Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. Molecular cloning, expression, and biochemical studies on oryzacystatin-II

Abstract

Oryzacystatin (oryzacystatin-I) is a proteinaceous cysteine proteinase inhibitor (cystatin) in rice seeds and is the first well defined cystatin of plant origin. In this study we isolated cDNA clones for a new type of cystatin (oryzacystatin-II) in rice seeds by screening with the oryzacystatin-I cDNA probe. The newly isolated cDNA clone encodes 107 amino acid residues whose sequence is similar to that of oryzacystatin-I (approximately 55% of identity). These oryzacystatins have no disulfide bonds, and so could be classified as family-I cystatins; however, the amino acid sequences resemble those of family-II members more than family-I members. Oryzacystatin-I and -II are remarkably distinct in two respects: 1) their specificities against cysteine proteinases; and 2) the expression patterns of their mRNAs in the ripening stage of rice seeds. Oryzacystatin-I inhibits papain more effectively (Ki 3.0 x 10(-8) M) than cathepsin H (Ki 0.79 x 10(-6) M), while oryzacystatin-II inhibits cathepsin H (Ki 1.0 x 10(-8) M) better than papain (Ki 0.83 x 10(-6) M). The mRNA for oryzacystatin-I is expressed maximally at 2 weeks after flowering and is not detected in mature seeds, whereas the mRNA for oryzacystatin-II is constantly expressed throughout the maturation stages and is clearly detected in mature seeds. Western blotting analysis using antibody to oryzacystatin-II showed that, as is the case with oryzacystatin-I, oryzacystatin-II occurs in mature rice seeds. Thus, these two oryzacystatin species are believed to be involved in the regulation of proteolysis caused by different proteinases.