Molecular cloning and characterization of oryzacystatin-III, a novel member of phytocystatin in rice (Oryza sativa L. japonica)

On the basis of cDNA sequences, we found that the calli of rice encodes an amino acid sequence that shares 56% and 89% identity, respectively, with oryzacystatin-I and oryzacystatin-11. This sequence differs from that of oryzacystatin-II in the N-terminal region (Gln 7 -Ala 19 in the oryzacystatin-III numbering), and this region contained a glycine residue (Gly 14 ), which is evolutionarily conserved in the cystatin superfamily. We named this novel protein oryzacystatin-III. Nucleotide sequencing of the 5'-flanking region of the oryzacystatin-III gene showed that it is highly homologous to the oryzacystatin-II gene but distinct from the oryzacystatin-II locus. Oryzacystatin-III inhibited papain, ficin, and human cathepsin B. The inhibition constants for papain and ficin differ from those of oryzacystatin-I and -II, and cathepsin B activity is affected only by oryzacystatin-III, showing differences in the interaction of these inhibitors with enzymes. These data suggest that the above three inhibitors may play unique physiological roles in the regulations of rice cysteine proteinases.