| Research Abstract |
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.
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