Identification, characterization and the interaction of Tollip and IRAK-1 in grass carp (Ctenopharyngodon idellus)

Abstract

Tollip and IRAK-1 are key components of the TLR/IL-1R signaling pathway in mammals, which play crucial roles as mediators of the TLR/IL-1R signal transduction pathways. Although several TLRs have been found in fish, molecular associations, protein-protein interactions or the role of the TLR signaling pathway in infection-induced immunity in fish has received little attention. In this study, Tollip and IRAK-1 sequences of grass carp were isolated from a head kidney cDNA library. Full length transcripts and sequences of promoter regions were obtained by 3' and 5' RACE and genome walking, respectively. Reporter gene-promoter constructs and real-time RT-PCR analysis was used to determine grass carp Tollip and IRAK-1 transcription pattern in tissues. Recombinant proteins were used for antibodies production. Phylogenetically, the grass carp loci clustered with previously reported Tollip and IRAK-1genes, respectively, and their sequences shared the highest identity with the genes of zebrafish (Danio rerio). The promoter region of grass carp Tollip and IRAK-1 proved to be active. After viral infection transcript levels of both loci were upregulated in most immune-related tissues in a time-dependent manner. Using antibodies produced in this study, immunofluorescence analysis indicated that Tollip and IRAK-1 were uniformly distributed and co-localized in the cytoplasm of CIK cells. After viral infection, however, Tollip and IRAK-1 both trended toward the cell membrane. Our results demonstrate the existence of Tollip and IRAK-1 proteins in teleost species, and suggest that Tollip-IRAK-1 complexes are being recruited to receptor complexes after stimulation with virus. These results provide novel insights into the role of the TLR signaling pathway in teleosts, especially the action of teleost Tollip and IRAK-1 and the interaction of these molecules as part of this pathway.