Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor with three cystatin domains from sunflower seeds.
Two cysteine proteinase inhibitors, cystatins Sca and Scb, were previously isolated from sunflower seeds [Kouzuma et al. J. Biochem. 119 (1996) 1106-1113]. A cDNA clone encoding a novel phytocystatin with three repetitive cystatin domains was isolated from a cDNA library of sunflower seeds using the Sca cDNA fragment as a hybridization probe. The cDNA insert comprises 1,093 bp and encodes 282 amino acid residues. The deduced amino acid sequences of the domains are highly similar to each other (66-81%), sharing 65-90% identical residues with Sca. The cDNA was expressed in Escherichia coli cells, and then the recombinant sunflower multicystatin (SMC) was purified and its inhibitory activity toward papain was examined. SMC exhibited strong inhibitory activity toward papain, with a stoichiometry of 1:3, indicating that each cystatin domain independently functions as a potent cysteine proteinase inhibitor. Proteolysis of SMC with Asn-specific proteinase suggested that post-translational processing by an Asn-specific proteinase may give rise to mature Sca-like phytocystatins.
Kouzuma
Y
Y
Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki Higashi-ku, Fukuoka 812-8581, Japan. kouzuma@agr.kyushu-u.ac.jp
Inanaga
H
H
Doi-Kawano
K
K
Yamasaki
N
N
Kimura
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D015891:Cystatins / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006368:Helianthus / Q000235:genetics*; D008969:Molecular Sequence Data; D010940:Plant Proteins; D017434:Protein Structure, Tertiary; D012639:Seeds / Q000235:genetics
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D010940:Plant Proteins; C413764:SMC protein, Helianthus annuus