Human cystatin A is inactivated by engineered truncation. The NH2-terminal region of the cysteine proteinase inhibitor is essential for expression of its inhibitory activity.
A series of NH2-terminal truncated forms of human cysteine proteinase inhibitor, cystatin A, was prepared by genetic engineering using Escherichia coli harboring mutated genes. Each variant of cystatin A was efficiently expressed as a fused protein with porcine adenylate kinase and released by CNBr degradation after exchange of the sole inner Met to Leu. The mutant cystatin A lacking an amino-terminal Met residue (called standard variant starting from Ile2, CystA2-98(M65L) showed the same inhibitory activity as authentic one isolated from human epidermis. Two-residue truncation scarcely influenced the activity, but further truncations deleting Pro3 and beyond conservative Gly4 and Gly5 caused a remarkable decrease of their inhibitory activity. But little effect was observed by a substitution of Pro3 with Leu. The loss of the activity by amino-terminal truncation was compensated slightly by engineered substitution of Gly75 with His on a second loop. In the two-dimensional 15N-1H HSQC NMR spectrum, four-residue truncation was found to cause changes in the chemical shifts of Val47 and Val48, which locate on a first loop and consist of a conservative QVVAG sequence. Furthermore, the truncation led to a change in fluorescence spectroscopic behavior of Trp75, which was introduced as a probe on the second loop. Fluorescence intensity of the Trp of the truncated (5-98) form was more affected by heating than the active standard variant. Conversely, fluorescence of Trp75 in 2-98 form was more quenched by acrylamide than the 5-98 variant. Thus, the amino-terminal region of cystatin A is essential for the expression of its inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Shibuya
K
K
Department of Chemistry, College of Science & Engineering, Aoyama Gakuin University, Tokyo, Japan.
Kaji
H
H
Itoh
T
T
Ohyama
Y
Y
Tsujikami
A
A
Tate
S
S
Takeda
A
A
Kumagai
I
I
Hirao
I
I
Miura
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015891:Cystatins / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism*; D004817:Epidermis / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D011994:Recombinant Proteins