Differential effect toward inhibition of papain and cathepsin C by recombinant human salivary cystatin SN and its variants produced by a baculovirus system

Ching Chung Tseng*, Ching Ping Tseng, Michael J. Levine, Libuse A. Bobek

*此作品的通信作者

研究成果: 期刊稿件文章同行評審

17 引文 斯高帕斯(Scopus)

摘要

Human salivary cystatin SN (CsnSN) is a member of the cystatin superfamily of cysteine proteinase inhibitors. In this study we used a baculovirus expression system to produce a full-length unaltered CsnSN and its variants. The variants were constructed with the changes in the three predicted proteinase-binding regions: The N-terminus (variant N12-13, G12A-G13A), β-hairpin loop I (variant L56-58, Q56G-T57G-V58G) and β-hairpin loop H (variant L106-107, P106G-W107G). The secreted CsnSNs were purified using sequential spiral cartridge ultrafiltration and DE-52 radial flow chromatography. The purified proteins were examined for papain and cathepsin C-inhibition. The wild-type CsnSN, and variants N12-13 and L106-107 bound tightly to papain (K(i) < 10 pM), whereas mutation in the loop I reduced binding affinity 5700-fold (K(i) = 57 nM). On the other hand, the wild-type CsnSN bound to cathepsin C less tightly (K(i) = 100 nM). The mutation in the N-terminus or loop I reduced binding affinity by 16 (K(i) = 1.6 μM)- and 19-fold (K(i) = 1.9 μM), respectively, while mutation in loop H resulted in an ineffective cathepsin C inhibitor (K(i) = 14 μM). Collectively, these results suggest that the N-terminal G12-G13 residues of CsnSN are not essential for papain inhibition but play a role in cathepsin C inhibition; residues Q56-T57-V58 in the loop I are essential for both papain and cathepsin C inhibitions, and residues P106-W107 in the loop H are not important for papain inhibition but essential for cathepsin C inhibition. These resuits demonstrated that CsnSN variants have different effects toward different cysteine proteinases. (C) 2000 Academic Press.

原文英語
頁(從 - 到)133-140
頁數8
期刊Archives of Biochemistry and Biophysics
380
發行號1
DOIs
出版狀態已出版 - 01 08 2000

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