Significance of the highly conserved Gly-4 residue in human cystatin A.
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 A (2-98)) was found to be almost identical with that of the wild type, and thereafter a mutation was introduced into this variant (Ctst A(2-98)), 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 A(2-98)[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 effect 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.
Shibuya
K
K
Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, Tokyo.
Kaji
H
H
Ohyama
Y
Y
Tate
S
S
Kainosho
M
M
Inagaki
F
F
Samejima
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D015891:Cystatins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009587:Nitrogen Isotopes; D010206:Papain / Q000037:antagonists & inhibitors; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014633:Valine / Q000378:metabolism
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D010206:Papain; D014633:Valine; D005998:Glycine