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Published on: November 13, 2012
Molecular characterisation, ontogeny and expression analysis of melanoma differentiation-associated factor 5 (MDA5)
Anutosh Paria1, M Makesh1, Aparna Chaudhari1
1ICAR-Central Institute of Fisheries Education (CIFE), Off-Yari Road, Versova, Mumbai -400 061, India.
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 alginolyticus, 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.
Insights
MDA5, a key immune receptor in Asian seabass, detects viral and bacterial molecules. Its expression is widespread and crucial for immune responses from early development through adulthood.
Area of Science:
- Immunology
- Molecular Biology
- Aquaculture
Background:
- MDA5 is a crucial retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) involved in innate immunity.
- RLRs recognize pathogen-associated molecular patterns (PAMPs), particularly nucleic acids, initiating immune responses.
Purpose of the Study:
- To identify and characterize the MDA5 gene in Asian seabass (Lates calcarifer).
- To investigate the expression patterns and immune regulatory roles of Asian seabass MDA5.
Main Methods:
- Full-length cDNA sequence of MDA5 was identified and cloned.
- AsMDA5 protein domains were predicted bioinformatically.
- mRNA expression was analyzed in various tissues and developmental stages.
- Expression was modulated by poly I:C, bacterial challenges (Vibrio alginolyticus, Staphylococcus aureus), and cell line treatments (LPS, PGN).
Main Results:
- The Asian seabass MDA5 (AsMDA5) gene comprises 3398 nucleotides, encoding a 978 AA protein with conserved RLR domains.
- AsMDA5 mRNA is constitutively expressed in most tissues, with highest levels in heart, gill, and skin.
- Expression is present from unfertilized eggs, suggesting maternal immunity.
- Poly I:C stimulation upregulated AsMDA5 expression in vivo and in vitro, particularly in the kidney.
- Bacterial challenges and PAMP treatments (LPS, PGN) also modulated AsMDA5 expression.
Conclusions:
- Asian seabass MDA5 functions as a vital receptor for detecting viral and bacterial PAMPs.
- AsMDA5 plays a significant antimicrobial role, potentially from early embryonic development onwards.
- This study enhances understanding of innate immunity in marine teleosts.
