Cloning and expression of flatfish (Paralichthys olivaceus) interferon cDNA

182

Biochimica et Biophysica Acta, 1174 (1993) 182-186 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00

BBAEXP 90532

Short Sequence-Paper

Cloning and expression of flatfish ( Paralichthys olivaceus) interferon cDNA Tadakazu Tamai a, Sanetaka Shirahata b, Tohru Noguchi b, Nobuyuki Sato a, Shouji Kimura a and Hiroki Murakami b a Taiyo Central R&D Institute, Taiyo Fishery Co. Ltd., Ibaraki (Japan) and b Graduate School of Genetic Resources Technology, Kyushu University, Fukuoka (Japan) (Received 26 February 1993)

Key words: Interferon; cDNA; Expression; Antiviral protein; (Flatfish)

Fish interferon (IFN) cDNA was first cloned from the cDNA library of immortalized flatfish leukocytes. The clone contains an open reading frame that encodes a 138 amino acid polypeptide including a glycosylation site and a signal peptide containing 30 amino acids. BHK-21 cells transfected with the INF-expression plasmid produced active recombinant IFN (about 16 kDa) which was then purified by WGA agarose affinity chromatography. This recombinant IFN inhibited infection of fish cells with the Hirame (flatfish) rhabdovirus.

Over the last two decades, aquaculture has grown into a very important industry in many parts of the world. However, in farms which raise their fishes in high density tanks, the risk of infection from feral diseases caused by bacteria or viruses is great. So far, no effective therapy for the control of fish viral diseases has been found [1]. Interferon (IFN) is a general name of proteins or glycoproteins of approx. 20 kDa which show antiviral properties via specific receptors on the cell membrane [2]. IFNs are expected to be a source of potential drugs for the prevention and treatment of various kinds of viral fish diseases. There has been no previous report on fish IFN gene cloning. We have purified a flatfish IFN protein from the cultured medium of immortalized flatfish leukocytes [3]. The protein is a glycoprotein of about 16 kDa in size. Here, we report the cloning of the flatfish INF cDNA, the production of the protein in mammalian cells. An immortalized flatfish leukocyte cell line, HL-8, was established by cotransfecting human c-Ha-ras and human c-fos, as described previously [3]. HL-8 cells, carp epitherial cells (EPC) and Chinook salmon em-

Correspondence to: S. Shirahata, Graduate School of Genetic Resources Technology, Kyushu University, Fukuoka 812, Japan. The sequence data reported in this paper have been submitted to the E M B L / G e n b a n k Data Libraries under the accession number D 13040.

bryo cells (CHSE) were cultured in E R D F medium (Kyokuto Seiyaku, Japan) supplemented with 5% fetal bovine serum (FBS) in a humidified 5% CO 2 atmosphere at 15°C. BHK-21 cells were also cultured in the same conditions except that the culture temperature was increased to 37°C. For a bioassay of fish IFN, samples were added to EPC cells (8- 105 cells/ml) in 96 well microplates. The next day, Hirame (flatfish) rhabdovirus (HRV) [4] was added to the EPC cells with a multiplicity of infection (m.o.i.) of 50. On day 7 after infection, the cell viability was determined by the neutral red uptake method [3]. For the cloning of flatfish IFN cDNA, total RNA was isolated using guanidinium thiocyanate [5] from HL-8 cells, which had been infected with HRV. Poly(A) R N A was then purified with an oligo(dT) column (Pharmacia LKB Biotechnology, Japan). Poly(A) RNAs were fractionated by size using 10-30% sucrose density gradient centrifugation [6]. The IFN activity of each fraction was examined with an in vitro translation system using rabbit reticulocyte lysate (Amersham, UK). The cDNAs were synthesized from poly(A) RNA of the active fraction using a cDNA synthesis kit (Amersham, UK) and ligated into a vector plasmid, pBluescript. The recombinant plasmids were then transfected to Escherichia coli strain XL1 blue to construct a cDNA library. The cDNA library was screened by the IFN bioassay using an in vitro transcription system (Stratagene, USA) with T3 and T7 R N A poly-

183 merase and an in vitro translation system. The nucleotide sequence of flatfish IFN cDNA was determined by a Sequenase sequencing system (United States Biochemical, USA). Flatfish IFN cDNA cut out from pBlue-IFN was inserted into the E c o R I - P s t I site of the pcDL-SRa296 mammalian expression vector containing the SRa promoter [7]. The plasmid was termed pSRalFN. The p S R a l F N plasmid was introduced into BHK-21 cells using the transfectam method (IBF Biotechnics, USA). Stable transformed cell lines producing recombinant flatfish IFN were screened via the IFN bioassay with the cultured supernatant of transfected cells plated into 96-well microplates and cloned by limiting dilution performed twice. The recombinant flatfish IFN, secreted into the cultured medium of transformed BHK-21 cells, was purified using WGA agarose chromatography. The tissue culture infectious dose (TCIDs0) showing the antiviral activity of each fraction was examined by the method described previously [3]. The active fraction was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (see the legend of Fig. 4). Because the N-terminal amino acid residue of the flatfish IFN protein was blocked by a chemical modification, the protein was treated with CNBr [8]. This produced a N-terminal peptide composed of eight amino acids (GlyGlySerLeuPheArgLysMet) which was isolated by reverse phase chromatography. The oligopeptide was analyzed by an amino acid analyzer (Hitachi 835 type, Japan). The complete nucleotide sequence of flatfish IFN cDNA is shown in Fig. 1. The DNA sequence contains a single large open reading frame encoding 138 amino

acids before a terminal codon TAA appears. The Nterminus of the deduced amino acid sequence was started with a hydrophobic sequence. AIa-28 and Ser-30 residues satisfied the - 1 and - 3 rule for signal peptidase [9], suggesting that the signal peptide would be cleaved at the position between Ser-30 and Gly-31. The predicted signal sequence contains nine hydrophobic amino acid residues (Ala, Leu, Ile and Met), meeting the criteria that signal peptides should contain more than seven or eight hydrohobic residues. Since the N-terminus of the protein had been blocked with a chemical modification, the protein was treated with CNBr to cleave the polypeptide at two positions, Met-38 and Met-40, producing an N-terminal oligopeptide composed of eight amino acids (GlyGlySerLeuPheArgLysMet) and one larger polypeptide. The oligopeptide was isolated using reverse phase chromatography. The amino acid composition of the oligopeptides were completely coincident with the predicted nucleotide sequence (2Gly, Ser, Leu, Phe, Arg, Lys and Met) (data not shown), indicating that the peptide was cleaved between Ser-30 and Gly-31. Flatfish IFN is a glycoprotein [3]. An asparagine type glycosylation site (Asn-Thr-Ser) was found positioned from amino acid 63 to amino acid 65 in the deduced amino acid sequence of flatfish IFN. In fact, purified flatfish IFN gave a positive result on the phenol-sulfuric acid test for the detection of carbohydrates [3]. Mammalian IFNs are known to have a broad molecular weight range of about 15-30 kDa [10]. The molecular size of flatfish IFN deduced from the nucleotide sequence is 11657 Da, which is smaller than that of purified flatfish IFN (about 16 kDa) obtained from the cultured medium of HL-8 ceils as determined

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300 310 320 330 340 350 360 T C A A C T G G A T T T ~ ~ T G C T T A R ~ T ~ A C LysP ro SerCysAsnSe rPheGlnLeuAspLeuLeuLeuAla SerSerAlaTrpThrLeuLeuThrAlaArgLeuLeuAsnTyrP roTyr 370 380 390 400 410 CCTGCTGTTT TACTCTCTGCTGGTGTTGCT TCGGTAGTTT TAGTGCAAGTCCCATAAACTAATA P roAlaValLeuLeuSerAlaGlyValAla SerValValLeuValGlnValPro* ** Fig. 1. The nucleotide sequence of the flatfish IFN c D N A and the deduced amino acid sequence of flatfish IFN. The signal peptide sequence and an asparagine-type glycosylation site are underlined.

184 by SDS-PAGE. The difference in the size may be caused by the glycosylation of flatfish IFN. Glycosylated mammalian IFNs are known to show a 10% to 50% higher molecular weight than unglycosylated IFNs as demonstrated by analysis using SDS-PAGE [11,12]. All mammalian type I IFNs including IFNa and IFN/3 are thought to have similar three-dimensional structures [13]. Mammalian type II IFN like IFN7 has also been reported to have a striking similarity to the chain-folding topology of IFN/3 [14,15]. Flatfish IFN antiviral properties were exhibited when EPC fish cells were preincubated with IFN a day before being infected with HRV. However, IFN showed little antiviral effect when flatfish INF was preincubated with HRV alone for a day before infecting EPC ceils (data not shown). These results suggested that the flatfish IFN

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did not inactivate fish virus mimicking toxic substances or neutralizing antibodies, but instead, gave fish cells resistance against virus, thus behaving like mammalian IFN. As shown in Fig. 2, the amino acid sequence predicted from the nucleotide sequence of the flatfish IFN cDNA was aligned with 37 amino acid gaps to increase the similarity to mammalian IFNa and IFN/3. When the sequence of the flatfish IFN was compared with human IFNaN [16], bovine IFNaA [17], horse IFNa2 [18], rat IFNal [19], human IFNfl [20] and mouse IFN/3 [21], the sequences were 24%, 20%, 19%, 18%, 14% and 12% identical without counting the gaps, respectively. If the amino acid substitutions were allowed among the four IFNa listed in Fig. 2, the sequence of flatfish IFN would show a 30% similarity with the other four mammalian IFNa. This is shown

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Fig. 2. Comparison of amino acid sequence between flatfish IFN and mammalian I F N a s and IFN/3s. Amino acid sequence of flatfish IFN from Fig. 1 is shown together with mammalian IFNas and IFN~s, specifically from human I F N a N (HumIFNaN) [16], bovine I F N a A (BovlFNaA) [17], horse I F N a 2 (HrslFN2) [18], rat IFNcal (RatlFN1) [19], human IFNfl (HumIFN/~) [20] and mouse IFNf~ (MusIFN~) [21]. The protein has four Cys residues (boxed) which are conserved in all subtypes of IFNa. The highly conserved region of IFNa, the area from amino acid 115 to amino acid 151 is denoted by shadowing. Flatfish INF amino acid residues that are identical to at least one residue among the other four mammalian IFNas are shown by asterisks.

185 highlighted with asterisks in Fig. 2. The amino acid sequence homology between flatfish IFN and human IFNy [22] is very low. Although all observed mammalian IFNas have four Cys residues in identical positions, the flatfish INF has only one Cys residue in common with the mammalian INFas. The area between amino acid 115 to amino acid 151 in the mammalian IFNa protein is highly conserved between mammalian species. However, concerning this region, no significant homology exists between the flatfish and mammalian INF. Homology of amino acid sequences of flatfish IFN and mammalian IFNs analyzed by the computer program 'ALIGN' with Dayhoff's mutation matrix (250 PAMs) gave Z values (higher values indicate higher homology). Very low Z values of - 0 . 4 to 1.0 were obtained for flatfish IFN and mammalian IFNs listed in Fig. 2 indicating very low homology. Z values between mammalian IFNas were as high as 41.7 to 46.8. The Z value between human and mouse IFN/3s was 23.2. Even between mammalian IFNas and IFN/3s, high Z values of 9.8 to 12.8 were found. Computer analysis also revealed that there is no homology between the amino acid sequences of flatfish IFN and mammalian IFNs. Furthermore, IFNas were stable at pH 2, but the flatfish IFN was unstable [3] at that pH. Human IFNa and IFNy demonstrated no IFN activity on EPC and CHSE ceils infected with HRV, infectious pancreatic necrosis virus (IPNV) and infectious hematopoietic necrosis virus (IHNV) (data not shown). Anti human IFNa and IFN/3 (Tago, USA) did not cross-react with the flatfish IFN (data not shown). These results suggested that the flatfish IFN is a different molecule from mammalian IFNs. In order to obtain a large amount of glycosylated recombinant flatfish IFN, an expression plasmid (pSRodFN), for mammalian cells was constructed. The plasmid was introduced into BHK-21 cells and a stable transformed cell line producing flatfish IFN was established. The cultured supernatant from transformed BHK-21 cells showed distinct IFN activity on EPC cells when challenged with HRV, as shown in Fig. 3. On the other hand, cultured media from untransfected and vector only transfected BHK cells exhibited no antiviral activity. The recombinant flatfish IFN could then be easily purified from the cultured supernatant of the recombinant BHK cells using WGA agarose affinity column chromatography (Fig. 4a). The SDS-PAGE results from the active fraction separated via WGA agarose column chromatography showed a main band of about 16 kDa (Fig. 4b). This suggests that the recombinant flatfish IFN produced by transformed BHK cells is coincident to that of the natural IFN produced by immortalized flatfish leukoc~tes [3], which also has a molecular size of about 16 kDa. The protein that was extracted from

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BHK Sup (%) Fig. 3. Antiviral activity of the recombinant flatfish IFN produced by BHK-21 cells. BHK21 cells (5"105 cells/ml) were transfected with pSRalFN using the transfectam method. Varying concentrations of the cultured medium of the transfected BHK cells was added to EPC cell suspension (8.105 cells/ml). The next day, cells were infected with HRV in various m.o.i, doses ranging from 5 to 500. On the day 7, the viability of EPC cells was assayed by uptake of neutral red. The viability of EPC cells (%) was calculated by the ratio of absorbance of each test well to that of the control well which contained uninfected cells. Open marks show the culture medium of recombinant BHK-21 cells transfected with pSRalFN. The solid shapes denote the cultured medium of BHK-21 cells transfected with vector pcDL-SRa296. M.o.i. of HRV: 5, © and o; 50, zx and • 500, [] and B.

the gel (method described previously [3]) showed IFN activity. To demonstrate that the 16 kDa protein was actually flatfish IFN, anti flatfish IFN mouse monoclonal antibodies were generated. Mice were immunized with natural flatfish IFN which was purified from the cultured medium of HL-8 cells. The 16 kDa protein was blotted on a nitrocellulose membrane that reacted against the anti flatfish IFN monoclonal antibody (data not shown). A glycosylation detection reaction revealed that both the recombinant protein as well as the natural flatfish IFN had been glycosylated. The amino acid composition of the N-terminal oligopeptide from purified recombinant IFN, after treatment with CNBr, showed an exact homology with the predicted nucleotide sequence of flatfish IFN. These results demonstrated that the glycoprotein secreted from transformed BHK-21 cells is the product of the flatfish IFN cDNA introduced into these ceils. Fish IFN cDNA was first cloned from the cDNA library from an immortalized flatfish leukocyte cell line and the recombinant flatfish IFN produced by BHK-21 ceils has been purified. Field tests have been promising. The properties of recombinant flatfish IFN include a wide range of antiviral action on various kinds of fish challenged with various fish viruses in vivo, results to be published. The authors thank Dr. Y. Takebe, AIDS Research Center, National Institute of Health, Tokyo, for his generous gift of the pcDL-SRa296 vector. We also thank Dr. S. Kuhara, Graduate School of Genetic Resources Technology, Kyushu University, for his use-

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Fig. 4. Purification of recombinant flatfish IFN by WGA agarose column chromatography. (a) Culture medium (100 ml) of recombinant BHK cells was applied to a column of WGA agarose (1 ml of bed volume) at a flow rate of 30 ml/h. Adsorbed materials were eluted following a stepwise increase of N-acetylglucosamine (GlucNAc) (0-100 mM). The TCIDs0 which shows the antiviral activity of each fraction was assayed using HRV (m.o.i. = 1) and shown by the bar. The circles show the absorbance of each fraction at 280 nm and the thin line demonstrates the stepwise increase of GlucNAc. (b) The fractions of WGA agarose chromatography (0.1 mg protein), dialized against PBS and lyophilized, were applied to a SDS-PAGE using a 15% separating gel. After electrophoresis, the gel was silverstained. Lane 1, unadsorbed fraction; lane 2, fraction No. 10; lane 3, fraction No. 11. The arrow on the right side shows a protein band (lane 3) which exhibited IFN activity after extraction from the gel.

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