Abstract
The expression system for human recombinant cystatin A has already been established to be a fusion protein with porcine adenylate kinase in Escherichia coli [Kaji et al. (1990) Biol. Chem. Hoppe-Seyler 371, Suppl., 145-150]. After cyanogen bromide cleavage of the fused protein expressed in E. coli, the cystatin portion could be readily isolated. The inhibitory activity of the obtained variant (Cyst A2-98)was found to be almost identical with that of the wild type, and thereafter a mutation was introduced into this variant (Cyst A2-98)called the standard variant. To elucidate the role of the Gly-4 residue, which is completely conserved in all cystatin species, this residue was substituted with 17 other amino acids by means of cassette mutagenesis. Thus 17 variants (Cyst A2-98[G4X]) obtained were examined as to their inhibitory activity towards papain. As the side chain of the substituted amino acid residue became more bulky, the inhibitory activity of the variant markedly decreased. Variants whose side chains were bulkier than a Val residue showed almost no inhibitory activity towards papain. Consequently, it was deduced that the large side chain of a substituted amino acid may cause steric hindrance, which may be responsible for the decrease in inhibitory activity. Thus, we could conclude that the 4th (Gly) residue on cystatin A must be small, because amino acids which existed on the N-terminal side of this residue could interact with a papain molecule.
| Original language | English |
|---|---|
| Pages (from-to) | 635-642 |
| Number of pages | 8 |
| Journal | Journal of Biochemistry |
| Volume | 118 |
| Issue number | 3 |
| Publication status | Published - 1995 Sep |
| Externally published | Yes |
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Keywords
- Cassette mutagenesis
- Cystatin A
- Cysteine proteinase inhibitor
- Inhibitory activity
- Papain
ASJC Scopus subject areas
- Statistics, Probability and Uncertainty
- Applied Mathematics
- Physiology (medical)
- Radiology Nuclear Medicine and imaging
- Molecular Biology
- Biochemistry
Cite this
Significance of the highly conserved gly-4 residue in human cystatin A. / Shibuya, Kazunori; Kaji, Hiroyuki; Ohyama, Yukihito; Tate, Shin ichi; Kainosho, Masatsune; Inagaki, Fuyuhiko; Samejima, Tatsuya.
In: Journal of Biochemistry, Vol. 118, No. 3, 09.1995, p. 635-642.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Significance of the highly conserved gly-4 residue in human cystatin A
AU - Shibuya, Kazunori
AU - Kaji, Hiroyuki
AU - Ohyama, Yukihito
AU - Tate, Shin ichi
AU - Kainosho, Masatsune
AU - Inagaki, Fuyuhiko
AU - Samejima, Tatsuya
PY - 1995/9
Y1 - 1995/9
N2 - The expression system for human recombinant cystatin A has already been established to be a fusion protein with porcine adenylate kinase in Escherichia coli [Kaji et al. (1990) Biol. Chem. Hoppe-Seyler 371, Suppl., 145-150]. After cyanogen bromide cleavage of the fused protein expressed in E. coli, the cystatin portion could be readily isolated. The inhibitory activity of the obtained variant (Cyst A2-98)was found to be almost identical with that of the wild type, and thereafter a mutation was introduced into this variant (Cyst A2-98)called the standard variant. To elucidate the role of the Gly-4 residue, which is completely conserved in all cystatin species, this residue was substituted with 17 other amino acids by means of cassette mutagenesis. Thus 17 variants (Cyst A2-98[G4X]) obtained were examined as to their inhibitory activity towards papain. As the side chain of the substituted amino acid residue became more bulky, the inhibitory activity of the variant markedly decreased. Variants whose side chains were bulkier than a Val residue showed almost no inhibitory activity towards papain. Consequently, it was deduced that the large side chain of a substituted amino acid may cause steric hindrance, which may be responsible for the decrease in inhibitory activity. Thus, we could conclude that the 4th (Gly) residue on cystatin A must be small, because amino acids which existed on the N-terminal side of this residue could interact with a papain molecule.
AB - The expression system for human recombinant cystatin A has already been established to be a fusion protein with porcine adenylate kinase in Escherichia coli [Kaji et al. (1990) Biol. Chem. Hoppe-Seyler 371, Suppl., 145-150]. After cyanogen bromide cleavage of the fused protein expressed in E. coli, the cystatin portion could be readily isolated. The inhibitory activity of the obtained variant (Cyst A2-98)was found to be almost identical with that of the wild type, and thereafter a mutation was introduced into this variant (Cyst A2-98)called the standard variant. To elucidate the role of the Gly-4 residue, which is completely conserved in all cystatin species, this residue was substituted with 17 other amino acids by means of cassette mutagenesis. Thus 17 variants (Cyst A2-98[G4X]) obtained were examined as to their inhibitory activity towards papain. As the side chain of the substituted amino acid residue became more bulky, the inhibitory activity of the variant markedly decreased. Variants whose side chains were bulkier than a Val residue showed almost no inhibitory activity towards papain. Consequently, it was deduced that the large side chain of a substituted amino acid may cause steric hindrance, which may be responsible for the decrease in inhibitory activity. Thus, we could conclude that the 4th (Gly) residue on cystatin A must be small, because amino acids which existed on the N-terminal side of this residue could interact with a papain molecule.
KW - Cassette mutagenesis
KW - Cystatin A
KW - Cysteine proteinase inhibitor
KW - Inhibitory activity
KW - Papain
UR - http://www.scopus.com/inward/record.url?scp=0029119009&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029119009&partnerID=8YFLogxK
M3 - Article
C2 - 8690729
AN - SCOPUS:0029119009
VL - 118
SP - 635
EP - 642
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 3
ER -