Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of ∼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.

中文翻译：

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来自狗绦虫细粒棘球绦虫的单域 Kunitz 蛋白 EgKU-7 是一种高亲和力胰蛋白酶抑制剂，具有两个相互作用位点


典型的 Kunitz 蛋白（MEROPS 数据库的 I2 家族，Kunitz-A 家族）是后生动物丝氨酸肽酶的竞争性抑制剂，可形成 1:1 化学计量的紧密复合物，模仿底物。细粒棘球绦虫是一种引起人类和牲畜囊性包虫病的狗绦虫，它编码一个扩展的单域 Kunitz 蛋白家族，其中一些蛋白被分泌到狗宿主界面。 Kunitz 蛋白 EgKU-7 除了具有包含关键精氨酸的抗肽酶环的 Kunitz 结构域外，还包含约 20 个氨基酸的 C 端延伸。使用 EgKU-7（一种 C 末端截短变体和一种关键精氨酸被丙氨酸取代的突变体）进行的动力学、电泳和质谱研究表明，EgKU-7 是牛和犬胰蛋白酶的紧密抑制剂，具有不寻常的特性具有两个而不是一个与肽酶相互作用的位点的特性。一个位点位于抗肽酶环中，可被牛胰蛋白酶部分水解，但不能被犬胰蛋白酶水解，表明对目标酶具有特异性。另一个位点位于 C 端延伸部分。这种延伸可以在特定的精氨酸中被阳离子牛和犬胰蛋白酶水解，但不能被阴离子犬胰蛋白酶水解。据我们所知，这是第一次报道单域 Kunitz-A 蛋白与其靶标有两个相互作用位点。考虑到 EgKU-7 的推定直系同源物存在于其他绦虫中，我们的发现揭示了肽酶-抑制剂相互作用的新片段，并为进化宿主-寄生虫协奏曲添加了新的音符。