Investigation on inhibitory characterization of different segments of tarocystatin from Colocasia esculenta

Tarocystatin (CeCPI), a group-2 cysteine proteinase inhibitor in planta, is a defensive protein against phytopathogenic nematodes and fungi. This protein is made up of 205 amino acids, including N terminal segment (Nt peptide) of 98 amino acids and C terminal segment (Ct peptide) of 107 amino acids. The full length (FL), Nt peptide and Ct peptide segments of tarocystatin from Colocasia esculenta (Kaohsiung No.1), were separately amplified by PCR and expressed as GST fusion proteins in E. coli. Kinetic analysis of FL, Nt peptide and Ct peptide on papain activity revealed that FL exhibited mixed type inhibition (Kia =0.098 µM and Kib =0.252 µM) and Nt peptide showed competitive inhibition (Ki, 0.057 µM), whereas Ct peptide enhanced papain activity. Moreover, FL exhibited stronger antipapain activity than Nt peptide. But the antifungal activity of Nt peptide appears to be greater than that of FL, and Ct peptide did not show any antifungal activity indicating that antifungal effect is not related to proteinase inhibition. A shift in the inhibition pattern from competitive inhibition of Nt peptide segment alone to mixed type inhibition of FL implied that Ct peptide has got an influence on FL function, and Nt peptide can determine the fate of CeCPI function. Based on the inhibitory kinetics of FL and fragments of CeCPI on papain activity, a model for group-2 phytocystatin inhibitory mechanism has been proposed. The physiological significance for two varied types of proteins is also discussed.