Molecular characterization, expression patterns, and functional analysis of toll-interacting protein (Tollip) in Japanese eel Anguilla japonica

Abstract

Toll-interacting protein (Tollip) is a key negative regulator of TLR-mediated innate immune responses. The structure and function of Tollip have been well identified in mammals, but the information about Tollip is still limited in teleost fishes. In the present study, the homologue of Tollip was cloned from Japanese eel. It contained an open reading frame encoding a polypeptide of 276 amino acids which shared high identities with other homologues from different species. Multiple alignment of the amino acid sequence showed that the AjTollip protein has the typical conserved domains including an N-terminal Target of Myb1 (Tom1) binding domain (TBD), a central conserved 2 (C2) domain, and a C-terminal coupling of ubiquitin to endoplasmic reticulum degradation (CUE) domain. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed a broad expression for AjTollip in a wide range of tissues, with the highest expression in the liver, a relatively high expression in the spleen, kidney, gills, skin and intestine, and a low expression in the heart and muscle. The AjTollip expressions in the liver and kidney were significantly induced following injection with the bacterial mimic LPS, the viral mimic poly I:C, and Aeromonas hydrophila infection. In vitro, the AjTollip transcripts of Japanese eel liver cells were significantly enhanced by the treatment of LPS, poly I:C, CpG-DNA, and PGN or the stimulation of high concentration of Aeromonas hydrophila (1 × 107 cfu/mL and 1 × 108 cfu/mL). Subcellular localization study showed that AjTollip was mainly distributed in the cytoplasm in a condensed state. When AjTollip was co-transfected with AjMyD88 into HEK293 cells, the luciferase activities of NF-κB were significantly decreased compared with that of AjMyD88 single-transfection groups in natural state or under the stimulation of LPS and poly I:C. These results collectively suggested that AjTollip functions as a negative regulator of MyD88-dependent TLR signaling and plays an important role in fish defense against viral and bacterial infections.