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

钙蛋白酶是一种胞质钙<2+>调节的半胱氨酸蛋白酶。蛋白酶直接与细胞内蛋白相互作用，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calpain基因，其中大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与特定组织的特定功能有关，因此，它们的生理功能引起了人们的极大兴趣。nCL-2/-2'和nCL-4这两种组织特异性钙蛋白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。因此，这些calpain的功能被认为与粘液分泌机制有关。为了阐明这些钙蛋白的生理功能，我们构建了nCL-2: C105S敲入小鼠，在内源性表达控制下表达nCL-2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Cys105到Ser的错义突变，并在C105S突变附近插入带有loxP序列的新霉素耐药基因（neoR）。获得同源靶向小鼠后，与表达Cre-recombinase的转基因小鼠杂交，切除neoR，得到nCL-2: C105S敲入小鼠。利用这些敲入小鼠及其组织，进行了各种生化、细胞生物学和分子生物学分析。结果表明，nCL-2不仅可以形成单体，还可以通过类似c2结构域（结构域III）形成同聚低聚物。这种新颖的结构表明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.