities to distinct polypeptides.

Gross and colleagues have assigned an activity found in myocardium (21-24), to a 40-kDa protein that exists as a 350-kDa complex by gel permeation chromatography (25). Moreover, the level of calcium-independent activity was found to increase during induced myocardial ischemia (22). In contrast, others failed to observe the same activation under similar experimental conditions; indeed, a decrease in calcium-independent activity was observed with prolonged ischemia (26).

Since the 40-kDa species copurified with an 85-kDa phosphofructokinase isoform, it was suggested that the activity exists as a complex of the 40-kDa species and four units of the 85-kDa polypeptide. The activity was irreversibly inactivated by a bromoenol lactone (BEL; (E)-6-(bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one) (27), a suicide inhibitor originally designed to inhibit the serine esterase, alpha -chymotrypsin (28). Similar calcium-independent BEL-sensitive activities were reported to exist in pancreatic islets (29), aortic smooth muscle cells (30), RAW 264.7 cells (31), and rat hippocampus (32). The existence of a 40-kDa PLA2 or a functional high molecular weight complex was not demonstrated in these cell lines except in the case of pancreatic islets (33). The activity was stimulated by the presence of ATP (23) and had a slight preference, 2-fold, for 1-ether over 1-acylphospholipid substrates as well as a 3-fold preference for arachidonic over palmitic acid at the sn-2 position (21), but there were no significant phospholipase A1 or lysophospholipase activities associated with the complex.

Dennis and colleagues (34) have identified an activity from a macrophage cell line, P388D1, correlating with a single 80-kDa polypeptide by SDS-polyacrylamide gel electrophoresis (PAGE) where the phospholipase activity was substantially enhanced in the presence of detergent (Triton X-100), giving a specific activity 5 µmol/min/mg. Moreover, this preparation showed a 2-fold selectivity for hydrolysis of palmitic acid over arachidonic acid, favored 1-acyl- over 1-ether-containing phospholipids, and has an intrinsic lysophospholipase activity in a mixed micelle assay. However, analogous to the 40-kDa species studied by Gross and co-workers (21-25), the 80-kDa activity exists as a 337-kDa complex that was stimulated by ATP (and other nucleoside triphosphates), reversibly inhibited by palmitoyl and arachidonyl trifluoromethyl ketones, and irreversibly inhibited by BEL and methyl arachidonyl fluorophosphonate (35-37). Notably, in the absence of detergent, i.e. a vesicle assay, the substrate selectivity at the sn-2 position was reversed compared with the mixed micelle assay, favoring arachidonic over palmitic acid. Also, there was no stimulation of activity by ATP when phospholipid was presented in a vesicle. Recently, the 80-kDa activity has been implicated in phospholipid remodeling in the cell line P388D1 (38, 39).

More recently, a 28-kDa polypeptide has been purified to homogeneity from rabbit kidney cortex that preferentially hydrolyzes arachidonyl-containing plasmalogen phospholipids in a vesicle assay (40).

In our efforts to molecularly define the cytosolic enzymes that release fatty acid from membrane phospholipids, we describe the purification of an 85-kDa cytosolic calcium-independent phospholipase A2, iPLA2, and expression of a cDNA encoding this activity. The deduced amino acid sequence has one lipase motif (GXSXG) containing the putative active site serine and eight ankyrin repeat sequences and lacks homology with any other known phospholipase A2 enzymes.


EXPERIMENTAL PROCEDURES

Assays

1,2-[1-14C]Dipalmitoyl-sn-glycero-3-phosphorylcholine (PC) (112 Ci/mol), 1-palmitoyl-2-[1-14C]palmitoyl-sn-glycero-3-phosphorylcholine (55.5 Ci/mol), 1-stearoyl-2-[1-14C]arachidonyl-sn-glycero-3-phosphoryl-choline (56 Ci/mol), and 1-palmitoyl-2-[1-14C]arachidonyl-sn-glycero-3-phosphorylcholine (56 Ci/mol) were obtained from Amersham Corp.; 1-stearoyl-2-[1-14C]arachidonyl-sn-glycero-3-phosphorylinositol (PI) (50 Ci/mol), 1-O-hexadecyl-2-[5,6,8,9,11,12,14,15-3H]arachidonyl-sn-glycero-3-phosphorylcholine (210 Ci/mmol), 1-palmitoyl-2-[1-14C]arachidonyl-sn-glycero-3-phosphorylethanolamine (53 Ci/mol), 1-palmitoyl-2-[1-14C]oleyl-sn-glycero-3-phosphorylcholine (58 Ci/mol), 1-palmitoyl-2-[1-14C]linoleyl-sn-glycero-3-phosphorylcholine (50 Ci/mol), 1-[1-14C]palmitoyl-2-hydroxy-sn-glycero-3-phosphorylcholine (57 Ci/mol), 1,2-dipalmitoyl-sn-[U-14C]glycero-3-phosphatidic acid (PA) (144 Ci/mol), and 1-O-hexadecyl-2-[3H]acetyl-sn-glycero-3-phosphorylcholine (7.1 Ci/mmol) were obtained from DuPont NEN. Unlabeled 1-O-hexadecyl-2-arachidonyl PC and PAF were obtained from Biomol and Avanti Polar Lipids, respectively. The lipid (10 µM) was dried under a stream of N2 and resuspended in 100 mM Tris-HCl, pH 7.5, 50 or 500 µM Triton X-100, 5 mM EDTA, and 10% glycerol (v/v) and was sonicated for 2 × 30 s at 4 °C. Substrate was incubated with aliquots of iPLA2 at 37 °C for defined time periods, and released fatty acid was measured as described (41). The release of [3H]acetyl from [acetyl-3H]PAF was measured as described (42). Alternatively, quantification of products by tlc (34) was used for determining regioselectivity of iPLA2 and hydrolysis of 1,2-dipalmitoyl PA (43), where the lipids were separated in the basic solvent system, chloroform:methanol:ammonium hydroxide:water (45:30:3:5, v/v/v/v) or the acidic system (34), respectively.

Purification Procedure

Chinese hamster ovary (CHO) cells were harvested at about 2.4 × 106 cells/ml (44) as a by-product of large scale cultures for the production of secreted proteins. The level of calcium-independent activity in these cell lines was equivalent to the parental CHO-DUKX cell cultured as described (44). CHO cells were collected by centrifugation, washed with PBS, rapidly frozen in liquid nitrogen, and stored at -80 °C. Frozen pellets (3.5 kg, about 1.4 × 1012 cells) were resuspended in 20 mM imidazole, pH 7.5, 0.25 M sucrose, 2 mM EDTA, 2 mM EGTA, 1 µg/ml leupeptin, 5 µg/ml aprotinin, 5 mM DTT, and 1 mM phenylmethylsulfonyl fluoride (extraction buffer). The cells were lysed by N2 cavitation at 600 p.s.i., and the lysate was clarified by centrifugation at 10,000 × g for 30 min followed by 100,000 × g for 60 min. The lysate was made 10% (v/v) in glycerol and diluted to 5 mg/ml total protein with 20 mM imidazole, pH 7.5, 5 mM DTT, 1 mM EDTA, 1 mM EGTA, and 10% glycerol (v/v) (buffer A). Lysate (10 g of total protein) was applied (16 ml/min) to a DEAE toyopearl (TosoHaas) column (1-liter volume) equilibrated with buffer A. The column was developed with a gradient of 0.0-0.5 M NaCl. Active fractions (peak I) were diluted to 3 mg/ml with buffer A and made 0.5 M in (NH4)2SO4 and loaded (4 g of total protein) onto a phenyl toyopearl column (300-ml volume) at 10 ml/min. The column was developed with a reverse gradient of 0.5-0.2 M followed by 0.2-0.0 M (NH4)2SO4 over 15 min and 85 min, respectively, and at this point the activity was still bound. The activity was eluted with buffer A at 1.5 ml/min for 18 h and loaded directly onto a heparin toyopearl column (10-ml volume) connected in tandem to the phenyl column. The phenyl column was disconnected, and the activity was eluted from the heparin column with 0.5 M NaCl in buffer A at 2 ml/min. Active fractions were exhaustively dialyzed against 20 mM Bis-Tris, pH 7, 1 M urea, 5 mM DTT, and 10% glycerol (v/v) and applied (1 ml/min) to a Mono P 10/30 (Pharmacia Biotech Inc.) column equilibrated with the same buffer. Activity was eluted by establishing a pH gradient with 10% polybuffer 74, pH 5, 1 M urea, 5 mM DTT, and 10% glycerol (v/v) (1 ml/min). The combined active fractions were loaded onto a heparin toyopearl column (10 mg of protein/ml of resin) and eluted as above. Portions of active fractions were further purified using two TSK G3000SWXL (TosoHaas) columns (7.8 mm × 30 cm) in tandem, developed in buffer A with 150 mM NaCl at 0.3 ml/min.

Recombinant iPLA2 was partially purified from the CHO line c4.20.5s (6 × 109 cells; see below) in a similar manner except that the Mono P step was omitted.

Generation of Tryptic Peptides

Active fractions from the Mono P/heparin step (63 µg of total protein) were made 10% in SDS and concentrated in a Microcon-30 (Amicon). The sample was loaded onto a 4-20% gradient SDS-PAGE minigel (Novex) run under reducing conditions. The gel was stained (45), and the 85-kDa band was excised. Tryptic peptides were generated as described previously (8).

cDNA Library Screening

Three tryptic peptides were used to design 17 residue oligonucleotides: DNMEMIK, pools 1 and 2, 8- and 12-fold degenerate, respectively; MKDEVF, pool 3, 32-fold degenerate; and EFGEHT, pool 4, 64-fold degenerate. Poly(A)+ mRNA was prepared from 2 × 108 CHO-DUKX cells using RNAgents Total RNA kit and Poly(A)Tract mRNA Isolation System (Promega). cDNA was prepared by the Superscript Choice System (Life Technologies, Inc.), ligated into lambda  ZAP11/EcoRI and packaged into phage particles with Gigapack Gold packaging extracts (Stratagene). 4 × 105 recombinant phage were screened with pools 1 and 2 under tetramethylammonium chloride hybridization conditions (46). Twelve positive phage were identified, replated, and probed with pools 2, 3, and 4. Plasmid DNA was prepared for four clones (clones 9, 17, 31, and 49) that were positive with all three probes. All four clones contained a 3.0-kb insert, and one (clone 9) was sequenced.

A mouse/rat multiple tissue Northern blot (Clonetech) was probed with a 2-kb SmaI fragment (2 × 106 cpm/ml) of clone 31 randomly primed to about 109 cpm/µg, at 60 °C in 5 × Denhardt's solution, 5 × SSC, 0.1% SDS, and 50 µg/ml yeast tRNA and washed at 55 °C in 0.5 × SSC and 0.1% SDS.

1 × 106 plaques from a human Burkitt lymphoma (RAJI) library (Clonetech) were screened with the 2-kb SmaI probe. Ten positive clones were isolated upon replating positive plaques. One of these, number 19, was subcloned as two EcoR I fragments, A (2 kb) and B (1.3 kb), and sequenced.

Expression of iPLA2

Clone 9 was excised from the lambda  vector and subcloned into the SalI site of pED (47), yielding pC9. iPLA2 in pC9 was transiently expressed in COS cells as described (48). A stable CHO cell line was established, c4.20.5, by selection and amplification of iPLA2 in 20 nM methotrexate as described (44). c4.20.5 was adapted to suspension culture by changing the growth media from alpha  (Life Technologies) to R1 (Mediatech) and initially maintaining the cell density at 3 × 105 cells/ml. Activity levels were determined by detaching transfected COS or c4.20.5 cells with phosphate-buffered saline, 2 mM EDTA, collecting the cells by centrifugation, and washing with phosphate-buffered saline. The cell pellet was resuspended in extraction buffer (1 × 107 cells/ml) and lysed by 20 strokes in a glass Dounce homogenizer, and the supernatant was collected by centrifugation. The supernatant was analyzed for activity by the release of radiolabeled fatty acid from phospholipid. The expression of iPLA2 was analyzed by immunoblot with a polyclonal antibody raised against iPLA2 at 1:200 dilution or, in the case of the Flag-tagged proteins, the anti-Flag M2 (IBI) monoclonal antibody (0.75 µg/ml) and visualized with the ECL System (Amersham).

DNA Constructs

A glutathione S-transferase fusion was generated by ligating the StuI/NotI fragment of pC9 (amino acids 437-752) into pGEX-5x-3 (Pharmacia Biotech Inc.). The amino-terminal N-Flag construct was formed by ligating a HincII/XbaI fragment of pC9 with synthetic oligonucleotides encoding the Flag epitope (IBI) and the first eight amino acids of iPLA2 in the pED expression vector. The carboxyl-terminal C-Flag construct was prepared similarly by ligating a SalI/HindIII fragment of pC9 with synthetic oligonucleotides encoding the last seven amino acids of iPLA2 and the Flag epitope. The first 150 amino acids and ankyrin repeats were deleted by ligating the TfiI/XbaI fragment of pC9 (amino acids 422-752) with synthetic oligonucleotides encoding the Flag epitope and iPLA2 residues (406-421, Delta a; 411-421, Delta b; 416-421, Delta c) into the pED vector. The serine 252 and 465 to alanine mutations were performed with the Chameleon Mutagenesis kit (Stratagene) using oligonucleotides CATGGGACCCGGCTTTCC and GGCAGGAACCACTGGGGGC, respectively.

Antibody Generation

Cultures expressing the glutathione S-transferase fusion protein were prepared as described (Pharmacia). The cells were pelleted at 2000 g, resuspended in phosphate-buffered saline (25 ml), and lysed by French press, and the pellet was collected at 30,000 × g. The pellet was resuspended in 50 mM Tris-HCl, pH 8, and passed five times through a 22-gauge needle, and the insoluble material was collected at 5000 × g. The glutathione S-transferase fusion was dissolved in 7 M guanidine HCl, 50 mM Tris-HCl, pH 8, 1 mM EDTA, and 10 mM DTT (3 mg/ml), 10 mg was loaded onto a C-4 reverse-phase column (Vydac), and the fusion protein was eluted with acetonitrile (~35%). Antiserum was generated by injecting rabbits with this material (Pocono Rabbit Farm).


RESULTS

Purification of a Cytosolic iPLA2 from CHO Cells

In order to identify calcium-independent phospholipase A2 activities, we surveyed a variety of cell lines and tissues under conditions where the activity of endogenous cPLA2 is low, i.e. pH 7, 10% glycerol in the absence of Ca2+. Using 1-palmitoyl-2-[14C]arachidonyl PC (PAPC) as the substrate, higher activity was observed in a mixed micelle assay in the presence of Triton X-100 than in a vesicle assay. Under these conditions (10 µM PAPC, 500 µM Triton X-100), the activity is widely distributed in extracts of brain, heart, and a variety of heart cell lines (data not shown). This wide distribution encouraged us to examine CHO cells, which can be readily grown in large quantities. Extracts of CHO cells had similar activity to heart cell lines and tissue extracts, and consequently iPLA2 was purified from this source.

Table I summarizes the purification of iPLA2 from 500 liters of CHO cells. The majority of the activity was found in the supernatant upon lysis by nitrogen cavitation. DEAE column chromatography of the cell lysate gave two broad peaks of activity eluting at 75 mM and 300 mM NaCl (data not shown). Our efforts focused on purifying the activity from the 75 mM salt peak, since attempts to further purify the high salt peak were unsuccessful due to a lack of reproducible chromatographic behavior. The 75 mM NaCl eluate was loaded onto a hydrophobic interaction (phenyl-TSK) column, and calcium-independent PLA2 activity eluted as a broad peak in 0 M salt wash following a reverse gradient. To minimize losses of iPLA2, the activity was concentrated by an in-line heparin column. Subsequent chromatofocusing on a Mono P column, using a gradient from pH 7 to 5, resulted in a substantial purification (Table I). SDS-PAGE of Mono P fractions indicated that the amount of iPLA2 activity (Fig. 1A, fractions 9-13) correlated with the staining intensity of an 85-kDa protein. To demonstrate that iPLA2 activity corresponded to the 85-kDa species, a small portion of pooled fractions from the Mono P column was further purified using gel permeation chromatography. Interestingly, all the activity eluted in the 250-350-kDa range (Fig. 1B, fractions 55-58), suggesting that the native enzyme is an oligomer of 85-kDa monomers. Indeed, SDS-PAGE of these fractions (Fig. 1B) confirmed the correlation of iPLA2 activity with the intensity of the 85-kDa protein. The estimated specific activity of the iPLA2 is approximately 1 µmol/min/mg in a mixed micelle assay (10 µM PAPC, 500 µM Triton X-100) based on a purity of 10-20% in the size exclusion fractions.

Table I.

Purification of iPLA2 from cytosol of Chinese hamster ovary cells


Step Protein Activitya Specific activity Purification Recovery

mg nmol/min nmol/min/mg -fold %
Cytosolb 126,000 2050 0.016 100
DEAE 16,000 1264 0.079 5 60
Phenyl/ 193 90 0.46 30 4.5
Heparin
Mono P 0.1 14 140 8000 0.7

a Hydrolysis of 10 µM 1-palmitoyl-2-[14C]arachidonyl PC in the presence of 500 µM Triton X-100 over 30 min at 37 °C.
b Activity in cytosolic extract after centrifugation.


Fig. 1. Active fractions from Mono P and gel permeation chromatography examined by SDS-PAGE. A, aliquots of active fractions eluted from the Mono P column were analyzed by 4-20% SDS-PAGE and visualized with silver stain. B, a portion of the activity (A) was applied to a G3000SWXL column, and active fractions were examined as for A. Molecular size markers for the gel permeation column were bovine thyroglobulin and gamma  globulin (670 and 158 kDa, respectively).
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iPLA2 Contains One Lipase Motif and Eight Ankyrin Repeats

The Mono P/heparin fractions containing the activity were separated by SDS-PAGE, and the 85-kDa polypeptide was excised and digested in situ with trypsin. Reverse-phase high pressure liquid chromatography followed by Edman degradation yielded several peptide sequences, three of which were used to design degenerate oligonucleotide pools for screening a CHO cell cDNA library. Several full-length clones were obtained, and the amino acids of one of these were sequenced (clone 9) (Fig. 2A).



Fig. 2. Amino acid sequence and alignment of CHO and human iPLA2 with putative C. elegans proteins T04B2.5 and F47A4.5. A, the deduced amino acid sequences and alignment of CHO and human iPLA2. Boxed/shaded and shaded sequences indicate amino acid identity and similarity, respectively. Gaps are shown by a dashes, and X denotes the uncertainty in the amino acid sequence due to a lack of consensus splice acceptor and donor site in clone 19. The ankyrin repeats are underlined, the lipase motif is overlined, and arrows indicate Ser252 and Ser465. B, sequence alignment of CHO and human iPLA2 after the eighth ankyrin repeat with the equivalent regions in T04B2.5 and F47A4.5. C, sequence alignment of CHO and human iPLA2 from the amino terminus to the end of the eighth ankyrin repeat with the putative ankyrin-containing region of F47A4.5. The cDNA sequences for CHO iPLA2, T04B2.5, and F47A4.5 can be found in the GenBankTM data base under accession numbers Il5470 (U.S. patent 5466595), Cet04b2_7, and Cef47a4_5, respectively. Sequences were aligned using the GeneWorks progressive alignment method.
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The cDNA encodes a polypeptide of 752 amino acids with a calculated molecular mass of 85 kDa and a neutral isoelectric point. iPLA2 does contain a consensus motif, GXS465XG, commonly found in serine proteases and lipases (14, 15). Notably, there are seven ankyrin repeat sequences and one less conserved repeat from amino acids 251-280 (Fig. 2A), a domain that is widely distributed through proteins with diverse functions (49, 50). A partial human cDNA (clone 19) was isolated from a Burkitt lymphoma library by screening with a CHO cDNA labeled probe. Although clone 19 was the correct size for a full-length cDNA, it was found to contain potential unspliced intron sequences (data not shown). Based on sequence comparison with the CHO iPLA2, the putative coding sequence of clone 19 encodes 90% of human iPLA2 and is 90% identical to the CHO protein in the region of overlap (Fig. 2A).

Importantly, iPLA2 lacks homology to cPLA2, the secreted phospholipases, or the 85-kDa phosphofructokinase that was reported to be associated with the 40-kDa myocardial calcium-independent phospholipase (25). However two putative proteins of unknown function from Caenorhabditis elegans, T04B2.5 (408 amino acids) and F47A4.5 (1071 amino acids), have identical lipase motifs and significant sequence similarity in the region surrounding the lipase motif (Fig. 2B). Moreover, closer inspection of the F47A4.5 sequence amino-terminal to the putative lipase motif revealed stretches of homology throughout the equivalent region in CHO and human iPLA2, with conservation of the ankyrin repeats (Fig. 2C).

Finally, Northern analysis of several rat/mouse tissues (Fig. 3A) and cell lines (data not shown) indicates ubiquitous expression with the highest levels in testis and liver.


Fig. 3. Northern analysis of iPLA2. A, a poly(A)+ mRNA mouse/rat multiple tissue blot was probed with a 32P-labeled cDNA encompassing all of the coding sequence of iPLA2 except the last 60 amino acids, as described under "Experimental Procedures." B, the same blot as in A reanalyzed with a beta -actin probe.
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iPLA2 Encodes an 85-kDa Cytosolic Calcium-independent PLA2

To prove the purified 85-kDa protein is an iPLA2, clone 9 was subcloned into the mammalian expression plasmid, pED, and transiently expressed in COS cells. Cytosolic extracts of the COS cells had over 300-fold more iPLA2 activity than mock-transfected COS cells (Fig. 4A; Cos and iPLA2cos). We also established stable CHO cell lines expressing iPLA2 through selection and amplification in methotrexate. One of these, c4.20.5, expressed 50-fold higher levels of iPLA2 activity than the parental line, CHO-DUKX (Fig. 4A, CHO and iPLA2CHO).


Fig. 4. Transient and stable expression of iPLA2 and various mutants. A, 10 µM 1-palmitoyl-2-[14C]arachidonyl PC dispersed in 500 µM Triton X-100 was incubated with cytosolic extracts of COS cells transiently transfected with vector alone (Cos), clone 9 (iPLA2cos), an NH2- or COOH-terminal Flag fusion of iPLA2 (N-Flag and C-Flag), a COOH-terminal Flag fusion with amino acids 406-752 of iPLA2 (Del 1), and an NH2-terminal Flag fusion with Ser252 or Ser465 of iPLA2 changed to Ala (A252 and A465), CHO cells (CHO), and the iPLA2-overexpressing CHO cell line c4.20.5 (iPLA2CHO). All assays were in duplicate. B, aliquots of cytosolic extracts (same order as in A) were separated by SDS-PAGE on a 4-20% gel. Proteins were transferred to nitrocellulose, probed with the anti-iPLA2 polyclonal sera, and visualized as described under "Experimental Procedures." C, cytosolic extracts were analyzed as in B except that the nitrocellulose blot was probed with an anti-Flag monoclonal antibody.
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To study the function of iPLA2, a polyclonal antibody was generated to a glutathione S-transferase fusion of the carboxyl-terminal half of the protein, amino acids 437-752, containing the lipase consensus sequence. This antibody specifically recognizes an 85-kDa polypeptide expressed in COS and CHO cells transfected with clone 9 (Fig. 4B, iPLA2cos and iPLA2CHO) compared with the parental cell lines (Fig. 4B, Cos and CHO).

Although during the purification we did not observe a 40-kDa polypeptide correlating with activity (Fig. 1, A and B), we considered the possibility the the 85-kDa polypeptide might be the precursor to a 40-kDa species equivalent to the one purified by Gross and colleagues (21). To address this hypothesis, two fusion proteins of iPLA2 tagged at either the amino or carboxyl termini with the Flag epitope, N- and C-Flag, respectively, were expressed transiently in COS cells. Both constructs yielded levels of iPLA2 activity comparable with clone 9 (Fig. 4A, iPLA2cos, N-Flag, and C-Flag). Moreover, polypeptides of 40 kDa were not observed by immunoblot when probed with either an anti-Flag monoclonal antibody (Fig. 4C) or with the anti-iPLA2 polyclonal antibody (Fig. 4B). These data demonstrate that the amino acid sequence in Fig. 2A corresponds to a novel cytosolic calcium-independent PLA2.

We considered the possibility that the ankyrin repeats might be required for activity, since this motif is postulated to be involved in protein-protein interactions (49, 50). To test this hypothesis, the carboxyl-terminal half of iPLA2, lacking the first 150 amino acids and the eight ankyrin repeats but retaining the lipase consensus sequence, was expressed as a fusion protein with the Flag epitope at the amino terminus. Although expression of the truncated form was similar to full-length iPLA2 (Fig. 4, B and C), it lacked activity (Fig. 4A). Similar results were obtained when amino acids 411-752 or 416-752 of iPLA2 where expressed as fusion proteins (data not shown).

iPLA2 contains two serine residues that could be at the active site. The first, GXS465XG, is a perfect consensus sequence found in many lipases (14, 15). The other potential active site is located in the sequence GPSGF, although this is a poor site due to the presence of the large Phe residue in the sequence (Fig. 2A). To identify the catalytic serine, Ser252 right-arrow Ala and Ser465 right-arrow Ala were expressed in COS cells as amino-terminal fusions with the Flag epitope. Both constructs were expressed at levels comparable with that of the full-length N-Flag iPLA2 (Fig. 4, B and C), indicating that there were no gross conformational changes in the proteins due to the respective mutations. However, detailed studies will be required to confirm the structural integrity of the mutants. Ser465 right-arrow Ala had no activity above background levels, i.e. 1-2% of residual activity compared with wild-type iPLA2, whereas Ser252 right-arrow Ala exhibited activity comparable with that of the full-length N-Flag iPLA2 (Fig. 4A). These data are consistent with Ser465 being the active site nucleophile and with the 85-kDa polypeptide as the catalytic unit of the 250-350-kDa complex.

iPLA2 Selectively Hydrolyzes Phospholipids at the sn-2 Position

We investigated the positional selectivity of iPLA2 using material purified from the overexpressing CHO cell line, c4.20.5s, and the substrate 1,2-dipalmitoyl PC presented in a Triton X-100 mixed micelle. Initially, the hydrolysis of 1,2-dipalmitoyl PC was followed by measuring the release of palmitic acid, 1- and 2-palmitoyl lysophosphatidylcholine, where both the sn-1 and sn-2 fatty acids were labeled. The results in Fig. 5A show that the ratio of palmitic acid to lyso products was 1:1 over 30 min. This observation suggests that iPLA2 has little lysophospholipase activity under the assay conditions. Based on these results, we examined the regioselectivity of hydrolysis using 1,2-dipalmitoyl PC labeled at the sn-2 fatty acid. Over a 30-min time period, we observed that 5-fold more palmitate was formed compared with 2-palmitoyl lysophosphatidylcholine (Fig. 5B), demonstrating that iPLA2 is selective for the hydrolysis of sn-2 fatty acids in phospholipid substrates.


Fig. 5. Regioselectivity of iPLA2. A, 10 µM 1-[14C]palmitoyl-2-[14C] palmitoyl PC dispersed in 500 µM Triton X-100 was incubated with iPLA2. Aliquots were removed over 30 min, and lipids and fatty acids were extracted (33) and then separated by tlc using the basic solvent system (see "Experimental Procedures"). Radioactive spots were excised and counted. All points were in duplicate. B, 1-palmitoyl-2-[14C]palmitoyl PC was incubated with iPLA2, and products were quantified as in A.
[View Larger Version of this Image (39K GIF file)]


Fatty Acid Selectivity of iPLA2 Depends upon Substrate Presentation

We investigated the preference for the fatty acid at the sn-2 position when the phospholipid substrate (10 µM) was dispersed in either 50 or 500 µM Triton X-100. These concentrations of detergent were chosen, since we observed an abrupt drop in activity at the critical micelle concentration of Triton X-100 (0.24 mM in water) when substrate concentration was kept constant (10 µM) and the detergent concentration was varied (data not shown). This phenomenon has also been observed for cPLA2 (58). As the results in Table II indicate, iPLA2 failed to demonstrate any significant preference for a particular fatty acid, although the rate of hydrolysis of unsaturated fatty acids was greater at 50 µM Triton. Additionally, the overall rates of hydrolysis were, at least for some substrates, very sensitive to lipid presentation, e.g. 1,2-dipalmitoyl PC was 4 times better when substrate was dispersed in 500 µM Triton, whereas 1-palmitoyl-2-arachidonyl (linoleyl or oleyl) PC were equivalent in both assays (Table II).

Table II.

Relative substrate selectivity of CHO iPLA2


Substratea Activityb Activityc

pmol/min pmol/min
1,2-Dipalmitoyl-PC 13.7 3.4
1,2-Dipalmitoyl-PAd 12.1 68.3
1-Palmitoyl-2-linoleyl-PC 18.7 14.8
1-Palmitoyl-2-oleyl-PC 12.4 14.0
1-Palmitoyl-2-arachidonyl-PC 5.1 8.6
1-Palmitoyl-2-arachidonyl-PE 6.2 8.4
1-Stearoyl-2-arachidonyl-PC 3.0 9.8
1-Stearoyl-2-arachidonyl-PI 0.66 1.8
1-Hexadecyl-2-arachidonyl-PCe 2.1 17.4
1-Hexadecyl-2-acetyl-PCf 3.0 8.7
1-Palmitoyl-2-lyso-PCg 1.4 10.7

a sn-2 fatty acid 14C-labeled.
b Rate of hydrolysis in the presence of 500 µM Triton X-100, 10 µM labeled substrate over 15 min at 37 °C.
c Rate of hydrolysis in the presence of 50 µM Triton X-100, 10 µM labeled substrate over 15 min at 37 °C.
d Glycerol backbone 14C-labeled.
e [3H]Arachidonyl; specific activity adjusted to 128 × 103 dpm/nmol with unlabeled lipid.
f [3H]Acetyl; specific activity adjusted to 125 × 103 dpm/nmol with unlabeled lipid.
g Hydrolysis of 1-[14C]palmitoyl monitored.

We also observed an intrinsic lysophospholipase activity with the substrate 1-palmitoyl lysophosphatidylcholine where the relative rate of hydrolysis was lower with 500 µM than 50 µM Triton X-100, 1.4 and 10.7 pmol/min, respectively (Table II). We posed the question of whether the lysophospholipase/phospholipase A1 activity of iPLA2, at the low concentration of Triton, was required for cleavage of an sn-2 fatty acid, as opposed to 500 µM Triton, where it is not required (Fig. 5). This is clearly not the case, since 1-hexadecyl-2-arachidonyl PC, containing a nonhydrolyzable ether-linked sn-1 fatty acid, was readily cleaved at twice the rate compared with 1-palmitoyl-2-arachidonyl PC (Table II). Significantly, we found the sn-1 ether-linked substrate PAF to be a substrate for iPLA2 in both assay formats (Table II), indicating that the phospholipase A2 activity of iPLA2 is not restricted to long chain fatty acids. This is in contrast to cPLA2 and sPLA2, which have no significant PAF acetylhydrolase activity under either assay condition in the presence of calcium (data not shown).

Finally, we investigated the effect of the phospholipid head group on relative rates of hydrolysis. The enzyme showed no preference between choline and ethanolamine head groups for the substrates tested (Table II). Yet there were clear differences in rates of hydrolysis between choline and negatively charged head groups, such as PI and PA (Table II). For instance, the rate of hydrolysis of 1-stearoyl-2-arachidonyl PI was 5-fold lower than the corresponding PC substrate in both assay systems. Conversely, the immediate precursor to the mitogen lysophosphatidic acid, 1,2-dipalmitoyl PA, was hydrolyzed at a 20-fold higher rate than 1,2-dipalmitoyl PC when the lipid was dispersed in 50 µM Triton, but no significant difference in rates was observed at 500 µM Triton. Indeed, for the substrates 1,2-dipalmitoyl PC and PA, at least part of this rate difference is attributable to the charge of the head group, since the hydrolysis of 1,2-[14C]dipalmitoyl PA (10% molar fraction) in a bilayer of 1,2-dipalmitoyl PC (90% molar fraction) is reduced 2-fold compared with 1,2-dipalmitoyl PA alone. In contrast, 1,2-[14C]dipalmitoyl PC (10% molar fraction) is hydrolyzed 5-fold faster in a background of 1,2-dipalmitoyl PA (90% molar fraction) (data not shown).


DISCUSSION

We have demonstrated the existence of a novel cytosolic calcium-independent iPLA2 in the cytosol of CHO cells whose sequence is not related to any other phospholipase. We found that the iPLA2 activity correlated with an 85-kDa species in active fractions from two consecutive chromatographic steps. However, we observed that iPLA2 in the native state exists as a oligomeric complex of molecular mass 250-350 kDa, a result that has been found for other calcium-independent phospholipase activities (23, 25, 34). We obtained several full-length cDNA CHO clones, and one of these was sequenced and found to encode a 752-amino acid polypeptide. Expression in COS cells generates a single 85-kDa species and 300-fold increase of iPLA2 activity in cytosolic extracts.

Although the inferred amino acid sequences of CHO and human proteins are 90% identical, they lack homology to cPLA2, to any sPLA2, or to the 85-kDa phosphofructokinase isoform associated with the 40-kDa myocardial calcium-independent phospholipase (25, 33) but are homologous to the murine iPLA2 isolated from P388D1 cells (accompanying manuscript (Ref. 59)). Significantly, iPLA2 is homologous to two putative proteins from C. elegans, T04B2.5 and F47A4.5, through two distinct sets of sequence motifs. Amino acids surrounding the consensus lipase motif and the relative position of the motif (GTS465TG) to the carboxyl termini are conserved among CHO, human, and C. elegans proteins. Indeed, mutagenesis of Ser465 right-arrow Ala in CHO iPLA2 abrogates activity, while Ser252 right-arrow Ala in a partial lipase motif, which is not conserved from CHO to human, had no effect. Second, iPLA2 contains eight consecutive ankyrin repeats of approximately 30 amino acids in length. Likewise, one of the C. elegans proteins (F47A4.5) has the two-domain structure of iPLA2 with eight putative ankyrin repeats followed by the region containing the lipase motif. In contrast, T04B2.5 (408 amino acids) has no ankyrin repeats and corresponds to the carboxyl-terminal half of iPLA2 (340 amino acids). This raises the possibility that F47A4.5 is the C. elegans homolog of iPLA2. More speculatively, T04B2.5, which has a theoretical molecular size of 45 kDa, may be a related lipase that does not require the ankyrin repeat motif for lipolytic activity and, potentially, could be related to the 40-kDa calcium-independent activities observed by others (21, 25).

The presence of ankyrin repeats in iPLA2 is a novel and intriguing result, since this structural motif has been found in many proteins of diverse function (49, 50), including transcriptional regulation, cell cycle control, and cell differentiation. Indeed, the predominant function of ankyrin repeats, although poorly defined, appears to be protein-protein interactions in three broad categories: first, the coupling of the spectrin network on the cytoplasmic face of the plasma membrane in neural cells and erythrocytes with the cytoplasmic domain of various integral membrane proteins (51); second, the formation of functionally active protein complexes, e.g. the Ets family of nuclear proteins (52); and third, the masking of regulatory signals, e.g. the retention of NF-kappa B by Ikappa Balpha or the p105 precursor of p50 in the cytoplasm (53, 54).

The demonstration that iPLA2, with or without the Flag epitope, is not posttranslationally modified to a smaller catalytically active protein, at least when expressed in COS or CHO cells, implies the ankyrin repeats are not masking activity. This observation is supported by the loss of lipolytic function when the ankyrin repeats and the first 150 amino acids before the repeats are deleted, although the lipase consensus sequence is retained. Although further deletion experiments will be required to fully define the functional boundaries of the domains. However, the results imply that some or all of the ankyrin repeats are required for function, possibly to enable the ordered association of 85-kDa subunits to give the 250-350-kDa oligomeric complex found by gel permeation chromatography. Also, this region may contain amino acid residues that support catalytic function at the active site of iPLA2. Overall these results suggest that iPLA2 is not the precursor to a 40-kDa species analogous to the one found by others (21, 25), but rather that the 85-kDa protein is similar to the 80-kDa activity from the murine macrophage-like cell line, P388D1 (34). Indeed, the 80-kDa protein is recognized by the anti-iPLA2 polyclonal sera and has the same electrophoretic mobility as the 85-kDa species (accompanying manuscript (Ref. 59)).

In preliminary experiments we found the phospholipid selectivity of iPLA2 and the relative reactivity for a particular substrate to be highly dependent upon substrate presentation and detergent concentration. Indeed, our results parallel those found for the 80-kDa activity from P388D1 cells, including the 5:1 regioselectivity for sn-2 versus sn-1 fatty acids (34). At 500 µM Triton X-100, the apparent substrate preference is 1,2-dipalmitoyl PC > 1-palmitoyl-2-arachidonyl PC > 1-hexadecyl-2-arachidonyl PC. Conversely, at 50 µM Triton X-100, the order of preference for these three substrates was reversed, analogous to the substrate selectivity exhibited by the 40-kDa myocardial activity in a vesicle assay (21). iPLA2 does exhibit an intrinsic lysophospholipase and PLA1 activity, depending on the assay conditions. However we found no stimulation of lipolysis by the presence of ATP in either assay system compared with others (23, 34).

Notably, iPLA2 is a PAF acetylhydrolase indicating a unique flexibility at the active site in being able to accommodate not only long but also short chain fatty acids. This is in contrast to the known intracellular PAF acetylhydrolases that are specific for short chain sn-2 substituents (42, 55, 56). Further, the enzyme has a marked preference, at 50 µM Triton X-100, for the phosphatidic acid substrate 1,2-dipalmitoyl PA producing the lysophospholipid, lysophosphatidic acid, a second messenger implicated in a number biological events (2, 3). Only two other distinct PA-specific phospholipase A2 activities have been identified, neither of which appears to be biochemically or biophysically similar to iPLA2; the first isolated from activated platelets is calcium-dependent (43), and the second, a 58-kDa protein from rat brain, is calcium-independent and specific for phosphatidic acid over other phospholipid head groups (57).

Additionally, iPLA2 has similar sensitivity to inhibition by BEL, palmitoyl trifluoromethyl ketone, and methyl arachidonyl fluorophosphonate (accompanying manuscript (Ref. 59)) as the P388D1 iPLA2 (35-37). However, although BEL at 30 µM completely inhibits iPLA2 and has no effect on cPLA2 activity in cell lysates, caution should be exercised in using this reagent on intact cells. For instance, the above concentration of BEL used to inhibit iPLA2 will suppress cPLA2-mediated arachidonic acid release by 50% using a CHO cell line overexpressing cPLA2, E5 (16). Similarly, calcium ionophore-induced production of prostaglandin E2 from COS cells transiently transfected with cPLA2 and COX-2 is reduced by 70%.2

In summary, we have described the isolation, molecular cloning, and characterization of a novel 85-kDa cytosolic calcium-independent phospholipase A2, iPLA2. The unanticipated discovery is the presence of the ankyrin repeats, an important structural and functional motif for many proteins. Clearly, there is ample precedent to suggest that this motif has two roles that are not mutually exclusive: first, to cause the self-association of iPLA2 to form a catalytically competent species that can bind a lipid bilayer, and second, to mediate the interaction with other proteins that may regulate or direct lipolytic activity in particular intracellular locations. Defining these interactions and determining whether the PAF acetylhydrolase activity, as well as the selectivity for phosphatidic acid are biologically relevant will answer the question of involvement of iPLA2 in the inflammatory cascade, the production of lipid second messengers, and general lipid metabolism.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Genetics Institute, 87 Cambridge Park Dr., Cambridge, MA 02140. Tel.: 617-876-1170; Fax: 617-498-8993; E-mail: sjones{at}genetics.com.
1   The abbreviations used are: PAF, platelet-activating factor; sPLA2, secretory phospholipase A2; cPLA2, cytosolic calcium-dependent phospholipase A2; iPLA2, cytosolic calcium-independent phospholipase A2; BEL, bromoenol lactone; PAGE, polyacrylamide gel electrophoresis; PC, phosphorylcholine; PI, phosphorylinositol; PA, phosphatidic acid; CHO, Chinese hamster ovary; DTT, dithiothreitol; PAPC, 1-palmitoyl-2-[14C]arachidonyl PC; kb, kilobase pair(s).
2   S. Jones, unpublished observations.

ACKNOWLEDGEMENTS

We thank Steve Venuti and Richard Zollner for the CHO cells; Jim Vath for mass spectrometric analysis of tryptic peptides; Kerry Kelleher, Kevin Bean, and Heather Finnerty for DNA sequencing; the DNA Synthesis Group for synthetic oligonucleotides; and Xiao-Jia Chang for purifying the GST-iPLA2 fusion protein. We are especially grateful to Lih-Lin Lin, James Clark, and John Knopf for critically reviewing the manuscript and for many valuable discussions.


Note Added in Proof

It came to our attention during review of this paper that the gene described herein was isolated from CHO cells using the sequence we deposited in GenBankTM, accession number I15470[GenBank] (U. S. Patent 5466595, filed Apr. 14, 1995) by Wolf and Gross (Wolf, M. J., and Gross, R. W. (1996) J. Biol. Chem. 271, 30879-30885). We would like to correct the statement in the paper of Wolf and Gross that the cDNA we isolated was transiently expressed in the same cell line from which it was obtained, i.e. Chinese hamster ovary cells, The cDNA was transiently expressed in the monkey kidney cell line, COS, demonstrating the cDNA encodes a bona fide catalytic activity rather than an activator of an endogenous activity as was suggested in their paper.


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