Stomach-specific calpain, nCL-2, localizes in mucus cells and proteolyzes the β-subunit of coatomer complex, β-COP

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 two-hybrid screening identified several nCL-2-intracting molecules. Of these, the β-subunit of coatomer complex (β-COP) occurs in the stomach pit cells and is proteolyzed by nCL-2 in vitro. Furthermore, β-COP and nCL-2 co-expressed in COS7 cells co-localized in the Golgi, and Ca2+-ionophore stimulation caused the proteolysis of β-COP near the linker region, resulting in the dissociation of β-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.

intracellular Ca 2+ -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 (1,2,3).
Ubiquitous µand m-calpains, two major calpains in mammals, form a heterodimer composed of a distinct 80 kDa catalytic large subunit (abbreviated to "µCL" and "mCL", respectively) 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 2+ /phospholipid-binding domain (III), the penta-EF-hand domains (IV and VI), and the Gly-clustering domain (V) (4,5).In the absence of Ca 2+ , the protease domain (II) is further divided into two subdomains, IIa and IIb, which form a single active domain upon binding to Ca 2+ (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).
However, our understanding of the specific molecular mechanisms underlying calpain functions in vivo is far from complete.
One clue to the molecular actions of calpain is their tissue-specificity, because this should reflect their specific cellular functions, which are highly differentiated and specialized in the corresponding tissues.
Indeed, a defect in skeletal-muscle-specific calpain causes muscular dystrophy, as mentioned above.nCL-2, also called calpain 8a, is another tissue-specific calpain, which is predominantly expressed in the stomach (16).The domain structure of nCL-2 is identical to that of mCL described above.By alternative splicing, the gene for nCL-2, Capn8, generates nCL-2´ (also called calpain 8b), which lacks domains III and IV (16,17).Possible functions have been reported for nCL-2 in the pituitary gland and for its Xenopus laevis homologue, XCL-2, in embryogenesis (18,19).However, the specific targets and functions of nCL-2 are still unclear.
Another tissue-specific calpain, nCL-4, the expression of which is predominantly in the digestive tract, also occurs in the stomach.
It is reported to be down-regulated in a subset of gastric cancer patients and is involved in the suppression of tumorigenesis (20,21,22).µ-Calpain is also involved in the acid secretory process by cleaving ezrin, a membrane cytoskeletal protein, in parietal cells (23).How these data relate to the physiological functions of nCL-2/-2´ has yet to be elucidated.
The stomach is a highly differentiated organ with specific functions and complex programmed glandular structures, organized into diverse types of epithelial cells.The rodent stomach is composed of four regions: fundus, cardia, corpus, and pylorus, the latter three of which have numerous tubular pits with glands deep within them (24).The pits are lined by pit cells that secrete neutral mucus to protect the epithelium from acid (25).The oxyntic glands in the corpus consist of acid-secreting parietal cells, pepsinogen-secreting chief cells, and hormone-secreting enteroendocrine cells (25,26).Pylorocytes and gastrin-secreting G cells are mainly found in the pyloric glands of the pylorus (27).All these cells originate from proliferative progenitor cells located under the pits and undergo continuous renewal.
The progenitor cells follow a specific differentiation process during their migration upwards, downwards, or both, to their final programmed destinations (25).
Therefore, to understand nCL-2 functions, it is essential first to determine its localization in the stomach.In this study, in situ hybridization (ISH) and immunohistochemical analysis of nCL-2/-2´ were performed to localize them precisely.
Surprisingly, nCL-2 was strictly localized in the pit cells, in sharp contrast to the expression of the ubiquitous calpains.
Furthermore, yeast two-hybrid screening followed by biochemical analysis demonstrated the proteolytic interaction between nCL-2 and the β-subunit of the COPI coatomer complex (β-COP), strongly suggesting novel functions for nCL-2 that involve the membrane trafficking of mucus cells by interacting with coat proteins.The stomach was analyzed using anti-nCL2/2´ (reactive to both nCL-2 and -2´)

Materials
and anti-nCL2C (reactive only to nCL-2) antibodies (Fig. 3A).Firstly, western blot analysis using protein extract from the gastric mucosa showed that the anti-nCL2/2´ antibody recognized nCL-2 but not nCL-2´ (Fig. 3B), indicating undetectable or no expression of nCL-2´ protein in the gastric mucosa, even though the amounts of nCL-2 and -2´ mRNAs in the stomach were similar (see Fig. 4A) (16).The localization of nCL-2 protein was consistent with that observed by ISH (Figs. 3C-F).Significantly, nCL-2 signals were positive in the relatively smaller cells in the pits, and were absent from larger cells (Fig. 3F, arrowheads), which are considered to be the parietal cells located in the pit region (25).
To confirm this, stomach sections were doubly immunostained with antibody directed against H + /K + -ATPase (green cells in Fig. 3G), a marker protein for parietal cells, or one directed against proliferating-cell nuclear antigen (PCNA) (green in Fig. 3H), a marker for progenitor cells.These signals did not overlap with that of nCL-2, indicating that nCL-2 is localized specifically in the pit cells and is thus implicated in pit cell functions.
nCL-2 is expressed in a subset of goblet cells in the small intestine -Northern blot analysis of human and mouse tissues with long exposures revealed that the gene for nCL-2 is expressed at low levels in the trachea and intestines (Fig. 4A).Therefore, these tissues were treated with ISH to detect the expression of nCL-2 and nCL-4 mRNAs.Signals were observed in some epithelial cells scattered in the villi of the duodenum (Figs.4B, C).
Furthermore, the nCL-2-positive cells were scattered in the upper half of the villi close to the luminal surface (Fig. 4B), whereas most of the nCL-4-positive cells were in the lower regions close to the crypt (Fig. 4C).

Immunohistochemical analysis using
anti-nCL2/2´ antibody showed patterns consistent with those obtained with ISH (Fig. 4D).The nCL2/2´-positive cells had an expanded shape in the apical region and were stained in the basal region.clones (30).
A two-hybrid assay using full-length nCL-2 and 30K showed no binding under the same conditions used when µCL and mCL bound 30K (unpublished data), strongly suggesting that nCL-2 exists as a monomer/homo-oligomer without 30K, as is the case for p94 (31).
Among the molecules identified, Although goblet cells are reported to contain several different types of cells (45,46), the distributions of these calpains suggest cell-stage, rather than cell-type, specificity, i.e., nCL-2 functions at late stages, when cells are terminally differentiated, whereas nCL-4 functions at early to mid stages, in processes including the proliferation and differentiation of cells.In the stomach, the localizations of nCL-2 and nCL-4 do not differ, except that the distribution of nCL-4 is slightly broader than that of nCL-2.This is consistent with the idea discussed above that nCL-4 functions in those cells close to the stem cells.
However, the precise roles shared by these calpains must be examined further.present.However, it should be noted that conventional calpains and skeletal-muscle-specific calpain, p94, proteolyze HSP60, a major chaperon protein in the mitochondria (47).Proteolysis of HSP60 was observed not only in vitro, but also in living cultured cells, suggesting a physiological significance (29).Therefore, the interaction between nCL-2 and TOM70 implies a novel but as yet unidentified function of the calpains in mitochondria.
An in vitro assay using a COS7 cell expression system showed that β-COP, but not Eya2, is a proteolytic substrate for nCL-2.
This suggests that Eya2 is instead a regulatory The different levels of expression of nCL-2 in the intestines and trachea, in which the mucosa is also very important, possibly explain the requirement for this extra secretion system in the stomach, mediated by 14 nCL-2, in addition to the usual system.
It is also possible that, like Eya2, β-COP functions as a scaffold protein for nCL-2 in its proteolysis of substrates as yet unidentified.One of the most important questions is how calpain selects specific substrates for proteolysis.A scaffold for proteolysis, like that observed in various kinase cascades, is a strong possibility, although there are few reports of scaffold proteins for calpain (30,58).These possibilities can be explored in mice genetically modified at the gene for nCL-2.
This research is now proceeding in our laboratory.
(to HS) and 02J07250 (to SH), by a Research Grant (14B-4) for Nervous and Mental Disorders from the Ministry of Health, Labor and Welfare, and by Ground-Based Research Announcement for Space Utilization promoted by JSF (to HS).

RESULTS nCL- 2
is expressed specifically in the pit cells of the gastric mucosa -To investigate the localization of nCL-2/-2´ expression in the stomach, in situ hybridization (ISH) analysis of the corpus and pylorus of the mouse stomach was performed.nCL-2/-2´ was expressed around the upper quarter of the corpus (oxyntic) mucosa and the upper half of the pyloric mucosa, where the mucus-secreting pit cells are abundant (Figs.1A, B, 2A, B), whereas µCL and mCL transcripts were detected in all cells including proliferative cells of the pyloric mucosa (Figs 1D, E, 2D, E).The digestive-tract-specific calpain, nCL-4, showed expression similar to but a little wider than that of nCL-2 (Figs.1C, 2C).
Fig. 8B, one major proteolytic fragment (60 kDa) of endogenous β-COP was detected only when COS cells were transfected with wild-type nCL-2 (lane 4, closed arrowhead).The molecular mass of the fragment coincided with that detected by in vitro proteolysis (Fig. 8C, closed arrowhead).The 60 kDa fragment was not observed when -2 and nCL-4 in the oxyntic mucosa, conventional ubiquitous µand m-calpains show rather diffuse and very little expression in this region (see Figs. 1D, especially abundantly in the pit and the isthmus (see Figs. 2D, E).These sharp contrasts strongly suggest that the functions of the tissue-specific calpains in gastrointestinal tissues cannot be compensated by the conventional calpains, at least in the oxyntic mucosa.Yeast two-hybrid screening followed by in vitro pull-down analysis identified several nCL-2-interacting proteins, including TOM70, GPS1, nm23-M2, nm23-M4, Eya2, and β-COP.ISH analysis showed that, of these, only Eya2 and β-COP are expressed in the pit cells of the oxyntic and pyloric mucosae, suggesting an in vivo interaction with nCL-2.Thus, the physiological significance of the observed interactions between nCL-2 and TOM70, GPS1, nm23-M2, and nm23-M4 is not clear at and/or scaffold protein for nCL-2.Eya2 was first identified as one of the highly conserved vertebrate homologues of the Drosophila eyes absent gene product, required for the proper development of compound eyes.It is expressed in the cytoplasm of various tissues relatively late in mammalian development.Following its translocation to the nucleus, Eya2 forms a synergistic complex with the DNA-binding homeodomain-containing Sine oculis 1 (Six1) protein and a transcription factor, Dachshund 2 (Dach2).It thus acts as a transcriptional co-activator and its tyrosine phosphatase activity regulates specific target genes controlling cell proliferation, differentiation, and cell death during mammalian organogenesis (48,49).This study shows, for the first time, the limited expression of Eya2 in the progenitor and pit cells of the stomach.It is possible that nCL-2, binding to Eya2, is involved in gene regulation during pit cell maturation in the stomach.The proteolytic interaction and co-localization of nCL-2 and β-COP in the Golgi strongly suggests novel functions for nCL-2 in intracellular membrane trafficking.β-COP is a subunit of the COPI coatomer complex, which coats the Golgi-derived vesicles involved in protein transport from the Golgi to the ER and within the Golgi.When a COPI-coated vesicle reaches the acceptor membrane, GTP hydrolysis by the small GTPase, ARF1, triggers coat disassembly, allowing vesicle fusion (50).The COPI complex can be reversibly dissociated into two subcomplexes, F-COPI composed of β-, γ-, δ-, and ζ-COPs, and B-COPI composed of α-, β´-, and ε-COPs (51).Sequence similarities, as well as biochemical and structural biological properties, indicate that the F-and B-COPI complexes are significantly similar to the adaptin and clathrin complexes, respectively, other major vesicle-coating proteins involved in transport between the trans Golgi and the plasma membrane (42).β-COP, like γ-COP and the adaptins, can be divided into three regions: an N-terminal α-helical domain called the "adaptin N-terminal domain", a flexible linker region, and a C-terminal ear domain (see Fig. 5D).It should be noted that the nCL-2-binding site of β-COP is in the ear domain, and that the proteolytic site of β-COP (Ser528-Ser529) is located near the boundary between the N-terminal and the linker domains (see Fig. 5D).The linker domains of βand γ-COP bind to the WD40 repeat domains in β´-COP and α-COP, facilitating the assembly of the F-and B-COPI subcomplexes, and regulatory or accessory proteins such as AP180, eps15, amphiphysin, and epsin interact with the ear domains of the adaptins and probably with that of β-COP (42,52,53).Although the mechanisms of the disassembly of the COPI and clathrin-adaptin complexes are not fully understood, the interaction between nCL-2 and β-COP provides a new model for COPI disassembly: nCL-2, binding to the ear domain of β-COP, cleaves the linker region, causing β-COP to dissociate from the Golgi, and resulting in the irreversible disassembly of the coat proteins (Fig. 10).This nCL-2-mediated COPI disassembly model is consistent with the report that calpain may be involved in the maturation of secretory granules via clathrin-adaptin uncoating by the limited proteolysis of clathrin and the adaptins (54).Moreover, the relationship between membrane trafficking and the calpains has been confirmed recently, e.g., calpain localization in the ER and Golgi (55), in lipid rafts (56), and in multivesicular bodies (57) etc., suggesting a general involvement of various calpains in a variety of membrane retrograde, retrieval transport system.The stomach is an "extreme" environment where the pH is extremely low and many infective microorganisms enter, and extensive protection by a tough mucosa is thus essential.To ensure the quality and amount of glycoproteins secreted from the mucosa, such as mucins, a secretion system that includes retrograde transport has to function at full capacity.This may be one reason why the stomach has an additional COPI disassembly system involving nCL-2.

Fig. 3 .
Fig. 3. Specific expression of nCL-2 in the pit cells.(A) Schematic structures of nCL-2 and nCL-2´, and the locations of epitopes for the antibodies used.(B) Protein (20 µg) from mouse gastric mucosa was analyzed by western blotting probed with anti-nCL2/2´ antibody.The arrowhead indicates the band corresponding to nCL-2.nCL-2´ (43 kDa) was not detected.(C-F) Sections of rat stomach oxyntic mucosa were immunostained with anti-nCL2/2´ (C, E, and F) and anti-nCL2C (D) antibodies.Signals were visualized by Alexa 594 (C and D) or HRP methods (E and F).(F) A magnified image of the area indicated by the broken square in (E).Arrowheads in (F) indicate nCL-2-negative parietal cells.(G and H) Rat oxyntic mucosa sections immunostained with anti-nCL2/2´ (red) and anti-H + /K + -ATPase (green) antibodies (G), or with anti-nCL2/2´ (red) and anti-PCNA (green) antibodies (H).Staining with secondary antibody alone yielded negligible background signal in all experiments (unpublished data).Bars, 100 µm.

(
B and C) ISH analysis of mouse duodenum sections was performed using riboprobes for nCL-2 (B) and nCL-4 (C).The lower two panels are magnified images of the upper panels.(D) Immunostaining of the rat duodenum using anti-nCL2/2´ antibody.Arrows indicate nCL-2 immunoreactive goblet cells characterized by their expanded shape in the apical region.L, lumen; E, epithelium; LP, lamina propria.Bars, 100 µm (B and C), 50 µm (D).

Fig. 5 .Fig. 6 .
Fig. 5. Identification of nCL-2-interacting proteins.Schematic structures of nCL-2-binding proteins identified by yeast two-hybrid screening using domain IV and full-length nCL-2 as baits: Eya2 (A), GPS1 (B), TOM70 (C), β-COP (D).Thick lines below the schematic structures indicate the positions and lengths of the identified prey clones (see table1).Thin lines in (A) indicate two truncated constructs used to examine the binding site for Eya2; + and -stand for the binding and unbinding of the fragment to nCL-2, respectively, examined with a yeast two-hybrid system.The closed arrowhead in (D) indicates the proteolytic site of β-COP (see Fig.7).(E) HA-tagged GPS1 (lanes 1 and 2), β-COP (lanes 3 and 4), nm23-M2 (lanes 5 and 6), nm23-M4 (lanes 7 and 8), and Eya 2 (lanes 9 and 10) were transfected with FLAG-tagged nCL-2 (odd lanes) or mock vector (even lanes) into COS7 cells.The cell lysates were immunoprecipitated with anti-FLAG agarose, and subjected to western blot analysis using anti-FLAG and anti-HA monoclonal antibodies.(F) GST (lane 2) or GST-fusion protein (lane 3) of the C-terminal region of TOM70 corresponding to the identified two-hybrid clone (see (A)) was incubated with FLAG-tagged nCL-2:C105S expressed in COS7 cells, and pulled-down by glutathione-Sepharose.Lane 1 shows 10% of the input of FLAG-nCL-2:C105S.

Fig. 10 .
Fig. 10.Schematic illustration of a proposed model for COPI disassembly.A model of the disassembly of the COPI subcomplex with proteolysis by nCL-2, modified from the model proposed by Hoffman et al. (42).nCL-2 binds to the ear domain of β-COP through domain IV and also to possible regulatory molecule(s) such as Eya2.Upon stimulation with Ca 2+ , nCL-2 proteolyzes β-COP close to the C-terminal end of the adaptin N-terminal domain to dissociate the polymeric COPI complex to the single complex.

Table 1 .
Summary of the results of yeast two-hybrid screening For in vitro interactions, see Figs. 5E and F. b n.d., not determined. a