MOLECULAR CHARACTERIZATION AND FUNCTIONAL INSIGHT TO MITOCHONDRIAL GLUTATHIONE REDUCTASE HOMOLOG ISOLATED FROM RED LIP MULLET Liza haematocheila

Glutathione reductase (GSHR) is a biologically important enzyme involved in the conversion of oxidized glutathione (GSSG) into its reduced form, reduced glutathione reductase (GSH) with the catalytic activity of NADPH. The precise GSH: GSSG ratio is very much important to a cell in detoxifying a variety of potentially harmful electrophilic compounds such as reactive nitrogen and oxygen species (ROS, RNS). Therefore GSHR which keeps this ratio exactly so is considered to be an imperative enzyme in the cell. Most of the aquatic organisms including fish are fortified with the above enzyme system to neutralize the oxidative stress arises due to toxic substances. In the current study, the sequence of mullet GSHR was identified from the cDNA library followed by the in-silico analysis of the protein using appropriate bioinformatics tools and software. cDNA samples synthesized from twelve different tissues were subjected to quantitative real-time PCR in determining the tissue-specific expression of the gene. cDNA was synthesized from selected tissues in certain time points after the immune challenge of fish with lipopolysaccharide (LPS), poly I:C, Lactococcus garvieae and Phosphate buffered saline (PBS/control) respectively and the recombinant protein purified from the pMAL™ Protein Fusion and Purification System was used in the GSHR activity assay.

Complete ORF of the mullet GSHR consisted of    1527 base pairs encoding 508 amino acids with a predicted molecular weight of 55.43 kDa. Multiple sequence alignment revealed the conservation of the authoritative amino acids within the fish species and the phylogenetic analysis demonstrated clustering of mullet GSHR with other fish GSHR complements with the highest evolutionary relationship. The highest expression of GSHR was observed in the gills tissue of according to the tissue distribution data. This may happened because relatively high concentration of H₂O₂ is accumulated in the gills as H₂O₂ is removed from the gills of fish. Significant up regulation of the gene could be observed in the gills tissue of the fish immunized with L. garvieae after 72 hours of immunization. The recombinant LhGSHR activity assay demonstrated a linear graph confirming proper activity of the enzyme with a specific activity of 35.2 U/mg. Experimental results suggest the significant role of GSHR enzyme in counterbalancing the oxidative stress arises due to pathogenic attack of fish.