Molecular cloning, functional expression, and mutagenesis of cDNA encoding a cysteine proteinase inhibitor from sunflower seeds.
Sunflower cystatin Scb differs from other phytocystatins in that it is a highly basic protein with a pI value of 9.6 and includes six additional amino acids (Arg30-Leu-Gln-Arg-Thr34, Thr37) in the middle region as compared with other phytocystatins [Kouzuma et al. (1996) J. Biochem. 119, 1106-1113]. We identified and sequenced a complete cDNA encoding the Scb; the cDNA of Scb consists of 645 nucleotides and includes an open reading frame encoding a polypeptide of 123 amino acids. On the basis of these findings, Scb appears to be synthesized as a prepeptide consisting of a signal sequence of 22 amino acids and a mature protein of 101 amino acids. A recombinant Scb (rScb) was produced by expression in Escherichia coli and purified by gel filtration on Sephacryl S-200 followed by ion-exchange column chromatography on a S-Sepharose column. rScb exhibited almost the same inhibitory activity toward papain as the authentic Scb did, but its inhibition profile toward cathepsins B, L, and H was slightly different. Scb mutant proteins, in which selected N-terminal residues or the additional amino acids were deleted, were subsequently constructed and characterized with respect to their inhibitory activities toward papain. The result revealed that the additional sequence (Arg30-Leu-Gln-Arg-Thr34) in Scb is not essential for papain-inhibitory activity, while the N-terminal amino acids (Ile1-Pro2) as well as the N-terminal glycine residues Gly3 and/or Gly4 play an important role in manifesting the inhibitory activity toward papain.
Doi-Kawano
K
K
Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Higashi-ku, Fukuoka, 812-8581, Japan.
Kouzuma
Y
Y
Yamasaki
N
N
Kimura
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D015891:Cystatins / Q000235:genetics*; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics*; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D006368:Helianthus / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins; D012639:Seeds / Q000737:chemistry
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D010940:Plant Proteins; C117023:Scb protein, Helianthus annuus