Purified Group X Secretory Phospholipase A2 Induced Prominent Release of Arachidonic Acid from Human Myeloid Leukemia Cells
1999; Elsevier BV; Volume: 274; Issue: 48 Linguagem: Inglês
10.1074/jbc.274.48.34203
ISSN1083-351X
AutoresKohji Hanasaki, Takashi Ono, Akihiko Saiga, Yasuhide Morioka, Minoru Ikeda, Keiko Kawamoto, Kenichi Higashino, Kazumi Nakano, Katsutoshi Yamada, Jun Ishizaki, Hitoshi Arita,
Tópico(s)Neuropeptides and Animal Physiology
ResumoGroup X secretory phospholipase A2 (sPLA2-X) possesses several structural features characteristic of both group IB and IIA sPLA2s (sPLA2-IB and -IIA) and is postulated to be involved in inflammatory responses owing to its restricted expression in the spleen and thymus. Here, we report the purification of human recombinant COOH-terminal His-tagged sPLA2-X, the preparation of its antibody, and the purification of native sPLA2-X. The affinity-purified sPLA2-X protein migrated as various molecular species of 13–18 kDa on SDS-polyacrylamide gels, andN-glycosidase F treatment caused shifts to the 13- and 14-kDa bands. NH2-terminal amino acid sequencing analysis revealed that the 13-kDa form is a putative mature sPLA2-X and the 14-kDa protein possesses a propeptide of 11 amino acid residues attached at the NH2 termini of the mature protein. Separation with reverse-phase high performance liquid chromatography revealed that N-linked carbohydrates are not required for the enzymatic activity and pro-sPLA2-X has a relatively weak potency compared with the mature protein. The mature sPLA2-X induced the release of arachidonic acid from phosphatidylcholine more efficiently than other human sPLA2groups (IB, IIA, IID, and V) and elicited a prompt and marked release of arachidonic acid from human monocytic THP-1 cells compared with sPLA2-IB and -IIA with concomitant production of prostaglandin E2. A prominent release of arachidonic acid was also observed in sPLA2-X-treated human U937 and HL60 cells. Immunohistochemical analysis of human lung preparations revealed its expression in alveolar epithelial cells. These results indicate that human sPLA2-X is a unique N-glycosylated sPLA2 that releases arachidonic acid from human myeloid leukemia cells more efficiently than sPLA2-IB and -IIA. Group X secretory phospholipase A2 (sPLA2-X) possesses several structural features characteristic of both group IB and IIA sPLA2s (sPLA2-IB and -IIA) and is postulated to be involved in inflammatory responses owing to its restricted expression in the spleen and thymus. Here, we report the purification of human recombinant COOH-terminal His-tagged sPLA2-X, the preparation of its antibody, and the purification of native sPLA2-X. The affinity-purified sPLA2-X protein migrated as various molecular species of 13–18 kDa on SDS-polyacrylamide gels, andN-glycosidase F treatment caused shifts to the 13- and 14-kDa bands. NH2-terminal amino acid sequencing analysis revealed that the 13-kDa form is a putative mature sPLA2-X and the 14-kDa protein possesses a propeptide of 11 amino acid residues attached at the NH2 termini of the mature protein. Separation with reverse-phase high performance liquid chromatography revealed that N-linked carbohydrates are not required for the enzymatic activity and pro-sPLA2-X has a relatively weak potency compared with the mature protein. The mature sPLA2-X induced the release of arachidonic acid from phosphatidylcholine more efficiently than other human sPLA2groups (IB, IIA, IID, and V) and elicited a prompt and marked release of arachidonic acid from human monocytic THP-1 cells compared with sPLA2-IB and -IIA with concomitant production of prostaglandin E2. A prominent release of arachidonic acid was also observed in sPLA2-X-treated human U937 and HL60 cells. Immunohistochemical analysis of human lung preparations revealed its expression in alveolar epithelial cells. These results indicate that human sPLA2-X is a unique N-glycosylated sPLA2 that releases arachidonic acid from human myeloid leukemia cells more efficiently than sPLA2-IB and -IIA. phospholipase A2 prostaglandin leukotriene secretory PLA2 group IV cytosolic PLA2 group IB sPLA2 group IIA sPLA2 group IID sPLA2 group V sPLA2 group X sPLA2 PLA2receptor phosphatidylcholine antibody bovine serum albumin 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 1-palmitoyl-2-palmitoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phospho-choline 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanol-amine polymerase chain reaction COOH-terminal His-tagged sPLA2-X fetal calf serum SDS-polyacrylamide gel electrophoresis enzyme-linked im- munosorbent assay phosphate-buffered saline phosphatidylethanolamine sodium deoxycholate Chinese hamster ovary high performance liquid chromatography Phospholipase A2(PLA2)1 comprises a diverse family of lipolytic enzymes that hydrolyze thesn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acid and lysophospholipids (1Vadas P. Pruzanski W. Lab. Invest. 1986; 55: 391-404PubMed Google Scholar, 2Arita H. Nakano T. Hanasaki K. Prog. Lipid Res. 1989; 28: 273-301Crossref PubMed Scopus (160) Google Scholar). PLA2s participate in pathophysiological processes by releasing arachidonic acid from membrane phospholipids, leading to the production of various types of proinflammatory lipid mediators, such as prostaglandins (PGs) and leukotrienes (LTs) (3Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar). Over the past two decades, a number of PLA2s have been identified and characterized. From their biochemical features, these PLA2s are classified into several families (4Dennis E.A. Trends Biol. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (754) Google Scholar), including secretory PLA2(sPLA2) (5Seilhamer J.J. Pruzanski W. Vadas P. Plant S. Miller J.A. Kloss J. Johnson L.K. J. Biol. Chem. 1989; 264: 5335-5338Abstract Full Text PDF PubMed Google Scholar, 6Kramer R.M. Hession C. Johansen B. Hayes G. McGray P. Chow E.P. Tizard R. Pepinsky R.B. J. Biol. Chem. 1989; 264: 5768-5775Abstract Full Text PDF PubMed Google Scholar, 7Chen J. Engle S.J. Seilhamer J.J. Tischfield J.A. J. Biol. Chem. 1994; 269: 2365-2368Abstract Full Text PDF PubMed Google Scholar, 8Chen J. Engle S.J. Seilhamer J.J. Tischfield J.A. J. Biol. Chem. 1994; 269: 23018-23024Abstract Full Text PDF PubMed Google Scholar, 9Cupillard L. Koumanov K. Mattei M.-G. Lazdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15745-15752Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 10Ishizaki J. Suzuki N. Higashino K. Yokota Y. Ono T. Kawamoto K. Fujii N. Arita H. Hanasaki K. J. Biol. Chem. 1999; 274: 24973-24979Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 11Valentin E. Koduri R.S. Scimeca J.-C. Carle G. Gelb M.H. Lazdunski M. Lambeau G. J. Biol. Chem. 1999; 274: 19152-19160Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), arachidonoyl-specific cytosolic PLA2 (cPLA2) (12Clark 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, 13Sharp 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), and Ca2+-independent PLA2 (14Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Low molecular mass sPLA2s (13–18 kDa) have several features including a high disulfide bond content, a requirement for millimolar concentrations of Ca2+ for catalysis, and a broad specificity for phospholipids with different polar head groups and fatty acyl chains (15Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 16Lambeau G. Lazdunski M. Trends Pharmacol. Sci. 1999; 20: 162-170Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Mammalian sPLA2s are classified into different groups depending on the primary structure characterized by the number and positions of cysteine residues. At present, five types of functional sPLA2s (group IB, IIA, IID, V, and X) have been identified in humans (10Ishizaki J. Suzuki N. Higashino K. Yokota Y. Ono T. Kawamoto K. Fujii N. Arita H. Hanasaki K. J. Biol. Chem. 1999; 274: 24973-24979Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 15Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar), whereas group IIC sPLA2 found in rodents is a pseudogene in humans (17Tischfield 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. Genomics. 1996; 32: 328-333Crossref PubMed Scopus (90) Google Scholar). Among them, group IIA sPLA2 (sPLA2-IIA) is thought to play a pivotal role in the progression of inflammatory conditions, since its local and systemic levels are elevated in numerous inflammatory diseases (18Gronroots J.M. Nevalainen T.J. Digestion. 1992; 52: 232-236Crossref PubMed Scopus (71) Google Scholar, 19Green J.-A. Smith G.M. Buchta R. Lee R. Ho K.Y. Rajkovic I.A. Scott K.F. Inflammation. 1991; 15: 355-367Crossref PubMed Scopus (149) Google Scholar). However, some inbred mouse strains have a natural frameshift mutation in the sPLA2-IIA gene (20MacPhee M. Chepenik K.P. Liddell R.A. Nelson K.K. Siracusa L.D. Buchberg A.M. Cell. 1995; 81: 957-966Abstract Full Text PDF PubMed Scopus (525) Google Scholar, 21Kennedy B.P. Payette P. Mudgett J. Vadas P. Pruzanski W. Kwan M. Tang C. Rancourt D.E. Cromlish W.A. J. Biol. Chem. 1995; 270: 22378-22385Crossref PubMed Scopus (306) Google Scholar). The phenotype of these deficient mice is similar to that of sPLA2-IIA-expressing mouse strains in their responses to various inflammatory challenges that initiate arthritis (22Brackertz D. Mitchell G.F. Mackay I.R. Arthritis Rheum. 1977; 20: 841-850Crossref PubMed Scopus (252) Google Scholar, 23Wooley P.H. Luthra H.S. Griffiths M.M. Stuart J.M. Huse A. David C.S. J. Immunol. 1985; 135: 2443-2451PubMed Google Scholar). In addition, we have recently shown that indoxam, one of the potent sPLA2 inhibitors (24Hagishita S. Yamada M. Shirahase K. Okuda T. Murakami Y. Ito Y. Matsuura T. Wada M. Kato T. Ueno M. Chikazawa Y. Yamada K. Ono T. Teshirogi I. Ohtani M. J. Med. Chem. 1996; 39: 3636-3658Crossref PubMed Scopus (198) Google Scholar), suppressed the endotoxin-induced elevation of plasma tumor necrosis factor-α levels, with a similar potency for sPLA2-IIA-expressing and sPLA2-IIA-deficient mouse strains (25Yokota Y. Hanasaki K. Ono T. Nakazato H. Kobayashi T. Arita H. Biochim. Biophys. Acta. 1999; 1438: 213-222Crossref PubMed Scopus (61) Google Scholar). Transgenic mice expressing the human sPLA2-IIA gene do not develop any overt inflammatory conditions (26Grass D.S. Felkner R.H. Chiang M.-Y. Wallace R.E. Nevalainen T.J. Bennet C.F. Swanson M.E. J. Clin. Invest. 1996; 97: 2233-2241Crossref PubMed Scopus (159) Google Scholar). These findings point to the need to reevaluate the contribution of sPLA2-IIA in inflammatory diseases and suggest that other types of sPLA2 may play pivotal roles in place of or in concert with sPLA2-IIA. Group IB sPLA2 (sPLA2-IB) has been thought to act as a digestive enzyme, given its abundance in digestive organs (27de Haas G.H. Postema N.M. Nieuwenhuizen W. van Deenen L.L.M. Biochim. Biophys. Acta. 1968; 159: 118-129Crossref PubMed Scopus (146) Google Scholar). However, a series of our studies have identified a variety of biological responses induced by sPLA2-IB via binding to its specific receptor, the PLA2-receptor (PLA2R) (28Arita H. Hanasaki K. Nakano T. Oka S. Matsumoto K. J. Biol. Chem. 1991; 266: 19139-19141Abstract Full Text PDF PubMed Google Scholar, 29Hanasaki K. Arita H. J. Biol. Chem. 1992; 267: 6414-6420Abstract Full Text PDF PubMed Google Scholar, 30Ishizaki J. Hanasaki K. Higashino K. Kishino J. Kikuchi N. Ohara O. Arita H. J. Biol. Chem. 1994; 269: 5897-5904Abstract Full Text PDF PubMed Google Scholar, 31Ohara O. Ishizaki J. Arita H. Prog. Lipid Res. 1995; 34: 117-138Crossref PubMed Scopus (65) Google Scholar). Recent studies with PLA2R-deficient mice have demonstrated its potential role in the production of inflammatory cytokines during the progression of endotoxic shock (32Hanasaki K. Yokota Y. Ishizaki J. Itoh T. Arita H. J. Biol. Chem. 1997; 272: 32792-32797Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Group V sPLA2 (sPLA2-V) has been reported to be involved in the release of lipid mediators in P388D1 murine macrophages and mouse bone marrow-derived mast cells based on antisense experiments (33Reddy 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 (153) Google Scholar, 34Balboa 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 (197) Google Scholar). Recent studies have shown that this type of sPLA2 hydrolyzes phosphatidylcholine (PC) more effectively than sPLA2-IIA (35Han S.-K. Yoon E.T. Cho W. Biochem. J. 1998; 331: 353-357Crossref PubMed Scopus (52) Google Scholar). We have recently cloned a cDNA encoding a novel type of sPLA2, termed IID (sPLA2-IID) (10Ishizaki J. Suzuki N. Higashino K. Yokota Y. Ono T. Kawamoto K. Fujii N. Arita H. Hanasaki K. J. Biol. Chem. 1999; 274: 24973-24979Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The sPLA2-IID is most similar to sPLA2-IIA with respect to the number and positions of cysteine residues as well as overall sequence identity (10Ishizaki J. Suzuki N. Higashino K. Yokota Y. Ono T. Kawamoto K. Fujii N. Arita H. Hanasaki K. J. Biol. Chem. 1999; 274: 24973-24979Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 11Valentin E. Koduri R.S. Scimeca J.-C. Carle G. Gelb M.H. Lazdunski M. Lambeau G. J. Biol. Chem. 1999; 274: 19152-19160Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The expression of its mRNA dramatically changes upon challenge with endotoxin in rats and sPLA2-IIA-deficient mice, suggesting its potential role in the progression of inflammatory processes. Human group X sPLA2 (sPLA2-X) has been cloned from fetal lung based on sPLA2-related sequences from DNA data bases (9Cupillard L. Koumanov K. Mattei M.-G. Lazdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15745-15752Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). The sPLA2-X cDNA clone suggests a mature sPLA2 protein of 123 amino acids and the presence of a signal peptide with 32 amino acids. The presence of an arginine doublet and other polar residues preceding the mature sPLA2-X protein indicates that the signal sequence is a prepropeptide, although there is no biochemical evidence for the cleavage sites. sPLA2-X is the most acidic (pI 5.3) among human sPLA2s thus far identified and contains one potential glycosylation site. This sPLA2 possesses 16 cysteine residues located at positions characteristic of both sPLA2-IB and sPLA2-IIA/IID and also has an amino acid COOH-terminal extension that is typical of sPLA2-IIA and -IID. A 1.5-kilobase transcript coding for sPLA2-X was detected in human spleen and thymus, suggesting its potential role in the immune system and/or inflammation (9Cupillard L. Koumanov K. Mattei M.-G. Lazdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15745-15752Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 16Lambeau G. Lazdunski M. Trends Pharmacol. Sci. 1999; 20: 162-170Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar), although there are no data available with respect to its functional role in physiological and pathological conditions. To define the biochemical properties of human sPLA2-X, we prepared the antibody (Ab) and purified the protein. We found that human sPLA2-X releases arachidonic acid from PC more efficiently than other sPLA2 groups and also induces a rapid and marked release of arachidonic acid from several human myeloid leukemia cells compared with sPLA2-IB and -IIA. We also present evidence for the expression of sPLA2-X in human alveolar epithelial cells by immunohistochemical analysis. All oligonucleotides were purchased from Kokusai Shiyaku KK (Kobe, Japan). Recombinant human sPLA2-IB was prepared as described previously (28Arita H. Hanasaki K. Nakano T. Oka S. Matsumoto K. J. Biol. Chem. 1991; 266: 19139-19141Abstract Full Text PDF PubMed Google Scholar), and recombinant human sPLA2-IIA was a generous gift from Dr. Ruth Kramer (Eli Lilly, Indianapolis, IN). Diheptanoylthio-PC was synthesized as described previously (24Hagishita S. Yamada M. Shirahase K. Okuda T. Murakami Y. Ito Y. Matsuura T. Wada M. Kato T. Ueno M. Chikazawa Y. Yamada K. Ono T. Teshirogi I. Ohtani M. J. Med. Chem. 1996; 39: 3636-3658Crossref PubMed Scopus (198) Google Scholar). AACOCF3 was obtained from Cayman Chemicals. 5,5′-Dithiobis(2-nitrobenzoic acid) was obtained from Wako Chemicals, Japan. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-palmitoyl-sn-glycero-3-phosphocholine (PPPC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline (POPC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (PAPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphate, and 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) were purchased from Avanti Polar Lipids. 1-Palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (PLPE) and bovine serum albumin (BSA) were obtained from Sigma. The coding region of human sPLA2-X cDNA was obtained by polymerase chain reaction (PCR) using human placenta cDNA as a template. The sequences of the upstream and downstream primers were: hGX-S, (5′-CTGTGTACGCGTCCACCATGCTGCTCCTGCTACTGCCGTC-3′; and hGX-AS, 5′-TCAAGTGCGGCCGCTCAGTCACACTTGGGCGAGTCC-3′, respectively. hGX-S had a Kozak sequence and an MluI recognition site. hGX-AS possessed a NotI recognition site. The PCR conditions were 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 2 min, for 35 cycles. The PCR-amplified fragment was digested withMluI and NotI followed by the insertion into the modified pBS-SK(−). After sequencing confirmation, COOH-terminal His-tagged sPLA2-X (sPLA2-X-HisTag) was constructed by PCR using hGX-S primer and hGX-H6AS primer: 5′-TCAAGTGCGGCCGCTCAATGGTGATGGTGATGATGGTCACACTTGGGCGAGTCCGGCT-3′ (the underlined sequence corresponds to the His tag). The PCR-amplified fragment was digested with NotI andXhoI, which had a recognition site in the coding region of sPLA2-X followed by exchange of the corresponding region in the native sPLA2-X plasmid. The sequence of the PCR-amplified region was confirmed, and the cDNAs were inserted into the mammalian cell expression vector under SR-α promoter. The sPLA2-V cDNA was amplified by PCR from human heart cDNA as a template using the primers 5′-AAAGAACGCGTCCACCATGAAAGGCCTCCTCCCACTGGCT-3′ and 5′-CTCGCTGCGGCCGCCTAGGAGCAGAGGATGTTGGGAAA-3′. The upstream primer had a Kozak sequence and an MluI recognition site. The downstream primer possessed a NotI recognition site. The amplified fragment was digested with MluI andNotI followed by subcloning in the modified pBS-SK(−). The sPLA2-V expression plasmid was constructed by the same method as described above. The preparation of sPLA2-IID cDNA and its expression plasmid were performed as described previously (10Ishizaki J. Suzuki N. Higashino K. Yokota Y. Ono T. Kawamoto K. Fujii N. Arita H. Hanasaki K. J. Biol. Chem. 1999; 274: 24973-24979Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Spectrometric PLA2 assay was performed according to the method of Reynolds et al. (36Reynolds L.J. Hughes L.L. Dennis E.A. Anal. Biochem. 1992; 204: 190-197Crossref PubMed Scopus (95) Google Scholar). Briefly, 180 μl of the reaction mixture solution containing 1 mm diheptanoylthio-PC, 0.3 mm Triton X-100, 0.12 mm5,5′-dithiobis(2-nitrobenzoic acid), 10 mmCaCl2, 0.1 m KCl, and 0.1% BSA in 25 mm Tris-HCl buffer (pH 7.5) was preincubated in a 96-well plate for 15 min at 37 °C. The reactions were initiated by the addition of 20 μl of the enzyme preparation and continued for an appropriate time at 37 °C. The reaction was monitored by the absorbance at 405 nm. Recombinant plasmid containing sPLA2-X-HisTag and 0.5 μg of hygromycin B-resistant gene were co-transfected into SV40-transformed human embryonic kidney 293 (293T) cells with LipofectAMINE reagent (Life Technologies, Inc.), and the stably expressing clones were generated by selection against hygromycin B (250 μg/ml). Expression of the recombinant protein was determined with the chromogenic PLA2 assay. The conditioned medium (10% fetal calf serum (FCS)) was applied to a Ni2+-charged chelating Sepharose fast flow column (Amersham Pharmacia Biotech) equilibrated with 10 mm imidazole, 0.5 m NaCl, 20 mmsodium phosphate buffer (pH 7.4), and the bound materials were eluted with 500 mm imidazole, 0.5 m NaCl, 20 mm sodium phosphate buffer (pH 7.4). Peak fractions of PLA2 activity were dialyzed against 1 mmphenylmethylsulfonyl fluoride, 20 mm Tris-HCl (pH 7.4) and loaded on a HiTrap Q column (Amersham Pharmacia Biotech). The bound proteins were eluted with a gradient of NaCl from 0 to 0.5m. The sPLA2-X-HisTag fractions was then applied to a reverse-phase HPLC column (Cosmosil, 5C18 300AR, 4.6 × 150 mm, Nacarai Tesque, Japan) with a gradient of acetonitrile from 20 to 95% in 0.05% trifluoroacetic acid. The chromogenic PLA2 assay revealed that sPLA2-X-HisTag was eluted at 43% acetonitrile. The eluted materials were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in 15–25% acrylamide gradient gels (Daiichi Chemicals, Co.). In separate experiments, the purified protein was treated with 0.2 unit ofN-glycosidase F (E-5003; Oxford GlycoSystems) in 0.5% SDS, 5% 2-mercaptoethanol, 3% Nonidet P-40 for 18 h at 37 °C. After SDS-PAGE, electroblotting of proteins to an Immobilion-P membrane (Millipore Co.) was performed as described previously (37Hanasaki K. Powell L.D. Varki A. J. Biol. Chem. 1995; 270: 7543-7550Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). After staining with Coomassie Brilliant Blue, the protein bands were excised, and NH2-terminal amino acid sequencing was performed with an Applied Biosystems Procise Sequencer. Recombinant plasmid containing sPLA2-V or sPLA2-IID was transfected into CHO cells with LipofectAMINE reagent, and stably expressing clones were generated by selection against G418 (1 mg/ml; Life Technologies, Inc.). In the case of sPLA2-IID, the recombinant enzyme was partially purified with a heparin-Sepharose column (Amersham Pharmacia Biotech), as described previously (10Ishizaki J. Suzuki N. Higashino K. Yokota Y. Ono T. Kawamoto K. Fujii N. Arita H. Hanasaki K. J. Biol. Chem. 1999; 274: 24973-24979Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). After immunization of rabbits with purified sPLA2-X-HisTag (200 μg), the anti-serum was prepared, and the IgG fractions were purified with a HiTrap protein G-column (Amersham Pharmacia Biotech). The specificity and titer of the anti-serum and purified Ab were determined by enzyme-linked immunosorbent assay (ELISA) as follows. The 96-well plates (Nunc, Immuno MaxiSorp plate) coated with purified sPLA2 proteins (100 ng) were blocked with Block-Ace solution (Dainippon Pharmaceuticals Co.) and incubated with anti-sPLA2-X Ab, anti-human sPLA2-IB Ab (Seikagaku Corp.), and anti-human sPLA2-IIA serum (Cayman Chemical Co.) for 2 h. After washing with PBS, 0.05% Tween 20, the plates were incubated with peroxidase-conjugated goat anti-rabbit IgG (Immunotech) followed by the addition of 0.5 mg/ml o-phenylenediamine in 50 mm sodium citrate buffer (pH 5.3) containing 0.01% (v/v) H2O2. The reaction was stopped by addition of 4n H2SO4, and the plates were read at 492 nm. In separate experiments, the plates were coated with sPLA2-X-HisTag or Axl-HisTag proteins and assayed as described above. The inhibitory effects of anti-sPLA2-X Ab on PLA2 activity were examined with the chromogenic assay. Purified sPLA2 proteins (50 ng), partially purified sPLA2-IID, and the conditioned medium from the sPLA2-V-expressing CHO cells were incubated with anti-sPLA2-X Ab for 2 h and then added to the reaction mixture solution, as described above. The inhibitory potency of anti-sPLA2-X Ab was examined at the times when each sPLA2 reaction reached the sub-maximum at 37 °C. Transfection with sPLA2-X expression plasmid and preparation of stably expressing clones were performed as described above, and the established CHO cells were grown in 10% FCS, Dulbecco's modified Eagle's medium. After reaching confluence, the medium was changed to serum-free PM-1000 medium (Eiken Co., Japan), and the cells were incubated for 3 days. The conditioned medium was then applied to the anti-sPLA2-X Ab-conjugated affinity column, which had been prepared by coupling of anti-sPLA2-X IgG (30 mg) to HiTrapN-hydroxysuccinimide-activated column (5 ml; Amersham Pharmacia Biotech) according to the manufacturer's instructions. After washing with 40 mm sodium phosphate buffer (pH 7.4), the bound protein was eluted with 0.1 m glycine-HCl (pH 1.7). For further separation, the affinity-purified materials were applied to a reverse-phase HPLC column with a gradient of 20 to 40% acetonitrile in 0.1% trifluoroacetic acid. The three major fractions were collected, dried, and dissolved in 50 mm Tris-HCl (pH 7.4) containing 0.3 m NaCl. Human sPLA2s were subjected to individual reactions with 12 types of commercially available phospholipids as a substrate. These sPLA2 materials were also subjected to reactions with the mixed phospholipids composed of four types of PCs or three types of phosphatidylethanolamines (PEs) as the substrates. The enzymatic activity was measured using mixed micelles of 3 mm sodium deoxycholate (DOC) and 1 mm of each substrate or micelles of 3 mm DOC and mixed PCs or PEs consisting of 0.25 mm concentrations of each substrate in a total volume of 100 μl. The assay mixture contained 10 mmCaCl2, 1 mg/ml BSA, 150 mm NaCl, and 100 mm Tris-HCl (pH 8.0). After the incubation at 40 °C, the reaction was stopped by the addition of Dole's reagent (heptane, 2-propanol, 2 n sulfuric acid = 10:40:1, v/v/v), and the released fatty acids were quantified according to the method of Tojo et al. (38Tojo H. Ono T. Okamoto M. J. Lipid Res. 1993; 34: 837-844Abstract Full Text PDF PubMed Google Scholar). Human THP-1 cells were grown in RPMI 1640, 10% FCS and pretreated with 1.3% dimethyl sulfoxide for 2 days before the assay (39Kubin M. Chow J.M. Trinchieri G. Blood. 1994; 83: 1847-1855Crossref PubMed Google Scholar). After washing with PBS, the cells were suspended in Hanks' buffered saline (pH 7.6) containing 0.1% BSA at a density of 5.56 × 106 cells/ml. Aliquots of cell suspension (0.45 ml) were preincubated for 15 min at 37 °C and stimulated with sPLA2 proteins (0.05 ml). To analyze the released fatty acids, the reaction was stopped with 2 ml of Dole's reagent, and 3 nmol of margaric acid (Nu-Chek-Prep, Inc.) was added as an internal standard. After the addition of 1.2 ml of heptane and 0.8 ml of water, the heptane phase was removed and dried in vacuo, and the fatty acids in this fraction were labeled with 9-anthryldiazomethane (Funakoshi Co.), as described by Tojo et al. (38Tojo H. Ono T. Okamoto M. J. Lipid Res. 1993; 34: 837-844Abstract Full Text PDF PubMed Google Scholar). The sample was analyzed by reverse-phase HPLC on a LiChroCART 125–4 Superspher 100 RP-18 column (Merck) with acetonitrile/water (97:3, v/v) as the solvent. The column oven temperature was 26 °C, and the column effluents were monitored by the fluorescence intensity at the wavelengths of excitation and emission of 365 and 412 nm, respectively. To measure PGE2and LTC4, the reaction was stopped by cooling on ice. The supernatant was then collected after centrifugation at 1,000 ×g for 5 min at 4 °C, and the released PGE2and LTC4 were quantified with a radioimmunoassay kit (NEN Life Science Products) and an ELISA kit (Cayman Chemicals Co.), respectively. Human U937 and HL60 cells (5 × 105 cells/ml) grown in 10% FCS, RPMI 1640 were labeled with [3H]arachidonic acid (0.5 μCi/ml; Amersham Pharmacia Biotech) for 18 h. After washing three times, the cells were resuspended in 10% FCS, RPMI 1640. Aliquots of cell suspension (5 × 105 cells) were stimulated with sPLA2 proteins in a total volume of 400 μl at 37 °C. After the reaction, the supernatant was collected by centrifugation at 3,000 rpm for 2 min at 4 °C, and the released radioactivity in the supernatant (100 μl) was counted. Total incorporated radioactivity into the cells used for one assay was 1.04 × 106 and 1.02 × 106 dpm in U937 and HL60 cells, respectively. Preparations of normal adult human lung tissue were purchased from Novagen Inc. (Madison, WI). The tissue slides were dewaxed, incubated in methanol containing 0.3% H2O2 for 30 min, and then treated with 5% normal rabbit serum for 20 min. The slides were incubated with anti-sPLA2-X Ab (6 μg/ml) in PBS containing 0.1% BSA for 14 h at 4 °C. After washing with PBS, they were incubated with biotin-conjugated goat anti-rabbit IgG for 30 min followed by treatment with horseradish peroxidase avidin-biotin complex reagent (Vector Laboratories). After washing, the peroxidase activity was visualized with 10-min incubation in 50 mm Tris-HCl (pH 7.6) containing 200 μg/ml diaminobenzidine and 0.006% H2O2. After th
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