Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. Molecular cloning, expression, and biochemical studies on oryzacystatin-II.
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.
Kondo
H
H
Department of Agricultural Chemistry, University of Tokyo, Japan.
Abe
K
K
Nishimura
I
I
Watanabe
H
H
Emori
Y
Y
Arai
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D015891:Cystatins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012275:Oryza / Q000235:genetics*; D010957:Plasmids; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012639:Seeds / Q000032:analysis*; D012689:Sequence Homology, Nucleic Acid
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D015345:Oligonucleotide Probes; D012313:RNA; D004247:DNA