Molecular Cloning and Phylogenetic Analysis of Inflammatory Cytokines of the Ferret (Mustela putorius furo)

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1 FULL PAPER Immunology Molecular Cloning and Phylogenetic Analysis of Inflammatory Cytokines of the Ferret (Mustela putorius furo) Makoto NAKATA 1), Takuya ITOU 1) and Takeo SAKAI 1) * 1) Nihon University Veterinary Research Center, 1866 Kameino, Fujisawa, Kanagawa , Japan (Received 3 October 2007/Accepted 30 January 2008) ABSTRACT. The present study determined the cdna and deduced amino acid sequences of ferret (Mustela putorius furo) inflammatory cytokines, interferon (IFN)-γ, interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α. The homologies of the nucleotide sequences of IFN-γ, IL-1β, IL-6, IL-8 and TNF-α of the ferret to those from other mammalian species ranged from %, , %, % and %, respectively. As distinctive amino acid residues constituting various motifs and ligandbinding sites and cysteine residues were highly conserved in ferret inflammatory cytokine proteins, ferret cytokines may have fundamentally similar functions to those of other mammals. Phylogenetic analyses based on the deduced amino acid sequences revealed that all ferret inflammatory cytokines were more closely related to those of the Carnivora order, specifically dog and cat, than to other species. KEY WORDS: ferret, inflammatory cytokines, molecular cloning,, phylogenetic analysis. J. Vet. Med. Sci. 70(6): , 2008 While cytokines play important roles in the normal maintenance of homeostasis, they are overabundantly produced under inflammatory conditions and are related to development of disorders and persistence resulting from oversecretion. When inflammation occurs, pre-inflammatory cytokines, such as Interleukin (IL)-1 and IL-6, are produced in the regions of local lesions as part of an early host defense response to infection. T lymphocytes or monocytes infiltrating into the lesion region are then activated by antigens, secreting characteristic cytokines, such as IL-1 and tumor necrosis factor (TNF)-α, and enhancing leukocyte migration [18]. Both IL-1 and TNF-α activate metabolism of numerous immune cells, introduce adhesive factors and increase the permeability of vascular endothelial cells [8, 12]. The IL-6 produced by monocytes/macrophages in the acute phase of immune reactions plays a critical role as a central mediator of inflammatory response [1, 15]. Interferon (IFN)-γ is involved in regulation of nearly all phases of the immune and inflammatory responses, including activation and differentiation of T cells, B cells, NK cells, macrophages and other types of cells [7]. IL-8, produced by different cell types, is a multifunctional member of the α- chemokine family of chemotactic CXC cytokines. IL-8 is a potent stimulator of neutrophil activation and chemotaxis, and its upregulation is associated with numerous acute and chronic inflammatory disorders [27]. Thus, these inflammatory cytokines are involved in host defense via a complicated network. These inflammatory cytokines have been molecularly characterized in many domestic species [3, 4, 6, 10, 14, 19 22, 25, 26, 34]. Recently, several complete and partial nucleotide sequences of ferret cytokines have been determined for investigation of their mrna expressions during viral infection [28]. However, fundamental analyses *CORRESPONDENCE TO: Prof. SAKAI, T., Nihon University Veterinary Research Center, 1866 Kameino, Fujisawa, Kanagawa , Japan. sakai.takeo@nihon-u.ac.jp of their deduced amino acid sequences and protein structures have not been fully demonstrated. The ferret (Mustela putorius furo) belongs to the Carnivora order, and the number of domestic ferrets kept as companion animals has recently increased. The ferret also serves as an animal model for several inflammatory human diseases, such as influenza and severe acute respiratory syndrome [2, 5, 17, 30, 31]. Despite the importance of the ferret in the field of veterinary and human medicine, there have been few reports analyzing the molecular basis of inflammatory cytokines in this animal. The aim of this study was to identify the structures and functions of ferret inflammatory cytokines by analyzing nucleotide and deduced amino acid sequences, comparing them with those of other mammals and investigating their phylogenetic relationships. MATERIALS AND METHODS The animals used in this study were clinically healthy male ferrets (n=4) between 10 months and 3 years of age. They were obtained from Marshall Pet Products (New York City, NY, U.S.A.) and were maintained at Nihon University Veterinary Research Center (NUVERC). This study was approved by the Ethical Committee for Animal Experimentation, NUVERC. Peripheral blood samples (6 ml) were drawn from the anterior vena cava into heparin-anticoagulated tubes. After dextran sedimentation, the upper layer containing the leukocyte-rich fraction was centrifuged at 100 g for 10 min. The pellet was suspended with 0.2% hypotonic NaCl solution, gently mixed for 30 sec and then added to an equal volume of 1.6% hypertonic NaCl solution. After centrifugation at 100 g for 10 min, the cell pellet was washed twice with PBS ( ) and finally resuspended in RPMI 1640 medium containing 10% fetal bovine serum. The cell suspension ( cells/ml) was incubated at 37 C for 4 hr in 24-well microplates, and concanavalin A

2 544 M. NAKATA, T. ITOU AND T. SAKAI (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added to produce a final concentration of 7.5 mg/ml. After incubation, total RNA was extracted from leukocytes using ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer s instructions. Ferret leukocyte cdna was synthesized using oligo dt primers (Perkin-Elmer, Foster City, CA, U.S.A.) and Moloney murine leukemia virus (MMLV) reverse transcriptase (Clontech, Palo Alto, CA, U.S.A.) and was amplified by the method described by Inoue et al. [11]. The primers used to amplify the ferret cytokine genes were designed based on highly conserved regions of other mammalian cytokine sequences registered in GenBank; the relevant accession numbers (ANs) are NM_ (human), M29867 (bovine) and AF (canine) for IFN-γ; DQ (canine) for IL-1β; M54894 (human), X57317 (bovine) and U12234 (canine) for IL-6; Y00787 (human), S74436 (sheep) and U19849 (macaque) for IL-8; and X01394 (human) and DQ (canine) for TNF-α. The primer sequences and annealing temperatures are shown in Table 1. PCR was performed for 35 cycles of denaturation at 94 C for 1 min, annealing at each temperature for 1 min and extension at 72 C for 1 min. The amplified cdnas were electrophoresed on 2% agarose gel, visualized with ethidium bromide under UV light and then purified with a Wizard SV Gel and PCR Clean-Up System Kit (Promega, Madison, WI, U.S.A.). The purified cdnas were directly sequenced using a Big Dye Terminator ver.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, U.S.A.), and the primers described in Table 1 were analyzed on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). For each cytokine, cdnas were isolated from 4 ferrets, and the analyzed cdna sequences were identical. The determined nucleotide and deduced amino acid sequences of the complete region coding for ferret inflammatory cytokine proteins were analyzed using GENETYX-WIN version 7.0 (Software Development Co., Ltd., Tokyo, Japan). The homology of each ferret cytokine nucleotide and deduced amino acid sequence with those of dog, cat, human, pig, horse, cattle, sheep and mouse were determined using the FASTA (NCBI) and ClustalX 1.8 programs. Multi-alignments were performed with ClustalX 1.8 and arranged with BioEdit Version (Ibis Therapeutic, Carlsbad, CA, U.S.A.). Phylogenetic trees were generated by the neighbor-joining method and drawn with Tree View (Molecular Evolution) after processing by ClustalX 1.8, and the mouse was used as an out-group. RESULTS Sequence homology The complete nucleotide sequences encoding the cytokine proteins of IFN-γ, IL-1β, IL-6, IL-8 and TNF-α consisted of 501, 813, 633, 306 and 702 bp, respectively. The respective, deduced amino acid sequences made up 166, 270, 210, 101 and 233 amino acid residues. The ferret IFNγ, IL-1β and IL-8 cdna sequences were deposited in the GenBank database (ANs: AB300566, AB and AB300565). The ferret IL-6 and TNF-α sequences were completely identical to those previously reported (ANs: EF368209, EF368211) [28]. The IFN-γ sequence determined in the present study could not be compared with registered sequence (AN: EF368214) because it was partial sequence. The percentages of identifiable nucleotide and deduced amino acid sequences for ferret inflammatory cytokines compared with those from other mammals are given in Table 2. The homologies of the ferret IFN-γ, IL-1β, IL-6, IL-8 and TNF-α nucleotide (deduced amino acid) sequences encoding proteins to the dog were 92.9% (86%), 83.9% (74%), 84.8% (73%), 89.7% (89%) and 95.0% (97%), respectively. The nucleotide and amino acid sequences of the ferret cytokines showed close homology to those of Carnivora, such as the dog and cat. In contrast, the homologies of the ferret cytokine nucleotide (deduced amino acid) sequences to the mouse were lower compared with other mammals and were 64.3% (54%), 73.0% (57%), 58.1% (39%), 58.1% (41%) and 79.0% (79%) for IFN-γ, IL- 1β, IL-6, IL-8 and TNF-α, respectively. The multi-alignments and characterization of the deduced amino acid sequences of ferret cytokines Structural analysis of mature proteins: The multi-alignments of the deduced amino acid sequences are shown in Fig. 1. The mature ferret proteins of IFN-γ, IL-1β, IL-6, IL- 8 and TNF-α were predicted to start at Gln 20, Cys 36, Thr 28, Table 1. Primer sequences used to amplify cytokine cdnas from the ferret Cytokine Primer sequences (5-3 ) Annealing temp. IFN-γ F 5 -CAGGAGCTACCGATTTCAAC C R 5 -AAATTCAAATATTGCAGGCA-3 IL-1β F 5 -TTTCTAAAGCAGCCATGGCA-3 54 C R 5 -CTTCTACTCCCTTTCCATCAG-3 IL-6 F 5 -ACGAAAGAGAGCTCCATCTG-3 50 C R 5 -AGGAGGGAATGCCCAKGAAC-3 IL-8 F 5 -CAAGAGCCAGGAAGAAACC C R 5 -TGATTCTTGGATACCA-3 TNF-α F 5 -GAAGACGCCATGAGCACTGA-3 53 C R 5 -AATTCTCTTTCTAAGCCTGA-3 The primers were designed based on highly conserved regions of the cytokine sequences of other species reported in GenBank.

3 INFLAMMATORY CYTOKINES OF THE FERRET 545 Table 2. Percentages of identifiable nucleotide amino acid sequences of various inflammatory cytokines from several mammals Mammalian Nucleotide (amino acid) homology % species FN-γ IL-1β IL-6 IL-8 TNF-α Dog 92.9 (86) 83.9 (74) 84.8 (73) 89.7 (89) 95.0 (97) Cat 88.1 (83) 83.4 (73) 83.0 (75) 86.2 (85) 93.0 (92) Human 78.4 (63) 77.3 (62) 74.3 (59) 82.3 (75) 88.9 (89) Pig 82.8 (75) 73.8 (58) 79.8 (69) 81.8 (86) 84.2 (85) Horse 85.0 (80) 79.1 (61) 78.5 (68) 79.1 (77) 87.4 (87) Cattle 82.9 (76) 73.7 (57) 73.5 (53) 83.9 (83) 82.8 (78) Sheep 82.2 (76) 73.1 (56) 73.5 (51) 83.1 (85) 82.9 (78) Mouse 64.3 (54) 73.0 (57) 58.1 (39) 58.1 (41) 79.0 (79) Fig. 1. Alignment of deduced amino acid sequences of inflammatory cytokines from the ferret and several mammals. The amino acid residues identical to the ferret sequences are indicated by dots. A dash denotes the absence of amino acids. The starting point for the amino acids of each mature protein are indicated by an asterisk (*). The conserved Cys residues or potential N-linked glycosylation sites are delineated by solid boxes (A-E). In Fig. 1B (IL-1β), the amino acids forming a discontinuous binding site for the type I IL-1 receptor are marked with a star ( ), and two residues maintaining the conformation of IL-1β are marked with a black triangle ( ). The IL-1β carboxyl-terminal endpoint for active protein (tripeptide of Thr-Asp-Phe: TDF) is indicated by an arrow ( ). In Fig. 1D (IL-8), ELR motif is indicated by a double circle ( ). In Fig. 1E (TNF-α), the amino acids involved in binding for the two TNF-α receptors are indicated by a star ( ). The amino acids of the binding of neutralizing monoclonal antibodies are marked with a white triangle ( ). Glu 21 and Val 77 and comprised 147, 235, 183, 81 and 157 amino acids, respectively. Cysteine residues were conserved at Cys 23 in ferret IFN-γ protein (Fig. 1A), with seven residues at Cys 36, 83, 98, 110, 126, 179 and 189 in IL-1β protein (Fig. 1B), four residues at Cys 72, 78, 101 and 111 in IL-6 protein (Fig. 1C), four residues at Cys 34, 36, 61 and 77 in IL-8 protein (Fig. 1D) and two residues at Cys 145 and 177 in TNF-α protein (Fig. 1E), respectively. Ferret IFN-γ had two N-glycosylation sites at Asn 39 -Ser 41 and Asn 106 -Ser 108 (Fig. 1A), and ferret IL-1β had four N-glycosylation sites at Asn 41 -Ser 43, Asn 60 - Ser 62, Asn 150 -Thr 152 and Asn 220 -Thr 222 (Fig. 1B). Characterization of mature proteins related to the function of ferret inflammatory cytokines: Seven amino acids (Arg 120, Leu 122, Phe 162, Ile 172, Lys 208, Lys 218 and Glu 220 ) are

4 546 M. NAKATA, T. ITOU AND T. SAKAI clustered in the human IL-1β molecule. These amino acids form a discontinuous binding site for the type I IL-1 receptor in humans [16]. These amino acids were conserved in ferret IL-1β at Arg 122, Leu 124, Phe 164, Ile 174, Lys 210, Lys 221 and Glu 223. Moreover, two residues (Met 160 and Val 174 ) may be important in maintaining the conformation of human IL-1β. These amino acids were retained in ferret IL-1β (Met 162 and Val 176 ). In addition, ferret IL-1β carboxyl-terminal endpoints for active protein (tripeptide of Thr-Asp-Phe: TDF) were found at the same positions in other mammals, except for the mouse (Fig. 1B). In ferret IL-8 protein, glutamic acid: E, leucine: L, arginine: R, ELR motif that is supposed to act on neutrophil chemotactic properties, was specifically conserved at Gln 31 - Arg 33. In the ferret TNF-α, Arg 207, which is involved in the binding of neutralizing monoclonal antibodies, was retained. In addition, several amino acids (Arg 82, Glu 97, Glu 99, Arg 107,

5 INFLAMMATORY CYTOKINES OF THE FERRET 547 Arg 108, Lys 141, Arg 143, Val 150, Ala 160, Tyr 163, Tyr 191, Phe 220, Ala 221 and Glu 222 ) that were supposed to correlate with the binding for two TNF-α receptors were identified in ferret TNF-α (Fig. 1E). Phylogenetic trees Phylogenetic trees were constructed amino acid sequences of ferret cytokines the using the neighbor-joining method in this study; other mammalian data were obtained from GenBank. Since the tree patterns of these five cytokines were similar, only that of IFN-γ is shown in Fig. 2. The mammalian cytokines can be classified into five groups: Primates, Artiodactyla, Carnivora and Perissodactyla, with Rodentia as the out-group. The inflammatory cytokines of the ferret were more closely related to the dog and cat, which belong to the Carnivora group. DISCUSSION The nucleotide and amino acid sequences of ferret cytokines showed close homology to those of Carnivora. These results suggest the sequences of inflammatory cytokines defined in this study could be classified according to species. Moreover, the homology of TNF-α in all species was highest compared with the other inflammatory cytokines investigated in this study. The starting positions of the mature ferret proteins of IFN-γ, IL-1β, IL-6, IL-8 and TNF-α were based on the N- terminal amino acid sequences of the secreted proteins [23]. The starting positions of the mature proteins for human, murine, bovine and ovine IFN-γ were at Gly 20, Gly 19, Gly 23 and Gly 23, respectively [4, 9, 24, 25] and were at Gly 20 for ferrets, dogs and cats. The cysteine residue is related to disulfide bonds. Seven cysteine residues in mature ferret IL-1β protein were conserved in different positions for each mammal. In ferret IL-

6 548 M. NAKATA, T. ITOU AND T. SAKAI 72, 78, 101 and 6, four cysteine residues were conserved at Cys 111, and these were conserved in the same positions in all mammals compared. IL-8 is classified as a CXC chemokine, one of four chemokines classified according to the arrangement of cysteine residues. In humans, CXC chemokines have four highly conserved cysteine residues and are characterized by a single amino acid between the number 1 and 2 cysteines as a CXC motif [35]. Ferret IL-8 has four cysteine residues, the same as other mammals, including humans [21, 26]. Ferret TNF-α had two cysteine residues at Cys 145 and 177. These cysteine residues are conserved in all mammals. N-linked glycosylation sites are also referred to as sequons. Glycosylation of asparagine (N) requires the amino acid pattern N-X-S, where X can be any amino acid followed by serine (S) or threonine (T). The biological roles of N-linked glycosylation sites are recognition and adhesion, protein folding, metabolism and transport and maintenance of cell and protein structures [33]. Ferret IFN-γ had two N-glycosylation sites in the same positions as the dog, cat, horse and mouse, which is in contrast to humans, which have two different sites at Asn 48 -Thr 51 and Asn 120 -Ser 122. Ferret IL-1β had four potential N-linked glycosylation sites; mature human IL-1β does not. The ferret IL-6 amino acid sequence had no glycosylation sites, like cats and dogs; however humans have two conserved amino acid sequences at amino acids Asp 73 -Ser 75 and Asp 172 -Ser 174. The carbohydrate moieties of the glycoprotein contribute to the solubility and thermal stability of proteins [33]. The difference in the presence of N-linked glycosylation sites between the other mammals may be related to these functions. Ferrets have several amino acids that, from the discontinuous binding site for the type I IL-1 receptor, maintain the conformation of IL-1β and have carboxyl-terminal endpoints for active protein (TDF) that are conserved in the same positions, as seen in previous reports [10, 14, 16, 19, 20]. In ferret IL-1β, comparison of each residue suggests that these conserved amino acids have the same function as those of human IL-1β. CXC chemokines, including IL-8, can be further subdivided depending on whether they have an ELR motif just in front of the first cysteine residue. These chemokines all attract neutrophils and use CXC chemokine receptor 2. Ferret IL-8 is considered to stimulate neutrophils and to be an important chemotactic factor. Arg 207 in human TNF-α, which binds neutralizing monoclonal antibodies [29], is retained in almost all other mammals including the ferret, except for the horse and mouse. This amino acid may be important in almost all mammals for this function. Thirteen amino acids in human TNF-α, which supposedly correlate with binding for two TNF-α receptors [13], were substituted in ferret TNF-α (Gln 97 Glu and Gln 143 Arg). However, other positions in these amino acids were substituted by several amino acids in every species in the present study. Accordingly, it is still unknown whether these substitutions affect species-specific affinity for the receptors. In this study, we defined the molecular sequences of ferret cytokines, and the results suggest that each of the inflammatory cytokine proteins had a specific structure similar to those of other mammals, including the human. This suggests that the ferret can be utilized as a beneficial animal model for human inflammatory disease. In addition, phylogenetic analysis in the present study showed that all ferret inflammatory cytokines have a close relationship to the dog and cat. This phylogenetic similarity suggests that the ferret is immunologically similar to dog and cat, implying that cytokine therapeutics would be applicable to inflammatory

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