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

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Abstract

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 95(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- 1HSQC 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. We concluded that the N-terminal region of cystatin A contributes to not only contact and distinguish cognate cysteine proteinases, but also maintain conformational integrity of tripartite reactive wedge of cystatin A.

Original languageEnglish
Pages (from-to)12185-12192
Number of pages8
JournalBiochemistry
Volume34
Issue number38
Publication statusPublished - 1995
Externally publishedYes

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Cystatin A
Cysteine Proteinase Inhibitors
Fluorescence
Substitution reactions
Genetic engineering
Adenylate Kinase
Genetic Engineering
Cysteine Proteases
Acrylamide
Chemical shift
Epidermis
Heating
Escherichia coli
Ion exchange
Swine
Genes
Nuclear magnetic resonance
Degradation

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "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",
abstract = "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 95(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- 1HSQC 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. We concluded that the N-terminal region of cystatin A contributes to not only contact and distinguish cognate cysteine proteinases, but also maintain conformational integrity of tripartite reactive wedge of cystatin A.",
author = "Kazunori Shibuya",
year = "1995",
language = "English",
volume = "34",
pages = "12185--12192",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "38",

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TY - JOUR

T1 - 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

AU - Shibuya, Kazunori

PY - 1995

Y1 - 1995

N2 - 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 95(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- 1HSQC 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. We concluded that the N-terminal region of cystatin A contributes to not only contact and distinguish cognate cysteine proteinases, but also maintain conformational integrity of tripartite reactive wedge of cystatin A.

AB - 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 95(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- 1HSQC 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. We concluded that the N-terminal region of cystatin A contributes to not only contact and distinguish cognate cysteine proteinases, but also maintain conformational integrity of tripartite reactive wedge of cystatin A.

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