Molecular characterisation, ontogeny and expression analysis of melanoma differentiation-associated factor 5 (MDA5) from Asian seabass, Lates calcarifer.

Abstract

MDA5 is the pivotal member of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and is reported to play a crucial role in type I IFN-mediated responses against pathogen-associated molecular patterns (PAMPs), especially nucleic acids. In this study, we have identified and cloned the full-length cDNA sequence of MDA5, which comprises 3398 nucleotides and encodes for a putative protein of 978 AA length, in Asian seabass, Lates calcarifer. From the putative amino acid sequence of AsMDA5, four different conserved domains could be predicted: two N-terminal CARD domains, a DExDc domain, a HELICc domain and a C-terminal RIG-1_C-RD domain. The mRNA transcript of AsMDA5 could be detected in all the 11 tissues tested in healthy animals with the highest expression in heart followed by gill and skin. The ontogenetic expression profile showed constitutive expression in developmental stages starting from unfertilized eggs, which implies the possibility of maternally acquired immunity of RLRs in offspring. The viral analogue poly I:C could modulate the AsMDA5 expression both in vivo and in vitro. In all the tissues, AsMDA5 expression was found to be highly regulated following injection with poly I:C with the highest expression observed in kidney. The expression level of AsMDA5 was found to be modulated at different time-points following challenge with Gram-negative bacterium, Vibrio alginoly- ticus, and Gram-positive bacterium, Staphylococcus aureus. Similarly, noticeable change in AsMDA5 expression was detected in SISK cell line induced with either LPS or PGN. The observations made in this study suggest that in euryhaline marine teleosts like Asian seabass, MDA5 gene serves as one of the pivotal receptor for the detection of viral and bacterial PAMP, and might play an important antimicrobial role during early embryonic development.