Abstract
Calpains constitute a superfamily of Ca2+-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit) cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8−/− and Capn9−/− mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8−/− stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9−/−. Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed “G-calpain,” in which both were essential for activity. This is the first example of a “hybrid” calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S “knock-in” (Capn8CS/CS) mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8−/− stomach, that of the Capn8CS/CS mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric mucosal defense, and they point to G-calpain as a potential target for gastropathies caused by external stresses.
Highlights
Calpain (Clan CA-C2, EC 3.4.22.17) is a family of intracellular Ca2+-regulated cysteine proteases found in almost all eukaryotic and some bacterial, and all the members of this family share homology in the protease domain [1,2]
These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric mucosal defense, and they point to G-calpain as a potential target for gastropathies caused by external stresses
The functions of tissue-specific calpains are most likely relevant to the tissues expressing them; there is an advantage to studying them over ubiquitous calpains, which are indispensable in embryogenesis or show functional redundancy [22,24,25]
Summary
Calpain (Clan CA-C2, EC 3.4.22.17) is a family of intracellular Ca2+-regulated cysteine proteases found in almost all eukaryotic and some bacterial, and all the members of this family share homology in the protease domain [1,2]. Calpains play indispensable roles in biological processes including the cell cycle, apoptosis, platelet aggregation, and myoblast fusion, through the limited proteolytic cleavage of diverse substrates. The mammalian calpains include both ubiquitous and tissuespecific members [2]. The gastrointestinal-tract (GI) expresses two major ubiquitous calpains, m- and m-calpain, and two GI-specific ones, calpain 8/nCL-2 and calpain 9/nCL-4 [9,10]. The m- and m-calpains each form a heterodimer consisting of a distinct 80kDa catalytic subunit (calpain 1/mCL and calpain 2/mCL, respectively) and a common 30-kDa regulatory subunit (CAPNS1/30K), which acts as a molecular chaperone for calpains 1 and 2. Calpains 8 and 9 have a typical domain structure like that of calpains 1 and 2: a regulatory N-terminal domain (domain I), protease domain (II, consisting of sub-domains IIa and IIb), C2like Ca2+/phospholipid-binding domain (III), and penta-EF-hand (PEF) domain (IV) (Figure 1A)
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