FUNCTIONAL CAPABILITIES OF TWO THIOREDOXIN-LIKE GENES IN BLACK ROCKFISH Sebastes schlegelii: PROTECTION AGAINST OXIDATIVE STRESS.  

Thioredoxins are major disulfide oxidoreductases, are well known in all life forms    from archaebacteria to human; they are functioning as a system which comprising the thioredoxin reductase (TXNDR) and NADPH. Thioredoxin-like 1 (TXN-like 1) and Mitochondrial thioredoxin-like (MtTXN-like) are two different isoforms in thioredoxin superfamily, which play a vital role in numerous physiological processes. One of the main regulatory functions of both TXNs is to maintain the intracellular environment in a highly reduced state via their antioxidant property. Oxidative stress can be encountered due to the elicited levels of the reactive oxygen species (ROS), during the pathogen invasion. TXNs are regulating the oxidative stress in the intracellular environment by maintaining the dithiol/disulfide balance of oxidative damaged proteins via thiol-disulfide exchange reaction.

In the present study, the genes coding for two thioredoxin isoforms were identified from the cDNA database of black rockfish Sebastes schlegelii designated as SsTXN-like 1 and SsMtTXN-like, respectively. Molecular characteristics of SsTXN-like 1 and SsMtTXN-like were retrieved using various bioinformatics tools, whereas the transcriptional modulation was assessed in vivo with various biotic and abiotic stimuli. To affirm the functional properties of the both SsTXN-like 1 and SsMtTXN-like, recombinant proteins were expressed in the Escherichia coli system. Thereafter, recombinant proteins were subjected to insulin disulfide reduction (IDR) assay, metal catalyzed oxidation protection (MCO) assay and free radical scavenging activity assay to determine the relevant functional properties in vitro.

The estimated molecular weights of SsTXN-like 1 and SsMtTXN-like were 32.0 kDa and 19.2 kDa, respectively, and the SsMtTXN-like was identified with an N-terminal mitochondrial localization signal. For temporal expression analysis, fish were challenged with poly I:C, LPS and Streptococcus iniae, and significant upregulation were noticed against all three stimuli in the different post-injection time points. Classical insulin disulfide reduction assay proved that the antioxidant activity of rSsTXN-like 1 and rSsMtTXN-like behave in a concentration-dependent manner where the specific enzyme activity of rSsMtTXN-like comparatively higher than rSsTXN-like 1. Also, we reported excellent radical scavenging properties of both rSsTXN-like 1 and rSsMtTXN-like against DPPH, hydroxyl and alkyl radicals. The present study suggests that the up-regulation of both gene expressions during various immune challenges endorsed their vital role in redox homeostasis. Further, insulin disulfide reduction activity assay and radical scavenging activity assay have proved that the SsTXN-like 1 and SsMtTXN-like are being a potent antioxidant system to regulate the redox homeostasis in rockfish.