Functional analysis of stomach-specific calpain.

钙蛋白酶是一种胞质Ca2+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14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。其中一种组织特异性钙蛋白酶nCL-2/-2′主要在胃中表达，特别是在上皮“坑”细胞中。胃窝细胞分泌粘液保护胃免受胃酸和细菌的侵害。因此，nCL-2的功能被认为与粘液分泌机制有关。为了阐明nCL-2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。因此，β - cop是COPI复合体的亚基之一，在高尔基到内质网逆行膜运输中起作用，已被确定为nCL-2的底物。此外，在β - cop的情况下，nCL-2被证明在高尔基体中定位。这些结果有力地表明nCL-2在膜运输的背景下起作用，这是钙蛋白酶作用方式的全新概念。

Calpains are a cytosolic Ca2+-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 14 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. One of the tissue-specific calpain, nCL-2/-2', is predominantly expressed in the stomach, especially, in the epithelial "pit" cells. The pit cells secrete mucus to protect the stomach from acid and bacteria. Therefore, nCL-2 functions are considered to be related with mucus secretion mechanisms.To elucidate physiological functions of nCL-2, 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, beta-COP, which is one of the subunits of COPI complex functioning in the Golgi-to-ER retrograde membrane trafficking, has been identified as a substrate for nCL-2. Moreover, nCL-2 was shown to localize in the Golgi as in the case of beta-COP. These results strongly suggest that nCL-2 functions in the context of membrane trafficking, which is brand-new concept for mode of action of calpains.