Takahashi S, Nakagawa T, Kasai K, Banno T, Duguay S J, Van de Ven W J, Murakami K, Nakayama K
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
J Biol Chem. 1995 Nov 3;270(44):26565-9. doi: 10.1074/jbc.270.44.26565.
Furin is a Golgi membrane-associated endoprotease that is involved in cleavage of various precursor proteins predominantly at Arg-X-Lys/Arg-Arg sites. Furin itself is synthesized as an inactive precursor, which is activated through intramolecular autocatalytic cleavage at an Arg-X-Lys-Arg site. We previously found that human colon carcinoma LoVo cells have a frameshift mutation within the homo B domain of furin and thereby lack processing activity toward Arg-X-Lys/Arg-Arg sites. In this study, however, we identified a second furin mutation in this cell line. The mutation, a replacement of a conserved Trp residue within the homo B domain with Arg, results in lack of processing activity of the mutant furin. The combination of both mutations can account for the recessive nature of the processing incompetence of LoVo cells. Immunofluorescence analysis with three distinct anti-furin monoclonal antibodies revealed that neither furin mutant underwent the autocatalytic activation or left the endoplasmic reticulum for the Golgi. These data indicate that the homo B domain as well as the catalytic domain is required for autocatalytic activation of furin.
弗林蛋白酶是一种与高尔基体膜相关的内切蛋白酶，主要参与各种前体蛋白在精氨酸- X -赖氨酸/精氨酸-精氨酸位点的切割。弗林蛋白酶本身作为无活性前体被合成，通过在精氨酸- X -赖氨酸-精氨酸位点的分子内自催化切割而被激活。我们之前发现，人结肠癌细胞系LoVo在弗林蛋白酶的同源B结构域内存在移码突变，因此缺乏对精氨酸- X -赖氨酸/精氨酸-精氨酸位点的加工活性。然而，在本研究中，我们在该细胞系中鉴定出了第二个弗林蛋白酶突变。该突变是同源B结构域内一个保守的色氨酸残基被精氨酸取代，导致突变型弗林蛋白酶缺乏加工活性。这两种突变的组合可以解释LoVo细胞加工无能的隐性性质。用三种不同的抗弗林蛋白酶单克隆抗体进行的免疫荧光分析显示，两种弗林蛋白酶突变体均未经历自催化激活，也未离开内质网前往高尔基体。这些数据表明，同源B结构域以及催化结构域是弗林蛋白酶自催化激活所必需的。
