Production and characterization of two variants of human cystatin SA encoded by two alleles at the CST2 locus of the type 2 cystatin gene family.
Production and characterization of two variants of human cystatin SA encoded by two alleles at the CST2 locus of the type 2 cystatin gene family.
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DOI：
10.1006/abbi.1997.0609
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发表时间：
1998-04
影响因子：
3.9
通讯作者：
E. Saitoh;K. Minaguchi;O. Ishibashi
中科院分区：
文献类型：
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作者：
E. Saitoh;K. Minaguchi;O. Ishibashi
在大肠杆菌中表达了由2型人胱抑素基因家族的CST 2位点的两个等位基因编码的两种胱抑素SA变体。一种称为半胱氨酸蛋白酶抑制剂SA 1，与半胱氨酸蛋白酶抑制剂SA相同[S. Isemura，E. Saitoh和K. Sanada J.Biochem.102，693-704，1987]。另一种称为半胱氨酸蛋白酶抑制剂SA 2，携带两个氨基酸取代（59 Gly->Asp; 120 Glu->Asp），其中一个在所谓的QXVXG区域（第一个发夹环），另一个在分子的C末端部分。从大肠杆菌的周质组分中纯化了四种重组半胱氨酸蛋白酶抑制剂[全长半胱氨酸蛋白酶抑制剂SA 1、两种N端截短的四残基半胱氨酸蛋白酶抑制剂SA 1（WSPQ）和六残基半胱氨酸蛋白酶抑制剂SA 1（WSPQEE）以及全长半胱氨酸蛋白酶抑制剂SA 2]。coli细胞。两个N-末端截短的重组半胱氨酸蛋白酶抑制剂SA 1抑制牛组织蛋白酶C的Ki值比全尺寸的低2至20倍。然而，在木瓜蛋白酶和无花果蛋白酶的抑制中，N-末端截短的半胱氨酸蛋白酶抑制剂SA 1的Ki值比全尺寸的高10倍。在抑制木瓜蛋白酶，无花果蛋白酶，重组人组织蛋白酶K，重组半胱氨酸蛋白酶抑制剂SA 2分别显示，3826-，1090-，和30倍的Ki值相比，SA 1。重组半胱氨酸蛋白酶抑制剂SA 2抑制牛组织蛋白酶C的Ki值比SA 1低50倍。重组半胱氨酸蛋白酶抑制剂SA 1不抑制人组织蛋白酶H，但SA 2对其有轻微抑制作用（Ki = 528 nM）。两种重组变体均不抑制牛组织蛋白酶B。我们的数据提供了证据表明，半胱氨酸蛋白酶抑制剂超家族的第一个发夹环的氨基酸序列在抑制木瓜蛋白酶、无花果蛋白酶、组织蛋白酶C、组织蛋白酶H和组织蛋白酶K中是重要的。
Two variants of cystatin SA encoded by two alleles at the CST2 locus of the type 2 human cystatin gene family were expressed in Escherichia coli. One, termed cystatin SA1, is identical to cystatin SA [S. Isemura, E. Saitoh, and K. Sanada J. Biochem. 102, 693-704, 1987]. Another, termed cystatin SA2, carries two amino acid substitutions (59Gly-->Asp; 120Glu-->Asp), one of which is in the so-called QXVXG region (the first hairpin loop) and another in the C-terminal portion of the molecule. Four recombinant cystatins [full-sized cystatin SA1, two N-terminally truncated cystatin SA1 lacking four residues (WSPQ) and six residues (WSPQEE), and full-sized cystatin SA2] were purified from the periplasmic fractions of E. coli cells. Two N-terminally truncated recombinant cystatin SA1 inhibited bovine cathepsin C with 2- to 20-fold lower Ki values than that of the full-sized one. In the inhibition of papain and ficin, however, both of the N-terminally truncated cystatin SA1 displayed a 10-fold higher Ki value than that of full-sized one. In the inhibition of papain, ficin, and recombinant human cathepsin K, recombinant cystatin SA2 showed, respectively, 3826-, 1090-, and 30-fold higher Ki values compared with those of SA1. Recombinant cystatin SA2 inhibited bovine cathepsin C with a 50-fold lower Ki value compared with that of SA1. Recombinant cystatin SA1 did not inhibit human cathepsin H but SA2 inhibited it slightly (Ki = 528 nM). Neither of the recombinant variants inhibited bovine cathepsin B. Our data supply evidence indicating that the amino acid sequence of the first hairpin loop of the cystatin superfamily is important in the inhibition of papain, ficin, cathepsin C, cathepsin H, and cathepsin K.