Cloning and expression of rat lung acidic Ca2+-independent PLA2 and its organ distribution

Tae-Suk Kim1, Chandra Dodia1, Xi Chen1, Brian B. Hennigan1, Mahendra Jain2, Sheldon I. Feinstein1, and Aron B. Fisher1

1 Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; and 2 Department of Chemistry and Biochemistry, Newark, Delaware 19716

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

A clone for a rat acidic Ca2+-independent phospholipase A2 (aiPLA2) was isolated from a cDNA library prepared from rat granular pneumocytes with a probe based on the human aiPLA2 sequence (T. S. Kim, C. S. Sundaresh, S. I. Feinstein, C. Dodia, W. R. Skach, M. K. Jain, T. Nagase, N. Seki, K. Ishikawa, N. Nomura, and A. B. Fisher. J. Biol. Chem. 272: 2542-2550, 1997). In addition, a consensus sequence for mouse aiPLA2 was constructed from several mouse cDNA clones in the GenBank and dbEST databases. Each sequence codes for a 224-amino acid protein with 88% identity of the amino acids among the three species and conservation of a putative lipase motif (GDSWG). Translation of mRNA produced from the rat clone in a wheat germ system resulted in expression of PLA2 activity with properties similar to those of the human enzyme, i.e., acidic pH optimum and Ca2+ independence. The localization of aiPLA2 in rat tissues was studied with the human cDNA probe, polyclonal and monoclonal antibodies, and aiPLA2 activity. aiPLA2 is present in the lung as evidenced by high levels of mRNA and protein expression and by enzymatic activity that is inhibited by anti-PLA2 antibody and by the transition state analog 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33). Immunocytochemistry showed the presence of aiPLA2 in alveolar type II cells, alveolar macrophages, and bronchiolar epithelium. In the brain, heart, liver, kidney, spleen, and intestine, aiPLA2 mRNA content was <50% of that in the lung, immunoreactive protein was not detectable, and enzymatic activity was not inhibited by MJ33 or aiPLA2 antibody. These results show marked enrichment of aiPLA2 in the lung compared with the other organs and suggest translational control of aiPLA2 expression.

phospholipase A2; calcium ion; mouse; liposome; lipase; 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol; active serine; in vitro translation; type II cell complementary deoxyribonucleic acid expression library

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE UPTAKE AND CLEARANCE of lung surfactant phospholipid from the alveolar space are followed by extensive degradation of the internalized phosphatidylcholine by intracellular phospholipases, predominantly phospholipase A2 (PLA2) (12, 13). PLA2 represents a large superfamily of enzymes that catalyze the hydrolysis of the sn-2 fatty acyl or alkyl bond of phospholipids, liberating free fatty acids and lysophospholipids. These enzymes have been purified from different sources such as the mammalian pancreas, lung, platelets, and synovial fluid and from insect and snake venoms; they are classified into several groups according to their molecular characteristics, cellular location, and Ca2+ requirement for catalysis (2). A specific subset of the PLA2 family shows activity that is similar in the presence or absence of Ca2+ and has been termed Ca2+-independent PLA2 (iPLA2) (2). iPLA2 activity in rat lung homogenates was first described ~25 years ago (22), and iPLA2 with an acidic pH optimum (aiPLA2) was subsequently demonstrated in the rabbit lung (16), rat granular pneumocytes (lung alveolar type II cells) (8), and rat and human alveolar macrophages (12, 14). Activity of aiPLA2 has been localized further to the lung lamellar bodies (the surfactant secretory organelle) and the lysosomal fraction (12, 16). Under our assay conditions, lung aiPLA2 activity is inhibited by a phospholipid transition state analog, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), whereas other PLA2 activities in the lung homogenate are insensitive (12, 13).

Kim et al. (18) previously reported the purification of aiPLA2 from rat lung and the cloning of the cDNA for this enzyme from a human myeloblast cell line. They showed that the protein expressed by this cDNA is aiPLA2 and identified a putative "lipase" motif, GDSWG, which matches the consensus sequence GXSXG found in serine proteases and some lipases (11). The molecular composition and spectrum of activity for aiPLA2 clearly differentiates it from other Ca2+-independent phospholipases A2 that have been described to date. To facilitate study of the regulation of expression of this enzyme in rat and mouse models, we have cloned and sequenced a cDNA for aiPLA2 isolated from a cDNA library prepared from an enriched rat type II cell preparation. We also constructed a mouse cDNA clone sequence by retrieval of sequences previously entered into several databases.

Although the presence of the enzyme in the lung has been demonstrated by previous studies (3, 18), the distribution of aiPLA2 in other organs is not known. To evaluate distribution in rat tissues, we used the human cDNA probe, a polyclonal antibody against an internal peptide of rat lung aiPLA2, a polyclonal antiserum against the Escherichia coli-expressed human homolog of the enzyme, and assay of MJ33-inhibitable activity. Results by these criteria indicate marked enrichment of aiPLA2 in the lung and especially in alveolar type II cells compared with other organs.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials. Male Sprague-Dawley rats weighing ~200 g were obtained from Charles River Breeding Laboratories (Kingston, NY). The KG-1 human myeloblast cell line was obtained from the American Type Culture Collection (Rockville, MD). HA0683, the human aiPLA2 cDNA clone from the KG-1 cell line, was provided by Kazusa DNA Research Institute (Kisarazu, Chiba, Japan) as previously reported (18). Lipids were obtained from Avanti Polar Lipids (Birmingham, AL). All radiochemicals and X-ray film were purchased from NEN Life Sciences (Boston, MA). Arachidonyltrifluoromethyl ketone (AACOCF3) was from Calbiochem (La Jolla, CA); bromoenol lactone (BEL) was from Cayman Chemicals (Ann Arbor, MI); and p-bromophenacyl bromide (pBPB) and diethyl p-nitrophenyl phosphate (DENP) were from Sigma (St. Louis, MO). MJ33 was synthesized as previously described (17). Molecular-mass standards for SDS-PAGE and the protein dye-binding kit were from Bio-Rad (Hercules, CA). Nitrocellulose and supported nitrocellulose membrane (Optitran) were from Schleicher & Schuell (Keene, NH). Horseradish peroxidase-conjugated goat anti-rabbit IgG and enhanced chemiluminescence kit were purchased from Amersham (Arlington Heights, IL). The SuperScript Plasmid System (for cDNA library construction) and Library Efficiency Competent Cells were obtained from GIBCO BRL (Life Technologies, Grand Island, NY). Klenow enzyme was from Boehringer Mannheim (Indianapolis, IN). pCR 2.1 vector was from Invitrogen (San Diego, CA). In vitro transcription and wheat germ in vitro translation kits were from Ambion (Austin, TX). Oligonucleotides were obtained from CyberSyn (Lenni, PA). PCR Kits were from Perkin-Elmer Applied Biosystems (Foster City, CA).

Rat tissue homogenates. The rats were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg), ventilated through a cannula inserted via a tracheostomy, and then exsanguinated by transection of the abdominal aorta. Lungs were cleared of blood in situ by perfusion through the pulmonary artery with saline. Other organs (brain, heart, liver, kidney, spleen, and intestine) were harvested and rinsed with saline. All organs were extensively homogenized in 10 volumes of saline with a Polytron with a PT10 probe (Brinkmann, Westbury, NY) followed by a Potter-Elvehjem vessel (Thomas, Philadelphia, PA) with a motor-driven pestle.

Isolation of rat lung cells and preparation of RNA. A preparation enriched in rat type II cells was prepared by elastase digestion of lungs and overnight culture on plastic dishes as previously described (9). Alveolar macrophages were obtained by lung lavage. For enzyme assay, the cells were disrupted by sonication as previously described (12). Total RNA was extracted from freshly isolated type II cells (before the panning step and containing ~67% type II cells) with the acid guanidinium thiocyanate-phenol-chloroform extraction method (10). Poly(A)+ RNA was purified from the total RNA preparation with the FastTrack mRNA isolation system (Invitrogen, San Diego, CA).

cDNA library construction. A cDNA library was constructed in the pSPORT 1 vector with the SuperScript Plasmid System (Life Technologies). Double-stranded cDNA was synthesized from 4 µg of the poly(A)+ RNA and cloned unidirectionally into the pSPORT 1 plasmid with Sal I (5') and Not I (3') adaptors. The plasmid DNA was transformed into E. coli strain DH5alpha . The cDNA library containing ~310,000 independent clones was amplified and stored as five separate plasmid pools. The number of clones in each cDNA pool ranged from 40,000 to 100,000. The average insert size was ~1.1 kb.

DNA sequencing. DNA sequencing was carried out by the DNA Sequencing Facility of the University of Pennsylvania (Philadelphia) with fluorescent dye-labeled dideoxy terminator cycle Perkin-Elmer sequencing kits, and the products were analyzed on ABI automated sequencers (Perkin-Elmer Applied Biosystems).

Isolation of a partial clone by hybridization screening of cDNA library. Plasmid DNA from the type II cell cDNA library was transformed into E. coli strain HB101 and screened for positive colonies by standard methods (5). To reduce the background caused by vector-to-vector hybridization, we generated a probe by RT-PCR with primers designed from human aiPLA2 cDNA. The sequence of the upstream primer, 5'-GTTTCCACGACTTTCTGGGAGAC-3', was located at positions 114-136 of the human sequence (18). The sequence of the downstream primer, 5'-AGCGGAGGTATTTCTTGCCAG-3', was located at positions 681-702 of the human sequence (18). RT-PCR was performed with these primers with human placental poly(A)+ RNA (Clontech, Palo Alto, CA) according to the manufacturer's protocol (Perkin-Elmer Applied Biosystems). The 589-bp PCR product was then labeled with [32P]dCTP (3,000 Ci/mmol) by random priming with Klenow enzyme and used as a probe. A total of 220,000 clones were screened by standard methods (5). One hybridizing clone was identified after autoradiography on X-ray film at -80°C for 16-18 h. Sequencing showed that the 530 bp of DNA [excluding poly(A)+ and linkers] in this clone contained the 3' terminus for aiPLA2, including 366 bp of the coding region, the putative translational termination codon (TAA), 161 bp of the 3'-untranslated sequence, and a 15-bp poly(A)+ tract.

Cloning the 5' terminus by PCR. The 5' terminus of the cDNA clone was obtained from the rat cDNA library by PCR with an upstream primer (5'-TACGCCAAGCTCTAATACGACTCAC-3') corresponding to the pSPORT 1 vector and a downstream primer (5'-TGCCTTTTTCATCCTTCTCTCCT-3') complementary to a region near the 5'-end of the partial clone obtained from the library. The PCR product was subcloned into pCR 2.1 vector (Invitrogen, San Diego, CA), and the nucleotide sequence was determined. This PCR-generated cDNA insert contained 399 bp from the cDNA sequence corresponding to the 5' region of rat aiPLA2, of which 73 bp overlapped with the 3' clone.

Reconstruction of complete cDNA. The complete cDNA clone was constructed from the two partial clones with a single BamH I site in the overlapping region. On digestion with BamH I, the partial clone containing both the 3' terminus of cDNA for aiPLA2 (530 bp) and a BamH I fragment of the pSPORT 1 polylinker (20 bp) was ligated into the pCR2.1 vector containing the 5' terminus of the cDNA clone that also had been cleaved by BamH I, leaving 326 bp. The total length of the complete cDNA, exclusive of the poly(A)+ tract and linkers was, therefore, 856 bp. Large-scale preparations of plasmid containing the full-length cDNA clone were obtained with a Qiagen maxiprep column (Qiagen, Chatsworth, CA) according to the manufacturer's protocol. Sequencing of the full-length cDNA clone was carried out as described in DNA sequencing.

Construction of mouse sequence. BLAST searches with the rat cDNA sequence for aiPLA2 identified 15 expressed sequence tags (ESTs) in the GenBank and dbEST databases (4). These ESTs corresponded to mouse cDNA clones from the I.M.A.G.E. Consortium collection (19). Their GenBank accession numbers are AA166588, AA105054, AA039135, W70913, W13509, AA240416, AA073086, AA172277, AA242505, W71818, AA048916, W59407, W83606, AA049385, and W83464. Clone AA166588 had the most upstream sequence but differed considerably from the other clones and was not included in the final sequence. Sequences from the other individual clones were edited and assembled with Sequencher software (GeneCodes, Ann Arbor, MI). Estimated error/ambiguity rate in the overall sequence was 0.1%. The CLUSTALW multiple program (27), as well as manual alignment, was used to generate Fig. 1.

In vitro transcription and translation. The rat cDNA clone was linearized by digestion with Hind III, which cleaves downstream from the cDNA insert, and was used as a template for the synthesis of capped mRNA employing T7 RNA polymerase with 7mGpppG as previously described (18). The RNA was subsequently used as a template for in vitro translation in wheat germ lysate using previously described methods (18). Each experiment included a negative control in which no RNA was added and a positive control in which XeF-1alpha RNA, encoding Xenopus elongation factor 1-alpha [relative molecular weight (Mr) 50,300] provided by the supplier, was added. The translated products were then assayed for aiPLA2 activity.

Autoradiography of translated protein with [35S]methionine. Labeling of the translation product in wheat germ extract was performed with the addition of 29.7 µCi of translation grade [35S]methionine ([35S]Met; 1,174 Ci/mmol) and 1 mM of the other amino acids in a reaction mixture as previously described (18). Small aliquots of translated proteins were analyzed on 15% SDS-PAGE followed by autoradiography as previously described (18).

Northern blot. Total RNA was extracted from rat tissues with the acid guanidinium thiocyanate-phenol-chloroform method (10). RNA was dissolved in diethyl pyrocarbonate-treated water, quantified by absorbance at 260 nm, and stored frozen at -80°C. Extracted RNA samples were electrophoresed in a 1% agarose gel containing formaldehyde, blotted, and hybridized to 32P-labeled HA0683 or rat cDNA probes generated by random priming with Klenow enzyme (Boehringer Mannheim) (5). Membranes were air-dried and exposed to X-ray film, and the radioactivity was measured with an AMBIS radioisotopic detector (Scanalytics, Billerica, MA). Normalization of aiPLA2 mRNA was carried out by stripping and rehybridizing the membrane with a 28S RNA oligonucleotide probe (20).

Production of antisera. Affinity-purified polyclonal antiserum, commercially produced in rabbits against a peptide (CLSILYPATTGRNFDE) bearing an internal sequence of rat 26-kDa aiPLA2, has been described previously (3). This antibody is designated as antibody to rat aiPLA2 synthetic peptide (anti-raiPLA144-1582), but it should be noted that this peptide sequence is identical to the deduced human enzyme. Mouse antiserum was also produced to the expressed human protein. The human cDNA clone of aiPLA2 was expressed in E. coli with the pET28C plasmid (Novagen, Madison, WI). The expressed protein containing a His tag was purified on a nickel column and showed a single immunoreactive band by Western blot analysis with anti-raiPLA144-1582 antibody. The purified expressed protein was used for production of monoclonal antisera by the University of Pennsylvania Cell Center with the use of standard methods. Polyclonal antiserum was obtained from the mice at the time of death and is designated as antiserum to expressed human aiPLA2 (anti-haiPLA2). Twenty-three clones showed a positive reaction to the immunogen by ELISA assay. The clone 8H11 was selected for immunohistochemistry based on a strong fluorescence signal with isolated type II cells.

Western blot. Proteins (40 µg of tissue homogenate protein or 60 µg of KG-1 cell line protein) were solubilized with gel loading buffer (50 mM Tris · HCl, 10% glycerol, 1% SDS, 2% mercaptoethanol, and 0.01% bromphenol blue, pH 6.8) and boiled for 4 min. The samples were subjected to 12% SDS-PAGE at 100-V constant voltage and then transferred overnight to nitrocellulose membrane at 20-mA constant current. The membrane was blocked at room temperature with 2% gelatin in Tris-buffered saline (TBS; 0.15 M NaCl and 0.01 M Tris · HCl, pH 7.5) for 1 h and then incubated with 1:1,000 anti-raiPLA144-1582 antibody or anti-haiPLA2 antiserum in 1% gelatin-TBS-T (TBS-T is 1:2,000 Tween 80 in TBS) for 2 h. After three washes with TBS-T, the membrane was incubated with 1:4,000 horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG in 1% gelatin-TBS-T for 1 h. The membranes were washed two times with TBS-T for 5 min each and two times with TBS for 5 min each. Antibody binding was detected by enhanced chemiluminescence.

Assay of PLA2 activity. Enzyme activity was measured at pH 4 (40 mM acetate buffer with 5 mM EDTA) with a liposomal-based radiochemical assay as previously described (12). The substrate was 1-palmitoyl,2-[9,10-3H]palmitoyl-sn-glycerol-3-phosphocholine ([3H]DPPC) added to a lipid mixture of DPPC-egg phosphatidylcholine-egg phosphatidylglycerol-cholesterol in a molar ratio of 10:5:2:3. The specific activity for [3H]DPPC in the lipid mixture was 4,400 dpm/nmol. Lipids were dried under N2, resuspended in isotonic saline, subjected to repeated freeze-thaw cycles by alternating liquid N2 and warm H2O, and then extruded through a polycarbonate membrane (0.1-µm pore size) to generate unilamellar liposomes. The 1-ml PLA2 assay reaction volume contained 1 µmol total lipid. Incubation was at 37°C, generally for 1 h. The reaction was stopped by addition of CHCl3-CH3OH. Lipids were extracted, and radiolabeled free fatty acids were separated by TLC as previously described (12). Authentic palmitic acid was cochromatographed. The free fatty acid spots were identified with I2 vapor, scraped from the plate, and analyzed by scintillation counting with an internal standard for quench correction. The value obtained for recovered disintegrations per minute was corrected for blank values obtained in the absence of enzyme. PLA2 activity was calculated based on the specific radioactivity of DPPC. For evaluation of MJ33, the inhibitor was added (usually at 3 mol%) to the lipid mixture before generation of the liposomal substrate. The effects of the potential PLA2 inhibitors AACOCF3, BEL, and DENP were determined after preincubation of the enzyme with inhibitor at pH 4 for 30 min at 37°C. To determine the effect of pBPB, the protein was preincubated with inhibitor at pH 7.4 in 25 mM Tris containing 0.5 mM EDTA. The concentrations selected for the testing of inhibitors have been shown previously to produce maximal effect in other systems (1, 3, 15).

Immunohistochemistry. Rat lungs were cleared of blood and fixed by perfusion with 4% paraformaldehyde in PBS. Tissue pieces were degassed in vacuum, cryoprotected with increasing sucrose concentrations, and frozen with dry ice and isopentane. Tissue sections were cut with a cryostat, treated with 0.1% NaBH4 followed by a blocking solution consisting of 5% bovine serum albumin, 10% goat serum, and 0.3% Triton X-100 in PBS. The sections were exposed to monoclonal antibody 8H11 antiserum (1:100 dilution) as the primary antibody and Texas Red-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) as the secondary antibody. Control slides were treated identically except that the primary antibody was replaced with nonimmune serum. Slides were air-dried, a coverslip was applied with Mowiol (Calbiochem, San Diego, CA), and sections were examined with a Nikon Diaphot inverted microscope with an attached MetaMorph image-analysis system (Universal Imaging, West Chester, PA). Freshly isolated type II alveolar epithelial cells and alveolar macrophages were allowed to attach to coverslips for 1 h and were then fixed and processed as described for tissue.

Protein determination. Protein was quantitated with the Coomassie blue binding assay kit (Bio-Rad, Hercules, CA) with bovine gamma -globulin as the standard.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Identification of rat and mouse cDNA clones. We have isolated an 856-bp [exclusive of poly(A)+] cDNA clone encoding rat aiPLA2 from the type II cell-enriched cDNA library. An open reading frame runs from the 5'-end of the clone through nucleotide 692. Initiation of translation at the putative ATG start codon, 21 nucleotides from the 5'-end of the clone, would result in a coding region of 672 nucleotides that would be translated into a 224-amino acid polypeptide.

To assemble the sequence of the mouse clone, we identified 15 ESTs in GenBank and dbEST databases by BLAST searches (4) using the rat aiPLA2 cDNA sequence. A consensus sequence from these mouse clones was generated by assembling and editing as described in METHODS.

Comparison within the coding region for the rat aiPLA2 cDNA sequence shows 87% identity with the previously cloned human cDNA (18) and 93% identity with the assembled mouse sequence (Fig. 1A). More than 85% of the nucleotides in the coding region are identical in all three species. cDNAs from all three species code for a 224-amino acid protein. The rat protein (calculated Mr 24,820) shows 92% identity to deduced amino acids from the human cDNA for aiPLA2 (calculated Mr 25,032) and 94% to the mouse sequence (calculated Mr 24,825; Fig. 1B). Eighty-eight percent of the amino acids are identical in all three species, including the putative lipase motif (GDSWG). In addition to the protein coding sequence, the rat clone contains 20 bp of the upstream and 161 bp of the downstream sequence. Comparisons among the three species show more divergence in the upstream and downstream sequences than in the coding region.


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Fig. 1.   Sequence comparisons among mouse (M), rat (R), and human (H) acidic Ca2+-independent phospholipase A2 (aiPLA2). A: alignment of M, R, and H nucleotide sequences for aiPLA2. * Nucleotides that are identical in all 3 species. Putative translational initiation codon (ATG) and termination codon (TAA) are underlined. Numbering follows rat sequence. B: alignment of M, R, and H amino acid sequences for aiPLA2. * Amino acids that are identical in all 3 species. Putative lipase motif (GXSXG) is indicated by a double underline. Peptide (amino acid numbers 144-158) used for polyclonal antibody production is indicated in boldface.

Expression of rat cDNA in wheat germ in vitro translation system. To determine whether the rat cDNA actually encodes a translatable protein with a molecular mass of the predicted value, the rat cDNA was transcribed in vitro with T7 RNA polymerase and translated in vitro with wheat germ extract. Based on the structure of the cDNA clone and vector, it was expected that translation might begin either at an ATG codon in the vector or at the putative start site for the actual PLA2 (Fig. 2). Initiation at the first upstream start codon present in the multiple cloning site of the vector, 159 bp upstream of the actual PLA2 start site (calculated Mr of additional amino acids 5,902) would be expected to produce a polypeptide with a molecular mass of ~30.7 kDa.


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Fig. 2.   Schematic outline of pCR2.1 vector construct containing a full-length cDNA insert for rat aiPLA2 (RaiPLA2). T7 in vector construct indicates T7 promoter. T7 promoter sequence is underlined. Upstream region shown includes sequences from pCR2.1 vector, pSPORT 1 vector, Sal I adapter, and 5'-untranslated region of RaiPLA2. Two potential upstream translational start sites located in pCR 2.1 vector are shown in boldface. Sequence from RaiPLA2 cDNA is double underlined, and actual start site in cDNA clone is in boldface. Translation beginning at 1st ATG in vector would preserve reading frame and result in an extra 5.9-kDa segment.

SDS-PAGE with autoradiography of the translated [35S]Met-labeled protein showed two bands ~24.9 and 30.1 kDa in size (Fig. 3). The smaller product is similar in size to that of the predicted protein and to the PLA2 enzyme isolated from rat lung (18). The size of the larger product is consistent with it being translated from the first ATG codon in the vector or possibly from a second ATG codon in the vector that is just 18 nucleotides downstream from the first (Fig. 2). [35S]Met-labeled protein expressed by the wheat germ extract increased as input RNA increased and appeared to saturate at ~1.5 µg (Fig. 3). Control RNA (XeF-1alpha ) was translated into a protein with a major band at 50 kDa as expected. Incubation in the absence of cRNA (indicated as 0 µg in Fig. 3) showed no signal on autoradiography.


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Fig. 3.   SDS-PAGE of in vitro translated RaiPLA2. In vitro transcribed complementary RNA (cRNA), in amounts ranging from 0 to 2 µg, was translated with wheat germ lysate in presence of [35S]methionine, and translation products were analyzed by 15% SDS-PAGE. Nos. at right, migration of prestained molecular-mass markers (in kDa) that were electrophoresed in an adjacent lane (not shown). Nos. at bottom, counts/min (CPM; minus background) measured on an Ambis 4000 imager. Electrophoresis of translated products resulted in 2 bands ~32 and 26 kDa in size. Expression of both products increased with increasing amounts of input cRNA, saturating at ~1.5 µg. Control RNA (XeF-1alpha ) was translated into a protein with a major band at 50 kDa, as expected.

Characterization of aiPLA2 activity. The in vitro translated rat enzyme was analyzed for activity by PLA2 assay at pH 4 in both the presence and absence of Ca2+. Activity of the expressed rat PLA2 was not affected by the addition of Ca2+ (Table 1). The activity at pH 6 was decreased by ~93% compared with the activity at pH 4 (Table 1).

                              
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Table 1.   Effects of pH and Ca2+ on activity of in vitro translated rat aiPLA2

Potential inhibitors AACOCF3 (100 µM), BEL (100 µM), and pBPB (20 µM) were tested for their effects on the activity of the in vitro translated rat aiPLA2 (Table 2). ATP (10 mM), an activator of myocardial iPLA2 (31), was also evaluated. Enzymatic activity was insensitive to these effectors. As expected, the tetrahedral mimic MJ33 (3 mol%) showed a significant inhibitory effect on the activity of aiPLA2 (Table 2). Because of the presence of the lipase motif, the effect of a serine protease inhibitor (DENP, 0.5 mM) was evaluated and found to significantly inhibit aiPLA2 activity (78% inhibition; Table 2). A 1:50 dilution of the polyclonal antibody anti-raiPLA144-1582 decreased activity of in vitro translated rat protein by ~50% compared with the control value (Table 2).

                              
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Table 2.   Effect of inhibitors on activity of in vitro translated rat aiPLA2

aiPLA2 mRNA in rat tissues. The expression of aiPLA2 in rat lung and granular pneumocytes was investigated by Northern blot analysis with rat cDNA as a probe. The mRNA corresponding to this clone, ~1.7 kb in size, was expressed in both rat lung and isolated rat granular pneumocytes with approximately twofold enrichment in the cells (Fig. 4).


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Fig. 4.   Northern analysis of aiPLA2 in rat lung and granular pneumocytes. Total RNA from rat lung and granular pneumocytes (10 µg) and cRNA (10 pg), transcribed in vitro from RaiPLA2 clone with T7 RNA polymerase, was electrophoresed on a formaldehyde-agarose gel, transferred to a nitrocellulose membrane, and hybridized with an RaiPLA2 cDNA probe labeled with [32P]dCTP by random priming. aiPLA2 mRNA was detected as a single band of ~1.7 kb in lung and granular pneumocytes. Hybridization of cRNA with rat cDNA probe resulted in a smaller band due to shorter length of 3'-untranslated region. 18S and 28S, 18S and 28S rRNA, respectively.

We next evaluated the distribution of aiPLA2 in selected rat tissues. Northern blot analysis with equal RNA loading in all lanes demonstrated expression of aiPLA2 mRNA (~1.7 kb) in all tissues examined (brain, lung, heart, liver, kidney, spleen, and intestine; Fig. 5). Normalization with 28S rRNA as an internal standard demonstrated a twofold or greater enrichment of mRNA level in the lung compared with other tissues (Fig. 5).


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Fig. 5.   Northern blot analysis of aiPLA2 in rat tissues. Total RNA (20 µg) from rat organs was electrophoresed on a 1% formaldehyde-agarose gel and transferred to nitrocellulose membrane. Lanes 1-7, brain, lung, heart, liver, kidney, spleen, and intestine, respectively. A: hybridization with aiPLA2 probe labeled with [32P]dCTP by random priming. A single band of ~1.7 kb was detected. B: hybridization with oligonucleotide complementary to 28S rRNA as a control for RNA loading. C: relative levels of aiPLA2 mRNA normalized to 28S rRNA levels.

Immunological detection of aiPLA2. Western blot analysis showed a single immunoreactive band at 26 kDa in rat lung homogenate, corresponding to the size of rat aiPLA2 (Fig. 6). Results obtained with rabbit anti-raiPLA144-1582 polyclonal antibody and anti-haiPLA2 polyclonal antiserum from mice were similar. No immunoreactive bands were detected in the homogenates of other tissues (brain, heart, liver, kidney, spleen, and intestine) with either of the two antibodies (Fig. 6). The amount of protein loaded for all lanes was similar. Because of the somewhat surprising lack of immunoreactivity in tissues other than the lung, we evaluated the KG-1 human myeloblast cell line, the source of the original aiPLA2 cDNA isolation. Cells were grown to confluence, and RNA and soluble protein were isolated. mRNA for aiPLA2 was detected on Northern blot as expected, but no immunoreactive band was seen on Western blot with the antiraiPLA144-1582 antibody (data not shown).


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Fig. 6.   Detection of proteins recognized by aiPLA2 antibodies [antibody to RaiPLA2 synthetic peptide (antiRaiPLA144-1582; A) or anti-HaiPLA2 antiserum (B)] in rat tissue homogenates by Western blot analysis. Samples from tissue homogenates (40 µg) were dissolved in gel loading buffer, boiled for 4 min, and then loaded onto 12% SDS-PAGE. Proteins were transferred overnight to nitrocellulose membrane, blotted with 1:1,000 dilution of anti-RaiPLA144-1582 antibody or anti-HaiPLA2 antiserum, and detected with enhanced chemiluminescence. Nos. at left and right, position of molecular-mass markers (in kDa).

aiPLA2 activity in rat organs. Rat organs were surveyed for the presence of Ca2+-independent PLA2 activity at pH 4. Activity in lung homogenate was 18.8 ± 0.5 nmol · h-1 · mg protein-1 and was inhibited 88% by MJ33 (Fig. 7), similar to our previous reports (12, 13). Anti-raiPLA144-1582 antibody inhibited activity ~70% at a 1:50 dilution, whereas nonimmune serum had no effect (Fig. 7). The antibody demonstrated concentration-dependent inhibition of activity of the lung enzyme in the range of 1:400 to 1:50 dilutions (data not shown). The serine protease covalent inactivator DENP (0.5 mM) decreased activity in lung homogenate by 80% (Fig. 7) as previously described for aiPLA2 translated in vitro (18) and for bovine enzyme (3).


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Fig. 7.   aiPLA2 activity in rat tissues. aiPLA2 activity was measured in homogenates of different rat tissues by incubation at 37°C for 1 h with 1-palmitoyl,2-[9,10-3H]palmitoyl-sn-glycerol-3-phosphocholine as substrate in mixed unilamellar liposomes in Ca2+-free buffer (1 ml) at pH 4. Rat tissue homogenates (200 µg) were preincubated with anti-RaiPLA144-1582 antibody (Ab; 1:50 dilution) for 2 h at 4°C or with diethyl p-nitrophenyl phosphate (DENP; 0.5 mM) for 30 min at 37°C. 1-Hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33; 3 mol%) was cosonicated with liposomal substrate. Values are means ± SE for control and MJ33; n = 4 rats. Values are means ± range for Ab and DENP; n = 2 rats.

PLA2 activities at pH 4 (minus Ca2+) in homogenates of brain, heart, liver, kidney, spleen, and intestine varied from 2.1 to 7.3 nmol · h-1 · mg protein-1 (Fig. 7); thus specific activity was <40% compared with the lung homogenate. DENP inhibited activity at pH 4 in each of these tissues, although only by 40-75% (Fig. 7). These assay results indicate low-level but widespread distribution of serine-dependent iPLA2 activity in rat organs. However, in contrast to the effects on lung homogenate, neither MJ33 nor anti-raiPLA144-1582 antibody at a 1:50 dilution had any effect on aiPLA2 activity in the other organs (Fig. 7) or in the KG-1 cell line (data not shown). Thus aiPLA2 activity as characterized by sensitivity to MJ33 and the anti-raiPLA144-1582 antibody so far is limited to the lung, which is in good agreement with the results of Western blots.

aiPLA2 in rat lung cells. Western blot with antiraiPLA144-1582 antibody showed immunoreactivity at 26 kDa for both granular pneumocytes and alveolar macrophages (Fig. 8). Assay for aiPLA2 showed high specific activity in sonicated granular pneumocytes that was markedly inhibited by MJ33 (Table 3). MJ33-inhibitable specific activity in alveolar macrophages was only 27% of type II cell activity. No significant activity was demonstrated in the homogenates of major bronchi.


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Fig. 8.   Western blot with RaiPLA2 antibody of type II cells in primary culture after overnight incubation and freshly isolated alveolar macrophages. Each lane was loaded with 25 µg of protein and analyzed as in Fig. 6. Nos. on left, position of molecular-mass markers (in kDa).

                              
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Table 3.   aiPLA2 activity of rat lung cells

In sections of rat lung, immunofluorescence with monoclonal antibody 8H11 was prominent in granular pneumocytes (type II epithelial cells) and in bronchiolar epithelium but was not demonstrated in membranous (type I) epithelium or capillary endothelium (Fig. 9) or in major bronchi (data not shown). Isolated rat type II epithelial cells and alveolar macrophages both demonstrated positive immunofluorescence (Fig. 10).


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Fig. 9.   Immunofluorescence of rat lung using anti-HaiPLA2 monoclonal Ab 8H11 with Texas Red-conjugated goat anti-mouse IgG secondary antibody in alveolar region (A) and bronchiolar region (C). Comparison with control sections (primary antibody replaced by nonimmune serum) in alveolar region (B) and bronchiolar region (D) indicates specific immunostaining of type II alveolar epithelial cells and bronchiolar epithelium. All panels are at same magnification. Bar, 10 µm.


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Fig. 10.   Immunofluorescence of freshly isolated rat type II alveolar epithelial cells (A, B, E, and F) and alveolar macrophages (C, D, G, and H). Cells were attached to coverslips and immunostained as in Fig. 9. A-D: immunofluorescence (A and C) and corresponding phase-contrast images (B and D) for monoclonal antibody 8H11. E-H: immunofluorescence (E and G) and phase-contrast images (F and H) for cells reacted with nonimmune serum. All panels are at same magnification. Bar, 10 µm.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Ca2+-independent phospholipases A2 have been shown to be widely distributed in most mammalian tissues, underlining their potential importance in cellular functions (2). However, there is scant molecular information about these enzymes due to difficulties associated with purification and relatively low yield. Thus the structure of the Ca2+-independent phospholipases A2, their mechanism of action, and their relationship to other phospholipases A2 is not well described. Recently, we have isolated a 26-kDa aiPLA2 from rat lung, which corresponds to a cDNA clone from the human male myeloblast cell line KG-1 (18). There have been no other reports of sequence information for lysosomal type iPLA2, although two other members of the iPLA2 family, a cytosolic iPLA2 (25) and a low-density lipoprotein-associated PLA2 (26) that appears to be identical to platelet-activating factor (PAF) hydrolase (29), have been cloned and sequenced. Here we describe the cloning, sequencing, and characterization of a cDNA for rat aiPLA2.

Sequence similarity to other proteins. As expected, the deduced amino acids from the nucleotide sequence of the clone isolated from a rat type II cell cDNA library is identical to the sequence obtained previously by amino acid sequencing (35 residues) of tryptic digests of the 26-kDa protein isolated from rat lung (18). Searches for similarity to the rat cDNA sequence at the protein (SwissProt database) and DNA (GenBank database) levels did not identify any known phospholipases other than the 1,653-bp cDNA clone from a human myeloblast cell line (GenBank accession no. D14662) that we previously demonstrated to be aiPLA2 (18). This search also revealed a sequence consisting of 300 nucleotides from a rat PC-12 cell cDNA library (GenBank accession no. H32258) that shows at least 99 and 96% identity at DNA and amino acid levels, respectively, to the nucleotide and the predicted amino acid sequences for rat aiPLA2. This sequence probably represents a partial clone for this enzyme.

We also assembled a composite amino acid sequence from 14 mouse cDNA clones that showed similarity to the rat cDNA-encoded protein. The consensus sequence derived from these mouse clones showed even higher identity to the rat sequence at both the DNA and amino acid levels than did the human sequence. In general, the variant amino acids were located at positions that were also variant in the human clone (Fig. 1B). This high conservation in the amino acid and nucleotide sequences between species suggests that this protein is likely to have an important function that is very sensitive to amino acid sequence variation.

The predicted amino acid sequences of mouse, rat, and human acidic Ca2+-independent phospholipases A2 contain a 5-amino acid sequence, GDSWG (double underlined in Fig. 1B), that fits the consensus GXSXG that is common to the active site of serine proteases and neutral lipases (11). This motif is present in the three iPLA2 enzymes, aiPLA2, cytosolic iPLA2 (25), and low-density lipoprotein-associated PLA2 (or PAF acetylhydrolase) (26, 29), that have been cloned to date. Recently, it has been shown that the predicted amino acid sequence of cDNA for phosphatidylserine-specific PLA1 also contains the lipase motif (24). Of note, the lipase motif has not been observed in the secreted or cytosolic classes of PLA2 but has been demonstrated in lysophospholipase (30).

Evidence for aiPLA2 activity of expressed protein. Given the strong sequence similarity between the rat and human cDNA clones and previously isolated rat lung enzyme, it was expected that the protein product of the rat cDNA clone would have properties similar to those found previously (18). Expression of rat cDNA with the wheat germ system demonstrated maximal PLA2 activity at pH 4 and Ca2+ independence. Thus protein encoded by this cDNA clone has enzymatic properties that correspond to the partially purified rat lung enzyme and the expressed human enzyme previously reported (18), as well as the characteristics previously demonstrated in homogenates of rat lungs and granular pneumocytes (12, 13). Northern analysis with the rat cDNA clone as a probe confirmed that this gene is highly expressed in rat lung and granular pneumocytes (Fig. 4) as shown previously with human cDNA (18).

Properties of the predicted protein. The rat cDNA encodes a mature protein of 224 amino acids with a calculated molecular mass of 24.8 kDa. We have shown in a wheat germ in vitro translation system that mRNA transcribed from this clone results in the expression of a protein of 24.9 kDa in size, in good agreement with the predicted mass of the deduced amino acid sequence and with the estimated mass of the aiPLA2 previously described (18). The predicted protein has 31 (13.8%) negatively charged and 27 (12.1%) positively charged residues, a predicted pI of 5.7, and no predicted charge clusters (7). Nonpolar residues account for 108 (48.2%) of the 224 amino acids in the predicted sequence. A hydrophobicity plot of the predicted protein did not indicate any extended regions of high hydrophobicity, consistent with the fact that the rat enzyme was originally isolated as a soluble protein.

mRNA for aiPLA2. The size of the mRNA predicted by the cDNA sequence of rat aiPLA2 is <0.9 kb (Fig. 1). However, in the Northern blot analysis, the size of the band is closer to 1,700 bp, in agreement with the size of the human cDNA clone. Both clones contain what appears to be a full-length coding region; however, the rat clone we have isolated has a much shorter 3'-untranslated region. This short clone could be due to the use of an upstream polyadenylation signal. The putative polyadenylation signal for the short clone would be ATTAAA, which differs by one base from the more conventional AATAAA signal. Processing and polyadenylation of mRNA from such a signal may be inefficient. In fact, RNA of this class is relatively rare because it was not detected on a Northern blot. Alternatively, the short mRNA could be an artifact of the cloning process caused by binding of oligo(dT) used for priming to an internal A-rich region instead of the poly(A)+ sequences. This appears less likely because the corresponding region of the human clone, which is almost identical to the 3'-end of the rat clone (21 of 24 nucleotides; Fig. 1), does not contain an A-rich region that corresponds to the poly(A)+ region of the rat cDNA clone.

Various iPLA2 types have been previously isolated from heart (40 kDa), brain (58 kDa), kidney (28 kDa), plasma (45 kDa), and macrophage and ovarian cell lines (85 kDa) (6, 15, 23, 26, 28), but none of these demonstrates the acidic pH optimum determined for rat lung 26-kDa aiPLA2 (12, 13, 18). In addition to its unique molecular mass and pH profile, aiPLA2 can be differentiated from these other Ca2+-independent phospholipases A2 by its insensitivity to both bromoenol lactone and fluoromethyl ketone inhibitors, the failure of ATP to activate it, and its lack of PLA1, lysophospholipase, and PAF hydrolase activities (18). Finally, its molecular sequence is distinct (18). Thus aiPLA2 represents a different enzyme from previous iPLA2 enzymes that have been isolated.

With the use of polyclonal aiPLA2 antibodies, the human cDNA probe encoding the enzyme, and measurements of aiPLA2 activity, we studied the tissue and cellular localization of this novel enzyme. The polyclonal antibodies were generated to a 15-amino acid peptide sequence of the rat (and human) enzyme (3) and to the expressed human full-length sequence. On the basis of mRNA expression, immunoreactive protein and enzymatic activity, aiPLA2 was markedly enriched in the lung compared with other rat tissues. Activity was inhibited by the anti-peptide antibody and by the transition state analog MJ33. Remarkably, although mRNA was detected in tissues other than the lung, immunoreactive protein was not demonstrated with either of the polyclonal antibodies. Furthermore, the relatively low level of enzymatic activity in nonlung tissues was not sensitive to MJ33 or the polyclonal anti-peptide antibody. These findings suggest that the aiPLA2 activity in these tissues is due to another iPLA2 and could be similar to residual activity in the lung that is not inhibited by MJ33 or anti-raiPLA144-1582 antibody (3, 18).

The presence of message for aiPLA2 but the absence of protein in organs other than the lung suggests translational control of enzyme expression. The presence of an untranslated mRNA in a tissue has been noted before; for example, mRNA for the transcription factor DBP is present in several tissues, but the protein is found only in the liver (21). An example of translational control is the Fe-releasable block in translation that has been shown for the mRNA that codes for ferritin (32). An alternative possibility is that the aiPLA2 message is translated, but the protein is degraded more rapidly in other organs than in the lung. The possibility that the mRNA present in other tissues represents the message for an isoform of lung aiPLA2 seems unlikely because neither of the polyclonal antibodies recognized protein in tissue other than the lung. Although the protein was not detected in adult rat tissues, it may be stably expressed under some circumstances because human liver and red blood cell proteins (24-26 kDa) with NH2-terminal amino acid sequences of 14 and 12 amino acids, respectively, that are identical to aiPLA2 have been identified as discussed by Kim et al. (18). The mechanisms for control of aiPLA2 mRNA translation in the lung as well as in other tissues remain to be determined.

    ACKNOWLEDGEMENTS

We thank Dr. Henry Shuman and Kathleen Notarfrancesco (Institute for Environmental Medicine, Morphology and Imaging Core, Philadelphia, PA) for immunofluorescence studies, Dr. Vahe Bedian (Department of Genetics, University of Pennsylvania, Philadelphia) for assistance with the preparation of monoclonal antibodies and the homology search, Dr. Michael Beers for suggestions regarding use of antibodies, Dr. Meng Chen for suggestions for the manuscript, and Elaine Primerano for secretarial support. The rat acidic Ca2+-independent phospholipase A2 cDNA sequence has been submitted to GenBank (accession no. AF014009).

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-19737 and National Cancer Institute Grant CA-01614.

X. Chen and B. B. Hennigan were trainees of National Heart, Lung, and Blood Institute Institutional National Research Service Award HL-07748.

This work was presented in part at the Experimental Biology '97 Meeting, New Orleans, LA, April 1997, and the American Lung Association/American Thoracic Society International Conference, San Francisco, CA, May 1997.

Address for reprint requests: A. B. Fisher, Institute for Environmental Medicine, Univ. of Pennsylvania School of Medicine, 1 John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6068.

Received 7 August 1997; accepted in final form 10 February 1998.

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Top
Abstract
Introduction
Methods
Results
Discussion
References

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