Cytokine Property of Tongue Sole ISG15 Figure 1. The nucleotide and deduced amino acid sequences of CsISG15. The nucleotides and amino acids are numbered along the left margin. In the nucleotide sequence, the translation start and stop codons are in bold, and the polyadenylation signal is underlined. In the amino acid sequence, the two ubiquitin-like (UBL) domains are shaded, and the ubiquitin conjugation motif (LRGG) is boxed. doi:10.1371/journal.pone.0044884.g001 (TNF)-a and TNF-a mRNA expression from peripheral blood mononuclear cells in response to endotoxin [15]. Likewise, unconjugated extracellular ISG15, which are released from several types of human and murine cells, are known to possess cytokine-like activity [16–20]. ISG15-like sequences from a number of teleost species have been reported, most of which were found to be positively regulated in expression by microbial challenge [21–26]. Recent studies showed that the ISG15 homologues of Atlantic cod (Gadus morhua L.), Atlantic salmon (Salmo salar), and goldfish (Carassius auratus) are capable of forming conjugates with cellular proteins [27–29], and the ISG15 of red drum (Sciaenops ocellatus) exhibits immunoregulatory property [30]. However, except these few cases, the activity and biological function of teleost ISG15 are essentially unknown. In this study, we identified an ISG15 homologue, CsISG15, from tongue sole (Cynoglossus semilaevis) and analyzed its potential function. Our results showed that CsISG15 is a secreted cytokine-like protein that, unlike mammalian ISG15, possesses antiviral capacity in the unconju- gated form. Results Characterization of the sequence of CsISG15 The cDNA of CsISG15 is 1159 bp, which contains a 59-UTR of 57 bp, an open reading frame of 489 bp, and a 39-UTR of 613 bp (Fig. 1). The cDNA is terminated with a poly-A tail formed by 25 adenines. A putative polyadenylation signal, AATAAA, is located 16 bp upstream the poly-A tail. The deduced amino acid sequence of CsISG15 is 162-residue in length and has a calculated molecular mass of 18.3 kDa and a theoretical pI of 9.7. CsISG15 contains two tandem UBL domains (residues 1 to 76 and 81 to 151, respectively) and the conserved C-terminal LRGG motif, the latter being followed by seven residues that terminate the CsISG15 sequence. No putative signal peptide or glycosylation site was found in CsISG15. Blast analysis showed that CsISG15 shares 41.5–61.6% overall sequence identities with the ISG15 of the teleosts Oplegnathus fasciatus, Epinephelus coioides, Paralichthys olivaceus, Sebastes schlegelii, Anoplopoma fimbria, Channa argus, Oncorhynchus mykiss, Salmo salar, and Danio rerio (Figure S3). The overall sequence identities between CsISG15 and human and mouse ISG15 are 30.5% and 27.1% respectively. PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44884