J. Biochem. 110, 956-964 (1991)

Takashi Saku,' Hideaki Sakai," Yasnaki Shibata,* Kenji Yamamoto* * * *'

Yuzo Kato," and

'Department of Pathology, Niigata University School of Dentistry, Niigata, Niigata 951; Departments of "Pharmacology and '"Pathology, Nagasaki University School of Dentistry, Nagasaki, Nagasaki 852; and ""Department of Pharmacology, Kyushu University Faculty of Dentistry, Higashi-ku, Fukuoka, Fukuoka 812 Received for publication, July 18, 1991

Immunocytochemical localization of two distinct intracellular aspartic proteinases, cathepsins E and D, in human gastric mucosal cells and various rat cells was investigated by immunogold technique using discriminative antibodies specific for each enzyme. Cathepsin D was exclusively confined to primary or secondary lysosomes in almost all the cell types tested, whereas cathepsin E was not detected in the lysosomal system. The localization of cathepsin E varied with different cell types. Microvillous localization of cathepsin E was found in the intracellular canaliculi of human and rat gastric parietal cells, rat renal proximal tubule cells, and the bile canaliculi of rat hepatic cells. The immunolocalization of each enzyme in gastric cells were essentially the same in humans and rats. In the gastric feveolar epithelial cells and parietal cells, definite immunolabeling for cathepsin E was observed in the cytoplasmic matrix, the cisternae of the rough endoplasmic reticulum, and the dilated perinuclear envelope. In rat kidney, cathepsin E was detected only in the proximal tubule cells, while cathepsin D was found mainly in the lysosomes of the distal tubule cells but not in those of the proximal tubule cells. These results clearly indicate the distinct intracytoplasmic localization of cathepsins E and D and suggest the possible involvement of cathepsin E in extralysosomal proteolysis that is related to specialized functions of each cell type.

Mammalian cells are known to contain two types of intracellular aspartic proteinaatis. One is cathepsin D, which is a representative lysosomal aspartic proteinase and is known to be widely distributed in mammalian tissues and cells (1). The other is cathepsin E, which has been identified in limited sources (2-10) and is relatively poorly characterized. Although cathepsins E and D are clearly distinct proteins (11-13), they have many common biochemical and catalytic features such as pH optimum, substrate specificity, and susceptibility to various proteinase inhibitors (4, 14, 15). These observations raise the possibility that cathepsins E and D may share some physiological functions in cells. Nevertheless, the functional partnership of cathepsins E and D still remains to be determined. Cathepsin D, besides its lysosomal role, has been suggested to be involved in a variety of physiological and pathological processes: for example, in the processing of lysosomal enzymes (16-18) and inflammatory and neoplastic disease states (19-21). In contrast, information on the cellular function of cathepsin E is very limited. Our previous studies on cathepsin E in human erythrocytes, in which the enzyme is located on the cytoplasmic surface of the membrane, have suggested its potential importance in the removal of senescent or damaged erythrocytes from the 1

To whom all correspondence should be addressed. Abbreviations: PBS, phosphate-buffered saline; rER, rough endoplasmic reticulum.

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blood circulation (22). A study of human gastric cathepsin E also has suggested its possible role in development of well differentiated adenocarcinoma from intestinal metaplasia (23). We previously discovered that the distribution of cathepsin E in various rat tissues and blood cells is markedly different from that of cathepsin D by using the discriminative antibodies specific for each enzyme; cathepsin D was found in all of the tissues, whereas cathepsin E had a limited distribution in certain cells such as lymphoid tissues, gastrointestinal tracts, and urinary organs. We also found that the immunohistochemical localization of the two enzymes at the light microscopy level was also different. However, the exact intracellular localization of cathepsin E at the electron microscopy level has not yet been established. To gain further understanding of the cellular functions of cathepsin E, we analyzed its immunocytochemical localization in various cell types of human gastric mucosa and of rat tissues and compared it with that of cathepsin D; we used immunogold labeling techniques with antibodies specific for each enzyme. MATERIALS AND METHODS Preparations—The antisera against cathepsins E and D purified from rat spleen (4, 14) and the antiserum against cathepsin E purified from human erythrocyte membranes (8) were raised in rabbits by essentially the same procedure as that for rat spleen cathepsin D (11). The IgG J. Biochem.

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An Immunocytochemical Study on Distinct Intracellular Localization of Cathepsin E and Cathepsin D in Human Gastric Cells and Various Rat Cells

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1b Fig. 1. Immunocytochemical staining of cathepsin E in foveolar epithelial cells of the hnman stomach, (a) Gold particles indicating antigenetic sites for cathepsin E are accumulated in the cytoplasmic compartment. ER, endoplasmic reticulum; MD, mucinogen droplets; N, nucleus. Original magnification: X30,600. Bar=0.5//m. (b) Photomicrograph of the foveolar epithelial cells from the same section as (a) showing the accumulation of gold particles for cathepsin E in the cisternae of the rER and along the cytoplasmic processes of lateral cell walls (arrowheads). Original magnification: X82.600. Bar=0.2^m. Vol. 110, No. 6, 1991

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fraction from each of these antisera was prepared by ammonium sulfate fractionation followed by protein ASepharose affinity chromatography. These fractions were further purified by affinity chromatography using an enzyme (cathepsin E or cathepsin D)-coupled Sepharose 4B. The monspecific antibody for each enzyme eluted from the column with 0.1 M glycine-HCl buffer (pH2.8) was dialyzed overnight against 10 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl. Rat tissues were obtained from normal Wistar rats which were killed by intracardiac perfusion with isotonic saline, then with 1% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The minced tissue blocks were further fixed in the same fixative for 3 h at 4'C. After washes with phosphate-buffered saline (PBS) and dehydration with ethanol, tissue blocks were embedded in LR White acrylic Resin (London Resin, London). Polymerization in gelatin capsules was performed for 24 h at 60*C. Human gastric tissue specimens were obtained by gas-

troendoscopy from the normal mucosa of 6 Japanese patients with stomach-complaints at Nagasaki University Hospital. The tissue blocks were minced, fixed, and embedded in LR White resin as above. Ultrathin sections were mounted on 300 mesh nickel grids coated with Formvar. Rat tissues were also fixed in 95% ethanol and embedded in paraffin. Tissue sections were immunostained by the PAP method as previously described (25). Immunogold Staining—All incubation were performed in a moist chamber at room temperature by floating grids on 20 fi 1 of reagents, basically according to the method of Goto et al. (26). The grids were blocked with 3% normal goat serum in PBS for 15 min, then incubated with the primary antibodies (50 jug/ml) for 1 h. After washing with PBS, the grids were incubated with colloidal gold-labeled goat antirabbit IgG (gold particles: 5 or 10 nm in diameter; 1 : 20 dilution; Janssen Life Science Products, Olen, Belgium). As a control, the primary antibodies were replaced by normal rabbit IgG. The grids were then counterstained with

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Fig. 3. Immunocytochemical stainings of cathepsins E and D in parietal cells of the human stomach, (a) Gold particles for cathepsin E are found in the cytoplasmic matrix between plentiful mitochondria, as well as in the cytoplasmic processes of intracellular canaliculi (arrowheads). TV, tubulovesicular system; M, mitochondria. Original magnification: x 40,800. Bar=0.5 //m. (b) Gold particles for cathepsin D appear in lysosomes (arrows). ER, rough endoplasmic reticulum; M, mitochondria. Original magnification: X39.100. Bar=0.5 Fig. 2. Immunocytochemical stainings of cathepsins E and D in foveolar epithelial cells of the human stomach, (a) A similar section to Fig. 1, but showing a portion of dilated perinudear spaces which are continuous with the rER Arrows indicate rims of the isolation membranes. Gold partides for cathepsin E are accumulated in the dilated perinudear spaces. N, nucleus. Original magnification: X 33,400. Bar=0.5 ftm. (b) A portion of Fig. la is shown enlarged. N, nucleus. Original magnification: x 105,500. Bar=0.1 pm. (c) Gold particles for cathepsin D appear in lysosome-like bodies (arrows). L, lysosomes; M, mitochondria. Original magnification: X43.200. Bar=0.2 pm. Vol. 110, No. 6, 1991

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Immunocytochemical Localization of Cathepsins E and D

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The intracytoplasmic localization of cathepsin E presented a marked contrast to that of cathepsin D. Cathepsin D was exclusively confined to lysosomes, whereas cathepsin E was distributed diffusely in the cisternae of the rough endoplasmic reticulum (rER) and of the nuclear envelope and often in the cytoplasmic matrix. Microvilli were another area in which cathepsin E was concentrated. The immunocytochemical localization of cathepsins E and D in each cell type will hereafter be described in detail. Stomach—Immunolocalization of cathepsins E and D in the stomach was essentially the same in humans and rats, although the density of labeled gold particles was much higher in human gastric cells than in those of the rat. Among the gastric cells, Lhe foveolar epithelial cells and the parietal cells gave definite immunolabeling for both cathepsins E and D. In the foveolar epithelial cells, electron dense

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tft

cytoplasmic matrix was pushed aside to periphery by an accumulation of mucinogen droplets. Gold particles indicating cathepsin E antigenecity were scattered in the electron dense portion (Fig. la). It is apparent from Fig. lb that cathepsin E was accumulated in the cisternae of the rER in the surface epithelial cells. In addition, there was a small number of gold particles for cathepsin E on the cytoplasmic processes. Occasionally the foveolar epithelial cells had dilated perinuclear cisternae which were continuous with the rER. Such perinuclear spaces contained a great amount of gold particles for cathepsin E (Fig. 2, a and b). In contrast, cathepsin D was limited to the lysosome-like bodies which were located beneath the clusters of mucinogen droplets (Fig. 2c). The parietal cells presented distribution patterns of cathepsins E and D similar to those of the foveolar epithelial cells. Gold particles indicating cathepsin E antigenicity were notable within cistic spaces of the tubulovesicular system as well as around microvilli of intracellular canaliculi (Fig..3a). The labeling for cathepsin D was confined to lysosome-like granules (Fig. 3b). The density of gold particles for cathepsin D was higher in the lysosomes of gastric parietal cells than of any other type of gastric cells in the stomach. There was no apparent immunolabeling for cathepsins E and D in the Golgi apparatus of any types of gastric cells. Kidney—Cathepsin E was enriched in brush borders of rat proximal tubules at the light microscopy level (24). Our immunoelectron microscopy study using ultrathin frozen sections also revealed that gold particles for cathepsin E were concentrated along the microvilli of those cells (Fig. 4). At this level of resolution, the antigenic sites for cathepsin E appeared to be located on or within the microvilli with an intimate association with the plasma membranes. Such intense immunolabeling was obtained on ultrathin frozen sections. The labeling on resin-embedded sections was weak. Cathepsin D was localized exclusively in the lysosomes-like bodies of the epithelial cells of distal tubules (Fig. 5a), but hardly any gold particles for cathepsin

£1 Fig. 4. Ultrathin frozen sections showing immunocytochemical localization of cathepsin E in microvilli of rat renal proximal tubule cells. Gold particles for cathepsin E are concentrated along the microvilli. Original magnification: x39,000. Bar=0.5^m. J. Biochem.

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uranyl acetate and lead citrate in the usual manner and examined in a Hitachi H700 electron microscope at 100 kV. Cryo-Ultramicrotomy—Ultrathin frozen sections were utilized for the tissues in which antigenic sites for cathepsin E were poorly preserved after resin embedding. Tissue samples from rat liver and kidney were fixed in the PLP fixative [2% paraformaldehyde/0.75 M lysine/0.01 M NaIO 4 /0.0375 M sodium phosphate (pH6.2)] for 2 h at room temperature, cryoprotected by incubation with 2.3 M sucrose in 0.1 M phosphate buffer (pH 7.4) and then frozen in liquid propane cooled with liquid nitrogen. Ultrathin frozen sections were cut on a Reichert Ultracut E equipped with an FC4D cryoattachment, using the technique of Tokuyasu (27). Sections were mounted on 300 mesh grids coated with Formvar and carbon. After blocking and immunostaining in mostly the same manner as described above, the grids were postfixed with 1% glutaraldehyde and further with 1% osmium tetraoxide, stained with 4% uranyl acetate, dehydrated, and embedded in LR White acrylic resin, as described in detail elsewhere (28).

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Fig. 5. Immunocytochemical staining of cathepsin D in rat renal distal (a) and proximal (b) tubule cells, (a) Gold particles for cathepsin D are accumulated in lysosome-like bodies of the distal tubule cells (arrows). M, mitochondria. Original magnification: x35,700. Bar=0.5//m. (b) Hardly any gold particles for cathepsin D are detected in lysosome-like structures of the proximal tubule cells. Original magnification: X28.400. Bar=0.5//m.

D were detected in the lysosomes in those of proximal tubules (Fig. 5b). Liver—Figure 6a shows an immunoperoxidase staining pattern of cathepsin E in rat liver at the light microscopy level. Cathepsin E was sharply demonstrated around the bile canaliculi in the hepatic cell borders and at the cell membrane facing Disse's space. At the ultrastructural level, gold particles for cathepsin E were found around the microvilli of hepatic cells (Fig. 6b). Only cryo-ultramicrotomy allowed clear immunolocalization of cathepsin E along the microvilli. Cathepsin D was shown to be localized in lysosomes within sinusoidal Kupffer cells (not shown). Spleen—Only a small number of gold particles for cathepsin E was diffusely distributed in the cytoplasm of macrophages in the splenic cords (not shown), whereas cathepsin D was abundantly demonstrated in the lysosomal bodies of macrophages (Fig. 7). In Fig. 7, lysosomes that contain gold particles for cathepsin D are fused with or attached to the surface of some internalized erythrocytic debris. Cathepsin D was also accumulated in the phagosomes of various sizes. DISCUSSION The primary aim of the present study is to clarify the subcellular localization of two distinct intracellular aspartic Vol. 110, No. 6, 1991

proteinases, cathepsins E and D, in human gastric cells and in various cell types of rat tissues by using antibodies specific for each enzyme. To know the exact intracytoplasmic localization of cathepsin E, we employed ultrathin frozen sections, besides ultrathin sections of resin-embedded specimens, for the detection of cathepsin E. The ultrathin frozen sections were more suitable for detection of antigenetic sites for cathepsin E, which were poorly preserved after resin embedding. The microvillous localization of cathepsin E in the kidney and liver could be clearly demonstrated with these sections. The present results show that the subcellular localization of cathepsins E and D was noticeably different in all of the cell types examined. Cathepsin D was exclusively confined to lysosomes, although the concentration of the enzyme varied in each cell type. By contrast, cathepsin E was not localized to the lysosomal system and appeared to have a strategic localization in certain cell types. The microvillous localization of cathepsin E found in gastric parietal cells, renal proximal tubule cells, and hepatic cell borders suggests that the enzyme may be associated with specialized functions of these cell types, such as absorption and secretion. The existence of the membrane-associated form of cathepsin E has also been shown in erythrocytes (29). The present study also showed that cathepsin E was present in the cytosolic compartment of gastric foveolar epithelial

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Fig. 6. Tmnninnrtalnlngti of cathepsin E in the rat liver at the light (a) and electron (b) microscopy levels, (a) Cathepsin E is localized to the bile canaliculi in the hepatic cell borders. Original magnification: X244. Bar=50^m. (b) Ultrathin frozen sections showing immunocytochemical localization of cathepsin E in microvilli of the hepatic cells (arrows). Original magnification: x 49,400. Bar=0.2 fim.

cells and of parietal cells, suggesting that the enzyme may be involved in extralysosomal proteolysis in such cells, which have not only high absorptive and secretive activities but also a rapid regenerating activity. Rat neutrophils have also been shown to contain cathepsin E in the cytoplasmic compartment (9, 24, 30). It is of special interest that high levels of cathepsin E were found in the cisternae of the rER and the dilated nuclear envelope of the gastric cells. A similar accumulation of cathepsin E in the cisternae of the rER was observed in human gastric foveolar cells (31). At this time, it is unclear whether cathepsin E exerts its proteolytic activity in such neutral compartments. Since, however, it has been shown that cathepsin E from human erythrocyte membranes is stabilized by physiological concentrations of ATP or RNAs and exhibits its catalytic activity even at around neutral pH (32, 33), it seems likely that the enzyme is regulated by such cellular factors to play a part in intracellular extralysosomal proteolysis. Our present data give no information on the biosynthesis of cathepsin E. However, considering the findings that cathepsin E is a glycoprotein (4, 6, 34, 35) and has an oligosaccharide chain of the nigh-mannose type (36), the enzyme must be initially synthesized on membrane-bound ribosomes in a manner similar to lysosomal proteins. There is no evidence to

validate the intracellular transport of cathepsin E from the ER to the Golgi complex, since the labeling intensity in the Golgi complex was too low to be detected by the technique employed in this study. However, such low labeling in the Golgi complex has also been noted for cathepsins B and D in various rat tissues (37-39); nevertheless, these cathepsins, as well as other lysosomal enzymes, are thought to be transported to lysosomes via the Golgi complex. In addition, cathepsin E purified from rat stomach has been shown to contain galactose (36). Therefore, cathepsin E must be transported from the ER to the Golgi complex. It is unknown at present how cathepsin E as a non-lysosomal glycoprotein is segregated and transported into the cytosolic compartment. It is noteworthy that the renal cathepsin D was mainly contained in lysosomes of the distal tubule cells and that no significant positive staining for cathepsin D was observed in lysosomes of the proximal tubule cells. The results were in good agreement with those obtained with the immunohistochemical study at the light microscopy level (24). Yokota et al. (38) have shown that cathepsin D is immunocytochemically localized in lysosomes of the distal tubule cells in rat kidney. This is also quite compatible with our present results. Therefore, it seems likely that the involvement of cathepsin D in the lysosomal proteolysis in the proximal J. Biochem.

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tubule cells is lower than in the distal tubule cells. By contrast, gold particles positive for cathepsin E are concentrated in the brush borders of the proximal tubules and are very small in the distal tubule cells and the collecting tubule cells. The present study, therefore, indicates that the major expression sites for cathepsins D and E in rat kidney are clearly different, suggesting that the physiological significance of these two enzymes in the kidney is distinct. Since the proximal tubules are a major site for uptake and degradation of proteins that are filtered through the glomerulus, cathepsin E may be related to these specialized functions. REFERENCES 1. Barrett, A.J. (1977) in Proteinases in Mammalian Cells and Tissues (Barrett, A.J., ed.) pp. 209-248, North-Holland, Amsterdam 2. Lapresle, C. & Webb, T. (1962) Biochem. J. 84, 455-462 3. Turk, V., Kregar, I., &Lebez, D. (1968) EnzymologiaM, 89-100 4. Yamamoto, K., Kateuda, N., & Kato, K. (1978) Eur. J. Biochem. 92, 499-508 5. Roberts, N.B. & Taylor, W.H. (1978) Biochem. J. 169, 617-624 6. Kageyama, T. & Takahashi, K. (1980) J. Biochem. 87, 725-735 7. Muto, N., Murakami-Arai, K., & Tani, S. (1983) Biochim. Biophys. Acta 745, 61-69 8. Yamamoto, K. & Marchesi, V.T. (1984) Biochim. Biophys. Acta 790, 208-218 9. Ichimaru, E., Sakai, H., Saku, T., Kunimatsu, K., Kato, Y., Kato, I., & Yamamoto, K. (1990) J. Biochem. 108, 1009-1015 10. Yonezawa, S. & Nakamura, K. (1991) Biochim. Biophys. Acta 1073, 155-160 Vol. 110, No. 6, 1991

11. Yamamoto, K., Kamata, O., Katsuda, N., & Kato, K. (1980) J. Biochem. 87, 511-516 12. Puizdar, V., Lapresle, C, & Turk, V. (1985) FEBS Lett. 186, 236-238 13. Jupp, R.A., Richards, A.D., Kay, J., Dunn, B.M., Wyckoff, J.B., Samloff, I.M., & Yamamoto, K. (1988) Biochem. J. 254,895-898 14. Yamamoto, K., Katsuda, N., Himeno, M., & Kato, K. (1979) Eur. J. Biochem. 95, 459-467 16. Takeda, M., Ueno, E., Kato, Y., & Yamamoto, K. (1986) J. Biochem. 100, 1269-1277 16. Nishimura, Y. & Kato, K. (1988) Arch. Biochem. Biophys. 260, 712-718 17. Nishimura, Y., Kawabata, T., & Kato, K. (1988) Arch. Biochem. Biophys. 261, 64-71 18. Nishimura, Y., Kawabata, T., Furuno, K., & Kato, K. (1989) Arch. Biochem. Biophys. 271, 400-406 19. Capony, F., Morisset, M., Barrett, A.J., Capony, J.P., Broquet, P., Vignon, F., Chambon, M., Louisot, P., & Rochefort, H. (1987) J. Cell Biol. 104, 253-262 20. Capony, F., Rouquet, C, Montcourrier, P., Cavailles, V., Salazar, G., & Rochefort, H. (1989) Cancer Res. 49, 3904-3909 21. Poole, A.R. (1975) in Dynamics of Connective Tissue Macromolecules (Burleigh, P.M.C. & Poole, A.R., eds.) pp. 357-383, North-Holland, Amsterdam 22. Yamamoto, K., Yamada, M., & Kato, Y. (1989) J. Biochem. 105, 114-119 23. Saku, T., Sakai, H., Tsuda, N., Okabe, H., Kato, Y., & Yamamoto, K. (1990) Gut 31, 1250-1255 24. Sakai, H.( Saku T., Kato, Y., & Yamamoto, K. (1989) Biochim. Biophys. Acta 991, 367-375 25. Stemberger, L.A., Hardy, P.H., Cuculis, J.J., & Meyer, H.G. (1970) J. Histochem. Cytochem. 18, 315-333 26. Goto, M., Meyermann, R., &Wekerle, H. (1987) Histochemistry

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Fig. 7. Immunocytochemical staining of cathepsin D in macrophages of the rat splenic pnlp. Gold particles for cathepsin D are accumulated in lysosome-like structures of the macrophage. L, lysosome-Uke bodies; E, erythrocytic debris. Original magnification: x 30,800. Bar=0.5>/m.

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87, 201-207 27. Tokuyasu, K.T. (1986) J. Microsc. 143, 139-149 28. Saku, T. & Furthmayer, H. (1986) J. Biol. Chem. 264, 35143523 29. Ueno, E., Sakai, H., Kato, Y., & Yamamoto, K. (1989) J. Biochem. 105, 878-882 30. Yonezawa, S., Fujiki, K., Maejima, Y., Tamoto, K., Mori, Y., & Muto, N. (1988) Arch. Biochem. Biophys. 267, 176-183 31. Thomas, D.J., Richards, A.D., Jupp, R.A., Ueno, E., Yamamoto, K., Samloff, I.M., Dunn, B.M., & Kay, J. (1989) FEBS Lett. 243, 145-148 32. Yamamoto, K., Sakai, H., Ueno, E., & Kato, Y. (1991) in Structure and Function of the Aspartic Proteinases (Dunn, B.M., ed.) pp. 297-306, Plenum Press, New York 33. Fiocca, R., Villani, L., Tenti, P., Comaggia, M., Finzi, G., Riva,

T. Saku et al.

An immunocytochemical study on distinct intracellular localization of cathepsin E and cathepsin D in human gastric cells and various rat cells.

Immunocytochemical localization of two distinct intracellular aspartic proteinases, cathepsins E and D, in human gastric mucosal cells and various rat...
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