Multiple Splice Variants of the Human Calcium-independent Phospholipase A2 and Their Effect on Enzyme Activity*

Pontus K. A. LarssonDagger §, Hans-Erik ClaessonDagger , and Brian P. Kennedy§par

From the Dagger  Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institute, S-171 77 Stockholm, Sweden and the § Department of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, P. O. Box 1005, Pointe Claire-Dorval, Quebec H9R 4P8, Canada

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Recently, the cloning of a novel Ca2+-independent phospholipase A2 (iPLA2) from Chinese hamster ovary cells as well as from mouse and rat sources containing a C-terminal lipase motif and eight N-terminal ankyrin repeats has been described. In this report we describe the cloning of the human iPLA2 cDNA and its expression in B-cells and show that the iPLA2 gene undergoes extensive alternative splicing generating multiple isoforms that contribute to a novel mechanism to control iPLA2 activity. The full-length cDNA clone encodes a 806-amino acid protein with a calculated molecular mass of 88 kDa. The protein contains a lipase motif, GXSXG, and ankyrin repeats, as described for the hamster and rodent forms of the enzyme but has an additional 54-amino acid proline-rich insertion in the last of the eight ankyrin repeats (residues 395-449). Furthermore, at least three additional isoforms most likely due to alternative splicing were identified. One that is present as a partial cDNA in the expressed sequence tag data base is similar to iPLA2 but terminates just after the lipase active site, and two other isoforms contain only the iPLA2 ankyrin repeat sequence (ankyrin-iPLA2-1 and -2). Ankyrin repeats are involved in protein-protein interactions and because the purified iPLA2 enzyme exists as a multimeric complex of 270-350 kDa, the expression of just the ankyrin-iPLA2 sequence suggested that these may also interact with the iPLA2 oligomeric complexes and perhaps modulate PLA2 activity. Transfection of the human iPLA2 cDNA into COS cells resulted in a substantial increase in calcium-independent PLA2 activity in cell lysate. No activity above background was observed following ankyrin-iPLA2-1 cDNA transfection. However, co-transfection of the ankyrin-iPLA2-1 and the iPLA2 cDNAs resulted in a 2-fold reduction in activity compared with iPLA2 alone. A similar co-transfection of ankyrin-iPLA2-1 cDNA with the cPLA2 cDNA had no effect on PLA2 activity. These results suggest that the ankyrin-iPLA2 sequence can function as a negative regulator of iPLA2 activity and that the alternative splicing of the iPLA2 gene can have a direct effect on the attenuation of enzyme activity.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Phospholipases A2 (PLA2)1 are a rapidly growing family of diverse enzymes that hydrolyze fatty acids at the sn-2 position of phospholipids (1, 2). PLA2 enzymes can be subdivided into two classes, extracellular or intracellular, depending on the enzymes localization during catalysis (2). The intracellular PLA2s can be further categorized into calcium-dependent, best exemplified by the cytosolic phospholipase A2 (cPLA2) (3), and calcium-independent forms (iPLA2), which tend to be quite diverse and have until recently been less characterized at the molecular level. The calcium-independent PLA2s have a wide tissue distribution (4) and have been purified from human myocardium (5), bovine brain (6), P388D1 murine macrophages (7), and rabbit kidney (8). They all have distinct molecular masses, indicating the diversity of iPLA2s. Recently, an 85-kDa iPLA2 was purified and cloned from CHO cells (9), and its sequence was found to be analogous to the 85-kDa iPLA2 from P388D1 cells (10) as well as the sequence for iPLA2 from rat pancreatic islet (11). The amino acid sequence indicated the presence of eight ankyrin repeats and the GXSXG conserved catalytic sequence, as found in other lipases. Although there were apparent differences in ATP sensitivity among the enzymes, the biochemical, immunological, and sequence data indicate that these three enzymes are likely to be species variants of the same protein. In both P388D1 cells and in rat pancreatic islets it is thought that iPLA2 has a function in membrane phospholipid remodeling (12). It has been postulated that the rat islet iPLA2 may be involved in arachidonic acid release leading to activation of beta -cell ion channels (11).

Arachidonic acid is also the main precursor for important biological mediators such as leukotrienes (LT) (13). The oxygenation of arachidonic acid catalyzed by 5-lipoxygenase is the first step in the biosynthesis of leukotrienes and leads to the formation of LTA4, which can be further metabolized to LTB4 and LTC4. In our ongoing studies of leukotriene synthesis and phospholipase activity in human B lymphocytes, we have demonstrated conversion of arachidonic acid to LTB4 and expression of 5-lipoxygenase in human B lymphocytes (14, 15). Although cellular homogenates of B lymphocytes can release arachidonic acid from phospholipids in vitro, exogenous arachidonic acid is a prerequisite for leukotriene synthesis in intact cells (16). To elucidate the expression of PLA2(s) in human B lymphocytes, we have examined in this report the expression of the different PLA2s at the transcriptional level and describe the cDNA cloning of the human 85-kDa iPLA2 and its various isoforms and their effect on enzyme activity.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals and Reagents-- Chemicals were from Sigma or Aldrich. Cell culture medium and fetal bovine serum were from Life Technologies, Inc. Restriction enzymes and Taq DNA polymerase were from Boehringer Mannheim or Pharmacia Biotech Inc. The human B lymphocytic cell line BL-41 E95A was kindly provided by Dr. Klein (Karolinska Institute), whereas the Raji cell line was obtained from ATCC. All oligonucleotides were synthesized on an Applied Biosystems 380B DNA synthesizer.

Cell Culture Conditions-- The B-cell lines BL-41 E95A (17) and Raji were cultivated in RPMI 1640 supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin and grown in humidified atmosphere with 5% CO2 (16). The cultures were seeded at a cell density of 0.2 × 106 cells/ml and harvested at approximately 1 × 106 cells/ml.

RT-PCR Analysis of B-cell PLA2 Expression-- Total cellular RNA was isolated from 10-20 × 106 cells of the B-cell lines described above using Trizol Reagent (Life Technologies, Inc.) according to the instructions of the manufacturer. Reverse transcription of total RNA (2 µg) was performed using Expand Reverse transcriptase (Boehringer Mannheim) and priming with random hexamers or oligo(dT). The reverse transcription reaction was carried out at 42 °C for 60 min and then terminated by heating at 65 °C for 5 min. PCR was performed in a total volume of 50 µl containing 2 µl of the reverse transcription reaction mixture, 0.2 mM dNTPs, 0.5 µM of each primer, and 2 units of Taq polymerase in PCR buffer (50 mM KCl, 1.5 mM MgCl2, and 10 mM Tris-HCl, pH 9.0) (Pharmacia). The conditions for PCR reactions, if not indicated otherwise were: an initial denaturation step at 94 °C for 5 min, followed by 36 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and a final extension step at 72 °C for 10 min. Control reactions including minus reverse transcriptase and amplification of a glyceraldehyde 3-phosphate dehydrogenase fragment were also performed. Primers used for PCR were: cPLA2 (sense, 5'-CCAAAATGTCATTTATAGATCCTTA-3'); cPLA2 (antisense, 5'-CTGATTAGGATCCAAAATAAA-3'); iPLA2-1 (sense, 5'-AACGTTAACCTCAGGCCTCC-3'); iPLA2-2 (antisense, 5'-GAGAGTTTCTTCACCTTGTT-3'); iPLA2-33 (sense, 5'-CAGGGCTCTGCAGCGCCACATCAT-3'); iPLA2-34 (antisense, 5'-GGCCTTCTCGATGGCGATGAGGAG-3'); sPLA2(I) (sense, 5'-CCGTCATGAAACTCCTTGTGCTAGCT-3'); sPLA2(I) (antisense, 5'-CCAGATCTCAACTCTGACAATACTTCTT-3'); sPLA2(II) (sense, 5'-CCGAATTCATGAAGACCCTACTGTTGGCA-3'); sPLA2(II) (antisense, 5'-CCAGATCTCAGCAACGAGGGGTGCTCCCTCTG-3'); sPLA2(V) (sense, 5'-CCAGAGATGAAAGGCCTCCTC-3'); sPLA2(V) (antisense, 5'-GCCTAGGAGCAGAGGATGTTG-3'); lipoprotein-associated PLA2 (sense, 5'-TACATAAATCCTGTTGCCCA-3'); lipoprotein-associated PLA2 (antisense, 5'-GTTGTCATTGAACCAAAGAG-3'). The PCR products were analyzed on 1.5% low melt agarose gels.

Cloning of Human iPLAA2-- The CHO cell-derived iPLA2 amino acid sequence (9) was used to perform a TBLASTN data base search of GenBankTM. The sequences of two human expressed sequence tag (EST) clones, 46450 (accession number H10676) and 30643 (accession number R18691), were found to show considerable identity to the CHO cell sequence. Bacteria containing the EST clones were obtained from Research Genetics, plasmid DNA was prepared, and the cDNA insert was sequenced on both strands using an ABI 373A automated DNA sequencer and m13 forward and reverse, as well as gene-specific primers. The DNA sequence identified the ESTs as human homologs of the CHO cell iPLA2. To obtain a full-length human iPLA2 cDNA clone, 5'-rapid amplification of cDNA ends (RACE) was used to amplify the sequence from various cDNA sources (see Fig. 4). Amplification was carried out using a human iPLA2-specific 3' primer (iPLA2-2), a 5' anchor primer (CLONTECH), and Marathon Ready cDNAs (CLONTECH) as template. The Expand High Fidelity PCR system (Boehringer Mannheim) was used for the 5'-RACE using the following conditions: an initial denaturation step at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 2.5 min, and a final extension at 72 °C for 10 min. The amplified products were analyzed on 0.8% agarose gels, and DNA fragments were recovered (Qiagen). Taq polymerase was used to add a 3' A overhang to the fragment, which was then cloned into the pCR2.1 TA cloning vector (Invitrogen). Sequencing of both strands of the cloned 5'-RACE fragment was performed as described above.

A full-length human iPLA2 clone was obtained by combining the 3' end of the iPLA2 sequence in clone EST 46450 with the 5' iPLA2 sequence obtained by the RACE reaction. Briefly, the 5'-RACE fragment was released from pCR2.1 by a HindIII/HpaI double digest. The released fragment was gel purified, extracted, and sticky/blunt end ligated into HindIII/HpaI-digested Lafmid BA/EST 46450. Sequencing of the full-length iPLA2 was performed to verify the integrity of the sequence.

Transient Expression in COS-7 Cells-- Plasmid DNA (5 µg) containing iPLA2, ankyrin-iPLA2-1, or cPLA2 cDNAs cloned into the eukaryotic expression vector pcDNA 3.1+ (Invitrogen) was transfected into COS-7 cells using LipofectAMINE (Life Technologies, Inc.). Transfections consisted of various combinations of the PLA2 cDNAs and pcDNA 3.1 (see the legend to Fig. 6) to a total of 10 µg of DNA transfection. Briefly, both DNA and LipofectAMINE (60 µl) were each mixed with 800 µl of medium (Opti-MEM) and then combined and incubated for 45 min at 20 °C to allow formation of DNA-liposome complexes. Subsequently, 6.4 ml of medium was added to each tube, and the transfection mixture was transferred to washed COS-7 cells. Transfection was allowed to proceed for 5 h at 37 °C in 80-mm dishes and terminated by replacement of the transfection mixture with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.

PLA2 Enzyme Activity-- Enzyme activities were determined using a vesicular based assay containing equal concentrations (5 µM) of 1-palmitoyl 2-[1-14C]arachidonyl phosphatidylcholine (55 mCi/mmol) and 1-palmitoyl 2-[1-14C]arachidonyl phosphatidylethanolamine (55 mCi/mmol, NEN Life Science Products). The radioactive phospholipids were dried under nitrogen and resuspended in either 180 µl of calcium-free assay buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 1 mg/ml albumin) or calcium-containing assay buffer (80 mM glycine, pH 9.0, 5 mM CaCl2, 2 mM dithiothreitol, and 1 mg/ml albumin). Cells were collected 48 h after transfection by scraping, washed twice, and resuspended in homogenization buffer (50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.5 mM dithiothreitol, and 20% glycerol). Cells were then lysed by sonication (2 × 5 s), the resulting homogenate was centrifuged at 10,000 × g for 20 min at 4 °C, and the supernatant was collected. Calcium-dependent and -independent PLA2 activity in the supernatant was determined by the addition of 20 µl of supernatant to 180 µl of one of the above assay buffers, and the reaction was allowed to proceed for 20 min at 37 °C before termination with the addition of 400 µl of methanol containing 0.5% acetic acid and 10 µM arachidonic acid. The sample was then applied to a Sep-Pak Vac (Waters) C18 cartridge, and fatty acids were eluted in 500 µl of methanol. The [1-14C]arachidonate content of the eluate was analyzed by high pressure liquid chromatography using a Nova-Pak C18 column (3.9 × 150 mm) at a flow rate of 1 ml/min (the mobile phase was methanol/H2O/trichloroacetic acid (85:15:0.01 by volume), and radioactivity was detected using a Beckman 171 radioisotope detector coupled on-line to a Waters 996 diode array spectrophotometer. Peak area integration was performed using Millenium software (Waters).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

PLA2 Expression in B-cell Lines-- Human monoclonal B-cells express both FLAP and 5-lipoxygenase and have the ability to make LTs only after the addition of exogenous arachidonic acid and stimulation with calcium ionophore and a reducing agent (14, 15). The reason for the lack of cellular leukotriene biosynthesis in B-cells without these added factors is unclear. Furthermore, stimulation with various agents known to promote arachidonic acid release fails to induce a similar release in B-cells. However, B-cells possess phospholipase activity and sonicates of these cells release arachidonic acid (16). Therefore, to investigate the presence of PLA2 in B-cells, we have examined the expression of the various PLA2 enzymes at the transcriptional level using RT-PCR. Total RNA from the human monoclonal B-cell lines Raji and BL-41 E95A was used as template for the RT-PCR reaction, and primers were designed for the amplification of a fragment of the following PLA2 cDNAs: cPLA2 (3), iPLA2, sPLA2 types I, II, and V (2, 18), and lipoprotein-associated PLA2 (19). The sequence used for the design of the iPLA2 primers was obtained by a TBLASTN data base search using the CHO cell-derived amino acid sequence. Two human EST clones, 46450 and 30643, revealed 84 and 65% sequence identity, respectively, to the CHO cell-derived sequence, strongly suggesting that they represented the human form of the enzyme. Sequencing of the two clones revealed that EST 46450 contained a partial cDNA for the human homolog of iPLA2, whereas EST 30643, which also contained part of the human iPLA2 cDNA sequence, differed from EST 46450 at the 5' end (Fig. 1). The difference in sequence at the 5' end of these clones was not due to a misspliced intron because the 3' intron junction consensus sequence was not present. Therefore, it could be either a cDNA library artifact or the iPLA2 sequence is subject to alternative splicing.


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Fig. 1.   Alignment of CHO iPLA2 cDNA with DNA sequences of human EST clones 30643 and 46450. A comparison of the two EST sequences with the CHO iPLA2 cDNA indicated that these ESTs contained the 3' end of the human iPLA2 cDNA sequence. Each contained about a third of the full-length iPLA2 sequence as judged from the size of the CHO sequence. However, at the 5' end of these clones, EST 46450 aligned more closely to the CHO sequence than did EST 30643. Identical sequence is boxed, and the termination codon for the CHO cDNA is double underlined. The human iPLA2 PCR primers 1 and 2, which were based on the EST 46450 sequence, are underlined, and the polyadenylation signal is shown with asterisks. The dashes indicates the presence of gaps in the sequence.

The iPLA2 primers 1 and 2 (Fig. 1) along with the primers for the various PLA2s described above were used to amplify B-cell cDNA and revealed that the iPLA2 sequence was easily amplified from these cells. A weak signal for the cPLA2 sequence was detectable, but none of the other PLA2 sequences were detected (data not shown). The weak signal obtained for cPLA2 is in agreement with previous results showing that the cPLA2 mRNA (20, 21) or protein (22) was either not present or detectable at low levels in B-cells. The results from one typical RT-PCR using primers iPLA2-1 and -2 is shown in Fig. 2. At least three distinct amplified products were observed. The most abundant product was the expected 217-bp fragment, whose identity was confirmed by sequencing, as well as two additional fragments with the apparent sizes of 330 and 380 bp, respectively. The 380-bp fragment was sequenced and found to contain the 5' end of EST clone 30643 (Fig. 3A). This iPLA2 EST contains a 168-bp insert between iPLA2 primers 1 and 2, indicating that there are splice variants of iPLA2. The insertion introduces a premature termination codon that would result in the expression of a truncated iPLA2 (Fig. 3A). The nature of the 330-bp fragment is currently under investigation, although it is related to iPLA2 because it hybridizes to an iPLA2 oligonucleotide probe (data not shown).


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Fig. 2.   Expression of iPLA2 in human B-cell lines. RT-PCR analysis of total RNA from BL-41 E95A and Raji cell lines using iPLA2 primers 1 and 2 was performed as described under "Materials and Methods." A 10-µl aliquot of the PCR reaction was analyzed on a 1.5% agarose gel containing ethidium bromide. Reactions containing reverse transcriptase are indicated with a plus sign (+) and negative controls without reverse transcriptase with a minus sign (-). A glyceraldehyde 3-phosphate dehydrogenase (G3PDH) control amplification was also performed using the same reaction conditions. The DNA size markers are in bp.


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Fig. 3.  

DNA sequence alignment of iPLA2 and its multiple isoforms. A, the difference between EST 30643 and 46450 (human iPLA2 cDNA) is a 168-bp insertion between iPLA2 PCR primers 1 and 2, which are indicated in bold and underlined. If EST 30643 cDNA were translated in the same frame as iPLA2, it would terminate 44 bp into the insert at the termination codon that is boxed and in bold. B, comparison of iPLA2 with the ankyrin-iPLA2 isoforms showing the various insertions. The iPLA2 PCR primers 33 and 34 are indicated in bold and underlined, and the termination codons for the isoforms are shown in bold and dotted underlined. The lipase active site sequence is boxed and in bold. C, the in-frame 216-bp 5' deletion in ankyrin-iPLA2-2. The dots indicates the presence of gaps in the sequence.

Cloning of the Human iPLA2 cDNA-- The results from the RT-PCR indicated that iPLA2 is at least one of the more significant PLA2 enzymes in B-cells, and therefore we decided to characterize it more extensively. To obtain a full-length iPLA2 cDNA clone, 5'-RACE was performed on various cDNAs to amplify the remainder of the sequence. Fig. 4 shows the results from one such reaction and the substantial amplification of a 2.2-kilobase DNA fragment from testis cDNA. This fragment was subcloned, and sequence data were obtained for five different clones. Three clones had identical sequence and contained an open reading frame encoding the human iPLA2 cDNA sequence (Fig. 5). One of the clones had an insertion just before the iPLA2 catalytic domain that produced a frameshift, thus leading to a truncated iPLA2 without the catalytic domain (Fig. 3B). This clone was identified as ankyrin-iPLA2-1 to indicate that it coded for only the ankyrin repeats. Similarly, the other clone, ankyrin-iPLA2-2, had the identical 53-bp insertion as the previous isoform but also had an additional 52-bp insertion 80 bp 5' to the ankyrin-iPLA2-1 insertion, thus generating a different C terminus but again without the catalytic domain (Fig. 3B). In addition, this clone contained a 216-bp in-frame deletion in the 5' region of the cDNA resulting in the removal of amino acids 71-143 (Figs. 3C and 5). To prove that the observed different forms of iPLA2 were not due to artifacts in the 5'-RACE reaction, testis cDNA as well as two different testis cDNA libraries and B-cell cDNA were subjected to PCR using primers iPLA2-33 and -34 (Fig. 3B), which spanned the region of interest. Two detectable amplified products were obtained, one major product corresponding to iPLA2 and a minor one corresponding to ankyrin-iPLA2-1, whereas ankyrin-iPLA2-2 was not observed (data not shown). The absence of isoform 2 is not too surprising because it could be at levels that would require more rounds of amplification or the use of ankyrin-iPLA2-2-specific primers.


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Fig. 4.   Amplification of the iPLA2 cDNA. 5'-RACE was used to amplify the remainder of the iPLA2 cDNA as described under "Materials and Methods" using Marathon-Ready cDNAs (CLONTECH) derived from various human tissues and cell lines as indicated. 10 µl of the amplified material (50 µl) was analyzed on a 0.8% agarose gel. The DNA size markers are in bp.


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Fig. 5.  

Amino acid sequence and alignment of human iPLA2 and the various isoforms with the CHO sequence. A, the amino acid sequence of human and CHO cell iPLA2 as well as the various iPLA2 isoforms were compared using the Genetics Computer Group program GAP. The seven ankyrin repeats in the human sequence are overlined (I-VII), and the eighth repeat, which is underlined in the CHO sequence, is interrupted by an insertion in the human sequence. The active site (GTSTG) is indicated in bold, and termination is indicated by an asterisk. The EST 30643 sequence is the partial sequence from the EST clone. Shading denotes identity, and the dots indicate the presence of gaps in the sequence. B, a diagrammatic representation of the iPLA2 isoforms. The 54-amino acid insertion into the eighth ankyrin repeat of the human sequences and the 72-amino acid deletion in ankyrin-iPLA2-2 are illustrated. The active site serine is indicated for both the CHO and human sequence. The C-terminal hatched region in ankyrin-iPLA2-2 denotes nonidentical sequence, and the number of amino acids in each sequence is also shown.

A full-length iPLA2 cDNA sequence was obtained by combining the 3' end of EST 46450 with the appropriate 5'-RACE fragment (see "Materials and Methods"). The inferred human iPLA2 amino acid sequence was compared with the recently reported sequences and found to have an overall identity of 90% to hamster, rat, and mouse iPLA2 sequences (Fig. 5A). The major difference between the human sequence and the other species is the insertion of an additional 54 amino acids that would interrupt the last putative ankyrin repeat as defined in the hamster and rat iPLA2 sequences (9, 11) (Fig. 5). This sequence is also present in the other human iPLA2 isoforms. A search of GenBankTM with this additional 54-amino acid sequence did not reveal any significant homologies.

In addition to the human iPLA2 cDNA there are at least three other iPLA2 isoforms. The EST 30643 sequence, if it were full-length, could encode an iPLA2 that contained the ankyrin repeats and lipase active site but with a truncated C terminus (Fig. 5). The other two iPLA2 isoforms ankyrin-iPLA2-1 and -2 contain only the ankyrin repeats because they terminate before the lipase active site (Fig. 5). A diagrammatic representation of the iPLA2 isoforms is illustrated in Fig. 5B. The ankyrin-iPLA2-1 sequence is identical to the human iPLA2 up to the last three C-terminal amino acids and terminates about 40 residues before the active site serine. In contrast, although the ankyrin-iPLA2-2 sequence has the same iPLA2 ankyrin repeats, it also contains more structural and sequence variation than does ankyrin-iPLA2-1. First, there is the 72-amino acid deletion between residues 70-143, and second, the additional insertion in the iPLA2-2 cDNA (Fig. 3C) alters the sequence of the last 50 C-terminal amino acids (Fig. 5, A and B). The presence of these iPLA2 isoforms indicates that the iPLA2 sequence is subjected to a significant amount of alternative splicing.

Effect of Ankyrin-iPLA2 Sequence on iPLA2 Activity-- The ankyrin structural motif appears to have a function in the formation of various types of protein-protein interactions (23). Deletion of the ankyrin repeats of the CHO cell iPLA2 results in the loss of lipase activity, suggesting that this structure is required for enzyme activity (9). A possible function for the ankyrin repeats in iPLA2 is to participate in the formation of the large oligomeric structures (270-350 kDa) found for iPLA2 upon gel filtration (7, 9). If this is the case then the ankyrin-iPLA2 sequences (iPLA2-1 and -2) may also participate in the formation of the iPLA2 oligomeric structures in the cell and have some effect on iPLA2 activity. To test this, iPLA2 cDNA was co-transfected into COS cells with either pcDNA 3.1 vector or ankyrin-iPLA2-1 cDNA, and PLA2 activity was measured in the cell lysate in the presence and the absence of calcium (Fig. 6, A and B). A control transfection for PLA2 activity using the cPLA2 cDNA was also performed. Transfection of iPLA2 and control vector resulted in a substantial increase in PLA2 activity in cell lysate over control (vector alone) in the absence (Fig. 6A) or the presence of calcium (Fig. 6B), indicating that the iPLA2 cDNA sequence codes for an active enzyme. Increased PLA2 activity was also observed upon transfection of the cPLA2 cDNA but only in the presence of calcium, as would be expected (Fig. 6B). Transfection of just the ankyrin-iPLA2 cDNA sequence did not result in any PLA2 activity over background (data not shown). However, replacement of the vector control in the iPLA2 cDNA transfection with ankyrin-iPLA2-1 cDNA caused a 2-fold decrease in PLA2 activity in the cell lysate in both assays (Fig. 6, A and B). Including the ankyrin-iPLA2-1 cDNA in the cPLA2 cDNA transfection had no effect on PLA2 activity (Fig. 6B). These results would suggest that the ankyrin-iPLA2 sequences can specifically modulate iPLA2 activity.


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Fig. 6.   Effect of ankyrin-iPLA2-1 on iPLA2 activity in transfected COS-7 cells. PLA2 cDNAs were cloned into the eukaryotic expression vector pcDNA 3.1, and COS-7 cells were then transfected with 5 µg each of the following DNAs: transfection 1, iPLA2 cDNA and pcDNA 3.1 vector; transfection 2, iPLA2 and ankyrin-iPLA2-1 cDNAs; transfection 3, cPLA2 cDNA and pcDNA 3.1 vector; transfection 4, cPLA2 and ankyrin-iPLA2-1 cDNAs; and transfection 5, 10 µg of pcDNA 3.1. Cell lysates were prepared 48 h after transfection and PLA2 activity determined in the absence (A) and the presence (B) of calcium. Activity is defined as the release of [14C]arachidonic acid (cpm)/mg protein. The results are the means of duplicate samples from two different transfections.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The human iPLA2 sequence presented here contains the ankyrin and the GXSXG lipase motifs described for the hamster and rodent enzymes (9, 11). A major difference between the human enzyme and that of other species is the insertion of a 54-amino acid proline-rich sequence in the eighth ankyrin motif (residues 395-449). A recent report from Tang et al. (9), which describes a partial human iPLA2 cDNA clone, found at the exact same position in their clone some sequence that was not present in the CHO iPLA2, which they attributed to potential unspliced intron sequences. The insertion described here for human iPLA2, which was detected in all sequenced iPLA2 clones, does not contain any splice junction consensus sequence, nor does it disrupt the reading frame. Whether the human gene contains an additional exon or this form is also present in the other species but has not yet been cloned remains to be determined. The possibility that iPLA2 expression could give rise to splice variants was suggested by finding two partial iPLA2 cDNAs in the EST data base with different 5' ends. The difference was found to be due to a 168-bp insertion in EST 30643. Both EST 30643 and iPLA2 cDNA sequences were easily detected by RT-PCR of B-cell RNA suggesting that both iPLA2 isoforms are expressed. Presently, we are trying to obtain a full-length EST 30643 clone to confirm that this truncated isoform of iPLA2 is catalytically active. Two additional iPLA2 splice variants were also detected, ankyrin-iPLA2-1 and -2, which contain only the ankyrin repeats and no lipase catalytic site.

The existence of these truncated forms of iPLA2 is intriguing because the ankyrin motif has been shown to be involved in various types of protein-protein interactions (23-26). The truncated forms could function as negative regulatory proteins by docking to iPLA2 binding sites in the cell and thereby prevent docking of the catalytically active enzyme. Alternatively, they may interfere with the formation of the quaternary structure of iPLA2 and in this way alter enzyme activity. An oligomeric form of the enzyme may indeed be the active state of the enzyme because removal of ankyrin repeats results in loss of enzyme activity (9). The fact that ankyrin-iPLA2-1 can alter iPLA2 activity was shown by co-transfection of the iPLA2 and ankyrin-iPLA2-1 cDNAs into COS cells. The co-transfection of both constructs results in a 2-fold decrease in PLA2 enzyme activity compared with that observed for the co-transfection of iPLA2 cDNA and vector DNA. Co-transfection of ankyrin-iPLA2-1 cDNA with the cPLA2 cDNA had no effect on PLA2 enzyme activity. Thus the interaction of ankyrin-iPLA2-1 with iPLA2 results in a decrease in enzyme activity. The most likely explanation for this is a competition between ankyrin-iPLA2-1 and iPLA2 monomers to form the oligomeric species. In cells transfected with the iPLA2 cDNA alone, the iPLA2 oligomer is most likely composed entirely of active iPLA2 monomers. Co-transfection of both iPLA2 and ankyrin-iPLA2 cDNAs could result in iPLA2 oligomers that contain various combinations of both iPLA2 and ankyrin-iPLA2-1 monomers. What is not known is the stoichiometry of the active iPLA2 enzyme complex. Does it contain only iPLA2 monomers, or can it tolerate formations with ankyrin-iPLA2? The size of the iPLA2 complex, based on gel permeation chromatography, ranges from 270 to 350 kDa (7, 9), suggesting that there are at most four iPLA2 monomers/complex. If this is the case and we assume that there is equal expression of both subunits in the transfected cells and binding affinity is not changed between the different subunits, then iPLA2-(ankyrin-iPLA2-1) complexes should be enzymatically active, because enzyme activity was only decreased 2-fold in the co-transfected cells. However, a more thorough investigation using purified subunits will have to be done to confirm this. Although we can show that ankyrin-iPLA2 can have an effect on iPLA2 activity, its in vivo role remains to be determined. The level of ankyrin-iPLA2-1 expression is at least 10-fold lower than that of iPLA2,2 suggesting that if it participates in the iPLA2 enzyme complex, it must have a much more subtle effect on enzyme activity in the cell. Nevertheless, what we have shown is that alternative splicing of the iPLA2 gene can have a direct effect on iPLA2 activity.

In fact, alternative splicing of genes containing sequences that encode both an activity domain and some kind of protein binding domain appears to be a common mechanism to control activity. A similar situation to that of iPLA2 is the Ikappa Bgamma inhibition of NF-kappa B activity. Ikappa B proteins contain six or seven ankyrin-like repeats, which have been shown to be essential for retaining NF-kappa B in the cytoplasm and inhibit DNA binding by Rel/NF-kappa B (25, 27). Ikappa Bgamma is derived by alternative splicing of the murine p105 gene, which is the precursor for the p50 component of the NF-kappa B, p50-p65 heterodimer (28, 29). The N-terminal half of p105 contains p50, which is derived by proteolytic cleavage, whereas the C-terminal half has eight ankyrin repeats (29). In addition, there are multiple isoforms of Ikappa Bgamma , all derived by alternative splicing of the p105 gene and each with unique Ikappa B activities (30). The two ankyrin-iPLA2 isoforms described here do have structural and sequence differences, but whether or not they have unique inhibitory activities remains to be determined. Additional examples of alternative splicing having positive and negative effects on activity are two genes involved in programmed cell death: ich-1 (Caspase-2) (31), which encodes a cysteine protease, and bcl-x, a bcl-2-related regulatory gene (32). Again it is analogous to the iPLA2 situation described above in that both genes produce a long transcript that codes for a functional product that is inhibited by a truncated version encoded by a shorter alternatively spliced transcript (31-33).

In the two B-cell lines tested, the iPLA2 cDNA sequence was easily amplified, whereas only a weak signal was obtained for the cPLA2 cDNA. The sPLA2 groups I, II, and V and the lipoprotein-associated PLA2 sequences were not detected. The weak PCR signal obtained for cPLA2 is consistent with previous findings that showed that cPLA2 expression in B-cells was either very low or undetectable (20-22). Based on this it would suggest that iPLA2 may be one of the more significant PLA2s in B-cells. In fact the partial human iPLA2 cDNA sequence recently described (9) was cloned from a B-cell line cDNA library. However, no physiological stimuli is at present known to induce leukotriene synthesis in human B lymphocytes; hence they are dependent on exogenous arachidonic acid for leukotriene synthesis. Perhaps, what is required is the correct stimulus to activate the iPLA2 enzyme. It has been demonstrated that the majority of the 5-lipoxygenase enzyme in nonstimulated B lymphocytes is located at the nucleus (16). Thus, an increase in the intracellular calcium concentration, which renders translocation of 5-lipoxygenase to the nucleus, might not be a prerequisite for leukotriene synthesis in B lymphocytes, because the 5-lipoxygenase already is located at the nucleus. Therefore, it is tempting to speculate that some stimulus that activates the calcium-independent iPLA2 without increasing the intracellular calcium concentration may be sufficient for the induction of leukotriene synthesis in B lymphocytes. However, in a recent review, Balsinde and Dennis suggest that the major function of iPLA2 is in membrane remodeling and not in arachidonic acid release, although involvement in the latter cannot be completely ruled out (34). Therefore, the function and the regulation of iPLA2 in B lymphocytes and its role in leukotriene synthesis remain to be determined.

In conclusion, we describe in this report the cDNA sequence of the human iPLA2 and its various splice variants. We furthermore present data indicating that a splice variant of the iPLA2 containing only the ankyrin motifs and not the active site specifically modulates iPLA2 activity when the proteins are co-expressed in COS-7 cells. These findings suggest that alternative splicing of the iPLA2 pre-mRNA can result in the production of regulatory subunits that can modify iPLA2 in vivo activity.

    ACKNOWLEDGEMENTS

Paul Payette and Wanda Cromlish are acknowledged for helpful assistance and discussion. Dr. Ann English is acknowledged for critically reading the manuscript.

    FOOTNOTES

* This work was supported by Grants K97-03RM-12067/03X-7135 from the Swedish Medical Research Council and funds from the Swedish Foundation for Health Care Sciences and Allergy Research and the Swedish Society against Rheumatism.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.

To whom correspondence should be addressed: Dept. of Medical Biochemistry and Biophysics, Div. of Physiological Chemistry II, Doktorsringen 9A3, Karolinska Inst., 171 77 Stockholm, Sweden. Tel./Fax: 46-8-728-76-27; E-mail: Hans-Erik.Claesson{at}mbb.ki.se.

par To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, P. O. Box 1005, Pointe Claire-Dorval, Quebec H9R 4P8, Canada. Tel.: 514-428-8548; E-mail: brian_kennedy{at}merck.com.

1 The abbreviations used are: PLA2, phospholipase(s) A2; cPLA2, cytosolic PLA2; iPLA2, calcium-independent PLA2; sPLA2, secretory PLA2; CHO, Chinese hamster ovary; LT, leukotriene; RT, reverse transcriptase; PCR, polymerase chain reaction; EST, expressed sequence tag; RACE, rapid amplification of cDNA ends; bp, base pair(s).

2 P. K. A. Larsson, H.-E. Claesson, and B. P. Kennedy, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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