Cloning and Characterization of Novel Mouse and Human Secretory Phospholipase A2s*

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 (M r = 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 A 2 s (sPLA 2 s) are classified into several groups according to molecular structure and the localization of intramolecular disulfide bridges. Among them, group IIA sPLA 2 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 sPLA 2 gene is naturally disrupted by a frameshift mutation. Here, we report the cloning of a cDNA encoding a novel sPLA 2 expressed in the spleen of group IIA sPLA 2 -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 sPLA 2 genes. The human mature sPLA 2 protein consists of 125 amino acids (M r ‫؍‬ 14,500) preceded by a 20-residue prepeptide and is most similar to group IIA sPLA 2 with respect to the number and positions of cysteine residues as well as overall identity (48%). Based on these structural properties, the novel sPLA 2 should be categorized into group II, called group IID to follow the already identified IIA to IIC sPLA 2 s. When the cDNA was expressed in COS-7 cells, PLA 2 activity preferentially accumulated in the culture medium. It is maximally active at neutral to alkaline pH and with 2 mM Ca 2؉ . In assays with individual substrates, L-␣-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 sPLA 2 mRNA was elevated in the thymus after treatment with endotoxin in rats as well as in group IIA sPLA 2 -deficient mice, suggesting its functional role in the progression of the inflammatory process.
Phospholipase A 2 (PLA 2 ) 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 (1,2). PLA 2 s 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 (3). Over the past two decades along with advances in molecular biology, numerous PLA 2 s have been identified and characterized (4 -13). According to their biochemical features such as cellular localization, requirement of Ca 2ϩ , substrate specificity, and the primary structure, these PLA 2 s are classified into several families, including low molecular weight secretory PLA 2 (sPLA 2 ), Ca 2ϩ -sensitive arachidonoyl-specific 85-kDa cytosolic PLA 2 , Ca 2ϩ -independent PLA 2 , and platelet-activating factor-acetylhydrolase (14).
Low molecular mass sPLA 2 s (13-18 kDa) have several features distinct from other PLA 2 families, such as a high disulfide bond content, a requirement for millimolar concentration of Ca 2ϩ for catalysis, and a broad specificity for phospholipids with different polar head groups and fatty acyl chains (15). At present, mammalian sPLA 2 s 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 (12,14). Among them, group IIA sPLA 2 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 (16,17), and correlate well with disease severity in rheumatoid arthritis (18). Furthermore, the expression of group IIA sPLA 2 is enhanced by inflammatory cytokines such as interleukin 1␤ and tumor necrosis factor-␣ as well as lipopolysaccharide (LPS) in various cell types (19 -22). In some inbred mouse strains, however, the group IIA sPLA 2 gene is spontaneously inactivated by a point mutation (23,24). These deficient mice are susceptible to arthritis in antigeninduced models (25,26), and the mast cells derived from these mice exhibit normal prostaglandin production to ligand activation (27). The transgenic mice expressing the human group IIA sPLA 2 gene do not develop any overt inflammatory conditions (28). These findings point to the need to reassess the contribution of group IIA sPLA 2 in inflammatory diseases and suggest that other types of the sPLA 2 isoform play a pivotal role in place of or in concert with the group IIA sPLA 2 . For example, group V sPLA 2, one of the newly identified sPLA 2 isoforms (7), has been reported to be involved in the production of lipid mediators in P388D 1 murine macrophages and bone marrowderived mast cells based on antisense experiments (27,29). The most recently identified group X sPLA 2 (12) is another candidate. The involvement of group X sPLA 2 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 * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF112982 and AF112983.
‡ To whom correspondence should be addressed. Tel.: 81-6-6458-5861; Fax: 81-6-6458-0987; E-mail: kohji.hanasaki@shionogi.co.jp. 1 The abbreviations used are: PLA 2 , phospholipase A 2 ; sPLA 2 , secretory PLA 2 ; EST, expressed sequence tag; PCR, polymerase chain reaction; bp, base pairs; LPS, lipopolysaccharide; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PA, phosphatidic acid; PG, phosphatidylglycerol; kb, kilobase(s). sPLA 2 , group IB sPLA 2 , in the inflammatory response is also worth considering. This sPLA 2 has been thought to act as a digestive enzyme, given its abundance in digestive organs including the pancreas (30). However, a series of our studies have revealed group IB sPLA 2 -induced various biological responses, such as cell proliferation, smooth muscle contraction, and lipid mediator release, through the binding to its specific receptor, the PLA 2 receptor (31)(32)(33)(34)(35)(36). Furthermore, recent studies with mice deficient for both PLA 2 receptor and group IIA sPLA 2 demonstrated a potential role of group IB sPLA 2 /PLA 2 receptor-mediated responses in the progression of endotoxic shock, because the knock-out mice exhibit resistance to endotoxininduced lethality with reduced plasma levels of inflammatory cytokines (37). Besides previously identified sPLA 2 s, other low molecular weight PLA 2 s have been detected in various tissues including the brain and lung (38), suggesting the presence of novel sPLA 2 s that might play a compensatory role for the deficiency in group IIA sPLA 2 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 sPLA 2 isoform. Here, we report the cloning of a cDNA encoding a novel sPLA 2 expressed in the spleen of group IIA sPLA 2 -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 sPLA 2 -deficient mice.
Cloning of the Mouse sPLA 2 -tBLASTn search of the GenBank TM Data Base was performed (39) using an 11-amino acid sequence (DRC-CVTHDCCY) around the catalytic center of the mouse group IIA sPLA 2 (24). A cDNA fragment corresponding to the identified EST sequence was amplified by polymerase chain reaction (PCR). Primers for amplification were 5Ј-ctcctgaacctgaacaagatggtcacac-3Ј, 5Ј-cctgaacctgaacaagatggtcacacac-3Ј (sense) and 5Ј-agagtgggagcagcaagctgcaggac-3Ј, 5Ј-tccaggggacagacagagtggactcc-3Ј (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 5Ј and 3Ј 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, 5Ј-ataaggggctgcctgccttgct-3Ј and 5Јgaaagttgtttattaagagggctctt-3Ј. In each cloning step, sequences were determined with more than 10 individual clones to rule out the possibility of misincorporation during the PCR.
Cloning of Human sPLA 2 -Based on the mouse sPLA 2 cDNA se-quence, four primers (5Ј-acagactggtgctgtcagaa-3Ј, 5Ј-catgactgttgctatgccca-3Ј, 5Ј-acacagttgcctttcacacca-3Ј, and 5Ј-ttcacaccagctcccgttgtc-3Ј) 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 5Јand 3Ј-rapid amplification of cDNA ends were performed basically as described above using human sPLA 2 -specific primers and spleen cDNA. Chromosome Mapping-The chromosome localization of the human sPLA 2 was determined using the radiation hybrid mapping panel (Genebridge4, Research Genetics). 1 l of each DNA aliquot was subjected to PCR according to the manufacturer's protocol with primers (5Ј-aagggaagctggtgtgagcag-3Ј and 5Ј-ccgccagtagaaacgcagtcg-3Ј) which amplified the 108-bp PLA 2 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 TaqStart TM 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.
Recombinant Expression of the sPLA 2 s-Two primers, 5Ј-agtagttgatgcggccgccaccatgagactcgccctgctgtgtg-3Ј and 5Ј-taagcttttctagattagcatgctggagtcttgccttt-3Ј, were used for PCR amplification of the coding region of the mouse sPLA 2 cDNA. Those for the human sPLA 2 were 5Ј-agtagttgatgcggccgccaccatggaacttgcactgctgtgtg-3Ј and 5Ј-taagcttttctagactagcaccaggggtctgcccc-3Ј. Upstream primers have a NotI recognition site and Kozak sequence (italic). Downstream primers are with the XbaI recognition site. The sPLA 2 cDNA was amplified by PCR from mouse or human spleen cDNA followed by digestion with NotI and XbaI and inserted into pcDNA3.1(ϩ) (Invitrogen) to construct mouse and human sPLA 2 expression plasmids. After sequencing confirmation, 5 g of recombinant plasmid was transfected into 50% confluent COS-7 cells grown in 56-cm 2 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 mM Tris-HCl, pH 7.4, 2 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride and kept at Ϫ40°C until the assay. The expression plasmid without sPLA 2 cDNA was also introduced into COS-7 cells for the control.
PLA 2 Assays Using [ 3 H]Oleate-labeled E. coli Membranes-Preparation of autoclaved E. coli membranes and sPLA 2 assays were performed essentially as described previously (40). Unless otherwise specified, sPLA 2 assays were performed at 37°C in a total volume of 250 l consisting of 100 mM Tris-HCl, pH 7.4, 10 mM CaCl 2 , and 50,000 dpm of [ 3 H]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 l of culture medium containing novel sPLA 2 was incubated for 60 min to measure the PLA 2 activity. Control incubation in the absence of novel sPLA 2 was carried out in parallel and used to calculate the specific hydrolysis. The pH dependence of sPLA 2 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. PLA 2 Assay for Substrate Specificity-Chinese hamster ovary cells were transfected with human sPLA 2 expression 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 sPLA 2 activity was eluted with 1 M NaCl) 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 l. The assay mixture contained 10 mM CaCl 2 , 1 mg/ml bovine serum albumin, 150 mM NaCl, and 100 mM Tris-HCl, pH 8.0. The released fatty acids were quantified according to the method of Tojo et al. (41). Incubation times and sample volumes were adjusted to ensure hydrolysis rates within the linear range of enzymatic assays. Typically, 20 l of partially purified human sPLA 2 or 5 ng of purified human group IIA sPLA 2 was incubated for 30 min. The results were expressed as the percentage of hydrolyzed phospholipids within 30-min incubation.
Tissue Distribution of the mRNA-The coding region of the novel sPLA 2 cDNA was amplified by PCR and labeled with 32 P using Prime-It TM II random primer labeling kit (Stratagene). A multiple tissue Northern blot (CLONTECH) was hybridized with the probe in GMC buffer (250 mM Na 2 HPO 4 , 1 mM EDTA, 1% bovine serum albumin, 7% sodium dodecyl sulfate, pH 7.2) (42) (2.0 ϫ 10 6 cpm/ml) at 65°C overnight, then washed, and subjected to autoradiography. Subsequently, human group IIA sPLA 2 cDNA, which was isolated by PCR and ␤-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 Treatment-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 g of RNA) using mouse novel sPLA 2 or rat group IIA sPLA 2 cDNA 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.

Molecular
Cloning of Novel sPLA 2 and Chromosomal Localization of Its Gene-In searching for novel sPLA 2 s in the rapidly expanding data base, we identified a cDNA fragment (Gen-Bank TM accession number AA762051) by tBLASTn search using catalytically essential residues of sPLA 2 s as a query. This cDNA was an EST sequence originally cloned from thymus of C57BL/6J mouse (one of the group IIA sPLA 2 -deficient strains (23,24)) and theoretically could encode a portion of functional sPLA 2 s 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 5Ј and 3Ј surrounding regions were isolated using the PCR-based protocol. These separately isolated 5Ј, middle, and 3Ј cDNAs were assembled to yield one open reading frame that potentially encodes a functional sPLA 2 . The full-length cDNA was cloned with the 5Ј-and 3Ј-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 sPLA 2 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 sPLA 2 sequence under the assumption that the catalytically and functionally essential residues are conserved between the two animals. Through intensive search for the human PLA 2 cDNA 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 sPLA 2 cDNA and encoded 25 amino acid residues with a characteristic of sPLA 2 . 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 sPLA 2 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 sPLA 2 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 sPLA 2 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 hu-man/hamster hybrid clones gave clear amplification of the genomic fragment. The PCR results statistically assigned the sPLA 2 gene location to chromosome 1p36.12 at 3.77 centiray centromeric relative to the sequence tagged site WI-5273.
Structural Features of Novel sPLA 2 and Comparison with Other Mammalian sPLA 2 s-Amino acid sequences of novel mouse and human sPLA 2 s are shown in Fig. 1 aligned with those of other sPLA 2 s. 2 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 sPLA 2 does not have a propeptide. The calculated molecular masses of mouse and human novel sPLA 2 s are 14.3 and 14.5 kDa, respectively. There is one potential N-glycosylation site in each sPLA 2 ; Asn 99 (mouse) and Asn 89 (human). As shown in Fig. 1, the novel sPLA 2 s have about 40% identity with other isoforms and show preferential homology with group IIA sPLA 2 (47 and 48% in mouse and human, respectively). All of the previously published sPLA 2 s contain 12-16 cysteine residues to form 6 -8 intramolecular disulfide bonds by which each isoform is taxonomically characterized (15). In the mature portion of the novel sPLA 2 , mouse and human enzymes share identical distribution of 14 cysteine residues. Compared with the characteristic cysteine residues found in the known sPLA 2 sequences, the novel sPLA 2 possesses 50 -137 pairs, which is typical of group IIA sPLA 2 , but does not have the 11-77 and 86 -92 sets, which are characteristic of group IB and IIC sPLA 2 , respectively. In addition, novel sPLA 2 has an amino acid Cterminal extension, which is found in group IIA, IIC, and X sPLA 2 s, whereas it does not contain the pancreatic loop, a feature characteristic of the group IB sPLA 2 . Taken together, the novel sPLA 2 is most similar to group IIA sPLA 2 and should be categorized into group II based on the traditional grouping criteria proposed by Heinrikson (43). The sequence comparison clearly established the molecular identity of novel sPLA 2 distinguished from the PLA 2 s thus far cloned (groups I-X (12,14)). Therefore, we propose to assign the name of the novel sPLA 2 as group IID following so far identified IIA-IIC sPLA 2 s. Langlais et al. (44) identified PLA 2 activity in human spermatozoa and determined its 19 N-terminal amino acids, which lack Cys 11 . The novel sPLA 2 is distinct from the spermatic PLA 2 in its sequence.
Recombinant Expression of Novel sPLA 2 s and Characterization of sPLA 2 Activity-The deduced amino acid sequences from both mouse and human novel sPLA 2 cDNA contain all of the amino acids that are absolutely conserved in all functional sPLA 2 s including His 48 and Asp 49 . Therefore, they were expected to possess enzymatic activities, which should be exported extracellularly after cleavage from the presumed signal peptide. To confirm this, the mouse and human novel sPLA 2 cDNAs were subcloned into the eukaryotic expression plasmid and then transfected into COS-7 cells. As shown in Fig. 2A, PLA 2 activity was detected in the supernatant of the culture medium of mouse and human sPLA 2 recombinant cells, whereas cells transfected with the parent vector did not show enzymatic activity. Only 2-4% of the total PLA 2 activity was detected in the cell-associated fraction, indicating that novel sPLA 2 s were actively secreted from COS-7 cells. Similar results were obtained when human embryonic kidney 293 cells were used as a recombinant host (data not shown). As shown in Fig.  2B, sPLA 2 activity was completely dependent on Ca 2ϩ and required 2 mM Ca 2ϩ for the maximal level. Recombinant sPLA 2 was optimally active over a broad range of pH 6 -10 (Fig. 2C), whereas the activity of human group IIA sPLA 2 was optimal within pH 6 -9 and declined at pH 10 (data not shown). The Ca 2ϩ dependence and optimal pH profile were compatible with common features of sPLA 2 s (15).
The substrate preference of novel human sPLA 2 was determined individually with 13 types of commercially available phospholipids that possess palmitic acid at the sn-1 position and have different fatty acids at the sn-2 position as well as polar head groups. For this experiment, recombinant human sPLA 2 was partially purified by heparin affinity chromatography from the culture medium of Chinese hamster ovary cells that stably expressed the protein. The summary of the results (Table I) indicated the absence of the preference for the arachidonic acid-containing phospholipid. This lack of specificity toward arachidonate is also observed for the human group IIA sPLA 2 (Table I). This is a general feature of the sPLA 2 family (3) and is quite a contrast to the arachidonoyl-specific cytosolic PLA 2 . Among the phospholipids examined, L-␣-1-palmitoyl-2linoleoyl PE was most efficiently hydrolyzed. The PLA 1 activity was not detected when this substrate was used (data not shown). When compared within 1-palmitoyl-2-oleoyl phospholipids, novel sPLA 2 hydrolyzes PG and PE more efficiently than PC, whereas PS and PA are poorly hydrolyzed in our assay system. In contrast, group IIA sPLA 2 prefers PG to the other phospholipids as reported previously (45).
Tissue Expression of Novel sPLA 2 and Its Response to LPS Stimulation-The tissue expression pattern of the novel sPLA 2 was analyzed by probing several human multiple tissue Northern blots with labeled cDNA. As shown in Fig. 3, human novel sPLA 2 mRNAs were expressed in the pancreas and spleen and less abundantly in the colon, thymus, placenta, small intestine, and prostate. Two kinds of mRNA (2.0 and 1.0 kb) were detected and the ratio between these two transcripts varied among tissues. These size differences are probably due to the usage of a different site of initiation and/or termination of transcription or the result of an alternative splicing event, although we did not analyze the structure of each transcript. The distribution of the transcript of novel sPLA 2 contrasted with that of group IIA sPLA 2 , which is predominantly expressed in the prostate, small intestine, colon, and heart.
The expression of the novel sPLA 2 in the spleen and thymus indicates its involvement in the regulation of the immune system and inflammation. Its expression levels in endotoxin-challenged rats and mice were also examined. In untreated rat, one transcript (2.1 kb) of novel sPLA 2 was detected in the spleen, thymus, and lung. At 24 h after LPS injection, the expression level of the sPLA 2 mRNA was elevated 6-fold in the thymus (quantified after normalizing with the control glyceraldehyde-3-phosphate dehydrogenase transcript), whereas the signal was unchanged in the spleen and obviously decreased in the lung (Fig. 4A). In the case of group IIA sPLA 2 , a marked enhancement was observed in the thymus and lung in contrast to a slight decrease in the spleen. The elevation of group IIA sPLA 2 mRNA after LPS treatment was also detected in the small intestine, heart, kidney, pancreas, and liver, where the signal of novel sPLA 2 was not detected (data not shown). In group IIA sPLA 2 -deficient C57BL/6J mice, two transcripts (2.0 and 1.0 kb) were detected only in the spleen and thymus (data not shown). After challenge with LPS, the expression level of this sPLA 2 mRNA was elevated 3-fold in the thymus with no transcript of group IIA sPLA 2 (Fig. 4B). DISCUSSION Rapid increase of DNA data, especially from EST projects, has led to discoveries of a number of genes that had not been known. Among the PLA 2 molecules, group X sPLA 2 and a novel paralog of the cytosolic PLA 2 were successful outcomes of these genomic approaches (12,13). The initial retrieval of the EST sequence described in the present report is quite interesting, as The putative signal peptide and the mature portion are separated by a space in each sequence. Completely preserved residues among sPLA 2 family are indicated with capital letters in the consensus line. The characteristic cysteine by which sPLA 2 are categorized is indicated in lowercase letters. Catalytic site His is indicated by an asterisk. Amino acid sequence identities (%) between novel sPLA 2 and other sPLA 2 isoforms are shown on the right. References are: mouse IIA (24); mouse IIC (8); mouse V (56); human IIA (5); human IB (61); human V (7); human X (12); and mouse IB and mouse X (our unpublished data). The portion corresponding to the human cDNA fragment initially isolated based on the mouse sequence is underlined. A polymorphism (Gly or Ser) found in human novel sPLA 2 is doubleunderlined. the protein sequence coded by this cDNA fragment appears to be similar to but is distinct from the group IIA sPLA 2 . Upon isolation of this EST, we cloned its cDNA and identified the novel sPLA 2 from mouse and human. The novel sPLA 2 possesses biochemical characteristics common to the known sPLA 2 proteins in terms of molecular size, conservation of consensus sequence and distribution of disulfide-forming cysteine residue, the requirement of Ca 2ϩ as well as optimal pH range for catalysis, and the extracellular localization of the lipase activity. Thus, the newly cloned sPLA 2 is the sixth isoform of the sPLA 2 family found in rodents and the fifth in humans as a functional enzyme, because group IIC PLA 2 is thought to be a pseudogene in humans (15). Because the novel sPLA 2 is most similar to the group IIA sPLA 2 with respect to the number and positions of cysteine residues as well as overall identity, we propose to call the novel sPLA 2 as group IID following so far identified IIA to IIC sPLA 2 s.
The group IIA sPLA 2 is thought to be one of the key enzymes critically important for the pathogenesis of inflammatory diseases, because its expression level is enhanced under various inflammatory conditions (19 -22). The original EST corresponding to the novel mouse sPLA 2 has been cloned from the C57BL/6J strain in which the group IIA PLA 2 gene was naturally inactivated. Because these deficient mice are similar to group IIA sPLA 2 -expressing mouse strains in their inflammatory responses (25,26), the novel sPLA 2 might play a compensatory role for several functions of the group IIA sPLA 2 . In the three mammals used in this study, the expression of novel sPLA 2 was detected commonly in the spleen and thymus, which is a an expression profile similar to that of group X sPLA 2 in humans (12). Although the origin of cells producing this sPLA 2 is unknown at present, the tissue distribution pattern suggests its role in relation to the immune system and/or inflammation. Upon endotoxin challenge, the expression level of a novel sPLA 2 was elevated in the rat thymus along with the group IIA sPLA 2 transcript (Fig. 4). Enhanced expression of the novel sPLA 2 in the thymus was also observed in mice deficient for group IIA sPLA 2 . During the progression of sepsis, thymic atrophy is induced via apoptosis (46). Because several reports suggest an involvement of PLA 2 in thymocyte apoptosis (47,48), the novel sPLA 2 might play a role in this process. The expression of novel sPLA 2 mRNA was also changed in rat lung after LPS treatment, but its pattern was different from the case of group IIA sPLA 2 . In humans, the distribution of the transcript of novel sPLA 2 contrasted to that of group IIA sPLA 2 (Fig. 3), suggesting distinct biological functions for these two related sPLA 2 s in the physiological and pathological states. In addition to diverse tissue expression profiles, we observed a drastic difference between group IIA and novel sPLA 2 s in susceptibility to one of the 1-oxamoylindolidine derivatives (49). This sPLA 2 inhibitor has a strong inhibitory potency for group IIA sPLA 2 (IC 50 ϭ 1.2 nM), whereas more than 50% of the full activity of novel sPLA 2 remains even at 1000 nM. 3 Moreover, an antibody that neutralized the human group IIA sPLA 2 activity 3 T. Ono, Y. Yokota, and K. Hanasaki, unpublished data.  did not absorb the novel sPLA 2 activity (data not shown). These findings reflected the structural differences, especially around the active center, between two related sPLA 2 s, which could be relevant to their distinct functions.
In the C-terminal region of novel sPLA 2 , there is a relatively high content of basic amino acid residues, which is characteristic of heparin-binding PLA 2 s including group IIA and V sPLA 2 (50). In fact, the novel human sPLA 2 showed binding to a heparin-Sepharose column with successful purification of the enzyme. Murakami et al. (51) found correlation between the heparin binding ability of sPLA 2 and its potential contribution to the arachidonic acid release in the transfection/overexpression experiment. With this criterion, novel sPLA 2 could act on the cell surface proteoglycan, which leads to the arachidonic acid release at least under specific conditions. A heparin-sensitive PLA 2 activity responsible for the delayed phase prostaglandin D 2 synthesis was detected from mouse (C57BL/6J) bone marrow-derived mast cells (52). Because this mouse strain is deprived of group IIA sPLA 2 , the novel sPLA 2 is a conceivable candidate for the display of such activity. The possible contribution of novel sPLA 2 to the production of lipid mediators during the cell activation process deserves attention in future studies. sPLA 2 can participate in defensive action against invading bacteria through degradation of their phospholipids. For the effective breakdown of a pathogen, the substrate must be pretreated with bactericidal permeability increasing protein. PLA 2 activity toward bactericidal permeability increasing proteintreated E. coli varies greatly among the sPLA 2 family and apparently depends on a cluster of basic residues near the N terminus of the PLA 2 protein (53). In particular, the indispensability of Arg 7 and Lys 15 in human group IIA sPLA 2 was demonstrated by a site-directed mutagenesis experiment (54). The conservation of basic residues at these sites in novel sPLA 2 (Lys 7 and Lys 15 ) suggests its involvement in the antimicrobial activity. Its preferential hydrolysis of PE and PG (Table I) agrees well with this speculation, because they are major components of bacterial phospholipid.
Northern analysis detected an intense signal of the novel sPLA 2 transcript of relatively short size in the human pancreas, which displays abundant expression of group IB sPLA 2 . Cross-hybridization of the novel sPLA 2 probe with group IB sPLA 2 mRNA is not likely, because the calculated identity between these two cDNAs is only 53%. One possible function of the novel sPLA 2 is the digestion of phospholipid in nutrition. Another possibility is the involvement of the PLA 2 -specific receptor, which is known to be relevant to various biological reactions, because the pancreas is one of the tissues displaying ample expression of the PLA 2 receptor in humans (55). In this context, the receptor binding activity of the novel sPLA 2 should be evaluated in the future.
Radiation hybrid mapping analysis revealed the location of a human novel sPLA 2 gene on chromosome 1 in the vicinity of WI-5273. Around this region, group IIA, IIC, and V sPLA 2 genes have already been mapped (1p34-p36) (56), whereas group IB and X sPLA 2 genes are located on chromosome 12 and 16, respectively (12,57). These findings indicate that three sPLA 2 genes (IIA, V, and the novel type) and one pseudogene (IIC) constitute a gene cluster that is likely to have emerged from ancient gene duplication events. Some mammalian genes such as globin and apolipoprotein genes are known to form a gene cluster that also includes pseudogenes (58,59). It is interesting to note that some members of the apolipoprotein multigene family show similarities in the structural organization of their regulatory regions (60). A close linkage among sPLA 2 isoform genes suggests the possibility that they are under similar or overlapping transcriptional control. Of special interest is the similar up-regulation of expression between the novel sPLA 2 and the group IIA sPLA 2 in the thymus in the response to LPS challenge (Fig. 4A). In the promoter region of human and rat group IIA sPLA 2 gene, a putative interleukin-6-responsive element that is found in several acute phase genes was identified (19). 4 Analysis of the regulating region of the FIG. 3. Tissue distribution of novel sPLA 2 . Human multiple tissue Northern blots were hybridized with novel sPLA 2 , group IIA sPLA 2 , and ␤-actin probe. Approximately 2 g of poly(A) ϩ RNA was placed on each lane. The calculated size of the transcript detected is indicated at the right. PBL, peripheral blood leukocyte.
FIG. 4. Expression of novel sPLA 2 and group IIA sPLA 2 in LPS-treated animals. Rats were intravenously injected with E. coli LPS (A), or C57BL/6J mice were intraperitoneally injected with S. typhosa LPS (B). Control animals were treated with saline. After 24 h, the tissues indicated in the figure were isolated, and total RNAs were prepared. The RNA (20 g) was analyzed by Northern blotting as described under "Experimental Procedures." Two rats and mice were subjected to each experiment, and the typical result is shown. novel sPLA 2 gene should provide a clue to the functional significance of this isoform.
In conclusion, we isolated novel mouse and human sPLA 2 s (group IID) and characterized the activities and expression. This sPLA 2 shares common structural and catalytic features with previously known sPLA 2 isoforms and is especially related to group IIA sPLA 2 . Further studies are required to establish the precise physiological functions of this new sPLA 2 and to determine its role in disease states, especially in inflammatory conditions. Finally, the discovery of this novel sPLA 2 should enable more precise assignment of distinct functions of each isoform and should also broaden our understanding of the biochemical properties of the sPLA 2 family.