Leduc R, Molloy S S, Thorne B A, Thomas G
Vollum Institute, Oregon Health Sciences University, Portland 97201.
J Biol Chem. 1992 Jul 15;267(20):14304-8.
Human furin is a calcium-dependent serine endoprotease that can efficiently cleave many precursor proteins on the carboxyl side of the consensus cleavage sequence, -Arg-X-Lys/Arg-Arg-, both in vivo and in vitro. Analysis of furin proteins in extracts of cells infected with a vaccinia recombinant expressing human furin show that the enzyme is present as two prominent forms of 90 and 96 kDa. Because the structurally related bacterial subtilisins require endoproteolytic removal of the NH2-terminal pro-region by an autocatalytic intramolecular cleavage, we speculated that the size heterogeneity in the furin doublet similarly may result from a proteolytic removal of an NH2-terminal pro-region. Here we report identification of the 90-kDa furin NH2 terminus and, based on the reported sequence of the furin cDNA, demonstrate that this furin protein is derived from a larger precursor by an endoproteolytic cleavage on the COOH-terminal side of a consensus furin cleavage site, -Arg-Thr-Lys-Arg107-. Expression of mutant furin molecules containing an altered cleavage site (Arg104----Ala or Arg107----Gly) resulted in the production of only the 96-kDa furin protein. Assays of furin-dependent cleavage of a protein substrate in vitro showed that proteolytic activity was associated with the 90-kDa and not the 96-kDa furin protein, demonstrating that removal of the NH2-terminal pro-region is required for furin activity. Expression of a third furin construct containing a mutation of the active site aspartate (Asp153----Asn) similarly resulted in the expression of only the 96-kDa protein, suggesting that furin activation occurs by an autoproteolytic cleavage. Finally, the production of 90-kDa furin from either site-directed furin mutant could not be potentiated by overexpressing active furin, suggesting that the autoproteolytic activation was an intramolecular event.
人弗林蛋白酶是一种钙依赖性丝氨酸内切蛋白酶，在体内和体外均能有效地在共有切割序列-Arg-X-Lys/Arg-Arg-的羧基侧切割许多前体蛋白。对感染表达人弗林蛋白酶的痘苗重组体的细胞提取物中的弗林蛋白酶蛋白进行分析表明，该酶以90 kDa和96 kDa两种主要形式存在。由于结构相关的细菌枯草杆菌蛋白酶需要通过自催化分子内切割对内氨基端前肽区域进行内切蛋白水解去除，我们推测弗林蛋白酶双峰中的大小异质性可能同样是由于氨基端前肽区域的蛋白水解去除所致。在此，我们报告了对90 kDa弗林蛋白酶氨基端的鉴定，并根据已报道的弗林蛋白酶cDNA序列，证明该弗林蛋白酶蛋白是通过在共有弗林蛋白酶切割位点-Arg-Thr-Lys-Arg107-的羧基端进行内切蛋白水解切割从更大的前体衍生而来。表达含有改变的切割位点（Arg104→Ala或Arg107→Gly）的突变弗林蛋白酶分子仅产生96 kDa的弗林蛋白酶蛋白。体外对弗林蛋白酶依赖性蛋白底物切割的测定表明，蛋白水解活性与90 kDa而非96 kDa的弗林蛋白酶蛋白相关，这表明去除氨基端前肽区域是弗林蛋白酶活性所必需的。表达含有活性位点天冬氨酸突变（Asp153→Asn）的第三种弗林蛋白酶构建体同样仅产生96 kDa的蛋白，这表明弗林蛋白酶的激活是通过自蛋白水解切割发生的。最后，过表达活性弗林蛋白酶不能增强来自任何一种定点弗林蛋白酶突变体的90 kDa弗林蛋白酶的产生，这表明自蛋白水解激活是一个分子内事件。
