The NH2-terminal 21 amino acid residues are not essential for the papain-inhibitory activity of oryzacystatin, a member of the cystatin superfamily. Expression of oryzacystatin cDNA and its truncated fragments in Escherichia coli.
Oryzacystatin, a proteinaceous cysteine proteinase inhibitor (cystatin) in rice, is comprised of 102 residues (Met1-Ala102) (Abe, K., Emori, Y., Kondo, H., Suzuki, K., and Arai, S. (1987) J. Biol. Chem. 262, 16793-16797). We constructed an expression plasmid containing a full length oryzacystatin cDNA at the multi-cloning site of pUC18 and produced a lacZ'-oryzacystatin fusion protein in Escherichia coli. The partially purified expressed protein efficiently inhibits papain activity assayed using N-benzoyl-DL-arginine-2-naphthylamide as a substrate. We also constructed expression plasmids lacking the 5'- and 3'-regions of cDNAs that encode NH2- and COOH-terminally truncated oryzacystatins. An N-truncated oryzacystatin lacking Gly5 and retaining Gln53-Val54-Val55-Ala56-Gly57 inhibited papain as efficiently as the full length oryzacystatin, although both Gly5 and Gln53-Gly57 (oryzacystatin numbering) are conserved among members of most cystatin superfamilies. However, another N-truncated oryzacystatin lacking the NH2-terminal 38 residues was almost completely inactive. On the other hand, a COOH-terminally truncated oryzacystatin lacking the COOH-terminal 11 residues possesses potent papain-inhibitory activity, whereas another COOH-terminally truncated oryzacystatin lacking 35 residues shows much less inhibitory activity, although it retains the two well conserved features Gly5 and Gln53-Gly57. These results indicate that the NH2-terminal 21 residues containing Gly5 and the COOH-terminal 11 residues are not essential, suggesting that a portion of the polypeptide segment containing Gln53-Gly57 is necessary for oryzacystatin to elicite its papain-inhibitory activity efficiently.
Abe
K
K
Department of Agricultural Chemistry, University of Tokyo, Japan.
Emori
Y
Y
Kondo
H
H
Arai
S
S
Suzuki
K
K
D000595:Amino Acid Sequence; D015853:Cysteine Proteinase Inhibitors; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D010206:Papain / Q000037:antagonists & inhibitors*; D010446:Peptide Fragments / Q000096:biosynthesis; D011480:Protease Inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D013329:Structure-Activity Relationship
D015853:Cysteine Proteinase Inhibitors; D010446:Peptide Fragments; D011480:Protease Inhibitors; D004247:DNA; D010206:Papain