INSIGHTS INTO THE REDOX REGULATORY FUNCTIONS OF GLUTAREDOXIN-1 FROM BLACK ROCKFISH Sebastes schlegelii WITH MOLECULAR AND EXPRESSION ANALYSIS

Glutaredoxins (Grx) are versatile disulfide oxidoreductases with CXXC motifs  that belong to the thioredoxin  fold super-family.  They catalyze  the reduction of protein disulfides, glutathione ( GSH) and  protein mixed disulfides via  coupled reactions with NADPH, GSH and glutathione reductase . As these redox proteins serve  as an inevitable  member  for the  redox homeostasis, it is  implicated as a cellular protector against oxidative stress conditions .  Further, glutaredoxins involve in iron homeostasis and bind labile 2Fe-2S clusters .  The present study was carried out to characterize  rockfish glutaredoxin-1 (SsGrx1) in molecular, transcriptional insights  and  ultimately examine the functional aspects. Recombinant SsGrx1 (rSsGrx1 )  was  used to  perform  the  insulin disulfide reduction  assay. Transient transfection method and WST1 assay was used to study the SsGrx1  cell survival  functions  in FHM cells upon H₂O₂ exposure.

Amino acid sequence analysis  indicated the presence of  specific  motifs and amino acids required for the GSH binding, including T⁶⁹V⁷⁰P⁷¹ motif, G⁸³S⁸⁴D⁸⁵ motif, K²⁰ and Q⁵⁸.  The phylogenetic tree unveiled that a ll the Grx family members were emerged from a single ancestor and affirmed the evolutionary conserveness of  SsGrx1 with the other Grx1 counterparts. Larimichthys crocea  was identified as the evolutionary closest member . SsGrx1 and other vertebrate Grx1  showed higher sequence homology , especially the  two catalytic cysteine residues and the TVP motif for GSH binding were 100% conserved among the counterparts.  Tissue distribution analysis  confirmed an ubiquitous nature of the SsGrx1  basal expression  in naive rockfish tissues with the highest expression  found to be  in  kidney.  Immune  challenge experiment showed a  significant up-regulation of the SsGrx1 mRNA expression in blood and gills with the  injection of LPS,  S. iniae and poly I:C.  During the functional studies, rSsGrx1 was able to reduce the insulin disulphide bonds in a concentration dependent manner.  Further, t he cell survival assay against H₂O ₂ exposure, exhibited a  significant FHM cell  survival compared to the negative controls.

The presence of the  amino acids  and motifs including the TVP motif, K²⁰ and Q⁵⁸ implies the importance of the GSH mediated redox activity which was affirmed by the conservation in multiple sequence alignment.  The tissue distribution of SsGrx1 was found to  have a wide range in the rockfish tissues illustrating its  ubiquitous  functions in  different kind of cells and tissues. The significant upregulation of th e SsGrx1 after  immune challenge denotes the immune relevance of SsGrx1 to prevent the pathogenic attack. The functional studies  revealed  the SsGrx1 function in  insulin disulfide reduction and cell survival against H₂O₂ induces oxidative stress.  Collectively, the results obtained  in this study plausibly suggest the immune  relevance  of SsGrx1 and the relevance for oxidative protection.