Characterisation and expression analysis of interleukin 2 (IL-2) and IL-21 homologues in the Japanese pufferfish, Fugu rubripes, following their discovery by synteny

Abstract

This investigation provides the first conclusive evidence for the existence of the interleukin 2 (IL-2) and IL-21 genes in bony fish. The IL-2 and IL-21 sequences have been determined in Fugu rubripes by exploiting the conservation of synteny that is found between regions of the human and Fugu genomes. The predicted 149-amino acid IL-2 homologue contains the IL-2 family signature, has a predicted secondary structure of three alpha helixes and has the two cysteines important in disulphide-bond formation. It shows low amino acid identities (24-34%) with other known IL-2 sequences. The predicted 155-amino acid IL-21 homologue has a predicted secondary structure of four alpha helixes and has the four cysteines important in disulphide-bond formation. It shows low amino acid identities (29-31%) with other known IL-21 sequences. The gene organisation of Fugu IL-2 and IL-21 and the level of synteny between the human and Fugu genomes has been well conserved during evolution, with the order and orientation of the genes matching exactly to human Chromosome 4. Phytohaemagglutinin stimulation of Fugu kidney cells resulted in a large increase in the Fugu IL-2 and IL-21 transcripts. In vivo stimulation of Fugu with LPS and poly I:C showed IL-21 expression to be localised within mucosal tissues. The discovery of IL-2 and IL-21 in fish will now allow more detailed investigations into T-helper cell responses.

Functional Characterization of a Nonmammalian IL-21: Rainbow Trout Oncorhynchus mykiss IL-21 Upregulates the Expression of the Th Cell Signature Cytokines IFN-γ, IL-10, and IL-22

“…1). This gene organization is consistent with the tetraodon IL-21 organization (32), but different to that reported for fugu IL-21, where a five-exon/four-intron structure was found as seen in mammals (31). To gain further insight into the fish IL-21 gene organization, we analyzed another fish IL-21 gene from zebrafish where the genomic (accession number NW_001880637) and cDNA (accession number EF143579) sequences of this gene are available in the database.…”

“…The final two exons of IL-21 are equivalent to the final exon of its relatives IL-2 and IL-15. Analysis of the trout gene and other fish genes revealed that the fish IL-21 genes have a common six-exon/five-intron gene organization, with the exception of fugu IL-21 that has only five exons (31). Examination of the sizes of the exons suggests that exons 3 and 4 in fish IL-21 are equivalent to exon 3 in mammals (Fig.…”

“…1), secondary structure prediction, phylogenetic tree analysis (Fig. 3), and the linkage of IL-21 and IL-2 in other fish species (31,32) suggest that the tIL-21 molecule is indeed an ortholog of mammalian IL-21. In addition, the trout gene has a six-exon/five-intron structure in common with other fish species, except fugu where exons 5 and 6 appear fused.…”

“…This discrepancy might result from the low sensitivity of the RT-PCR used in the earlier study or perhaps the physiological state of the fish used. What is clear is that fish IL-21 is expressed in immune-related tissues including gills, intestine, skin, HK, and spleen, constitutively or under stimulation (31,32). Mammalian IL-21 is produced by activated CD4 + T cells and NKT cells (20).…”

“…The function of IL-21 has so far only been described in mammals, despite the recent identification of IL-21 molecules in pufferfish, birds, and amphibians genomes (31,32). In this report, we have sequenced and functionally analyzed the IL-21 molecule in rainbow trout (Oncorhynchus mykiss).…”