MOLECULAR CHARACTERIZATION OF THIOREDOXIN MITOCHONDRIAL-LIKE PROTEIN FROM BIG-BELLY SEAHORSE Hippocampus abdominalis IN RESPONSE TO IMMUNE STIMULATION

Oxidative stress is generally occurred due to imbalance of Reactive oxygen species molecules in cells. To maintain the homeostatic of ROS level, organisms have antioxidant defend systems including enzymatic & non- enzymatic antioxidants. Among those, Thioredoxin protein is a 12 kDa small redox protein with characteristic conserved WCGPC active site and ubiquitously expressed in all living organisms. This protein exists as either a cytosolic (TRx-1) or mitochondrial (TRx-2) isoform. TRx members are involved in wide range of biological functions for instance regulation of gene expression, control of apoptosis, protein folding and scavenging of ROS. In this present study, we examined molecular features and the biological functions of TRx-2 from Hippocampus abdominalis (HaTRx-2).

The cDNA & protein sequence of HaTRx-2 was analyzed molecular wise by using bioinformatics tools such as UGENE software, NCBI conserved domain database (CDD), MEGA 7, ExPASY PROSITE, SignalP 4.1 online server, ClustalW and etc. The qPCR was performed to determine the tissue specific distribution of HaTRx-2. Also, the mRNA expression profile of HaTRx-2 upon immune challenged with Poly I.C, LPS, Edwardsiella tarda & Streptococcus iniae was investigated. Further, the TRx-2 was recombinantly expressed in Escherichia coli BL21(DE3) and the antioxidant property of recombinant TRx-2 (rHaTRx-2) was investigated by carrying out the MCO assay.

The cDNA sequence of HaTRx-2 consists of 519 bp of open reading frame (ORF) which encoding 172 amino acids with a molecular weight of 18.8 kDa and calculated pI of 7.80. According to the phylogenetic tree analysis, HaTRx-2 is close to its ortholog from Fundulus heteroclitus. The qPCR results revealed that HaTRx-2 transcripts ubiquitously express in all examined tissues with a high expression in ovary. The mRNA expression levels in the blood showed significant up regulation with Poly I.C and E.tarda and significant down regulation  with  LPS & S.iniae at time post immune challenge. The metal-catalyzed oxidization assay results (Fig.1) elucidated an antioxidant property of HaTRx-2 by protecting super coiled DNA from oxidative damage. On the whole, our data indicated that HaTRx-2 may play an important role in host immune response and regulating oxidative stress.