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• W2128222537 abstract "Secreted phospholipases A2(sPLA2s) form a class of structurally related enzymes that are involved in a variety of physiological and pathological effects including inflammation and associated diseases, cell proliferation, cell adhesion, and cancer, and are now known to bind to specific membrane receptors. Here, we report the cloning and expression of a novel sPLA2 isolated from mouse thymus. Based on its structural features, this sPLA2 is most similar to the previously cloned mouse group IIA sPLA2 (mGIIA sPLA2). As for mGIIA sPLA2, the novel sPLA2 is made up of 125 amino acids with 14 cysteines, is basic (pI = 8.71) and its gene has been mapped to mouse chromosome 4. However, the novel sPLA2 has only 48% identity with mGIIA and displays similar levels of identity with the other mouse group IIC and V sPLA2s, indicating that the novel sPLA2 is not an isoform of mGIIA sPLA2. This novel sPLA2 has thus been called mouse group IID (mGIID) sPLA2. In further contrast with mGIIA, which is found mainly in intestine, transcripts coding for mGIID sPLA2 are found in several tissues including pancreas, spleen, thymus, skin, lung, and ovary, suggesting distinct functions for the two enzymes. Recombinant expression of mGIID sPLA2 in Escherichia coli indicates that the cloned sPLA2 is an active enzyme that has much lower specific activity than mGIIA and displays a distinct specificity for binding to various phospholipid vesicles. Finally, recombinant mGIID sPLA2 did not bind to the mouse M-type sPLA2 receptor, while mGIIA was previously found to bind to this receptor with high affinity. Secreted phospholipases A2(sPLA2s) form a class of structurally related enzymes that are involved in a variety of physiological and pathological effects including inflammation and associated diseases, cell proliferation, cell adhesion, and cancer, and are now known to bind to specific membrane receptors. Here, we report the cloning and expression of a novel sPLA2 isolated from mouse thymus. Based on its structural features, this sPLA2 is most similar to the previously cloned mouse group IIA sPLA2 (mGIIA sPLA2). As for mGIIA sPLA2, the novel sPLA2 is made up of 125 amino acids with 14 cysteines, is basic (pI = 8.71) and its gene has been mapped to mouse chromosome 4. However, the novel sPLA2 has only 48% identity with mGIIA and displays similar levels of identity with the other mouse group IIC and V sPLA2s, indicating that the novel sPLA2 is not an isoform of mGIIA sPLA2. This novel sPLA2 has thus been called mouse group IID (mGIID) sPLA2. In further contrast with mGIIA, which is found mainly in intestine, transcripts coding for mGIID sPLA2 are found in several tissues including pancreas, spleen, thymus, skin, lung, and ovary, suggesting distinct functions for the two enzymes. Recombinant expression of mGIID sPLA2 in Escherichia coli indicates that the cloned sPLA2 is an active enzyme that has much lower specific activity than mGIIA and displays a distinct specificity for binding to various phospholipid vesicles. Finally, recombinant mGIID sPLA2 did not bind to the mouse M-type sPLA2 receptor, while mGIIA was previously found to bind to this receptor with high affinity. phospholipase A2 secreted phospholipase A2 high performance liquid chromatography polyacrylamide gel electrophoresis rapid amplification of cDNA ends by polymerase chain reaction glutathione S-transferase 1,2-dioleoyl-sn-glycerol-3-phosphomethanol [3H]DPPC and [3H]DPPG, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine and 1,2-dipalmitoyl-sn-glycerol-3-phosphoglycerol (mixture of stereo isomers) ([3H] in the 9,10 positions of thesn-2 chain) 1,2-ditetradecyl-sn-glycero-3-phosphomethanol 1-palmitoyl-2-[1-14C]arachidonyl-sn-glycero-3-phosphoethanolamine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine 1-stearoyl-2-[1-14C]arachidonyl-sn-glycero-3-phosphocholine expressed sequence tag 5-dimethylaminonaphthalene-1-sulfonyl 1,2-dioleoyl-sn-glycero-3-phosphocholine Phospholipases A2(PLA2,1phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) are a family of enzymes that catalyze the hydrolysis of glycerophospholipids at thesn-2 position, producing free fatty acids and lysophospholipids, and the list of members is expanding (1Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar, 2Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 3Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-283Crossref PubMed Google Scholar, 4Cupillard L. Koumanov K. Mattéi M.G. Lazdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15745-15752Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 5Dennis E.A. Trends Biol. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar). Several mammalian intracellular and secreted PLA2s (sPLA2s) have been characterized and classified as different groups (5Dennis E.A. Trends Biol. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar). Intracellular PLA2s comprise the well known Ca2+-sensitive arachidonoyl-selective 85-kDa cPLA2 (6Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar) and a number of Ca2+-independent PLA2s (7Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Over the last decade, five different secreted PLA2s have been identified and classified as five distinct groups (4Cupillard L. Koumanov K. Mattéi M.G. Lazdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15745-15752Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 5Dennis E.A. Trends Biol. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar). Main common characteristics of these sPLA2s are a relatively low molecular mass (13–16 kDa), the presence of many disulfide bridges, a broad selectivity for phospholipids with different polar head groups and fatty acid chains, and an absolute catalytic requirement for millimolar concentrations of Ca2+ (1Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar, 8Gelb M.H. Jain M.K. Hanel A.M. Berg O.G. Annu. Rev. Biochem. 1995; 64: 653-688Crossref PubMed Scopus (226) Google Scholar). Group IB sPLA2 is known as pancreatic-type sPLA2 because of its initial purification from pancreatic juice (9Verheij H.M. Slotboom A.J. De Haas G. Rev. Physiol. Biochem. Pharmacol. 1981; 91: 91-203Crossref PubMed Scopus (494) Google Scholar). Subsequently, this sPLA2 was detected in other tissues including lung, spleen, kidney and ovary (10Seilhamer J.J. Randall T.L. Yamanaka M. Johnson L.K. DNA. 1986; 5: 519-527Crossref PubMed Scopus (213) Google Scholar), and its involvement in various physiological and pathophysiological responses such as cell proliferation, cell contraction, lipid mediator release, acute lung injury, and endotoxic shock has been proposed (11Ohara O. Ishizaki J. Arita H. Prog. Lipid Res. 1995; 34: 117-138Crossref PubMed Scopus (65) Google Scholar, 12Hanasaki K. Yokota Y. Ishizaki J. Itoh T. Arita H. J. Biol. Chem. 1997; 272: 32792-32797Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 13Kundu G.C. Mukherjee A.B. J. Biol. Chem. 1997; 272: 2346-2353Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Group IIA sPLA2 is also referred to as the inflammatory-type sPLA2, as it is expressed at high levels during inflammation and associated diseases (3Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-283Crossref PubMed Google Scholar, 14Vadas P. Browning J. Edelson J. Pruzanski W. J. Lipid Mediator. 1993; 8: 1-30PubMed Google Scholar). This sPLA2 is a potent mediator of inflammation (3Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-283Crossref PubMed Google Scholar, 14Vadas P. Browning J. Edelson J. Pruzanski W. J. Lipid Mediator. 1993; 8: 1-30PubMed Google Scholar) and a potent bactericidal agent (15Ganz T. Weiss J. Semin. Hematol. 1997; 34: 343-354PubMed Google Scholar, 16Harwig S.S. Tan L. Qu X.D. Cho Y. Eisenhauer P.B. Lehrer R.I. J. Clin. Invest. 1995; 95: 603-610Crossref PubMed Google Scholar, 17Qu X.D. Lehrer R.I. Infect. Immun. 1998; 66: 2791-2797Crossref PubMed Google Scholar). It is also expressed at high levels in various gastrointestinal cancers (18Ogawa M. Yamashita S. Sakamoto K. Ikei S. Res. Commun. Chem. Pathol. Pharmacol. 1991; 74: 241-244PubMed Google Scholar, 19Ohmachi M. Egami H. Akagi J. Kurizaki T. Yamamoto S. Ogawa M. Int. J. Oncol. 1996; 9: 511-516PubMed Google Scholar). More recently, it has been proposed that mouse group IIA (mGIIA) 2A comprehensive abbreviation system for the various mammalian sPLA2s was used: each sPLA2was abbreviated with a lowercase letter indicating the sPLA2 species (m, h, and r for mouse, human, and rat, respectively), followed by uppercase letters identifying the sPLA2 group (GIB, GIIA, GIIC, GIID, GV, and GX for group IB, IIA, IIC, V, and X sPLA2s, respectively). sPLA2serves as a tumor suppressor gene in colorectal cancer (20MacPhee M. Chepenik P.K. Liddel A.R. Nelson K.K. Siracusa D.L. Buchberg M.A. Cell. 1995; 81: 957-966Abstract Full Text PDF PubMed Scopus (531) Google Scholar, 21Cormier R.T. Hong K.H. Halberg R.B. Hawkins T.L. Richardson P. Mulherkar R. Dove W.F. Lander E.S. Nat. Genet. 1997; 17: 88-91Crossref PubMed Scopus (292) Google Scholar). Much less is known about the regulation and biological roles of group IIC, V, and X sPLA2s. Rat and mouse group IIC sPLA2s have been cloned (22Chen J. Engle S.J. Seilhamer J.J. Tischfield J.A. J. Biol. Chem. 1994; 269: 23018-23024Abstract Full Text PDF PubMed Google Scholar), but this sPLA2 appears to be a non-functional pseudogene in humans (23Tischfield J.A. Xia Y.-R. Shih D.M. Klisak I. Chen J. Engle S.J. Siakotos A.N. Winstead M.V. Seilhamer J.J. Allamand V. Gyapay G. Lusis A.J. Genomics. 1996; 32: 328-333Crossref PubMed Scopus (92) Google Scholar). Group V sPLA2 is highly expressed in heart (24Chen J. Engle S.J. Seilhamer J.J. Tischfield J.A. J. Biol. Chem. 1994; 269: 2365-2368Abstract Full Text PDF PubMed Google Scholar) and has been recently detected in murine macrophages and mastocytes, where it plays a role in lipid mediator production (25Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 26Reddy S.T. Winstead M.V. Tischfield J.A. Herschman H.R. J. Biol. Chem. 1997; 272: 13591-13596Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Group X sPLA2 has been cloned in humans and displays distinct structural features (4Cupillard L. Koumanov K. Mattéi M.G. Lazdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15745-15752Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). It is mainly expressed in tissues and cells of the immune system, suggesting a role related to inflammation and/or immunity (4Cupillard L. Koumanov K. Mattéi M.G. Lazdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15745-15752Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Besides their roles as enzymes, some sPLA2s have been shown to bind to specific membrane receptors (27Lambeau G. Lazdunski M. Trends Pharmacol. Sci. 1999; 20: 174-182Abstract Full Text Full Text PDF Scopus (347) Google Scholar). To date, two main types of high affinity sPLA2 receptors have been identified initially using venom sPLA2s as ligands. N-type receptors are highly expressed in brain membranes and display high affinities for neurotoxic venom sPLA2s but not for nontoxic venom sPLA2s, suggesting that these receptors play a role in the neurotoxic effects of sPLA2s (28Lambeau G. Barhanin J. Schweitz H. Qar J. Lazdunski M. J. Biol. Chem. 1989; 264: 11503-11510Abstract Full Text PDF PubMed Google Scholar, 29Nicolas J.P. Lin Y. Lambeau G. Ghomashchi F. Lazdunski M. Gelb M.H. J. Biol. Chem. 1997; 272: 7173-7181Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The physiological role(s) and the endogenous ligands of N-type receptors remain to be discovered. M-type receptors were identified in skeletal muscle (30Lambeau G. Schmid-Alliana A. Lazdunski M. Barhanin J. J. Biol. Chem. 1990; 265: 9526-9532Abstract Full Text PDF PubMed Google Scholar), and have now been cloned from different animal species (11Ohara O. Ishizaki J. Arita H. Prog. Lipid Res. 1995; 34: 117-138Crossref PubMed Scopus (65) Google Scholar, 27Lambeau G. Lazdunski M. Trends Pharmacol. Sci. 1999; 20: 174-182Abstract Full Text Full Text PDF Scopus (347) Google Scholar). These receptors may be involved in various biological effects of pancreatic group IB sPLA2 (11Ohara O. Ishizaki J. Arita H. Prog. Lipid Res. 1995; 34: 117-138Crossref PubMed Scopus (65) Google Scholar, 13Kundu G.C. Mukherjee A.B. J. Biol. Chem. 1997; 272: 2346-2353Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and the recent targeted disruption of the M-type receptor gene has indicated a role of this receptor in the inflammatory processes leading to endotoxic shock (12Hanasaki K. Yokota Y. Ishizaki J. Itoh T. Arita H. J. Biol. Chem. 1997; 272: 32792-32797Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Finally, studies with mammalian sPLA2s have shown that M-type receptors can be physiological targets for group IB and/or group IIA sPLA2s, depending on the animal species (11Ohara O. Ishizaki J. Arita H. Prog. Lipid Res. 1995; 34: 117-138Crossref PubMed Scopus (65) Google Scholar, 27Lambeau G. Lazdunski M. Trends Pharmacol. Sci. 1999; 20: 174-182Abstract Full Text Full Text PDF Scopus (347) Google Scholar, 31Cupillard L. Mulherkar R. Gomez N. Kadam S. Valentin E. Lazdunski M. Lambeau G. J. Biol. Chem. 1999; 274: 7043-7051Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). In light of the growing molecular diversity of mammalian sPLA2s, we have searched for novel sPLA2s in data bases and found a novel mouse sPLA2. This enzyme displays all the structural features of mammalian group IIA sPLA2s (7 disulfides and a C-terminal extension), and displays the best identity score (48%) with the previously cloned mGIIA sPLA2, also known as enhancing factor (32Mulherkar R. Rao R.S. Wagle A.S. Patki V. Deo M.G. Biochem. Biophys. Res. Commun. 1993; 195: 1254-1263Crossref PubMed Scopus (38) Google Scholar, 33Kadam S. Deshpande C. Coulier F. Mulherkar R. Indian J. Exp. Biol. 1998; 36: 553-558PubMed Google Scholar). However, the novel sPLA2 has similar levels of identity with mouse group IIC (43%) and group V (47%) sPLA2s, indicating that the novel sPLA2 is not significantly more related to mGIIA sPLA2 than to these two other sPLA2s and thus is not an isoform of mGIIA sPLA2. For these reasons, the novel sPLA2 is designated hereafter as mouse group IID (mGIID) sPLA2. Search for sPLA2 homologs in gene data bases stored at the National Center for Biotechnology by using the tBLASTn sequence alignment program (34Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar) resulted in the identification of an expressed sequence tag (EST) (IMAGE Consortium clone identification 1225779 5′, GenBank accession number AA762051) that was derived from mouse thymus and that encodes a partial sequence of a novel sPLA2. The 501-nucleotide EST sequence was then used to clone the entire cDNA sequence coding for this novel sPLA2 by 5′ RACE-PCR experiments. These experiments were performed as follows: 10 μg of total mouse thymus RNA were reverse-transcribed using oligo(dT) primers (Promega) and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). Second strand DNA synthesis was carried out using RNase H and DNA polymerase I for 2 h at 16 °C. After blunt-ending with T4 DNA polymerase, double-strand cDNA was precipitated and ligated to adaptors containing sequences for the universal primers Sp6 and KS and SalI and EcoRI restriction sites. A first PCR reaction using KS primer and a specific sPLA2 reverse primer corresponding to nucleotides 211–234 (Fig. 1) was followed by a second amplification using the same KS primer and a second specific reverse primer corresponding to nucleotides 89–111 (Fig. 1). PCR conditions were: 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, 35 cycles. PCR products were subcloned into the pGEM-T easy vector (Promega) and screened using a 32P-labeled primer corresponding to nucleotides 59–87 (Fig. 1). Positive clones were analyzed by restriction and sequenced by using an automatic sequencer (Applied Biosystems model 377). The CV panel of mouse X Chinese hamster somatic cell hybrids (35Williamson P. Holt S. Townsend S. Boyd Y. Mamm. Genome. 1995; 6: 429-432Crossref PubMed Scopus (23) Google Scholar) was used for the chromosomal assignment of mGIID sPLA2. For that purpose, a genomic fragment of 5.5 kilobase pairs was isolated by PCR and partially sequenced, and a set of specific primers was designed. A forward primer within an intron (5′-AAAGATTAGGTGGCTGGAACAACCA-3′) and an antisense primer within the exon coding for the active site region (5′-CATCCATCGATCTTCAGGTGGGCA-3′) were found to amplify a fragment of 230 nucleotides from a mouse CBA/H DNA template, while a product of 250 nucleotides was amplified with hamster V79TOR DNA as template. PCR reactions were performed in 25 μl containing 50 ng of DNA template, 0.5 μg of each primer, 1.5 mm MgCl2, and 0.25 units of Taqpolymerase (Eurobio, France). PCR conditions were: 94 °C for 2 min, followed by 5 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, followed by 25 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. PCR products were analyzed on a 2% agarose gel. Two Northern blots (Origene, catalog no. MB-1002 and MB-1012) and a mouse RNA Master blot (CLONTECH, catalog no. 7771-1) were probed with a randomly primed 32P-labeled mGIID cDNA fragment (nucleotides 58–436 in Fig. 1) in 50% formamide, 5× SSPE (0.9m NaCl, 50 mm sodium phosphate, pH 7.4, 5 mm EDTA), 5× Denhardt's solution, 0.1% SDS, 20 mm sodium phosphate, pH 6.5, and 250 μg/ml denatured salmon sperm DNA for 18 h at 42 °C and 50 °C, respectively. The membranes were washed to a final stringency of 0.2× SSC (30 mm NaCl, 3 mm trisodium citrate, pH 7.0) in 0.1% SDS at 60 °C and exposed to Kodak Biomax MS films with a Transcreen-HE intensifying screen. The full-length cDNA coding for mGIID sPLA2(nucleotides 1–435 in Fig. 1) was subcloned into the expression vector pCI-neo (Promega) and transfected into COS cells as described (4Cupillard L. Koumanov K. Mattéi M.G. Lazdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15745-15752Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Five days after transfection, cell medium was collected and analyzed for sPLA2 activity or loaded on a heparin-agarose column (Sigma) to concentrate sPLA2 activity. The column was washed with 0.1 m NaCl, eluted stepwise with 1m NaCl, and eluted fractions were assayed for sPLA2 activity using [3H]oleate-labeledE. coli membranes (36Ancian P. Lambeau G. Lazdunski M. Biochemistry. 1995; 34: 13146-13151Crossref PubMed Scopus (76) Google Scholar). A PCR fragment coding for a factor Xa cleavage site (Ile-Glu-Gly-Arg) followed by the mGIID mature protein was prepared with Pwo DNA polymerase (Roche Molecular Biochemicals) and subcloned in frame with a truncated glutathioneS-transferase (∼10 kDa) encoded by the modified pGEX-2T vector (pAB3), which was previously used for the expression of porcine pancreatic sPLA2 (37van den Berg B. Tessari M. de Haas G.H. Verheij H.M. Boelens R. Kaptein R. EMBO J. 1995; 14: 4123-4131Crossref PubMed Scopus (47) Google Scholar). Recombinant expression of the fusion protein was performed in E. coli BL21 host cells grown in 1 liter of Terrific broth containing ampicillin (100 μg/ml). Cells were grown to an OD600 ∼0.8, and induced with isopropyl-1-thio-β-d-galactopyranoside (1 mm) for 4 h at 37 °C. Cells were pelleted and resuspended in 40 ml of lysis buffer (50 mm Tris-HCl, pH 8.0, 50 mmNaCl, 2 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride, 1% Triton X-100, and 1% deoxycholate) for 1 h at 4 °C. The suspension was homogenized with a French press (SLM Aminco). Inclusion bodies were collected by centrifugation at 10,000 × g for 20 min and washed four times with lysis buffer without detergents. The resulting pellet was solubilized in 100 ml of 6 m guanidine-HCl, 0.3 mNa2SO3, 20 mm borate, pH 7.4, and proteins were sulfonated by addition of 0.05 volume of Thannhauser reagent (38Thannhauser T.W. Konishi Y. Scheraga H.A. Anal. Biochem. 1984; 138: 181-188Crossref PubMed Scopus (270) Google Scholar) for 1 h at room temperature. After overnight dialysis at 4 °C against 5 liters of 1% acetic acid, the precipitated protein was pelleted and resuspended at 0.2 mg/ml protein in 6m guanidine-HCl, 20 mm borate, pH 8.0. The denatured sPLA2 (200 ml) was refolded by dialysis against 8 liters of 0.9 m guanidine-HCl, 50 mm Tris-HCl, pH 8.0, 5 mm EDTA, and 5 mm cysteine for 24 h at 4 °C. The refolded protein was then dialyzed against 8 liters of 50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm CaCl2. The fusion protein solution was clarified by centrifugation and finally subjected to overnight digestion at 20 °C with 40 units of factor Xa (Amersham Pharmacia Biotech). The mixture was loaded at 2 ml/min onto a 5-ml Hi-Trap heparin-Sepharose column (Amersham Pharmacia Biotech) equilibrated with buffer A (100 mm NaCl, 20 mm Tris-HCl, pH 7.4). The column was washed with buffer A until OD280 dropped to zero and then eluted with a linear gradient of NaCl (0.1–1m NaCl, in 50 min at 1 ml/min). Fractions containing sPLA2 activity were pooled and directly loaded on a Nucleosil™ C18 reverse phase HPLC column (4.6 × 250 mm, 4.2 ml, 300 Å, 5 μm). Elution was performed at 1 ml/min using water/acetonitrile with 0.1% trifluoroacetic acid (10–30% acetonitrile over 20 min, followed by 30–60% acetonitrile over 110 min). N-terminal sequences were determined by automated Edman degradation with an Applied Biosystems Sequencer model A473. Ion spray mass spectrometry was performed on a simple-quadrupole mass spectrometer equipped with an ion-spray source and using polypropylene glycol for calibration. Small unilamellar vesicles of DOPM, POPC, POPE, POPG, and POPS (all from Avanti Polar Lipids) were prepared by sonication as described (39Jain M.K. Gelb M.H. Methods Enzymol. 1991; 197: 112-125Crossref PubMed Scopus (82) Google Scholar). Large unilamellar POPC vesicles were prepared by extrusion (40Bayburt T. Gelb M.H. Biochemistry. 1997; 36: 3216-3231Crossref PubMed Scopus (62) Google Scholar).N-Dansyl-1,2-dihexadecyl-phosphatidylethanolamine and DTPM were prepared as described (41Hixon M.S. Ball A. Gelb M.H. Biochemistry. 1998; 37: 8516-8526Crossref PubMed Scopus (61) Google Scholar). Initial velocities for the hydrolysis of these vesicles by mGIIA, mGIID, and hGIIA sPLA2s were monitored using a fluorescent fatty acid displacement assay (42Kinkaid A.R. Voysey J.E. Wilton D.C. Biochem. Soc. Trans. 1997; 25: 497SCrossref PubMed Scopus (5) Google Scholar). Reactions contained 40 μm phospholipid, 10 μg of rat liver fatty acid binding protein, 1 μm11-(dansylamino)-undecanoic acid (Molecular Probes, Eugene, OR) in 1 ml of 100 mm Tris-HCl, pH 8.0, 2.5 mmCaCl2 at 30 °C. mGIIA (2–100 ng, prepared as described in Ref. 31Cupillard L. Mulherkar R. Gomez N. Kadam S. Valentin E. Lazdunski M. Lambeau G. J. Biol. Chem. 1999; 274: 7043-7051Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), mGIID (1 μg), or hGIIA (2 ng to 1 μg, prepared as described in Ref. 43Snitko Y. Koduri R.S. Han S.K. Othman R. Baker S.F. Molini B.J. Wilton D.C. Gelb M.H. Cho W. Biochemistry. 1997; 36: 14325-14333Crossref PubMed Scopus (110) Google Scholar) sPLA2s was added to start the reaction in a stirred fluorescence cuvette with excitation at 350 nm and emission at 500 nm. For each type of phospholipids, assays were calibrated by measuring the fluorescence change following the addition of a known amount of oleic acid to reaction mixtures containing all components except enzyme. Competitive substrate specificity studies to examine the phospholipid headgroup preferences of mGIIA, mGIID, and hGIIA sPLA2s (43Snitko Y. Koduri R.S. Han S.K. Othman R. Baker S.F. Molini B.J. Wilton D.C. Gelb M.H. Cho W. Biochemistry. 1997; 36: 14325-14333Crossref PubMed Scopus (110) Google Scholar) were carried out with the dual radiolabel method (44Ghomashchi F. Yu B.Z. Berg O. Jain M.K. Gelb M.H. Biochemistry. 1991; 30: 7318-7329Crossref PubMed Scopus (94) Google Scholar) using pairs of competing and radiolabeled substrates present as minor components in sonicated DOPM vesicles. Reaction mixtures contained 40 μm DOPM containing ∼120,000 cpm of 3H-phospholipid and ∼20,000 cpm14C-phospholipid in 100 μl of the same buffer as was used in the kinetic studies described above. Lipids were mixed in chloroform, solvent was removed with a stream of N2 and then in vacuo for 30 min, and buffer was added followed by sonication. Mixtures were incubated at 30 °C for 30 min with sufficient enzyme to hydrolyze 10–20% of the preferred radiolabeled substrate (10–20 ng of mGIIA or hGIIA sPLA2s, ∼1 μg of mGIID sPLA2). Reactions were quenched, and liberated fatty acids were prepared for scintillation counting as described (45Ghomashchi F. Schuttel S. Jain M.K. Gelb M.H. Biochemistry. 1992; 31: 3814-3824Crossref PubMed Scopus (62) Google Scholar) to determine the relativek cat*/K m* values (44Ghomashchi F. Yu B.Z. Berg O. Jain M.K. Gelb M.H. Biochemistry. 1991; 30: 7318-7329Crossref PubMed Scopus (94) Google Scholar). The sources of radiolabeled phospholipids for these experiments are as follows: [3H]DPPC (89 Ci/mmol, NEN Life Science Products), [3H]DPPG (400 Ci/mol, prepared from [3H]DPPC by head group exchange with cabbage phospholipase D from Sigma as described in Ref. 46Schmitt J.D. Amidon B. Wykle R.L. Waite M. Chem. Phys. Lipids. 1995; 77: 131-137Crossref PubMed Scopus (2) Google Scholar), [14C]PAPE (53 Ci/mol, NEN Life Science Products), [14C]SAPC (53 Ci/mol, Amersham Pharmacia Biotech). Interfacial binding of sPLA2s to anionic vesicles was monitored using energy transfer measured with a spectrofluorimeter as described (47Yu B.Z. Ghomashchi F. Cajal Y. Annand R.R. Berg O.G. Gelb M.H. Jain M.K. Biochemistry. 1997; 36: 3870-3881Crossref PubMed Scopus (34) Google Scholar). The release of fatty acids from live RAW 264.7 cells (400,000 cells/ml) treated exogenously with sPLA2s was measured with the fatty acid binding protein assay as described (48Koduri R.S. Baker S.F. Snitko Y. Han S.K. Cho W. Wilton D.C. Gelb M.H. J. Biol. Chem. 1998; 273: 32142-32153Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Specific activities were calculated from the rates measured in the presence of two or three different amounts of enzyme under conditions where the rate was proportional to the amount of enzyme (5 ng to 6 μg, depending on the sPLA2). Cobra venom (Naja naja) sPLA2 was from Sigma. Competition binding assays with recombinant mouse M-type receptor expressed in COS cells were performed as described using125I-OS1 as labeled sPLA2 ligand (31Cupillard L. Mulherkar R. Gomez N. Kadam S. Valentin E. Lazdunski M. Lambeau G. J. Biol. Chem. 1999; 274: 7043-7051Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Briefly, membranes containing M-type receptor,125I-OS1, and unlabeled sPLA2s were incubated at 20 °C in 0.5 ml of binding buffer (140 mmNaCl, 0.1 mm CaCl2, 20 mm Tris-HCl, pH 7.4, and 0.1% bovine serum albumin). Incubations were started by addition of membranes and filtered after 60 min through GF/C glass fiber filters presoaked in 0.5% polyethyleneimine. Protein sequences of various sPLA2s were used to search for novel sPLA2s in gene data bases by using the tBLASTn sequence alignment program (34Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar). This resulted in the identification of an EST of 501 nucleotides (Fig.1) that was derived from a mouse thymus cDNA library, and that displayed high homology to sPLA2s. We thus postulated that this EST was a partial copy of a mRNA coding for a novel low molecular mass sPLA2. This thymus EST sequence was then used to search in data bases for other related EST sequences. A second EST derived from a mouse mammary gland cDNA library was identified and found to have identity to the 3′-end of the thymus EST sequence. On the other hand, no related sequence was found on human EST data bases. Since none of the mouse EST sequences were found to encode for the full-length sPLA2, PCR experiments were performed to clone the entire cDNA sequence. Primers were first designed from the thymus EST sequence and used in PCR experiments on different mouse tissue cDNAs. DNA products of the expected size were obtained from mouse BALB/c thymus cDNAs and found to have the same sequence as the original EST sequence (Fig. 1). 5′ RACE-PCR experiments were then performed on the same cDNAs to obtain the full-length cDNA (see “Experimental Procedures”). Screening of the amplified products with a specific oligonucleotide probe resulted in the identification of a DNA fragment of 128 nucleotides that is identical to the EST sequence in its 3′-end and contains in its 5′-end sequence all the expected features of a sPLA2 including a signal peptide sequence preceded by an initiator methionine. Based on this sequence, a new set of primers was designed to amplify the full-length sPLA2 cDNA from mouse BALB/c t" @default.
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• W2128222537 title "Cloning and Recombinant Expression of a Novel Mouse-secreted Phospholipase A2" @default.
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