Molecular Characterization of Cytosolic Phospholipase A2-beta *

Chuanzheng Song, Xiao Jia Chang, Kevin M. Bean, Mark S. Proia, John L. Knopf, and Ronald W. KrizDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

We have isolated a cDNA encoding a 1012-amino acid polypeptide cPLA2-beta , that has significant homology with cPLA2-alpha in both the calcium-dependent lipid binding domain as well as in the catalytic domain. Transient expression of cPLA2-beta cDNA in COS cells results in an increase in calcium-dependent phospholipase A1 (PLA1) and PLA2 activities compared with vector-transfected cells. cPLA2-beta is markedly less selective for cleavage at sn-2 as compared with cPLA2-alpha and cPLA2-gamma . Northern analysis reveals a cPLA2-beta transcript of 8 kilobase pairs that is expressed in all the human tissues examined. With the identification of cPLA2-beta , the newly defined cPLA2 family now comprises three members that may have dramatically different mechanisms for regulation of expression and enzymatic activation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Phospholipase A2 (PLA2,1 EC 3.1.1.4) comprises a large family of structurally and mechanistically distinct enzymes involved in membrane glycerophospholipid hydrolysis (1). Phospholipases A2 that reside in the cytosol are of particular interest because of their ability to interact directly with signal transduction elements such as G-proteins and kinases. The mammalian 85-kDa calcium-dependent cytosolic PLA2 (cPLA2-alpha ) is a well studied example of this class of enzyme (2, 3). It is a monomeric polypeptide of 749 amino acids containing an NH2-terminal calcium-dependent regulatory domain responsible for the translocation of the enzyme to membranes of the nuclear envelope upon activation (4-6). The most convincing evidence for the involvement of cPLA2-alpha in the release of arachidonic acid and the initiation of subsequent proinflammatory response comes from mice that have been made genetically deficient for cPLA2 (7, 8). Stimulation of cells derived from these mice with calcium ionophore or lipopolysaccharide results in little if any detectable increase in leukotrienes, prostaglandins, or platelet-activating factor. Importantly, these mice have a markedly reduced sensitivity to antigen induced bronchoconstriction believed to be mediated at least in part by leukotrienes.

cPLA2-alpha lacks sequence similarity to other members of the PLA2 super family. Although low-stringency hybridization of genomic DNA failed to identify related genes, two cPLA2- homologs, cPLA2-beta and cPLA2-gamma (9), have been identified in the EST data base using a cPLA2-alpha sequence as a query. In this report, we describe the molecular cloning and initial biochemical characterization of cPLA2-beta , which shares about 30% identity to cPLA2-alpha .

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Library Construction-- cDNA libraries were constructed from U937 cells using a Poly(A) Track kit for isolation of mRNA (Promega), a Superscript Choice kit for the generation of double-stranded cDNA (Life Technologies, Inc.), and a LambdaZapII phage cloning kit (Stratagene).

Molecular Cloning of Full-length cPLA2-beta -- Two cPLA2-beta -specific deoxyribonucleotides were designed based on the sequence of EST clone with GenBankTM accession number W92213 (5'-CCTCCTGCAGCCCACTCGGGAC-3' and 5'-GCTGACCAGAGGAAAGTGCAGC-3').

These oligonucleotides were used to screen a U937 cDNA library. One clone that hybridizes with both oligonucleotides, clone 52A, was examined for complete DNA sequence determination. It contains a 4-kilobase pair insert with a partial coding sequence that begins at nucleotide 1560 and continues to a stop codon at nucleotide 3894, representing 778 amino acids. The region on the DNA sequence 5' to nucleotide 1560 was determined to be unspliced intron sequence. A number of cDNAs isolated by hybridization have been examined in this region and also shown to contain intron sequence.

A rapid amplification of cDNA ends (RACE) PCR strategy was used to amplify the 5'-end sequence of cPLA2-beta lacking the intron sequence. Double-stranded human heart cDNA with ligated adapters were purchased from CLONTECH. The cDNA was amplified with primers NPCR1 (5'-GGCTGCAGGCCGTGGGCAGCCAGAGAGTCA-3') and AP1 (the outer primer of the CLONTECH RACE PCR kit), and the product was subsequently amplified with primers NPCR2 (5'-AGCAGTCAGAGGGGGTCACTAGGTCCTTAG-3') and AP2 (the inner primer from CLONTECH). A major PCR band of about 700 base pairs was purified on a 1% agarose gel and cloned into pCR-Blunt vector (Invitrogen). The sequence for this fragment lacks the intron sequence and extends the open reading frame by 234 additional amino acids. The two gene segments were spliced together using a BfaI restriction site in the region of overlap, creating a 3.4-kilobase pair cDNA in pBluescript coding for 1012 amino acids (pBlue-cPLA2-beta wt).

Northern Blot Analysis-- Membranes blotted with human tissue mRNA were purchased from CLONTECH and 32P-labeled probes derived from the coding region of clone 52A was used for hybridization. The washed membranes were exposed to x-ray film to reveal the expression pattern of cPLA2-beta .

Production of Polyclonal Antibodies to cPLA2-beta -- Two regions of cPLA2-beta , representing amino acids 299-537 and 681-1012 were subcloned into pGEX vectors for expression of glutathione S-transferase fusion proteins in Escherichia coli (Amersham Pharmacia Biotech). Inclusion body material was prepared and used to raise polyclonal antibodies in rabbits.

Construction of Expression Vectors to Produce cPLA2-beta Proteins in COS-7 Cells-- The cDNA insert of pBlue-cPLA2-beta -wt was subcloned into mammalian expression vector pEDDelta C to generate plasmid pEDDelta C-cPLA2-beta -wt. Transfection of this plasmid into COS cells produced a full-length cPLA2-beta protein of 110 kDa as revealed by Western blot analysis of the harvested cell lysates. To express cPLA2-beta protein lacking the N-terminal 231 amino acids, the 5' NcoI fragment of the cDNA insert in pBlue-cPLA2-beta -wt was deleted and the remaining cDNA insert was subcloned into pEDDelta C to produce plasmid pEDDelta C-cPLA2-beta -5'd2. Transfection of this plasmid into COS cells resulted in expression of protein cPLA2-beta MEK, which was identified as a 90-kDa band revealed by Western blot analysis.

Transfection cPLA2 Isoforms and Activity Assay-- 8 µg of pEDDelta C vector, pEDDelta C-cPLA2-beta -wt, pEDDelta -cPLA2-beta -5'd2, pEDdelta-cPLA2-gamma (9) or pED-cPLA2-alpha plasmid DNA was transfected into COS-7 cells on a 10-cm cell culture plate using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's protocol. At 66 h posttransfection, cells were washed twice with 10 ml of ice-cold Tris-buffered saline and scraped into 1 ml of Tris-buffered saline. Cell pellets were collected, resuspended in lysis buffer (10 mM HEPES, pH 7.5, 1 mM EDTA, 0.1 mM dithiothreitol, 0.34 M sucrose, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin), and lysed in a Parr-bomb (700 psi, 10 min) on ice. Protein concentration was measured by Bradford assay (Bio-Rad), and lysis buffer was used to adjust the samples to the same protein concentration. Phospholipase assays were carried out as described (9).

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Identification of cPLA2-beta and cDNA Cloning-- As described previously, search of the GenBankTM EST data base using the human 85-kDa cPLA2- protein sequence (cPLA2-alpha ) as a query yielded two ESTs with related sequences, which we named cPLA2-beta (GenBankTM accession number W92213) and cPLA2-gamma (GenBankTM accession number N56796). Through combined efforts of cDNA library screening and RACE PCR amplification, we obtained a cPLA2-beta cDNA clone containing a complete open reading frame encoding a polypeptide of 1012 amino acids with a calculated molecular mass of 110 kDa. A comparison of the amino acid sequence of cPLA2-beta with that of cPLA2-alpha (2) and cPLA2-gamma (9) revealed the following features. 1) All three enzymes share a homologous catalytic region which can be divided into domain A and domain B (Fig. 1A). Domain A contains 253, 251, and 237 amino acids for cPLA2-alpha , cPLA2-beta , and cPLA2-gamma , respectively. The lipase consensus sequence, GXSXG, is located at the N-terminal region of domain A (indicated in Fig. 1A). A signature of GGGXRAX5GX6EXGLLDX6GXSGSTWX4LY around the lipase consensus sequence can be assigned for the three cPLA2- isoforms (Fig. 1B). Catalytic domain B contains 196, 186, and 170 amino acids for cPLA2-alpha , cPLA2-beta , and cPLA2-gamma , respectively. The linker regions between domains A and B are from 91 amino acids (for cPLA2-beta ) to 133 amino acids (for cPLA2-alpha ) with very limited homology (Fig. 1B). It is interesting to note that the three residues in the putative catalytic triad of cPLA2-alpha (10) are all found in the conserved domains A (arginine and serine) and B (aspartate), whereas the phosphorylated serines that are believed to participate in cPLA2-alpha regulation (11, 12) are all located in the less conserved interdomain region and the COOH-terminal tail (Fig. 1, A and B). 2) Both cPLA2-beta and cPLA2-alpha have a calcium-dependent lipid binding (CaLB) domain that is lacking in cPLA2-gamma . However, cPLA2-beta has a 120 amino acid insert between the CaLB domain and catalytic domain A, whereas in cPLA2-alpha the two domains are adjacent to each other. In addition, cPLA2-beta has an NH2-terminal domain composed of 242 amino acids, which shares no homology to any protein sequence in the GenBankTM data bases.



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Fig. 1.   Sequence comparison of three cPLA2 isoforms. A, schematic diagram of homologous protein domains. The number of amino acids for each domain are indicated in the boxes. The percentage of identical amino acids to cPLA2-alpha is shown above the homologous domains in cPLA2-beta and cPLA2-gamma . The stars denote the conserved serines in the lipase consensus sequence shown above the catalytic domain A of each isoform. The phosphorylated serines of cPLA2-alpha and their positions are also shown. B, alignment of amino acid sequences of the three cPLA2-s. The conserved domains are marked by two arrows, and the three residues in the putative catalytic triad are denoted by stars. Identical amino acids are shaded. The sequence of cPLA2-beta has been submitted to GenBankTM under accession number AF121908.

When the multiple human tissue mRNA blots were hybridized with a probe derived from the cPLA2-beta cDNA containing sequences from the CaLB domain to the COOH terminus (see "Experimental Procedures"), a discrete band of about 8 kilobase pairs can be observed in all the tissues examined (Fig. 2). This indicates that cPLA2-beta mRNA is expressed ubiquitously, although at higher levels in pancreas, brain, heart, and liver tissues.


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Fig. 2.   Expression of cPLA2-beta mRNA in different human tissues by Northern analysis. Human multiple-tissue blots (CLONTECH) were hybridized with 32P-labeled cPLA2-beta -specific probes and washed under high stringency conditions. The same blots were also probed with beta -actin DNA and confirmed to have equal amounts of mRNA loaded in each lane (data not shown).

Expression of cPLA2-beta in COS Cells and PLA2 Activity Assay-- When full-length cPLA2-beta cDNA was subcloned into a eukaryotic expression vector and used to transfect COS-7 cells, a cPLA2-beta -specific protein of about 110 kDa was detected in cell lysates as revealed by Western analysis using polyclonal antibodies raised against bacteria-expressed cPLA2-beta peptide (data not shown). When the cell lysates were tested in a PLA2 activity assay using 1-palmitoyl-2-[14C]arachidonyl-PC (P*APC) as a substrate (see "Experimental Procedures"), the amount of arachidonic acid released by cPLA2-beta -transfected lysate is only twice as much as that by vector-transfected cell lysate (Fig. 3). We suspected that the relatively small increase of PLA2 activity might be due to the relative low level expression of a large protein, or the N-terminal domain of cPLA2-beta could be inhibiting the enzyme activity of the catalytic domains. Therefore, we constructed the cPLA2-beta MEK plasmid expressing cPLA2-beta protein lacking the N-terminal 236 amino acids (starting from Met-Glu-Lys sequence just upstream of the CaLB domain). Indeed, COS cell lysate transfected with cPLA2-beta MEK has an approximate 6-fold increase in PLA2 activity compared with vector-transfected cell lysate. However, Western analysis of cell lysates using cPLA2-beta antibodies followed by scanning quantification reveals that 5-fold more cPLA2-beta MEK protein was expressed compared with the full-length cPLA2-beta (data not shown). These results suggest that cPLA2-beta proteins do have PLA2 activity, and cPLA2-beta MEK has the same specific activity as cPLA-beta protein, indicating that the N-terminal domain is not negatively regulating the activity of the catalytic domains. When lysates from COS cells transfected with either cPLA2-beta or cPLA2-beta MEK were subjected to ultracentrifugation (100,000 × g centrifugal force), the majority (60-70%) of the cPLA2-beta protein and phospholipase activity was found in the supernatant fraction as detected by Western blot analysis and P*APC hydrolysis assay (data not shown), suggesting that cPLA2-beta is a cytosolic protein. In addition, when COS cell lysate transfected with a CaLB-containing cPLA2-beta construct was fractionated in the presence of calcium, nearly all the enzyme activity was found to be associated with the particulate fraction (data not shown). Therefore we predict cPLA2-beta may respond to a calcium-mobilizing agonist in similar fashion as cPLA2-alpha .


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Fig. 3.   Overexpression of cPLA2 isoforms in COS cells correlates with increased PLA2 activity. Lysates from COS cells transfected with either cPLA2-beta (), cPLA2-beta MEK (triangle ), or vector DNA (open circle ) were assayed for their ability to release arachidonic acid from P*APC at 37 °C for indicated times. Activity is expressed as percentage of radiolabel released (% conversion).

Relative Regiospecificity of cPLA2-beta -- Because of a higher level of expression, cPLA-beta MEK rather than the full-length cPLA2-beta protein was used to compare regioselectivity of the three cPLA2- isoforms. For this purpose, one of the three substrates, namely 1-[14C]palmitoyl-2-arachidonyl-PC (*PAPC), 1-palmitoyl-2-[14C]arachidonyl-PC (P*APC), and 1-O-hexadecyl-2-[3H]arachidonyl-PC (O-H*APC), was used in a vesicle assay system to measure radiolabeled fatty acids released by COS cell lysates transfected with either cPLA2-alpha , cPLA2-beta MEK, cPLA2-gamma , or vector plasmid DNA. Although all three enzymes were able to liberate the sn-2 arachidonic acid from P*APC, each enzyme has unique properties in releasing the radioactive labels in *PAPC and O-H*APC. cPLA2-beta MEK and cPLA2-gamma were able to liberate the sn-1 palmitic acid from *PAPC, although cPLA2-alpha had almost no activity in this regard (Fig. 4). On the other hand, cPLA2-alpha and cPLA2-gamma could release the sn-2 arachidonate from O-H*APC, but cPLA2-beta MEK showed very limited activity. Although full-length cPLA2-beta is expressed at low levels, preferences toward the three substrates are the same as cPLA2-beta MEK (data not shown). These results indicate that under conditions tested, cPLA2-alpha prefers sn-2 cleavage, cPLA2-beta prefers sn-1 cleavage, whereas cPLA2-gamma is able to efficiently cleave at either site. This implies that the observed PLA2 activity of cPLA2-beta (Fig. 3) is likely due to combined PLA1 activity and lysophospholipase activity (or transacylase activity). However, affirmative conclusion can only be made when extensive enzymology studies are conducted with pure cPLA2-beta proteins.


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Fig. 4.   Phospholipase A1/A2 activities of cPLA2 isoforms. Lysates from COS cells transfected with either cPLA2-alpha (A), cPLA2-beta MEK (B), cPLA2-gamma (C), or vector DNA were incubated with *PAPC (), P*APC (triangle ), and O-H*APC (open circle ) for the indicated time periods. Activity is expressed as percentage of conversion.

Phospholipase Activity of cPLA2-beta Is Calcium dependent-- Because cPLA2-beta has a CaLB domain, we tested whether its phospholipase activity is calcium-dependent. As can be seen from Fig. 5, when *PAPC (Fig. 5A) or P*APC (Fig. 5B) was used as substrate, release of the radiolabeled fatty acids by both cPLA2-beta and cPLA2-beta MEK is strictly dependent on the presence of calcium. When EGTA was included in the reaction, no phospholipase activity was detected. This experiment demonstrates that the CaLB domain in cPLA2-beta is functional. We were unable to determine the minimal calcium concentration required for enzymatic activation due to impurities of the cell lysates.


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Fig. 5.   Phospholipase activity of cPLA2-beta is calcium-dependent. COS lysates from cells transfected with cPLA2-beta , cPLA2-beta MEK, or vector were assayed for their ability to liberate radioactive labels from *PAPC (A) or P*APC (B) in the presence of 7 mM calcium (shaded bars) or 5 mM EGTA (open bars). Activity is measured as percentage of radiolabel released (% conversion).

In conclusion, we have cloned a cDNA for a 110-kDa calcium-dependent phospholipase, cPLA2-beta , which shares significant homology to cPLA2-alpha and cPLA2-gamma . Although the three members of cPLA2 family share similar catalytic domains and have the same conserved residues in the catalytic triad (10), initial biochemical characterization suggests that they have different regioselectivity and may also have different headgroup specificity. The mechanisms for membrane association are also different: cPLA2-beta , like cPLA2-alpha , seems to rely on the CaLB domain to gain access to aggregated phospholipids, whereas cPLA2-gamma may use lipid modification for membrane attachment. Furthermore, the fact that cPLA2-beta has additional protein domains suggests that the regulation of this enzyme may be dramatically different from that of cPLA2-alpha and cPLA2-gamma .

    ACKNOWLEDGEMENT

We thank Drs. James D. Clark and Simon Jones for advice and help in phospholipase activity assays. We also thank Kerrry Kelleher and Heather Finnerty for assistance in sequencing the cDNA clones.

    Note Added in Proof

Following the acceptance of this paper, a report appeared describing the cloning of cPLA2-beta (13).

    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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF121908.

Dagger To whom correspondence should be addressed: Genetics Institute, 87 Cambridge Park Dr., Cambridge, MA 02140. Tel.: 617-665-8978; Fax: 617-665-8993; E-mail: rkriz{at}genetics.com.

    ABBREVIATIONS

The abbreviations used are: PLA2, phospholipase A2; cPLA2-alpha , 85-kDa cytosolic PLA2; cPLA2-beta , 110-kDa cytosolic PLA2; cPLA2-gamma , 60.9-kDa cytosolic PLA2; EST, expressed sequence tag; CaLB, calcium-dependent lipid binding; RACE, rapid amplification of cDNA ends; PC, phosphatidylcholine; PCR, polymerase chain reaction; P*APC, 1-palmitoyl-2-[14C]arachidonyl-PC; *PAPC, 1-[14C]palmitoyl-2-arachidonyl-PC; O-H*APC, 1-O-hexadecyl-2-[3H]arachidonyl-PC.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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