Stomach-specific Calpain, nCL-2, Localizes in Mucus Cells and Proteolyzes the β-Subunit of Coatomer Complex, β-COP

Abstract

Calpain is a Ca2؉ -regulated cytosolic protease. Mammals have 14 calpain genes, half of which are predominantly expressed in specific organ(s); the rest are expressed ubiquitously. A defect in calpains causes lethality/pathogenicity, indicating their physiological indispensability. nCL-2/calpain-8a was identified as a stomach-specific calpain, whose physiological functions are unclear. To elucidate these, we characterized nCL-2 in detail. Unexpectedly, nCL-2 was localized strictly to the surface mucus cells in the gastric epithelium and the mucus-secreting goblet cells in the duodenum. Yeast twohybrid screening identified several nCL-2-intracting molecules. Of these, the ␤-subunit of coatomer complex (␤-COP) occurs in the stomach pit cells and is proteolyzed by nCL-2 in vitro. Furthermore, ␤-COP and nCL-2 co-expressed in COS7 cells co-localized in the Golgi, and Ca 2؉ -ionophore stimulation caused the proteolysis of ␤-COP near the linker region, resulting in the dissociation of ␤-COP from the Golgi. These results strongly suggest novel functions for nCL-2 that involve the membrane trafficking of mucus cells via interactions with coat protein.Calpain (EC 3.4.22.17) is an intracellular Ca 2ϩ -regulated cysteine protease, comprising a superfamily in most organisms, including some bacteria, budding yeasts, fungi, plants, and animals. Calpains proteolyze specific substrates at very limited sites to irreversibly modify/modulate their functions, structures, and activities, and are thus called "modulator proteases." Fourteen calpain genes have been identified in humans and mice and can be classified into two categories according to their expression, i.e. ubiquitous or tissue-specific (1-3). Ubiquitous -and m-calpains, two major calpains in mammals, form a heterodimer composed of a distinct 80-kDa catalytic large subunit (abbreviated to "CL" and "mCL," respectively) 2 and a common 30-kDa regulatory small subunit (30K). Calpain three-dimensional structures define four (I-IV) and two (V and VI) domains in the large and small subunits, respectively: the regulatory N-terminal domain (I), the protease domain (II), the C2-domain-like Ca 2ϩ /phospholipid-binding domain (III), the penta-EF-hand domains (IV and VI), and the Glyclustering domain (V) (4, 5). In the absence of Ca 2ϩ , the protease domain (II) is further divided into two subdomains, IIa and IIb, which form a single active domain upon binding to Ca 2ϩ (6, 7). Genetic studies in mammals and other organisms have clearly shown the physiological indispensability of the calpains. A gene knock-out mouse with a mutation in the gene for 30K, Capn4, exhibits embryonic lethality (8); mutations in the human gene for skeletal-muscle-specific p94/calpain 3, CAPN3, are responsible for limb-girdle muscular dystrophy type 2A (9); and a single nucleotide polymorphism in intron 3 of the human gene for calpain 10, CAPN10, is associated with type 2 diabetes (10). Mutations in the calpain genes of various other organisms, including yeasts, plants, nematodes, and Drosophila, result in sign...

Drug-Induced Alterations to Gene and Protein Expression in Intestinal Epithelial Cell 6 Cells Suggest a Role for Calpains in the Gastrointestinal Toxicity of Nonsteroidal Anti-Inflammatory Agents

“…We attempted to validate drug-induced changes in the expression of calpains 8, 2, and 1 as well as calpastatin using qRT-PCR and immunoblotting to assess expression of mRNA and protein. We focused on those signaling molecules, either because of their expression in the gastrointestinal tract or because of their association with signal transduction in migrating cells (Franco and Huttenlocher, 2005;Hata et al, 2006). The results of qRT-PCR validated our microarray findings that NS-398 induced down-regulation of calpain 8 and calpain 2 mRNA.…”

Activity-dependent calpain activation plays a critical role in synaptic facilitation and post-tetanic potentiation

“…It was suggested that in mucosa cells calpain dissociates b-coat protein and triggers vesicular trafficking (Hata et al 2006). Thus, it is conceivable that calpain participates in the process of membrane cycling and thereby may affect the rates of vesicle refilling.…”

Stomach-specific Calpain, nCL-2/Calpain 8, Is Active without Calpain Regulatory Subunit and Oligomerizes through C2-like Domains

“…Materials-Recombinant human -and m-calpains, antinCL2 polyclonal antibody (previously called anti-nCL2/2Ј), and mouse gastric mucosal protein were prepared as described previously (28,30). RP2-calpain 8 polyclonal antibody, anti-FLAG (clone M2), anti-hemagglutinin (HA) 2 (clone 6E2), anti-MYC, and anti-His monoclonal antibodies were purchased from Triple Point Biologics Inc. (OR), Sigma, Cell Signaling Technology Inc., Developmental Studies Hybridoma Bank (IA), and Novagen Inc., respectively.…”

“…Possible functions for nCL-2 in the pituitary gland and in the embryogenesis of Xenopus laevis have been reported (26,27). We recently showed that, in the stomach, nCL-2 as well as nCL-4/calpain 9, a digestive tract-specific calpain, are expressed specifically in the gastric mucus cells (pit cells), quite unlike the -and m-calpains, which are expressed diffusely in all cells, and that nCL-2 may be involved in the regulation of vesicle trafficking in the pit cells (28). These reports highlight potentially important nCL-2 functions that cannot be compensated for by -and m-calpains, not only in the stomach but also in other tissues.…”