Proc. Nad. Acad. Sci. USA Vol. 88, pp. 5418-5422, June 1991 Medical Sciences

Cloning of a phospholipase A2-activating protein (antisense DNA/Ieukotriene/arachidonic acid)

MIKE A. CLARK*tt§, LYNNE E. OZGURtt, THERESA M. CONWAY*, JANICE DISPOTO*, STANLEY T. CROOKE*, AND JOHN S. BOMALASKI¶ *Smith Kline and French Laboratories, King of Prussia, PA 19479; tWashington University School of Medicine, St. Louis, MO 63110; Research, Bloomfield, NJ 07003; and 1Veterans Affairs Medical Center, Medical College of Pennsylvania, Philadelphia, PA 19104

tSchering-Plough

Communicated by Pedro Cuatrecasas, February 27, 1991

England Nuclear. LTD4 was provided by Smith Kline and French. Molecular Cloning of PLAP. BC3H1 cells were treated with LTD4 (1 AM) for 2 min prior to extraction of total cellular RNA using guanidine isothiocyanate. A cDNA library was prepared as described by Gubler and Hoffman (23). EcoRI linkers were added to the cDNAs, which, in turn, were ligated to Agtll arms and packaged using extracts obtained from Vector Laboratories. The library was screened using anti-mellitin antibodies (8, 22) by the procedure of Young, Davis, and coworkers (24, 25). The cDNA clone was subcloned into M13 and sequenced in both directions using dideoxy methodologies (26). Antibody Production. Antibodies were generated to the recombinant PLAP using PLAP-f3-galactosidase (p-Gal) fusion protein as antigen as described (25). The fusion protein was isolated by preparative SDS/polyacrylamide electrophoresis utilizing a 6% gel. The band containing the PLAP-pB-Gal fusion protein was initially identified on the basis of its marked increase with isopropyl 3-D-thiogalactoside (IPTG) treatment and by Western blot analysis using anti-mellitin antibodies. This band, which was well resolved from other proteins, was excised, mixed with Freund's adjuvant, and injected into female New Zealand White rabbits ("100 .ug of protein per injection). Control antiserum to pl-Gal was purchased from Bethesda Research Laboratories. Affinity Purification of PLAP and Phospholipase A2 and PLAP Assays. PLAP was immunoaffinity purified as described (8, 22). Phospholipase A2 was assayed in cell-free preparations using 1-palmitoyl-2-[14C]arachidonyl-sn-glycero-3-phospho-[methyl-3H]choline as described (7, 22, 2729). PLAP activity was also assayed using the method we have described (8, 22). [3HJArachidonic Acid Release and Prostaglandin Radioimmunoassays. The release of [3H]arachidonic acid from smooth muscle and endothelial cells was assayed as described (8, 30). Radioimmunoassays were performed to quantitate the production of 6-keto-prostaglandin Fia and thromboxane B2 using radioimmunoassay kits purchased from New England Nuclear as described (12, 30). Synthesis of Single-Stranded Antisense DNA and PLAP Peptides. Single-stranded DNA was chemically synthesized with phosphoramidite chemistry using either a Beckman or a Pharmacia DNA synthesizer; reagents were purchased from Beckman or Pharmacia, respectively. The synthetic PLAP peptides were synthesized using t-butoxycarbonyl chemistry. Cell Culture. The BC3H1 murine smooth muscle cell line and the bovine endothelial cell line CPAE were obtained from the American Type Culture Collection. Cells were electro-

ABSTRACT Recently we have described the isolation and biochemical characterization of a phospholipase A2-activating protein (PLAP). We have cloned this protein and found it to be expressed as a 2.5-kilobase mRNA. The steady-state levels of PLAP mRNA are induced in smooth muscle and endothelial cells following treatment with leukotriene D4. The increased message levels coincide with increased amounts of PLAP. Synthetic antisense DNA was used to block the synthesis of PLAP and this treatment effectively blocked the activation of phospholipase A2 and the increased generation of prostanoids in smooth muscle and endothelial cells treated with leukotriene D4.

Many aspects of inflammation are mediated, or modulated, by the release of biologically active oxygenated eicosanoids (for review see refs. 1, 2). The rate-limiting step for eicosanoid production is the release of arachidonic acid from endogenous phospholipid storage depots by one or more phospholipases (3-6). Our laboratory (7, 8) and others (9-11) have implicated phospholipase A2 activation as the major pathway responsible for the release of arachidonic acid in various cells. Accordingly, extensive investigation has focused on the biochemical mechanisms responsible for the activation of phospholipase A2 after agonist stimulation. Several laboratories, including our own, have demonstrated that a number of agonists require RNA and protein synthesis for the physiological production of oxygenated eicosanoid metabolites (8-16). These results suggested the existence of a mammalian phospholipase-activating protein. Dramatic activation of phospholipase A2 activity has been observed with a number of natural protein products (17-21). Since protein constituents help augment the biochemical activity of venom phospholipase A2 in a number of species, we postulated that a similar protein might exist in mammalian cells that would activate phospholipase A2. Utilizing antimellitin antibodies, we purified a mammalian protein that was a potent activator of phospholipase A2 (8, 22). We have termed this protein phospholipase A2-activating protein (PLAP). In this report we describe the cloning of PLAP and the use of antisense DNA to demonstrate the relevance of this protein in the activation of phospholipase A2 and the subsequent generation of eicosanoids produced by smooth muscle and endothelial cells in response to the inflammatory mediator leukotriene D4 (LTD4).

MATERIALS AND METHODS Materials. All radioactive compounds including 1-palmit-

oyl-2-[14C]arachidonyl-sn-glycero-3-phospho-[methyl-3H]-

choline and [3H]arachidonic acid were obtained from New

Abbreviations: LTD4, leukotriene D4; .3-Gal, /3-galactosidase; ELISA, enzyme-linked immunosorbent assay; PLAP, phospholipase A2-activating protein; IPTG, isopropyl 13-D-thiogalactoside. §To whom reprint requests should be addressed at: Schering-Plough Research, 60 Orange St., Bloomfield, NJ 07003.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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porated using 100 ,ug of DNA per 106 cells and a Bio-Rad electroporation device using the setting of 250 V and 25 ,ud. Transfected cells were selected in growth medium supplemented with G418 (GIBCO). Enzyme-Linked Immunosorbent Assays (ELISAs) for PLAP. Cells were grown in 96-well plates for 2-3 days until they were 75% confluent (8). Cells were treated (in quadruplicate wells) with LTD4 (1 ,tM) for the indicated periods of time. The reactions were stopped by the addition of a concentrated solution of formalin such that the final concentration was 3% (vol/vol). The cells were then permeabilized (0.05% Tween 20) and the primary antibodies were then allowed to react with the cells for 1 hr. The amount of primary antibody bound was determined using peroxidase-conjugated second antibody (Vector Laboratories). Northern Blot Analysis for the Induction of PLAP mRNA. CPAE or BC3H1 cells were treated with LTD4 (1 ,M) for the indicated periods of time prior to the addition of hot (65°C) phenol, and total RNA was then extracted. The resulting RNA was electrophoresed on an agarose gel (10 jig per lane). The gel was blotted and probed using the PLAP cDNA clone that had been nick-translated with [32P~dCTP. Assay for PLAP-Neutralizing Activity. PLAP (10 units) was purified as described (22) and was incubated with 2 ,ul of antibody that had been purified by ammonium sulfate precipitation. Next, the PLAP/antibody solution was added to whole cell sonicates and phospholipase A2 activity was assayed.

AACGACGGCCAGTGAAATTCCGCCGGCTCGGGCCTGTGGACGAGTCTCGC GCTGTGCCCGGGGCGCGCGTCCGGATCCACGCTGGCCATGGCGAGCGCGC CTCCAGATACCGGCTGAGCTGCTCGCTACCGGGCCACGAACTGGACGTGA GGGGCCTGGTGTGCTGCCTCTACCCGCCGGAGCCTTTGTGTCTGTGTCCC GGGATCGACCACCCGCCTCTGGGCTCCAGACAGTCCTAACAGGGGCTTTA CAGAAATGCACTATATGAGCGGCCACTCTAAIT'TTGTGTCTTATGTGTGT M H Y M S G H S N F V S Y V C ATCATACCCTCAAGTGACATATATCCTCATGGACTGATTGCCACTGGAGG I I P S S D I Y P H G L I A T G G AAATGACCACAATATTTGCATTTTCTCGCTGGACAGTCCAATGCCACTTT N D H N I C I F S L D S P M P L Y ATATTTTAAAGGGTCACAAAGATACTG15TGTAGTCTTTCTTCTGGAAAA I L X G H K D T V C S L S S G K

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RESULTS Cloning of PLAP. We hypothesized that treatment of BC3H1 cells with LTD4 would increase the synthesis of PLAP mRNA. Therefore, we made a cDNA library in Agtll using mRNA isolated from BC3H1 cells that had been treated for 2 min with LTD4 (1 ,M). The resulting library was then screened using a previously described antibody that had been produced to the bee venom peptide mellitin (8, 22). The cDNA clone was sequenced using dideoxy methodologies (26) and the results are shown in Fig. 1. The predicted amino acid sequence for PLAP was deduced from the longest open reading frame contained within the cDNA clone. To identify the region within this clone having homology with mellitin, we utilized the University of Wisconsin Genetics Computer Group data base software and compared the known sequence of mellitin (19) with the predicted PLAP sequence. This region of homology is underlined in Fig. 1. Because the protein sequence for PLAP was not known, various techniques were used to demonstrate that the isolated cDNA clone encoded PLAP. Antibodies to the PLAP-f-Gal Fusion Protein Affinity Purify and Neutralize PLAP Activity. Although the PLAP-P-Gal fusion protein was devoid of phospholipase A2-stimulatory activity (data not shown), it was useful for generating antibodies that could be utilized to immunoaffinity purify PLAP activity. PLAP-f3-Gal fusion protein was isolated from Escherichia coli that had been lysogenized using the A cDNA clone. Antibodies prepared to the fusion protein were immobilized on cyanogen bromide-coupled Sepharose particles. Whole cell extracts made from CPAE cells were next applied to the immunoaffinity columns and subsequently eluted using low pH buffer (50 mM sodium acetate, pH 3.1). The eluted fractions were assayed for PLAP activity (Fig. 2). Next, the antibodies made using the PLAP-f3-Gal fusion protein as antigen were tested for their ability to neutralize PLAP activity. Antibodies were incubated with purified PLAP that had been isolated from CPAE cells. The antibodies plus PLAP were then added to whole cell sonicates and assayed for PLAP-stimulatory activity (Table 1). In control

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Proc. Natl. Acad. Sci. USA 88 (1991)

Medical Sciences: Clark et al.

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TACCTGTGGTGGACTGACCTCAACTAGGTAAATGTCCCTTGTCACGGAT GGCTAGTCGACGTAGATTTTGTCACTTGTAAATAAAGGGGTTTPTTCTTC GAGAATGGAAAC -T ATGIY;TTTATAATCCTTTTGACTTC CTTGAATTACCTTGACGTGGACTTICTCTTCTTCGATTGACTTCTGCTGAA TCATGAAGAACTTTTCTATGACAGGGACTAAACATTATTAAGGAGTCTTT CTTCTGTAACAGAAAGGACGTGAACTGTAAGAAGCGGATAGCTAATTCGT

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FIG. 1. Sequence of the PLAP cDNA. The PLAP cDNA was isolated from a cDNA library made from BC3H1 cell mRNA in Agt1l. The EcoRI insert was subcloned into M13 and sequenced using dideoxy methodologies. The predicted amino acid sequence of the largest open reading frame is shown. The region having the greatest homology with mellitin is underscored. The complementary antisense DNA used in Table 2 was synthesized using the region encoded between the two arrowheads (v).

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Proc. Natl. Acad Sci. USA 88 (1991) GIGAVLKVLTIGLPALISWIKRKRQQ

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Cloning of a phospholipase A2-activating protein.

Recently we have described the isolation and biochemical characterization of a phospholipase A2-activating protein (PLAP). We have cloned this protein...
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