Molecular identification and functional characterization of IRAK-3 from a teleost fish, the orange-spotted grouper (Epinephelus coioides).

Interleukin-1 receptor-associated kinase-3 (IRAK-3) is a unique IRAK family member, which negatively regulates the TLR-mediated immune response in mammals. However, the function of IRAK-3 remains to be elucidated in fish. In the present study, an IRAK-3 cDNA sequence (EcIRAK-3) with an ORF of 1776 bp encoding 591 amino acids was identified in the orange-spotted grouper (Epinephelus coioides). Sequence analysis indicated that EcIRAK-3 shared the conserved structure characteristics and functional sites of vertebrate IRAK-3, and has a high sequence identity and phylogenetic relationship with that of other fish species. The genomic EcIRAK-3 ORF contained 13 exons and 12 introns, which was similar to that of most other fish species. In healthy grouper, EcIRAK-3 was ubiquitously expressed in seven tested tissues with the highest expression in the gills. Following Cryptocaryon irritans infection, the EcIRAK-3 transcript was up-regulated in the gills during the course of the experiment, but down-regulated in the spleen at an earlier point in time. EcIRAK-3 was localized in both the cytoplasm and nucleus in a condensed form, and its cellular distribution was affected by the death domain and ProST domain. In addition, EcIRAK-3 significantly increased MyD88-mediated NF-κB activity, and its function was ProST domain and kinase domain dependent. Taken together, the results obtained here have contributed to the understanding of the function of IRAK-3 in fish.