Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor Sca from sunflower seeds.
Sunflower cystatin a (Sca) is distinguished from other phytocystatins by its lack of the N-terminal about 20 amino acids, resulting in the absence of the evolutionarily conserved Gly residue. The cDNA encoding Sca was amplified by PCR methods. The cDNA consists of 520 nucleotides and includes an open reading frame encoding a polypeptide of 98 amino acids. Comparison of the deduced amino acid sequence with the Sca protein sequence indicated that the deduced sequence has an extra 15 amino acids and one amino acid at the N- and C-termini, respectively. This result suggests that Sca is synthesized as a preprotein (preSca) and proteolytic cleavages at peptide bonds may give rise to the mature Sca. To address this assumption and also to investigate the significance of the N-terminal extension sequence to Sca for inhibitory activity, a recombinant pre-Sca (rpre-Sca), in which the N-terminal extension was fused to the matured Sca, and a recombinant matured Sca (rSca) were overproduced in Escherichia coli cells. Incubation of the rpre-Sca with a seed extract resulted in a mobility by SDS-PAGE that was the same as rSca, demonstrating a proteolytic cleavage by endogenous proteinases. The rSca and rpre-Sca proteins were further characterized with respect to inhibitory activity and sensorgrams of the interaction with papain. The result showed that rpre-Sca had stronger inhibitory activity than rSca, and that the increased activity toward papain was due to a lower dissociation rate constant. This finding indicates that the N-terminal region of rpre-Sca increases the inhibitory activity by stabilizing the rpre-Sca and papain complex.
Kouzuma
Y
Y
Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan. kouzuma@agr.kyushu-u.ac.jp
Tsukigata
K
K
Inanaga
H
H
Doi-Kawano
K
K
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 / Q000096:biosynthesis* / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D006368:Helianthus / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D010206:Papain / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D011994:Recombinant Proteins; D010206:Papain