Cathepsin E Deficiency Induces a Novel Form of Lysosomal Storage Disorder Showing the Accumulation of Lysosomal Membrane Sialoglycoproteins and the Elevation of Lysosomal pH in Macrophages
2006; Elsevier BV; Volume: 282; Issue: 3 Linguagem: Inglês
10.1074/jbc.m604143200
ISSN1083-351X
AutoresM Yanagawa, Takayuki Tsukuba, Tsuyoshi Nishioku, Yoshiko Okamoto, Kuniaki Okamoto, Ryosuke Takii, Yoshihiro Terada, Keiichi I. Nakayama, Tomoko Kadowaki, Kenji Yamamoto,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoCathepsin E, an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, has an important role in immune responses. However, little is known about the precise roles of cathepsin E in this system. Here we report that cathepsin E deficiency (CatE-/-) leads to a novel form of lysosome storage disorder in macrophages, exhibiting the accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 and the elevation of lysosomal pH. These striking features were also found in wild-type macrophages treated with pepstatin A and Ascaris inhibitor. Whereas there were no obvious differences in their expression, biosynthesis, and trafficking between wild-type and CatE-/- macrophages, the degradation rates of these two membrane proteins were apparently decreased as a result of cathepsin E deficiency. Because there was no difference in the vacuolar-type H+-ATPase activity in both cell types, the elevated lysosomal pH in CatE-/- macrophages is most likely due to the accumulation of these lysosomal membrane glycoproteins highly modified with acidic monosaccharides, thereby leading to the disruption of non-proton factors controlling lysosomal pH. Furthermore, the selective degradation of LAMP-1 and LAMP-2, as well as LIMP-2, was also observed by treatment of the lysosomal membrane fraction isolated from wild-type macrophages with purified cathepsin E at pH 5. Our results thus suggest that cathepsin E is important for preventing the accumulation of these lysosomal membrane sialoglycoproteins that can induce a new form of lysosomal storage disorder. Cathepsin E, an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, has an important role in immune responses. However, little is known about the precise roles of cathepsin E in this system. Here we report that cathepsin E deficiency (CatE-/-) leads to a novel form of lysosome storage disorder in macrophages, exhibiting the accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 and the elevation of lysosomal pH. These striking features were also found in wild-type macrophages treated with pepstatin A and Ascaris inhibitor. Whereas there were no obvious differences in their expression, biosynthesis, and trafficking between wild-type and CatE-/- macrophages, the degradation rates of these two membrane proteins were apparently decreased as a result of cathepsin E deficiency. Because there was no difference in the vacuolar-type H+-ATPase activity in both cell types, the elevated lysosomal pH in CatE-/- macrophages is most likely due to the accumulation of these lysosomal membrane glycoproteins highly modified with acidic monosaccharides, thereby leading to the disruption of non-proton factors controlling lysosomal pH. Furthermore, the selective degradation of LAMP-1 and LAMP-2, as well as LIMP-2, was also observed by treatment of the lysosomal membrane fraction isolated from wild-type macrophages with purified cathepsin E at pH 5. Our results thus suggest that cathepsin E is important for preventing the accumulation of these lysosomal membrane sialoglycoproteins that can induce a new form of lysosomal storage disorder. Cathepsin E is a endolysosomal aspartic proteinase of the pepsin superfamily, which is predominantly expressed in cells of the immune system (1Yamamoto K. Turk V. Proteases: New Perspectives. Birkhauser Verlag, Basel, Switzerland1999: 59-71Google Scholar). In antigen presenting cells, such as macrophages and microglia, cathepsin E is mainly localized in the endosomal compartment (2Sastradipura D.F. Nakanishi H. Tsukuba T. Nishishita K. Sakai H. Kato Y. Gotow T. Uchiyama Y. Yamamoto K. J. Neurochem. 1998; 70: 2045-2056Crossref PubMed Scopus (87) Google Scholar, 3Nishioku T. Hashimoto K. Yamashita K. Liou S.Y. Kagamiishi Y. Maegawa H. Katsube N. Peters C. Von Figura K. Saftig P. Katunuma N. Yamamoto K. Nakanishi H. J. Biol. Chem. 2002; 277: 4816-4822Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Previous studies have demonstrated that cathepsin E is important for exogenous antigen processing via the major histocompatibility complex class II presenting system (3Nishioku T. Hashimoto K. Yamashita K. Liou S.Y. Kagamiishi Y. Maegawa H. Katsube N. Peters C. Von Figura K. Saftig P. Katunuma N. Yamamoto K. Nakanishi H. J. Biol. Chem. 2002; 277: 4816-4822Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 4Bennett K. Levine T. Ellis J.S. Peanasky R.J. Samloff I.M. Kay J. Chain B.M. Eur. J. Immunol. 1992; 22: 1519-1524Crossref PubMed Scopus (186) Google Scholar, 5Medd P.G. Chain B.M. Semin. Cell Dev. Biol. 2000; 11: 203-210Crossref PubMed Scopus (24) Google Scholar) and is increasingly expressed in inflammatory cells within and nearby carcinomas (6Matsuo K. Kobayashi I. Tsukuba T. Kiyoshima T. Ishibashi Y. Miyoshi A. Yamamoto K. Sakai H. Hum. Pathol. 1996; 27: 184-190Crossref PubMed Scopus (57) Google Scholar), Clara cells, and the reactive type II pneumocytes (7Bosi F. Silini E. Luisetti M. Romano A.M. Prati U. Silvestri M. Tinelli C. Samloff I.M. Fiocca R. Am. J. Respir. Cell Mol. Biol. 1993; 8: 626-632Crossref PubMed Scopus (16) Google Scholar, 8Arbustini E. Morbini P. Diegoli M. Grasso M. Fasani R. Vitulo P. Fiocca R. Cremaschi P. Volpato G. Martinelli L. Vigano M. Samloff I.M. Solcia E. Am. J. Pathol. 1994; 145: 310-321PubMed Google Scholar). Recent gene or protein expression profiles have demonstrated the increased expression of cathepsin E in several types of carcinomas (9Ullmann R. Morbini P. Halbwedl I. Bongiovanni M. Gogg-Kammerer M. Rapotti M. Gabor S. Renner H. Ropper H.H. J. Pathol. 2004; 203: 798-807Crossref PubMed Scopus (54) Google Scholar, 10Fukushima N. Sato N. Prasad N. Leach S.D. Hruban R.H. Goggins M. Oncogene. 2004; 23: 9042-9051Crossref PubMed Scopus (94) Google Scholar, 11Lewis B.C. Klimstra D.S. Socci N.D. Xu S. Koutcher J.A. Varmus H.E. Mol. Cell. Biol. 2005; 25: 1228-1237Crossref PubMed Scopus (102) Google Scholar, 12Busquets L. Guillen H. DeFord M.E. Suckow M.A. Navari R.M. Castellino F.J. Prorok M. Tumor Biol. 2006; 27: 36-42Crossref PubMed Scopus (15) Google Scholar), which is significantly associated with survival. Furthermore, we recently reported that a deficiency of cathepsin E in mice caused atopic dermatitis, a pruritic inflammatory skin disease (14Tsukuba T. Okamoto K. Okamoto Y. Yanagawa M. Kohmura K. Yasuda Y. Uchi H. Nakahara T. Furue M. Nakayama K. Kadowaki T. Yamamoto K. Nakayama K.I. J. Biochem. (Tokyo). 2003; 134: 893-902Crossref PubMed Scopus (74) Google Scholar). Based on these observations, we have suggested that cathepsin E plays an important role in the maintenance of homeostasis by participating in host defense mechanisms. However, because physiological substrates for cathepsin E have not yet been identified, the precise function of this enzyme remains elusive. If cathepsin E plays a critical role in the catabolism of one or several of substrate proteins, its deficiency may cause an accumulation of the substrate proteins or their metabolites in lysosomal compartments, thereby manifesting a certain phenotype of lysosomal storage disorders. The endolysosome system represents the final destination for many endocytic, autophagic, and secretory molecules targeted for destruction or recycling (15Gruenberg J. Stenmark H. Nat. Rev. Mol. Cell. Biol. 2004; 5: 317-323Crossref PubMed Scopus (593) Google Scholar). This system therefore contributes to the maintenance of homeostasis via numerous functions, including the supply of nutrients, the turnover of cellular proteins, the elimination of defective or unfavorable molecules, and the down-regulation of surface receptors (16Kornfeld S. Mellman I. Annu. Rev. Cell Biol. 1989; 5: 483-525Crossref PubMed Scopus (1324) Google Scholar, 17Mullins C. Bonifacino J.S. Bioessays. 2001; 23: 333-343Crossref PubMed Scopus (168) Google Scholar). These organelles are extremely unique in containing a variety of acidic hydrolases able to degrade or modify the transported macromolecules. Accordingly, the limiting membranes of the endolysosomal organelles most likely not only protect other cellular constituents against the attack by these potent hydrolases but also serve to maintain the acidification of the endolysosomal lumen (16Kornfeld S. Mellman I. Annu. Rev. Cell Biol. 1989; 5: 483-525Crossref PubMed Scopus (1324) Google Scholar, 18Fukuda M. J. Biol. Chem. 1991; 266: 21327-21330Abstract Full Text PDF PubMed Google Scholar). Intriguingly, the endolysosomal membrane contains several unique integral proteins such as LAMP-1, 3The abbreviations used are: LAMP, lysosome-associated membrane protein; PBS, phosphate-buffered saline; CatE-/-, cathepsin E-deficient; LIMP, lysosomal integral membrane protein; LAP, lysosomal acid phosphatase; MPR300, cation-independent mannose 6-phosphate receptor; V-ATPase, the vacuolar-type H+-ATPase; MES, 4-morpholineethanesulfonic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. LAMP-2, and LIMP-2/LGP85, whose luminal domains are heavily N-glycosylated with complex poly-N-acetyllactosamines (19Peters C. Von Figura K. FEBS Lett. 1994; 346: 146-150Crossref PubMed Scopus (83) Google Scholar, 20Eskelinen E.L. Tanaka Y. Saftig P. Trends Cell Biol. 2003; 13: 137-145Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). Of further importance, these membrane proteins represent more than 50% of the total membrane proteins of endolysosomes (18Fukuda M. J. Biol. Chem. 1991; 266: 21327-21330Abstract Full Text PDF PubMed Google Scholar, 20Eskelinen E.L. Tanaka Y. Saftig P. Trends Cell Biol. 2003; 13: 137-145Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar) and their glycosylation constitutes about 60% (LAMP-1 and LAMP-2) and 20% (LIMP-2/LGP85) of the total mass of the respective molecules (21Hunziker W. Geuze H.J. Bioessays. 1996; 18: 379-389Crossref PubMed Scopus (240) Google Scholar), and for the most part are present on the intraluminal side of endosomes and lysosomes, suggesting their significance in protecting the membrane from degradation by lysosomal hydrolases. To better understand the role of cathepsin E in the endolysosomal system, therefore, it was of particular importance for us to investigate the effect of cathepsin E deficiency on expression, trafficking, localization, and turnover of these membrane proteins as well as lysosomal soluble enzymes. We herein report that cathepsin E deficiency leads to a novel form of lysosome storage disorder in macrophages, exhibiting the accumulation of major lysosomal membrane sialoglycoproteins, including LAMP-1, LAMP-2, and LIMP-2, and the elevation of lysosomal pH. We also found that the trafficking of soluble lysosomal proteins to lysosomes was partially impaired in CatE-/- macrophages. To address the mechanism underlying these consequences, we determined the synthesis, expression, and turnover of the lysosomal membrane proteins in CatE-/- macrophages in comparison to those of the wild-type cells. In addition, to determine whether cathepsin E is directly involved in the degradation of these lysosomal membrane proteins, the effects of its inhibitors on the cellular levels of lysosomal membrane proteins and the lysosomal pH in wild-type macrophages. Our results indicate that cathepsin E is essential for degradation of these lysosomal membrane proteins, and that its deficiency is implicated in the development of a new form of lysosomal storage disorder associated with the elevation of lysosomal pH. Materials—Pepstatin A was purchased from Peptide Institute Inc. (Osaka, Japan). Bafilomycin A1 and polyclonal antibodies to V-ATPase (A-subunit) were from Wako Pure Chemicals (Tokyo, Japan). [35S]Methionine was from Amersham Biosciences. Recombinant Ascaris pepsin inhibitor was kindly donated by Dr. Takashi Kageyama, Kyoto University. Antibodies to mouse LAMP-1 and LAMP-2 were from Southern Biotechnology Inc. (Birmingham, AL). Antibodies to rat LIMP-2/LGP85, LAP, and MPR300 were kindly donated by Dr. Masaru Himeno and Dr. Yoshitaka Tanaka, Kyushu University. Antibodies to actin were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibodies to rat cathepsins B and D have been described previously (22Nakanishi H. Tominaga K. Amano T. Hirotsu I. Inoue T. Yamamoto K. Exp. Neurol. 1994; 126: 119-128Crossref PubMed Scopus (157) Google Scholar). Animals—Wild-type and CatE-/- mice on C57BL/6 genetic background were used as described previously (14Tsukuba T. Okamoto K. Okamoto Y. Yanagawa M. Kohmura K. Yasuda Y. Uchi H. Nakahara T. Furue M. Nakayama K. Kadowaki T. Yamamoto K. Nakayama K.I. J. Biochem. (Tokyo). 2003; 134: 893-902Crossref PubMed Scopus (74) Google Scholar). All animals were maintained according to the guidelines of the Japanese Pharmacological Society. The animals and all experiments were approved by the Animal Research Committee of Graduate School of Dental Science, Kyushu University. Preparation of Peritoneal Macrophages—Thioglycolate-elicited peritoneal macrophages were isolated from mice as described previously (3Nishioku T. Hashimoto K. Yamashita K. Liou S.Y. Kagamiishi Y. Maegawa H. Katsube N. Peters C. Von Figura K. Saftig P. Katunuma N. Yamamoto K. Nakanishi H. J. Biol. Chem. 2002; 277: 4816-4822Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Briefly, 8–14-week-old mice were injected peritoneally with 4.05% thioglycolate (2 ml/mouse). Three and one-half days later, peritoneal exudate cells were isolated from the peritoneal cavity by washing with phosphate-buffered saline (PBS). The cells were incubated with RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (50 units/ml), and streptomycin (50 μg/ml) at 37 °C with 5% CO2. After incubation for 2 h, non-adherent cells were removed by washing with Ca2+/Mg2+-free PBS three times. Peritoneal macrophages isolated as adherent MAC-2-positive cells were obtained at a purity of greater than 95% by this procedure. Preparation of Media and Cell Lysates—The culture media of macrophages were collected after 24 h and centrifuged at 16,000 × g for 20 min. The supernatant fraction was concentrated at 10-fold using Centriprep-30 and Microcon-30 concentrators (Millipore Co. Bedford, MA). For the preparation of the cell lysates, the cells were washed twice with PBS, removed from the plates with a rubber scraper, and centrifuged at 300 × g for 5 min. The precipitated cells were resuspended in PBS containing 0.1% Triton X-100, and then subjected to sonication for 1 min at 4 °C followed by centrifugation at 100,000 × g for 1 h. The supernatant fraction is referred to as the cell lysate. For SDS-PAGE or immunoblot analyses, the supernatant was precipitated with trichloroacetic acid at a final concentration of 5% and centrifuged at 12,000 × g for 15 min after incubation on ice for 15 min. After washing with ice-cold acetone and evaporating with air, the pellets were suspended in the buffer for SDS-PAGE. The cells were washed twice with PBS, removed from the plates by pipetting, and then subjected to centrifugation at 300 × g for 5 min. The precipitated cells were suspended in PBS containing 0.05% Triton X-100, sonicated 3 times for 5 s at 4 °C, and subjected to centrifugation at 120,000 × g for 30 min at 4 °C, the supernatant fraction was used as the cell lysate. Preparation of Lysosomal Membranes from Macrophages— The lysosomal membrane fraction was isolated from wild-type macrophages according to the method by Ohsumi et al. (23Ohsumi Y. Ishikawa T. Kato K. J. Biochem. (Tokyo). 1983; 93: 547-556PubMed Google Scholar) with some modifications. Briefly, after suspension in a solution containing 0.25 m sucrose and 0.2 m KCl, wild-type macrophages were homogenized with Potter-Elvehjem type homogenizer. After centrifugation at 650 × g for 10 min, the supernatant was centrifuged at 11,000 × g for 20 min. The pellet was suspended with a dilute solution containing 25 mm sucrose and 20 mm KCl to be lysed, and then further centrifuged at 11,000 × g for 20 min. The supernatant was referred to as the crude lysosome fraction. After addition of CaCl2 (a final concentration of 10 mm), this fraction was further centrifuged at 1,500 × g for 10 min, and then the supernatant was centrifuged at 5,000 × g for 10 min. Then, the supernatant was centrifuged at 10,000 × g for 30 min and subsequently at 50,000 × g for 30 min. The supernatant was further centrifuged at 105,000 × g for 30 min, the resultant supernatant was referred to as the final lysosomal membrane fraction, which were almost free from mitochondria, peroxisomes, and endoplasmic reticulum (23Ohsumi Y. Ishikawa T. Kato K. J. Biochem. (Tokyo). 1983; 93: 547-556PubMed Google Scholar). Degradation of Lysosomal Membrane Proteins by Purified Cathepsin E—The lysosomal membrane fractionation (50 μg) was incubated with purified cathepsin E (0–2 μg) (24Yamamoto K. Katsuda N. Kato K. Eur. J. Biochem. 1978; 92: 499-508Crossref PubMed Scopus (111) Google Scholar) in 0.1 m sodium acetate buffer (pH 5.0) at 25 °C for 12 h. The reaction was stopped by addition of 2.0 m Tris-HCl buffer (pH 9.0) to give a final concentration of 0.2 m. Then, the samples were subjected to SDS-PAGE and Western blot analyses. Enzyme Assays—Cathepsins B and L were assayed as described previously (22Nakanishi H. Tominaga K. Amano T. Hirotsu I. Inoue T. Yamamoto K. Exp. Neurol. 1994; 126: 119-128Crossref PubMed Scopus (157) Google Scholar). β-Glucronidase was assayed by the method of Robins et al. (25Robins E. Hirsch H.E. Emmons S.S. J. Biol. Chem. 1968; 243: 4246-4252Abstract Full Text PDF PubMed Google Scholar). Cathepsin D activity was determined using MOCAc-Gly-Lys-Pro-Ile-Phe-Phe-Arg-Leu-Lys(Dnp)-d-Arg-NH2 (Peptide Institute, Inc., Osaka, Japan) as a substrate according to the method described previously (26Yasuda Y. Kageyama T. Akamine A. Shibata M. Kominami E. Uchiyama Y. Yamamoto K. J. Biochem. (Tokyo). 1999; 125: 1137-1143Crossref PubMed Scopus (138) Google Scholar). β-Hexosaminidase and α-mannnosidase were assayed with 4-methylumbelliferyl-β-d-glucopyranoside and 4-methylumbelliferyl-α-d-mannopyranoside as synthetic substrates essentially according to the method of Robins et al. (25Robins E. Hirsch H.E. Emmons S.S. J. Biol. Chem. 1968; 243: 4246-4252Abstract Full Text PDF PubMed Google Scholar). Pulse-Chase Analysis—Pulse-chase experiments were performed as described previously (27Tsukuba T. Ikeda S. Okamoto K. Yasuda Y. Sakai E. Kadowaki T. Sakai H. Yamamoto K. FEBS J. 2006; 273: 219-229Crossref PubMed Scopus (12) Google Scholar). Briefly, the cells were preincubated for 1 h at 37°C in Dulbecco's modified Eagle's medium without methionine supplemented with 10% fetal bovine serum and then pulse-labeled for 30 or 60 min with [35S]methionine (100 μCi/ml) and chased in fresh RPMI 1640 medium supplemented with 10% fetal bovine serum (1.5 ml/plate). At the times indicated, the cells were separated from the medium, washed twice with PBS, lysed in PBS containing 1% Triton X-100, 0.5% sodium deoxycholate, 0.02% sodium azide, and a proteinase inhibitor mixture (1 mg/ml of each inhibitor: antipain, chymostatin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride), and then subsequently sonicated for 1 min. Immunoprecipitation—The cell lysates and media were mixed with 40 μl of Pansorbin for 1 h at 4 °C to prevent non-specific binding to IgG-protein A beads, and then centrifuged at 6,500 × g for 30 min. For immunoprecipitation of LAMP-1 and LAMP-2, the supernatant fractions were incubated with 10 μl of each monoclonal antibody at 37 °C for 10 min, and then stored at 4 °C for 16 h. The mixtures were further incubated with 20 μg of goat anti-rat antibody at 37 °C for 10 min and stored at 4 °C for 3 h. For the immunoprecipitation of cathepsins B and D, the supernatant fractions were incubated with 10 μl of each polyclonal antibodies at 37 °C for 10 min, and then stored at 4 °C for 16 h. Immune complexes were adsorbed onto protein A-Sepharose beads (50% gel suspension) at 4 °C for 3 h with gentle agitation, followed by three washes with 0.1% SDS, 0.1% Triton X-100, 200 mm EDTA, 10 mm Tris-HCl (pH 7.5). The immunoprecipitates were washed 3 more times with the same buffer containing 1 m NaCl and 0.1% sodium lauryl sarcosinate and twice with 5 mm Tris-HCl (pH 7.0). The beads were boiled for 5 min at 100 °C with 50 μl of 0.1% SDS, 0.5 mm EDTA, 5% sucrose, 5 mm Tris-HCl (pH 8.0) with 2-mercaptoethanol. Gel Electrophoresis and Immunoblot Analysis—SDS-PAGE and immunoblotting were performed as described previously (27Tsukuba T. Ikeda S. Okamoto K. Yasuda Y. Sakai E. Kadowaki T. Sakai H. Yamamoto K. FEBS J. 2006; 273: 219-229Crossref PubMed Scopus (12) Google Scholar). The quantification of the immunoreactive bands was analyzed by LAS 1000 and Image Gauge software (Fuji Photo Film Co., Ltd., Tokyo, Japan). Two-dimensional Electrophoresis and Proteomic Analyses— The culture supernatant was applied to immobilized pH gradient gel strips (Amersham Biosciences) and then subjected to isoelectric focusing using a Multiphor II (Amersham Biosciences). After isoelectric focusing, the strips were equilibrated for 15 min in 50 mm Tris-HCl (pH 8.8), containing 6 m urea, 30% glycerol, 1% SDS, and 64 mm dithiothreitol and subsequently immersed for 15 min in the same buffer containing 135 mm iodoacetamide instead of dithiothreitol. The strips were then transferred onto 10% SDS-polyacrylamide gels. After eletrophoresis, the gels were silver-stained for proteins. Peptide mass mapping was performed by recording the peptide mass fingerprints of typical in-gel digests of the corresponding gel bands using matrix-assisted laser desorption ionization time-of-flight MS (AXIMA-CFR plus, Shimadzu, Tsukuba, Japan) and the subsequent use of the mascot search engine (Matrix Science, Tokyo, Japan). Measurement of Endolysosomal pH—The endolysosomal pH in macrophages was determined in situ by the method of Chen et al. (28Chen Q.R. Zhang L. Luther P.W. Mixson A.J. Nucleic Acids Res. 2002; 30: 1338-13453Crossref PubMed Scopus (75) Google Scholar) with some modifications. Briefly, after plating on a 96-well plate, macrophages were incubated with 500 μg/ml of an acidotropic probe, Lysosensor yellow/blue dextran (Molecular Probes, Eugene, OR) for 24 h and then washed with PBS. The fluorescence from the acidic compartments in the labeled cells was quantified with a fluorescence microplate reader at an emission wavelength of 430/535 nm with excitation at 340 nm (Wallac 1420 ARVOsx, PerkinElmer Inc., Wellesley, MA). To confirm the validity of a pH titration curve, Chinese hamster ovary cells were also incubated with Lysosensor yellow/blue dextran for 24 h and then treated with 10 μm monensin and 0.5 μm bafilomycin A1. These cells were treated for 20 min with the equilibration buffers consisting of 5 mm NaCl, 115 mm KCl, 1.2 mm MgSO4, and 25 mm MES varied between pH 4.5 and 7.0. After incubation, the fluorescence intensity was determined. Measurement of V-ATPase Activity by Immunoprecipitation— Both wild-type and CatE-/- macrophages (1 × 107 cells) were lysed with 0.1% Triton X-100 in PBS, and the total ATPase activity was then measured by the method of Ramirez-Montealegre and Pearce (29Ramirez-Montealegre D. Pearce D.A. Hum. Mol. Genet. 2005; 14: 3759-3773Crossref PubMed Scopus (74) Google Scholar). The cell lysates (50 μg) were mixed with 0–20 μl of anti-V-ATPase A subunit antibody, which was raised using a synthetic peptide corresponding to amino acid sequence 366AEMPADSGYPAYLGARS381 of bovine V-ATPase and exhibited cross-reaction with V-ATPase of most organisms such as animals, bacteria, and plant, but not with other ATPases such P-ATPase and F-ATPase, and incubated at 37 °C for 10 min, and stored at 4 °C for 3 h. The immune complexes were adsorbed onto 20 μl of protein A-Sepharose beads (50% gel suspension) at 4 °C for 3 h with gentle agitation. After centrifugation to remove immunoprecipitates, ATPase activity remaining in the supernatant was remeasured. The amount of V-ATPase could be calculated from the amount of ATPase activity remaining in the supernatant. The enzyme unit, U, was defined as micromole of Pi liberated per min per ml. mRNA Extraction and Quantitative Real-time PCR—The total mRNA of the cells was prepared using a RNeasy kit (Qiagen, Tokyo, Japan.) according to the manufacturer's instructions. Complementary DNA was synthesized using 1 μg of total RNA incubated with random hexamers, followed by a 50-μl reverse transcription reaction with 6.25 units of Multiscribe reverse transcriptase (Applied Biosystems) at 25 °C for 10 min for binding, at 48 °C for 30 min for reverse transcription, and at 95 °C for 5 min for inactivation, respectively. The cDNA products were amplified using the following oligonucleotide primers for mouse LAMP-1, LAMP-2, LIMP-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs; LAMP-1, 5′ primer = CAAGGCAGACATCAACAAAG and 3′ primer = TAGGGVATCAGGAACACTCA; LAMP-2, 5′ primer = GGTGCTGGTCTTTCAGGCTTGATT and 3′ primer = ACCACCCAATCTAAGAGCAGGACT; LIMP-2, 5′ primer = TGTTGAAACGGGAGACATCA and 3′ primer = TGGTGACAACCAAAGTCGTG; GAPDH, 5′ primer = ACTCCCACTCTTCCACCTTC and 3′ primer = TCTTGCTCAGTGTCCTTGC. The housekeeping gene GAPDH was also amplified in parallel as a reference for the quantification of LAMP-1, LAMP-2, and LIMP-2 transcripts. The cDNA samples (1 μl) were added to 12.5 μl of 2× SYBR Green PCR Master Mix, 1 μl of 10 μm for the 5′- and 3′-primers to give a total volume of 25μl. All reactions were performed in duplicate in the ABI PRISM 7000 Sequence Detector (Applied Biosystems). The thermal cycling was performed at 50 °C for 2 min, at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s plus at 56 °C for 1 min. Realtime PCR data were analyzed for at least three different experiments. Immunofluorescence Microscopy— The cells were grown on glass coverslips and briefly washed with PBS and then fixed with 4.0% paraformaldehyde in PBS for 30 min at room temperature. The fixed cells were washed and permeabilized with 50 mm NH4Cl and 0.3% Tween 20 in PBS. The cells were incubated with 1% bovine serum albumin plus 1% normal goat serum for 3 h, and subsequently incubated with the primary antibodies overnight at 4.0 °C followed by fluorescence-labeled secondary antibodies, and then inspected by microscopy using a laser-scanning confocal imaging system (Leica TCS, Leica-Microsystems, Heidelberg, Germany). Statistical Analysis—Quantitative data are presented as mean ± S.D. The statistical significance of differences between mean values was assessed by Student's t test. p values of <0.05 were considered statistically significant. Effect of Cathepsin E Deficiency on the Cellular Levels of Major Lysosomal Membrane Glycoproteins—To determine the effect of cathepsin E deficiency on intracellular levels of lysosomal membrane proteins, we performed SDS-PAGE and immunoblot analysis for the cell lysates of wild-type and CatE-/- macrophages. Two major lysosomal membrane sialoglycoproteins, LAMP-1 and LAMP-2, were highly increased in CatE-/- macrophages compared with the wild-type cells (Fig. 1). Another major lysosomal membrane sialoglycoprotein LIMP-2 was also increased, but not significantly, in CatE-/- macrophages. In contrast, cellular levels of other endolysosomal membrane glycoproteins, including lysosomal acid phosphatase (LAP), which is known to be synthesized and transported to lysosomes as an integral type I membrane protein and slowly released its luminal domain into the lysosomal lumen by proteolytic processing (19Peters C. Von Figura K. FEBS Lett. 1994; 346: 146-150Crossref PubMed Scopus (83) Google Scholar), V-ATPase A-subunit, which is an integral endolysosomal membrane protein essential for V-ATPase activity (30Forgac M. J. Biol. Chem. 1999; 274: 12951-12954Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 31Sun-Wada G.H. Wada Y. Futai M. Cell Struct. Funct. 2003; 28: 455-463Crossref PubMed Scopus (103) Google Scholar), and cation-independent mannose 6-phosphate receptor (MPR300), which is known as a type I integral membrane protein found mainly in the trans-Golgi network and plays a critical role in the intracellular trafficking of soluble lysosomal hydrolases (32Ghosh P. Dahms N.M. Kornfeld S. Nat. Rev. Mol. Cell. Biol. 2003; 4: 202-212Crossref PubMed Scopus (833) Google Scholar), were not different between wild-type and CatE-/- macrophages. Elevation of Lysosomal pH in CatE-/- Macrophages—The luminal acidic pH is essential for the normal function of intracellular acidic organelles including endosomes and lysosomes (16Kornfeld S. Mellman I. Annu. Rev. Cell Biol. 1989; 5: 483-525Crossref PubMed Scopus (1324) Google Scholar). Increasing evidence suggests that the accumulation of undegraded metabolites in lysosomal compartments induces an elevated lysosomal pH, thereby impairing the functions of the endolysosomal system (33Futerman A.H. van Meer G. Nat. Rev. Mol. Cell. Biol. 2004; 5: 554-565Crossref PubMed Scopus (655) Google Scholar). We therefore analyzed the lysosomal pH in CatE-/- macrophages using an acidotropic fluorescent probe, namely, Lysosensor yellow/blue dextran. The lysosomal pH of the wild-type cells was estimated to be 5.3 ± 0.4, which closely agreed with the findings previously reported in macrophages (34Lukacs G.L. Rotstein O.D. Grinstein S. J. Biol. Chem. 1991; 266: 24540-24548Abstract Full Text PDF PubMed Google Scholar), whereas that of CatE-/- macrophages were 6.4 ± 0.3 (Fig. 2A), indicating the strong induction of the elevated lysosomal pH as a result of cathepsin E deficiency. We further analyzed whether this elevation of lysosomal pH was due to the disruption of V-ATPase, which acts as a major regulating factor for the acidification of the lysosomal lumen (31Sun-Wada G.H. Wada Y. Futai M. Cell Struct. Funct. 2003; 28: 455-463Crossref PubM
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