Functional characterization of the N-terminal and C-terminal domains of a sesame group II phytocystatin.

BACKGROUND

Phytocystatins are natural inhibitors of cysteine protease, and may regulate endo- or exo-genous proteolytic activities in plants. They are classified into Group I and II differing by the presence of C-terminal extension of Group II. A cDNA fragment encoding a Group II phytosystatin, SiCYS was previously obtained from sesame seeds.

RESULTS

SiCYS as well as its two structural domains, N-terminal and C-terminal domains (SiCYS-N and SiCYS-C), was expressed in Escherichia coli. The recombinant SiCYS and SiCYS-N showed inhibitory activity against papain. The K i values of SiCYS and SiCYS-N were ~1.9 ×10 M and ~7.9 ×10 M, respectively. All the three recombinants possessed comparable ability to inhibit spore germination of Trichoderma reesei, Aspergillus sydowii, and Helminthosporium sesamum. Similar protein profile including proteases in germinating seeds was found in proteins purified by the SiCYS, SiCYS-N or SiCYS-C coupling affinity column.

CONCLUSION

SiCYS exhibited more effective papain-inhibitory activity than SiCYS-N; while SiCYS-C had almost no inhibitory activity. All displayed similar antifungal activities indicating that there is no correlation between antifungal and papain-inhibitory activities. Structural and sequence analyses suggest that the C-terminal domain of SiCYS may be originated from gene duplication to enhance its inhibitory activity.