MOLECULAR CHARACTERIZATION AND PEROXIDATION FUNCTIONAL ASSAY OF NATURAL KILLER CELL ENHANCING FACTOR IN SEAHORSE Hippocampus abdominalis  

Natural killer cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family which was originally isolated from human red blood cells. It can protect the DNA and protein against the oxidative damage via enhancing the cytotoxic activity of natural killar cells. Here, we examined the molecular characteristic features and the biological functions of the NKEF from big-belly seahorse (Hippocampus abdominalis) (HaNKEF). In silico characterization was conducted based on full-length of HaNKEF cDNA sequence using bioinformatics tools and webservers. In order to determine the cell viability under oxidative stress, MTT assay was conducted with the recombinant HaNKEF protein (rHaNKEF). Also the effect on the ROS production in the presence of the rHaNKEF protein was determined.

Putative open reading frame (ORF) of HaNKEF encoded 594 amino acids (aa) with 29.9 kDa polypeptide and a pI of 6.43. Two conserved domains (PRX_Tip2cys and Thioredoxin_like domain) and several active sites including, catalytic triad, dimer interface, decamer, peroxidatic and resolving cysteines reactive active sites were identified with the bioinformatics tools. The results of the MTT assay revealed that the presence of the rHaNKEF increased the cell viability % against the H₂O₂ oxidative stress. The activity was dose dependent and the highest cell viability percentage was gained with the 100 µg/ mL of rHaNKEF. The same concentration of the rHaNKEF was given the highest reduction of the ROS level in the human LNCaP cells against the H₂O₂ oxidative stress.