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FUNCTIONAL CHARACTERIZATION OF BIG-BELLY SEAHORSE Hippocampus abdominalis NATURAL KILLER CELL ENHANCING FACTOR-A (NKEF-A/Prx1)

G. I. Godahewa1,2*, N. C. N. Perera and Jehee Lee
E-mail: imarshana@gmail.com
 
1Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea
2Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea

Natural killer cell enhancing factor (NKEF) has been remarked as a cytosolic protein that enhances the natural killer cell cytotoxic activity. Apart from the cytotoxic activity of the NKEF, it has shown potential antioxidant function similar to that of peroxiredoxin members. Hence, natural killer cell enhancing factor A (NKEF-A) is also referred as peroxiredoxin 1. It protects the host cells from different oxidative damages such as hydrogen peroxide, alkyl hydroperoxide and heavy metals. In the present study, molecular features, functional properties and immune responses of Hippocampus abdominalis NKEF-A (HaNKEF-A) were assessed. Putative open reading frame encoded 594 amino acids with 29.9 kDa polypeptide and a pI of 6.43. Two conserved domains (Prx_tipical_2Cys and Thioredoxin_like) and several active sites including, catalytic triad, dimer interface, decamer, peroxidatic and resolving cysteines were identified through bioinformatics tools. It shared the highest identity (93.4%) and similarity (98%) with the Cyprinodon variegatus Prx1. Multiple sequence alignment revealed the conservation of functionally active peroxidatic and resolving cysteines among the other NKEF-A/Prx1 counterparts suggesting the common peroxidase activity. Metal catalyzed oxidative (MCO) stress, cleaved the pUC19 DNA from supercoiled state into nicked state where, rHaNKEF-A protein could protect the pUC19 DNA cleavage by MCO system in a concentration dependent manner. The rHaNKEF-A catalyze the insulin reduction activity with the presence of 1,4-Dithiothreitol (DTT) in a time dependent manner. The results of the MTT assay revealed that the presence of the rHaNKEF-A increased the cell viability% against the H2O2 oxidative stress. Moreover, the activity was dose dependent and the highest cell viability percentage was gained with the 100 µg/mL of rHaNKEF-A. 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. The HaNKEF-A transcripts were ubiquitously expressed in all examined tissues with highest expression in liver. Bacterial (Edwardsiella tarda, Streptococcus iniae and LPS) and viral (poly I:C) immune stimulated liver tissue showed significant HaNKEF-A mRNA expression after the post infection. Collectively, HaNKEF-A is belonging to the teleostean peroxiredoxin family member with its antioxidant function and potential immune responses upon bacterial and viral challenges. Also, it could be suggested that HaNKEF-A is an active member of seahorse antioxidant defense system.

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