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Molecular cloning and functional expression of bottle-nosed dolphin (Tursiops truncatus) interleukin-1 receptor antagonist
Veterinary Immunology and Immunopathology 78 (2001) 131±141
Molecular cloning and functional expression of bottle-nosed dolphin (Tursiops truncatus) interleukin-1 receptor antagonist Yuuki Inouea, Takuya Itoua, Tomoyo Jimboa, Youko Syoujia, Kenji Uedab, Takeo Sakaia,* a
Department of Preventive Veterinary Medicine and Animal Health, Nihon University School of Veterinary Medicine, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan b Department of Applied Biological Sciences, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan Received 23 May 2000; received in revised form 12 September 2000; accepted 17 November 2000
Abstract The bottle-nosed dolphin (Tursiops truncatus) interleukin-1 receptor antagonist IL-1ra cDNA was cloned from mitogen-stimulated peripheral blood mononuclear cells (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The sequence of this cDNA showed that dolphin IL-1ra clones contained open reading frames encoding 177 amino acids. Comparison of the deduced amino acids showed that dolphin IL-1ra sequence shared 87.6, 77.9, 77.4, 77.4, 76.4, and 75.8% similarity with the bovine, rabbit, equine, human, mouse, and rat IL-1ra sequences, respectively. Recombinant glutathione S-transferase (GST) dolphin IL-1ra produced in Escherichia coli (E. coli) was puri®ed. This protein suppressed the cytostatic activity of dolphin IL-1b on A375S2 cells, indicating that the dolphin IL-1ra cDNA obtained in the present study encodes biologically active dolphin IL-1ra. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Bottle-nosed dolphin; IL-1ra; IL-1b; Sequence; Expression
*
Corresponding author. Tel.: 81-466-84-3650; fax: 81-466-84-3650. E-mail address: [email protected] (T. Sakai). 0165-2427/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 0 ) 0 0 2 6 3 - 4
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1. Introduction Interleukin-1 receptor antagonist IL-1ra is a cytokine produced mainly by mononuclear phagocytes. There are two forms of IL-1ra, soluble (s) and intracellular (ic) IL-1ra that speci®cally inhibits IL-1 mediated proin¯ammatory actions, such as the expression of a variety of genes and synthesis of acute phase proteins. sIL-1ra was produced in monocytes, macrophages, lymphocytes, and neutrophils by stimulators such as IgG, lipopolysaccharide, and granulocyte-macrophage colony-stimulating factor (Arend, 1991; Poutsiaka et al., 1991; Krzesicki et al., 1993; Jenkins and Arend, 1993). icIL-1ra constitutively produced in keratinocytes and epithelial cells may block the binding of IL1a to nuclear DNA (Haskill et al., 1991; Arend, 1993). The primary amino acid homology of mature human IL-1b to IL-1ra is 26%, greater than that between IL-1a and IL-1b. Human IL-1a, IL-1b, and IL-1ra shared structural similarities are comprised of all-b strand molecules which form an open barrel-like structure, which bind to the IL-1 receptor type I (IL-1RI) (Priestle et al., 1989; Graves et al., 1990; Vigers et al., 1994). IL1b has two binding sites to IL-1RI. There is a primary binding site located at its barrel shape which is similar but not identical to that of IL-1a (Grutter et al., 1994; LambriolTomkins et al., 1993). IL-1ra also has two binding sites similar to those of IL-1b (Evans et al., 1994; Vigers et al., 1994). When IL-1ra binds to IL-1RI-bearing cells, it blocks the binding of both IL-1a and IL-1b without inducing a signal of its own (Dripps et al., 1991). Moreover, when it was injected intravenously into humans at dose 1,000,000-fold greater than that of IL-1a or IL-1b, IL-1ra has no agonist activity (Crown et al., 1991; Smith et al., 1991; Granowitz et al., 1992a,b). IL-1ra reduces in¯ammation in sepsis, arthritis, in¯ammatory bowel disease, and asthma. Therefore, recombinant human IL-1ra has been administered to patients with sepsis syndrome. Although, the results of a phase II trial were encouraging, two phase III trials have not con®rmed the promising bene®cial effects (Quezado et al., 1995). While bovine, equine, rabbit, and rat IL-1ra genes have been cloned and sequenced, the human and murine IL-1ra genes have been fully characterized (Carter et al., 1990; Eisenberg et al., 1990, 1991; Zahedi et al., 1991; Goto et al., 1992; Cominelli et al., 1994; Kato et al., 1997; Kirisawa et al., 1998). However, the cloning and sequencing of dolphin IL-1ra genes have never been reported, so we carried out research into cloning and sequencing of dolphin IL-1ra gene. The immune systems of terrestrial mammals such as human and mouse are well understood, while those of marine mammals, which encounter a wide variety of infectious agents in their environment in comparison with terrestrial mammals, are not. The dolphin IL-1a and IL-1b (Inoue et al., 1999c) gene sequences and ®ndings from killer whale IL-6 (King et al., 1996) and IL-2 (Ness et al., 1998) gene analysis showed high similarity with the bovine sequences. Moreover, the dolphin IL-4 and IFN-g (Inoue et al., 1999a,b) gene sequences were also similar to the bovine ones. The aim of our study is to clarify the mechanism of the IL-1ra activity in bottle-nosed dolphin (Tursiops truncatus), speci®cally its inhibition of IL-1 mediated proin¯ammatory actions. Also de®ning part of the cytokine network will provide useful information on in¯ammatory and immune responses in marine mammals. In this study, nucleotide and predicted amino acid sequences of the bottle-nosed dolphin IL-1ra are described and compared with those of other species, and recombinant dolphin IL-1ra activity is also investigated.
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2. Materials and methods 2.1. Animals and blood samples Peripheral blood samples were collected in heparinized vacutainer tubes from bottlenosed dolphins at the Minamichita Beachland Aquarium, Okuda, Aichi-ken, Japan and Shinagawa Aquarium, Sinagawa-ku, Tokyo, Japan. 2.2. Isolation of white blood cells White blood cells were isolated within 24 h after blood collection. A 10 ml of blood sample was diluted with a four-fold volume of MEM containing 2% calf serum and 10 mM HEPES, and layered onto 20 ml of Ficoll-paque (Amersham, Buckinghamshire, UK). After centrifugation, the peripheral white blood cells (WBCs)-enriched fraction was isolated and washed twice with RPMI 1640 containing 10% fetal bovine serum, 3 mM Lglutamine, 25 mM 2-mercaptoethanol, 0.1 mM non-essential amino acids (GIBCO-BRL, USA), 1 mM pyruvic acid, 100 IU/ml penicillin, and 100 mg/ml streptomycin. WBCs were counted and suspended in RPMI 1640 containing 7.5 mg/ml concanavalin-A (Wako Pure Chemicals Industries, Ltd., Osaka, Japan) adjusted to 106 cells/ml, and then incubated in 24-well micro-plates for 6 h at 378C before RNA extraction. 2.3. RNA extraction and RT-PCR for detection of dolphin IL-1ra RNA was extracted from cell pellets using ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer's instructions. The cDNA was synthesized using oligo (dT)18 primer (Perkin-Elmer, Foster City, CA) and RNA with Moloney-murine leukemia virus (MMLV) reverse transcriptase (Perkin-Elmer, Foster City, CA). The primers used to amplify IL-1ra gene were 50 -TCA GGA TCT GGG ATG TCA ACC AGA AGA TCT-30 (IL-1ra-F) and 50 -CTA CTA GTC CTG CTG GAA GTA GAA CTT GGT GAC-30 (IL-1ra-R). These primers were chosen from the conserved region of the coding sequences of human (Carter et al., 1990), bovine (Kirisawa et al., 1998), and murine (Eisenberg et al., 1990) IL-1ra genes. By using these primers, an approximately 400 bp fragment, including about 90% of the coding sequence of IL-1ra, was expected to be ampli®ed. A 50 ml of PCR mixture containing 3.5 mM MgCl2, 50 mM KCl, 10 mM Tris± HCl pH 8.0, 0.2 mM of each dNTP, 25 pmol of each primer and 2.5U Taq DNA polymerase (Perkin-Elmer, Foster City, CA). PCR conditions were 35 cycles at 948C for 1 min, 508C for 2 min, and 728C for 2 min. The chain elongation period at 728C was extended to 5 min after the ®nal cycle. The PCR products were separated by electrophoresis in 2% agarose gel and partially puri®ed by gel extraction (QIAGEN, Gmbh, Germany). 2.4. 50 and 30 rapid ampli®cation of cDNA ends To obtain complete dolphin IL-1ra sequences, 30 RACE was performed with a 30 -Full RACE Core Set (Takara, Tokyo, Japan) using primers based on the partial sequences of dolphin IL-1ra determined above. For 30 RACE, the ®rst strand cDNA was synthesized
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from the total RNA using the oligo dT-3site adaptor primer. The cDNA was ampli®ed by PCR using IL-1ra forward and reverse primers, 50 -CGC CTC AAG ATG CTG TCC AG30 and 50 -CTG ATC TAG AGG TAC CGG ATC C-30 , respectively. By using these primers, IL-1ra fragments of 1000 bp were ampli®ed. To determine the 50 end of dolphin IL-1ra sequence, 50 RACE was performed with a 50 -Full RACE Core Set (Takara, Tokyo, Japan) using primers based on the partial sequences of dolphin IL-1ra determined above. For 50 RACE, the ®rst strand cDNA was synthesized from the total RNA using the IL-1ra RT-primer (50 -[P] CCC GAG CTT GAT CTC ATC AC-30 ). The ®rst PCR primers to amplify the 50 end of IL-1ra gene were 50 -TGC AAG TAT CCA GCA ACT AG-30 (IL1ra-A1) and 50 -ATC CAC GGG GGG AAG CTG TG-30 (IL-1ra-S1). The second PCR primers to amplify the 50 end of IL-1ra gene were 50 -CTG GTT ATT TCT CAG GTA GA30 (IL-1ra-A2) and 50 -CCT GGC CTG TGT CAA GTC TG-30 (IL-1ra-S2). By using these primers, IL-1ra fragments of 350 bp were ampli®ed. 2.5. DNA sequencing and analysis The ampli®ed fragments were subcloned into pMOSBlue vector (Amersham, Buckinghamshire, UK) using the blunt-ended cloning method according to the manufacturer's instructions. Putative clones were then screened by PCR using the above primers under the same cycle conditions. The selected clones were puri®ed from transformed MOSBlue competent cells. The nucleotide sequences of the dolphin IL-1ra were con®rmed on both strands of several separate clones using T7 and U-19 primers and a thermo-sequence ¯uorescent-labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham, Buckinghamshire, UK) according to the manufacture's instructions. These clones had identical nucleotide sequences. DNA sequence analysis was carried out using the DNASIS Version 2.0 programs in combination with the GenBank and SWISS-PROT databases for comparison with other known gene sequences. 2.6. Expression of recombinant dolphin IL-1ra and IL-1b in E. coli Recombinant dolphin IL-1ra and IL-1b expressed in Escherichia coli (E. coli) BL21 cells were concentrated and used in A375S2 assays (Nakai et al., 1988) to investigate functional activity of the recombinant proteins. To obtain the mature secreted dolphin IL1ra and IL-1b cDNA, ampli®cation was performed with PCR using primers based on the partial sequences of dolphin IL-1ra and IL-1b determined previously (Inoue et al., 1999c). Enzymatic ampli®cation of the IL-1ra and IL-1b cDNAs was performed using primers: 50 -GGC GGA TCC TAT CCC CTG GGA AAG AGA-30 (mIL1ra-F), 50 -CGG CTC GAG CTA CTG GTC CTG CTG GAA-30 (mIL1ra-R), 50 -GGC GGA TCC GCG GCT GTG CAG TCG CTG-30 (mIL-1b-F) and 50 -CGG CTC GAG TTA GGG AGA GAT GAT TTC-30 (mIL-1b-R), where BamHI and XhoI restriction sites are indicated in bold. The ampli®ed cDNA fragments were digested with BamHI and XhoI under the condition recommended by the manufacturer (TaKaRa, Tokyo, Japan). The digested fragments were subcloned into the BamHI/XhoI restriction sites of the glutathione S-transferase (GST) fusion plasmid vector, pGEX-4T-1 (Amersham Pharmacia Biotech). E. coli BL21
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cells (Amersham Pharmacia Biotech) were then transformed with the resulting plasmids DNA and grown in LB broth containing ampicillin (50 mg/ml). Production of GSTdolphin IL-1ra and IL-1b fusion proteins was induced by the addition of isopropyl b-Dthiogalactoside (IPTG) to a ®nal concentration of 0.1 mM, and grown in LB broth containing ampicillin (50 mg/ml). Recombinant dolphin IL-1ra and IL1-b fusion proteins were cleaved by thrombin digestion and obtained by puri®cation of the supernatant with Bulk GST Puri®cation Module (Amersham Pharmacia Biotech). 2.7. Bioassay for dolphin IL-1ra The biological activities of the recombinant dolphin IL-1ra and IL-1b were determined by cytostatic or cytotoxic activity to human melanoma A375S2 cells. The recombinant dolphin IL-1ra (undiluted concentration, 40 mg/ml) was serially diluted into 100 ml of the basal medium containing 0.4 mg of recombinant dolphin IL-1b (undiluted concentration, 40 mg/ml) on 96-well micro-culture plates, and 100 ml of 5 103 A375S2 cell suspension was added into each well. After 96 h incubation in a 5% CO2 incubator at 378C, the cells were stained with 50 ml of 0.05% neutral red at 378C for 6 h. The cells were washed with phosphate-buffered saline (PBS) and treated with 50% ethanol in 0.05 M NaH2PO4 to extract dye incorporated into the viable cells. Cell viability was determined by measuring the absorbance at 540 nm of the solution in each well with a micro-plate reader. The percentage of growth inhibition was calculated from the formula of Nakai et al. (1988). 3. Results 3.1. Sequencing of dolphin IL-1ra cDNA Using mitogen-stimulated bottle-nosed dolphin peripheral WBCs cDNA as a template, we performed PCR ampli®cation with IL-1ra-F and IL-1ra-R primers to isolate partial fragments of dolphin IL-1ra. By using these primers, we ampli®ed about 400 bp fragments including 90% of the coding sequence of IL-1ra. These ampli®ed fragments were cloned into the pMOSBlue vector and sequenced. The cDNA insert was 442 bp long except for the 50 142 and 30 1128 nucleotides of IL-1ra. Complete dolphin IL-1ra sequence was obtained by 50 - and 30 -RACE. The sequence reported here is deposited in the GenBank database (accession no. AB038268). The dolphin IL-1ra coding region consisted of 534 nucleotides and the calculated 177 amino acids. Fig. 1 compares the deduced amino acid sequence of the dolphin IL-1ra protein with those of human, bovine, equine, rabbit, rat, and murine IL-1ra. The dolphin IL-1ra gene shared the following sequence homology: bovine (88.7%), equine (85.2%), human (83.9%), rabbit (81.7%), murine (79.9%), and rat (79.6%). Correspondingly, the deduced amino acid sequence of dolphin IL-1ra showed 87.6, 77.9, 77.4, 77.4, 76.4, and 75.8% similarity with that of bovine, rabbit, equine, human, mouse, and rat IL-1ra, respectively. The sequence of cDNA showed that dolphin IL-1ra clones contained open reading frames encoding 177 amino acids with a predicted molecular weight of 19,921 Da. The IL-1a, IL-1b, and IL1ra were indicated to bind to the same receptor causing the same biological functions
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Fig. 1. Alignment of dolphin, bovine, equine, human, rabbit, rat, and murine IL-1ra amino acid sequences. The amino acid residues that are identical to the dolphin sequence are indicated by dashes. Where the amino acids are absent, it is denoted by a slash. The predicted amino terminal of the mature protein is marked with an asterisk. The Cys residues are delineated by solid boxes. The potential N-glycosylation sites are delineated by dashed boxes.
(Priestle et al., 1989; Graves et al., 1990; Vigers et al., 1994). We compared the sequence of mature dolphin IL-1ra with that of mature dolphin IL-1b and found 30% homology between the antagonist and IL-1b, and 27% homology between the antagonist and mature IL-1a (Fig. 2). The C-terminal region of dolphin IL-1ra, IL-1a, and IL-1b showed the similarity to each other in the analysis of hydrophobicity/hydrophilicity plots that would enable it to bind to the IL-1 receptor as human IL-1 (Fig. 3). 3.2. Expression of recombinant dolphin IL-1ra To con®rm the cloned dolphin IL-1ra cDNA (amino acids 24±177 in dolphin IL-1ra) codes for the biologically active protein, we expressed the GST fusion vector pGEX-4T-1
Fig. 2. Comparison of deduced mature dolphin IL-1ra, IL-1b, and IL-1a amino acid sequences. Homologous residues in all sequences are indicated by an asterisk. Slashes indicate gaps introduced for maximal alignment.
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Fig. 3. Comparison of hydrophilicity plots for mature dolphin IL-1ra, IL-1b, and IL-1a. Positive values on the Yaxis indicate hydrophilic regions. Amino acid numbers of these positions are indicated at the bottom of each panel.
in E. coli and assayed on A357S2 cells. Recombinant dolphin IL-1ra fusion proteins were cleaved by thrombin digestion and obtained by puri®cation of the supernatant with Bulk Columns. Puri®ed dolphin IL-1ra had a molecular weight of 17 kDa based on polyacrylamide gel analysis and was stained with Coomassie brilliant blue (data not shown). Recombinant dolphin IL-1b (0.4 mg) had growth inhibitory activity (83.8%) on A375S2 cells (Fig. 4). As shown in Fig. 5, extracts from bacteria expressing the dolphin IL-1ra cDNA clearly inhibited the cytostatic or cytotoxic activity of recombinant dolphin IL-1b on A375S2 cells in a dose-dependent manner, whereas GST protein (1.6 mg)
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Fig. 4. Bioassay of recombinant dolphin IL-1b on A375S2 cells. The biological activity of the recombinant GST and dolphin IL-1b were determined by growth inhibitory activity on human melanoma A375S2 cells. Results represent mean of triplicate wells with standard deviation.
showed no inhibition of dolphin IL-1b activity, indicating that the dolphin IL-1ra cDNA obtained in this study was biologically active. 4. Discussion The dolphin IL-1ra cDNA was cloned from mitogen-stimulated PBMCs RNA utilizing RT-PCR. A short 50 untranslated region (UTR) of 31 nucleotides and a long 30 UTR of 1128 nucleotides were also present in this 1.7 kb cDNA. Furthermore, the 30 UTR of dolphin IL-1ra mRNA does not contain the AUUUA sequence that has been implicated in the shortening of the half-life of several cytokines and growth factors (Shaw and Kamen, 1986). According to the homology analysis of the hydrophobicity of the polypeptide and proposed consensus sequence for the processing of signal peptides, it is predicted that the start of the dolphin IL-1ra is at Tyr25. It encodes a 152 amino acid protein preceded by a classical 25 amino acid secretary leader sequence, indicating that this protein takes a straightforward pathway out of the cell. The dolphin IL-4, IFN-g, IL-1a, and IL-1b gene
Fig. 5. Bioassay of recombinant dolphin IL-1ra on A375S2 cells. The biological activity of the recombinant GST and dolphin IL-1ra were determined by growth inhibition of recombinant dolphin IL-1b on human melanoma A375S2 cells. Results represent mean of triplicate wells with standard deviation.
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sequences are also similar to the bovine ones (Inoue et al., 1999a,b,c). Moreover, the dolphin IL-1ra gene sequence showed high similarity with the bovine sequence as well as with those of the killer whale as in the case of IL-6 (King et al., 1996) and IL-2 (Ness et al., 1998) gene sequences. In the amino acid sequence, therefore, cetacean cytokines tend to resemble bovine ones. The N-linked glycosylation site, Asn-109-Thr-111, is conserved at the same position in every species (dolphin, human, bovine, rabbit, and murine). Recombinant human IL-1ra expressed by E. coli is not glycosylated but blocks the binding of IL-1 as equally well as the glycosylated natural form. The recombinant dolphin IL-1ra produced in E. coli had antagonistic activity of IL-1 on A375S2 cells like those of human, bovine, equine, and rabbit IL-1ras (Nakai et al., 1988; Goto et al., 1992; Kato et al., 1997; Kirisawa et al., 1998). The dolphin IL-1ra contains eight cysteine residues (Cys4, 6, 25, 33, 91, 94, 132 and 138) that are conserved in dolphin and human proteins except for Cys6. In humans, crystal structure analysis indicated that Cys94 and Cys132 are linked via a disul®de bond (Schreuder et al., 1995), and these two amino acids are conserved in human, bovine, equine, and rabbit IL-1ras. Dolphin IL-1ra also has Cys94 and Cys132, which would form an intrachain disul®de bridge. There are two IL-1 receptors recognized as IL-1R type I and type II, corresponding to the 80 and 68 kDa IL-1 binding proteins on T and B cells, respectively. IL-1ra binds to the type I receptor with the same af®nity as IL-1 but lacking the second binding site. IL-1RI binding sites are present on IL-1ra, consisting of residues Typ16, Gln20, Tyr34, Gln36, and Tyr147 in humans (Evans et al., 1995). These amino acids were conserved in dolphin IL-1ra (Typ16, Gln20, Tyr34, Gln36, and Tyr147). Healthy humans are the most sensitive indicators of IL-1 agonist activity; 1 ng/kg of intravenous IL-1b produces symptoms. In contrast, the intravenous infusion of 10 mg/kg of IL-1ra in healthy humans is without effect. The concentration of IL-1ra needed to interfere with IL-1 activity is 10- to 100-fold greater than that of IL-1 (Granowitz et al., 1992a,b). However, the dolphin recombinant IL-1ra activity, as determined by using A375S2 cells, indicated that a parallel concentration of IL-1ra was required to inhibit 50% of the dolphin IL-1b activity. This suggests that dolphin IL-1ra may have antagonistic activity to dolphin IL-1b or that there is a difference in receptor binding activity for the two molecules in the A357S2 growth inhibition assay. In humans, IL-1ra has been shown to decrease in¯ammation in experimental arthritis and to suppress enterocolitis. Clinical trials are in progress to evaluate IL-1ra in rheumatoid arthritis and in¯ammatory bowel disease (Quezado et al., 1995). Dolphins have a susceptibility to in¯ammatory disease such as pneumonitis and enteritis. Though, potential clinical applications of dolphin IL-1ra as a therapeutic agent have never been investigated, recombinant dolphin IL-1ra could be useful for the prevention and control of in¯ammatory diseases in dolphin. Acknowledgements We would like to thank Tatsuya Oike (Minamichita Beachland Aquarium, Aichi, Japan) and Tomoko Endo (Shinagawa Aquarium, Tokyo, Japan) for providing the dolphin blood samples, and Dr. Yasuo Nakayama (Otsuka Parmaceutical Co. Ltd., Japan) for providing human melanoma A375S2 cells. This work was supported in part by a grant
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Inoue, Y., Itou, T., Sakai, T., Oike, T., 1999a. Cloning and sequencing of a bottle-nosed dolphin (Tursiops truncatus) interleukin-4-encoding cDNA. J. Vet. Med. Sci. 61, 693±696. Inoue, Y., Itou, T., Oike, T., Sakai, T., 1999b. Cloning and sequencing of the bottle-nosed dolphin (Tursiops truncatus) interferon-g gene. J. Vet. Med. Sci. 61, 939±942. Inoue, Y., Itou, T., Ueda, K., Oike, T., Sakai, T., 1999c. Cloning and sequencing of the bottle-nosed dolphin (Tursiops truncatus) interleukin-1a and -1b complementary DNAs. J. Vet. Med. Sci. 61, 1317±1321. Jenkins, J.K., Arend, W.P., 1993. Interleukin-1 receptor antagonist production in human monocytes is induced by IL-1a, IL-3, IL-4 and GM-CSF. Cytokine 5, 407±415. Kato, H., Ohashi, T., Matsushiro, H., Watari, T., Goitsuka, R., Tsujimoto, H., Hasegawa, A., 1997. Molecular cloning and functional expression of equine interleukin-1 receptor antagonist. Vet. Immunol. Immunopathol. 56, 221±231. King, D.P., Schrenzel, M.D., McKnight, M.L., Reidarson, T.H., Hanni, K.D., Stott, J.L., Ferrick, D.A., 1996. Molecular cloning and sequencing of interleukin 6 cDNA fragments from the harbor seal (Phoca vitulina), killer whale (Orcinus orca), and southern sea otter (Enhydra lutris nereis). Immunogenetics 43, 190±195. Kirisawa, R., Fukuda, T., Yamanaka, H., Hagiwara, K., Goto, M., Obata, Y., Yoshino, T., Iwai, H., 1998. Enzymatic ampli®cation and expression of bovine interleukin-1 receptor antagonist cDNA. Vet. Immunol. Immunopathol. 62, 197±208. Krzesicki, R.F., Hat®eld, C.A., Bienkowski, M.J., McGuire, J.C., Winterrowd, G.E., Chapman, D.L., Berger, A.E., McEwan, R.N., Carter, D.B., Chosay, J.G., Tracey, D.E., Chin, J.E., 1993. Regulating of expression of IL-1 receptor antagonist protein in human synovial and dermal ®broblasts. J. Immunol. 150, 4008±4018. Lambriol-Tomkins, E., Chandran, C., Varnell, T.A., Madison, V.S., Ju, G., 1993. Structure±function analysis of human IL-1a: identi®cation residues required for binding to the human type I IL-1 receptor. Protein Eng. 6, 535±539. Nakai, S., Mizuno, K., Kaneko, M., Hirai, Y., 1988. A simple, sensitive bioassay for the detection of interleukin1 using human melanoma A375 cell line. Biochem. Biophys. Res. Commun. 154, 1189±1196. Ness, T.L., Bradlley, W.G., Reynolds III, J.E., Roess, W.B., 1998. Isolation and expression of the interleukin-2 gene from the killer whale, Orcinus orca. Marine Mammal Sci. 14, 531±543. Poutsiaka, D.D., Clark, B.D., Vannier, E., Dinarello, C.A., 1991. Production of interleukin-1 receptor antagonist and interleukin-1b by peripheral blood mononuclear cells is differentially regulated. Blood 78, 1275±1281. Ê Priestle, J.P., Schar, H.P., Grutter, M.G., 1989. Crystallographic re®nement of interleukin 1 beta at 2.0 A resolution. Proc. Natl. Acad. Sci. U.S.A. 86, 9667±9671. Quezado, Z., Banks, S., Natanson, C., 1995. New strategies for combating sepsis: the magic bullets missed the mark. . .but the search continues. Trends Biotechnol. 13, 56±63. Schreuder, H.A., Rondeau, J.M., Tardif, C., Sof®entini, A., Sarubbi, E., Akeson, A., Bowlin, T.L., Yanofsky, S., Barrett, R.W., 1995. Re®ned crystal structure of the interleukin-1 receptor antagonist. Presence of a disul®de link and a cis-proline. Eur. J. Biochem. 227, 838±847. Shaw, G., Kamen, R., 1986. A conserved AU sequence from the 30 untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46, 659±667. Smith, J.W., Urba, W.J., Curti, B.D., Elwood, L.J., Steis, R.G., Janik, J.E., Sharfman, W.H., Miller, L.L., Fenton, R.G., Conlon, K.C., Rossio, J., Kopp, W., Shimuzut, M., Oppenheim, J.J., Longo, D., 1991. Phase II trial of interleukin-1 alpha in combination with indomethacine in melanoma patients. Proc. Am. Soc. Clin. Oncol. 10, 293. Vigers, G.P., Caffes, P., Evans, R.J., Thompson, R.C., Eisenberg, S.P., Brandhuber, B.J., 1994. X-ray structure of Ê resolution. J. Biol. Chem. 269, 12874±12879. interleukin-1 receptor antagonist at 2.0 A Zahedi, K., Seldin, M.F., Rits, M., Ezekowitz, R.A.B., Whitehead, A.S., 1991. Mouse IL-1 receptor antagonist protein. Molecular characterization, gene mapping, and expression of mRNA in vitro and in vivo. Immunology 146, 4228±4233.
Molecular cloning and functional expression of bottle-nosed dolphin (Tursiops truncatus) interleukin-1 receptor antagonist Yuuki Inouea, Takuya Itoua, Tomoyo Jimboa, Youko Syoujia, Kenji Uedab, Takeo Sakaia,* a
Department of Preventive Veterinary Medicine and Animal Health, Nihon University School of Veterinary Medicine, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan b Department of Applied Biological Sciences, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan Received 23 May 2000; received in revised form 12 September 2000; accepted 17 November 2000
Abstract The bottle-nosed dolphin (Tursiops truncatus) interleukin-1 receptor antagonist IL-1ra cDNA was cloned from mitogen-stimulated peripheral blood mononuclear cells (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The sequence of this cDNA showed that dolphin IL-1ra clones contained open reading frames encoding 177 amino acids. Comparison of the deduced amino acids showed that dolphin IL-1ra sequence shared 87.6, 77.9, 77.4, 77.4, 76.4, and 75.8% similarity with the bovine, rabbit, equine, human, mouse, and rat IL-1ra sequences, respectively. Recombinant glutathione S-transferase (GST) dolphin IL-1ra produced in Escherichia coli (E. coli) was puri®ed. This protein suppressed the cytostatic activity of dolphin IL-1b on A375S2 cells, indicating that the dolphin IL-1ra cDNA obtained in the present study encodes biologically active dolphin IL-1ra. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Bottle-nosed dolphin; IL-1ra; IL-1b; Sequence; Expression
*
Corresponding author. Tel.: 81-466-84-3650; fax: 81-466-84-3650. E-mail address: [email protected] (T. Sakai). 0165-2427/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 0 ) 0 0 2 6 3 - 4
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1. Introduction Interleukin-1 receptor antagonist IL-1ra is a cytokine produced mainly by mononuclear phagocytes. There are two forms of IL-1ra, soluble (s) and intracellular (ic) IL-1ra that speci®cally inhibits IL-1 mediated proin¯ammatory actions, such as the expression of a variety of genes and synthesis of acute phase proteins. sIL-1ra was produced in monocytes, macrophages, lymphocytes, and neutrophils by stimulators such as IgG, lipopolysaccharide, and granulocyte-macrophage colony-stimulating factor (Arend, 1991; Poutsiaka et al., 1991; Krzesicki et al., 1993; Jenkins and Arend, 1993). icIL-1ra constitutively produced in keratinocytes and epithelial cells may block the binding of IL1a to nuclear DNA (Haskill et al., 1991; Arend, 1993). The primary amino acid homology of mature human IL-1b to IL-1ra is 26%, greater than that between IL-1a and IL-1b. Human IL-1a, IL-1b, and IL-1ra shared structural similarities are comprised of all-b strand molecules which form an open barrel-like structure, which bind to the IL-1 receptor type I (IL-1RI) (Priestle et al., 1989; Graves et al., 1990; Vigers et al., 1994). IL1b has two binding sites to IL-1RI. There is a primary binding site located at its barrel shape which is similar but not identical to that of IL-1a (Grutter et al., 1994; LambriolTomkins et al., 1993). IL-1ra also has two binding sites similar to those of IL-1b (Evans et al., 1994; Vigers et al., 1994). When IL-1ra binds to IL-1RI-bearing cells, it blocks the binding of both IL-1a and IL-1b without inducing a signal of its own (Dripps et al., 1991). Moreover, when it was injected intravenously into humans at dose 1,000,000-fold greater than that of IL-1a or IL-1b, IL-1ra has no agonist activity (Crown et al., 1991; Smith et al., 1991; Granowitz et al., 1992a,b). IL-1ra reduces in¯ammation in sepsis, arthritis, in¯ammatory bowel disease, and asthma. Therefore, recombinant human IL-1ra has been administered to patients with sepsis syndrome. Although, the results of a phase II trial were encouraging, two phase III trials have not con®rmed the promising bene®cial effects (Quezado et al., 1995). While bovine, equine, rabbit, and rat IL-1ra genes have been cloned and sequenced, the human and murine IL-1ra genes have been fully characterized (Carter et al., 1990; Eisenberg et al., 1990, 1991; Zahedi et al., 1991; Goto et al., 1992; Cominelli et al., 1994; Kato et al., 1997; Kirisawa et al., 1998). However, the cloning and sequencing of dolphin IL-1ra genes have never been reported, so we carried out research into cloning and sequencing of dolphin IL-1ra gene. The immune systems of terrestrial mammals such as human and mouse are well understood, while those of marine mammals, which encounter a wide variety of infectious agents in their environment in comparison with terrestrial mammals, are not. The dolphin IL-1a and IL-1b (Inoue et al., 1999c) gene sequences and ®ndings from killer whale IL-6 (King et al., 1996) and IL-2 (Ness et al., 1998) gene analysis showed high similarity with the bovine sequences. Moreover, the dolphin IL-4 and IFN-g (Inoue et al., 1999a,b) gene sequences were also similar to the bovine ones. The aim of our study is to clarify the mechanism of the IL-1ra activity in bottle-nosed dolphin (Tursiops truncatus), speci®cally its inhibition of IL-1 mediated proin¯ammatory actions. Also de®ning part of the cytokine network will provide useful information on in¯ammatory and immune responses in marine mammals. In this study, nucleotide and predicted amino acid sequences of the bottle-nosed dolphin IL-1ra are described and compared with those of other species, and recombinant dolphin IL-1ra activity is also investigated.
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2. Materials and methods 2.1. Animals and blood samples Peripheral blood samples were collected in heparinized vacutainer tubes from bottlenosed dolphins at the Minamichita Beachland Aquarium, Okuda, Aichi-ken, Japan and Shinagawa Aquarium, Sinagawa-ku, Tokyo, Japan. 2.2. Isolation of white blood cells White blood cells were isolated within 24 h after blood collection. A 10 ml of blood sample was diluted with a four-fold volume of MEM containing 2% calf serum and 10 mM HEPES, and layered onto 20 ml of Ficoll-paque (Amersham, Buckinghamshire, UK). After centrifugation, the peripheral white blood cells (WBCs)-enriched fraction was isolated and washed twice with RPMI 1640 containing 10% fetal bovine serum, 3 mM Lglutamine, 25 mM 2-mercaptoethanol, 0.1 mM non-essential amino acids (GIBCO-BRL, USA), 1 mM pyruvic acid, 100 IU/ml penicillin, and 100 mg/ml streptomycin. WBCs were counted and suspended in RPMI 1640 containing 7.5 mg/ml concanavalin-A (Wako Pure Chemicals Industries, Ltd., Osaka, Japan) adjusted to 106 cells/ml, and then incubated in 24-well micro-plates for 6 h at 378C before RNA extraction. 2.3. RNA extraction and RT-PCR for detection of dolphin IL-1ra RNA was extracted from cell pellets using ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer's instructions. The cDNA was synthesized using oligo (dT)18 primer (Perkin-Elmer, Foster City, CA) and RNA with Moloney-murine leukemia virus (MMLV) reverse transcriptase (Perkin-Elmer, Foster City, CA). The primers used to amplify IL-1ra gene were 50 -TCA GGA TCT GGG ATG TCA ACC AGA AGA TCT-30 (IL-1ra-F) and 50 -CTA CTA GTC CTG CTG GAA GTA GAA CTT GGT GAC-30 (IL-1ra-R). These primers were chosen from the conserved region of the coding sequences of human (Carter et al., 1990), bovine (Kirisawa et al., 1998), and murine (Eisenberg et al., 1990) IL-1ra genes. By using these primers, an approximately 400 bp fragment, including about 90% of the coding sequence of IL-1ra, was expected to be ampli®ed. A 50 ml of PCR mixture containing 3.5 mM MgCl2, 50 mM KCl, 10 mM Tris± HCl pH 8.0, 0.2 mM of each dNTP, 25 pmol of each primer and 2.5U Taq DNA polymerase (Perkin-Elmer, Foster City, CA). PCR conditions were 35 cycles at 948C for 1 min, 508C for 2 min, and 728C for 2 min. The chain elongation period at 728C was extended to 5 min after the ®nal cycle. The PCR products were separated by electrophoresis in 2% agarose gel and partially puri®ed by gel extraction (QIAGEN, Gmbh, Germany). 2.4. 50 and 30 rapid ampli®cation of cDNA ends To obtain complete dolphin IL-1ra sequences, 30 RACE was performed with a 30 -Full RACE Core Set (Takara, Tokyo, Japan) using primers based on the partial sequences of dolphin IL-1ra determined above. For 30 RACE, the ®rst strand cDNA was synthesized
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from the total RNA using the oligo dT-3site adaptor primer. The cDNA was ampli®ed by PCR using IL-1ra forward and reverse primers, 50 -CGC CTC AAG ATG CTG TCC AG30 and 50 -CTG ATC TAG AGG TAC CGG ATC C-30 , respectively. By using these primers, IL-1ra fragments of 1000 bp were ampli®ed. To determine the 50 end of dolphin IL-1ra sequence, 50 RACE was performed with a 50 -Full RACE Core Set (Takara, Tokyo, Japan) using primers based on the partial sequences of dolphin IL-1ra determined above. For 50 RACE, the ®rst strand cDNA was synthesized from the total RNA using the IL-1ra RT-primer (50 -[P] CCC GAG CTT GAT CTC ATC AC-30 ). The ®rst PCR primers to amplify the 50 end of IL-1ra gene were 50 -TGC AAG TAT CCA GCA ACT AG-30 (IL1ra-A1) and 50 -ATC CAC GGG GGG AAG CTG TG-30 (IL-1ra-S1). The second PCR primers to amplify the 50 end of IL-1ra gene were 50 -CTG GTT ATT TCT CAG GTA GA30 (IL-1ra-A2) and 50 -CCT GGC CTG TGT CAA GTC TG-30 (IL-1ra-S2). By using these primers, IL-1ra fragments of 350 bp were ampli®ed. 2.5. DNA sequencing and analysis The ampli®ed fragments were subcloned into pMOSBlue vector (Amersham, Buckinghamshire, UK) using the blunt-ended cloning method according to the manufacturer's instructions. Putative clones were then screened by PCR using the above primers under the same cycle conditions. The selected clones were puri®ed from transformed MOSBlue competent cells. The nucleotide sequences of the dolphin IL-1ra were con®rmed on both strands of several separate clones using T7 and U-19 primers and a thermo-sequence ¯uorescent-labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham, Buckinghamshire, UK) according to the manufacture's instructions. These clones had identical nucleotide sequences. DNA sequence analysis was carried out using the DNASIS Version 2.0 programs in combination with the GenBank and SWISS-PROT databases for comparison with other known gene sequences. 2.6. Expression of recombinant dolphin IL-1ra and IL-1b in E. coli Recombinant dolphin IL-1ra and IL-1b expressed in Escherichia coli (E. coli) BL21 cells were concentrated and used in A375S2 assays (Nakai et al., 1988) to investigate functional activity of the recombinant proteins. To obtain the mature secreted dolphin IL1ra and IL-1b cDNA, ampli®cation was performed with PCR using primers based on the partial sequences of dolphin IL-1ra and IL-1b determined previously (Inoue et al., 1999c). Enzymatic ampli®cation of the IL-1ra and IL-1b cDNAs was performed using primers: 50 -GGC GGA TCC TAT CCC CTG GGA AAG AGA-30 (mIL1ra-F), 50 -CGG CTC GAG CTA CTG GTC CTG CTG GAA-30 (mIL1ra-R), 50 -GGC GGA TCC GCG GCT GTG CAG TCG CTG-30 (mIL-1b-F) and 50 -CGG CTC GAG TTA GGG AGA GAT GAT TTC-30 (mIL-1b-R), where BamHI and XhoI restriction sites are indicated in bold. The ampli®ed cDNA fragments were digested with BamHI and XhoI under the condition recommended by the manufacturer (TaKaRa, Tokyo, Japan). The digested fragments were subcloned into the BamHI/XhoI restriction sites of the glutathione S-transferase (GST) fusion plasmid vector, pGEX-4T-1 (Amersham Pharmacia Biotech). E. coli BL21
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cells (Amersham Pharmacia Biotech) were then transformed with the resulting plasmids DNA and grown in LB broth containing ampicillin (50 mg/ml). Production of GSTdolphin IL-1ra and IL-1b fusion proteins was induced by the addition of isopropyl b-Dthiogalactoside (IPTG) to a ®nal concentration of 0.1 mM, and grown in LB broth containing ampicillin (50 mg/ml). Recombinant dolphin IL-1ra and IL1-b fusion proteins were cleaved by thrombin digestion and obtained by puri®cation of the supernatant with Bulk GST Puri®cation Module (Amersham Pharmacia Biotech). 2.7. Bioassay for dolphin IL-1ra The biological activities of the recombinant dolphin IL-1ra and IL-1b were determined by cytostatic or cytotoxic activity to human melanoma A375S2 cells. The recombinant dolphin IL-1ra (undiluted concentration, 40 mg/ml) was serially diluted into 100 ml of the basal medium containing 0.4 mg of recombinant dolphin IL-1b (undiluted concentration, 40 mg/ml) on 96-well micro-culture plates, and 100 ml of 5 103 A375S2 cell suspension was added into each well. After 96 h incubation in a 5% CO2 incubator at 378C, the cells were stained with 50 ml of 0.05% neutral red at 378C for 6 h. The cells were washed with phosphate-buffered saline (PBS) and treated with 50% ethanol in 0.05 M NaH2PO4 to extract dye incorporated into the viable cells. Cell viability was determined by measuring the absorbance at 540 nm of the solution in each well with a micro-plate reader. The percentage of growth inhibition was calculated from the formula of Nakai et al. (1988). 3. Results 3.1. Sequencing of dolphin IL-1ra cDNA Using mitogen-stimulated bottle-nosed dolphin peripheral WBCs cDNA as a template, we performed PCR ampli®cation with IL-1ra-F and IL-1ra-R primers to isolate partial fragments of dolphin IL-1ra. By using these primers, we ampli®ed about 400 bp fragments including 90% of the coding sequence of IL-1ra. These ampli®ed fragments were cloned into the pMOSBlue vector and sequenced. The cDNA insert was 442 bp long except for the 50 142 and 30 1128 nucleotides of IL-1ra. Complete dolphin IL-1ra sequence was obtained by 50 - and 30 -RACE. The sequence reported here is deposited in the GenBank database (accession no. AB038268). The dolphin IL-1ra coding region consisted of 534 nucleotides and the calculated 177 amino acids. Fig. 1 compares the deduced amino acid sequence of the dolphin IL-1ra protein with those of human, bovine, equine, rabbit, rat, and murine IL-1ra. The dolphin IL-1ra gene shared the following sequence homology: bovine (88.7%), equine (85.2%), human (83.9%), rabbit (81.7%), murine (79.9%), and rat (79.6%). Correspondingly, the deduced amino acid sequence of dolphin IL-1ra showed 87.6, 77.9, 77.4, 77.4, 76.4, and 75.8% similarity with that of bovine, rabbit, equine, human, mouse, and rat IL-1ra, respectively. The sequence of cDNA showed that dolphin IL-1ra clones contained open reading frames encoding 177 amino acids with a predicted molecular weight of 19,921 Da. The IL-1a, IL-1b, and IL1ra were indicated to bind to the same receptor causing the same biological functions
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Fig. 1. Alignment of dolphin, bovine, equine, human, rabbit, rat, and murine IL-1ra amino acid sequences. The amino acid residues that are identical to the dolphin sequence are indicated by dashes. Where the amino acids are absent, it is denoted by a slash. The predicted amino terminal of the mature protein is marked with an asterisk. The Cys residues are delineated by solid boxes. The potential N-glycosylation sites are delineated by dashed boxes.
(Priestle et al., 1989; Graves et al., 1990; Vigers et al., 1994). We compared the sequence of mature dolphin IL-1ra with that of mature dolphin IL-1b and found 30% homology between the antagonist and IL-1b, and 27% homology between the antagonist and mature IL-1a (Fig. 2). The C-terminal region of dolphin IL-1ra, IL-1a, and IL-1b showed the similarity to each other in the analysis of hydrophobicity/hydrophilicity plots that would enable it to bind to the IL-1 receptor as human IL-1 (Fig. 3). 3.2. Expression of recombinant dolphin IL-1ra To con®rm the cloned dolphin IL-1ra cDNA (amino acids 24±177 in dolphin IL-1ra) codes for the biologically active protein, we expressed the GST fusion vector pGEX-4T-1
Fig. 2. Comparison of deduced mature dolphin IL-1ra, IL-1b, and IL-1a amino acid sequences. Homologous residues in all sequences are indicated by an asterisk. Slashes indicate gaps introduced for maximal alignment.
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Fig. 3. Comparison of hydrophilicity plots for mature dolphin IL-1ra, IL-1b, and IL-1a. Positive values on the Yaxis indicate hydrophilic regions. Amino acid numbers of these positions are indicated at the bottom of each panel.
in E. coli and assayed on A357S2 cells. Recombinant dolphin IL-1ra fusion proteins were cleaved by thrombin digestion and obtained by puri®cation of the supernatant with Bulk Columns. Puri®ed dolphin IL-1ra had a molecular weight of 17 kDa based on polyacrylamide gel analysis and was stained with Coomassie brilliant blue (data not shown). Recombinant dolphin IL-1b (0.4 mg) had growth inhibitory activity (83.8%) on A375S2 cells (Fig. 4). As shown in Fig. 5, extracts from bacteria expressing the dolphin IL-1ra cDNA clearly inhibited the cytostatic or cytotoxic activity of recombinant dolphin IL-1b on A375S2 cells in a dose-dependent manner, whereas GST protein (1.6 mg)
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Fig. 4. Bioassay of recombinant dolphin IL-1b on A375S2 cells. The biological activity of the recombinant GST and dolphin IL-1b were determined by growth inhibitory activity on human melanoma A375S2 cells. Results represent mean of triplicate wells with standard deviation.
showed no inhibition of dolphin IL-1b activity, indicating that the dolphin IL-1ra cDNA obtained in this study was biologically active. 4. Discussion The dolphin IL-1ra cDNA was cloned from mitogen-stimulated PBMCs RNA utilizing RT-PCR. A short 50 untranslated region (UTR) of 31 nucleotides and a long 30 UTR of 1128 nucleotides were also present in this 1.7 kb cDNA. Furthermore, the 30 UTR of dolphin IL-1ra mRNA does not contain the AUUUA sequence that has been implicated in the shortening of the half-life of several cytokines and growth factors (Shaw and Kamen, 1986). According to the homology analysis of the hydrophobicity of the polypeptide and proposed consensus sequence for the processing of signal peptides, it is predicted that the start of the dolphin IL-1ra is at Tyr25. It encodes a 152 amino acid protein preceded by a classical 25 amino acid secretary leader sequence, indicating that this protein takes a straightforward pathway out of the cell. The dolphin IL-4, IFN-g, IL-1a, and IL-1b gene
Fig. 5. Bioassay of recombinant dolphin IL-1ra on A375S2 cells. The biological activity of the recombinant GST and dolphin IL-1ra were determined by growth inhibition of recombinant dolphin IL-1b on human melanoma A375S2 cells. Results represent mean of triplicate wells with standard deviation.
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sequences are also similar to the bovine ones (Inoue et al., 1999a,b,c). Moreover, the dolphin IL-1ra gene sequence showed high similarity with the bovine sequence as well as with those of the killer whale as in the case of IL-6 (King et al., 1996) and IL-2 (Ness et al., 1998) gene sequences. In the amino acid sequence, therefore, cetacean cytokines tend to resemble bovine ones. The N-linked glycosylation site, Asn-109-Thr-111, is conserved at the same position in every species (dolphin, human, bovine, rabbit, and murine). Recombinant human IL-1ra expressed by E. coli is not glycosylated but blocks the binding of IL-1 as equally well as the glycosylated natural form. The recombinant dolphin IL-1ra produced in E. coli had antagonistic activity of IL-1 on A375S2 cells like those of human, bovine, equine, and rabbit IL-1ras (Nakai et al., 1988; Goto et al., 1992; Kato et al., 1997; Kirisawa et al., 1998). The dolphin IL-1ra contains eight cysteine residues (Cys4, 6, 25, 33, 91, 94, 132 and 138) that are conserved in dolphin and human proteins except for Cys6. In humans, crystal structure analysis indicated that Cys94 and Cys132 are linked via a disul®de bond (Schreuder et al., 1995), and these two amino acids are conserved in human, bovine, equine, and rabbit IL-1ras. Dolphin IL-1ra also has Cys94 and Cys132, which would form an intrachain disul®de bridge. There are two IL-1 receptors recognized as IL-1R type I and type II, corresponding to the 80 and 68 kDa IL-1 binding proteins on T and B cells, respectively. IL-1ra binds to the type I receptor with the same af®nity as IL-1 but lacking the second binding site. IL-1RI binding sites are present on IL-1ra, consisting of residues Typ16, Gln20, Tyr34, Gln36, and Tyr147 in humans (Evans et al., 1995). These amino acids were conserved in dolphin IL-1ra (Typ16, Gln20, Tyr34, Gln36, and Tyr147). Healthy humans are the most sensitive indicators of IL-1 agonist activity; 1 ng/kg of intravenous IL-1b produces symptoms. In contrast, the intravenous infusion of 10 mg/kg of IL-1ra in healthy humans is without effect. The concentration of IL-1ra needed to interfere with IL-1 activity is 10- to 100-fold greater than that of IL-1 (Granowitz et al., 1992a,b). However, the dolphin recombinant IL-1ra activity, as determined by using A375S2 cells, indicated that a parallel concentration of IL-1ra was required to inhibit 50% of the dolphin IL-1b activity. This suggests that dolphin IL-1ra may have antagonistic activity to dolphin IL-1b or that there is a difference in receptor binding activity for the two molecules in the A357S2 growth inhibition assay. In humans, IL-1ra has been shown to decrease in¯ammation in experimental arthritis and to suppress enterocolitis. Clinical trials are in progress to evaluate IL-1ra in rheumatoid arthritis and in¯ammatory bowel disease (Quezado et al., 1995). Dolphins have a susceptibility to in¯ammatory disease such as pneumonitis and enteritis. Though, potential clinical applications of dolphin IL-1ra as a therapeutic agent have never been investigated, recombinant dolphin IL-1ra could be useful for the prevention and control of in¯ammatory diseases in dolphin. Acknowledgements We would like to thank Tatsuya Oike (Minamichita Beachland Aquarium, Aichi, Japan) and Tomoko Endo (Shinagawa Aquarium, Tokyo, Japan) for providing the dolphin blood samples, and Dr. Yasuo Nakayama (Otsuka Parmaceutical Co. Ltd., Japan) for providing human melanoma A375S2 cells. This work was supported in part by a grant
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