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

钙蛋白酶是一种胞内受钙离子调节的半胱氨酸水解酶。钙蛋白酶直接与细胞内的蛋白质相互作用，改变或改变其底物的功能和/或活性，因此被称为“调节蛋白水解酶”。哺乳动物有15个独立的钙调蛋白基因，其中大约一半是在组织中特异表达的。这些特定于组织的钙蛋白酶有望参与特定组织的特定功能，因此，它们的生理功能引起了人们的极大兴趣。组织特异性钙蛋白NCL-2/-2‘和NCL-4主要在消化道表达，特别是在上皮性粘液分泌细胞中表达。这些细胞保护胃和肠道免受酸性/蛋白水解酶和细菌的侵害。为了阐明这些钙蛋白酶的生理功能，我们构建了NCL-2：C105S“敲入”小鼠，在内源表达控制下表达NCL-2的蛋白失活突变体，而不是野生型的NCL-2。在小鼠基因组DNA中引入了与Cys105到Ser对应的错义突变，并在C105S突变附近插入了loxP序列两侧的新霉素抗性基因(New R)。获得同源靶标小鼠后，与表达Cre重组酶的转基因小鼠杂交，获得NCL-2：C105S基因敲除小鼠，利用这些敲入小鼠及其组织进行各种生化、细胞生物学和分子生物学分析。结果表明，NCL-2不仅形成单体，而且通过C2结构域样结构域(结构域III)形成均低聚物。这种新的结构提示NCL-2在消化道中具有独特的功能。

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.