A Novel Calcium-independent Phospholipase A2, cPLA2-gamma , That Is Prenylated and Contains Homology to cPLA2*

Kathryn W. UnderwoodDagger , Chuanzheng SongDagger , Ronald W. Kriz, Xiao Jia Chang, John L. Knopf, and Lih-Ling Lin§

From the Small Molecule Drug Discovery Group, Genetics Institute, Cambridge, Massachusetts 02140

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

We report the cloning and characterization of a novel membrane-bound, calcium-independent PLA2, named cPLA2-gamma . The sequence encodes a 541-amino acid protein containing a domain with significant homology to the catalytic domain of the 85-kDa cPLA2 (cPLA2-alpha ). cPLA2-gamma does not contain the regulatory calcium-dependent lipid binding (CaLB) domain found in cPLA2-alpha . However, cPLA2-gamma does contain two consensus motifs for lipid modification, a prenylation motif (-CCLA) at the C terminus and a myristoylation site at the N terminus. We present evidence that the isoprenoid precursor [3H]mevalonolactone is incorporated into the prenylation motif of cPLA2-gamma . Interestingly, cPLA2-gamma demonstrates a preference for arachidonic acid at the sn-2 position of phosphatidylcholine as compared with palmitic acid. cPLA2-gamma encodes a 3-kilobase message, which is highly expressed in heart and skeletal muscle, suggesting a specific role in these tissues. Identification of cPLA2-gamma reveals a newly defined family of phospholipases A2 with homology to cPLA2-alpha .

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Phospholipases A2 (PLA2)1 are a diverse group of enzymes that hydrolyze the sn-2 fatty acids from phospholipids and play a role in a wide range of physiological functions. Of particular interest is the role of these enzymes in the production of factors involved in mediating the inflammatory response. The phospholipases A2 family is large, and individual members can be classified according to localization (extracellular versus intracellular), sequence homology, and biochemical characteristics (1). Known PLA2 members include the secreted PLA2s and the cytosolic PLA2s. To date only two cytosolic PLA2 sequences have been reported: the calcium-dependent PLA2 (cPLA2-alpha ) and the calcium-independent PLA2 (iPLA2) (2-5). cPLA2-alpha has a predicted molecular mass of 85 kDa and contains two domains, a calcium-dependent lipid binding (CaLB) domain and a catalytic domain (2, 6). The catalytic domain contains a lipase consensus sequence and a novel catalytic triad that employs a serine, an aspartate, and an arginine instead of the usual serine, aspartate, and histidine found in many lipases and serine proteases (7-9). cPLA2-alpha activity is regulated by the activation of the CaLB domain in response to increased intracellular calcium (6). The activated CaLB domain translocates the enzyme to its substrate in the nuclear envelope and endoplasmic reticulum (10). cPLA2-alpha activity is also increased by the phosphorylation of a MAP kinase consensus site, in response to stimulation of cells with cytokines such as tumor necrosis factor and interleukin 1 (11, 12). These same cytokines have also been found to increase the expression of cPLA2-alpha (11, 12). Although there have been many studies that suggest the importance of cPLA2 in the generation of prostaglandins and leukotrienes, the most convincing data have come from studies using mice that are genetically deficient in cPLA2 (13, 14). Studies demonstrate that cPLA2-alpha is essential for both the calcium ionophore, A23187, and lipopolysaccharide-induced prostaglandin E2 and leukotriene B4 production in peritoneal monocytes (13, 14). The possible importance of cPLA2 in asthma was also shown (13).

The 85-kDa calcium-independent iPLA2, purified by two groups, shares no homology with cPLA2-alpha except, like other lipases, it contains the critical consensus sequence, GXSXG (4, 5, 15). Interestingly, iPLA2 contains a domain of eight ankyrin repeats, which may be involved in protein-protein interactions (4). iPLA2 possesses no clear preference for a fatty acid at the sn-2 position, and it is thought to play a role in the remodeling of phospholipids (16).

Although cPLA2-alpha and iPLA2 are the only intracellular PLA2s that have been cloned, many other PLA2 activities, which presently seem to be distinct from cPLA2-alpha and iPLA2, have been reported (17-19). The relationship of the enzymes responsible for these activities to the known PLA2 enzymes will be clear only upon sequence determination.

Our initial efforts to identify additional PLA2 enzymes failed using low stringency cross-hybridization techniques with cPLA2-alpha sequences.2 A search of the expressed sequence tag (EST) data base was quite successful, and two independent cPLA2-alpha related gene fragments were identified. Subsequent sequence analysis of the full-length clones revealed two novel homologs of cPLA2-alpha , designated cPLA2-beta and cPLA2-gamma . The characterization of cPLA2-beta will be described elsewhere.3 Here, we report the sequence and characterization of a novel 60.9-kDa calcium-independent, membrane-associated cPLA2, cPLA2-gamma .

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Culture and Antibodies-- COS cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 50 units/ml penicillin, 50 µg/ml streptomycin, and 1 mM glutamine. Cells were incubated in a 37 °C humidified atmosphere with 10% CO2. Chinese hamster ovary (CHO) cells were maintained in alpha medium (Life Technologies, Inc.) containing 50 units/ml penicillin, 50 µg/ml streptomycin, 1 mM glutamine, 1 mg/ml G418 (Life Technologies, Inc.), and 10% dialyzed fetal calf serum supplemented with 10 µg each of adenosine, deoxyadenosine, and thymidine per ml for parental cells, 5 nM methotrexate for cPLA2-gamma overexpressing cells, and 20 nM methotrexate for cPLA2-alpha overexpressing cells. Cells were incubated in a 37 °C humidified atmosphere with 5% CO2. Rabbit polyclonal antibodies 44282 and 44284, generated against peptides from cPLA2-gamma , amino acids 416-434, and amino acids 476-495, were used for immunoprecipitation and immunoblotting. Rabbit polyclonal anti-human Ras (Upstate Biotechnology) was used for immunoprecipitation.

Clone Identification-- The EST clone 258543 (GenBank accession N56796) was identified by searching the GenBank EST data base using the amino acid sequence of cPLA2-alpha . The 900-base pair EcoRI-NotI fragment from clone 258543 was used to screen 106 recombinates of the oligo(dT) primed human skeletal muscle library (Stratagene). Clone 19A, which is a phagemid DNA excised from the Lambda Uni-Zap XR phage vector, had its DNA sequence determined.

Northern Blot Analysis-- Northern analysis was performed on multiple human tissues blot (CLONTECH) using a random-primed 32P-labeled EcoRI-NotI fragment of cPLA2-gamma . The blot was washed under high stringency conditions (0.2 × SSC at 65 °C). Each lane comprises approximately 2 µg of poly(A)+ mRNA.

Construction of Expression Vectors-- Two oligonucleotides, 5'-GTTCACCTCATCCTCTCCTTCGAC-3' and 5'-TCGGGGTACCGAATTCGGGCCCTATGCCAAGCAGCAACTTCGGGCACT-3', corresponding to the 3'-end of cPLA2-gamma , were used to amplify the 3'-end coding region of cPLA2-gamma by polymerase chain reaction, using clone 19A DNA as a template. The polymerase chain reaction product was digested with XbaI and EcoRI and ligated with the XbaI-EcoRI fragment of clone 19A and the EcoRI-digested vector pEDDelta C. The resulted clone, named pEDDelta C-cPLA2-gamma WT, was sequenced to confirm the desired C-terminal coding sequence. Mutation of the C-terminal -CCLA was produced as above except using an oligonucleotide that introduced the mutation (C538S,C539S) to the 3' end of the sequence. The resulted clone, named pEDDelta C-cPLA2-gamma SSLA, was confirmed by sequencing to contain the desired C-terminal coding sequence.

Stable CHO Cell Lines Overexpressing cPLA2-gamma and cPLA2-alpha -- The plasmid of wild-type cPLA2-gamma was constructed as follows. The restriction fragment (EcoRI-EcoRI) containing the entire coding sequence was isolated from pEDDelta C-cPLA2-gamma WT and ligated to a controlled expression vector pHTOP. pHTOP was modified from pED6 vector (20) essentially by inserting a 289-base pair tetracycline operator sequence (21) at the XhoI site of pED6. The expression vector of transactivator (tTA) was generated similarly as described by Gossen and Bujard (21) using neomycin transferase as selection marker.

Stable CHO cell lines of cPLA2-gamma and cPLA2-alpha were generated by transfecting CHO cells that constitutively express tTA with pHTOP-cPLA2-gamma or pTOP6-cPLA2-alpha . Transfection was performed using Lipofectin as recommended by the manufacturer (Life Technologies, Inc.). Transfectants were then selected in growth medium containing methotrexate at a concentration of 5-100 nM.

Activity Assay-- Cell pellets (either from CHO cells overexpressing cPLA2-gamma or cPLA2-alpha or from COS cells transiently transfected with cPLA2-gamma or cPLA2-alpha ) were resuspended in lysis buffer (10 mM HEPES, pH 7.5, 1 mM EDTA or 1 mM EGTA, 0.1 mM dithiothreitol, 0.34 M sucrose, and 1 µg/ml leupeptin). Cells were lysed by nitrogen cavitation (750-1000 psi, 10 min) on ice. Approximately 5-15 µg of cell lysate (determined by Bradford assay, Bio-Rad) was used in the assay. 1-[14C]Palmitoyl-2-arachidonyl-phosphatidylcholine (PC) (57 Ci/mmol), 1-palmitoyl-2-[14C]arachidonyl-PC (55-58 Ci/mmol), 1-palmitoyl-2-[14C]oleoyl-PC (58 Ci/mmol), 1-palmitoyl-2-[14C]linoleoyl-PC (58 Ci/mmol), and 1-O-hexadecyl-2 [3H]arachidonyl-PC (200 Ci/mmol) were obtained from DuPont NEN. Unlabeled 1-O-hexadecyl-2-arachidonyl-PC was obtained from Biomol. The lipids were dried under N2 and sonicated with vesicle buffer (50 mM HEPES, pH 7.5, 1 mM EGTA or 5 mM EGTA, and 7 mM CaCl2 for regiospecificity assay, 30% glycerol, 1 mg/ml fatty acid-free bovine serum albumin, and 150 mM NaCl) adapted from Ghomashchi et al. (22). Aliquots of lysate were incubated with substrate (20 µM) at 37 °C for the indicated amount of time. Released fatty acid was measured as described (23).

Prenylation Assay-- 70-80% confluent COS cells (10-cm plate) were transfected with 8 µg of pMev (obtained from ATCC (24), to facilitate uptake of mevalonolactone) and either 8 µg of pEDDelta C-cPLA2-gamma WT, pEDDelta C-cPLA2-gamma SSLA, or pzflag (for vector control) or 4 µg of pCMV-Ras61L (obtained as a generous gift from R. Davis) using lipofectamine according to the manufacturer's instructions (Life Technologies, Inc.). Cells were grown for 2 days and then incubated with 20 µM mevistatin (Biomol) for 1 h. The cells were then incubated in 3 ml of growth media containing 40 µM mevistatin and 150 µCi of [3H]mevalonolactone (29 Ci/mmol) (DuPont NEN) for 14 h (25). Cells were washed twice in phosphate-buffered saline and scraped into 3 ml of phosphate-buffered saline. The cell pellets were lysed in 100 µl of lysis buffer (20 mM Tris-HCl, pH 7.5, 10% glycerol, 1% Triton X-100, 137 mM NaCl, 2 mM EDTA, 5 µg/ml aprotinin, 10 µg/ml leupeptin, and 2 mM phenylmethylsulfonyl fluoride for 10 min. A 20-µl aliquot was removed and centrifuged, and the pellet was resuspended in 50 µl of 2 × Laemmli's sample buffer. SDS to 1% was added to the remaining lysate and incubated for 10 min. Lysis buffer was added to the lysate to 1 ml to reduce the SDS to 0.1%, incubated for another 30 min, and then centrifuged 1 h at 100,000 × g, 4 °C. The supernatant was immunoprecipitated with 10 µl of anti-cPLA2-gamma antibody, 44282, or anti-Ras antibody (Upstate Biotechnology) for 3 h at 4 °C, then incubated with protein A-Sepharose (Amersham Pharmacia Biotech) saturated with 10% bovine serum albumin for 30 min. The beads were washed three times with lysis buffer containing 0.1% SDS and then resuspended in 50 µl of 2 × Laemmli's sample buffer. Samples were subjected to 4-20% SDS-PAGE (Novex), stained with Coomassie Blue, soaked in entensify (Amersham), and dried and exposed to Biomax MS film (Kodak) for 7 days. To monitor the expression levels of cPLA2-gamma , aliquots of the samples were subjected to 4-20% SDS-PAGE. Proteins were transferred to a nitrocellulose filter (Novex), immunoblotted for cPLA2-gamma with 44284 antibody, and detected by ECL (Amersham).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

cPLA2-gamma Is a Novel cPLA2-- The EST data base was searched to identify sequences similar to the human 85-kDa cPLA2 gene (cPLA2-alpha ). This analysis led to the identification of two related genes, named cPLA2-beta and cPLA2-gamma . The cPLA2-gamma EST clone 258543 was shown to contain a partial cDNA insert with sequence similarity to the C terminus of cPLA2-alpha . A full-length clone was isolated from a human skeletal muscle cDNA library. This clone contains a 541-amino acid open reading frame with predicted molecular mass of 60.9 kDa. Comparison of the amino acid sequences of cPLA2-gamma and cPLA2-alpha (Fig. 1) reveals 28.7% identity. Within this putative catalytic domain there exists two subdomains with greater sequence identity. Interestingly, the spacer region separating these two domains corresponds to an area in cPLA2-alpha considered to be an exposed hinge region containing many protease-accessible sites, as well as the MAP kinase activation site Ser-505 (6). cPLA2-gamma contains a sequence that is similar to the lipase consensus sequence, GLSGS, in cPLA2-alpha , which has been found to be critical for cPLA2-alpha activity (7). These sequences are very similar to the lipase consensus sequence, GXSXG, found in many lipases and serine proteases (9, 26). Also conserved in cPLA2-gamma are the amino acids that make up the putative catalytic triad of cPLA2-alpha (8). These amino acids correspond to serine 82, aspartate 385, and arginine 54 in cPLA2-gamma .


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Fig. 1.   Amino acid sequence of cPLA2-gamma and its alignment with cPLA2-alpha . The underline indicates the EST clone 258543 used to clone cPLA2-gamma . The dots represent the serines in cPLA2-alpha that are phosphorylated. The arrows indicate the amino acids in the putative catalytic triad of cPLA2-alpha and the conserved amino acids in cPLA2-gamma . The lipase consensus sequence in cPLA2-alpha and the corresponding sequence in cPLA2-gamma are overlined. Shaded amino acids are conserved, and boxed amino acids are identical. The cDNA sequence can be found in the GenBank data base under accession number AF058921.

cPLA2-alpha contains a CaLB or C-2 domain that has been shown to be important for calcium-dependent binding of the enzyme to membranes (6). cPLA2-gamma does not contain a CaLB domain and is therefore likely to be a novel calcium-independent phospholipase. Interestingly, a motif search of cPLA2-gamma reveals the presence of a C-terminal -CAAX box (-CCLA). This motif has been identified as a signal for prenylation, where C is the cysteine that becomes modified, A is an aliphatic amino acid, and X is any amino acid (27). The N terminus of cPLA2-gamma also contains a sequence that is a potential site for myristoylation (M-G-X-X-X-(S/small uncharged)-X) (Ref. 28 and the Prosite data base). Similar to other lipid-modified proteins, these putative lipid modifications may regulate the localization of cPLA2-gamma within the cell.

To determine the tissue distribution of cPLA2-gamma , Northern blot analysis was performed. Hybridization of the EST clone with RNA from various human tissues indicates that cPLA2-gamma mRNA is approximately a 3-kilobase transcript. Strikingly, cPLA2-gamma is most abundant in skeletal muscle and heart, with lower levels in spleen, brain, placenta, and pancreas (Fig. 2).


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Fig. 2.   Tissue distribution of cPLA2-gamma . Northern blot analysis was performed on various human tissues using a 32P-labeled 900-base pair EcoRI-NotI fragment of cPLA2-gamma . The blot was washed under high stringency conditions. Each lane is composed of approximately 2 µg of poly(A)+ mRNA.

cPLA2-gamma Encodes a Phospholipase A2-- To determine the regioselectivity of cPLA2-gamma , vesicle activity assays were performed using 1-[14C]palmitoyl-2-arachidonyl-PC, 1-palmitoyl-2-[14C]arachidonyl-PC, or 1-O-hexadecyl-2 [3H]arachidonyl-PC. Cell lysates prepared from COS cells transfected with either vector, cPLA2-gamma or cPLA2-alpha , were incubated with the various substrates for the indicated times (Fig. 3). Lysate from cPLA2-gamma transfected cells showed at least 4.4-fold more PLA2 activity than the lysate from vector transfected cells. Importantly, cPLA2-gamma readily liberated arachidonate when 1-O-alkyl phospholipid (1-O-hexadecyl-2 [3H]arachidonyl-PC) was utilized as a substrate, confirming its ability to cleave the sn-2 site. Comparing the activity of cPLA2-gamma in the presence of the substrates using 1-[14C]palmitoyl-2-arachidonyl-PC, 1-palmitoyl-2-[14C]arachidonyl-PC, or 1-O-hexadecyl-2 [3H]arachidonyl-PC, cPLA2-gamma seems to be as proficient at cleaving at the sn-1 site as the sn-2 site. This is dissimilar from cPLA2alpha (Fig. 3B), which under the conditions used has no apparent sn-1 activity. Importantly, the PLA1 and PLA2 activity of cPLA2-gamma does not seem to be sequential, as the radiolabeled fatty acid released from 1-palmitoyl-2-[14C]arachidonyl-PC and using 1-[14C]palmitoyl-2-arachidonyl-PC showed similar kinetics.


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Fig. 3.   Regioselectivity of cPLA2-gamma in a vesicle assay. Cell lysates prepared from COS cells transfected with cPLA2-gamma (A), cPLA2-alpha (B), or vector were incubated with 1-[14C]palmitoyl-2-arachidonyl-PC (*PAPC), 1-palmitoyl-2-[14C]arachidonyl-PC (P*APC), or 1-O-hexadecyl-2 [3H]arachidonyl-PC (O-H*APC) vesicles at 37 °C for the indicated times. The amount of released fatty acid was determined. Points are the average of duplicates (± range of the mean), with background activity from vector-transfected cells subtracted from the values. The amount of COS lysate used was approximately 6-7 µg/reaction. Lysates containing cPLA2-alpha were diluted 1: 50 with lysate from vector-transfected cells.

cPLA2-gamma Is Prenylated-- cPLA2-gamma contains the C-terminal sequence -CCLA, which is a motif for prenylation. Protein prenylation is mediated by the addition of either farnesyl (C-15) or geranylgeranyl (C-20) to the cysteine of the CAAX motif (29). The process is initiated by cleavage of the three most C-terminal amino acid residues -AAX, followed by methylation of the cysteine carboxyl group (29). The sequence of cPLA2-gamma also resembles the sequence CCXX, which is another motif for prenylation (27). This motif signals the addition of geranylgeranyl to the protein and occurs via mechanisms that differ from the CAAX modification. This motif is mostly found on the Rab family of proteins (29).

To investigate the utilization of the prenylation motif on cPLA2-gamma , COS cells were transfected with either cPLA2-gamma or cPLA2-gamma with the C terminus mutated from CCLA to SSLA. A plasmid encoding Ras was transfected as a positive control for prenylation. Approximately 48 h post-transfection, the cells were incubated for 14 h with the isoprenoid precursor, [3H]mevalonolactone. Cell lysates were prepared as described under "Experimental Procedures" and analyzed by SDS-polyacrylamide gel electrophoresis. Autoradiographic analysis reveals a band at 60 kDa in cells that were transfected with the wild-type cPLA2-gamma (Fig. 4A). Whereas cells transfected with plasmid encoding the mutant protein, vector only, or Ras show no bands at 60 kDa. Similar results were obtained when the cell lysate was immunoprecipitated with cPLA2-gamma antibody, 44282, confirming that the 60-kDa protein is cPLA2-gamma (data not shown). To determine if equal amounts of wild-type and mutant protein were expressed in the COS cells, Western blot analysis was performed on the samples and indicated equal expression of the two proteins (Fig. 4B). These data indicate that cPLA2-gamma is prenylated at its C terminus.


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Fig. 4.   Post-translational modification of cPLA2-gamma . COS cells were transiently transfected with the mevalonate transporter, pMev, and either wild-type cPLA2-gamma (WT), cPLA2-gamma -SSLA mutant (SSLA), vector, or Ras. Approximately 48 h post-transfection, cells were labeled with 150 µCi of [3H]mevalonolactone for 14 h. Cells were processed as stated under "Experimental Procedures." 1% Triton X-100 pellets were subjected to 4-20% SDS-PAGE, soaked in entensify and exposed to Biomax MS film for 7 days (A). To examine the expression levels of cPLA2-gamma , aliquots of samples were subjected to 4-20% SDS-PAGE, followed by Western blot analysis using anti-cPLA2-gamma antibody, 44284 (B).

cPLA2-gamma Is a Membrane-associated Protein-- To determine the subcellular localization of cPLA2-gamma , CHO cells stably transfected with cPLA2-gamma were lysed by nitrogen cavitation and centrifuged for 1 h at 100,000 × g. cPLA2-gamma was then detected by Western analysis. As shown in Fig. 5, cPLA2-gamma is found to localize to the particulate (pellet) fraction. Treatment of the particulate fraction with 1% Triton X-100 followed by centrifugation at 100,000 × g results in the majority of the enzyme being present in the supernatant. This is unlike cPLA2-alpha , which is found in the supernatant in the absence of calcium and in the pellet fraction following the addition of calcium (6, 10). Lipid modification may be responsible for the membrane association of cPLA2-gamma , as is the case for Ras. However, fractionation of cells transfected with cPLA2-gamma mutated at both the N- and C termini, to disrupt possible lipidation sites, revealed that this mutated protein remains in the membrane fraction (data not shown). This result indicates that there is another component that is involved in the association of cPLA2-gamma with the membrane.


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Fig. 5.   Localization of cPLA2-gamma in CHO cells stably transfected with cPLA2-gamma . CHO cells overexpressing cPLA2-gamma were lysed by nitrogen cavitation (CL) and spun for 1 h at 100,000 × g at 4 °C. The supernatant was collected (S). The pellet (P) was resuspended in lysis buffer containing 1% Triton X-100 and centrifuged for 1 h at 100,000 × g at 4 °C. The supernatant and pellet fractions were collected. Samples were subjected to 4-20% SDS-PAGE followed by Western blot analysis using anti-cPLA2-gamma antibody, 44282.

cPLA2-gamma Is Calcium-independent-- Unlike the secreted PLA2s, which require millimolar concentrations of calcium for activity, the catalytic domain of cPLA2-alpha does not require calcium for activity (6). However, cPLA2-alpha does require micromolar concentrations of calcium for membrane binding through its CaLB domain. We were interested in determining the requirement of calcium for cPLA2-gamma activity. Cell lysates prepared from COS cells transfected with cPLA2-gamma or cPLA2-alpha were incubated with 1-palmitoyl-2-[14C]arachidonyl-PC in the presence of 0 or 10 µM or 10 mM calcium (Fig. 6). cPLA2-gamma activity is unaffected by calcium, unlike cPLA2-alpha activity, which increases 14-fold in the presence of 10 µM calcium. This result demonstrates that cPLA2-gamma is a calcium-independent enzyme.


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Fig. 6.   Effect of calcium on cPLA2-gamma activity. Cell lysates prepared from COS cells transiently transfected with cPLA2-gamma (A) and cPLA2-alpha (B) were incubated with 1-palmitoyl-2-[14C]arachidonyl-PC vesicles in the presence of 1 mM EGTA (black-square), 10 µM (bullet ), or 10 mM (black-triangle) free calcium for the indicated times. The amount of released fatty acid was determined. Points are the average of duplicates (± range of the mean). The amount of cPLA2-gamma lysate used per reaction was 15 µg, and the amount of cPLA2-alpha lysate used per reaction was 8 µg.

Substrate Specificity at the sn-2 Position-- cPLA2-alpha selectively hydrolyzes arachidonic acid at the sn-2 position in several assay formats, whereas iPLA2 selectivity is significantly more assay dependent. To determine if cPLA2-gamma has a preference for the fatty acid at the sn-2 position, vesicle assays were performed using phosphatidylcholine that contains palmitoyl in the sn-1 position and radiolabeled arachidonyl, oleyl, linoleyl, or palmitoyl in the sn-2 position. The substrates were incubated for 15 min with lysates from CHO cells stably transfected with either cPLA2-gamma or cPLA2-alpha . cPLA2-gamma seems to prefer lipids that are unsaturated at the sn-2 position, and it does seem to prefer arachidonic acid approximately 3.5-fold over palmitic acid (Table I).

                              
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Table I
Fatty acid selectivity of cPLA2-gamma and cPLA2-alpha in CHO lysates
Lysates from CHO cells stably transfected with either cPLA2-gamma or cPLA2-alpha were incubated with PC vesicles that contain palmitoyl in the sn-1 position and [14C]arachidonyl, [14C]oleyl, [14C]linoleyl, or [14C]palmitoyl in the sn-2 position for 15 min at 37 °C. The amount of released fatty acid was determined. Points are the average of triplicates (±SD) and are corrected for nonenzymatic hydrolysis. 10 µg of CHO lysates overexpressing cPLA2-gamma (approximately 205 ng of cPLA2-gamma ) and 5 µg of CHO lysates overexpressing cPLA2-alpha (approximately 195 ng of cPLA2-alpha ) were used per reaction.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

We have identified a novel 60.9-kDa calcium-independent phospholipase A2, which we termed cPLA2-gamma . cPLA2-gamma contains 28.7% overall sequence identity with cPLA2-alpha and was identified by searching the EST data base for related proteins.

A common motif found in many lipases is the consensus sequence, GXSXG, which is essential for enzymatic activity (9, 26). cPLA2-gamma contains the sequence GVS82GS, which is similar but slightly different from the consensus. However, this sequence aligns with the sequence in cPLA2-alpha , and the change from glycine to serine also occurs in the corresponding region of cPLA2-alpha , GLS228GS (7). Mutation of serine-228 and aspartate-549 was shown to abolish cPLA2-alpha activity, consistent with their role in the putative catalytic triad (7, 8). Catalytic triads of lipases and serine proteases also frequently contain critical histidines; however, mutation of these residues in the catalytic domain of cPLA2-alpha had no affect on activity (8, 9, 26). Surprisingly, mutation of arginine-200, in what is thought to be the novel catalytic triad of cPLA2-alpha , abrogated cPLA2-alpha activity (8). These amino acids may serve as a catalytic triad, providing the active site for hydrolysis, or it is possible that the arginine may function in another but unknown critical role, such as in transition state stabilization (8). We have also shown that serine, aspartate, and arginine are conserved in cPLA2-gamma , providing further evidence that these amino acids are important and may indeed be part of a novel catalytic triad.

cPLA2-alpha is regulated by at least two post-translational mechanisms: 1) calcium-induced membrane association through its CaLB domain and 2) phosphorylation of serine-505 by a MAP kinase (6, 10, 30). The phosphorylation site and the CaLB domain of cPLA2-alpha are not conserved in the sequence of cPLA2-gamma , suggesting a different regulatory mechanism. Interestingly, cPLA2-gamma contains a potential prenylation motif at its C terminus and a putative signal for myristoylation at its N terminus. Initial studies have failed to indicate that the myristoylation site is utilized, whereas the prenylation site is indeed utilized. The isoprenoid precursor [3H]mevalonolactone is readily incorporated into cPLA2-gamma expressed in COS cells. We do not know, however, if the modifying isoprenoid is a farnesyl or a geranylgeranyl. Generally, in the consensus sequence CAAX, when the C-terminal (X) amino acid is a methionine, serine, glutamine, or alanine, this signals that the lipid will be farnesyl (27), whereas a leucine signals that the modifying lipid is a geranylgeranyl. However, there is also a motif XXCC, CXC, or CCXX found on the Rab family of proteins that modifies the two cysteines with geranylgeranyl (31). Because both of these motifs match the cPLA2-gamma sequence (-CCLA), we do not know which of these isoprenoids is modifying the protein.

One of the most striking differences between cPLA2-alpha and -gamma is the lack of a lipid binding CaLB domain in cPLA2-gamma . The presence of lipidation motifs suggests that these regions may function as the CaLB domain in cPLA2-alpha , localizing the enzyme to the membrane and being critical for activity. However, in a preliminary study, cPLA2-gamma mutant protein that disrupts the possible N- and C-terminal lipid modification sites did not affect its activity in a phopholipid vesicle assay.4 Moreover, this mutant protein fails to alter its association with the membrane fraction. However, it remains possible that lipid modification may be important in the subcellular localization of cPLA2-gamma and/or its ability to associate with other proteins. Interestingly, Ras shows increased affinity toward other proteins when it is prenylated compared with its nonprenylated form, and it has been shown that oncogenic forms of Ras need to be modified to transform cells (29). Therefore, it is possible that lipid modifications may play a role in regulating the activity of cPLA2-gamma in the cells.

cPLA2-gamma will hydrolyze fatty acids at the sn-1 and sn-2 position of phosphatidylcholine. This suggests that cPLA2-gamma contains PLA1 and PLA2 activity. The evidence that cPLA2-gamma contains PLA2 activity is also confirmed by its ability to cleave 1-O-hexadecyl-2-arachindonyl-phosphatidylcholine. However, we do not know whether the sn-1 cleavage is PLA1 activity or if it is cleavage of the lysophospholipid. The kinetics of the reactions suggest that it is PLA1 activity, as the hydrolysis of sn-1 would show a time-dependent lag as compared with sn-2 hydrolysis if sequential cleavage were taking place, unless cleavage of sn-1 from lysophospholipid occurs rapidly. Taken together, all of these data provide evidence that cPLA2-gamma is an enzyme with PLA2 activity and a probable PLA1 activity.

cPLA2-gamma prefers arachidonic acid to palmitic acid in the sn-2 position of phosphatidylcholine. However, this preference is modest in comparison to the strong preference that cPLA2-alpha displays for arachidonic acid, 3.5-fold versus 24.5-fold, respectively. The substrate specificity of cPLA2-gamma should be considered cautiously, however, because of the artificial nature of the substrate presentation. The selectivity of the enzyme using a natural membrane as a substrate may be a more relevant method to determine the preferred physiological substrate for this enzyme.

The preferred substrate for an enzyme provides a clue to its physiological role, as can its distribution within tissues. cPLA2-gamma is highly expressed in heart and skeletal muscle. The calcium independence of this enzyme may be important for its high expression in muscle, where contractions cause large fluxes in calcium concentrations. Therefore, it may be necessary in this environment to regulate a phospholipase in a calcium-independent manner, such as phosphorylation. As previously stated, cPLA2-alpha activity is also regulated by MAP kinase phosphorylation of serine-505. This serine is not conserved in the sequence of cPLA2-gamma ; however, there are several potential protein kinase C phosphorylation sites, which may be utilized to regulate the enzyme.

cPLA2-gamma may be highly expressed in these muscles because heart and skeletal muscle encounter physical stress upon increased load. It may be necessary to regulate the remodeling of the phospholipid bilayer when cells undergo stress. This speculation is substantiated by the reports of several calcium-independent phospholipases expressed in heart (17-19). Hazen et al. (18) and McHowat and Creer (19) have identified membrane-bound, calcium-independent PLA2 activity that prefers the myocardia-abundant lipid, plasmologen, as a substrate. They have shown increased hydrolysis of plasmologen under hypoxic conditions, such as in ischemia. It is believed that in ischemia, a PLA2 activity leads to the accumulation of lysophospholipids and subsequent injury to the heart tissue because of disruptions of the membrane. As the PLA2 activity of this myocardial membrane-bound enzyme was shown to increase under hypoxic conditions, it was suggested that this calcium-independent PLA2 is physiologically involved in ischemia (18). Because cPLA2-gamma is abundantly expressed in heart, and its properties (including calcium independence and membrane localization) are similar to that reported in heart muscle, it may be that cPLA2-gamma is involved in ischemia-induced injury to heart muscle.

In summary, we have described the molecular cloning and initial characterization of a novel 60.9-kDa membrane-associated, calcium-independent PLA2, cPLA2-gamma . This enzyme shares identity with cPLA2-alpha and contains the potential critical amino acids for the catalytic site but is missing the key elements that regulate the activity of cPLA2-alpha . This suggests that the mechanisms of regulation for cPLA2-gamma will be different from that of cPLA2-alpha and quite possibly employs the use of the lipid modification. Defining the mechanisms of regulation and the physiological substrate of cPLA2-gamma should shed some light on the physiological role of this newly identified PLA2.

    ACKNOWLEDGEMENTS

We thank Roger Davis for pCMV-RAS61L; Dianne Sako for the cPLA2-alpha :CHO cell line; Kevin Bean, Kerry Kelleher, and Heather Finnerty for DNA sequencing; and Mark Proia for screening the cDNA library. We are especially grateful to James Clark, Simon Jones, and Thomas Noland for many helpful discussions.

    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 Both authors contributed equally to this work.

§ To whom correspondence should be addressed: Genetics Institute, 87 Cambridge Park Dr., Cambridge, MA 02140. Tel.: 617-498-8934; Fax: 617-498-8993.

The abbreviations used are: cPLA2, cytosolic phospholipase A2CaLB, calcium-dependent lipid bindingiPLA2, cytosolic calcium-independent PLA2MAP, mitogen-activated proteinEST, expressed sequence tagPC, phosphatidylcholineCHO, Chinese hamster ovaryPAGE, polyacrylamide gel electrophoresis.

2 R. Kriz, unpublished results.

3 C. Song, X. J. Chang, K. Bean, M. Proia, J. L. Knopf, and R. W. Kriz, manuscript in preparation.

4 K. Underwood, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Dennis, E. A. (1997) Trends Biochem. Sci. 22, 1-2[CrossRef][Medline] [Order article via Infotrieve]
  2. Clark, J. D., Lin, L.-L., Kriz, R. W., Ramesha, C. S., Sultzman, L. A., Lin, A. Y., Milona, N., and Knopf, J. L. (1991) Cell 65, 1043-1051[Medline] [Order article via Infotrieve]
  3. Sharp, J. D., White, D. L., Chiou, X. G., Goodson, T., Gamboa, G. C., McClure, D., Burgett, S., Hoskins, J., Skatrud, P. L., Sportsman, J. R., Becker, G. W., Kang, L. H., Roberts, E. F., and Kramer, R. M. (1991) J. Biol. Chem. 266, 14850-14853[Abstract/Free Full Text]
  4. Tang, J., Kriz, R., Wolfman, N., Shaffer, M., Seehra, J., and Jones, S. (1997) J. Biol. Chem. 272, 8567-8575[Abstract/Free Full Text]
  5. Balboa, M., Balsinde, J., Jones, S., and Dennis, E. (1997) J. Biol. Chem. 272, 8576-8580[Abstract/Free Full Text]
  6. Nalefski, E. A., Sultzman, L. A., Martin, D. M., Kriz, R. W., Towler, P. S., Knopf, J. L., and Clark, J. D. (1994) J. Biol. Chem. 269, 18239-18249[Abstract/Free Full Text]
  7. Sharp, J. D., Pickard, R. T., Chiou, X. G., Manetta, J. V., Kovacevic, S., Miller, J. R., Varshavsky, A. D., Roberts, E. F., Strifler, B. A., Brems, D. N., and Kramer, R. M. (1994) J. Biol. Chem. 269, 23250-23254[Abstract/Free Full Text]
  8. Pickard, R., Chiou, X., Strifler, B., DeFelippis, M., Hyslop, P., Tebbe, A., Yee, Y., Reynolds, L., Dennis, E., Kramer, R., and Sharp, J. D. (1996) J. Biol. Chem. 271, 19225-19231[Abstract/Free Full Text]
  9. Derewenda, Z., and Sharp, A. (1993) Trends Biochem. Sci. 18, 20-25[CrossRef][Medline] [Order article via Infotrieve]
  10. Schievella, A., Regier, M., Smith, W., and Lin, L.-L. (1995) J. Biol. Chem. 270, 30749-30754[Abstract/Free Full Text]
  11. Lin, L.-L., Lin, A. Y., and DeWitt, D. L. (1992) J. Biol. Chem. 267, 23451-23454[Abstract/Free Full Text]
  12. Hoeck, W. G., Ramesha, C. S., Chang, D. J., Fan, N., and Heller, R. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4475-4479[Abstract]
  13. Uozumi, N., Kume, K., Nagase, T., Nakatani, N., Ishii, S., Tashiro, F., Komagata, Y., Maki, K., Ikuta, K., Ouchi, Y., Miyazaki, J., and Shimizu, T. (1997) Nature 390, 618-622[CrossRef][Medline] [Order article via Infotrieve]
  14. Bonventre, J., Huang, Z., Taheri, M., O'Leary, E., Li, E., Moskowitz, M., and Sapirstein, A. (1997) Nature 390, 622-625[CrossRef][Medline] [Order article via Infotrieve]
  15. Ackermann, E., and Dennis, E. (1995) Biochim. Biophys. Acta 1259, 125-136[Medline] [Order article via Infotrieve]
  16. Balsinde, J., Balboa, M., and Dennis, E. (1997) J. Biol. Chem. 272, 29317-29321[Abstract/Free Full Text]
  17. Hazen, S., Stuppy, R., and Gross, R. (1990) J. Biol. Chem. 265, 10622-10630[Abstract/Free Full Text]
  18. Hazen, S., Ford, D., and Gross, R. (1991) J. Biol. Chem. 266, 5629-5633[Abstract/Free Full Text]
  19. McHowat, J., and Creer, M. (1997) Am. J. Physiol. 272, H1972-H1980[Abstract/Free Full Text]
  20. Davies, M., and Kaufman, R. (1992) J. Virol. 66, 1924-1932[Abstract]
  21. Gossen, M., and Bujard, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5547-5551[Abstract]
  22. Ghomashchi, F., Schuttel, S., Jain, M. K., and Gelb, M. H. (1992) Biochemistry 31, 3814-3824[Medline] [Order article via Infotrieve]
  23. Clark, J. D., Milona, N., and Knopf, J. L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7708-7712[Abstract]
  24. Kim, C., Goldstein, J., and Brown, M. S. (1992) J. Biol. Chem. 267, 23113-23121[Abstract/Free Full Text]
  25. Otto, J. C., and Casey, P. J. (1996) J. Biol. Chem. 271, 4569-4572[Abstract/Free Full Text]
  26. Ollis, D. L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S. M., Harel, M., Remington, S. J., Silman, I., Schrag, J., Sussman, J. L., Verschueren, K. H. G., and Goldman, A. (1992) Protein Eng. 5, 197-211[Abstract]
  27. Clarke, S. (1992) Annu. Rev. Biochem. 61, 355-386[CrossRef][Medline] [Order article via Infotrieve]
  28. Johnson, R. D., Bhatnagar, R. S., Knoll, L. J., and Gordon, J. I. (1994) Annu. Rev. Biochem. 63, 869-914[CrossRef][Medline] [Order article via Infotrieve]
  29. Zhang, F., and Casey, P. (1996) Annu. Rev. Biochem. 65, 241-269[CrossRef][Medline] [Order article via Infotrieve]
  30. Lin, L.-L., Wartmann, M., Lin, A. Y., Knopf, J. L., Seth, A., and Davis, R. J. (1993) Cell 72, 269-278[Medline] [Order article via Infotrieve]
  31. Shen, F., and Seabra, M. C. (1996) J. Biol. Chem. 271, 3692-3698[Abstract/Free Full Text]


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