The Genomic Organization, Complete mRNA Sequence, Cloning, and Expression of a Novel Human Intracellular Membrane-associated Calcium-independent Phospholipase A2
2000; Elsevier BV; Volume: 275; Issue: 14 Linguagem: Inglês
10.1074/jbc.275.14.9937
ISSN1083-351X
AutoresDavid J. Mancuso, Christopher M. Jenkins, Richard W. Gross,
Tópico(s)Biochemical Acid Research Studies
ResumoDuring the sequencing of the long arm of chromosome 7 in the Human Genome Project, a predicted protein product of 40 kDa was identified, which contained two ∼10-amino acid segments homologous to the ATP and lipase consensus sequences present in the founding members of a family of calcium-independent phospholipases A2. Detailed inspection of the identified sequence (residues 79,671–109,912 GenBankTM accession no. AC005058) demonstrated that it represented only a partial sequence of a larger undefined polypeptide product. Accordingly, we identified the complete genomic organization of this putative phospholipase A2through analyses of previously published expressed sequence tags, PCR of human heart cDNA, and 5′-rapid amplification of cDNA ends. Polymerase chain reaction and Northern blotting demonstrated a 3.4-kilobase message, which encoded a polypeptide with a maximum calculated molecular weight of 88476.9. This 3.4-kilobase message was present in multiple human parenchymal tissues including heart, skeletal muscle, placenta, brain, liver, and pancreas. Cloning and expression of the protein encoded by this message in Sf9 cells resulted in the production of two proteins of apparent molecular masses of 77 and 63 kDa as assessed by Western analyses utilizing immunoaffinity-purified antibody. Membranes from Sf9 cells expressing recombinant protein released fatty acid from sn-2-radiolabeled phosphatidylcholine and plasmenylcholine up to 10-fold more rapidly than controls. The initial rate of fatty acid release from the membrane fraction was 0.3 nmol/mg·min. The recombinant protein was entirely calcium-independent, had a pH optimum of 8.0, was inhibited by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (IC50 = 3 μm), and was predominantly present in the membrane-associated fraction. Collectively, these results describe the genomic organization, complete mRNA sequence, andsn-2-lipase activity of a novel intracellular calcium-independent membrane-associated phospholipase A2. During the sequencing of the long arm of chromosome 7 in the Human Genome Project, a predicted protein product of 40 kDa was identified, which contained two ∼10-amino acid segments homologous to the ATP and lipase consensus sequences present in the founding members of a family of calcium-independent phospholipases A2. Detailed inspection of the identified sequence (residues 79,671–109,912 GenBankTM accession no. AC005058) demonstrated that it represented only a partial sequence of a larger undefined polypeptide product. Accordingly, we identified the complete genomic organization of this putative phospholipase A2through analyses of previously published expressed sequence tags, PCR of human heart cDNA, and 5′-rapid amplification of cDNA ends. Polymerase chain reaction and Northern blotting demonstrated a 3.4-kilobase message, which encoded a polypeptide with a maximum calculated molecular weight of 88476.9. This 3.4-kilobase message was present in multiple human parenchymal tissues including heart, skeletal muscle, placenta, brain, liver, and pancreas. Cloning and expression of the protein encoded by this message in Sf9 cells resulted in the production of two proteins of apparent molecular masses of 77 and 63 kDa as assessed by Western analyses utilizing immunoaffinity-purified antibody. Membranes from Sf9 cells expressing recombinant protein released fatty acid from sn-2-radiolabeled phosphatidylcholine and plasmenylcholine up to 10-fold more rapidly than controls. The initial rate of fatty acid release from the membrane fraction was 0.3 nmol/mg·min. The recombinant protein was entirely calcium-independent, had a pH optimum of 8.0, was inhibited by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (IC50 = 3 μm), and was predominantly present in the membrane-associated fraction. Collectively, these results describe the genomic organization, complete mRNA sequence, andsn-2-lipase activity of a novel intracellular calcium-independent membrane-associated phospholipase A2. phospholipase(s) A2 calcium-independent PLA2 calcium-dependent PLA2 1-O-(Z)-hexadec-1′-enyl-2-[9,10-3H]octadec-9′-enoyl-sn-glycero-3-phosphocholine (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one polymerase chain reaction rapid amplification of cDNA ends 3-(cyclohexylamino)propanesulfonic acid expressed sequence tag nucleotide Phospholipases A2 catalyze the esterolytic cleavage of fatty acids from the sn-2-position of phospholipids, thereby regulating the release of lipid second messengers (e.g.eicosanoids and lysophospholipids), growth factors (lysophosphatidic acid), and membrane physical properties (1.Samuelsson B. Goldyne M. Granstrom E. Hamberg M. Hammarstrom S. Malmsten C. Annu. Rev. Biochem. 1978; 47: 997-1029Crossref PubMed Scopus (970) Google Scholar, 2.Samuelsson B. 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Decades of painstaking research eventually illuminated several distinguishing kinetic and physical characteristics of the families of phospholipases A2 that facilitated their categorization into several broad classes of enzymes based upon their requirement for calcium ion in in vitro activity assays (i.e.millimolar, nanomolar, or no calcium dependence) (e.g. see Refs. 5.Demel R.A. Geurts van Kessel W.S. Zwaal R.F. Roelofsen B. van Deenen L.L. Biochim. Biophys. Acta. 1975; 406: 97-107Crossref PubMed Scopus (482) Google Scholar and 12.Ballou L.R. DeWitt L.M. Cheung W.Y. J. Biol. Chem. 1986; 261: 3107-3111Abstract Full Text PDF PubMed Google Scholar, 13.Loeb L.A. Gross R.W. J. Biol. Chem. 1986; 261: 10467-10470Abstract Full Text PDF PubMed Google Scholar, 14.Alonso F. Henson P.M. Leslie C.C. Biochim. Biophys. Acta. 1986; 878: 273-280Crossref PubMed Scopus (97) Google Scholar, 15.Wolf R.A. Gross R.W. J. Biol. 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A second group of calcium-facilitated phospholipases A2 (i.e. the cPLA2 family) did not absolutely require calcium ion for hydrolysis, although nanomolar amounts of calcium ion dramatically augmented their in vitro activity (13.Loeb L.A. Gross R.W. J. Biol. Chem. 1986; 261: 10467-10470Abstract Full Text PDF PubMed Google Scholar, 18.Kramer R.M. Checani G.C. Deykin A. Pritzker C.R. Deykin D. Biochim. Biophys. Acta. 1986; 878: 394-403Crossref PubMed Scopus (68) Google Scholar) and facilitated their translocation to subcellular membrane targets (19.Glover S. de Carvalho M.S. Bayburt T. Jonas M. Chi E. Leslie C.C. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). Finally, a third group of enzymes were identified that were entirely calcium-independent in in vitro activity assays (i.e. the iPLA2 family) (15.Wolf R.A. Gross R.W. J. Biol. Chem. 1985; 260: 7295-7303Abstract Full Text PDF PubMed Google Scholar, 20.Miyake R. Gross R.W. Biochim. Biophys. Acta. 1992; 1165: 167-176Crossref PubMed Scopus (36) Google Scholar, 21.Hirashima Y. Farooqui A.A. Mills J.S. Horrocks L.A. J. Neurochem. 1992; 59: 708-714Crossref PubMed Scopus (126) Google Scholar) and could be distinguished by their exquisite sensitivity to inhibition by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) at 1–2 μm concentration (22.Hazen S.L. Zupan L.A. Weiss R.H. Getman D.P. Gross R.W. J. Biol. Chem. 1991; 266: 7227-7232Abstract Full Text PDF PubMed Google Scholar, 23.Lehman J.J. Brown K.A. Ramanadham S. Turk J. Gross R.W. J. Biol. Chem. 1993; 268: 20713-20716Abstract Full Text PDF PubMed Google Scholar). Initial application of molecular biologic approaches to the phospholipase A2 field identified founding members and mechanistic insights into each of these three types of phospholipase A2 catalytic activities (16.Evenberg A. Meyer H. Gaastra W. Verheij H.M. De Haas G.H. J. Biol. 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For example, the secretory PLA2s employ a calcium ion to polarize thesn-2-carbonyl for attack by a histidine-activated H2O molecule, while the intracellular phospholipases employ a nucleophilic serine (17.Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 22.Hazen S.L. Zupan L.A. Weiss R.H. Getman D.P. Gross R.W. J. Biol. Chem. 1991; 266: 7227-7232Abstract Full Text PDF PubMed Google Scholar, 24.Clark J.D. Lin L-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1453) Google Scholar, 25.Sharp J.D. White D.L. Chiou X.G. Goodson T. Gamboa G.C. McClure D. Burgett S. Hoskins J. Skatrud P.L. Sportsman J.R. Becker G.W. Kang L.H. Roberts E.F. Kramer R.M. J. Biol. Chem. 1991; 266: 14850-14853Abstract Full Text PDF PubMed Google Scholar, 26.Andrews D.L. Beames B. Summers M.D. Park W.D. Biochem. J. 1988; 252: 199-206Crossref PubMed Scopus (206) Google Scholar, 27.Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S.S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Moreover, the cPLA2family is readily distinguished from the iPLA2 family by the presence of a GXSGS consensus lipase motif, while the iPLA2 family utilizes a GXSTG consensus motif. In addition, the iPLA2 (but not the cPLA2) gene family possesses a consensus sequence for nucleotide binding (26.Andrews D.L. Beames B. Summers M.D. Park W.D. Biochem. J. 1988; 252: 199-206Crossref PubMed Scopus (206) Google Scholar, 27.Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S.S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). These insights have greatly accelerated our progress in the understanding of the molecular identities of the polypeptides responsible for phospholipase A2 catalysis and their mechanisms of regulation in normal and disease states. More recently, global efforts aimed at identifying the complete human genome sequence have yielded a vast array of sequence information that has further delineated the role of individual phospholipases in biologic processes. For example, two recently described phospholipases A2(i.e. cPLA2β and cPLA2γ) were identified from initial insights gleaned from protein and nucleotide data bases (28.Underwood K.W. Song C. Kriz R.W. Chang X.J. Knopf J.L. Lin L-L. J. Biol. Chem. 1998; 273: 21926-21932Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 29.Pickard R.T. Strifler B.A. Kramer R.M. Sharp J.D. J. Biol. Chem. 1999; 274: 8823-8831Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). During the sequencing of the long arm of chromosome 7 in the Human Genome Sequencing Project, a predicted protein product of 40 kDa was identified, which contained two ∼10-amino acid segments homologous to the ATP and lipase consensus sequences present in the founding members of calcium-independent phospholipases A2 (i.e.iPLA2α (26.Andrews D.L. Beames B. Summers M.D. Park W.D. Biochem. J. 1988; 252: 199-206Crossref PubMed Scopus (206) Google Scholar) and iPLA2β (27.Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S.S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar)). However, close inspection of the Human Genome Sequencing Project sequence demonstrated that it represented only the partial sequence of a larger undefined polypeptide product. Herein, we report the entire genomic organization, complete mRNA sequence, cloning, expression, and initial activity analyses of the protein encoded by this gene, which we term iPLA2γ. [α-32P]dCTP (6000 Ci/mmol) and ECL detection reagents were purchased from Amersham Pharmacia Biotech. A human heart cDNA library was purchased from Stratagene, Inc. Human heart Marathon cDNA, QuickClone human skeletal cDNA, and human MTN multiple tissue Northern blots were purchased fromCLONTECH. For PCR, a Perkin-Elmer Thermocycler was employed, and all PCR reagents were purchased from PE Biosystems. The pGEM-T vector and the TnT Quick Coupled Transcription/Translation System were obtained from Promega. Vector pcDNA1.1 was purchased from Invitrogen. Culture media, Cellfectin and LipofectAMINE reagents for transfection of baculovirus vectors, and competent DH110BacEscherichia coli cells were purchased from Life Technologies, Inc. and used according to the manufacturer's protocol. QIAfilter plasmid kits and QIAquick gel extraction kits were obtained from Qiagen, Inc. Keyhole limpet hemocyanin was obtained from Pierce.l-α-Dipalmitoyl-2-[1-14C]palmitoyl phosphatidylcholine,l-α-1-palmitoyl-2-[1-14C]oleoyl phosphatidylcholine,l-α-1-palmitoyl-2-[1-14C]linoleoyl phosphatidylcholine, andl-α-1-palmitoyl-2-[1-14C]arachidonyl phosphatidylcholine were purchased from NEN Life Science Products. 1-O-(Z)-Hexadec-1′-enyl-2-[9,10-3H]oleoylsn-glycerol-3-phosphocholine was synthesized and purified as described previously (30.Han X. Zupan L.A. Hazen S.L. Gross R.W. Anal. Biochem. 1992; 200: 119-124Crossref PubMed Scopus (45) Google Scholar). BEL was obtained from Calbiochem. Most other reagents were obtained from Sigma. Searches of EMBL and NCBI data bases were performed using the Basic Local Alignment Search Tool (BLAST) (NCBI). Alignments of all sequences were performed with the MultAlign computer program (31.Corpet F. Nucleic Acids Res. 1988; 16: 10881-10890Crossref PubMed Scopus (4229) Google Scholar). For typical PCR analysis, a 30-cycle program was employed with steps at 53 °C for 30 s, 72 °C for 2 min, and 94 °C for 30 s per cycle. iPLA2γ was amplified utilizing oligonucleotides that flanked the predicted 5′- and 3′-coding region, M444 (5′-TTTTGTCGACATGTCTATTAATCTGACTGTAGATA-3′) and M449 (5′-GCATACTCGAGTCACAATTTTGAAAAGAATGGAAGTCC-3′), respectively. PCR screening was performed utilizing human skeletal muscle cDNA (0.5 ng), human heart Marathon cDNA (0.5 ng), and a human heart cDNA library (∼1 × 109 plaque-forming units) as templates. To directly compare differences between our sequences and those previously reported, PCR amplification of the iPLA2γ sequence present in the original BAC genomic clone RG054 DO4 (Research Genetics) was used as template, and PCR was performed with primers M452 (5′-GTACATACGGTGGACAAGCCTA-3′) and M446 (5′-CATTCCTCTCCCTTTCACTGGATCCACATAGCC-3′). All PCR products were resolved by 1% agarose gel electrophoresis. Candidate bands were extracted from the agarose gel using a QIAquick Gel extraction kit followed by blunt end ligation into the pGEM-T Vector (Promega) by standard procedures (32.Sambrook J. Fritsch E. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Following bacterial transformation and growth of transformants, plasmids were purified using a QIAfilter plasmid kit (CLONTECH) and subjected to automated sequence analysis using either an ABI 373S or 377XL automated DNA sequencer (PE Biosystems). Full-length iPLA2γ amplified by PCR was prepared for use as a probe by radiolabeling with [32P]dCTP for 1 h at 37 °C in the presence of Ready-To-Go labeling beads (Amersham Pharmacia) according to instructions provided by the manufacturer. The radiolabeled probe was purified by gel filtration employing a 1-ml Sepharose G-25 spin column. For Northern analysis, an MTN blot (CLONTECH) containing 2 μg of poly(A)+ RNA/lane from human brain, heart, pancreas, liver, lung, and placenta tissue was prehybridized at 68 °C for 30 min in hybridization buffer, hybridized for 1 h at 68 °C with radiolabeled iPLA2γ (2 × 106 cpm/ml), and washed in 2× SSC and 0.1% SDS twice for 30 min, followed by two washes with 0.1× SSC and 0.1% SDS for 40 min each at 50 °C as per the manufacturer's instructions. Hybridized sequences were identified by autoradiography for 16 h. For 5′-RACE, a 45-cycle program with steps at 58 °C for 30 s, 72 °C for 2 min, and 94 °C for 30 s per cycle was employed. Human heart Marathon-Ready cDNA was used as template (0.5 ng), and primer AP1 (CLONTECH) was paired with M460 (5′-GAAAACCTCTTTGTAGACTGATGTGGCTTATCCTCCAG-3′) to amplify products. Products were analyzed by electrophoresis utilizing a 1% agarose gel and visualized by ethidium bromide staining. PCR products were excised from the gel, purified with a QIAquick gel extraction kit, and subcloned into pGEM-T vector (Promega) for sequencing and alignment with the iPLA2γ sequence. A full-length iPLA2γ construct in pcDNA1.1 (1 μg) was used in a coupled transcription/translation rabbit reticulocyte lysate system (Promega) with RNA synthesis from the T7 promoter of pcDNA1.1 using T7 RNA polymerase and translation in the presence of 20 μCi of [35S]methionine for 90 min according to the manufacturer's instructions. Labeled protein products were resolved on a 10% SDS-polyacrylamide gel followed by autoradiographic visualization. Anti-iPLA2γ polyclonal antibodies were made by immunizing rabbits with the synthetic peptide CENIPLDESRNEKLDQ. The peptide was conjugated to maleimide-activated keyhole limpet hemocyanin by incubation for 2 h at 22 °C followed by dialysis according to the manufacturer's instructions. After two booster injections of the peptide conjugate spaced 2 weeks apart, serum was collected, and antibodies against the peptide were affinity-purified using a thiopropyl-Sepharose column to which the peptide had been covalently coupled according to the instructions of the manufacturer. PCR amplification with the primer pair m444 (5′-TTTTGTCGACATGCTATTAATCTGACTGTAGATA-3′) and m458 (5′-GCATAGCATGCTCACAATTTTGAAAAGAATGGAAGTCC-3′) was used to engineer appropriate restriction sites onto iPLA2γ for subsequent subcloning into SalI/SphI restriction sites of a pFASTBAC vector (Life Technologies, Inc.). The iPLA2γ and flanking sequences in pFASTBAC were sequenced in their entirety on both strands to verify the integrity of the sequence. Sf9 cells were grown and infected as described previously in detail (33.Wolf M.J. Gross R.W. J. Biol. Chem. 1996; 271: 30879-30885Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In brief, Spodoptera frugiperda (Sf9) cells were cultured in 100-ml flasks equipped with a magnetic spinner containing supplemented Grace's medium (34.O'Reilly D.R. Miller L.K Luckow V.A. Baculovirus Expression Vectors: A Laboratory Manual. W. H. Freeman and Co., New York1992Google Scholar). Sf9 cells at a concentration of 1 × 106 cells/ml were prepared in 50 ml of growth medium and incubated at 27 °C for 1 h prior to infection with either wild-type virus or recombinant virus containing human iPLA2γ cDNA. After 48 h, cells were pelleted by centrifugation, resuspended in ice-cold phosphate-buffered saline, and repelleted. All subsequent operations were performed at 4 °C. The supernatant was decanted, and the cell pellet was resuspended in 5 ml of homogenization buffer (25 mmimidazole, pH 8.0, 1 mm EGTA, 1 mmdithiothreitol, 0.34 m sucrose, 20 μmtransepoxysuccinyl-l-leucylamido-(4-guanidino) butane, and 2 μg/ml leupeptin). Cells were lysed at 0 °C by sonication (20 1-s bursts utilizing a Vibra-cell sonicator at a 30% output) and centrifuged at 100,000 × g for 1 h. The supernatant was saved (cytosol), and the membrane pellet was washed with homogenization buffer and resuspended using a Teflon homogenizer in 6 ml of homogenization buffer. After brief sonication (10 1-s bursts), the mixture was subjected to recentrifugation at 100,000 × g for 1 h. After removal of the supernatant, the membrane pellet was resuspended in 1 ml of homogenization buffer using a Teflon homogenizer and subsequently sonicated at 0 °C for 5 × 1-s bursts. Sf9 cell cytosol and membrane proteins were separated by SDS-polyacrylamide gel electrophoresis (35.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) and transferred to Immobilon-P membranes by electroelution in 10 mm CAPS, pH 11, containing 10% methanol. Dry powdered milk (5% (w/v) in 20 mm Tris·HCl, pH 7.4, containing 137 mm NaCl and 0.1% Tween 20) was used to block nonspecific binding sites before incubation with the primary antibody (prepared as described above). Secondary antibody (anti-rabbit F(ab′)2IgG-horseradish peroxidase conjugate) was incubated with the blot for 1 h, and immunoreactive bands were visualized utilizing an ECL detection system. Calcium-independent phospholipase A2 activity was measured by quantitating the release of radiolabeled fatty acid from various radiolabeled phospholipid substrates in the presence of membrane fractions from Sf9 cells infected with wild-type or recombinant human iPLA2γ containing baculovirus. Reactions (200 μl) were incubated for up to 5 min at 37 °C in reaction buffer (100 mm Tris acetate, pH 8.0, containing 1 mm EGTA) prior to their termination by the addition of 100 μl of 1-butanol and vortexing. Phospholipids and fatty acids extracted into the butanol phase were separated by thin layer chromatography using Whatman Silica 60A prescored plates employing a mobile phase of 70:30:1 petroleum ether/ethyl ether/glacial acetic acid (v/v/v). Radiolabeled fatty acids were identified by staining of an overlaid fatty acid standard by exposure to iodine vapor. Regions corresponding to fatty acids were scraped into scintillation vials and subsequently quantitated by scintillation spectrometry after the addition of fluor. For experiments employing BEL, reactions were preincubated in reaction buffer for 3 min in the presence of selected concentrations of BEL or vehicle (EtOH) prior to the addition of radiolabeled substrate. Inspection of the sequence encoding the putative 40-kDa phospholipase reported by the Human Genome Sequencing Project (BAC clone RG054D04; GenBankTM accession no.AC005058) demonstrated that it did not begin with an initiator methionine codon. Accordingly, we performed a TBLASTN data base search (36.Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (68368) Google Scholar) of GenBankTM to find expressed sequence tags (ESTs) that could align with the 5′-end of the putative iPLA2γ sequence. EST clones vz36b01.ri Soares 2NbMT Mus musculus cDNA IMAGE:1328521 and Rattus norvegicus cDNA UI-R-C0-hp-c-06–0-UI (accession nos.AA915561 and AA998901, respectively) were found to overlap with the iPLA2γ sequence, thereby extending the known 5′ sequence an additional 360 nt upstream (Fig. 1). Four other EST clones (Stratagene Homo sapienscolon cDNA clone IMAGE:588479, accession no. AA143503; StratageneH. sapiens cDNA clone IMAGE:647744, accession no.AA205258; normalized rat ovary, Bento Soares Rattus sp. cDNA clone ROVAA46, accession no. AA801084; and NCI_CGAP_GCB1H. sapiens cDNA clone IMAGE:825005, accession numberAA504219) were also present in the data base and are in close spatial proximity with the putative PLA2. However, when aligned with the BAC clone sequence, the 3′-end of the EST AA504219 sequence and the 5′-end of EST AA998901 are separated by a 150-nt gap (Fig. 1). Moreover, when the EST AA998901 sequence is back-translated through this gap and into EST AA504219, a continuous reading frame results. Thus, by overlapping known EST sequences with the 5′-end of iPLA2γ and back-translation through the 150-nt gap, the sequence could be extended approximately 1.2 kb upstream from the predicted GenBankTM protein. The furthest upstream ATG codon that remained in frame with the coding sequence was located 1210 nt upstream from the originally reported sequence. Translation of the reported gene sequence further 5′ from nt 122761 in BAC clone RG054D04 results in stop codons in all three reading frames. We performed PCR analysis using primers corresponding to the most 5′ candidate initiator methionine and the known 3′ stop codon (nt 79,673) in the gene sequence. PCR of human heart cDNA human and skeletal muscle libraries utilizing primers M444 and M949 gave rise to a single band, which was 2.4 kb in length (Fig. 2).Figure 2PCR amplification of human iPLA2 γ. PCR was performed using human heart cDNA (0.5 ng) (lane 1), a cDNA library prepared from human heart (lane 2), human skeletal muscle cDNA (lane 3), and a blank control (lane 4) as templates as described under "Experimental Procedures." PCR primers M444 and were utilized with M449 positioned at the 5′- and 3′-ends of iPLA2γ coding sequence (respectively) in 30 cycles of amplification (53 °C for 30 s, 72 °C for 2 min, and 94 °C for 30 s). PCR products were analyzed on a 1% agarose gel and visualized by ethidium bromide staining. Molecular size markers are shown on the left in kb. The arrow indicates the size of the major PCR band (2.4 kb).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The PCR product was subcloned into pGEM-T and sequenced in both directions (Fig.3). Based on amino acid residue 1 being the initiator methionine, the message encoded a 782-amino acid polypeptide with a calculated molecular weight of 88,476.9. Contained within this sequence were an ATP binding motif (amino acid residues 449–454) and a lipase consensus sequence (amino acid residues 481–485) as well as multiple potential cAMP phosphorylation sites, PKC phosphorylation sites, CK2 phosphorylation sites, and a microbody C-terminal targeting sequence as determined by a Prosite pattern search (37.Hofmann K. Bucher P. Falquet L. Bairoch A. Nucleic Acids Res. 1999; 27: 215-219Crossref PubMed Scopus (1000) Google Scholar, 38.Bucher P. Bairoch A. IS
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