Channa striatus TNFR-1: Molecular cloning, characterization and gene expression

Abstract

Tumor necrosis factor receptor (TNFR) superfamily entails in various biological functions including cell proliferation, differentiation, survival and providing co-stimulatory signals for programmed cell death or apoptosis. In this report, we have identified and characterized TNFR-1 from Channa striatus (Cs) at molecular level. In silico analyses revealed that the CsTNFR-1 cDNA sequence was 1200 bp in length, which includes an open reading frame of 1197 bp that encoded a putative protein of 399 amino acids (45 kDa). It contains three ‘Cys’ rich domains (CRDs) at CRD1, CRD2 and CRD3 in the extracellular region, also it possessed a putative TRAF6-binding site in the cytoplasmic tail. In addition, CsTNFR-1 cytoplasmic region contained a death domain between 297 and 388, which is important for apoptosis induction. It shared a maximum identity with its homologous from Paralichthys olivaceus (82%). Real-time PCR findings revealed that CsTNFR-1 was expressed predominantly in head kidney, further it is up-regulated upon fungal (Aphanomyces invadans) and bacterial (Aeromonas hydrophila) infection. Overall, the gene expression results indicated that CsTNFR-1 involvement in the innate immune response against pathogenic infections. The recombinant CsTNFR-1 protein was over-expressed and purified using an Escherichia coli expression vector system. The purified recombinant CsTNFR-1 derivative binds with TNF-α at high affinity and inhibits its cytotoxic activity in vitro in a dose dependent manner. CsTNFR-1 was also analyzed for respiratory burst activity upon two different ROS inducers such as opsonized zymosan and phorbol 12-myristate 13-acetate (PMA). The results showed that the recombinant CsTNFR-1 protein alone did not produce any ROS, but upon induction with opsonized zymosan, it considerably increased the ROS production in a concentration dependent manner. Overall, the findings showed the potential involvement of CsTNFR-1 in the pathogen-induced inflammatory process of C. striatus.