Cloning and Expression of Group IB Phospholipase A2 Isoforms in the Red Sea Bream, Pagrus major1 N. Iijima*, Y. Fujikawa, Y. Tateishi, Y. Takashima, S. Uchiyama, and M. Esaka Faculty of Applied Biological Science, Hiroshima University, Higashihiroshima 739-8528, Japan
ABSTRACT: Two cDNA encoding red sea bream DE-1 and DE-2 phospholipases A2 (PLA2) were cloned from the hepatopancreas of red sea bream, Pagrus (Chrysophrys) major. The cDNA of DE-1 PLA2 encoded a mature protein of 125 amino acid residues with an apparent signal peptide of 20 residues and propeptide of 5 residues, and that of DE-2 PLA2, a mature protein of 126 amino acid residues with an apparent signal peptide of 17 residues and propeptide of 6 residues. Comparison of the predicted amino acid sequences for mature DE-1 and DE-2 PLA2 showed that both proteins contain 14 cysteines including Cys 11 and 77 and a pancreatic loop, which are commonly conserved in group IB PLA2; however, the identity in amino acid sequence between DE-1 and DE-2 PLA2 was low (47%). A previous report concerning the cDNA cloning of red sea bream gill G-3 PLA2 and the present results represent the first cloning and sequencing of three distinct isoforms of group IB PLA2 in a single fish species, red sea bream. Reverse transcription-polymerase chain reaction analysis showed that DE-1 PLA2 mRNA was expressed in the hepatopancreas, pyloric ceca, intestine, spleen, gonad, stomach, and kidney, whereas gill G-3 PLA2 mRNA was expressed only in the gills and gonad. The expression of DE-2 PLA2 mRNA was detected in all of the tissues analyzed. These results indicate that three distinct isoforms of group IB PLA2, DE-1 and DE-2 PLA2 in hepatopanceas and gill G-3 PLA2, are expressed in a tissue-specific manner in red sea bream. Paper no. L8702 in Lipids 36, 499–506 (May 2001).
Phospholipase A2 (phosphatide 2-acyl hydrolase, EC 3.1.1.4) (PLA2) comprises a diverse family of enzymes that catalyze the hydrolysis of a fatty-acyl ester bond at the sn-2 position of glycerophospholipids to liberate free fatty acids and lysophospholipids. PLA2 plays a central role in cellular processes as diverse as phospholipid digestion and metabolism, host defense, and signal transduction (1–3). Secretory PLA2 (sPLA2) are Ca2+-dependent, low molecular mass enzymes (13–18 kDa) with five to eight disulfide bridges and a broad specificity for the phospholipid headgroup and fatty acids (3). At present, they are classified into six groups, I, II, III, V, IX and X, de1 The sequences of DE-1 and DE-2 PLA2 and β-actin presented in this article have been submitted to DDBJ/EMBL/GenBank with accession numbers AB050632, AB009286, and AB050670, respectively. *To whom correspondence should be addressed at Faculty of Applied Biological Science, Hiroshima University, 1-4-4 Kagamiyama, Higashihiroshima 739-8528, Japan. E-mail:
[email protected] Abbreviations: bp, base pair(s); dNTP, deoxynucleoside triphosphate; DTT, dithiothreitol; kb, kilobase(s); KOD Dash, Pyrococcus kodakaraensis KOD1; MMLV, Moloney murine leukemia virus; nt, nucleotides; PCR, polymerase chain reaction; PLA2, phospholipase(s) A2; RACE, rapid amplification of cDNA ends; RAV, Rous-associated virus; RT, reverse transcription.
Copyright © 2001 by AOCS Press
pending on the primary structure characterized by the number and positions of cysteine residues (2). Group IB PLA2 has been referred to as pancreatic-type PLA2 because it is abundant in the pancreatic juice in many mammals (4,5). However, group IB PLA2 mRNA and protein have been recently found in nondigestive organs, including the spleen, lung, kidney, and ovary (6–16). The discovery of specific receptors for group IB PLA2 from various mammalian tissues and cells has led to the notion that group IB PLA2 evokes various biological responses by binding to the receptor in addition to its digestive function (17–19). This implies a physiological role for group IB PLA2 in nondigestive tissues and cells, in addition to the function of digestive lipolysis in the digestive system. Compared with the amount of information available on mammalian group IB PLA2, little is known about the primary structure and enzymology of fish PLA2. PLA2 has been partially purified or purified from rainbow trout (Salmo gairdneri) liver (20,21) and cod (Gadus morhua) muscle (22,23), but its primary structure has not yet been determined. Zambonino Infante and Cahu (24) recently obtained a cDNA clone encoding group IB PLA2 from seabass (Dicentrarchus labrax) and found that the mRNA level of PLA2 in seabass larvae increased in culture with diets containing higher lipid levels. Previously, we detected PLA2 in the pancreatic acinar cells and secretory materials of certain epithelial cells in the pyloric ceca of red sea bream, by immunohistochemical analysis using an antiserum against Naja naja venom PLA2 (25). We have further purified six low molecular weight Ca2+-dependent PLA2 from the pyloric ceca (26), hepatopancreas (27,28) and gills (29), and classified hepatopancreas DE-1 and DE-2 PLA2 and three gill G-1, G-2 and G-3 PLA2 as group I PLA2, based on the analysis of N-terminal amino acid sequence and enzyme properties. In addition, we have very recently cloned a cDNA encoding red sea bream gill G-3 PLA2 and classified gill G-3 PLA2 as a group IB PLA2, based on the amino acid sequence deduced from nucleotide sequence of the cDNA (29). On the other hand, the primary structure of red sea bream hepatopancreas DE-1 and DE2 PLA2 remains to be established. In the present paper, we describe the cloning and sequencing of the cDNA for red sea bream hepatopancreas DE-1 and DE-2 PLA2 as well as the three distinct isoforms that exist in a single fish species, red sea bream. We further investigated the distribution of mRNA for hepatopancreas DE-1, DE-2 PLA2, and gill G-3 PLA2 in various tissues by reverse transcription-polymerase chain reaction (RT-PCR) to better understand the structure–function relationship of the three isoforms.
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MATERIALS AND METHODS Extraction of poly (A)+ RNA. The hepatopancreas and other tissues were removed immediately from freshly killed red sea bream and were stored in liquid nitrogen. Total RNA was extracted from the hepatopancreas using Isogen (Nippon Gene, Tokyo, Japan), and poly (A)+ RNA was isolated using Oligotex-dT30 Super (Roche Japan, Tokyo, Japan), according to the manufacturer’s protocol. cDNA amplification of DE-2 PLA2. Primers P-1 (5′GC(A/G/C/T)TT(A/G/C/T)AATCAGTTTTG(A/G/C/T)CAGATGAT-3′) and P-2 (5′-CGGTCGCAGTTGCAGATGAA3′) were derived from possible cDNA sequences corresponding to a part of the amino acid sequence of red sea bream hepatopancreas DE-2 PLA2 (28), ALNQFRQM (1st–8th in Fig. 1), and that of the conserved amino acid sequences of mammalian pancreatic PLA2 (30), FICNCD (96th–101th in Fig. 3), respectively. Hepatopancreas total RNA (1 µg) was reverse-transcribed using P-2 primer, 1 mM deoxynucleoside triphosphate (dNTP) and 0.25 units of Rous-associated virus (RAV)-2 reverse transcriptase (Takara, Tokyo, Japan) in a reaction buffer [50 mM Tris-HCl, 75 mM KCl, 8 mM MgCl2, 10 mM dithiothreitol (DTT), pH 8.3] at 50°C for 15 min, and then denatured at 99°C for 5 min. After the addition of P-1 primer and 2.5 units of Taq DNA polymerase (Takara) to the reaction buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, pH 8.3), PCR was carried out for 28 cycles of 30 s denaturation at 94°C, 30 s annealing at 60°C, and 90 s extension at 72°C. Then the PCR product was purified with 2% agarose gel and sequenced. From the nucleotide sequence of the internal cDNA, the new primers P-3 [5′-CTAGGATCCGCAATAGCAGCCATAGTCAGC-3′, complementary to nucleotides (nt) 136–159 in Fig. 1], P-4 (5′-CATCTGTCCAGATCATCCACGGGTGTG-3′, complementary to nt 174–200 in Fig. 1), and P-5 (5′-CTGTCTAGATGCTGCCAAGTGCACGA-3′, identical to nt 196–215 in Fig. 1) were designed for 5′- and 3′-end amplifications. The 5′RACE (rapid amplification of cDNA ends) method was performed using 5′-AmpliFINDER™ RACE Kit (Clontech, Palo Alto, CA). For 5′-end amplification of PLA2 cDNA, a singlestranded cDNA was synthesized with 2 µg of red sea bream hepatopancreas poly (A)+ RNA, 25 units of avian myeloblastosis virus (AMV) reverse transcriptase, and primer P-4. The resulting 5′-end PLA2 cDNA was ligated with an AmpliFINDER anchor using T4 RNA ligase and was then amplified with 10 mM dNTP, P-3 primer, AmpliFINDER anchor primer, and 2.5 units of AmpliTaq DNA polymerase in a reaction buffer (10 mM Tris-HCl, 50 mM KCl, 2 mM MgCl2, 0.1% (wt/vol) gelatin, pH 8.3). For PCR, 28 cycles of 45 s denaturation at 94°C, 45 s annealing at 60°C, and 2 min extension at 72°C were carried out. Then the PCR product was separated on 1% agarose gel, and the DNA of expected size was isolated and sequenced. For 3′-end amplification of PLA2 cDNA, red sea bream hepatopancreas total RNA (1 µg) was used to prepare first-stranded cDNA with an RNA PCR kit (Takara), employing 10 mM dNTP, Oligo (dT)20 adaptor Lipids, Vol. 36, no. 5 (2001)
primer (Takara), and 20 units of Moloney murine leukemia virus (MMLV)reverse transcriptase. The resulting dT primed single-stranded cDNA was amplified with 2.5 units of Taq polymerase (Takara), Oligo (dT)20 adaptor primer, and P-1 primer as described above. After the first PCR, the products were used as a template for the second PCR. The primers used in the second PCR were P-5 primer, Oligo (dT)20 adaptor primer, and 0.5 units of Ex Taq polymerase (Takara), and the PCR conditions were the same as those used in the first PCR. Then the PCR product was separated and sequenced. cDNA amplification of DE-1 PLA2. Primers P11, 5′-TGGCA(A/G)TT(C/T)GGIAA(C/T)ATGATICA-3′, and P2a, 5′GC(A/G)GCCTT(C/T)CTGTCGCACTC(A/G)CA-3′,were derived from possible cDNA sequences corresponding to a part of the amino acid sequence of red sea bream hepatopancreas DE-1 PLA2 (28), WQFGNMIQ (3rd–10th), and that of conserved amino acid sequences of mammalian pancreatic PLA2 (30) and red sea bream DE-2 PLA2, CECDRKAA (98th–105th in Fig. 1), respectively. The cDNA amplification of DE-1 PLA2 by PCR was performed essentially as described previously (29). One microgram of red sea bream hepatopancreas poly (A)+ RNA was used to prepare firststranded cDNA with an RNA PCR kit (Takara) employing 20 units of MMLV reverse transcriptase. An internal cDNA fragment encoding PLA2 was generated by PCR from firststranded cDNA of red sea bream hepatopancreas using primers P11 and P2a, 0.5 mM dNTP, and 1.25 units of Pyrococcus kodakaraensis KOD1 (KOD Dash) (Toyobo, Tokyo, Japan) in a reaction buffer (20 mM Tris-HCl, 7.5 mM DTT,1.8 mM MgCl2, pH 8.3). After an initial denaturation for 160 s at 94°C, 30 cycles of amplification were carried out with 30 s denaturation at 94°C, 10 s annealing at 58°C, and 30 s extension at 74°C. Then the PCR product was separated on 2% agarose gel, and the DNA of an expected size was isolated and sequenced. From the determined nucleotide sequence of the internal cDNA, new primers AP11 (5′GGAAGGTCAGCGACAGCCGTGCATCCAGG-3′, complementary to nt 247–275 in Fig. 2) and P15 (5′-GACGTGGATGCGTGCTGTAAGG-3′, identical to nt 190–211 in Fig. 2) were designed for 5′- and 3′-end amplifications, respectively. First-stranded and second-stranded cDNA were synthesized with 1 µg of red sea bream hepatopancreas poly (A)+ RNA and Marathon cDNA Amplification kit (Clontech) according to the manufacturer’s instruction. The resulting second-stranded cDNA was precipitated and ligated to a Marathon cDNA adaptor using T4 DNA Ligase for 40 min at room temperature. The 5′-end amplification of PLA2 cDNA was carried by PCR with adaptor-ligated double-stranded cDNA, adaptor primer (5′-CCA TCCTAA TAC GAC TCA CTA TAG GGC-3′), AP11 primer, and 1.25 units of KOD Dash (Toyobo) in the above reaction buffer. PCR conditions were: an initial denaturation for 160 s at 94°C, followed by 30 cycles of amplification, with 30 s denaturation at 94°C, 10 s annealing at 58°C, and 30 s extension at 74°C. The resulting PCR products were subcloned into pGEM-T vector (Promega, Madison, WI) and transformed into JM109 cells,
GROUP IB PHOSPHOLIPASE A2 ISOFORMS IN RED SEA BREAM
and positive clones were selected on LB/ampicillin/IPTG/ X-Gal plates according to the manufacturer’s protocol. Plasmid DNA was purified from positive clones with QIAprep Spin Miniprep Kit (Qiagen, Tokyo, Japan) and sequenced. For 3′-end amplification of PLA2 cDNA, PCR was carried out with adaptor-ligated double-stranded cDNA, adaptor primer, P15 primer, and 1.25 units of KOD Dash, and the resulting PCR products were subcloned into pGEM-T vector as described above and sequenced. Sequencing of PCR products. The sequences of DNA fragments were determined with an Applied Biosystems 373A DNA sequencer using the Dye terminator cycle sequencing kit (PerkinElmer, Norwalk, CT), according to the manufacturer’s protocol. RT-PCR. Total RNA was isolated from the hepatopancreas, pyloric ceca, intestine, spleen, gill, gonad, heart, brain, stomach, kidney, and muscle of three fishes (200 g in body weight) using Isogen. Three micrograms of total RNA was used as the template to synthesize the first-stranded cDNA using oligo (dT)20-M4 adaptor primer (5′-GTTTTCCCAGTCACGACTTTTTTTTTTTTTTTTTTTT-3′) and MMLV reverse transcriptase, RNase H Minus (Point mutant) (Promega). The cDNA fragments containing coding and noncoding regions of DE-1, DE-2, and gill G-3 PLA2 cDNA were amplified by PCR from the first-stranded cDNA. The primers used were: DE-1 PLA2, 5′-GCCTTATGGCAGTTTGGGAACA-3′ (identical to nt 76–97 in Fig. 2) and 5′-CTCTAACTTCAACAAATCAGG-3′ (complementary to nt 540–560 in Fig. 2); DE-2 PLA2, 5′-GCACTCAACCAGTTCAGACAG-3′ (identical to nt 70–92 in Fig. 1) and 5′-TAGTAGGGAATGATGGATGGC-3′ (complementary to nt 572–592 in Fig. 1); gill G-3 PLA2 (29), 5′-GCTATATGGCAGTTTGGGGACA-3′ (identical to nt 73–94) and 5′-ATGCTGAACTGATTGGACACA-3′ (complementary to nt 1009–1029). A pair of primers (5′-CGGGATCCACTACCTCATGAAGATCCTG-3′ and 5′-CCGCTCGAGTTGCTGATCCACATCTGCTG-3′) specific for red sea bream heart β-actin gene (DDBJ, accession number AB050670) was used for amplifying a 478 base pair (bp) fragment of red sea bream β-actin as an internal control. PCR conditions were: an initial denaturation for 2 min at 94°C, followed by 35 cycles of amplification, with 30 s denaturation at 94°C, 5 s annealing at 56°C for DE-1 and DE-2 PLA2 and at 60°C for gill G-3 PLA2, and 10 s extension at 74°C. The reaction products were electrophoresed on a 1% agarose gel.
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DE-2 PLA2 cDNA included a 447 bp open reading frame that encoded a prepropeptide of 23 amino acids, followed by a mature protein of 126 amino acids (Fig. 1). The calculated molecular mass and isoelectric point of the mature protein were 14,422 Da and 4.16, respectively. The sequence AATAAA, which is the conserved sequence of a polyadenylation signal, was found 23 bp upstream of the poly A tail. The nucleotide sequence of the DE-1 PLA2 cDNA included a 450 bp open reading frame that encoded a putative prepropeptide and mature protein of 25 and 125 amino acids, respectively (Fig. 2). The calculated molecular mass and isoelectric point of the mature protein were 13,634 Da and 5.63, respectively. The 3′-noncoding region contained two putative polyadenylation signals located 17 and 34 bp upstream of the poly A tail. The nucleotide sequences of DE-1 and DE-2 PLA2 encoded no putative sites of N-glycosylation (Asn-XSer/Thr, where X is any amino acid) (Figs. 1 and 2). The alignment of amino acid sequences for mature protein
RESULTS Isolation and characterization of cDNA clones for DE-1 and DE-2 PLA2. Total RNA and poly (A)+ RNA were prepared from the hepatopancreas of red sea bream. Primers for PCR were designed according to the amino-terminal amino acid sequence of purified hepatopancreas DE-2 PLA2 and the highly conserved amino acid sequences among mammalian pancreatic PLA2. A full-length cDNA clone was isolated by RT-PCR and RACE methods. The nucleotide sequence of the
FIG. 1. Nucleotide and deduced amino acid sequences of red sea bream hepatopancreas DE-2 phospholipase A2 (DE-2 PLA2). The predicted preprosegment is boxed and a possible initiator methionine is shown in bold. The putative polyadenylation signal is underlined and shown in bold. An asterisk shows the termination codon.
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FIG. 2. Nucleotide and deduced amino acid sequences of red sea bream hepatopancreas DE-1 PLA2 (DE-1 PLA2). The predicted preprosegment is boxed and a possible initiator methionine is shown in bold. The putative polyadenylation signals are underlined and shown in bold. An asterisk shows the termination codon.
and prepropeptide of red sea bream hepatopancreas DE-1 and DE-2 PLA2 with the sequences of red sea bream gill G-3 PLA2, seabass (D. labrax) PLA2, human and porcine group IB PLA2, and N. naja atra group IA PLA2 are presented in Figures 3 and 4, respectively (6,24,29,31,32). In amino acid sequence, DE-1 and DE-2 PLA2 have characteristics in common with mammalian pancreatic type, group IB PLA2, including the presence of Cys11 and 77 and the alignment of other Cys residues; residues of N-terminal helix Gln4, Phe5, and Ile9, and the presence of the absolutely conserved activesite His48, Tyr52, Tyr73, and Asp101; the “pancreatic loop” of residues 63–67 that are conserved in group IB PLA2; and the conserved sequence of the calcium-binding segment Tyr25-Gly35, except for the substitution of Trp for Tyr 28 in DE-1 PLA2 (Fig. 3). Insertion of lysine doublet at positions 82 and 83 was found in DE-2 and seabass PLA2. The degree of identity for amino acid sequence between red sea bream hepatopancreas DE-1 and DE-2 PLA2 was low (47%) (Table 1). DE-2 PLA2 shows high identity to gill G-3 PLA2 Lipids, Vol. 36, no. 5 (2001)
(65%), and DE-1 PLA2 shares higher identity to seabass PLA2 (87%). SignalP computer analysis (33) for the potential cleavage positions in the signal sequence suggested that DE-1 and DE-2 PLA2 contain five and six residues of propeptide preceding the mature enzyme, respectively (Fig. 4). In addition, signal peptide sequences of DE-1 and DE-2 PLA2 were homologous to those of gill G-3 and seabass PLA2, respectively. A phylogenetic tree was derived from an alignment of known protein sequences between fish PLA2 and secretory PLA2, using the CLUSTAL W program (34) and Tree view (35). The sequences of mouse group IIA, IIC, IID, IIE, IIF and V PLA2, and rat group X PLA2 were obtained from the DNA Data Bank of Japan. The others are the following: porcine (31), cow (36), dog and rat (37), human (38), guinea pig (39), horse (40), and rabbit (41) pancreatic group IB PLA2, Oxyuranus scuttus scutatus OS 1 group IB″ PLA2 and OS 2 group IA PLA2 (42), Notechis scutatus scutatus II-1 and II-5 (43,44), Pseudochis australis (45), Pseudonaja texistilis (46), Haemachatus hemachatus (47), N. melanoleuca DE II and DE III (48,49), N. naja naja (50), N. naja atra (32) and N. naja kaouthia (51) group IA PLA2, and Bitis gabonica group IIB PLA2 (52). Subdivision of snake venom and mammalian group I and group II PLA2 shown in Figure 5 are based on the proposals of Danse et al. (42) and Valentin et al. (15). This tree shows that fish PLA2 were placed in the branch of mammalian group IB PLA2 but were separated into two subgroups, red sea bream DE-1 and gill G-3 PLA2, and DE-2 and seabass PLA2, respectively. DE-2 and seabass PLA2 are more distantly related to mammalian group IB PLA2 than DE-1 and gill G-3 PLA2. RT-PCR. The mRNA expressions of hepatopancreas DE-1 and DE-2 PLA2 and gill G-3 PLA2 were compared among various tissues of red sea bream by RT-PCR. As shown in Figure 6, amplification of β-actin mRNA produced a band of 478 bp, providing a positive control. The expected cDNA of DE-1 PLA2 (483 bp) was amplified in the hepatopancreas, pyloric ceca, intestine, gonad, spleen, stomach, and kidney. The DE-2 PLA2 cDNA (522 bp) was detected in the hepatopancreas, pyloric ceca, intestine, spleen, gill, gonad, heart, brain, stomach, kidney, and muscle, suggesting that this PLA2 isoform is ubiquitously expressed. On the other hand, gill G-3 PLA2 (957 bp) was expressed only in the gills and gonad.
TABLE 1 Identity (%) of Amino Acid Sequence in Mature Protein Among Fish, Mammalian, and Snake Venom Group I Phospholipase A2 (PLA2)a Group I PLA2
DE-2
G-3
Seabass
pGIB
nGIA
DE-1 DE-2 G-3 Seabass pGIB
46.7
64.5 54.5
47.5 87.3 54.5
48.4 54.0 53.2 57.3
48.4 48.8 48.7 47.2 52.8
a
DE-1, red sea bream hepatopancreas DE-1 PLA2; DE-2, red sea bream hepatopancreas DE-2 PLA2; G-3, red sea brim gill G-3 PLA2; seabass, seabass group 1B PLA2; pGIB, porcine pancreatic group IB PLA2; nGIA, Naja naja naja venom group 1A PLA2.
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FIG. 3. Alignment of the amino acid sequences of red sea beam hepatopancreas DE-1 and DE-2 PLA2, gill G-3 PLA2 and seabass PLA2 with mammalian and snake venom group I PLA2. Sequences of mature PLA2 proteins are shown: DE-1, red sea bream hepatopancreas DE-1 PLA2; DE-2, red sea bream hepatopancreas DE-2 PLA2; G-3, red sea bream gill G-3 PLA2; seabass, seabass group IB PLA2; hGIB, human pancreatic group IB PLA2; pGIB, porcine pancreatic group IB PLA2; nGIA, Naja naja atra venom group IA PLA2. Asterisks indicate the amino acid residues identical to those of red sea bream hepatopancreas DE-1 PLA2. Cysteines that are conserved among all PLA2 are indicated in boxes.
Thus, the three distinct isoforms of red sea bream group IB PLA2, DE-1, DE-2 and G-3 PLA2, were expressed in a tissuespecific manner. DISCUSSION Group I PLA2 include mammalian pancreatic PLA2 and those forms from elapid and hydrophid snake venoms. This group possesses 14 cysteines including a disulfide bridge formed by
Cys 11 and 77 and can be further subdivided to group IA and group IB PLA2, based on the presence or absence of the pancreatic loop (30,42). In a previous report, we showed that red sea bream hepatopancreas contains two enzymatically distinct group I PLA2 isoforms, DE-1 and DE-2 PLA2 (28). In the present study, we reported the complete amino acid sequences of red sea bream hepatopancreas DE-1 and DE-2 PLA2, as deduced from their cDNA sequences. From the comparison of amino acid sequences of mature mammalian and snake Lipids, Vol. 36, no. 5 (2001)
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FIG. 4. Alignment of amino acid sequences in signal peptides and propeptides of red sea beam hepatopancreas DE-1 and DE-2 PLA2, gill G-3 PLA2, and seabass PLA2 with mammalian and snake venom group I PLA2. The potential cleavage site of the signal peptidase is indicated with a question mark. For abbreviations see Figure 3.
venom group I PLA2, DE-1 and DE-2 PLA2 were found to contain 14 cysteines including Cys 11 and Cys 77 and a pancreatic loop of residues 63–67, which are commonly conserved in group IB PLA2 (Fig. 3). In addition, both DE-1 and DE-2 PLA2 are predicted to have five and six propeptides preceding the mature protein, respectively (Fig. 4). These results indicate that red sea bream hepatopancreas DE-1 and DE-2 PLA2 belong to the mammalian pancreatic type, group IB PLA2, similar to red sea bream gill G-3 PLA2 (1). This work and a previous report (1) represent the first cloning and sequencing of three distinct isoforms of group IB PLA2 in a single fish species. From the results of phylogenetic tree, fish PLA2 were classified into the branch containing mammalian group IB PLA2; however, they were further divided into two subgroups, DE-1
FIG. 5. Phylogenetic tree of fish group IB PLA2, and mammalian and snake venom secretory PLA2. PLA2 belonging to the same group are denoted as IB, IB″, IA, and IIA-X, as described in the text.
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and gill G-3 PLA2, and DE-2 and seabass PLA2, respectively. In addition, signal peptide sequences of DE-1 PLA2 and DE-2 PLA2 are also homologous to those of gill G-3 PLA2 and seabass PLA2, respectively. As DE-2 and seabass PLA2 are more distantly related to mammalian group IB PLA2 than DE-1 and gill G-3 PLA2, a duplication event would have occurred to generate DE-1 and gill G-3 PLA2 and another subgroup, DE-2 and seabass PLA2, similar to mammalian group IB PLA2. From the result of RT-PCR analysis, both DE-1 and DE-2 PLA2 were expressed in digestive tissues such as the hepatopancreas, pyloric ceca, and intestine. In mammals, pancreatic type group IB PLA2 were synthesized as the preproPLA2 and were processed with signal peptidase in the rough
FIG. 6. Reverse transcription-polymerase chain reaction (RT-PCR) analysis for the tissue distribution of hepatopancreas DE-1 and DE-2 PLA2 and gill G-3 PLA2. Total RNA from various tissues of red sea bream were used as templates for RT-PCR. DE-1, DE-2 and G-3 PLA2 were amplified with a single round of PCR as described in the Materials and Methods section. β-Actin was used as an internal standard. The calculated size of the transcript detected is indicated at the left. 1, Hepatopancreas; 2, pyloric ceca; 3, intestine; 4, spleen; 5, gill; 6, gonad; 7, heart; 8, brain; 9, stomach; 10, kidney; 11, muscle.
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endoplasmic reticulum of pancreatic acinar cells (5). They were then stored as an inactive proPLA2 in the zymogen granules. After being secreted in the intestinal lumen, proPLA2 was activated by limited tryptic proteolysis and digest dietary phospholipids. As described above, red sea bream hepatopancreas DE-1 and DE-2 PLA2 have prepro sequences; both preproPLA2 may be processed and secreted in the digestive tract from the hepatopancreas, pyloric ceca, intestine, and stomach, similar to mammalian pancreatic group IB PLA2. Red sea bream hepatopancreas DE-1 and DE-2 PLA2 were expressed also in the nondigestive tissues; DE-1 PLA2 was expressed in the spleen and gonad, and DE-2 PLA2 in spleen, gill, gonad, heart, brain, kidney, and muscle. These results may indicate that DE-1 and DE-2 PLA2 have different physiological functions in the nondigestive tissues and cells of red sea bream, except for the digestion of dietary phospholipids. Mammalian group IB PLA2 are expressed in spleen, lung, and gonad of human (14), rat (8), and mouse (15,16). At present, two main types of PLA2 receptor, M-type and N-type, have been identified in mammals. Group IB PLA2 elicits a variety of biological responses, including cell proliferation, cell migration, hormone release, and eicosanoid production by interacting with the M-type receptor in various tissues and cells (17,53). In addition, neurotoxic snake venom PLA2 and bee venom PLA2 are said to bind to the N-type receptor, which is abundantly distributed in the brain (19). From the above results, it is reasonable to consider that the PLA2 receptor, reacted specifically to group IB PLA2, exists also in red sea bream. However, it still remains unclear whether DE-1 and DE-2 PLA2 express as a funtional enzyme in the above tissues. Although PLA2 activity was detected in the spleen, gonad, heart, kidney, and muscle (29), we do not yet analyze the distribution of both enzymes in these tissues. In order to demonstrate the above question, it is necessary to investigate the distribution of DE-1 and DE-2 PLA2 by immunoblotting and immunohistochemistry. We are now trying to make recombinant DE-1 and DE-2 PLA2, for preparing monoclonal antibodies and for the analysis of PLA2 receptor. Gill G-3 PLA2 was expressed only in the nondigestive tissues, gills, and gonad, as shown by RT-PCR analysis (Fig. 6). However, we had found previously that PLA2 activity in the gills was extremely high compared with that in the gonad (29). In addition, gill PLA2 was detected only in the gills, especially the mucous cells and pavement cells located on the surface of gill epithelia, by immunoblotting and immunohistochemistry using monoclonal antibody raised against gill PLA2 (Uchiyama, S., Fujikawa, Y., Uematsu, K., Matsuda, H., Aida, S., and Iijima, N., unpublished data). These aspects indicate that gill PLA2 is mainly expressed in the gills and the expression of gill PLA2 is extremely low in the gonad. REFERENCES 1. Tischfield, J.A. (1997) A Reassessment of the Low Molecular Weight Phospholipase A2 Gene Family in Mammals, J. Biol. Chem. 272, 17247–17250. 2. Balsinde, J., Balboa, M.A., Insel, P.A., and Dennis, E.A. (1999)
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