Characterization of rat cathepsin E and mutants with changed active-site residues and lacking propeptides and N-glycosylation, expressed in human embryonic kidney 293T cells Takayuki Tsukuba1, Shinobu Ikeda2, Kuniaki Okamoto2, Yoshiyuki Yasuda1, Eiko Sakai2, Tomoko Kadowaki1, Hideaki Sakai2 and Kenji Yamamoto1 Department of Pharmacology, Graduate School of Dental Science, Kyushu University, Fukuoka, Japan Department of Oral Molecular Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University, Japan Keywords aspartic proteinase; cathepsin E; mutation; processing; sorting Correspondence K Yamamoto, Department of Pharmacology, Graduate School of Dental Science, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan Fax: +81 92 642 6342 Tel: +81 92 642 6337 E-mail: kyama@dent.kyushu-u.ac.jp (Received 10 October 2005, revised November 2005, accepted 14 November 2005) doi:10.1111/j.1742-4658.2005.05062.x To study the roles of the catalytic activity, propeptide, and N-glycosylation of the intracellular aspartic proteinase cathepsin E in biosynthesis, processing, and intracellular trafficking, we constructed various rat cathepsin E mutants in which active-site Asp residues were changed to Ala or which lacked propeptides and N-glycosylation Wild-type cathepsin E expressed in human embryonic kidney 293T cells was mainly found in the LAMP-1positive endosomal organelles, as determined by immunofluorescence microscopy Consistently, pulse–chase analysis revealed that the initially synthesized pro-cathepsin E was processed to the mature enzyme within a 24 h chase This process was completely inhibited by brefeldin A and bafilomycin A, indicating its transport from the endoplasmic reticulum (ER) to the endosomal acidic compartment Mutants with Asp residues in the two active-site consensus motifs changed to Ala and lacking the propeptide (Leu23-Phe58) and the putative ER-retention sequence (Ser59-Asp98) were neither processed nor transported to the endosomal compartment The mutant lacking the ER-retention sequence was rapidly degraded in the ER, indicating the importance of this sequence in correct folding The single (N92Q or N324D) and double (N92Q ⁄ N324D) N-glycosylation-deficient mutants were neither processed into a mature form nor transported to the endosomal compartment, but were stably retained in the ER without degradation These data indicate that the catalytic activity, propeptides, and N-glycosylation of this protein are all essential for its processing, maturation, and trafficking Cathepsin E (EC 3.4.23.24) is an intracellular aspartic proteinase of the A1 family, which consists of two identical subunits of 42 kDa (reviewed in [1,2]) Evidence suggests that it is initially synthesized as a preproenzyme and is targeted to the correct destination after proteolytic processing and carbohydrate modification Differing from the definite localization of the analogous lysosomal aspartic proteinase, cathepsin D, the intracellular localization of cathepsin E appears to vary with cell type [3–5] In antigen-presenting cells such as macrophages and microglia, cathepsin E is mainly found in the endosomal compartment as a mature form which is N-glycosylated mostly with complex-type oligosaccharides [6] In erythrocytes [7–9], gastric cells [10–13], renal proximal tubule cells [13], and osteoclasts [14], cathepsin E is exclusively confined Abbreviations DMEM, Dulbecco’s modified Eagle’s medium; ER, endoplasmic reticulum; HEK, human embryonic kidney; LAMP, lysosome-associated membrane protein FEBS Journal 273 (2006) 219–229 ª 2005 The Authors Journal compilation ª 2005 FEBS 219 Recombinant cathepsin E and its mutants T Tsukuba et al to the plasma membrane The enzyme from erythrocytes [15] is N-glycosylated mostly with complex-type oligosaccharide chains, whereas that from gastric cells [16] has high mannose-type oligosaccharides Cathepsin E is also detected in the ER and Golgi complex in various cell types, including gastric cells [12,13], human M cells [5], Langerhans cells, and interdigitating reticulum cells [4] However, fundamental information on the biosynthesis, processing, and intracellular trafficking of this protein as well as its physiological significance remains elusive Given the variability in cellular localization, we decided that the roles of the catalytic activity, propeptides, and N-glycosylation of cathepsin E in its processing, maturation, and trafficking must be of particular importance In this study, to understand the molecular basis of the processing and intracellular trafficking of cathepsin E, we constructed a variety of mutants as well as wild-type enzyme These included mutants with Asp residues in the two active-site DTG motifs changed to Ala residues and mutants lacking the propeptide, putative ER-retention sequence, and N-glycosylation of this protein These mutant proteins, as well as wild-type cathepsin E, were expressed in human embryonic kidney (HEK)-293T cells We report that the catalytic activity, propeptide, ER-retention sequence, and N-glycosylation of this protein are all essential for processing, maturation, and trafficking Results Expression and cellular localization of recombinant rat cathepsin E expressed in HEK-293T cells In this study, we used HEK-293T cells to express wildtype cathepsin E and its mutants and to follow the processing, maturation, and trafficking of these proteins, as these cells have no detectable endogenous cathepsin E To establish the expression system, we transfected rat cathepsin E cDNA (wild-type) into HEK-293T cells and analyzed the molecular forms of the expressed proteins by SDS ⁄ PAGE under reducing conditions followed by immunoblotting with polyclonal antibodies to mature rat cathepsin E or rat pro-cathepsin E (Fig 1) The transfected cells gave two intense bands with apparent molecular masses of 46 and 42 kDa with antibodies to mature cathepsin E Antibodies to pro-cathepsin E, however, reacted with only the 46-kDa form, indicating that the 46-kDa and 42-kDa bands are pro-cathepsin E and mature cathepsin E, respectively To follow the biosynthesis and processing of wildtype cathepsin E, the transfected cells were labeled 220 A B Fig Immunoblot analysis of the cell extract of HEK-293T cells expressing wild-type cathepsin E HEK-293T cells were transfected with rat cathepsin E cDNA The cell lysate was subjected to SDS ⁄ PAGE under reducing conditions followed by immunoblotting with antibody to either mature cathepsin E (A) or pro-cathepsin E (B) with [35S]methionine for 30 and chased in complete Dulbecco’s modified Eagle’s medium (DMEM) containing unlabeled methionine for different periods of time up to 24 h At the end of the chase, cells and culture media were collected separately The cell lysate and culture medium were subjected to immunoprecipitation with antibodies to mature cathepsin E, and the immunoprecipitates were then analyzed by SDS ⁄ PAGE followed by fluorography (Fig 2A) After 30-min pulse labeling, cathepsin E was observed mainly as a 46-kDa precursor with a small amount of the 42-kDa mature form A small amount of the proenzyme was released into the culture medium as a 48-kDa form and accumulated during the chase period The difference in the molecular mass between the extracellular and intracellular proforms is probably due to the difference in their carbohydrate modifications, because the former has endoglycosidase Hsensitive high-mannose-type oligosaccharide chains, whereas the latter possesses endoglycosidase H-resistant complex-type oligosaccharide chains (Fig 6A) The 42-kDa mature form in the cells was time-dependently increased, and the 46-kDa pro-cathepsin E was almost completely converted into the 42-kDa mature cathepsin E after a 24-h chase These processing events were strongly inhibited by the fungal metabolite brefeldin A FEBS Journal 273 (2006) 219–229 ª 2005 The Authors Journal compilation ª 2005 FEBS T Tsukuba et al A B C Fig Pulse–chase analysis of wild-type cathepsin E expressed in HEK-293T cells (A) The transfected cells were metabolically labeled with [35S]methionine ⁄ cysteine for 30 and chased for the times indicated Cathepsin E in the cell lysate and culture medium was immunoprecipitated with antibodies to mature rat cathepsin E, and analyzed by SDS ⁄ PAGE under reducing conditions and fluorography (B, C) The transfected cells were preincubated at 37 °C for h in the presence of lgỈmL)1 brefeldin A (B) or 0.5 lM bafilomycin A1 (C) The cells were labeled with [35S]methionine ⁄ cysteine for 30 and chased for the times indicated in the continued presence of the drugs The cell lysate and the culture medium were immunoprecipitated with antibodies to mature rat cathepsin E The immunoprecipitates were analyzed by SDS ⁄ PAGE under reducing conditions followed by fluorography (Fig 2B), which is known to inhibit export of newly synthesized proteins from the ER to the Golgi complex [17] and also to cause reversible redistribution of Golgi resident proteins into the ER [18] The strong inhibition of the processing of pro-cathepsin E into the mature enzyme by brefeldin A indicates that the processing and maturation of this protein occur in the post-Golgi compartment In addition, this agent prevented the secretion of subspecies of the precursor, probably mediated by an alternative secretory pathway at the exit from the ER Available evidence indicates that internal acidification of some compartments in the vacuolar system, such as endosomes and lysosomes, is important in biosynthesis, sorting, transport, and degradation of proteins and other macromolecules [19–21] When the transfected cells were treated with bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, the processing and maturation of pro-cathepsin E was completely inhibited, with a concomitant increase in a secreted form of pro-cathepsin E in the culture Recombinant cathepsin E and its mutants medium (Fig 2C) Given that bafilomycin A1 effectively inhibits acidification of intracellular acidic organelles including endosomes, lysosomes, and phagosomes without perturbation of the formation of intracellular organelles and without alteration of the morphology of vacuolar compartments, resulting in the profound inhibition of the endosomal ⁄ lysosomal degradation of macromolecules and targeting of lysosomal acid hydrolases and cholesterol to the lysosome and processing of various secretory proteins including prohormones in the trans-Golgi network [22], our results indicate that acidification of intracellular acidic compartments is necessary for the processing and maturation of procathepsin E To determine the intracellular localization of wildtype cathepsin E expressed in HEK-293T cells, indirect immunofluorescence staining was performed (Fig 3A) With antibodies to mature cathepsin E, the transfected cells showed mainly punctate staining and partly reticular staining over the whole cytoplasm, consistent with staining of the lysosome-associated membrane protein LAMP-1 and the ER-associated molecular chaperon Bip, respectively In agreement with the data of the pulse–chase analysis, the results indicate that the proteolytic maturation of pro-cathepsin E occurs in the endosomal acidic compartment Importance of the catalytic activity of cathepsin E in acid-dependent autoactivation In vitro studies have shown that pro-cathepsin E is rapidly converted into mature cathepsin E by a brief acid treatment [9,23] However, whether the catalytic activity of cathepsin E is essential for its processing and maturation in vivo has not been elucidated To throw light on this, we constructed an active-site mutant by changing Asp residues in the two active-site motifs (DTG) to Ala residues using site-directed mutagenesis (D98A ⁄ D283A) This mutant protein was expressed in HEK-293T cells We confirmed that the D98A ⁄ D283A mutant had no catalytic activity on either protein or synthetic substrates, as described in our previous study, in which the single mutants D98A and D283A, as well as the double mutant, exhibited no catalytic activity in vitro [24] The autoprocessing capability of this mutant protein was analyzed by pulse–chase experiments with [35S]methionine (Fig 4) In contrast with wild-type cathepsin E, the D98A ⁄ D283A mutant in the cells was neither processed nor matured even after a 24-h chase period, but stably remained as a 46-kDa precursor, indicating that the catalytic activity is essential for the processing and maturation of this protein FEBS Journal 273 (2006) 219–229 ª 2005 The Authors Journal compilation ª 2005 FEBS 221 Recombinant cathepsin E and its mutants T Tsukuba et al Fig Immunofluorescence microscopy of wild-type cathepsin E and its mutants in the transfected HEK-293T cells (A) At 48 h after posttransfection with wild-type cathepsin E cDNA in HEK-293T cells, the cells were fixed and allowed to react with antibodies to mature rat cathepsin E, Bip, or LAMP-1 After being washed, the cells were incubated for h with fluorescein isothiocyanate-conjugated or tetramethylrhodamine isothiocyanate-conjugated secondary antibodies, and then visualized by confocal laser microscopy Wild-type cathepsin E is found mainly in the LAMP-1-positive endosomal organelles and partly in the Bip-positive ER (B, C) The subcellular localization of the deletion mutants lacking the propeptide (Dpro) (B) and the ER-retention motif (DER ret.) expressed in HEK-293T cells was analyzed under the same conditions as described in (A) Both mutants were exclusively confined to the ER, but not to the LAMP-1-positive organelles Role of the propeptides of cathepsin E in processing and maturation Recent studies have suggested that the propeptides of aspartic proteinases are necessary for correct folding and cellular sorting [25–27] and that activation of their 222 zymogens is initiated by a dramatic conformational rearrangement of the zymogen propeptides [28,29] This process is often triggered by acidic pH, resulting in the proteolytic removal of propeptides [29] More recently, using chimeric DNAs encoding the cathepsin E propeptide fused to mature cathepsin D tagged FEBS Journal 273 (2006) 219–229 ª 2005 The Authors Journal compilation ª 2005 FEBS T Tsukuba et al A Recombinant cathepsin E and its mutants A B B C C Fig Pulse–chase analysis of the active-site mutant with Asp residues in the two active-site motifs changed to Ala residues (A) Schematic representation of the structures of wild-type cathepsin E and its active-site mutant Asp residues in the two active-site consensus motifs (98DTG100 and 273DTG275) are substituted by Ala residues in the mutant (B, C) HEK-293T cells expressing wild-type cathepsin E (B) and the active-site mutant (C) were pulse-labeled with [35S]methionine ⁄ cysteine for 30 and chased for the times indicated The cell lysate and the culture medium were immunoprecipitated with antibodies to mature rat cathepsin E The immunoprecipitates were analyzed by SDS ⁄ PAGE under reducing conditions followed by fluorography with haemagglutinin at the C-terminus and encoding the cathepsin D propeptide fused to mature cathepsin E, we have demonstrated that the propeptide of cathepsin E, likewise those of other aspartic proteinases, plays an important role in the correct folding, maturation, and targeting of this protein to its final destination [30] To establish further the role of the propeptide in the processing and maturation of cathepsin E, we constructed a mutant lacking the propeptide (Leu23-Phe58) and expressed it in HEK-293T cells The transfected cells were pulse-labeled with [35S]methionine for 30 and chased for different periods up to 24 h (Fig 5B) In the cells, this mutant protein was first synthesized as a 42-kDa precursor but was neither processed nor matured even after a 24-h chase period Little protein was released into the medium Consistent with these data, immunofluorescence microscopy revealed that this mutant protein was found mostly in the Bip-positive ER compartment, but not LAMP-1-positive organelles (Fig 3B) Finley & Kornfeld [31] expressed various chimeric proteins between cathepsin E and pepsinogen in Fig Pulse–chase analysis of the mutants lacking the propeptide and ER-retention sequence (A) Schematic representation of the structures of the mutants lacking the propeptide and most of the ER-retention sequence (B, C) The transfected cells expressing the mutants lacking the propeptide (Dpro) (B) and the ER-retention sequence (DER-ret.) (C) were pulse-labeled with [35S]methionine ⁄ cysteine for 30 and chased for the times indicated The cell lysate and the culture medium were immunoprecipitated with antibodies to mature rat cathepsin E The immunoprecipitates were analyzed by SDS ⁄ PAGE under reducing conditions followed by fluorography The arrows indicate degradation products of the DER-ret mutant monkey Cos cells and analyzed their targeting and subcellular localization They showed that the aminoacid sequence 1–48 of human mature cathepsin E, which corresponds to 55–103 of the signal sequence [32], appeared to be essential for the retention of cathepsin E in the ER We thus examined whether this putative ER-retention sequence is also required for the processing, maturation, and trafficking of cathepsin E to the appropriate destination using a mutant lacking most of the ER-retention sequence (Ser59-Asp98) Pulse–chase analysis revealed that this mutant protein was initially synthesized as a 41-kDa precursor but not processed into a mature form Importantly, this mutant protein was rapidly degraded in the cells during a chase period up to h without any detectable formation of its mature form (Fig 5C) In addition, we found that the level of expression of this mutant FEBS Journal 273 (2006) 219–229 ª 2005 The Authors Journal compilation ª 2005 FEBS 223 Recombinant cathepsin E and its mutants T Tsukuba et al was low compared with wild-type-cathepsin E and other mutant proteins It was found in the Bip-positive ER compartment, but not in LAMP-1-positive organelles (Fig 3C) These results strongly suggest that the putative ER-retention sequence is absolutely required for the correct folding, processing maturation, and targeting of cathepsin E to the endosomal compartment Characterization of N-linked oligosaccharide chains of cathepsin E B We have previously demonstrated that cathepsin E from human erythrocyte membranes [9,15] and rat microglia [6] is N-glycosylated with complex-type oligosaccharides, whereas the enzyme from rat spleen [9,33] and rat stomach [16] has high-mannose-type oligosaccharide chains These results suggest that the nature of the N-glycosylation of cathepsin E varies with cell type or its cellular localization We thus analyzed the nature of the oligosaccharide chains of rat cathepsin E in the transfected cells and then assessed the role of N-glycosylation in its folding, processing, maturation, and subcellular trafficking HEK-293T cells expressing wild-type cathepsin E were pulse-labeled with [35S]methionine for 30 and chased with unlabeled methionine for 24 h The intracellular and extracellular cathepsin E were immuoprecipitated with antibodies to mature rat cathepsin E, and the immunoprecipitates were treated with endoglycosidase H and then analyzed by SDS ⁄ PAGE followed by fluorography (Fig 6A) Both newly synthesized pro-cathepsin E (30-min pulse) and the fully matured enzyme (24-h chase) in the cells were sensitive to endoglycosidase H A decrease in the molecular mass of 2–2.5 kDa resulted, indicating the presence of at least a single highmannose-type oligosaccharide chain In contrast, the extracellular cathepsin E molecules at 30-min pulse labeling and after 24-h chase were resistant to endoglycosidase H, indicating that they were secreted after modification with complex-type oligosaccharides Cathepsin D, like other lysosomal enzymes, is phosphorylated in a portion of the oligosaccharide chains during passage through the Golgi complex [34,35] This process is considered to be important for the recognition of cathepsin D by mannose 6-phosphate receptors and its delivery to the prelysosomal compartment To determine whether the oligosaccharides of cathepsin E are phosphorylated, the transfected cells were labeled with [32P]phosphate for 12 h, and then wild-type cathepsin E as well as endogenous cathepsin D was immunoprecipitated by antibodies specific for each enzyme (Fig 6B) Whereas the oligosaccharide chains of cathepsin D were, at least in part, clearly phosphorylated, and this 224 A Fig Characterization of oligosaccharide chains of wild-type cathepsin E and endogenous cathepsin D (A) HEK-293 cells expressing wild-type cathepsin E were pulse-labeled with [35S]methionine ⁄ cysteine for 30 and chased for 24 h The cell lysate and the culture medium were immunoprecipitated with antibodies to mature rat cathepsin E The immunoprecipitates were then incubated at 37 °C for 18 h with or without endoglycosidase H The mixtures were analyzed by SDS ⁄ PAGE under reducing conditions followed by fluorography (B) The transfected cells were pulse-labeled with [32P]orthophosphate for 12 h The cell lysate and the culture medium were immunoprecipitated with antibodies to either mature rat cathepsin E or rat cathepsin D The immunoprecipitates were incubated at 37 °C for 18 h with or without endoglycosidase H The mixtures were analyzed by SDS ⁄ PAGE under reducing conditions followed by fluorography phosphorylation had disappeared after endoglycosidase H treatment, wild-type cathepsin E was not phosphorylated at all, suggesting that this may serve to sort cathepsin E from lysosomal enzymes Role of the N-glycosylation of cathepsin E in processing and maturation To determine the role of N-glycosylation in the correct folding, processing, maturation, and subcellular trafficking of cathepsin E, we constructed mutants lacking one or two N-glycosylation sites by site-directed mutagenesis We have previously shown that the FEBS Journal 273 (2006) 219–229 ª 2005 The Authors Journal compilation ª 2005 FEBS T Tsukuba et al N-glycosylation null mutant of cathepsin E (N92Q ⁄ N374D) was less stable to temperature and pH than glycosylated cathepsin E, although the catalytic properties of the mutant were equivalent to those of the wild-type enzyme [15] In this study, we performed pulse–chase experiments with HEK-293 cells expressing N-glycosylation mutants The results show that two single N-glycosylation mutants (N92Q and N324D), as well as the double N-glycosylation mutant (N92Q ⁄ N374D), were stably retained in the cells, but not processed to the mature form even after a 24-h chase period (Fig 7) Immunofluorescence microscopy indicated that all of the N-glycosylation mutants were exclusively confined to the Bip-positive ER compartment (data not shown) Therefore, our data indicate that N-glycosylation of cathepsin E plays an important role in its processing, maturation, and trafficking to the appropriate destination in the cells, but is not necessarily essential for its correct folding A B C D Fig Pulse–chase analysis of the mutants lacking N-glycosylation (A) Schematic representation of the structures of the mutants lacking single (N92Q or N324D) and double N-glycosylation sites (N92Q ⁄ N324D) Asn residues in the two potential N-glycosylation sites (92NFT94 and 324NVT326) are substituted by Gln and ⁄ or Asp residues in the mutants (B–D) HEK-293 cells expressing each mutant as well as wild-type cathepsin E were pulse-labeled with [35S]methionine ⁄ cysteine for 30 and chased with nonradiolabeled medium for the times indicated The cell lysate and culture medium were immunoprecipitated with antibodies to mature rat cathepsin E The immunoprecipitates were analyzed by SDS ⁄ PAGE under reducing conditions followed by fluorography (B) N92Q; (C) N324D; (D) N92Q ⁄ N324D Recombinant cathepsin E and its mutants Discussion To assess the roles of the catalytic activity, propeptides, and N-glycosylation of cathepsin E in its processing, maturation, and targeting to its final destination, we constructed wild-type rat cathepsin E and a variety of its mutants, and successfully expressed them in heterologous HEK-293T cells The results provide the first experimental evidence that the catalytic activity, propeptide, ER-retention motif, and N-glycosylation are all essential for its processing, maturation, and intracellular trafficking to the endosomal compartment This conclusion is based on several lines of evidence First, the pulse–chase analysis showed that the wild-type enzyme in the transfected cells was processed from pro-cathepsin E to the mature form within a 24-h chase period Consistent with this finding, immunofluorescence microscopy revealed that wild-type cathepsin E was found mostly in LAPM-1-positive organelles and significantly in the ER Given that cathepsin E in rat microglia is mainly localized in endosome-like vacuoles distinct from typical lysosomes, as determined by immunoelectron microscopy [6] and that the localization of this enzyme in mouse microglia is consistent with that of LAMP-2-positive organelles [36], wild-type cathepsin E in the transfected cells is probably targeted to the endosomal compartment However, the processing of wild-type cathepsin E in the transfected cells appears to be slower than that of natural cathepsin E in rat microglia [6] This is probably due to the difference in the level of expression of cathepsin E between the transfected cells and the primary cultured microglia In contrast, none of the mutants were processed or targeted to the endosomal compartment Interestingly, the intracellular localization of recombinant cathepsin E appears to vary with the cell type used In mouse L cell and monkey Cos cells, the expressed human cathepsin E is localized in the ER as a precursor form [31] Similarly, rat cathepsin E expressed in normal rat kidney cells and monkey Cos cells is exclusively confined to the ER (T Tsukuba, T Kadowaki and K Yamamoto, unpublished work) Meanwhile, human cathepsin E expressed in Chinese hamster ovary cells is found in various subcellular compartments such as the vacuolar endolysosomal system, the ER, and the cytosol [37] The present results thus indicate that HEK-293T cells are useful for studying the biosynthesis, processing, maturation, and trafficking to the correct destination of cathepsin E Secondly, the mutant lacking the catalytic activity of cathepsin E in the transfected cells failed to be converted to the mature form, indicating that the catalytic FEBS Journal 273 (2006) 219–229 ª 2005 The Authors Journal compilation ª 2005 FEBS 225 Recombinant cathepsin E and its mutants T Tsukuba et al activity is necessary for processing and maturation Although pro-cathepsin D is capable of acid-dependent autoactivation to yield catalytically active pseudo-cathepsin D [38–40], lysosomal cysteine proteinase(s) is required to accomplish proteolytic removal of the entire propeptide [29,41] BACE ⁄ memapsin, a membrane-type aspartic proteinase, is also processed by furin-like convertase(s) [42–44] In contrast, secretory-type aspartic proteinases, such as pepsin A, chymosin and gastricsin, are capable of acid-dependent autoactivation to yield their mature forms without the aid of other proteinases [45,46] Therefore, it is of interest that cathepsin E undergoes acid-dependent autoproteolysis to yield mature cathepsin E in a similar manner to secretory aspartic proteinases rather than nonsecretory enzymes Thirdly, whereas the cathepsin D mutant lacking its propeptide expressed in mouse Ltk– cells was rapidly degraded, probably in the ER [47], the propeptidedeletion cathepsin E mutant in HEK-293T cells was relatively stable in the ER but was neither processed nor targeted to the endosomal compartment, indicating the importance of the propeptide in the processing, maturation, and trafficking of this protein However, the cathepsin E mutant lacking the putative ER-retention sequence was rapidly degraded in the ER, suggesting that the ER-retention sequence is necessary for correct folding In addition, this mutant did not undergo processing, maturation, and trafficking to the endosomal compartment Finally, we have shown that intracellular wild-type cathepsin E was N-glycosylated with high-mannosetype oligosaccharides, as demonstrated by endoglycosidase H sensitivity, whereas the extracellular enzyme had endoglycosidase H-resistant oligosaccharides Previous studies have shown that modification of oligosaccharide chains of cathepsin E differs between cell types Therefore, it is difficult to explain the processing, maturation, and targeting of cathepsin E by the nature of its oligosaccharide chains We thus constructed three N-glycosylation mutants and expressed them in HEK-293T cells to determine the importance of N-glycosylation in the processing, maturation, and trafficking of this protein Pulse–chase analysis and immunofluorescence microscopy revealed that neither single (N92Q, N324D) nor double (N92Q ⁄ N374D) N-glycosylation mutants were processed into the mature forms, but were stably retained in the ER without degradation after a 24-h chase period As all of the N-glycosylation mutants were converted into the active enzyme on acidification (data not shown), each the oligosaccharide chain appears to be not necessarily required for correct folding but to be essential for processing, maturation, and trafficking 226 Moreover, we show for the first time that wild-type cathepsin E is not phosphorylated on either the polypeptide backbone or the oligosaccharide chains Previous studies have shown that mannose 6-phosphate receptors participate in general in the sorting of individual lysosomal proteins, albeit with variable efficiency [35,48] The enzyme phosphotransferase, which transfers phosphate to the high-mannose oligosaccharide chain of individual lysosomal proteins, recognizes conformational determinants on the proenzymes [49] The phosphorylation of cathepsin D occurs mainly at the large oligosaccharide chain of two glycosylation sites [50] In agreement with previous studies, this work indicates that cathepsin D is apparently phosphorylated on its oligosaccharide chains Therefore, we conclude that the sorting and segregation of cathepsin E from other secretory proteins is independent of mannose 6-phosphate mechanisms Experimental procedures Materials [35S]Methionine ⁄ cysteine and Protein A–Sepharose were purchased from Amersham Biosciences (Piscataway, NJ, USA) Pansorbin was from Calbiochem (La Jolla, CA, USA) The pcDNA 3.0 ⁄ Amp plasmid, DMEM, methionine-free DMEM, and Opti-Mem were from Invitrogen (Carlsbad, CA, USA) Endoglycosidase H was from Boehringer Mannheim (Mannheim, Germany) Centriprep-30 and Microcon-30 concentrators were from Millipore (Bedford, MA, USA) Fluorescein isothiocyanate-conjugated and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies were from BD Bioscience (San Jose, CA, USA) Vectashield was from Vector Laboratory Inc (Burlingame, CA, USA) Antibodies to Bip (GRP 78) and Lamp1 were from Stressgen Bioreagents (Victoria, BC, Canada) Antibodies to rat cathepsin E and to rat cathepsin D were raised in rabbit and purified as described previously [24] Polyclonal antibodies to the synthetic peptide Ser-Gln-Leu-Ser-Glu-Phe-Trp-Lys-Ser-His-Asn-Leu-Asp-Met, which corresponds to the prosequence comprising residues Ser23-Met36 of human procathepsin E, were raised in rabbits and purified on the peptide–Sepharose affinity column as described previously [24) Plasmid construction and mutagenesis The pBluescript II SK plasmid containing full-length wildtype rat cathepsin E cDNA was described previously [15,24] Deletion mutants lacking the ER retention sequence (DER ret.) and the propeptide (Dpro) were constructed by the method of Kunkel [51] with some modifications The oligonucleotide primers used in the mutagenesuis reaction FEBS Journal 273 (2006) 219–229 ª 2005 The Authors Journal compilation ª 2005 FEBS T Tsukuba et al were as follows: primer 1, 5¢-ATGATCGAATTCACGGG CGGCTCA-3¢ (for DER ret.); primer 2, 5¢-CAGGC CCAAGGGGTGAGCGAGTCCTGT-3¢ (for Dpro) All constructs were verified as correct by DNA sequence analyses The cDNAs of wild-type cathepsin E and its mutants were transfected into pcDNA 3.0 plasmid for heterologous expression in mammalian cells Tissue culture and transfection HEK-293T cells were maintained in DMEM supplemented with 10% fetal calf serum, 50 mL)1 penicillin and 50 lgỈmL)1 streptomycin (complete DMEM) in a 37 °C incubator with 5% CO2 Wild-type cathepsin E and its mutants were expressed in HEK-293T cells after transfection by the calcium phosphate precipitation method using 10 lg expression plasmid in a 100-mm tissue culture plate [24] Cells were grown to  90% confluency by overnight incubation in complete DMEM Preparation of cell lysate and culture medium The tissue culture media were collected after 48 h of transfection and subjected to centrifugation at 16 000 g for 20 The supernatants were concentrated 10-fold to 500 lL ⁄ plate using Centriprep-30 and Microcon-30 concentrators The cells were washed twice with NaCl ⁄ Pi, removed from the plates with a rubber scraper, and subjected to centrifugation at 300 g for The sedimented cells were suspended in NaCl ⁄ Pi containing 0.1% Triton X-100, sonicated for at °C, and subjected to centrifugation at 100 000 g for h; the supernatant fraction is referred to as the cell lysate Pulse–chase analysis The transfected cells were preincubated for h at 37 °C in DMEM without methionine supplemented with 10% dialyzed fetal bovine serum The cells were pulse-labeled for 30 with [35S]methionine ⁄ cysteine (100 lCiỈmL)1 per dish), and then chased in fresh serum-free Opti-MEM (1.5 mL ⁄ plate) At the times indicated, the cells were separated from the medium, washed twice with NaCl ⁄ Pi, lysed in NaCl ⁄ Pi containing 1% Triton X-100, 0.5% sodium deoxycholate, 0.02% sodium azide, and a proteinase inhibitor cocktail, and subsequently sonicated for The suspension was centrifuged at 6500 g for 10 to obtain the cell lysate fraction Immunoprecipitation The cells and media were mixed with 40 lL Pansorbin for h at °C to prevent nonspecific binding to IgG–Protein A beads, and then centrifuged at 6500 g for 30 The supernatants fractions were incubated with 15 lL anti-(rat Recombinant cathepsin E and its mutants cathepsin E) IgG at 37 °C for 10 min, and then stored at °C for 16 h The immunoprecipitates were mixed with 40 lL Protein A–Sepharose beads (50% gel suspension) for h at °C with gentle agitation The beads were washed times with 0.1% SDS ⁄ 0.1% Triton X-100 ⁄ 200 mm EDTA ⁄ 10 mm Tris ⁄ HCl (pH 7.5), washed another times with the same buffer containing m NaCl and 0.1% sodium lauryl sarcosinate, then washed twice with mm Tris ⁄ HCl (pH 7.0) The beads were boiled for at 100 °C with 50 mL 0.1% SDS ⁄ 0.5 mm EDTA ⁄ 5% sucrose ⁄ mm Tris ⁄ HCl (pH 8.0) with 2-mercaptoethanol SDS ⁄ PAGE and immunoblotting SDS ⁄ PAGE and immunoblotting were performed as described previously [24] Endoglycosidase digestion Radiolabeled immunoprecipitates were dissolved by being boiled for at 100 °C in 10 lL mm Tris ⁄ HCl (pH 8.0) containing 0.2% SDS To this was added 90 lL 50 mm sodium acetate buffer (pH 6.0) containing 0.75% Triton X-100 and 100 lgỈmL)1 protease inhibitor cocktail and 10 mU endoglycosidase H, and then incubated at 37 °C for 18 h Reactions were stopped by boiling the samples in SDS ⁄ PAGE sample buffer Immunofluorescence microscopy The transfected cells were grown on glass coverslips The cells were briefly washed with NaCl ⁄ Pi and fixed with 3.7% formaldehyde in NaCl ⁄ Pi for 10 at room temperature The fixed cells were washed and permeabilized with 0.2% Triton X-100 in NaCl ⁄ Pi The cells were incubated with the indicated primary antibodies for h at room temperature Antibodies to mature rat cathepsin E, rat pro-cathepsin E, rat Bip, and rat LAMP-1 were used as primary antibodies After being washed, the cells were incubated for h with fluorescein isothiocyanate-conjugated or tetramethylrhodamine isothiocyanate-conjugated secondary antibodies After treatment with Vectashield, the cells were observed by confocal microscopy (Leica Microsystems) References Yamamoto K (1995) Cathepsin E and cathepsin D: biosynthesis, processing and subcellular location In Aspartic Proteinases: Structure, Function, Biology, and Biomedical Implications (Takahashi K, 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lysosome J Biol Chem 267, 21738– 21745 48 Pohlmann R, Boeker MW & von Figura K (1995) The two mannose 6-phosphate receptors transport distinct complements of lysosomal proteins J Biol Chem 270, 27311–27318 49 Cuozzo JW, Tao K, Wu QL, Young W & Sahagian GG (1995) Lysine-based structure in the proregion of procathepsin L is the recognition site for mannose phosphorylation J Biol Chem 270, 15611–15619 50 Hasilik A & Neufeld EF (1980) Biosynthesis of lysosomal enzymes in fibroblasts Phosphorylation of mannose residues J Biol Chem 255, 4946–4950 51 Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection Proc Natl Acad Sci USA 82, 488–492 FEBS Journal 273 (2006) 219–229 ª 2005 The Authors Journal compilation ª 2005 FEBS 229 ... haemagglutinin at the C-terminus and encoding the cathepsin D propeptide fused to mature cathepsin E, we have demonstrated that the propeptide of cathepsin E, likewise those of other aspartic proteinases,... maturation of cathepsin E, we constructed a mutant lacking the propeptide (Leu23-Phe58) and expressed it in HEK -293T cells The transfected cells were pulse-labeled with [35S]methionine for 30 and. .. Pulse–chase analysis of wild-type cathepsin E expressed in HEK -293T cells (A) The transfected cells were metabolically labeled with [35S]methionine ⁄ cysteine for 30 and chased for the times indicated