Inhibition of Cytosolic Phospholipase A2 by Annexin I
2001; Elsevier BV; Volume: 276; Issue: 19 Linguagem: Inglês
10.1074/jbc.m009905200
ISSN1083-351X
AutoresSeung Wook Kim, Hae Jin Rhee, Jesang Ko, Yeo Jeong Kim, Hyung Gu Kim, Jai Myung Yang, Eung Chil Choi, Doe Sun Na,
Tópico(s)Vitamin K Research Studies
ResumoAnnexins (ANXs) display regulatory functions in diverse cellular processes, including inflammation, immune suppression, and membrane fusion. However, the exact biological functions of ANXs still remain obscure. Inhibition of phospholipase A2(PLA2) by ANX-I, a 346-amino acid protein, has been observed in studies with various forms of PLA2. "Substrate depletion" and "specific interaction" have been proposed for the mechanism of PLA2 inhibition by ANX-I. Previously, we proposed a specific interaction model for inhibition of a 100-kDa porcine spleen cytosolic form of PLA2(cPLA2) by ANX-I (Kim, K. M., Kim, D. K., Park, Y. M., and Na, D. S. (1994) FEBS Lett. 343, 251–255). Herein, we present an analysis of the inhibition mechanism of cPLA2 by ANX-I in detail using ANX-I and its deletion mutants. Deletion mutants were produced in Escherichia coli, and inhibition of cPLA2 activity was determined. The deletion mutant ANX-I-(1–274), containing the N terminus to amino acid 274, exhibited no cPLA2 inhibitory activity, whereas the deletion mutant ANX-I-(275–346), containing amino acid 275 to the C terminus, retained full activity. The protein-protein interaction between cPLA2 and ANX-I was examined using the deletion mutants by immunoprecipitation and mammalian two-hybrid methods. Full-length ANX-I and ANX-I-(275–346) interacted with the calcium-dependent lipid-binding domain of cPLA2. ANX-I-(1–274) did not interact with cPLA2. Immunoprecipitation of A549 cell lysate with anti-ANX-I antibody resulted in coprecipitation of cPLA2. These results are consistent with the specific interaction mechanism rather than the substrate depletion model. ANX-I may function as a negative regulator of cPLA2 in cellular signal transduction. Annexins (ANXs) display regulatory functions in diverse cellular processes, including inflammation, immune suppression, and membrane fusion. However, the exact biological functions of ANXs still remain obscure. Inhibition of phospholipase A2(PLA2) by ANX-I, a 346-amino acid protein, has been observed in studies with various forms of PLA2. "Substrate depletion" and "specific interaction" have been proposed for the mechanism of PLA2 inhibition by ANX-I. Previously, we proposed a specific interaction model for inhibition of a 100-kDa porcine spleen cytosolic form of PLA2(cPLA2) by ANX-I (Kim, K. M., Kim, D. K., Park, Y. M., and Na, D. S. (1994) FEBS Lett. 343, 251–255). Herein, we present an analysis of the inhibition mechanism of cPLA2 by ANX-I in detail using ANX-I and its deletion mutants. Deletion mutants were produced in Escherichia coli, and inhibition of cPLA2 activity was determined. The deletion mutant ANX-I-(1–274), containing the N terminus to amino acid 274, exhibited no cPLA2 inhibitory activity, whereas the deletion mutant ANX-I-(275–346), containing amino acid 275 to the C terminus, retained full activity. The protein-protein interaction between cPLA2 and ANX-I was examined using the deletion mutants by immunoprecipitation and mammalian two-hybrid methods. Full-length ANX-I and ANX-I-(275–346) interacted with the calcium-dependent lipid-binding domain of cPLA2. ANX-I-(1–274) did not interact with cPLA2. Immunoprecipitation of A549 cell lysate with anti-ANX-I antibody resulted in coprecipitation of cPLA2. These results are consistent with the specific interaction mechanism rather than the substrate depletion model. ANX-I may function as a negative regulator of cPLA2 in cellular signal transduction. annexins phospholipase A2 secretory phospholipase A2 cytosolic phospholipase A2 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine polymerase chain reaction glutathioneS-transferase bovine serum albumin maltose-biding protein multivesicular body Annexins (ANXs)1 are structurally related, calcium-dependent, phospholipid-binding proteins that have been implicated in diverse cellular roles, including anti-inflammation, membrane fusion, differentiation, exocytosis, calcium channels, and interaction with cytoskeletal proteins (reviewed in Refs. 1Raynal P. Pollard H.B. Biochim. Biophys. Acta. 1994; 1197: 63-93Crossref PubMed Scopus (1030) Google Scholar and 2Buckingham J.C. Br. J. Pharmacol. 1996; 118: 1-19Crossref PubMed Scopus (67) Google Scholar). These proteins are defined structurally by a conserved core domain that contains either four or eight repeating units of ∼70 amino acids each (3Weng X. Luecke H. Song I.S. Kang D.S. Kim S.H. Huber R. Protein Sci. 1993; 2: 448-458Crossref PubMed Scopus (175) Google Scholar, 4Liemann S. Huber R. Cell. Mol. Life. Sci. 1997; 53: 516-521Crossref PubMed Scopus (94) Google Scholar). The conserved repeats account for the shared abilities of ANXs to bind phospholipids in a calcium-dependent manner, whereas the specific functions of each ANX are probably related to their type-specific N-terminal regions. Despite definitive structural characterization, the relationship between structure and function or precise biological function has not been well defined for any of the ANXs. ANX-I, a 37-kDa member of the family, has been proposed as a mediator of the anti-inflammatory actions of glucocorticoids (5Flower R.J. Melli M. Parente L. Cytokines and Lipocortins in Inflammation and Differentiation. Wiley-Liss, Inc., New York1990: 11-25Google Scholar). These anti-inflammatory properties have been related to the ability of ANX-I to inhibit phospholipase A2 (PLA2) activity. PLA2 represents a growing family of enzymes with the common function of catalyzing the release of fatty acids from thesn-2-position of membrane phospholipids, thereby providing production of bioactive lipid metabolites and cytoprotective functions (6Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 7Serhan C.N. Haeggstrom J.Z. Leslie C.C. FASEB J. 1996; 10: 1147-1158Crossref PubMed Scopus (370) Google Scholar). PLA2 enzymes can be subdivided into several groups based on their structure and enzymatic characteristics. Secretory PLA2 (sPLA2) enzymes are low molecular mass (14–18 kDa) enzymes with little fatty acid specificity that require a millimolar calcium concentration for catalysis. Types IIA and V sPLA2 isozymes are known to play a role in arachidonic acid release by certain stimuli (8Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). On the other hand, type VI Ca2+-independent PLA2 has been proposed to participate in fatty acid release associated with phospholipid remodeling (9Balsinde J. Barbour S.E. Bianco I.D. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11060-11064Crossref PubMed Scopus (127) Google Scholar, 10Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8527-8531Crossref PubMed Scopus (256) Google Scholar). In contrast, type IV cytosolic PLA2(cPLA2) is a ubiquitously distributed 85–100-kDa enzyme, the activation of which has been shown to be tightly regulated by growth factors and pro-inflammatory cytokines. cPLA2requires a submicromolar Ca2+ concentration for effective hydrolysis of its substrate, arachidonic acid-containing glycerophospholipids (11Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.R. Sultsman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1462) Google Scholar, 12Sharp 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. Robert E.F. Kramer R.M. J. Biol. Chem. 1991; 266: 14850-14853Abstract Full Text PDF PubMed Google Scholar). This requirement is associated with the C2 domain in the N terminus of cPLA2 that mediates calcium-dependent phospholipid binding and translocation of cPLA2 from the cytosol to membranes (13Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (742) Google Scholar). In addition to the calcium-dependent translocation, cPLA2 is phosphorylated by kinases of the mitogen-activated protein kinase family (14Nemenoff R.A. Winitz S. Qian N.X. Putten V.V. Johnson G.L. Heasley L.E. J. Biol. Chem. 1993; 268: 1960-1964Abstract Full Text PDF PubMed Google Scholar), which is one of the important regulatory mechanisms forin vivo activation of cPLA2 (15Lin L.-L. Wartmann M. Lin A.Y. Knopf J. Steh A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1657) Google Scholar, 16Durstin M. Durstin S. Molski T.F.P. Becker E.L. Sha'afi R.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3142-3146Crossref PubMed Scopus (135) Google Scholar). The mechanism by which ANX-I inhibits PLA2 is not fully understood. Most studies, which have been performed using a 14-kDa sPLA2, supported the "substrate depletion" model rather than the "specific interaction" model (17Davison F.F. Lister M.D. Dennis E.A. J. Biol. Chem. 1990; 265: 5602-5609Abstract Full Text PDF PubMed Google Scholar). In the presence of calcium, ANX-I tightly binds to negatively charged phospholipid substrates, which results in substrate depletion and apparent cPLA2 inhibition (18Comera C. Rothhut B. Russo-Marie F. Eur. J. Biochem. 1990; 188: 139-146Crossref PubMed Scopus (36) Google Scholar). To the contrary, our recent study using cPLA2 isolated from porcine spleen showed that ANX-I inhibited cPLA2 by specific interaction (19Kim K.M. Kim D.K. Park Y.M. Na D.S. FEBS Lett. 1994; 343: 251-255Crossref PubMed Scopus (83) Google Scholar). An increasing number of reports have suggested that cPLA2is a key enzyme responsible for signal transduction in inflammation, cytotoxicity, and mitogenesis (6Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 7Serhan C.N. Haeggstrom J.Z. Leslie C.C. FASEB J. 1996; 10: 1147-1158Crossref PubMed Scopus (370) Google Scholar). ANX-I suppresses cPLA2 activity not only in vitro (21Saris C.J. Kristensen T. D'Eustachio P. Hicks L.J. Noonan D.J. Hunter T. Tack B.F. J. Biol. Chem. 1987; 262: 10663-10671Abstract Full Text PDF PubMed Google Scholar, 22Frohlich M. Motte P. Galvin K. Takahashi H. Wands J. Ozturk M. Mol. Cell. Biol. 1990; 10: 3216-3223Crossref PubMed Scopus (72) Google Scholar), but also in cultured cells (23Solito E. Rahuenes-Nicol C. de Coupade C. Bisagni-Faure A. Russo-Marie F. Br. J. Pharmacol. 1998; 124: 1675-1683Crossref PubMed Scopus (45) Google Scholar, 24Oh J. Rhee H.J. Kim S. Kim S.B. You H. Kim J.H. Na D.S. FEBS Lett. 2000; 477: 244-248Crossref PubMed Scopus (25) Google Scholar). Thus, ANX-I may function as an endogenous negative regulator of cPLA2. Herein, we have studied the inhibition of cPLA2 by ANX-I in detail. Deletion mutants of ANX-I were constructed, and enzymatic studies were performed. Also, the protein-protein interaction between cPLA2 and ANX-I, a prerequisite for the specific interaction mechanism, was investigated. 1-Stearoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine (2-AA-PC; 56.0 mCi/mmol) was purchased from Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom) and used as the substrate. Unlabeled 2-AA-PC was purchased from Sigma. Scintillation fluid (Aquasol-2) was obtained from Molecular Probes, Inc. (Eugene, OR). Rabbit antiserum was raised against human ANX-I produced in Escherichia coli. Mouse anti-ANX-I antibody was purchased from Transduction Laboratories (Lexington, KY). Mouse anti-cPLA2antibody was a product of Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Cloning of ANX-I cDNA and expression in E. coli have been described (3Weng X. Luecke H. Song I.S. Kang D.S. Kim S.H. Huber R. Protein Sci. 1993; 2: 448-458Crossref PubMed Scopus (175) Google Scholar,25Huh K.R. Park S.H. Kang S.M. Song I.S. Lee H.Y. Na D.S. J. Biochem. Mol. Biol. 1990; 23: 459-464Google Scholar). Briefly, ANX-I cDNA was selected by colony hybridization from a human placenta cDNA library. ANX-I cDNA was then cloned into plasmid pET-28a(+) (Novagen, Madison, WI) and expressed in E. coli. Full-length ANX-I and N-terminally deleted ANX-I were cloned into the NcoI and SalI sites of pET-28a by cloning procedures that do not involve PCR. The deletion mutants ANX-I-(1–274) and ANX-I-(1–196) were cloned by PCR amplification of the DNA fragment, followed by insertion into the NcoI andSalI sites of pET-28a. The DNA fragment encoding amino acids 1–274 was amplified using primers TTAccatggCAATGGTATCAG and TTAgtcgacTCATTTGCTTGTGGCGCA (lowercase letters represent the restriction enzyme sites). The DNA fragment encoding amino acids 1–196 was amplified using primers TTAccatggCAATGGTATCAG and TTAgtcgacTCATTCATTCACACCAAA. PCR-amplified clones were verified by nucleotide sequencing. ANX-I and the mutants were purified according to methods previously described (25Huh K.R. Park S.H. Kang S.M. Song I.S. Lee H.Y. Na D.S. J. Biochem. Mol. Biol. 1990; 23: 459-464Google Scholar). Various deletion mutants of ANX-I were constructed as GST fusion proteins by inserting the full-length or deleted ANX-I cDNA into plasmid pGEX-5X-1 (Amersham Pharmacia Biotech). Portions of the ANX-I cDNA were isolated by PCR and subcloned into theBamHI and XhoI sites (full-length and C-terminal deletion mutants) or the EcoRI and XhoI sites (N-terminal deletion mutants) of pGEX-5X-1. The sequences of the PCR primers for GST-ANX-I-(1–346) were ATTggatccTTATGGCAATGGTATC (primer F1) and TTActcgagGTTTCCTCCACAAAG (primer R1). To clone the C-terminal deletion mutants, primer F1 was used as a common forward primer, and the reverse primers were as follows: GST-ANX-I-(1–274), TTActcgagTTTGCTTGTGGCGCA; GST-ANX-I-(1–196), TTActcgagTTCATTCACACCAAA; GST-ANX-I-(1–114), TTActcgagAGTTTTTAGCAGAGC; and GST-ANX-I-(1–33), TTActcgagTCCGGGACCACCTTT. To clone the N-terminal deletion mutants, primer R1 was used as a common reverse primer, and the forward primers were as follows: GST-ANX-I-(34–346), ATTgaattcTCAGCGGTGAGCCCCTAT; GST-ANX-I-(115–346), ATTgaattcCCAGCGCAATTTGATGCT; GST-ANX-I-(197–346), ATTgaattcGACTTGGCTGATTCAGAT; and GST-ANX-I-(275–346), ATTgaattcCCAGCTTTCTTTGCAGAG. All PCR-amplified clones were verified by nucleotide sequencing. GST fusion proteins were expressed and purified according to the manufacturer's instructions. The E. coli lysate was bound to a glutathione-Sepharose 4B column (Amersham Pharmacia Biotech) and washed three times with phosphate-buffered saline. GST fusion proteins were eluted with 10 mm reduced glutathione in 50 mm Tris-HCl (pH 8.0) and dialyzed against Tris-buffered saline containing 10% glycerol. The protein concentration was determined by the Bradford method (26Bradford M.M. Anal. Biochem. 1970; 72: 248-254Crossref Scopus (216357) Google Scholar). Bee venom sPLA2 was purchased from Sigma. A 100-kDa cPLA2was partially purified from porcine spleen according to previously described methods (27Kim D.K. Bonventre J.V. Biochem. J. 1993; 294: 261-270Crossref PubMed Scopus (49) Google Scholar). Since purification of cPLA2 from porcine spleen is laborious and time-consuming, we cloned cPLA2 cDNA into the baculovirus vector pFastBacHTa (Life Technologies, Inc.) and produced cPLA2 in Sf9 cells. cPLA2 cDNA was cloned into plasmid pGEMT (Promega, Madison, WI) by reverse transcription-PCR from U937 cell mRNA using primers ATTgtcgacATGTCATTTATAGATCC and TAAaagcttCTATGCTTTGGGTTTACTTAG. The nucleotide sequence was verified by DNA sequencing. Plasmid pGEMT-cPLA2 was digested with SalI andHindIII, and the insert was cloned into the SalI and HindIII sites of pFastBacHTa to produce pBac-cPLA2. To induce transposition between pBac-cPLA2 and Autographa californica nuclear polyhedrin virus DNA, pBac-cPLA2 was transformed into E. coli DH10Bac (maximum efficiency, Life Technologies, Inc.), which harbors A. californica nuclear polyhedrin virus bacmid. Transformed E. coli was incubated on LB plates containing 50 μg/ml kanamycin, 7 μg/ml gentamycin, 10 μg/ml tetracycline, 100 μg/ml 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal), and 40 μg/ml isopropyl-β-d-thiogalactopyranoside for 24 h. Recombinant bacmid PLA2-containing white colonies were then isolated. Bacmid cPLA2 was transfected into Sf9 cells using CellFectin (Life Technologies, Inc.) and cultured for 72 h, and recombinant virus cPLA2production was confirmed by PCR. The titer of the recombinant virus cPLA2 in the culture medium was determined by a plaque assay. Fresh Sf9 cells were infected with this culture medium and cultured for 72 h. Cells from a 50-ml culture were lysed in 1 ml of buffer containing 20 mm Tris-HCl (pH 7.4), 10% glycerol, 1% Nonidet P-40, 0.1% BSA, 0.1 mmphenylmethylsulfonyl fluoride, CompleteTM EDTA-free protease inhibitor mixture (Roche Molecular Biochemicals), and 0.1 μm Ca2+ and used as the source of Sf9 cPLA2. PLA2activity was assayed using sonicated liposomes prepared as described previously (19Kim K.M. Kim D.K. Park Y.M. Na D.S. FEBS Lett. 1994; 343: 251-255Crossref PubMed Scopus (83) Google Scholar, 28Favier-Perron B. Lewit-Bentley A. Russo-Marie F. Biochemistry. 1996; 35: 1740-1744Crossref PubMed Scopus (66) Google Scholar). A stock solution of the substrate was prepared as follows. The substrate (10–20 nmol) was dried under nitrogen and then suspended in 0.5–1.0 ml of distilled water by sonication (3 × 10 s) in a bath-type sonicator (Ultrasonik 300, The J. M. Ney Company, Broomfield, CT). The standard reaction mixture (200 μl) for the PLA2 assay contained 0.33 nmol (1.65 μm) of radioactive substrate (∼39,000 cpm), 200 μg of fatty acid-free BSA, and 10 ng (or an equivalent amount when partially purified enzyme was used) of PLA2 in 75 mmTris-HCl (pH 7.5). Ten ng of purified cPLA2 yielded ∼3000 cpm of the product under the standard conditions with 1 μm Ca2+. When partially purified porcine cPLA2 or the total cell lysate of Sf9 cPLA2 cells was used, the amount of cPLA2 was estimated from the activity. The reaction was started by addition of the enzyme to the reaction mixture. Assays were incubated at 37 °C for 1 h and then stopped by adding 1.25 ml of 2% NH2SO4, 20% n-heptane, and 78% isopropyl alcohol. Non-esterified fatty acid was extracted as follows. First, 0.55 ml of water was added, and the sample was Vortex-mixed and centrifuged at 5600 × g for 5 min. Then, 0.75 ml of the upper phase was transferred to a new tube, to which 100 mg of silica gel and 0.75 ml of n-heptane were added. The samples were Vortex-mixed and centrifuged again for 5 min. The supernatant was dried using a SpeedVac freeze drier, and the lipid was resuspended in chloroform/methanol (1:1, v/v) that contained unlabeled arachidonic acid (1 μg/μl) in methanol. Phospholipid and neutral lipid were separated by migration on layers of Silica Gel 60 F254plates (Merck, Darmstadt, Germany) in petroleum ether/ethyl ether/acetic acid (80:20:1, v/v/v). After drying, the plates were subjected to iodine vapor, and lipids were identified by their comigration with unlabeled arachidonic acids. Products were quantified by scraping their corresponding spots into counting vials containing 2 ml of Aquasol-2. Radioactivity was determined using a Packard Tri-Carb scintillation spectrophotometer. For analysis in which the substrate concentration dependence was determined, unlabeled phospholipid was added to the labeled phospholipid to produce a designated final concentration. For accurate control of the Ca2+concentration, a CaCl2/EGTA buffering system was used (29Durham A.C. Cell Calcium. 1983; 4: 33-46Crossref PubMed Scopus (64) Google Scholar). In all analyses, samples were tested in triplicate and adjusted for nonspecific release by subtracting a control value in which preparation of the enzyme was omitted. For inhibition assays, 5–100 nmANX-I was added to the reaction mixture. All analyses were performed in triplicate and repeated at least three times. The effect of ANX-I was represented by the percentage of cPLA2activity compared with the control value. All data shown are means ± S.E. The effect of ANX-I and its deletion mutants on cPLA2 activity was determined by the percentage of PLA2 activity using the following equation: % of PLA2 activity = (cpm test/cpm control) × 100. Portions of cPLA2and ANX-I cDNAs were cloned into the mammalian version of the bait and prey vectors, pM for GAL4 fusion and pVP16 for VP16 fusion (CLONTECH, Palo Alto, CA). To generate GAL4 fusion, ANX-I cDNA was subcloned into the BamHI andXbaI sites (full-length and C-terminal deletion mutant) or the EcoRI and XbaI sites (N-terminal deletion mutant) of pM. To generate VP16 fusion, cPLA2 cDNA was subcloned into the SalI and HindIII sites of pVP16. Portions of cPLA2 cDNA containing amino acids 1–80 or 81–793 were amplified with primers ATTgtcgacATGTCATTTATAGATCC and TAAaagcttATCCAAAATAAATTCAAA or, in the case of amino acids 81–793, primers ATTgtcgacCTTAATCAGGAAAATGTT and TAAaagcttTGCTTTGGGTTTACTTAG. The pG5CAT reporter was purchased fromCLONTECH. Chloramphenicol acetyltransferase assays (Promega) were performed according to the manufacturer's instructions. The 43 amino acids spanning amino acids 38–80 of the C2 domain of cPLA2 (ΔC2) were amplified by PCR using cPLA2 cDNA as a template and two oligonucleotide primers: ATAggatccATGCTTGATACTCCA and TAAaagcttATCCAAAATAAATTCAAA. After digestion with BamHI and HindIII, the amplified fragment was inserted into the BamHI andHindIII sites of pMAL-P2X, a maltose-biding protein (MBP) fusion vector (New England Biolabs Inc., Beverly, MA), to produce pMAL-ΔC2. The MBP fusion protein MBP-ΔC2 was expressed in E. coli and purified using amylose resin (New England Biolabs Inc.) (30Lee K.H. Na D.S. Kim W.H. FEBS Lett. 1999; 442: 143-146Crossref PubMed Scopus (12) Google Scholar) according to the manufacturer's instructions. The binding mixture contained 2 μg of either ANX-I or deletion mutant, 2 μg of MBP-ΔC2, and 20 μl of amylose resin in 300 μl of 20 mm Tris-HCl (pH 8.0), 30 mm NaCl, 1 mg/ml BSA, 0.1% Triton X-100, 0.1 mm phenylmethylsulfonyl fluoride, and 0.1 μm Ca2+. The mixture was incubated for 12 h at 4 °C and centrifuged at 14,00 × gfor 15 s at 4 °C. The beads were washed three times with 1 ml of the binding buffer and subjected to 12% SDS-polyacrylamide gel electrophoresis for Western blotting as follows. Proteins were transferred to a nitrocellulose membrane (Schleicher & Schüll), probed with monoclonal antibody against ANX-I, and visualized using the ECL system (Amersham Pharmacia Biotech). Coprecipitation of ANX-I in A549 cell lysate and purified MBP-ΔC2 was examined. A549 cells (2 × 107) were lysed in 200 μl of lysis buffer (20 mm Tris-HCl (pH 7.4), 10% glycerol, 1% Nonidet P-40, 0.1 mm phenylmethylsulfonyl fluoride, 0.1 μmCa2+, and CompleteTM EDTA-free protease inhibitor mixture). The binding mixture comprised 100 μl (∼100 μg of protein) of A549 cell lysate, 2 μg of MBP-ΔC2, 2 μg of anti-MBP antibody, and 100 μl of binding buffer (75 mmTris-HCl (pH 7.4), 1 mg/ml BSA, and 0.1 μmCa2+). After a 4-h incubation at 4 °C, the immune complexes were precipitated with protein A-agarose (Santa Cruz Biotechnology Inc.). The pellet was boiled in the gel loading buffer, and proteins were analyzed by SDS-polyacrylamide gel electrophoresis, followed by Western blotting using anti-ANX-I antibody. Coprecipitation of cPLA2 in the Sf9 cPLA2 cell lysate and purified GST-ANX-I was examined. Total cell lysate of Sf9 cPLA2 cells was prepared in the same way as described above for the preparation of A549 cell lysate. The binding mixture contained 100 μg of Sf9 cPLA2 cell lysate, 2 μg of ANX-I, 2 μg of anti-GST monoclonal antibody, and 100 μl of binding buffer. The immune complexes were analyzed by Western blotting using anti-cPLA2 antibody (31Raynal P. Hullin F. Ragab-Thomas J.M. Fauvel J. Chap H. Biochem. J. 1993; 292: 759-765Crossref PubMed Scopus (49) Google Scholar). Existence of the cPLA2·ANX-I complex in cells was examined using A549 cell lysates. A549 cell lysate was prepared as described above. One-hundred μl of A549 cell lysate was precipitated with anti-ANX-I antibody and protein A-agarose. cPLA2 activity in the precipitate or supernatant was determined in a standard buffer containing 5 mmCaCl2. cPLA2 in the pellet was also analyzed by Western blotting. In the previous experiments of cPLA2 inhibition by ANX-I, we used cPLA2 purified from porcine spleen (19Kim K.M. Kim D.K. Park Y.M. Na D.S. FEBS Lett. 1994; 343: 251-255Crossref PubMed Scopus (83) Google Scholar). Since purification of cPLA2 from porcine spleen is laborious and results are often inconsistent, human cPLA2cDNA was cloned into a baculovirus vector and expressed in Sf9 cells. cPLA2 produced in Sf9 cPLA2 cells was characterized by the following methods using porcine spleen cPLA2 as a reference: 1) size determination by Western blot analysis using anti-cPLA2antibody, 2) activity in the presence of dithiothreitol, and 3) Ca2+ concentration dependence of PLA2 activity. Although sPLA2 activity was sensitive to dithiothreitol, both cPLA2 enzymes from porcine spleen and Sf9 cells were essentially insensitive to the dithiothreitol concentration (data not shown). Sf9 cPLA2 was active at Ca2+concentrations as low as 0.1 μm and showed nearly identical activity compared with porcine spleen cPLA2 at all Ca2+ concentrations. On the other hand, at least 0.1 mm Ca2+ was necessary for sPLA2activity (data not shown). Therefore, Sf9 cPLA2exhibited nearly identical activity compared with porcine spleen cPLA2. The effects of ANX-I and its deletion mutants on cPLA2 activity were determined. Full-length ANX-I (ANX-I-(1–346)) and the deletion mutants ANX-I-(33–346), ANX-I-(1–274), and ANX-I-(1–196) were cloned, expressed in E. coli, and purified to near homogeneity (Fig.1 A). The effects of ANX-I and its mutants on cPLA2 from porcine spleen were determined at various concentrations of ANX-I and Ca2+ using 2-AA-PC as a substrate. The reaction mixtures were incubated at 37 °C for 1 h, and radiolabeled arachidonic acid, produced by the hydrolyzing reaction of cPLA2, was measured. An incubation time of 1 h was chosen for the following reasons. First, measuring initial rates was prone to more errors due to the small counts/min of the product; and second, the ANX-I inhibition pattern was nearly identical at all time points until 2 h. The substrate concentration was 1.65 μm, which was significantly greater than the enzyme (0.5 nm) and ANX-I (5–100 nm) concentrations. Fig. 1 B shows the results of experiments carried out at 1 μm Ca2+. The percentage of the remaining 2-AA-PC hydrolyzing activity in the presence of ANX-I was plotted against the ANX-I concentration. In the presence of either ANX-I-(1–346) or ANX-I-(33–346), PLA2activity decreased, indicating inhibition of enzymatic activity. On the other hand, ANX-I-(1–274) and ANX-I-(1–196) had no effect on cPLA2 activity. The calcium concentration dependence of inhibition by 20 nm ANX-I was determined at 0.1, 1, 10, and 100 μm and 1 and 10 mm Ca2+. The inhibition by both ANX-I-(1–346) and ANX-I-(33–346) was greatest at 0.1 μm (and 1 μm) calcium and least at 10 mm calcium (Fig. 1 C). Both ANX-I-(1–274) and ANX-I-(1–196) had no effect on cPLA2 activity at any calcium concentration. To rule out the possibility that the cPLA2 inhibition by ANX-I-(1–346) or ANX-I-(33–346) was due to substrate depletion, the substrate concentration was varied from 1.65 to 33 μm while holding the other components constant. As shown in Fig. 1 D, the inhibition of cPLA2 by both ANX-I-(1–346) and ANX-I-(33–346) was essentially independent of the substrate concentration. The results shown in Fig. 1 demonstrate that the N-terminal 32 amino acids are not important for the cPLA2inhibitory activity of ANX-I, whereas the C-terminal 72 amino acids are crucial for the inhibitory activity. To further investigate the important region of ANX-I, various deletion mutants were constructed. Attempts to produce ANX-I-(275–346) or ANX-I-(197–346) in E. coli failed due to very low expression levels. Therefore, use of the GST-ANX-I fusion protein was evaluated. The validity of Sf9 cPLA2 instead of porcine spleen cPLA2 was also evaluated. GST-ANX-I-(1–346) exhibited effects identical to those of ANX-I-(1–346) on the activity of cPLA2 from porcine spleen and Sf9 cPLA2 cells (data not shown). Therefore, various ANX-I mutants were constructed as GST fusion proteins and used for inhibition studies. Fig.2 A shows a schematic representation of various GST-ANX-I deletion mutants. All mutants were produced in E. coli, purified on a glutathione-Sepharose 4B column to near homogeneity, and used without cutting off the GST tail (Fig. 2 B). Fig. 3 shows the effects of GST-ANX-I mutants on cPLA2 activity. No C-terminal deletion mutants exhibited any inhibitory activity (Fig.3 A), whereas all N-terminal deletion mutants exhibited full inhibitory activity (Fig. 3 B). Therefore, the inhibitory activity is located in amino acids 275–346. Inhibition of cPLA2 by GST-ANX-I-(1–346) or GST-ANX-I-(275–346) was further characterized at various GST-ANX-I, substrate, and calcium concentrations. GST-ANX-I showed similar patterns with ANX-I (Fig. 1 versus Fig. 4). GST-ANX-I-(275–346) also showed similar patterns, except that the activity was independent of the calcium concentration (Fig.4 C).Figure 3Inhibition of cPLA2 by GST-ANX-I and its deletion mutants. The cell lysate of Sf9 cPLA2 cells was used as a cPLA2 source. GST-ANX-I mutants (shown schematically in Fig. 2) were used. Other details are as described in the legend to Fig. 1. A, C-terminal deletion mutants; B, N-terminal deletion mutants.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Inhibition of cPLA2 by GST-ANX-I-(1–346) and GST-ANX-I-(275–346). Assays were performed at various concentrations of GST-ANX-I (A), substrate (B), or calcium (C). Other details are as described in the
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