Differential effect toward inhibition of papain and cathepsin C by recombinant human salivary cystatin SN and its variants produced by a baculovirus system.
Differential effect toward inhibition of papain and cathepsin C by recombinant human salivary cystatin SN and its variants produced by a baculovirus system.
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DOI：
10.1006/abbi.2000.1909
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发表时间：
2000-08
影响因子：
3.9
通讯作者：
C. Tseng;C. Tseng;M. Levine;L. Bobek
中科院分区：
文献类型：
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作者：
C. Tseng;C. Tseng;M. Levine;L. Bobek
人唾液半胱氨酸蛋白酶抑制剂 SN (CsnSN) 是半胱氨酸蛋白酶抑制剂半胱氨酸蛋白酶抑制剂超家族的成员。在这项研究中，我们使用杆状病毒表达系统来产生全长未改变的 CsnSN 及其变体。这些变体是通过三个预测的蛋白酶结合区的变化构建的：N末端（变体N（12-13），G12A-G13A），β-发夹环I（变体L（56-58），Q56G-T57G-V58G）和β-发夹环II（变体L（106-107），P106G-W107G）。使用连续螺旋滤芯超滤和 DE-52 径向流色谱纯化分泌的 CsnSN。检查纯化蛋白质的木瓜蛋白酶和组织蛋白酶 C 抑制作用。野生型 CsnSN 以及变体 N(12-13) 和 L(106-107) 与木瓜蛋白酶紧密结合 (K(i) < 10 pM)，而环 I 中的突变使结合亲和力降低 5700 倍 (K(i) = 57 nM)。另一方面，野生型 CsnSN 与组织蛋白酶 C 的结合不太紧密 (K(i) = 100 nM）。 N 末端或环 I 的突变使结合亲和力分别降低 16 倍 (K(i) = 1.6 microM) 和 19 倍 (K(i) = 1.9 microM)，而环 II 的突变导致组织蛋白酶 C 抑制剂无效 (K(i) = 14 microM)。总的来说，这些结果表明 CsnSN 的 N 端 G12-G13 残基对于木瓜蛋白酶抑制不是必需的，但在组织蛋白酶 C 抑制中发挥作用；环 I 中的残基 Q56-T57-V58 对于木瓜蛋白酶和组织蛋白酶 C 抑制都是必需的，环 II 中的残基 P106-W107 对于木瓜蛋白酶抑制并不重要，但对于组织蛋白酶 C 抑制是必需的。这些结果表明，CsnSN 变体对不同的半胱氨酸蛋白酶具有不同的作用。
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 N(12-13), G12A-G13A), beta-hairpin loop I (variant L(56-58), Q56G-T57G-V58G) and beta-hairpin loop II (variant L(106-107), 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 N(12-13) and L(106-107) 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 microM)- and 19-fold (K(i) = 1.9 microM), respectively, while mutation in loop II resulted in an ineffective cathepsin C inhibitor (K(i) = 14 microM). 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 II are not important for papain inhibition but essential for cathepsin C inhibition. These results demonstrated that CsnSN variants have different effects toward different cysteine proteinases.