Cloning and Characterization of Novel Mouse and Human Secretory Phospholipase A2s
1999; Elsevier BV; Volume: 274; Issue: 35 Linguagem: Inglês
10.1074/jbc.274.35.24973
ISSN1083-351X
AutoresJun Ishizaki, Noriko Suzuki, Kenichi Higashino, Yasunori Yokota, Takashi Ono, Keiko Kawamoto, Noriko Fujii, Hitoshi Arita, Kohji Hanasaki,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoMammalian secretory phospholipase A2s (sPLA2s) are classified into several groups according to molecular structure and the localization of intramolecular disulfide bridges. Among them, group IIA sPLA2 has been thought to be one of the key enzymes in the pathogenesis of inflammatory diseases owing to its augmented expression under various inflammatory conditions. However, in a number of inbred mouse strains, the group IIA sPLA2 gene is naturally disrupted by a frameshift mutation. Here, we report the cloning of a cDNA encoding a novel sPLA2 expressed in the spleen of group IIA sPLA2-deficient mouse. We also cloned its human homolog and mapped its gene location on chromosome 1p36.12 near the loci of group IIA and V sPLA2 genes. The human mature sPLA2 protein consists of 125 amino acids (Mr = 14,500) preceded by a 20-residue prepeptide and is most similar to group IIA sPLA2 with respect to the number and positions of cysteine residues as well as overall identity (48%). Based on these structural properties, the novel sPLA2 should be categorized into group II, called group IID to follow the already identified IIA to IIC sPLA2s. When the cDNA was expressed in COS-7 cells, PLA2 activity preferentially accumulated in the culture medium. It is maximally active at neutral to alkaline pH and with 2 mm Ca2+. In assays with individual substrates,l-α-1-palmitoyl-2-linoleoyl phosphatidylethanolamine was more efficiently hydrolyzed than the other phospholipids examined. An RNA blot hybridized with the cDNA exhibited two transcripts (2.0 and 1.0 kb) in human spleen, thymus, and colon. The expression of a novel sPLA2 mRNA was elevated in the thymus after treatment with endotoxin in rats as well as in group IIA sPLA2-deficient mice, suggesting its functional role in the progression of the inflammatory process. Mammalian secretory phospholipase A2s (sPLA2s) are classified into several groups according to molecular structure and the localization of intramolecular disulfide bridges. Among them, group IIA sPLA2 has been thought to be one of the key enzymes in the pathogenesis of inflammatory diseases owing to its augmented expression under various inflammatory conditions. However, in a number of inbred mouse strains, the group IIA sPLA2 gene is naturally disrupted by a frameshift mutation. Here, we report the cloning of a cDNA encoding a novel sPLA2 expressed in the spleen of group IIA sPLA2-deficient mouse. We also cloned its human homolog and mapped its gene location on chromosome 1p36.12 near the loci of group IIA and V sPLA2 genes. The human mature sPLA2 protein consists of 125 amino acids (Mr = 14,500) preceded by a 20-residue prepeptide and is most similar to group IIA sPLA2 with respect to the number and positions of cysteine residues as well as overall identity (48%). Based on these structural properties, the novel sPLA2 should be categorized into group II, called group IID to follow the already identified IIA to IIC sPLA2s. When the cDNA was expressed in COS-7 cells, PLA2 activity preferentially accumulated in the culture medium. It is maximally active at neutral to alkaline pH and with 2 mm Ca2+. In assays with individual substrates,l-α-1-palmitoyl-2-linoleoyl phosphatidylethanolamine was more efficiently hydrolyzed than the other phospholipids examined. An RNA blot hybridized with the cDNA exhibited two transcripts (2.0 and 1.0 kb) in human spleen, thymus, and colon. The expression of a novel sPLA2 mRNA was elevated in the thymus after treatment with endotoxin in rats as well as in group IIA sPLA2-deficient mice, suggesting its functional role in the progression of the inflammatory process. phospholipase A2 secretory PLA2 expressed sequence tag polymerase chain reaction base pairs lipopolysaccharide phosphatidylcholine phosphatidylethanolamine phosphatidylserine phosphatidic acid phosphatidylglycerol kilobase(s) Phospholipase A2(PLA2)1 comprises a diverse family of lipolytic enzymes that hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acid and lysophspholipids (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 (161) Google Scholar). PLA2s participate in a wide variety of physiological processes, including phospholipid digestion, remodeling of cell membranes, and host defense, and also take part in pathophysiological processes by producing precursors of various types of biologically active lipid mediators, such as prostaglandins, leukotrienes, thromboxanes, and platelet-activating factor (3Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar). Over the past two decades along with advances in molecular biology, numerous PLA2s have been identified and characterized (4Seilhamer 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, 5Kramer 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, 6Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. 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Koumanov K. Mattei M.-G. Lazdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15745-15752Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 13Underwood K.W. Song C. Kriz R.E. Chang X.J. Knopf J.L. Lin L.-L. J. Biol. Chem. 1998; 273: 21926-21932Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). According to their biochemical features such as cellular localization, requirement of Ca2+, substrate specificity, and the primary structure, these PLA2s are classified into several families, including low molecular weight secretory PLA2 (sPLA2), Ca2+-sensitive arachidonoyl-specific 85-kDa cytosolic PLA2, Ca2+-independent PLA2, and platelet-activating factor-acetylhydrolase (14Dennis E.A. Trends Biol. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar). Low molecular mass sPLA2s (13–18 kDa) have several features distinct from other PLA2 families, such as a high disulfide bond content, a requirement for millimolar concentration 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 (270) Google Scholar). At present, mammalian sPLA2s are classified into five different groups (groups IB, IIA, IIC, V, and X), depending on the primary structure characterized by the number and positions of cysteine residues (12Cupillard L. Koumanov K. Mattei M.-G. Lazdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15745-15752Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 14Dennis E.A. Trends Biol. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar). Among them, group IIA sPLA2 has been a focus of attention as a potent mediator of the inflammatory process, because its local and systemic levels are elevated in numerous inflammatory diseases, including sepsis, Crohn's disease, and acute pancreatitis (16Gronroots J.M. Nevalainen T.J. Digestion. 1992; 52: 232-236Crossref PubMed Scopus (71) Google Scholar, 17Green 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 (150) Google Scholar), and correlate well with disease severity in rheumatoid arthritis (18Pruzanski W. Keystone E.C. Sternby B. Bombardier C. Snow K.M. Vadas P. J. Rheumatol. 1988; 15: 1351-1355PubMed Google Scholar). Furthermore, the expression of group IIA sPLA2 is enhanced by inflammatory cytokines such as interleukin 1β and tumor necrosis factor-α as well as lipopolysaccharide (LPS) in various cell types (19Crowl R.M. Stoller T.J. Conroy R.R. Stoner C.R. J. Biol. Chem. 1991; 266: 2647-2651Abstract Full Text PDF PubMed Google Scholar, 20Nakano T. Ohara O. Teraoka H. Arita H. FEBS Lett. 1990; 261: 171-174Crossref PubMed Scopus (175) Google Scholar, 21Ohara O. Ishizaki J. Nakano T. Arita H. Teraoka H. Nucleic Acids Res. 1990; 18: 6997-7002Crossref PubMed Scopus (25) Google Scholar, 22Murakami M. Kudo I. Inoue K. J. Biol. Chem. 1993; 268: 839-844Abstract Full Text PDF PubMed Google Scholar). In some inbred mouse strains, however, the group IIA sPLA2 gene is spontaneously inactivated by a point mutation (23MacPhee 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 (528) Google Scholar, 24Kennedy 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: 22378-22385Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). These deficient mice are susceptible to arthritis in antigen-induced models (25Brackertz D. Mitchell G.F. Mackay I.R. Arthritis Rheum. 1977; 20: 841-850Crossref PubMed Scopus (258) Google Scholar, 26Wooley P.H. Luthra H.S. Griffiths M.M. Stuart J.M. Huse A. David C.S. J. Immunol. 1985; 135: 2443-2451PubMed Google Scholar), and the mast cells derived from these mice exhibit normal prostaglandin production to ligand activation (27Reddy 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). The transgenic mice expressing the human group IIA sPLA2 gene do not develop any overt inflammatory conditions (28Grass 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 (160) Google Scholar). These findings point to the need to reassess the contribution of group IIA sPLA2 in inflammatory diseases and suggest that other types of the sPLA2 isoform play a pivotal role in place of or in concert with the group IIA sPLA2. For example, group V sPLA2, one of the newly identified sPLA2isoforms (7Chen J. Engle S.J. Seilhamer J.J. Tischfield J.A. J. Biol. Chem. 1994; 269: 2365-2368Abstract Full Text PDF PubMed Google Scholar), has been reported to be involved in the production of lipid mediators in P388D1 murine macrophages and bone marrow-derived mast cells based on antisense experiments (27Reddy 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, 29Balboa 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). The most recently identified group X sPLA2 (12Cupillard L. Koumanov K. Mattei M.-G. Lazdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15745-15752Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) is another candidate. The involvement of group X sPLA2 in inflammatory responses is suggested by its restricted expression in immune tissues such as the spleen and thymus, although there is no direct evidence for its commitment to the pathological conditions. A possibility of the involvement of the most classical sPLA2, group IB sPLA2, in the inflammatory response is also worth considering. This sPLA2 has been thought to act as a digestive enzyme, given its abundance in digestive organs including the pancreas (30de 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 revealed group IB sPLA2-induced various biological responses, such as cell proliferation, smooth muscle contraction, and lipid mediator release, through the binding to its specific receptor, the PLA2receptor (31Arita H. Hanasaki K. Nakano T. Oka S. Teraoka H. Matsumoto K. J. Biol. Chem. 1991; 266: 19139-19141Abstract Full Text PDF PubMed Google Scholar, 32Hanasaki K. Arita H. J. Biol. Chem. 1992; 267: 6414-6420Abstract Full Text PDF PubMed Google Scholar, 33Tohkin M. Kishino J. Ishizaki J. Arita H. J. Biol. Chem. 1993; 268: 2865-2871Abstract Full Text PDF PubMed Google Scholar, 34Kishino J. Ohara O. Nomura K. Kramer R.M. Arita H. J. Biol. Chem. 1994; 269: 5092-5098Abstract Full Text PDF PubMed Google Scholar, 35Ishizaki 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, 36Ohara O. Ishizaki J. Arita H. Prog. Lipid Res. 1995; 34: 117-138Crossref PubMed Scopus (65) Google Scholar). Furthermore, recent studies with mice deficient for both PLA2 receptor and group IIA sPLA2demonstrated a potential role of group IB sPLA2/PLA2 receptor-mediated responses in the progression of endotoxic shock, because the knock-out mice exhibit resistance to endotoxin-induced lethality with reduced plasma levels of inflammatory cytokines (37Hanasaki 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). Besides previously identified sPLA2s, other low molecular weight PLA2s have been detected in various tissues including the brain and lung (38Gray N.C. Strickland K.P. Can. J. Biochem. 1982; 60: 108-117Crossref PubMed Scopus (39) Google Scholar), suggesting the presence of novel sPLA2s that might play a compensatory role for the deficiency in group IIA sPLA2 or an independently functional role in the inflammatory processes. During a survey of the DNA data base, we encountered an expressed sequence tag (EST) that could represent part of a new sPLA2isoform. Here, we report the cloning of a cDNA encoding a novel sPLA2 expressed in the spleen of group IIA sPLA2-deficient mice. We also describe the cloning of its human homolog, the characterization of recombinant protein and its expression profile in humans, as well as in endotoxin-treated rats and group IIA sPLA2-deficient mice. All oligonucleotides were purchased from Kokusai Shiyaku KK (Kobe, Japan). LPS (Escherichia coli: 055:B5) was purchased from Sigma, LPS (Salmonella typhosa 0901) was from Difco Laboratories, and G418 was from Life Technologies, Inc. Recombinant human group IIA sPLA2 was a generous gift from Dr. Ruth Kramer (Eli Lilly, Indianapolis, IN). 1-Palmitoyl-2-oleoyl-sn-glycero-3-PG, 1-palmitoyl-2-palmitoyl-sn-glycero-3-PG, 1-palmitoyl-2-palmitoyl-sn-glycero-3-PC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-PC, 1-palmitoyl-2-linoleoyl-sn-glycero-3-PC, 1-palmitoyl-2-oleoyl-sn-glycero-3-PC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-PE, 1-palmitoyl-2-oleoyl-sn-glycero-3-PS, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-PA, and 1-palmitoyl-2-oleoyl-sn-glycero-3-PA were purchased from Avanti Polar Lipids. 1-Palmitoyl-2-docosahexaenoyl-sn-glycero-3-PC and 1-palmitoyl-2-linoleoyl-sn-glycero-3-PE were obtained from Sigma. Computational analyses on the isolated cDNA and related sequences were performed by using the GENETYX program (Software Development Co., Ltd.). tBLASTn search of the GenBankTM Data Base was performed (39Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59933) Google Scholar) using an 11-amino acid sequence (DRCCVTHDCCY) around the catalytic center of the mouse group IIA sPLA2 (24Kennedy 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: 22378-22385Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). A cDNA fragment corresponding to the identified EST sequence was amplified by polymerase chain reaction (PCR). Primers for amplification were 5′-ctcctgaacctgaacaagatggtcacac-3′, 5′-cctgaacctgaacaagatggtcacacac-3′ (sense) and 5′-agagtgggagcagcaagctgcaggac-3′, 5′-tccaggggacagacagagtggactcc-3′ (antisense). Two rounds of amplifications (nested PCR) were carried out with these primers and ExTaq (Takara, Japan). Reverse transcribed cDNAs from various mouse tissues were used as templates. Amplification conditions were 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 3 min for 30 cycles. The PCR products were separated on agarose gel, and the DNA of the expected size was isolated. The recombinant plasmid was then constructed with pCRII cloning vector (Invitrogen), purified with GFX Micro Plasmid Prep Kit (Amersham Pharmacia Biotech), and sequenced with Applied Biosystems PRISM 310 genetic analyzer. From the determined DNA sequence, four primers were designed for the isolation of 5′ and 3′ portions of the cDNA. The cloning of these remaining parts was carried out with rapid amplification of the cDNA ends protocol using mouse spleen marathon-ready cDNA (CLONTECH) according to the manufacturer's manual with a slight modification in the choice of polymerase; ExTaq was used instead of KlenTaq polymerase. The full-length cDNA was isolated by PCR with primers, 5′-ataaggggctgcctgccttgct-3′ and 5′-gaaagttgtttattaagagggctctt-3′. In each cloning step, sequences were determined with more than 10 individual clones to rule out the possibility of misincorporation during the PCR. Based on the mouse sPLA2 cDNA sequence, four primers (5′-acagactggtgctgtcagaa-3′, 5′-catgactgttgctatgccca-3′, 5′-acacagttgcctttcacacca-3′, and 5′-ttcacaccagctcccgttgtc-3′) were prepared for amplification of the middle part of the human homolog cDNA. Using human spleen marathon-ready cDNA (CLONTECH) as a template, two consecutive rounds of PCR were performed with two pairs of primers in the nested manner. The PCR conditions were 94 °C for 1 min, 45 °C for 1 min, and 72 °C for 3 min for 30 cycles with ExTaq. The amplified product was separated on agarose gel, and the DNA of the expected size (117 base pairs (bp)) was isolated and sequenced. The 5′- and 3′-rapid amplification of cDNA ends were performed basically as described above using human sPLA2-specific primers and spleen cDNA. The chromosome localization of the human sPLA2 was determined using the radiation hybrid mapping panel (Genebridge4, Research Genetics). 1 μl of each DNA aliquot was subjected to PCR according to the manufacturer's protocol with primers (5′-aagggaagctggtgtgagcag-3′ and 5′-ccgccagtagaaacgcagtcg-3′) which amplified the 108-bp PLA2 encoding genomic DNA fragment. The PCR was initiated at 94 °C for 2 min and then followed by 94 °C for 1 min, 62 °C for 1 min, and 72 °C for 1 min for 30 cycles with ExTaq and TaqStartTM Antibody (CLONTECH) using the hot start technique. The PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The presence or absence of the product in each of the hybrid clones was scored. With the screening result, mapping was performed on the server computer at the Whitehead Institute/MIT center for Genome Research. Two primers, 5′-agtagttgatgcggccg ccacc atgagactcgccctgctgtgtg-3′ and 5′-taagcttttctagattagcatgctggagtcttgccttt-3′, were used for PCR amplification of the coding region of the mouse sPLA2cDNA. Those for the human sPLA2 were 5′-agtagttgatgcggccg ccacc atggaacttgcactgctgtgtg-3′ and 5′-taagcttttctagactagcaccaggggtctgcccc-3′. Upstream primers have a Not I recognition site and Kozak sequence (italic). Downstream primers are with the Xba I recognition site. The sPLA2 cDNA was amplified by PCR from mouse or human spleen cDNA followed by digestion with Not I and Xba I and inserted into pcDNA3.1(+) (Invitrogen) to construct mouse and human sPLA2 expression plasmids. After sequencing confirmation, 5 μg of recombinant plasmid was transfected into 50% confluent COS-7 cells grown in 56-cm2 Petri dishes with LipofectAMINE reagent (Life Technologies, Inc.). At 72 h after transfection, culture media were collected. The washed cells were harvested and disrupted by sonication in 1 ml of 20 mmTris-HCl, pH 7.4, 2 mm EDTA, and 1 mmphenylmethylsulfonyl fluoride and kept at −40 °C until the assay. The expression plasmid without sPLA2 cDNA was also introduced into COS-7 cells for the control. Preparation of autoclaved E. coli membranes and sPLA2 assays were performed essentially as described previously (40Furue S. Hori Y. Kuwabara K. Ikeuchi J. Onoyama H. Yamamoto M. Tanaka K. Gut. 1997; 41: 826-831Crossref PubMed Scopus (15) Google Scholar). Unless otherwise specified, sPLA2 assays were performed at 37 °C in a total volume of 250 μl consisting of 100 mm Tris-HCl, pH 7.4, 10 mm CaCl2, and 50,000 dpm of [3H]oleate-labeled E. coli membranes. Incubation times and sample volumes were adjusted to ensure hydrolysis rates within the linear range of the enzymatic assays. Typically, 50 μl of culture medium containing novel sPLA2 was incubated for 60 min to measure the PLA2 activity. Control incubation in the absence of novel sPLA2 was carried out in parallel and used to calculate the specific hydrolysis. The pH dependence of sPLA2 activity was performed in the presence of 100 mm sodium acetate buffer at pH range 4.5–6.0, 100 mm Tris-HCl buffer at pH range 7.0–9.0, or 100 mm glycine-HCl at pH 10.0. Chinese hamster ovary cells were transfected with human sPLA2expression plasmid, and stably expressing clones were generated by selection against G418 (1 mg/ml). From the culture medium of the established cell lines, the recombinant enzyme was partially purified by heparin-Sepharose affinity chromatography (Amersham Pharmacia Biotech; the sPLA2 activity was eluted with 1 mNaCl) and then subjected to individual reactions with 13 types of commercially available phospholipid as the substrate. The enzymatic activity was measured using mixed micelles of 1 mm of each substrate and 3 mm sodium deoxycholate in a total volume of 100 μl. The assay mixture contained 10 mmCaCl2, 1 mg/ml bovine serum albumin, 150 mmNaCl, and 100 mm Tris-HCl, pH 8.0. The released fatty acids were quantified according to the method of Tojo et al. (41Tojo H. Ono T. Okamoto M. J. Lipid. Res. 1993; 34: 837-844Abstract Full Text PDF PubMed Google Scholar). Incubation times and sample volumes were adjusted to ensure hydrolysis rates within the linear range of enzymatic assays. Typically, 20 μl of partially purified human sPLA2 or 5 ng of purified human group IIA sPLA2 was incubated for 30 min. The results were expressed as the percentage of hydrolyzed phospholipids within 30-min incubation. The coding region of the novel sPLA2 cDNA was amplified by PCR and labeled with 32P using Prime-ItTM II random primer labeling kit (Stratagene). A multiple tissue Northern blot (CLONTECH) was hybridized with the probe in GMC buffer (250 mm Na2HPO4, 1 mm EDTA, 1% bovine serum albumin, 7% sodium dodecyl sulfate, pH 7.2) (42Church G.M. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1991-1995Crossref PubMed Scopus (7267) Google Scholar) (2.0 × 106 cpm/ml) at 65 °C overnight, then washed, and subjected to autoradiography. Subsequently, human group IIA sPLA2 cDNA, which was isolated by PCR and β-actin probe, were used for the hybridization of the same membrane. The sizes of the transcripts are calculated from the standard molecular size markers. LPS (E. coli: 055:B5) was injected intravenously into Harlan Sprague Dawley rat tail at a dosage of 5 mg/kg. In C57BL/6J mice, LPS (10 mg/kg; Salmonella typhosa 0901) was injected intraperitoneally. Total RNA was extracted from several tissues using RNeasy Mini Kit (Qiagen) at 24 h after LPS injection and subjected to Northern analysis (20 μg of RNA) using mouse novel sPLA2 or rat group IIA sPLA2cDNA probe. The intensity of the signals was quantified with BAS 2000 image analyzer (Fuji Photo Film) and normalized against the glyceraldehyde-3-phosphate dehydrogenase control. In searching for novel sPLA2s in the rapidly expanding data base, we identified a cDNA fragment (GenBankTM accession number AA762051) by tBLASTn search using catalytically essential residues of sPLA2s as a query. This cDNA was an EST sequence originally cloned from thymus of C57BL/6J mouse (one of the group IIA sPLA2-deficient strains (23MacPhee 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 (528) Google Scholar, 24Kennedy 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: 22378-22385Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar)) and theoretically could encode a portion of functional sPLA2s previously uncharacterized. We amplified the cDNA corresponding to this EST sequence from reverse transcribed RNA samples extracted from various mouse tissues including the spleen, liver, and small intestine. Using splenic cDNA fragment as a probe, an expression profile was examined by Northern analysis of multiple tissues originated from Balb/c mice, which detected two transcripts (1.2 and 2.4 kb) expressed most abundantly in the spleen among the tissues examined (data not shown). Using the spleen cDNA library as a source, the 5′ and 3′ surrounding regions were isolated using the PCR-based protocol. These separately isolated 5′, middle, and 3′ cDNAs were assembled to yield one open reading frame that potentially encodes a functional sPLA2. The full-length cDNA was cloned with the 5′- and 3′-most primers by PCR from spleen cDNA to confirm the existence of the consecutive transcript and its sequence. The 1233-bp cDNA thus identified encoded a novel sPLA2 consisting of 144 amino acids. To clone its human homolog, we first attempted to amplify the middle portion of the cDNA by PCR using primers designed from the mouse sPLA2 sequence under the assumption that the catalytically and functionally essential residues are conserved between the two animals. Through intensive search for the human PLA2cDNA fragments after PCR using several combinations of primers, one of the amplified cDNA fragments was found to have a nucleotide sequence 70% identical to that of the corresponding region of the mouse sPLA2 cDNA and encoded 25 amino acid residues with a characteristic of sPLA2. Using this sequence information, we were able to clone the full-length cDNA from a human spleen cDNA library with the rapid amplification of cDNA ends protocol. The human sPLA2 cDNA clone consists of 878 bp with one long open reading frame encoding 145 amino acids. The coding region has 79% nucleotide sequence identity with the mouse counterpart. To assign the chromosome localization of the human novel sPLA2 gene, we performed radiation hybrid mapping. The PCR using two primers described under "Experimental Procedures" gave a 108-bp fragment from human genomic DNA as well as from the isolated sPLA2 cDNA, which means that the corresponding gene sequence is not separated by interrupting introns. With this pair of primers, the radiation hybrid mapping panel was screened. 25 of 93 DNA aliquots derived from human/hamster hybrid clones gave clear amplification of the genomic fragment. The PCR results statistically assigned the sPLA2 gene location to chromosome 1p36.12 at 3.77 centiray centromeric relative to the sequence tagged site WI-5273. Amino acid sequences of novel mouse and human sPLA2s are shown in Fig.1 aligned with those of other sPLA2s. 2Mouse group IB and X sPLA2 cDNAs were isolated based on rat group IB sPLA2 and human group X sPLA2 sequence, respectively (N. Suzuki, H. Nakazato, and K. Hanasaki, unpublished data). The sequence similarity and hydropathy profiles (data not shown) suggest that the N-terminal 19 (mouse) and 20 (human) residues are signal peptides. Judging from the length of the predicted signal peptide and absence of basic amino acids preceding the N terminus of the mature protein, this sPLA2 does not have a propeptide. The calculated molecular masses of mouse and human novel sPLA2s are 14.3 and 14.5 kDa, respectively. There is one potential N-glycosylation site in each sPLA2; Asn99 (mouse) and Asn89 (human). As shown in Fig. 1, the novel sPLA2s have about 40% identity with other isoforms and show preferential homology with group IIA sPLA2 (47 and 48% in mouse and human, respectively). All of the previously published sPLA2s contain 12–16 cysteine residues to form 6–8 intramolecular disulfide bonds by which each isoform is taxonomically characterized (15Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). In the mature portion of the novel sPLA2, mouse and human enzymes share identical distribution of 14 cysteine residues. Compared with the characteristic cysteine residues found in the known sPLA2 sequences, the novel sPLA2 possesses 50–137 pairs, which is typical of group IIA sPLA2, but does not have the 11–77 and 86–92 sets, which are characteristic of group IB and IIC sPLA2, respectively. In addition, novel sPLA2 has an amino acid C-terminal extension, which is found in group IIA, IIC, and X sPLA2s, whereas it does not contain the pancreatic loop, a feature characteristic of the group IB sPLA2. Taken together, the novel sPLA2 is most similar to group IIA sPLA2 and should be categorized in
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