Functional analysis of digestive tract-specific calpains in terms of functional foods.

钙蛋白酶是一种胞质Ca^<2+>调节的半胱氨酸蛋白酶.钙蛋白酶直接与细胞内蛋白质相互作用以修饰或转化其底物的功能和/或活性，因此被称为“调节蛋白酶”。哺乳动物有15个独立的钙蛋白酶基因，其中约一半是组织特异性表达的。这些组织特异性钙蛋白酶被认为参与了特定组织中的特定功能，因此，它们的生理功能受到了极大的关注。两种组织特异性钙蛋白酶nCL-2/-2'和nCL-4主要在消化道中表达，尤其是在上皮粘液分泌细胞中。这些细胞保护胃和肠免受酸/蛋白酶和细菌的侵害。为了阐明这些钙蛋白酶的生理功能，我们构建了nCL-2：C105 S“敲入”小鼠，该小鼠在内源性表达控制下表达nCL-2的蛋白酶失活突变体而不是野生型nCL-2。在小鼠基因组DNA中引入对应于Cys 105至Ser的错义突变，并且在C105 S突变附近插入侧翼为loxP序列的新霉素抗性基因（neoR）。在获得同源靶向小鼠后，将它们与表达Cre重组酶的转基因小鼠杂交以切除neoR，从而产生nCL-2：C105 S敲入小鼠。结果显示，nCL-2不仅形成单体，而且通过C2结构域样结构域（结构域III）形成同源寡聚体。这种新的结构表明nCL-2在消化道中的独特功能。

Calpains are a cytosolic Ca^<2+>-regulated cysteine protease. Calpains directly interact with intracellular proteins to modify or transform functions and/or activities of their substrates, and are thus called "modulator protease". Mammals have 15 independent genes for calpains, about half of which are expressed tissue specifically. These tissue-specific calpain are expected to be involved in specific functions developed in the specific tissues, and, therefore, their physiological functions are of great interest. Two of the tissue specific calpains, nCL-2/-2' and nCL-4, are predominantly expressed in digestive tracts, especially, in the epithelial mucus secreting cells. These cells protect the stomach and intestines from acid/proteases and bacteria. Therefore, these calpains' functions are considered to be related with mucus secretion mechanisms.To elucidate physiological functions of these calpains, we constructed nCL-2: C105S "knock in" mice, which express a protease inactive mutant of nCL-2 instead of wild type nCL-2 under endogenous expression controls. A missense mutation corresponding to the Cys105 to Ser was introduced in the mouse genomic DNA, and neomycin-resistance gene (neoR) flanked by loxP sequences was inserted in the vicinity of the C105S mutation. After homologously targeted mice were obtained, they were crossed with Cre-recombinase expressing transgenic mice to excise neoR, resulting in nCL-2: C105S knock-in mice.Using these knock-in mice and their tissues, various biochemical, cell-biological, and molecular biological analyses have been performed. As a result, nCL-2 was shown to form not only monomer but also homo-oligomers via C2-domain like domain (domain III). This novel structure suggests unique functions for nCL-2 in digestive tracts.