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A new approach for distinguishing cathepsin E and Dactivity in antigen-processing organellesNousheen Zaidi1, Timo Herrmann1,5, Daniel Baechle2, Sabine Schleicher3, Jeannette Gogel4,Christoph Driessen4, Wolfgang Voelter5and Hubert Kalbacher1,51 Medical and Natural Sciences Research Centre, University of Tu¨bingen, Germany2 PANATecs GmbH, Tu¨bingen, Germany3 Children’s Hospital Department I, University of Tu¨bingen, Germany4 Department of Medicine II, University of Tu¨bingen, Germany5 Interfacultary Institute of Biochemistry, University of Tu¨bingen, GermanyCathepsin E (CatE; EC 3.4.23.34) and D (CatD; EC3.4.23.5) are the major intracellular aspartic protein-ases. They have similar enzymatic properties, e.g.susceptibility to various proteinase inhibitors such aspepstatin A and similar substrate preferences, asboth prefer bulky hydrophobic amino acids at P1and P1¢ positions [1]. In addition, both enzymeshave approximately the same acidic pH optimumtowards various protein substrates such as hemo-globin [2,3].However, these enzymes have different tissue distri-bution and cellular localization, suggesting that theymight have more specific physiological functions. CatEis a nonlysosomal proteinase with a limited distribu-tion in certain cell types, including gastric epithelialcells [4], but is mainly present in cells of the immuneKeywordsantigen-presenting cells; cathepsin D;cathepsin E; enzyme activity assay;fluorescent substrateCorrespondenceH. Kalbacher, Ob dem Himmelreich 7,72074 Tu¨bingen, GermanyFax: +49 7071 294507Tel: +49 7071 2985212E-mail: kalbacher@uni-tuebingen.deWebsite: http://www.kalbacher.uni-tuebingen.de(Received 27 March 2007, revised 24 April2007, accepted 25 April 2007)doi:10.1111/j.1742-4658.2007.05846.xCathepsin E (CatE) and D (CatD) are the major aspartic proteinases in theendolysosomal pathway. They have similar specificity and therefore it isdifficult 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 investigationsbecause it detects both CatE and CatD in antigen-processing organelles.Therefore it could be used to study the involvement of these proteinases inprotein 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-NH2[where MOCAcis (7-methoxycoumarin-4-yl)acetyl and Dnp is dinitrophenyl], is presented.This substrate is digested by both proteinases and therefore can be used todetect total aspartic proteinase activity in biological samples. After deple-tion of CatE by immunoprecipitation, the remaining activity is due toCatD, and the decrease in activity can be assigned to CatE. The activity ofCatE and CatD in cytosolic, endosomal and lysosomal fractions of B cells,dendritic cells and human keratinocytes was determined. The data clearlyindicate that CatE activity is mainly located in endosomal compartments,and that of CatD in lysosomal compartments. Hence this assay can also beused to characterize subcellular fractions using CatE as an endosomal mar-ker, whereas CatD is a well-known lysosomal marker. The highest totalaspartic proteinase activity was detected in dendritic cells, and the lowestin B cells. The assay presented exhibits a lower detection limit than com-mon antibody-based methods without lacking the specificity.AbbreviationsCatD, 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 FEBSsystem, such as macrophages [5], lymphocytes [5],microglia [6] and dendritic cells [7]. It is reported to belocalized in different cellular compartments, such asplasma membranes [8], endosomal structures [6], endo-plasmic reticulum and Golgi apparatus [6,9,10]. Incontrast, CatD is a typical lysosomal enzyme widelydistributed in almost all mammalian cells [5,9,11,12].Studies with CatE-deficient and CatD-deficient micehave provided additional evidence of the associationof 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 arefound to develop atopic dermatitis-like skin lesions[15]. It was reported recently that CatE-deficient miceshow increased susceptibility to bacterial infectionassociated with decreased expression of multiple cellsurface Toll-like receptors [16]. According to a veryrecent study [17], CatE deficiency induces a novel formof lysosomal storage disorder in which there is anaccumulation 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 isexpressed in pancreatic ductal adenocarcinoma [21],and its presence in pancreatic juice is reported to be adiagnostic marker for this cancer [22]. Increased con-centrations of CatE in neurons and glial cells of agedrats are suggested to be related to neuronal degener-ation and re-activation of glial cells during the normalaging process of the brain [23].CatE and CatD both play an important role in theMHC class II pathway. CatD is reported to beinvolved in processing MHC II-associated invariantchain [24] in antigen processing and presentation[25,26]. CatE is also reported to be involved in antigenprocessing by B cells [27,28] microglia [29] and murinedendritic cells [7].Several studies have determined the subcellularlocalization of CatE and CatD in different cell types,but there are few reports on the activity of theseenzymes in organelles relevant to antigen-processing[5,30]. Previous reports have described highly selectivesubstrates for aspartic proteinases, but none of thesubstrates described is exclusively specific for CatE orCatD [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 specificallydetermine CatD activity, a CatD digest and pull-downassay has been described [30]. Other studies have util-ized a specific inhibitor of CatE, the Ascaris pepsininhibitor, which inhibits pepsins and CatE [33], butdoes not affect other types of aspartic proteinasesincluding CatD [31,34]. This inhibitor was originallyisolated from the round worm Ascaris lumbricoides[35]. However, it is not commercially available.In the present study, CatE and CatD activities weredetermined in subcellular fractions (lysosomal, endo-somal and cytosolic) of antigen-presenting cells. Formeasuring total aspartic proteinase activity (TAPA) inbiological samples, the previously described peptidesubstrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2[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 peptidederivative in which the fluorescent signal of the fluoro-phore MOCAc is quenched by the chromophoric resi-due Dnp. After cleavage of the peptide, the quenchingefficiency is decreased, resulting in an increase influorescence. The activity determined in subcellularfractions was completely inhibited by pepstatin A.Therefore, this activity can be only attributed to aspar-tic proteinases and represents TAPA. For the specificdetermination of CatE and CatD activity, CatE wasspecifically depleted by immunoprecipitation. Theremaining activity is due to CatD, and the decrease inactivity is assigned to CatE. This approach allows thespecific and highly sensitive measurement of both CatEand CatD activities in biological samples.Results and DiscussionExpression of CatE mRNA in different cell linesTo determine the expression of CatE at the mRNAlevel in different cell lines, RT-PCR was performedusing RNA extracted from DCs (monocyte-derivedhuman dendritic cells), WT100 [Epstein–Barr virus(EBV)-transformed B-cell line] and HaCaT (immortal-ized human keratinocyte cell line). PCR products fromthe cell lines were analyzed by gel electrophoresis andfound to contain a band of the expected size (241 bp)(Fig. 1). As these cell lines were found to be positivefor CatE mRNA, they were used to determine theenzymatic activity of CatE and CatD. Previous studieshave also shown that murine dendritic cells [7] as wellas another EBV-transformed B-cell line (Fc7) are pos-itive for CatE mRNA [27].Determination of antibody specificityThe monospecific antibody for CatE was raised againstthe antigenic peptide SRFQPSQSSTYSQPG (CatEN. Zaidi et al. Determination of cathepsin E and D activityFEBS Journal 274 (2007) 3138–3149 ª 2007 The Authors Journal compilation ª 2007 FEBS 3139118–132). This peptide was selected from the CatEsequence using laser gene software (dnastar, Madi-son, WI, USA) for antigenicity and surface probabil-ity. blast tool analysis showed that the selectedpeptide sequence does not exhibit significant homologywith sequences in CatD or any other known protein,therefore it is specifically present in CatE. Figure 2Ashows sequence alignment of CatE and CatD.The antiserum obtained was further purified byaffinity chromatography on CH-activated Sepharosecontaining the peptide SRFQPSQSSTYSQPG immobi-lized via stable peptide bonds.To determine the specificity and cross-reactivity ofthe resulting CatE antibody, indirect ELISA, competit-ive inhibition ELISA (CI-ELISA) and western blotanalysis were performed.The results of indirect ELISA (Fig. 2B) showed thatthe antibody specifically recognized CatE and the anti-genic peptide SRFQPSQSSTYSQPG used to generatethe antibody, and gave a complete negative reactiontowards CatD.CI-ELISA was performed to further enhance thespecificity of the antibody. The antibody was preincu-bated with different concentrations of CatE and CatD,before a standard ELISA was performed to detect theantigenic peptide SRFQPSQSSTYSQPG. Preincuba-tion of CatE with the antibody showed a dose-depend-ent inhibition of antibody binding (IC50¼ 48.6 ng;Fig. 2C). Increasing concentrations of CatD did notaffect antibody binding. This experiment shows thatCatE specifically binds to the monospecific antibody ina free system.Western blot analysis also confirmed that the mono-specific antibody specifically recognizes CatE and notCatD (data not shown).Characterization of subcellular fractionsTo 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 andwell-established marker for endosomal ⁄ lysosomalcompartments [36]. Table 1 shows the activity ofNAG in subcellular fractions of different cell lines.As expected, all cell lines showed highest NAGactivity in lysosomal fractions with lower activity inendosomal fractions. Cytosolic fractions had verylow NAG activity.Western blot analysis of subcellular fractionsfrom different cell lines used for CatE and CatDdeterminationFor immunochemical determination of subcellularlocalization of CatE and CatD, western blot analysiswas performed. No CatE was recovered from any sub-cellular fraction of WT100. Endosomal fractions ofDCs and HaCaT contained a significantly largeramount of CatE than the respective lysosomal frac-tions, but no CatE was found in the cytosolic fractionsof any of the cell lines (Fig. 3). As expected, higheramounts of CatD were detectable in lysosomal frac-tions. No CatD was detected by western blotting inthe cytosolic fraction of any of the three cell types(Fig. 3).Specific inhibition of CatE byimmunoprecipitationTo determine the specificity of our immobilized CatEantibody in depleting CatE from the samples, we tes-ted it with CatE and CatD. CatE (recombinant) wascompletely immunoprecipitated by the antibodyagainst CatE (Fig. 4A), whereas it had almost noeffect on CatD activity (Fig. 4B). This approach fordepleting proteinase activity from complex biologicalsamples is flexible and can be used for other proteinasesas well.Activity of Cat E and CatD in subcellular fractionsof different cell typesThe activity of CatE and CatD was determined insubcellular fractions of different cell types using acombination of the peptide substrate, aspartic prote-inase inhibitor (pepstatin A) and depletion of CatEby immunoprecipitation. Activities were determinedby linear regression using a minimum of fivemeasurement points as described in ExperimentalCatE (241bp)MNegativeControlDCsHaCaTWT100Fig. 1. CatE expression at mRNA level in different cell lines. TotalRNA was extracted from HaCaT, WT100 and DCs. Equal amountsof total RNA (2 lg) from each sample were used for RT-PCR. Afterreverse transcription, specific primers for human CatE were usedto 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 FEBSprocedures. The activity in all subcellular fractionsof these different cell types was completely inhibitedwhen the samples were preincubated with pepsta-tin A (TAPA).For differential measurements of CatE and CatDactivity, samples were subjected to immunoprecipita-tion of CatE. The decrease in activity after immuno-precipitation is attributed to CatE, and theremaining 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]. Incontrast, 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 inendosomal fractions of all three cell types. InHaCaT and DCs, a low level of CatE activity wasfound in lysosomal fractions. In the EBV-trans-formed B-cell line (WT100) an almost equal level ofCatE and CatD activity was found in the lysosomalfraction, probably because of overlapping subcellularFig. 2. (A) Sequence alignment of CatE andCatD. The alignment was performed usinga conventionalBLAST search engine. Onlythe small region of CatE containing thesequence SRFQPSQSSTYSQPG (antigenicpeptide, CatE 118–132, which was used forgenerating monospecific antibody) wasincluded during theBLAST operation(sequence can be seen underlined in thefigure). This peptide was selected from theCatE sequence using laser gene software(DNASTAR, Madison, WI, USA) for antige-nicity and surface probability.BLAST tool ana-lysis showed that the selected peptidesequence does not exhibit significant homol-ogy with sequences in CatD or any otherknown protein, therefore it is specificallypresent in CatE. (B) Determination of specif-icity of monospecific antibody (raisedagainst SRFQPSQSSTYSQPG) by indirectELISA. The purified monospecific antibodyspecifically recognized CatE (10 ng) and theantigenic peptide (SRFQPSQSSTYSQPG),and gave a complete negative reactiontowards the same amount of CatD (10 ng).Values are mean ± SD, n ¼ 3. (Insertion:10 ng CatE and CatD and 1 ng antigenicpeptide were incubated on an ELISA plate.CatE and CatD antibodies were used for thedetection at dilutions of 1 : 10000 and1 : 5000.) (C) Competitive inhibition of anti-body (raised against SRFQPSQSSTYSQPG)binding to SRFQPSQSSTYSQPG-coatedplates by CatE. Immunoplates werecoated with antigenic peptide(SRFQPSQSSTYSQPG; 0.1 lg ⁄ well).Monospesific antibodies were preincubatedwith different concentrations of CatE orCatD, before standard ELISA. ELISA wasperformed as described in Experimental pro-cedures. The increasing concentration ofCatE caused inhibition of antibody bindinggiving the IC50value of 48.6 ng. The sameconcentrations of CatD had no effecton antibody binding. Data points aremean ± SD, n ¼ 2.N. Zaidi et al. Determination of cathepsin E and D activityFEBS Journal 274 (2007) 3138–3149 ª 2007 The Authors Journal compilation ª 2007 FEBS 3141fractions. Cytosolic fractions of all three cell typesshowed very low CatE activity, and no CatD activ-ity. Moreover, the overall activity in the subcellularfractions of the three cell types tested varied substan-tially, as did CatE and CatD activity. DCs showedthe highest, and WT100 cells, the lowest overallactivity.As shown in Table 2, endosomal fractions ofHaCaT showed  5.5-fold higher CatE activity thanthe corresponding fractions of WT100, whereas endo-somal fractions of DCs showed  19 times higher CatEactivity than the endosomal fractions of WT100.Table 2 also shows that the lysosomal fraction ofHaCaT had 7.2 times higher CatD activity than thecorresponding fraction of WT100, and this subcellularfraction from DCs had  16.6 times higher CatD activ-ity than that from WT100.Analysis of peptide fragments obtained bydigestion of the fluorogenic substrate withsubcellular fractions, CatE or CatD, usingRP-HPLC and MALDI-MSTo further confirm that the activity measured in thesubcellular fractions by the fluorescence assay was onlydue to aspartic proteinases, the peptide substrate wasdigested by CatE, CatD or subcellular fractions (asdescribed in Experimental procedures) The peptidefragments thus generated were separated by RP-HPLCusing fluorescence detection (kex¼ 350, kem¼ 450)and identified by MALDI-MS (Table 3). This methodallowed 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-NH2as a negativecontrol. The fluorescence signal is quenched as a resultof resonance energy transfer between the fluorophoreTable 1. NAG activity (fluorescence per min per lg protein) in sub-cellular fractions of different cell lines. Activities were determinedby linear regression analysis taking at least seven measurementpoints. Values are mean ± SD (n ¼ 3).Cell line Subcellular fraction NAG activityHaCaT Cytosolic 0.5 ± 0.05Lysosomal 15.2 ± 0.3Endosomal 7.0 ± 0.37WT100 Cytosolic 0.4 ± 0.02Lysosomal 9.6 ± 0.1Endosomal 2.2 ± 0.07DCs Cytosolic 0.7 ± 0.07Lysosomal 25.2 ± 0.07Endosomal 8.0 ± 0.2Fig. 3. CatE and CatD expression at protein level in relevant anti-gen-processing organelles of different cell lines. Equal amounts oftotal protein (50 lg) from each sample were applied for SDS ⁄ PAGEfollowed by western blot analysis. Representative immunoblotswith the monospecific CatE antibody and reprobe of the same blotwith 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 thefluorogenic peptide substrate (1 lM) by 10 ng CatE in 50 mMsodium acetate buffer (pH 4) at 37 °C. (m) Incubation with pepsta-tin A for 15 min at 37 °C before hydrolysis reaction inhibited theactivity of CatE completely. (d ) immunoprecipitation of CatE beforehydrolysis reaction also completely inhibited the activity of CatE.(B) (j) Hydrolysis of the fluorogenic peptide substrate (1 lM)by10 ng CatD in 50 mM sodium acetate buffer (pH 4) at 37 °C. (m)Incubation with pepstatin A for 15 min at 37 °C before hydrolysisreaction 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 FEBSand the quencher group. Figure 6B shows the resultsof digestion of the substrate with CatE, leading to onlyone cleavage product, because only the Phe-Phe bondis susceptible to cleavage by CatE or CatD [31]. Thepeak with a retention time of 25.54 min correspondsto the fragment, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe, asanalyzed by MALDI-MS (Table 3). Figure 6C showsdigestion of the substrate with CatD, giving a profilesimilar to that of CatE, i.e. only one peak is visiblewith the same retention time. However, when digestedwith the lysosomal fraction of HaCaT (Fig. 6E), anadditional peak with a retention time of 22.87 min wasobserved. Digestion of substrate with the endosomalfraction of HaCaT (Fig. 6F) gave a similar RP-HPLCprofile to the lysosomal fraction.Digestion of the substrate with lysosomal and endo-somal fractions (Fig. 6H,I) was completely inhibitedby pepstatin A, confirming that the activity observedin our assay was solely due to aspartic proteinases.The additional peak observed after digestion of thesubstrate 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 carboxypeptidaseactivity can only occur after aspartic proteinases havecreated cleavage products, as the undigested substratecontains a protective d-Arg residue at the C-terminus.Substrate digestion by the lysosomal fraction(Fig. 6K) after immunoprecipitation of CatE had almostno effect on the RP-HPLC profile. This indicates thatthe activity observed in the lysosomal fraction wasmainly due to CatD. Digestion by the endosomal frac-tion (Fig. 6L) was inhibited after immunoprecipitationof CatE, indicating that the activity in this fraction wasprimarily CatE activity. No cleavage was indicated in thecytosolic fraction, hence no CatE or CatD activity wasobserved by RP-HPLC. This agrees with the results fromthe fluorescence assay, in which only very low activitywas determined in the cytosolic fraction. Digestion ofsubstrate with subcellular fractions of DCs and WT100gave similar RP-HPLC profiles (data not shown).In conclusion, the combination of methods describedhere facilitates the specific and parallel measurement ofCatE and CatD activity in antigen-processing organ-elles. The data clearly show that our approach fordetecting CatE and CatD is more sensitive than immu-nodetection by western blot analysis. It allows detec-tion of CatE activity in subcellular fractions ofWT100, as compared to western blot analysis by whichno CatE was detectable in any WT100 fraction. It wasalso possible to discriminate between CatD activity inendosomal and lysosomal fractions, whereas the distri-bution of CatD in lysosomal and endosomal fractionswas not significantly distinguishable when detected bywestern 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 butwas markedly increased by the use of a monospecificantibody used to deplete CatE. This type of assay isflexible and can be used to discriminate activity ofother proteinases with similar enzymatic properties.This approach distinguishes between the activities ofthe enzymatically similar proteinases, CatE and CatD,and can therefore be used to investigate the involvementof these enzymes in antigen processing and presentation.Experimental proceduresEnzymes and chemicalsCatD (bovine kidney) was purchased from Calbiochem(Darmstadt, Germany) and stored as a 300 UÆmL)1stocksolution 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)1stock solution in50 mm sodium citrate buffer, pH 6.5, containing 150 mmTable 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 andWT100, n ¼ 3; where n is the number of individual experiments performed). ND, not detectable.Cell line Activity Cytosolic fraction Lysosomal fraction Endosomal fractionHaCaT TAPA 13.4 ± 2.15 106.3 ± 6.94 82.0 ± 11.33Cat E 13.4 ± 2.15 25.3 ± 14.01 65.4 ± 11.22Cat D ND 80.9 ± 12 16.6 ± 8.40WT100 TAPA 0.49 ± 0.19 20.9 ± 1.99 13.7 ± 0.69Cat E 0.49 ± 0.19 9.5 ± 0.54 11.8 ± 1.23Cat D ND 11.3 ± 2.54 1.9 ± 0.77DCs TAPA 0.76 ± 0.24 210.7 ± 18.40 232.8 ± 49.65Cat E 0.76 ± 0.24 23.2 ± 20.54 225.7 ± 50.38Cat D ND 187.5 ± 2.19 7.1 ± 0.72N. Zaidi et al. Determination of cathepsin E and D activityFEBS Journal 274 (2007) 3138–3149 ª 2007 The Authors Journal compilation ª 2007 FEBS 3143NaCl at )20 °C. Pepstatin A (Calbiochem) was dissolved inmethanol. Activated CH Sepharose 4B was purchased fromAmersham Biosciences (Munich, Germany). The substrateMOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-d-Arg-NH2[31] was obtained from Bachem (Weil am Rhein,Germany).Generation and immobilization of a monospecificCatE antibodyThe antigenic peptide SRFQPSQSSTYSQPG (CatE 118–132) was selected from the protein sequence using the lasergene software (dnastar, Madison, WI, USA) andcontrolled for specificity to CatD. It was synthesized as asingle peptide and as a multiple antigen peptide, (SRFQPS-QSSTYSQPG)8-(Lys)4-(Lys)2-Lys-Gly-OH, using standardFmoc ⁄ tBu [37] chemistry on a multiple peptide synthesizer,Syro II (MultiSynTech, Witten, Germany). The peptideswere purified using RP-HPLC and the identity was con-firmed using ESI-MS. Peptide purities were determined byanalytical RP-HPLC and were >90%. The single peptidewas coupled to key hole limpet hemocycanin using the glu-tardialdehyde method. The antiserum was obtained afterrepeated immunization of a rabbit with a 1 : 1 mixture ofthe peptide–key hole limpet hemocycanin conjugate and themultiple antigen peptide. This antiserum was further puri-fied by affinity chromatography on a CH-activated Seph-arose 4B column (Amersham Biosciences) containing thepeptide immobilized via a stable peptide bond. Peptideimmobilization was performed as described by the manu-facturer. The antiserum was applied to the column at0.5 mLÆmin)1and recycled overnight. The column waswashed with 20 column volumes of NaCl ⁄ Pi(Gibco LifeTechnologies, Paisley, UK). Elution was performed with 10volumes of 0.1 m glycine ⁄ HCl (pH 2.5). Antibody-contain-ing fractions were immediately neutralized with 1 mTris ⁄ HCl (pH 8.5) and then concentrated on a 20-kDamembrane. The resulting antibody was retested by ELISAand showed the expected specificity to the peptide epitopesand the CatE protein, but a completely negative reaction toCatD. The purified monospecific antibody was immobilizedon CH-activated Sepharose as described by the manufac-turer. After coupling for 3 h at room temperature, the gelwas deactivated with 0.1 m Tris ⁄ HCl, pH 8.0, for an addi-tional 2 h at room temperature. To block any remainingactive sites, the material was further incubated with 5%BSA for an additional 2 h. After a wash with NaCl ⁄ Pi, theimmobilized antibody was stored in NaCl ⁄ Picontaining0.02% (w ⁄ v) NaN3at 4 °C.ELISAThe 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 ⁄ Piin a final volume of 100 lL ⁄ well at 4 °Covernight. The plates were washed three times with 200 lLwashing buffer (NaCl ⁄ Pi⁄ 0.05% Tween 20, pH 7.0) andblocked with blocking buffer (NaCl ⁄ Pi⁄ 0.05% Tween 20,pH 7.0, containing 2% BSA) for 2 h at 37 °C. After awash, the plates were treated for 1 h at 37 °C with ourmonospecific CatE antibody (diluted in NaCl ⁄ Pi⁄ 0.05%Tween 20, pH 7.0, containing 0.5% BSA) or commercialCatD antibody. After a wash, the plates were incubatedwith horseradish peroxidase-conjugated goat anti-rabbit Ig(Dianova, Hamburg, Germany; 1 : 5000 diluted inNaCl ⁄ Pi⁄ 0.05% Tween 20 ⁄ 0.5% BSA). Then 100 lL azino-diethylbenzthiazoline sulfonate ⁄ H2O2in substrate buffer0 20 40 60 80 100 120EndosomesLysosomesCytosolEndosomesLysosomesCytosolEndosomesLysosomesCytosolpmol MOCAc liberated/min/20µg total protein0 50 100 150 200 250 300pmol MOCAc liberated/min/20µg total protein0 5 10 15 20 25pmol MOCAc liberated/min/20µg total proteinTAPACatE activityCaD activityTAPACatE activityCaD activityTAPACatE activityCaD activityABCFig. 5. Distribution of TAPA, CatE and CatD activity in subcellularfractions of the cell lines (A) HaCaT, (B) WT100 and (C) DCs. Equalamounts of total protein (20 lg) were used for the determination ofCatE and CatD activities, determined by linear regression analysisusing a minimum of five measurement points. Values aremean ± SD (DCs, n ¼ 2; HaCaT and WT100, n ¼ 3; where n is thenumber 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 of405 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 peptideSRFQPSQSSTYSQPG (0.1 lg ⁄ well).Cell cultureThe EBV-transformed human B-cell line, WT100, and theimmortalized human keratinocyte cell line, HaCaT, werecultured in RPMI 1640 medium (Gibco Life Technol-ogies) supplemented with 10% (v ⁄ v) heat-inactivated fetalcalf serum (Gibco), penicillin (final concentration100 UÆmL)1; Gibco) and streptomycin (final concentration0.1 mgÆmL)1; Gibco) at 37 °C in tissue culture flasks(Nunc).Peripheral blood mononuclear cells were isolated byFicoll ⁄ Paque (PAA Laboratories, Pasching, Austria) den-sity gradient centrifugation of heparinized blood obtainedfrom buffy coats. Isolated peripheral blood mononuclearcells were plated (1 · 108cells ⁄ 8 mL flask) into 75 cm2Cellstar 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 ofincubation at 37 °C, nonadherent cells were removed andadherent cells were cultured in complete culture mediumsupplemented with granulocyte ⁄ macrophage colony-stimu-lating factor (Leukomax; Sandoz, Basel, Switzerland) andinterleukin 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 flowcytometry (BD FACSCalibur, Heidelberg, Germany).Determination of CatE mRNA expression levelsusing RT-PCRRNA was extracted from DCs, WT100 and HaCaT cellsusing the TRIazol reagent as described by the manufacturer(Invitrogen, Karlsruhe, Germany). Reverse transcription of2 lg total RNA was initialized by 200 U Superscript IIreverse 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 of20 lL. After incubation at room temperature for 10 min,the reaction mixtures were set to 42 °C for 1 h. Thenamplification was carried out, adding 5 lL generatedcDNA to 45 lL reaction mixture [11.0% (v ⁄ v) 10-foldPCR buffer (Roche, Basel, Switzerland), 3.3% (v ⁄ v) bothprimers (5¢-CATGATGGAATTACGTT-3¢ and 5¢-GAATGATCCAGGTACAGCAT-3¢)10lm each (OperonTechnologies 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 cycleseach for 35 s at 94 °C, 30 s at 50 °C, and 60 s at 72 °C.Single PCR amplicons were analysed using agarose gelelectrophoresis.Subcellular fractionation and western blotanalysisCell fractionation was performed as previously described bySchroter et al. [39]. Briefly, (4–8) · 107cells were harvested,resuspended in 1.5 mL fractionation buffer (10 mmTris ⁄ 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 theendolysosomal fractions were separated by ultracentrifuga-tion at 100 000 g for 5 min (Beckman TL100 ultracentri-fuge, Palo Alto, CA, USA). Finally, lysosomes wereseparated 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 BeckmanTL100 ultracentrifuge. Lysosomal material was releasedinto the supernatant, and endosomes remained in thepellet. Total protein content was determined as describedby 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 productsRetention time(min)Expected mass[M +H]+[M +H]+DDaCatE MOCAc-GLPILF-OH 25.54 890.80 890.77 0.03CatD MOCAc-GLPILF-OH 25.19 890.80 891.76 0.96Lysosomal fraction MOCAc-GLPILF-OH 25.90 890.80 890.90 0.1MOCAc-GLPIL-OH 22.87 743.70 743.70 0.0Endosomal fraction MOCAc-GLPILF-OH 25.44 890.80 890.80 0.0MOCAc-GLPIL-OH 22.38 743.70 743.80 0.1N. Zaidi et al. Determination of cathepsin E and D activityFEBS Journal 274 (2007) 3138–3149 ª 2007 The Authors Journal compilation ª 2007 FEBS 3145Subcellular fractions were separated by SDS ⁄ PAGE(50 lg total protein per lane) on a 12% separating geland transferred to a poly(vinylidene difluoride) membrane(Amersham Biosciences, Freiburg, Germany). Membraneswere then blocked for 1 h using NaCl ⁄ Tris [0.15 mNaCl, 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 tohuman CatE was diluted 1 : 2000. Western blots weredeveloped according to the ECL protocol of AmershamBiosciences.Detection of NAG activityNAG activity was measured as described by Schmid et al.[36]. Briefly, 1 lg protein from each fraction was added to100 lL 0.1 m citrate buffer, pH 5, containing 0.8 mm4-methylumbelliferyl-N-acetyl-b-d-glucosaminide (Sigma,Deisenhofen Germany) and 0.1% Triton X-100. Fluores-cence (kex¼ 360 nm, kem¼ 465 nm) was measured every5 min at 37 °C using a fluorescence reader (Tecan SpectraFluor, 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 + CatDRelative Fluorescence Relative Fluorescence Relative Fluorescence25.54510152025303525.195101520253035510152025303551015202530355101520253035510152025303522.8725.90510152025303522.3825.44510152025303522.9125.92510152025303551015202530355101520253035Elution time (min)D Substrate + CF E Substrate + LF F Substrate + EFG Substrate + CF + PepA H Substrate + LF + PepA I Substrate + EF + PepAJ Substrate + CF (IP) K Substrate + LF (IP) L Substrate + EF (IP)5101520253035Fig. 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 lM) 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 FEBSParallel detection of CatE and CatD activityTAPA and specific catalytic activities of CatE and CatDwere determined fluorimetrically by hydrolysis of thesubstrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-d-Arg-NH2. Appropriate amounts of CatE,CatD or subcellular fraction (20 lg total protein) wereadded to 80 lL digestion buffer (50 mm sodium acetatebuffer, pH 4.0), and the reaction was started by the addi-tion of 1 lL substrate solution (stock solution 2 mm indimethyl sulfoxide). Fluorescent product formation wasrecorded using a fluorescence reader (Tecan Spectra Fluor)on kinetic mode at 37 ° C(kex¼ 340, kem¼ 405). Activitieswere determined by linear regression analysis using a mini-mum of five measurement points. All the experiments wereperformed in triplicate yielding TAPA, i.e. CatE and CatDactivity. Aspartic proteinase activity could be completelyinhibited using 1 lL1mm pepstatin A solution in meth-anol (1 lL methanol showed no inhibitory effect).For the specific determination of CatE activity, sampleswere subjected to immunoprecipitation of CatE before theabove assay. Then 20 lg total protein from each subcellularfraction 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 influorescence intensity is exclusively caused by CatD. Thedifference between total aspartic proteinase and CatD activ-ity can be assigned to CatE activity.Analytical RP-HPLCThe 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 (withor without pepstatin A treatment or after immunoprecipita-tion of CatE). The reaction was terminated by additionof 25 lL stop solution [5% (v ⁄ v) acetonitrile, 1% (v ⁄ v) tri-fluoroacetic acid] in water. Then 5 lL of the reaction mixturewas 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. Elutionwas performed using a linear gradient from 5% to 80% sol-vent B within 35 min. Fluorescence detection was carried outat kem.¼ 350 and kex¼ 450. Appropriate fractions werecollected and analysed by MALDI-MS.MALDI-MSFirst, 0.5 lL each RP-HPLC fraction was mixed with0.5 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 forMALDI-MS using a MALDI-TOF system (Reflex IV,serial number 26159.00007; Bruker Daltonics, Bremen, Ger-many). Signals were generated by accumulating 120–210laser shots. Raw data were analyzed using the softwareFlex Analysis 2.4 (Bruker Daltonics).AcknowledgementsWe gratefully acknowledge Andreas Dittmar and Flo-rian Kramer for their technical assistance. This workwas supported by Deutsche Forschungsgemeinschaft(SFB 685), Higher Education Commission Pakistan,and German Academic Exchange Service (DAAD),Germany.References1 Kay J & Dunn BM (1992) Substrate specificity and inhi-bitors of aspartic proteinases. Scand J Clin Lab InvestSuppl 210, 23–30.2 Yamamoto K, Katsuda N, Himeno M & Kato K(1979) Cathepsin D of rat spleen. Affinity purificationand properties of two types of cathepsin D. Eur JBiochem 95, 459–467.3 Takeda M, Ueno E, Kato Y & Yamamoto K (1986)Isolation, and catalytic and immunochemical propertiesof cathepsin D-like acid proteinase from rat erythro-cytes. J Biochem (Tokyo) 100, 1269–1277.4 Muto N, Yamamoto M, Tani S & Yonezawa S (1988)Characteristic distribution of cathepsin E which immu-nologically cross-reacts with the 86-kDa acid proteinasefrom rat gastric mucosa. J Biochem (Tokyo) 103, 629–632.5 Sakai H, Saku T, Kato Y & Yamamoto K (1989)Quantitation and immunohistochemical localizationof cathepsins E and D in rat tissues and blood cells.Biochim Biophys Acta 991, 367–375.6 Sastradipura DF, Nakanishi H, Tsukuba T, NishishitaK, Sakai H, Kato Y, Gotow T, Uchiyama Y &Yamamoto K (1998) Identification of cellular com-partments involved in processing of cathepsin E inprimary cultures of rat microglia. J Neurochem 70,2045–2056.7 Chain BM, Free P, Medd P, Swetman C, Tabor AB &Terrazzini N (2005) The expression and function ofcathepsin E in dendritic cells. 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Biotinylated fluorescent peptide substrates for the sensitive and specific determination of cathepsin D activity J Pept Sci 11, 166–174 31 Yasuda Y, Kageyama T, Akamine A, Shibata M, Kominami E, Uchiyama Y & Yamamoto K (1999) Characterization of new fluorogenic substrates for the rapid and sensitive assay of cathepsin E and cathepsin D J Biochem (Tokyo) 125, 1137–1143 32 Yasuda Y, Kohmura K, Kadowaki...Determination of cathepsin E and D activity 10 11 12 13 14 15 16 17 18 19 20 N Zaidi et al intracellular localization of cathepsin E and cathepsin D in human gastric cells and various rat cells J Biochem (Tokyo) 110, 956–964 Tsukuba T, Hori H, Azuma T, Takahashi T, Taggart RT, Akamine A, Ezaki M, Nakanishi H, Sakai H & Yamamoto K (1993) Isolation and characterization of recombinant human cathepsin. .. Schmid H, Lindmeier I, Schmitt H, Eissele R, Neuhaus G & Wehrmann M (1993) Nephrotoxicity of cyclosporine A in the rat II Reversible changes in intranephronal and urinary catalytic activities of N-acetyl-beta -D- glucosaminidase Ren Physiol Biochem 16, 222–232 Determination of cathepsin E and D activity 37 Chan WC & White PD (2000) Basic procedure In Fmoc Solid Phase Peptide Synthesis: A Practical Approach. .. Cathepsin E- deficient mice show increased susceptibility to bacterial infection associated with the decreased expression of multiple cell surface Toll-like receptors J Biochem (Tokyo) 140, 57–66 Yanagawa M, Tsukuba T, Nishioku T, Okamoto Y, Okamoto K, Takii R, Terada Y, Nakayama KI, Kadowaki T & Yamamoto K (2007) Cathepsin E deficiency induces a novel form of lysosomal storage disorder showing the accumulation... chain inovalbumin-immunized mice Immunology 100, 13–20 25 Hewitt EW, Treumann A, Morrice N, Tatnell PJ, Kay J & Watts C (1997) Natural processing sites for human cathepsin E and cathepsin D in tetanus toxin: implications for T cell epitope generation J Immunol 159, 4693–4699 26 Rhodes JM & Andersen AB (1993) Role of cathepsin D in the degradation of human serum albumin by peritoneal macrophages and. .. of cathepsin E in pancreas: a possible tumor marker for pancreas, a preliminary report Int J Cancer 67, 492–497 22 Uno K, Azuma T, Nakajima M, Yasuda K, Hayakumo T, Mukai H, Sakai T & Kawai K (2000) Clinical significance of cathepsin E in pancreatic juice in the diagnosis of pancreatic ductal adenocarcinoma J Gastroenterol Hepatol 15, 1333–1338 23 Nakanishi H, Tominaga K, Amano T, Hirotsu I, Inoue T... Mossmann H, Koster A, Hess B, Evers M, von Figura K, et al (1995) Mice deficient for the lysosomal proteinase cathepsin D exhibit progressive atrophy of the intestinal mucosa and profound destruction of lymphoid cells EMBO J 14, 3599–3608 Koike M, Nakanishi H, Saftig P, Ezaki J, Isahara K, Ohsawa Y, Schulz-Schaeffer W, Watanabe T, Waguri S, Kametaka S, et al (2000) Cathepsin D deficiency induces lysosomal... accumulation of lysosomal membrane sialoglycoproteins and the elevation of lysosomal pH in macrophages J Biol Chem 282, 1851–1862 Hara I, Miyake H, Yamanaka K, Hara S & Kamidono S (2002) Serum cathepsin D and its density in men with prostate cancer as new predictors of disease progression Oncol Rep 9, 1379–1383 Duffy MJ, Brouillet JP, Reilly D, McDermott E, O’Higgins N, Fennelly JJ, Maudelonde T & Rochefort... (Chan WC & White PD, eds), pp 41–74 Oxford University Press, Oxford 38 Lautwein A, Burster T, Lennon-Dumenil AM, Overkleeft HS, Weber E, Kalbacher H & Driessen C (2002) In ammatory stimuli recruit cathepsin activity to late endosomal compartments in human dendritic cells Eur J Immunol 32, 3348–3357 39 Schroter CJ, Braun M, Englert J, Beck H, Schmid H & Kalbacher H (1999) A rapid method to separate endosomes . material was releasedinto the supernatant, and endosomes remained in thepellet. Total protein content was determined as describedby Bradford [40].Table. (Insertion:10 ng CatE and CatD and 1 ng antigenicpeptide were incubated on an ELISA plate.CatE and CatD antibodies were used for thedetection at dilutions of
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