Molecular Characterization and In Silico Analysis of Defensin From Tor putitora (Hamilton).

Antimicrobial peptides (AMPs) are vital constituents of innate immune system. In fish, one of the most important AMP is β-defensin that has a potential to activate the adaptive immune system. β-defensin is a cysteine-arginine-rich cationic AMP with broad spectrum activity against microbes. In this study, we identified cloned and characterized β-defensin 1 from Tor putitora, a cold water fish species also known as mahseer. An open reading frame of β-defensin 1 was amplified, cloned and sequenced which encodes a peptide of 67 amino acid residues. The pro-peptide includes a signal peptide comprising 24 amino acids as predicted by Signal P along with a mature peptide of 43 amino acid residues. Tor putitora β-defensin 1 (TP-βdf1) has a molecular weight of 4.6 kDa with a pI of 8.35. Six cysteine residues are present in the mature peptide which is a characteristic feature of defensins. All six cysteine residues are involved in the formation of three intra-molecular disulfide bonds. Three-dimensional modeling of mature peptide of TP-βdf1 was carried out using Modeller 9.10, and validated TP-βdf1 model revealed three β-sheets. The cysteine residues form three disulfide bonds in the pattern of Cys(1)-Cys(5), Cys(2)-Cys(4), Cys(3)-Cys(6) stabilizing the β-sheet. Structural analysis revealed three β-strands and an α-helix at the N terminus. Phylogenetic analysis revealed that the β-defensin 1 of Tor putitora was close to Megalobrama amblycephala which possibly suggests that TP-βdf1 peptide sequence is quite similar to β-defensin peptide sequences of carps and minnows.