J Biol Chem, Vol. 274, Issue 35, 24973-24979, August 27, 1999

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

Jun Ishizaki, Noriko Suzuki, Ken-ichi Higashino, Yasunori Yokota, Takashi Ono, Keiko Kawamoto, Noriko Fujii, Hitoshi Arita, and Kohji Hanasaki

From the Shionogi Research Laboratories, Shionogi and Co., Ltd., Sagisu 5-12-4, Fukushima-ku, Osaka 553-0002, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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--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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (1, 2). 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 (3). Over the past two decades along with advances in molecular biology, numerous PLA₂s have been identified and characterized (4-13). 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 (14).

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 (15). 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 (12, 14). 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 (16, 17), and correlate well with disease severity in rheumatoid arthritis (18). Furthermore, the expression of group IIA sPLA₂ 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 sPLA₂ gene is spontaneously inactivated by a point mutation (23, 24). These deficient mice are susceptible to arthritis in antigen-induced 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 sPLA₂ gene do not develop any overt inflammatory conditions (28). 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 sPLA_2, one of the newly identified sPLA₂ isoforms (7), 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 (27, 29). The most recently identified group X sPLA₂ (12) 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 (30). 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 PLA₂ receptor (31-36). Furthermore, recent studies with mice deficient for both PLA₂ receptor and group IIA sPLA₂ demonstrated 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 (37). Besides previously identified sPLA₂s, other low molecular weight PLA₂s have been detected in various tissues including the brain and lung (38), 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 sPLA₂ isoform. 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- 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.).

Cloning of the Mouse sPLA₂-- tBLASTn search of the GenBank^TM Data Base was performed (39) using an 11-amino acid sequence (DRCCVTHDCCY) around the catalytic center of the mouse group IIA sPLA₂ (24). 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.

Cloning of Human sPLA₂-- 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.

Chromosome Mapping-- 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 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 sPLA₂s-- Two primers, 5'-agtagttgatgcggccgccaccatgagactcgccctgctgtgtg-3' and 5'-taagcttttctagattagcatgctggagtcttgccttt-3', were used for PCR amplification of the coding region of the mouse sPLA₂ cDNA. Those for the human sPLA₂ were 5'-agtagttgatgcggccgccaccatggaacttgcactgctgtgtg-3' and 5'-taagcttttctagactagcaccaggggtctgcccc-3'. Upstream primers have a NotI recognition site and Kozak sequence (italic). Downstream primers are with the XbaI recognition site. The sPLA₂ cDNA was amplified by PCR from mouse or human spleen cDNA followed by digestion with NotI and XbaI 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 mM Tris-HCl, pH 7.4, 2 mM EDTA, and 1 mM phenylmethylsulfonyl 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.

PLA₂ Assays Using [³H]Oleate-labeled E. coli Membranes-- Preparation of autoclaved E. coli membranes and sPLA₂ assays were performed essentially as described previously (40). 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.

PLA₂ Assay for Substrate Specificity-- Chinese hamster ovary cells were transfected with human sPLA₂ 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 sPLA₂ 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 µl. The assay mixture contained 10 mM CaCl₂, 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 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.

Tissue Distribution of the mRNA-- The coding region of the novel sPLA₂ cDNA was amplified by PCR and labeled with ³²P 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 Na₂HPO₄, 1 mM EDTA, 1% bovine serum albumin, 7% sodium dodecyl sulfate, pH 7.2) (42) (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 -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 µg of RNA) using mouse novel sPLA₂ or rat group IIA sPLA₂ 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Molecular Cloning of Novel sPLA₂ and Chromosomal Localization of Its Gene-- In searching for novel sPLA₂s in the rapidly expanding data base, we identified a cDNA fragment (GenBank^TM 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 (23, 24)) 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 PLA₂ 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 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.

Structural Features of Novel sPLA₂ and Comparison with Other Mammalian sPLA₂s-- Amino acid sequences of novel mouse and human sPLA₂s are shown in Fig. 1 aligned with those of other sPLA₂s.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 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 (15). 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 into group II based on the traditional grouping criteria proposed by Heinrikson (43). The sequence comparison clearly established the molecular identity of novel sPLA₂ distinguished from the PLA₂s thus far cloned (groups I-X (12, 14)). Therefore, we propose to assign the name of the novel sPLA₂ as group IID following so far identified IIA-IIC sPLA₂s. Langlais et al. (44) identified PLA₂ activity in human spermatozoa and determined its 19 N-terminal amino acids, which lack Cys¹¹. The novel sPLA₂ is distinct from the spermatic PLA₂ in its sequence.

View larger version (62K): [in this window] [in a new window]   Fig. 1.   Sequence alignment of mouse (A) and human (B) sPLA2. The putative signal peptide and the mature portion are separated by a space in each sequence. Completely preserved residues among sPLA2 family are indicated with capital letters in the consensus line. The characteristic cysteine by which sPLA2 are categorized is indicated in lowercase letters. Catalytic site His is indicated by an asterisk. Amino acid sequence identities (%) between novel sPLA2 and other sPLA2 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 sPLA2 is double-underlined.

Recombinant Expression of Novel sPLA₂s and Characterization of sPLA₂ Activity-- The deduced amino acid sequences from both mouse and human novel sPLA₂ cDNA contain all of the amino acids that are absolutely conserved in all functional sPLA₂s including His⁴⁸ and Asp⁴⁹. 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 sPLA₂ cDNAs were subcloned into the eukaryotic expression plasmid and then transfected into COS-7 cells. As shown in Fig. 2A, PLA₂ activity was detected in the supernatant of the culture medium of mouse and human sPLA₂ recombinant cells, whereas cells transfected with the parent vector did not show enzymatic activity. Only 2-4% of the total PLA₂ activity was detected in the cell-associated fraction, indicating that novel sPLA₂s 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, sPLA₂ activity was completely dependent on Ca²⁺ and required 2 mM Ca²⁺ for the maximal level. Recombinant sPLA₂ was optimally active over a broad range of pH 6-10 (Fig. 2C), whereas the activity of human group IIA sPLA₂ was optimal within pH 6-9 and declined at pH 10 (data not shown). The Ca²⁺ dependence and optimal pH profile were compatible with common features of sPLA₂s (15).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Recombinant expression of novel sPLA2 cDNA in COS-7 cells. A, sPLA2 activity measured in cell supernatants and cell lysates of COS-7 cells transiently transfected with the full-length novel human and mouse sPLA2 cDNAs. sPLA2 activity in the culture medium or cell lysates prepared at 72 h post transfection was measured by hydrolysis of [3H]oleate-labeled E. coli membranes as described under "Experimental Procedures." Results are expressed as the mean values ± S.E. of triplicate determinations. B, Ca2+ dependence of novel human sPLA2 activity. Enzymatic activity was determined in the presence of 2 mM EDTA (Ca2+-free) or of increasing concentrations of CaCl2. C, pH dependence of human sPLA2 activity. Specific sPLA2 activity was determined as described under "Experimental Procedures."

The substrate preference of novel human sPLA₂ 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 sPLA₂ 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 sPLA₂ (Table I). This is a general feature of the sPLA₂ family (3) and is quite a contrast to the arachidonoyl-specific cytosolic PLA₂. Among the phospholipids examined, L--1-palmitoyl-2-linoleoyl PE was most efficiently hydrolyzed. The PLA₁ activity was not detected when this substrate was used (data not shown). When compared within 1-palmitoyl-2-oleoyl phospholipids, novel sPLA₂ hydrolyzes PG and PE more efficiently than PC, whereas PS and PA are poorly hydrolyzed in our assay system. In contrast, group IIA sPLA₂ prefers PG to the other phospholipids as reported previously (45).

Tissue Expression of Novel sPLA₂ and Its Response to LPS Stimulation-- The tissue expression pattern of the novel sPLA₂ was analyzed by probing several human multiple tissue Northern blots with labeled cDNA. As shown in Fig. 3, human novel sPLA₂ 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 sPLA₂ contrasted with that of group IIA sPLA₂, which is predominantly expressed in the prostate, small intestine, colon, and heart.

View larger version (55K): [in this window] [in a new window]   Fig. 3.   Tissue distribution of novel sPLA2. Human multiple tissue Northern blots were hybridized with novel sPLA2, group IIA sPLA2, and -actin probe. Approximately 2 µg of poly(A)+ RNA was placed on each lane. The calculated size of the transcript detected is indicated at the right. PBL, peripheral blood leukocyte.

The expression of the novel sPLA₂ 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 sPLA₂ was detected in the spleen, thymus, and lung. At 24 h after LPS injection, the expression level of the sPLA₂ 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 sPLA₂, a marked enhancement was observed in the thymus and lung in contrast to a slight decrease in the spleen. The elevation of group IIA sPLA₂ mRNA after LPS treatment was also detected in the small intestine, heart, kidney, pancreas, and liver, where the signal of novel sPLA₂ was not detected (data not shown). In group IIA sPLA₂-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 sPLA₂ mRNA was elevated 3-fold in the thymus with no transcript of group IIA sPLA₂ (Fig. 4B).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Expression of novel sPLA2 and group IIA sPLA2 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 µg) 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.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 PLA₂ molecules, group X sPLA₂ and a novel paralog of the cytosolic PLA₂ 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 protein sequence coded by this cDNA fragment appears to be similar to but is distinct from the group IIA sPLA₂. Upon isolation of this EST, we cloned its cDNA and identified the novel sPLA₂ from mouse and human. The novel sPLA₂ possesses biochemical characteristics common to the known sPLA₂ proteins in terms of molecular size, conservation of consensus sequence and distribution of disulfide-forming cysteine residue, the requirement of Ca²⁺ as well as optimal pH range for catalysis, and the extracellular localization of the lipase activity. Thus, the newly cloned sPLA₂ is the sixth isoform of the sPLA₂ family found in rodents and the fifth in humans as a functional enzyme, because group IIC PLA₂ is thought to be a pseudogene in humans (15). Because the novel sPLA₂ is most similar to the group IIA sPLA₂ with respect to the number and positions of cysteine residues as well as overall identity, we propose to call the novel sPLA₂ as group IID following so far identified IIA to IIC sPLA₂s.

The group IIA sPLA₂ 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 sPLA₂ has been cloned from the C57BL/6J strain in which the group IIA PLA₂ gene was naturally inactivated. Because these deficient mice are similar to group IIA sPLA₂-expressing mouse strains in their inflammatory responses (25, 26), the novel sPLA₂ might play a compensatory role for several functions of the group IIA sPLA₂. In the three mammals used in this study, the expression of novel sPLA₂ was detected commonly in the spleen and thymus, which is a an expression profile similar to that of group X sPLA₂ in humans (12). Although the origin of cells producing this sPLA₂ 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 sPLA₂ was elevated in the rat thymus along with the group IIA sPLA₂ transcript (Fig. 4). Enhanced expression of the novel sPLA₂ in the thymus was also observed in mice deficient for group IIA sPLA₂. During the progression of sepsis, thymic atrophy is induced via apoptosis (46). Because several reports suggest an involvement of PLA₂ in thymocyte apoptosis (47, 48), the novel sPLA₂ might play a role in this process. The expression of novel sPLA₂ mRNA was also changed in rat lung after LPS treatment, but its pattern was different from the case of group IIA sPLA₂. In humans, the distribution of the transcript of novel sPLA₂ contrasted to that of group IIA sPLA₂ (Fig. 3), suggesting distinct biological functions for these two related sPLA₂s in the physiological and pathological states. In addition to diverse tissue expression profiles, we observed a drastic difference between group IIA and novel sPLA₂s in susceptibility to one of the 1-oxamoylindolidine derivatives (49). This sPLA₂ inhibitor has a strong inhibitory potency for group IIA sPLA₂ (IC₅₀ = 1.2 nM), whereas more than 50% of the full activity of novel sPLA₂ remains even at 1000 nM.³ Moreover, an antibody that neutralized the human group IIA sPLA₂ activity did not absorb the novel sPLA₂ activity (data not shown). These findings reflected the structural differences, especially around the active center, between two related sPLA₂s, which could be relevant to their distinct functions.

In the C-terminal region of novel sPLA₂, there is a relatively high content of basic amino acid residues, which is characteristic of heparin-binding PLA₂s including group IIA and V sPLA₂ (50). In fact, the novel human sPLA₂ 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 sPLA₂ and its potential contribution to the arachidonic acid release in the transfection/overexpression experiment. With this criterion, novel sPLA₂ could act on the cell surface proteoglycan, which leads to the arachidonic acid release at least under specific conditions. A heparin-sensitive PLA₂ activity responsible for the delayed phase prostaglandin D₂ synthesis was detected from mouse (C57BL/6J) bone marrow-derived mast cells (52). Because this mouse strain is deprived of group IIA sPLA₂, the novel sPLA₂ is a conceivable candidate for the display of such activity. The possible contribution of novel sPLA₂ to the production of lipid mediators during the cell activation process deserves attention in future studies.

sPLA₂ 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. PLA₂ activity toward bactericidal permeability increasing protein-treated E. coli varies greatly among the sPLA₂ family and apparently depends on a cluster of basic residues near the N terminus of the PLA₂ protein (53). In particular, the indispensability of Arg⁷ and Lys¹⁵ in human group IIA sPLA₂ was demonstrated by a site-directed mutagenesis experiment (54). The conservation of basic residues at these sites in novel sPLA₂ (Lys⁷ and Lys¹⁵) 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 sPLA₂ transcript of relatively short size in the human pancreas, which displays abundant expression of group IB sPLA₂. Cross-hybridization of the novel sPLA₂ probe with group IB sPLA₂ mRNA is not likely, because the calculated identity between these two cDNAs is only 53%. One possible function of the novel sPLA₂ is the digestion of phospholipid in nutrition. Another possibility is the involvement of the PLA₂-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 PLA₂ receptor in humans (55). In this context, the receptor binding activity of the novel sPLA₂ should be evaluated in the future.

Radiation hybrid mapping analysis revealed the location of a human novel sPLA₂ gene on chromosome 1 in the vicinity of WI-5273. Around this region, group IIA, IIC, and V sPLA₂ genes have already been mapped (1p34-p36) (56), whereas group IB and X sPLA₂ genes are located on chromosome 12 and 16, respectively (12, 57). These findings indicate that three sPLA₂ 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 sPLA₂ 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 sPLA₂ and the group IIA sPLA₂ in the thymus in the response to LPS challenge (Fig. 4A). In the promoter region of human and rat group IIA sPLA₂ 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 novel sPLA₂ gene should provide a clue to the functional significance of this isoform.

In conclusion, we isolated novel mouse and human sPLA₂s (group IID) and characterized the activities and expression. This sPLA₂ shares common structural and catalytic features with previously known sPLA₂ isoforms and is especially related to group IIA sPLA₂. Further studies are required to establish the precise physiological functions of this new sPLA₂ and to determine its role in disease states, especially in inflammatory conditions. Finally, the discovery of this novel sPLA₂ should enable more precise assignment of distinct functions of each isoform and should also broaden our understanding of the biochemical properties of the sPLA₂ family.

	ACKNOWLEDGEMENTS

We thank Dr. Ruth Kramer for a generous gift of recombinant human group IIA sPLA₂. We are grateful to Kazumi Nakano and Hitoshi Nakazato for excellent technical assistance and to Dr. Kiyoshi Nagata for continuous support throughout the work.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The 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/EMBL 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.

² Mouse 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).

³ T. Ono, Y. Yokota, and K. Hanasaki, unpublished data.

⁴ J. Ishizaki, O. Ohara, T. Nakano, H. Arita, and H. Teraoka, unpublished data.

	ABBREVIATIONS

The abbreviations used are: PLA₂, phospholipase A₂; sPLA₂, secretory PLA₂; 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).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES