A new approach for distinguishing cathepsin E and D activity in antigen-processing organelles Nousheen Zaidi 1 , Timo Herrmann 1,5 , Daniel Baechle 2 , Sabine Schleicher 3 , Jeannette Gogel 4 , Christoph Driessen 4 , Wolfgang Voelter 5 and Hubert Kalbacher 1,5 1 Medical and Natural Sciences Research Centre, University of Tu ¨ bingen, Germany 2 PANATecs GmbH, Tu ¨ bingen, Germany 3 Children’s Hospital Department I, University of Tu ¨ bingen, Germany 4 Department of Medicine II, University of Tu ¨ bingen, Germany 5 Interfacultary Institute of Biochemistry, University of Tu ¨ bingen, Germany Cathepsin E (CatE; EC 3.4.23.34) and D (CatD; EC 3.4.23.5) are the major intracellular aspartic protein- ases. They have similar enzymatic properties, e.g. susceptibility to various proteinase inhibitors such as pepstatin A and similar substrate preferences, as both prefer bulky hydrophobic amino acids at P1 and P1¢ positions [1]. In addition, both enzymes have approximately the same acidic pH optimum towards various protein substrates such as hemo- globin [2,3]. However, these enzymes have different tissue distri- bution and cellular localization, suggesting that they might have more specific physiological functions. CatE is a nonlysosomal proteinase with a limited distribu- tion in certain cell types, including gastric epithelial cells [4], but is mainly present in cells of the immune Keywords antigen-presenting cells; cathepsin D; cathepsin E; enzyme activity assay; fluorescent substrate Correspondence H. Kalbacher, Ob dem Himmelreich 7, 72074 Tu ¨ bingen, Germany Fax: +49 7071 294507 Tel: +49 7071 2985212 E-mail: kalbacher@uni-tuebingen.de Website: http://www.kalbacher.uni- tuebingen.de (Received 27 March 2007, revised 24 April 2007, accepted 25 April 2007) doi:10.1111/j.1742-4658.2007.05846.x Cathepsin E (CatE) and D (CatD) are the major aspartic proteinases in the endolysosomal pathway. They have similar specificity and therefore it is difficult to distinguish between them, as known substrates are not exclu- sively specific for one or the other. In this paper we present a substrate- based assay, which is highly relevant for immunological investigations because it detects both CatE and CatD in antigen-processing organelles. Therefore it could be used to study the involvement of these proteinases in protein degradation and the processing of invariant chain. An assay combi- ning a new monospecific CatE antibody and the substrate, MOCAc-Gly- Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-d-Arg-NH 2 [where MOCAc is (7-methoxycoumarin-4-yl)acetyl and Dnp is dinitrophenyl], is presented. This substrate is digested by both proteinases and therefore can be used to detect total aspartic proteinase activity in biological samples. After deple- tion of CatE by immunoprecipitation, the remaining activity is due to CatD, and the decrease in activity can be assigned to CatE. The activity of CatE and CatD in cytosolic, endosomal and lysosomal fractions of B cells, dendritic cells and human keratinocytes was determined. The data clearly indicate that CatE activity is mainly located in endosomal compartments, and that of CatD in lysosomal compartments. Hence this assay can also be used to characterize subcellular fractions using CatE as an endosomal mar- ker, whereas CatD is a well-known lysosomal marker. The highest total aspartic proteinase activity was detected in dendritic cells, and the lowest in B cells. The assay presented exhibits a lower detection limit than com- mon antibody-based methods without lacking the specificity. Abbreviations CatD, cathepsin D; CatE, cathepsin E; EBV, Epstein–Barr virus; NAG, N-acetyl-b- D-glucosaminidase; TAPA, total aspartic proteinase activity. 3138 FEBS Journal 274 (2007) 3138–3149 ª 2007 The Authors Journal compilation ª 2007 FEBS system, such as macrophages [5], lymphocytes [5], microglia [6] and dendritic cells [7]. It is reported to be localized in different cellular compartments, such as plasma membranes [8], endosomal structures [6], endo- plasmic reticulum and Golgi apparatus [6,9,10]. In contrast, CatD is a typical lysosomal enzyme widely distributed in almost all mammalian cells [5,9,11,12]. Studies with CatE-deficient and CatD-deficient mice have provided additional evidence of the association of these enzymes with different physiological effects. CatD-deficient mice develop massive intestinal necrosis [13], thromboembolia [13], lymphopenia [13], and neur- onal ceroid lipofuscinosis [14]. CatE-deficient mice are found to develop atopic dermatitis-like skin lesions [15]. It was reported recently that CatE-deficient mice show increased susceptibility to bacterial infection associated with decreased expression of multiple cell surface Toll-like receptors [16]. According to a very recent study [17], CatE deficiency induces a novel form of lysosomal storage disorder in which there is an accumulation of lysosomal membrane sialoglycopro- teins and an increase in lysosomal pH in macrophages. CatD has also been suggested to play a role in deter- mining the metastatic potential of several types of can- cer; high levels of CatD have been found in prostate [18], breast [19] and ovarian cancer [20]. CatE is expressed in pancreatic ductal adenocarcinoma [21], and its presence in pancreatic juice is reported to be a diagnostic marker for this cancer [22]. Increased con- centrations of CatE in neurons and glial cells of aged rats are suggested to be related to neuronal degener- ation and re-activation of glial cells during the normal aging process of the brain [23]. CatE and CatD both play an important role in the MHC class II pathway. CatD is reported to be involved in processing MHC II-associated invariant chain [24] in antigen processing and presentation [25,26]. CatE is also reported to be involved in antigen processing by B cells [27,28] microglia [29] and murine dendritic cells [7]. Several studies have determined the subcellular localization of CatE and CatD in different cell types, but there are few reports on the activity of these enzymes in organelles relevant to antigen-processing [5,30]. Previous reports have described highly selective substrates for aspartic proteinases, but none of the substrates described is exclusively specific for CatE or CatD [30–32]. In most of the studies, additional meth- ods or inhibitors are used to measure the specific activ- ity of CatE or CatD. For example, to specifically determine CatD activity, a CatD digest and pull-down assay has been described [30]. Other studies have util- ized a specific inhibitor of CatE, the Ascaris pepsin inhibitor, which inhibits pepsins and CatE [33], but does not affect other types of aspartic proteinases including CatD [31,34]. This inhibitor was originally isolated from the round worm Ascaris lumbricoides [35]. However, it is not commercially available. In the present study, CatE and CatD activities were determined in subcellular fractions (lysosomal, endo- somal and cytosolic) of antigen-presenting cells. For measuring total aspartic proteinase activity (TAPA) in biological samples, the previously described peptide substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg- Leu-Lys(Dnp)-D-Arg-NH 2 [where MOCAc is (7-meth- oxycoumarin-4-yl)acetyl and Dnp is dinitrophenyl] [31] was used, which is digested by both CatE and CatD. It is an intramolecularly quenched fluorogenic peptide derivative in which the fluorescent signal of the fluoro- phore MOCAc is quenched by the chromophoric resi- due Dnp. After cleavage of the peptide, the quenching efficiency is decreased, resulting in an increase in fluorescence. The activity determined in subcellular fractions was completely inhibited by pepstatin A. Therefore, this activity can be only attributed to aspar- tic proteinases and represents TAPA. For the specific determination of CatE and CatD activity, CatE was specifically depleted by immunoprecipitation. The remaining activity is due to CatD, and the decrease in activity is assigned to CatE. This approach allows the specific and highly sensitive measurement of both CatE and CatD activities in biological samples. Results and Discussion Expression of CatE mRNA in different cell lines To determine the expression of CatE at the mRNA level in different cell lines, RT-PCR was performed using RNA extracted from DCs (monocyte-derived human dendritic cells), WT100 [Epstein–Barr virus (EBV)-transformed B-cell line] and HaCaT (immortal- ized human keratinocyte cell line). PCR products from the cell lines were analyzed by gel electrophoresis and found to contain a band of the expected size (241 bp) (Fig. 1). As these cell lines were found to be positive for CatE mRNA, they were used to determine the enzymatic activity of CatE and CatD. Previous studies have also shown that murine dendritic cells [7] as well as another EBV-transformed B-cell line (Fc7) are pos- itive for CatE mRNA [27]. Determination of antibody specificity The monospecific antibody for CatE was raised against the antigenic peptide SRFQPSQSSTYSQPG (CatE N. Zaidi et al. Determination of cathepsin E and D activity FEBS Journal 274 (2007) 3138–3149 ª 2007 The Authors Journal compilation ª 2007 FEBS 3139 118–132). This peptide was selected from the CatE sequence using laser gene software (dnastar, Madi- son, WI, USA) for antigenicity and surface probabil- ity. blast tool analysis showed that the selected peptide sequence does not exhibit significant homology with sequences in CatD or any other known protein, therefore it is specifically present in CatE. Figure 2A shows sequence alignment of CatE and CatD. The antiserum obtained was further purified by affinity chromatography on CH-activated Sepharose containing the peptide SRFQPSQSSTYSQPG immobi- lized via stable peptide bonds. To determine the specificity and cross-reactivity of the resulting CatE antibody, indirect ELISA, competit- ive inhibition ELISA (CI-ELISA) and western blot analysis were performed. The results of indirect ELISA (Fig. 2B) showed that the antibody specifically recognized CatE and the anti- genic peptide SRFQPSQSSTYSQPG used to generate the antibody, and gave a complete negative reaction towards CatD. CI-ELISA was performed to further enhance the specificity of the antibody. The antibody was preincu- bated with different concentrations of CatE and CatD, before a standard ELISA was performed to detect the antigenic peptide SRFQPSQSSTYSQPG. Preincuba- tion of CatE with the antibody showed a dose-depend- ent inhibition of antibody binding (IC 50 ¼ 48.6 ng; Fig. 2C). Increasing concentrations of CatD did not affect antibody binding. This experiment shows that CatE specifically binds to the monospecific antibody in a free system. Western blot analysis also confirmed that the mono- specific antibody specifically recognizes CatE and not CatD (data not shown). Characterization of subcellular fractions To control the quality of subcellular fractions, N-acetyl-b-d-glucosaminidase (NAG; EC 3.2.1.52) activity was determined, as it is a wide-spread and well-established marker for endosomal ⁄ lysosomal compartments [36]. Table 1 shows the activity of NAG in subcellular fractions of different cell lines. As expected, all cell lines showed highest NAG activity in lysosomal fractions with lower activity in endosomal fractions. Cytosolic fractions had very low NAG activity. Western blot analysis of subcellular fractions from different cell lines used for CatE and CatD determination For immunochemical determination of subcellular localization of CatE and CatD, western blot analysis was performed. No CatE was recovered from any sub- cellular fraction of WT100. Endosomal fractions of DCs and HaCaT contained a significantly larger amount of CatE than the respective lysosomal frac- tions, but no CatE was found in the cytosolic fractions of any of the cell lines (Fig. 3). As expected, higher amounts of CatD were detectable in lysosomal frac- tions. No CatD was detected by western blotting in the cytosolic fraction of any of the three cell types (Fig. 3). Specific inhibition of CatE by immunoprecipitation To determine the specificity of our immobilized CatE antibody in depleting CatE from the samples, we tes- ted it with CatE and CatD. CatE (recombinant) was completely immunoprecipitated by the antibody against CatE (Fig. 4A), whereas it had almost no effect on CatD activity (Fig. 4B). This approach for depleting proteinase activity from complex biological samples is flexible and can be used for other proteinases as well. Activity of Cat E and CatD in subcellular fractions of different cell types The activity of CatE and CatD was determined in subcellular fractions of different cell types using a combination of the peptide substrate, aspartic prote- inase inhibitor (pepstatin A) and depletion of CatE by immunoprecipitation. Activities were determined by linear regression using a minimum of five measurement points as described in Experimental CatE (241bp) M Negative Control DCs HaCaT WT100 Fig. 1. CatE expression at mRNA level in different cell lines. Total RNA was extracted from HaCaT, WT100 and DCs. Equal amounts of total RNA (2 lg) from each sample were used for RT-PCR. After reverse transcription, specific primers for human CatE were used to amplify CatE cDNA. Determination of cathepsin E and D activity N. Zaidi et al. 3140 FEBS Journal 274 (2007) 3138–3149 ª 2007 The Authors Journal compilation ª 2007 FEBS procedures. The activity in all subcellular fractions of these different cell types was completely inhibited when the samples were preincubated with pepsta- tin A (TAPA). For differential measurements of CatE and CatD activity, samples were subjected to immunoprecipita- tion of CatE. The decrease in activity after immuno- precipitation is attributed to CatE, and the remaining activity is assigned to CatD. As expected, the highest CatD activity was determined in the lyso- somal fractions of all three cell types tested [30]. In contrast, CatE activity was mainly detected in endo- somal fractions, as indicated in Table 2 and Fig. 5. A low level of CatD activity was determined in endosomal fractions of all three cell types. In HaCaT and DCs, a low level of CatE activity was found in lysosomal fractions. In the EBV-trans- formed B-cell line (WT100) an almost equal level of CatE and CatD activity was found in the lysosomal fraction, probably because of overlapping subcellular Fig. 2. (A) Sequence alignment of CatE and CatD. The alignment was performed using a conventional BLAST search engine. Only the small region of CatE containing the sequence SRFQPSQSSTYSQPG (antigenic peptide, CatE 118–132, which was used for generating monospecific antibody) was included during the BLAST operation (sequence can be seen underlined in the figure). This peptide was selected from the CatE sequence using laser gene software ( DNASTAR, Madison, WI, USA) for antige- nicity and surface probability. BLAST tool ana- lysis showed that the selected peptide sequence does not exhibit significant homol- ogy with sequences in CatD or any other known protein, therefore it is specifically present in CatE. (B) Determination of specif- icity of monospecific antibody (raised against SRFQPSQSSTYSQPG) by indirect ELISA. The purified monospecific antibody specifically recognized CatE (10 ng) and the antigenic peptide (SRFQPSQSSTYSQPG), and gave a complete negative reaction towards the same amount of CatD (10 ng). Values are mean ± SD, n ¼ 3. (Insertion: 10 ng CatE and CatD and 1 ng antigenic peptide were incubated on an ELISA plate. CatE and CatD antibodies were used for the detection at dilutions of 1 : 10000 and 1 : 5000.) (C) Competitive inhibition of anti- body (raised against SRFQPSQSSTYSQPG) binding to SRFQPSQSSTYSQPG-coated plates by CatE. Immunoplates were coated with antigenic peptide (SRFQPSQSSTYSQPG; 0.1 lg ⁄ well). Monospesific antibodies were preincubated with different concentrations of CatE or CatD, before standard ELISA. ELISA was performed as described in Experimental pro- cedures. The increasing concentration of CatE caused inhibition of antibody binding giving the IC 50 value of 48.6 ng. The same concentrations of CatD had no effect on antibody binding. Data points are mean ± SD, n ¼ 2. N. Zaidi et al. Determination of cathepsin E and D activity FEBS Journal 274 (2007) 3138–3149 ª 2007 The Authors Journal compilation ª 2007 FEBS 3141 fractions. Cytosolic fractions of all three cell types showed very low CatE activity, and no CatD activ- ity. Moreover, the overall activity in the subcellular fractions of the three cell types tested varied substan- tially, as did CatE and CatD activity. DCs showed the highest, and WT100 cells, the lowest overall activity. As shown in Table 2, endosomal fractions of HaCaT showed  5.5-fold higher CatE activity than the corresponding fractions of WT100, whereas endo- somal fractions of DCs showed  19 times higher CatE activity than the endosomal fractions of WT100. Table 2 also shows that the lysosomal fraction of HaCaT had 7.2 times higher CatD activity than the corresponding fraction of WT100, and this subcellular fraction from DCs had  16.6 times higher CatD activ- ity than that from WT100. Analysis of peptide fragments obtained by digestion of the fluorogenic substrate with subcellular fractions, CatE or CatD, using RP-HPLC and MALDI-MS To further confirm that the activity measured in the subcellular fractions by the fluorescence assay was only due to aspartic proteinases, the peptide substrate was digested by CatE, CatD or subcellular fractions (as described in Experimental procedures) The peptide fragments thus generated were separated by RP-HPLC using fluorescence detection (k ex ¼ 350, k em ¼ 450) and identified by MALDI-MS (Table 3). This method allowed detection of only N-terminal fragments con- taining the fluorophore MOCAc. Figure 6A shows the chromatogram of the undigest- ed peptide substrate MOCAc-Gly-Lys-Pro-Ile-Leu- Phe-Phe-Arg-Leu-Lys(Dnp)-d-Arg-NH 2 as a negative control. The fluorescence signal is quenched as a result of resonance energy transfer between the fluorophore Table 1. NAG activity (fluorescence per min per lg protein) in sub- cellular fractions of different cell lines. Activities were determined by linear regression analysis taking at least seven measurement points. Values are mean ± SD (n ¼ 3). Cell line Subcellular fraction NAG activity HaCaT Cytosolic 0.5 ± 0.05 Lysosomal 15.2 ± 0.3 Endosomal 7.0 ± 0.37 WT100 Cytosolic 0.4 ± 0.02 Lysosomal 9.6 ± 0.1 Endosomal 2.2 ± 0.07 DCs Cytosolic 0.7 ± 0.07 Lysosomal 25.2 ± 0.07 Endosomal 8.0 ± 0.2 Fig. 3. CatE and CatD expression at protein level in relevant anti- gen-processing organelles of different cell lines. Equal amounts of total protein (50 lg) from each sample were applied for SDS ⁄ PAGE followed by western blot analysis. Representative immunoblots with the monospecific CatE antibody and reprobe of the same blot with the CatD antibody are shown. C, Cytosolic fraction; L, lyso- somal fraction; E, endosomal fraction. Fig. 4. Effect of immunoprecipitation of CatE and pepstatin A treat- ment on (A) CatE and (B) CatD activities. (A) (j) Hydrolysis of the fluorogenic peptide substrate (1 l M) by 10 ng CatE in 50 mM sodium acetate buffer (pH 4) at 37 °C. (m) Incubation with pepsta- tin A for 15 min at 37 °C before hydrolysis reaction inhibited the activity of CatE completely. (d ) immunoprecipitation of CatE before hydrolysis reaction also completely inhibited the activity of CatE. (B) (j) Hydrolysis of the fluorogenic peptide substrate (1 l M)by 10 ng CatD in 50 m M sodium acetate buffer (pH 4) at 37 °C. (m) Incubation with pepstatin A for 15 min at 37 °C before hydrolysis reaction inhibited the activity of CatD completely. (d) immunopre- cipitation of CatE before hydrolysis has no effect on CatD activity, hence immunoprecipitation was specific for CatE only. Determination of cathepsin E and D activity N. Zaidi et al. 3142 FEBS Journal 274 (2007) 3138–3149 ª 2007 The Authors Journal compilation ª 2007 FEBS and the quencher group. Figure 6B shows the results of digestion of the substrate with CatE, leading to only one cleavage product, because only the Phe-Phe bond is susceptible to cleavage by CatE or CatD [31]. The peak with a retention time of 25.54 min corresponds to the fragment, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe, as analyzed by MALDI-MS (Table 3). Figure 6C shows digestion of the substrate with CatD, giving a profile similar to that of CatE, i.e. only one peak is visible with the same retention time. However, when digested with the lysosomal fraction of HaCaT (Fig. 6E), an additional peak with a retention time of 22.87 min was observed. Digestion of substrate with the endosomal fraction of HaCaT (Fig. 6F) gave a similar RP-HPLC profile to the lysosomal fraction. Digestion of the substrate with lysosomal and endo- somal fractions (Fig. 6H,I) was completely inhibited by pepstatin A, confirming that the activity observed in our assay was solely due to aspartic proteinases. The additional peak observed after digestion of the substrate with these fractions (Fig. 6E,F) was a C-ter- minal-truncated peptide (MOCAc-Gly-Lys-Pro-Ile- Leu), as analyzed by MALDI-MS. This carboxypeptidase activity can only occur after aspartic proteinases have created cleavage products, as the undigested substrate contains a protective d-Arg residue at the C-terminus. Substrate digestion by the lysosomal fraction (Fig. 6K) after immunoprecipitation of CatE had almost no effect on the RP-HPLC profile. This indicates that the activity observed in the lysosomal fraction was mainly due to CatD. Digestion by the endosomal frac- tion (Fig. 6L) was inhibited after immunoprecipitation of CatE, indicating that the activity in this fraction was primarily CatE activity. No cleavage was indicated in the cytosolic fraction, hence no CatE or CatD activity was observed by RP-HPLC. This agrees with the results from the fluorescence assay, in which only very low activity was determined in the cytosolic fraction. Digestion of substrate with subcellular fractions of DCs and WT100 gave similar RP-HPLC profiles (data not shown). In conclusion, the combination of methods described here facilitates the specific and parallel measurement of CatE and CatD activity in antigen-processing organ- elles. The data clearly show that our approach for detecting CatE and CatD is more sensitive than immu- nodetection by western blot analysis. It allows detec- tion of CatE activity in subcellular fractions of WT100, as compared to western blot analysis by which no CatE was detectable in any WT100 fraction. It was also possible to discriminate between CatD activity in endosomal and lysosomal fractions, whereas the distri- bution of CatD in lysosomal and endosomal fractions was not significantly distinguishable when detected by western blot. Theses experimental conditions are also more speci- fic than previous assays, because specificity of detec- tion was not only based on the peptide sequence but was markedly increased by the use of a monospecific antibody used to deplete CatE. This type of assay is flexible and can be used to discriminate activity of other proteinases with similar enzymatic properties. This approach distinguishes between the activities of the enzymatically similar proteinases, CatE and CatD, and can therefore be used to investigate the involvement of these enzymes in antigen processing and presentation. Experimental procedures Enzymes and chemicals CatD (bovine kidney) was purchased from Calbiochem (Darmstadt, Germany) and stored as a 300 UÆmL )1 stock solution in 0.1 m sodium citrate buffer, pH 4.5, at )20 °C. CatE was purchased from R&D systems (Wiesbaden, Germany) and stored as a 0.1 mgÆmL )1 stock solution in 50 mm sodium citrate buffer, pH 6.5, containing 150 mm Table 2. CatE and CatD activity (pmol MOCAc liberated per min per 20 lg total protein) in subcellular fractions of different cell lines. Activit- ies were determined by linear regression analysis taking at least five measurement points. Values are mean ± SD (DCs, n ¼ 2; HaCaT and WT100, n ¼ 3; where n is the number of individual experiments performed). ND, not detectable. Cell line Activity Cytosolic fraction Lysosomal fraction Endosomal fraction HaCaT TAPA 13.4 ± 2.15 106.3 ± 6.94 82.0 ± 11.33 Cat E 13.4 ± 2.15 25.3 ± 14.01 65.4 ± 11.22 Cat D ND 80.9 ± 12 16.6 ± 8.40 WT100 TAPA 0.49 ± 0.19 20.9 ± 1.99 13.7 ± 0.69 Cat E 0.49 ± 0.19 9.5 ± 0.54 11.8 ± 1.23 Cat D ND 11.3 ± 2.54 1.9 ± 0.77 DCs TAPA 0.76 ± 0.24 210.7 ± 18.40 232.8 ± 49.65 Cat E 0.76 ± 0.24 23.2 ± 20.54 225.7 ± 50.38 Cat D ND 187.5 ± 2.19 7.1 ± 0.72 N. Zaidi et al. Determination of cathepsin E and D activity FEBS Journal 274 (2007) 3138–3149 ª 2007 The Authors Journal compilation ª 2007 FEBS 3143 NaCl at )20 °C. Pepstatin A (Calbiochem) was dissolved in methanol. Activated CH Sepharose 4B was purchased from Amersham Biosciences (Munich, Germany). The substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-d- Arg-NH 2 [31] was obtained from Bachem (Weil am Rhein, Germany). Generation and immobilization of a monospecific CatE antibody The antigenic peptide SRFQPSQSSTYSQPG (CatE 118– 132) was selected from the protein sequence using the laser gene software (dnastar, Madison, WI, USA) and controlled for specificity to CatD. It was synthesized as a single peptide and as a multiple antigen peptide, (SRFQPS- QSSTYSQPG) 8 -(Lys) 4 -(Lys) 2 -Lys-Gly-OH, using standard Fmoc ⁄ tBu [37] chemistry on a multiple peptide synthesizer, Syro II (MultiSynTech, Witten, Germany). The peptides were purified using RP-HPLC and the identity was con- firmed using ESI-MS. Peptide purities were determined by analytical RP-HPLC and were >90%. The single peptide was coupled to key hole limpet hemocycanin using the glu- tardialdehyde method. The antiserum was obtained after repeated immunization of a rabbit with a 1 : 1 mixture of the peptide–key hole limpet hemocycanin conjugate and the multiple antigen peptide. This antiserum was further puri- fied by affinity chromatography on a CH-activated Seph- arose 4B column (Amersham Biosciences) containing the peptide immobilized via a stable peptide bond. Peptide immobilization was performed as described by the manu- facturer. The antiserum was applied to the column at 0.5 mLÆmin )1 and recycled overnight. The column was washed with 20 column volumes of NaCl ⁄ P i (Gibco Life Technologies, Paisley, UK). Elution was performed with 10 volumes of 0.1 m glycine ⁄ HCl (pH 2.5). Antibody-contain- ing fractions were immediately neutralized with 1 m Tris ⁄ HCl (pH 8.5) and then concentrated on a 20-kDa membrane. The resulting antibody was retested by ELISA and showed the expected specificity to the peptide epitopes and the CatE protein, but a completely negative reaction to CatD. The purified monospecific antibody was immobilized on CH-activated Sepharose as described by the manufac- turer. After coupling for 3 h at room temperature, the gel was deactivated with 0.1 m Tris ⁄ HCl, pH 8.0, for an addi- tional 2 h at room temperature. To block any remaining active sites, the material was further incubated with 5% BSA for an additional 2 h. After a wash with NaCl ⁄ P i , the immobilized antibody was stored in NaCl ⁄ P i containing 0.02% (w ⁄ v) NaN 3 at 4 °C. ELISA The wells of microtiter plates (Nunc Brand Products, Maxi- Sorb surface, Wiesbaden, Germany) were coated with CatE (10 ng), CatD (10 ng) or the peptide SRFQPSQSSTYSQPG (1 ng) in NaCl ⁄ P i in a final volume of 100 lL ⁄ well at 4 °C overnight. The plates were washed three times with 200 lL washing buffer (NaCl ⁄ P i ⁄ 0.05% Tween 20, pH 7.0) and blocked with blocking buffer (NaCl ⁄ P i ⁄ 0.05% Tween 20, pH 7.0, containing 2% BSA) for 2 h at 37 °C. After a wash, the plates were treated for 1 h at 37 °C with our monospecific CatE antibody (diluted in NaCl ⁄ P i ⁄ 0.05% Tween 20, pH 7.0, containing 0.5% BSA) or commercial CatD antibody. After a wash, the plates were incubated with horseradish peroxidase-conjugated goat anti-rabbit Ig (Dianova, Hamburg, Germany; 1 : 5000 diluted in NaCl ⁄ P i ⁄ 0.05% Tween 20 ⁄ 0.5% BSA). Then 100 lL azino- diethylbenzthiazoline sulfonate ⁄ H 2 O 2 in substrate buffer 0 20 40 60 80 100 120 Endosomes Lysosomes Cytosol Endosomes Lysosomes Cytosol Endosomes Lysosomes Cytosol pmol MOCAc liberated/min/20µg total protein 0 50 100 150 200 250 300 p mol MOCAc liberated/min/20 µg total p rotein 0 5 10 15 20 25 pmol MOCAc liberated/min/20µg total protein TAPA CatE activity CaD activity TAPA CatE activity CaD activity TAPA CatE activity CaD activity A B C Fig. 5. Distribution of TAPA, CatE and CatD activity in subcellular fractions of the cell lines (A) HaCaT, (B) WT100 and (C) DCs. Equal amounts of total protein (20 lg) were used for the determination of CatE and CatD activities, determined by linear regression analysis using a minimum of five measurement points. Values are mean ± SD (DCs, n ¼ 2; HaCaT and WT100, n ¼ 3; where n is the number of individual experiments). Determination of cathepsin E and D activity N. Zaidi et al. 3144 FEBS Journal 274 (2007) 3138–3149 ª 2007 The Authors Journal compilation ª 2007 FEBS (100 mm sodium citrate buffer, pH 4.5) was added per well, and the colour development analyzed at a wavelength of 405 nm. For competitive inhibition ELISA, antiserum was prein- cubated with different concentrations of CatE or CatD (40 min, room temperature) and then used as primary anti- body for standard ELISA to detect the antigenic peptide SRFQPSQSSTYSQPG (0.1 lg ⁄ well). Cell culture The EBV-transformed human B-cell line, WT100, and the immortalized human keratinocyte cell line, HaCaT, were cultured in RPMI 1640 medium (Gibco Life Technol- ogies) supplemented with 10% (v ⁄ v) heat-inactivated fetal calf serum (Gibco), penicillin (final concentration 100 UÆmL )1 ; Gibco) and streptomycin (final concentration 0.1 mgÆmL )1 ; Gibco) at 37 °C in tissue culture flasks (Nunc). Peripheral blood mononuclear cells were isolated by Ficoll ⁄ Paque (PAA Laboratories, Pasching, Austria) den- sity gradient centrifugation of heparinized blood obtained from buffy coats. Isolated peripheral blood mononuclear cells were plated (1 · 10 8 cells ⁄ 8 mL flask) into 75 cm 2 Cellstar tissue culture flasks (Greiner Bio-One GmbH, Fric- kenhausen, Germany) in RPMI 1640 under the same cul- ture conditions as for WT100 and HaCaT. After 1.5 h of incubation at 37 °C, nonadherent cells were removed and adherent cells were cultured in complete culture medium supplemented with granulocyte ⁄ macrophage colony-stimu- lating factor (Leukomax; Sandoz, Basel, Switzerland) and interleukin 4 (R&D systems) for 6 days as described previ- ously [38]. This resulted in a cell population consisting of  70% DCs (data not shown), as determined by flow cytometry (BD FACSCalibur, Heidelberg, Germany). Determination of CatE mRNA expression levels using RT-PCR RNA was extracted from DCs, WT100 and HaCaT cells using the TRIazol reagent as described by the manufacturer (Invitrogen, Karlsruhe, Germany). Reverse transcription of 2 lg total RNA was initialized by 200 U Superscript II reverse transcriptase (Invitrogen), 4 lL synthesis buffer (fivefold concentrated; Invitrogen), 2.5 lL Random Primers (10 mm; Promega, Mannheim, Germany), 1 lL dithiothrei- tol (100 mm; Invitrogen), 1 lL dNTP mix (10 mm; Prome- ga) and 0.5 lL rRNAsin (Promega) in a final volume of 20 lL. After incubation at room temperature for 10 min, the reaction mixtures were set to 42 °C for 1 h. Then amplification was carried out, adding 5 lL generated cDNA to 45 lL reaction mixture [11.0% (v ⁄ v) 10-fold PCR buffer (Roche, Basel, Switzerland), 3.3% (v ⁄ v) both primers (5¢-CATGATGGAATTACGTT-3¢ and 5¢-GA ATGATCCAGGTACAGCAT-3¢)10lm each (Operon Technologies Alameda, CA, USA), 2.2% (v ⁄ v) dNTP mix (10 mm; Promega) in molecular-grade water and 1.1% (v ⁄ v) Taq DNA polymerase (Roche)] and running 35 cycles each for 35 s at 94 °C, 30 s at 50 °C, and 60 s at 72 °C. Single PCR amplicons were analysed using agarose gel electrophoresis. Subcellular fractionation and western blot analysis Cell fractionation was performed as previously described by Schroter et al. [39]. Briefly, (4–8) · 10 7 cells were harvested, resuspended in 1.5 mL fractionation buffer (10 mm Tris ⁄ HCl buffer, 250 mm sucrose, pH 6.8), and then homo- genized using a cell cracker (HGM Laboratory Equipment, Heidelberg, Germany). Then debris was separated by cen- trifugation at 8000 g for 10 min with a Minifuge RF 2150 (Heraeus, Osterode, Germany). Mitochondria and the endolysosomal fractions were separated by ultracentrifuga- tion at 100 000 g for 5 min (Beckman TL100 ultracentri- fuge, Palo Alto, CA, USA). Finally, lysosomes were separated from endosomes by hypotonic lysis with double- distilled water ( 2.5-fold of the pellet volume for keratino- cytes and DCs, and fivefold of the pellet volume for B cells) and centrifugation at 100 000 g for 5 min with a Beckman TL100 ultracentrifuge. Lysosomal material was released into the supernatant, and endosomes remained in the pellet. Total protein content was determined as described by Bradford [40]. Table 3. Peptides after digestion of fluorogenic peptide substrate by CatE, CatD and subcellular fractions of HaCaT identified by MALDI-MS. Retention times allude to those in Fig. 6. Sample Digestion products Retention time (min) Expected mass [M +H] + [M +H] + DDa CatE MOCAc-GLPILF-OH 25.54 890.80 890.77 0.03 CatD MOCAc-GLPILF-OH 25.19 890.80 891.76 0.96 Lysosomal fraction MOCAc-GLPILF-OH 25.90 890.80 890.90 0.1 MOCAc-GLPIL-OH 22.87 743.70 743.70 0.0 Endosomal fraction MOCAc-GLPILF-OH 25.44 890.80 890.80 0.0 MOCAc-GLPIL-OH 22.38 743.70 743.80 0.1 N. Zaidi et al. Determination of cathepsin E and D activity FEBS Journal 274 (2007) 3138–3149 ª 2007 The Authors Journal compilation ª 2007 FEBS 3145 Subcellular fractions were separated by SDS ⁄ PAGE (50 lg total protein per lane) on a 12% separating gel and transferred to a poly(vinylidene difluoride) membrane (Amersham Biosciences, Freiburg, Germany). Membranes were then blocked for 1 h using NaCl ⁄ Tris [0.15 m NaCl, 10 mm Tris ⁄ HCl, 0.05% (v ⁄ v) Tween 20, pH 8.0] containing 10% (v ⁄ v) RotiÒ Block (Roth, Karlsruhe, Germany). Rabbit antibody to human CatD (Calbio- chem) was diluted 1 : 5000, and rabbit antibody to human CatE was diluted 1 : 2000. Western blots were developed according to the ECL protocol of Amersham Biosciences. Detection of NAG activity NAG activity was measured as described by Schmid et al. [36]. Briefly, 1 lg protein from each fraction was added to 100 lL 0.1 m citrate buffer, pH 5, containing 0.8 mm 4-methylumbelliferyl-N-acetyl-b-d-glucosaminide (Sigma, Deisenhofen Germany) and 0.1% Triton X-100. Fluores- cence (k ex ¼ 360 nm, k em ¼ 465 nm) was measured every 5 min at 37 °C using a fluorescence reader (Tecan Spectra Fluor, Crailsheim, Germany). NAG activity was deter- mined by linear regression using a minimum of seven meas- urement points. A Undigested substrate B Substrate + CatE C Substrate + CatD Relative Fluorescence Relative Fluorescence Relative Fluorescence 25.54 5 10 15 20 25 30 35 25.19 5 10 15 20 25 30 35 5 10 15 20 25 30 35 5 10 15 20 25 30 35 5 10 15 20 25 30 35 5 10 15 20 25 30 35 22.87 25.90 5 10 15 20 25 30 35 22.38 25.44 5 10 15 20 25 30 35 22.91 25.92 5 10 15 20 25 30 35 5 10 15 20 25 30 35 5 10 15 20 25 30 35 Elution time (min) D Substrate + CF E Substrate + LF F Substrate + EF G Substrate + CF + PepA H Substrate + LF + PepA I Substrate + EF + PepA J Substrate + CF (IP) K Substrate + LF (IP) L Substrate + EF (IP) 5 10 15 20 25 30 35 Fig. 6. RP-HPLC profiles of peptide fragments obtained after digestion of the substrate with CatE, CatD or subcellular fractions of HaCaT. Fluorogenic peptide substrate (10 l M) was incubated at 37 °C in digestion buffer (50 mM sodium acetate buffer, pH 4.0) containing CatE (10 ng), CatD (10 ng) or a subcellular fraction (20 lg). (A) Undigested fluorogenic substrate, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu- Lys(Dnp)- D-Arg-NH2. Substrate digested with (B) CatE, (C) CatD, (D) cytosolic fraction (CF), (E) lysosomal fraction (LF), (F) endosomal fraction (EF), (G) cytosolic fraction after pepstatin A treatment, (H) lysosomal fraction after pepstatin A treatment, (I) endosomal fraction after pepsta- tin A treatment, (J) cytosolic fraction after immunoprecipitation of CatE, (K) lysosomal fraction after immunoprecipitation of CatE, and (L) endosomal fraction after immunoprecipitation of CatE. Determination of cathepsin E and D activity N. Zaidi et al. 3146 FEBS Journal 274 (2007) 3138–3149 ª 2007 The Authors Journal compilation ª 2007 FEBS Parallel detection of CatE and CatD activity TAPA and specific catalytic activities of CatE and CatD were determined fluorimetrically by hydrolysis of the substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu- Lys(Dnp)-d-Arg-NH 2 . Appropriate amounts of CatE, CatD or subcellular fraction (20 lg total protein) were added to 80 lL digestion buffer (50 mm sodium acetate buffer, pH 4.0), and the reaction was started by the addi- tion of 1 lL substrate solution (stock solution 2 mm in dimethyl sulfoxide). Fluorescent product formation was recorded using a fluorescence reader (Tecan Spectra Fluor) on kinetic mode at 37 ° C(k ex ¼ 340, k em ¼ 405). Activities were determined by linear regression analysis using a mini- mum of five measurement points. All the experiments were performed in triplicate yielding TAPA, i.e. CatE and CatD activity. Aspartic proteinase activity could be completely inhibited using 1 lL1mm pepstatin A solution in meth- anol (1 lL methanol showed no inhibitory effect). For the specific determination of CatE activity, samples were subjected to immunoprecipitation of CatE before the above assay. Then 20 lg total protein from each subcellular fraction was incubated with 20 lL monospecific CatE anti- body immobilized on CH-activated Sepharose at 4 °C over- night. With this method, the measured increase in fluorescence intensity is exclusively caused by CatD. The difference between total aspartic proteinase and CatD activ- ity can be assigned to CatE activity. Analytical RP-HPLC The fluorogenic peptide substrate (1 mm in dimethyl sulfox- ide; 1 lL) was incubated at 37 °Cin80lL digestion buffer (50 mm sodium acetate buffer, pH 4.0) containing the appro- priate amount of CatE, CatD or a subcellular fraction (with or without pepstatin A treatment or after immunoprecipita- tion of CatE). The reaction was terminated by addition of 25 lL stop solution [5% (v ⁄ v) acetonitrile, 1% (v ⁄ v) tri- fluoroacetic acid] in water. Then 5 lL of the reaction mixture was separated by analytical RP-HPLC using a C8 column (150 · 2 mm; Reprosil 100; Dr Maisch GmbH, Tu ¨ bingen, Germany) with the following solvent systems: (A) 0.055% (v ⁄ v) trifluoroacetic acid in water and (B) 0.05% (v ⁄ v) tri- fluoroacetic acid in 80% (v ⁄ v) acetonitrile in water. Elution was performed using a linear gradient from 5% to 80% sol- vent B within 35 min. Fluorescence detection was carried out at k em. ¼ 350 and k ex ¼ 450. Appropriate fractions were collected and analysed by MALDI-MS. MALDI-MS First, 0.5 lL each RP-HPLC fraction was mixed with 0.5 lL DHB-matrix [10 mgÆmL )1 (w ⁄ v) 2,5-dihydroxy- benzoic acid in 60% (v ⁄ v) ethanol containing 0.1% (v ⁄ v) trifluoroacetic acid] and applied to a gold target for MALDI-MS using a MALDI-TOF system (Reflex IV, serial number 26159.00007; Bruker Daltonics, Bremen, Ger- many). Signals were generated by accumulating 120–210 laser shots. 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