Research Article 2035 A role for E in the processing of mast-cell A

Frida Henningsson1, Kenji Yamamoto2, Paul Saftig3, Thomas Reinheckel4, Christoph Peters4, Stefan D. Knight5 and Gunnar Pejler1,* 1Swedish University of Agricultural Sciences, Department of Molecular Biosciences, The Biomedical Centre, Box 575, 751 23 Uppsala, Sweden 2Department of Pharmacology, Graduate School of Dental Science, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan 3Biochemisches Institut, Christian-Albrechts-Universität Kiel, Olshausenstr. 40, 24098 Kiel, Germany 4Institut fur Molekulare Medizin und Zellforschung, Albert Ludwigs Universitat Freiburg, 79106 Freiburg, Germany 5Swedish University of Agricultural Sciences, Department of Molecular Biology, Uppsala Biomedical Center, Box 590, SE-753 24 Uppsala, Sweden *Author for correspondence (e-mail: [email protected])

Accepted 16 February 2005 Journal of Cell Science 118, 2035-2042 Published by The Company of Biologists 2005 doi:10.1242/jcs.02333

Summary Mast-cell is stored in the secretory Immunohistochemical analysis revealed staining for granule and is released, together with a range of other cathepsin E in mast cells from normal mice but not in mast inflammatory mediators, upon mast-cell degranulation. cells from mice lacking heparin, indicating that cathepsin Carboxypeptidase A, like all mast-cell , is stored E is bound to heparin proteoglycan within mast-cell in the granule as an active (i.e. with its propeptide granules. In accordance with this notion, affinity removed). Although the processing mechanisms for the chromatography showed that recombinant cathepsin E other classes of mast-cell proteases (in particular the bound strongly to heparin under acidic conditions (the chymases) have been clarified to some extent, the conditions prevailing in mast-cell granules) but not at processing of procarboxypeptidase A is poorly neutral pH. Moreover, mast-cell degranulation resulted in characterized. Here, we show that mast cells from mice the release of cathepsin E. Taken together, our results lacking the aspartic cathepsin E display an indicate that cathepsin E is located in mast-cell secretory accumulation of procarboxypeptidase A, indicating a granules in complex with heparin proteoglycans, and that defect in carboxypeptidase-A processing. By contrast, it has a role in the processing of procarboxypeptidase A mast cells lacking B, L or D have into active protease. normal carboxypeptidase-A processing. Furthermore, recombinant cathepsin E was found to process Journal of Cell Science recombinant procarboxypeptidase A in vitro, under Key words: Mast cells, Carboxypeptidase A, Cathepsin E, Heparin, conditions resembling those found in mast-cell granules. Proteoglycan

Introduction have implicated CPA in the generation and degradation of Mast cells are well known for their harmful effects during angiotensin II (Lundequist et al., 2004), and the degradation of inflammatory conditions such as asthma and allergy, but they apolipoprotein B (Kokkonen et al., 1986), and it has been are also important for our innate defence against bacterial suggested that CPA could be involved in the metabolism of infections and parasites (Metcalfe et al., 1997; Stevens and leukotrienes (Reddanna et al., 2003). CPA, like mast-cell Austen, 1989; Wedemeyer et al., 2000). During these tryptases and chymases, is stored and released in fully active conditions, the mast cell is activated (e.g. by antigen-mediated form (i.e. with its propeptide removed). The mechanisms for cross-linking of IgE molecules bound to their high-affinity processing of the pro forms of the respective mast-cell receptor FcεRI) and thereby undergoes degranulation. Upon proteases into active have been studied to some degranulation, various preformed inflammatory mediators are extent. It was shown recently that mice lacking dipeptidyl released, including histamine, cytokines, proteoglycans and a peptidase I (DPPI; also known as cathepsin C) fail to process range of proteases [tryptases, chymases and carboxypeptidase the pro forms of the chymases mouse mast-cell protease 4 A (CPA)]. (mMCP-4) and mMCP-5, demonstrating an essential role for Previous studies aiming at understanding the function of DPPI in the generation of active chymases (Wolters et al., the mast cell proteases have mainly focused on tryptases and 2001). In the same study, it was observed that the levels of chymases, whereas relatively little is known concerning active tryptase mMCP-6 was reduced in DPPI–/– mast cells. the biology of CPA. Mast-cell CPA is a zinc-containing However, active mMCP-6 was clearly detectable also in metalloprotease with activity (Everitt and DPPI–/– cells, indicating that DPPI has a role but is not essential Neurath, 1980) and is synthesized as a proenzyme with a 94- for the processing of protryptase into active protease (Wolters amino-acid activation peptide (Reynolds et al., 1989). The et al., 2001). A role for DPPI has also been suggested in the biological function of CPA is uncertain but previous studies processing of human β-tryptase (Sakai et al., 1996). 2036 Journal of Cell Science 118 (9)

Unlike tryptase and chymase, the activation mechanism for blocking with 5% milk powder in TBS containing 0.1% Tween 20 for CPA has been poorly studied. However, it has been shown that 20 minutes to 1 hour at 20°C. Next, the membranes were incubated the processing of pro-CPA to the active enzyme occurs in the with rabbit antiserum towards CPA, mMCP-4, mMCP-5 or mMCP-6, mast-cell secretory granule (Rath-Wolfson, 2001; Springman diluted 1:200-1:1000 in 5% milk powder in TBS containing 0.1% et al., 1995) and studies using protease inhibitors have Tween 20, at 4°C overnight. For analysis of cathepsin E, a goat anti- implicated cysteine-protease activity in the process cathepsin-E antibody (R&D systems, Abingdon, UK) was used at a 1:500 dilution in 5% milk powder in TBS containing 0.1% Tween 20, (Springman et al., 1995). The family includes at 4°C overnight. After washing extensively with TBS containing several lysosomal cathepsins that are widely distributed among 0.1% Tween 20, the membranes were incubated with horseradish- living organisms (for a review, see Turk et al., 2000) – peroxidase-conjugated donkey anti-rabbit secondary antibody (for cathepsins B, C, F, H, K, L, O, S, V, W and X. In addition to CPA, mMCP-4, mMCP-5 and mMCP-6; Amersham Biosciences, cysteine proteases, the cathepsin family of proteases includes Uppsala, Sweden) or horseradish-peroxidase-conjugated rabbit anti- two aspartic proteases, cathepsins D and E. The cathepsins goat secondary antibody (for cathepsin E; Sigma-Aldrich, Stockholm, require a slightly acidic environment for optimal activity, a Sweden) diluted 1:3000 in TBS containing 0.1% Tween 20. After 45 condition that is prevalent in lysosomes but also in other minutes of incubation at 20°C, the membranes were washed cellular compartments such as the secretory granule of mast extensively with TBS containing 0.1% Tween 20 followed by washing cells. Traditionally, cathepsins are believed to carry out with TBS without detergent. The membranes probed with anti- cathepsin-E antibody were further incubated with a third antibody, unspecific bulk inside the lysosome (Barrett, 1992) donkey anti-rabbit horseradish-peroxidase conjugate. The membranes but there is growing evidence for specific, non-redundant in were developed with the enhanced chemiluminescence (ECL) system vivo functions of lysosomal cathepsins (Chapman et al., 1997; (Amersham Biosciences, Uppsala, Sweden) according to the protocol Turk et al., 2000), and several knockout mouse strains have provided by the manufacturer. provided important insight into their biological functions (Guicciardi et al., 2000; Halangk et al., 2000; Nakagawa et al., 1998; Nakagawa et al., 1999; Potts et al., 2004; Reinheckel et Culturing and activation of BMMCs al., 2001; Roth et al., 2000; Saftig et al., 1998; Wolters et al., BMMCs were obtained as described (Henningsson et al., 2002) by 2001). culturing C57BL/6 mouse (female, 12-15 weeks old) femur and tibia bone-marrow cells in Dulbecco’s modified Eagle’s medium (DMEM; Because it was shown that DPPI is involved in the SVA, Uppsala, Sweden) supplemented with 10% heat-inactivated processing of chymases and tryptases (see above), and because foetal calf serum (Gibco, Stockholm, Sweden), 50 µg ml–1 gentamicin the processing of pro-CPA was previously found to be sulfate (SVA, Uppsala, Sweden), L-glutamine (SVA, Uppsala, dependent on cysteine-protease activity (Springman et al., Sweden) and 50% WEHI-3B conditioned medium (which contains 1995), we investigated the role of DPPI in the processing of interleukin 3) for 3 weeks. The cells were kept at a concentration of CPA (Henningsson et al., 2003). However, we found that DPPI 500,000 cells ml–1 and the cell-culture medium was changed every was not involved in the processing of pro-CPA into active third day. To degranulate BMMCs, 1.2107 cells were pelleted by protease. In fact, the levels of active CPA in bone-marrow- centrifugation at 300 g for 10 minutes and resuspended in 12 ml serum-free culture medium. 2 ml aliquots of the suspension were derived mast cells (BMMCs) from DPPI-deficient mice were µ higher than in wild-type cells, and the same tendency was seen added to six culture wells. To three of the wells, 2 l 2 mM solution Journal of Cell Science (in H2O) of the calcium ionophore A23187 (Sigma; 2 µM final in BMMCs from mice deficient in . The most likely concentration) were added and to the remaining wells an equal volume explanation for these findings is that CPA is degraded at a (2 µl) of H2O was added. The cells were incubated at 37°C, 5% CO2 higher rate in normal BMMCs than in the DPPI-null and for 10 minutes, 30 minutes or 120 minutes. Subsequently, the cells cathepsin-S-null cells. were pelleted (centrifugation at 300 g, 10 minutes) and solubilized in The aim of the present study was to gain further insight into 100 µl SDS-PAGE sample buffer. The supernatants (conditioned the processing mechanisms for pro-CPA. The strategy was to medium) were concentrated approximately 50 times using Amicon take advantage of existing knockout models in which various Ultra-4 centrifugal filter devices (Millipore, Solna, Sweden) and cathepsins have been targeted. We report here that mast cells thereafter mixed with 0.25 volumes 4 SDS-PAGE sample buffer. deficient in cathepsin E display defective processing of pro- Cell extracts and concentrated conditioned media were analysed for CPA into active enzyme. presence of cathepsin E and CPA protein by western blotting.

Cloning, expression and purification of recombinant-mouse Materials and Methods mast-cell pro-CPA Mice Total RNA was isolated from mouse BMMCs using the NucleoSpin Mice deficient in cathepsins B (Halangk et al., 2000), L (Roth et al., RNA II kit (Macherey Nagel, Düren, Germany) and was used for first- 2000), D (Saftig et al., 1995) and E (Tsukuba et al., 2003), and N- strand cDNA synthesis using SuperScriptII for reverse transcription deacetylase/N-sulfotransferase-2 (NDST-2) (Forsberg et al., 1999; PCR (RT-PCR) using a CPA-specific reverse primer (5′-GT- Humphries et al., 1999) were as described previously. All of the CCTCGAGCAAGGGCAATTCATTAGGAAGTATTCTTGA-3′). A knockout strains were backcrossed into C57BL/6 background. PCR approach was used to produce a construct for expression of recombinant pro-CPA in a mammalian system. The 5′ primer used (5′- CACGAATTCCACCATCACCATCACCATGACGATGACGATAAG- Western-blot analysis ATTGCTCCTGTCCACTTTG-3′) in the PCR introduced a 6 His Peritoneal cells were obtained by peritoneal washing with 20 ml ice- tag, to facilitate purification. In addition, an enterokinase-susceptible cold PBS. Cells were counted and dissolved in SDS-PAGE sample peptide (Asp-Asp-Asp-Asp-Lys) was inserted between the His tag buffer. Samples of these mixtures were subjected to SDS-PAGE on and the pro-CPA, replacing the natural activation peptide. The 12% polyacrylamide gels under reducing conditions. Proteins were enterokinase site enables subsequent removal of the His residues. The subsequently blotted onto nitrocellulose membranes, followed by PCR product was ligated into the pCEP-Pu2 vector as described Processing of mast-cell carboxypeptidase A 2037

(Hallgren et al., 2000) and transformed into competent Escherichia eluted with PBS, pH 6.0 or pH 7.4, with increasing concentrations of coli. Preparations of plasmid DNA were made using QIAprep Spin NaCl (0.05 M, 0.14 M, 0.3 M, 0.5 M, 1.0 M). For every elution step, mini-prep (Qiagen, Stockholm, Sweden) and positive clones were four 100-µl fractions were collected and 30 µl of each fraction was confirmed by sequencing using Big Dye Sequencing kit (Applied analysed by SDS-PAGE using 12% polyacrylamide gels followed by Biosystems Prism, Foster City, CA) and sequenced in a 310 Genetic anti-cathepsin-E western-blot analysis. Analyzer (Applied Biosystems Prism, Foster City, CA). The human embryonic-kidney-cell line 293-EBNA expresses the for the Epstein-Barr nuclear antigen 1 (EBNA-1) protein. This Three-dimensional modelling of mouse cathepsin E protein is a DNA binding protein that interacts with OriP, which is A homology model was constructed using the coordinates for human the Epstein-Barr virus (EBV) origin of replication. OriP is located in cathepsin E (PDB code 1TZS) (Ostermann et al., 2004). The sequences the pCEP-Pu2 vector and is required for a stable episomal of mouse and human cathepsin E were aligned using ClustalW. The maintenance of the vector in the 293-EBNA cell line. The pCEP-Pu2 sequences are 82% identical, with no gaps or insertions in the vector containing the pro-CPA insert was transfected into 293-EBNA alignment. A model for mouse cathepsin E was generated using the cells using the Lipofectamin2000 kit (Invitrogen). The cells were kept computer program SOD (Kleywegt et al., 2001) and the molecular in DMEM (SVA, Uppsala, Sweden) supplemented with 10% heat- graphics program O (Jones et al., 1991). Side-chain rotamers were inactivated foetal calf serum (Gibco) and 2 mM L-glutamine. selected to correspond as closely as possible to those in the experimental Gentamicin (50 µg ml–1) was included as antibiotic and, for selection, models except when a different rotamer had to be chosen to avoid steric puromycin (0.5-5.0 µg ml–1; Sigma-Aldrich, Stockholm, Sweden) clashes. Limited energy minimization was carried out with REFMAC5 was used. Conditioned medium was harvested, centrifuged and stored (Murshudov, 1997). Surface representations were generated using at –20°C. For purification, conditioned medium (750 ml) was added PYMOL (http://www.pymol.org/). to a 10 ml Poly-Prep column containing 300 µl Ni-NTA agarose (Qiagen, Stockholm, Sweden). After washing the column, the pro- CPA fusion protein was eluted with 100 mM imidazole in eight 300 Results µl fractions. The fractions were analysed by SDS-PAGE and subsequent Coomassie Brilliant Blue staining or anti-CPA western- Analysis of CPA and other mast-cell proteases in mast blot analysis. cells from mice lacking cathepsins A previous report implicated cysteine-protease activity in the processing of pro-CPA into active protease (Springman et al., Activation of pro-CPA by recombinant cathepsins 1995), and so we assessed whether either cathepsins B or L To activate recombinant cathepsin E (R&D Systems, Abingdon, UK), µ µ were involved in this processing. Peritoneal cells were 0.15 g recombinant protein was incubated in 100 l 50 mM sodium recovered from mice deficient in cathepsins B and L, as well acetate, 0.1 M NaOH (pH 3.5) at 20°C for 15 minutes. Thereafter, 1.5 ng or 15 ng activated cathepsin E were mixed with ~20 ng as from the corresponding wild-type or heterozygous controls. recombinant pro-CPA and the pH was adjusted to 5.5 with 50 mM Cellular extracts were prepared and were subjected to western- sodium acetate, 0.1 M NaOH (pH 5.5). In addition, recombinant pro- blot analysis using an antibody that detects both the pro and CPA was incubated with 300 ng or 2 µg recombinant active forms of CPA (Henningsson et al., 2002). In wild-type (Sigma). The samples were incubated at 37°C for 30 minutes and peritoneal cells, only the active form of CPA (~35 kDa) was thereafter mixed with SDS-PAGE sample buffer and subjected to detected (Fig. 1A-D). Similarly, only the active form of CPA Journal of Cell Science SDS-PAGE on a 12% polyacrylamide gel. Subsequently, western-blot was detected in extracts from peritoneal cells deficient in either analysis for pro-CPA/CPA was performed, as described above. or , indicating that neither of these cathepsins is involved in the processing of pro-CPA (Fig. Immunohistochemistry 1A,B). Furthermore, we have previously established that Peritoneal cells were collected from NDST-2–/– and NDST-2+/– neither cathepsin C nor cathepsin S is involved in pro-CPA littermates. Cytospin slides were prepared and stained with a processing into active enzyme (Henningsson et al., 2003). polyclonal goat anti-mouse-cathepsin-E antibody (R&D Systems). A Cathepsins are traditionally thought to be located mainly in biotinylated horse anti-goat-IgG antibody (affinity purified; Vector lysosomes. However, it was recently found that one cathepsin, Laboratories, Burlingame, CA) was used as secondary antibody. the aspartic-protease cathepsin D, was located in the secretory Staining was performed by a standard protocol using the biotin/avidin- granule of rat basophilic leukemia (RBL) cells. We therefore based Vectastain Elite (Vector Laboratories) and DAB (3,3′- considered the possibility that cathepsin D or another aspartic diaminobenzidine) for detection of the secondary antibody. As a cathepsin, cathepsin E, is present in peritoneal mast cells and negative control, affinity-purified IgG purified from goat serum (SVA, Uppsala, Sweden) was used. To visualize mast cells, slides were participates in pro-CPA processing. Western-blot analysis of counterstained with May-Grünwald/Giemsa. First, the slides were cell extracts from cathepsin-D-deficient peritoneal cells did not fixed in methanol for 3 minutes and thereafter stained with May- reveal any defects in pro-CPA processing (Fig. 1C). By striking Grünwald (Merck, Sollentuna, Sweden) for 15 minutes. After contrast, peritoneal-cell extracts from cathepsin-E-deficient washing with water, the slides were stained with 5% Giemsa (Merck) animals displayed readily detectable levels of pro-CPA (~55 in H2O for 10 minutes. kDa), indicating defective pro-CPA processing with an accompanying accumulation of the proenzyme (Fig. 1D). This Affinity chromatography clearly suggests a role for cathepsin E in pro-CPA processing. However, active CPA was also detected, although at reduced Affinity chromatography was performed on a 10 ml Poly-Prep column containing ∼100 µl heparin-Sepharose (Amersham Biosciences, levels, indicating that cathepsin E is not essential for the Uppsala, Sweden). The column was equilibrated with PBS, 0.05 M processing. NaCl, pH 6.0 or pH 7.4. Recombinant cathepsin E (2.3 µg) (R&D Next, the possibility that cathepsin E also influenced the Systems) was loaded onto the column and left in the column for 15 processing of other mast-cell proteases was addressed. minutes at 20°C. Thereafter, the recombinant protein was stepwise Peritoneal mast cells from C57BL/6 mice produce, in addition 2038 Journal of Cell Science 118 (9)

Fig. 2. Western-blot analysis of mast-cell proteases in cathepsin-E–/– cells. Cell extracts were prepared from wild-type or cathepsin-E-null peritoneal cells and were subjected to western-blot analysis using specific antisera for the chymase mMCP-4 (A), the chymase mMCP- 5 (B) and the tryptase mMCP-6 (C).

both the pro form and the active form of CPA (Fig. 3A, right) (Henningsson et al., 2002). In addition, the recombinant pro- Fig. 1. Western-blot analysis of CPA and pro-CPA in peritoneal cells CPA was incubated with recombinant cathepsin D in order to from cathepsin-deficient mice. Peritoneal-cell extracts from mice assess whether this could also catalyse the deficient in cathepsin B (A), cathepsin L (B), cathepsin D (C) or processing of pro-CPA into active enzyme. However, there was cathepsin E (D), and extracts from corresponding wild-type or no clear increase in the level of active CPA after incubation of heterozygous mice, were subjected to western-blot analysis using a an antiserum that recognizes both pro-CPA (~55 kDa) and processed pro-CPA with cathepsin D (Fig. 3B). CPA (~35 kDa). Cat, cathepsin. Location of cathepsin E in mast-cell secretory granules Journal of Cell Science to CPA, the chymases mMCP-4 and mMCP-5 as well as the The data presented above indicate that cathepsin E has a role in tryptase mMCP-6. However, western-blot analysis did not the processing of pro-CPA, implying its presence in mast cells. demonstrate any differences in the levels of these proteases Further, the processing of mast-cell pro-CPA has previously between cathepsin-E+/+ and cathepsin-E–/– cells, nor was there been suggested to occur within the mast-cell secretory granule, any accumulation of their respective proforms (Fig. 2). Thus, suggesting that cathepsin E might actually be located in this cathepsin E appears to be specifically involved in the cellular compartment. To clarify these issues, we stained processing of pro-CPA, without affecting the processing of peritoneal cells with an anti-cathepsin-E antibody. As seen in secretory granule chymases or tryptases. Fig. 4A, cathepsin E was readily detected in cells with a mast- cell-like morphology, indicating that cathepsin E is indeed present in mast cells. A control antibody did not produce any In vitro studies of cathepsin-E-dependent CPA activation detectable staining in the peritoneal-cell population (not shown). The results above indicate that pro-CPA processing into active Intense staining was observed in granular structures, supporting protease is dependent on cathepsin E. This dependence could a location within the secretory granule. To further address the be either direct (i.e. pro-CPA is a substrate for cathepsin E) or possibility that cathepsin E is located in the secretory granule, indirect. To investigate whether pro-CPA processing can we used peritoneal cells taken from a mouse strain deficient in actually be catalysed by cathepsin E, we expressed NDST-2. The knockout of NDST-2 results in impaired synthesis recombinant pro-CPA. To ensure proper folding and of heparin in mast cells, accompanied by a dramatic defect in glycosylation conditions, we expressed it in a mammalian formation of mature mast-cell secretory granule (Forsberg et al., system. The recombinant protein was incubated with 1999; Humphries et al., 1999). As shown in Fig. 4C, no staining recombinant mouse cathepsin E at pH 5.5, the pH that prevails for cathepsin E was seen in the NDST-2-null peritoneal cells, in mast-cell granules (De Young et al., 1987). Indeed, western- giving further support for cathepsin E being located in the blot analysis showed that the recombinant cathepsin E heparin-containing secretory granule of peritoneal mast cells processed recombinant ~55 kDa pro-CPA into the ~35 kDa (Fig. 4C). If cathepsin E were indeed located in mast-cell active form (Fig. 3A). Extracts from BMMCs (derived from secretory granule, mast-cell degranulation would be expected to C57BL/6 mice) were included in the western blot as a marker result in release of cathepsin E together with other secretory- for the pro and active forms of CPA; these cells always contain granule components. To test this possibility, BMMCs were Processing of mast-cell carboxypeptidase A 2039 positive for cathepsin E also showed strong staining with May- Grünwald/Giemsa, further supporting that the cathepsin-E- positive cells were indeed mast cells (Fig. 4B). By contrast, the NDST-2–/– peritoneal cells displayed a complete lack of staining with May-Grünwald/Giemsa, in agreement with a defective proteoglycan synthesis (Fig. 4D).

Heparin-binding properties of cathepsin E The results above indicate that cathepsin E is stored in mast- cell granules and that this storage is heparin dependent. Further experiments were therefore conducted to investigate the possible heparin-binding properties of cathepsin E. To this end, recombinant cathepsin E was subjected to affinity chromatography on heparin-Sepharose. When the affinity chromatography was performed at pH 6.0 (a similar pH to that found in the mast-cell secretory granule), cathepsin E bound with remarkably high affinity, with 0.5 M NaCl being required for elution from the column (Fig. 6A). By striking contrast, very weak binding to heparin-Sepharose was seen when the chromatography was performed at neutral pH, with most of the cathepsin E being eluted either in the flow-through fraction or at 0.05 M NaCl (Fig. 6B). Because native cathepsin E is a disulfide-linked dimer (Tatnell et al., 1997), we investigated the Fig. 3. In vitro processing of recombinant pro-CPA by purified possibility that the dimerization contributes to the high affinity cathepsins. Activated recombinant cathepsin E (1.5 ng or 15 ng; A) µ µ for heparin (e.g. through multivalent interactions). However, or cathepsin D (300 ng or 2 g; B) was added to 1 l recombinant monomerization of the cathepsin-E dimer by reduction with CPA at pH 5.5. As a control, pro-CPA was incubated without added cathepsins. Samples were incubated at 37°C for 30 minutes and dithiothreitol did not result in any reduction in the affinity for subjected to western-blot analysis using specific antiserum for CPA heparin (data not shown). and pro-CPA. As a control, cell extracts prepared from wild-type BMMCs were included in the western-blot analysis. The BMMC extracts contains both the pro and the active forms of CPA. Discussion Mast-cell CPA is the most anonymous of the mast-cell proteases and there are relatively few reports describing its activated by a mast-cell-degranulating agent, the calcium basic biological properties. It has been known for a Journal of Cell Science ionophore A23187. Conditioned media were collected after considerable time that CPA is stored as an active fully various times and analysed for the presence of cathepsin E and processed form, with its 94-amino-acid propeptide removed CPA. In the absence of added A23187, only low levels of (Dikov et al., 1994), but the mechanism(s) involved in this cathepsin E and CPA were detected in the culture medium (Fig. processing have been poorly characterized. In a previous study, 5). However, addition of A23187 resulted in time- dependent release of both cathepsin E and CPA into the cell culture medium. Importantly, the release of both components appeared to follow similar kinetics (Fig. 5). Thus, mast-cell degranulation is accompanied by the release of both CPA and cathepsin E, further supporting a location of cathepsin E within mast-cell secretory granules. Peritoneal-cell populations normally contain 2- 4% mast cells and these can be visualized using May-Grünwald/Giemsa staining, which stains the negatively charged proteoglycans in the mast-cell granules. As can be seen in Fig. 4B, NDST-2+/– cells of similar morphology to those that were

Fig. 4. Identification of cathepsin E in mast-cell secretory granules. Peritoneal cells from wild-type (A) and NDST-2–/– (C) mice were fixed on cytospin slides and stained with an antibody specific for cathepsin E. The slides were counterstained with May- Grünwald/Giemsa (B,D) 2040 Journal of Cell Science 118 (9) cathepsin D, did not affect pro-CPA processing, indicating that the ability to process pro-CPA is not a general feature of aspartic proteases but rather a specific property of cathepsin E. It is important to realize that the lack of cathepsin E did not cause a complete inability of the mast cells to produce active CPA. This could imply that another, as yet unidentified, protease contributes to the pro-CPA processing in normal mast cells. Alternatively, the knockout of cathepsin E could unleash compensating mechanisms that partly rescue the inability to process pro-CPA into active enzyme in cathepsin-E-null cells. Cathepsin E has previously been identified in various locations, such as lymphoid tissue, the and urinary organs (Muto et al., 1988; Sakai et al., 1989), but Fig. 5. Release of cathepsin E and CPA after mast-cell degranulation. BMMCs from this is to our knowledge the first C57BL/6 mice were degranulated using the calcium ionophore A23187. Cell extracts report demonstrating the presence of and 50 concentrated conditioned media were subjected to western-blot analysis immunoreactive cathepsin E in mast cells. The using antibodies specific for cathepsin E and CPA. subcellular location of cathepsin E in the mast-cell is intriguing. Cathepsin E, like all it was shown that addition of the cysteine-protease inhibitor e- cathepsins, is normally confined to lysosomes, but the 64d to the mast-cell line KiSV-MC14 caused an accumulation immunohistochemical staining performed here clearly pointed of pro-CPA, indicating that cysteine-protease activity might be to a location within the secretory granule of the mast cells. A involved in its processing (Springman et al., 1995). However, granular location for cathepsin E was also strongly supported the identity of this cysteine protease was not revealed. In the by the lack of cathepsin-E staining in peritoneal cells lacking present study, we assessed the role of two specific cysteine NDST-2. Targeting of NDST-2 was previously shown to proteases, cathepsins B and L, in the processing of pro-CPA, interfere specifically with the biosynthesis of mast-cell heparin, but neither of these cysteine proteases appeared to have any one of the compounds present specifically in mast-cell significant role in pro-CPA processing. Furthermore, we have secretory granules, without affecting any other cell type shown previously that neither of two other cysteine proteases (Forsberg et al., 1999; Humphries et al., 1999). As a [cathepsin C (DPPI) and cathepsin S] is involved in the consequence, NDST-2-deficient mast cells display an almost Journal of Cell Science processing of pro-CPA (Henningsson et al., 2003). These complete inability to assemble normal densely packed granules studies were thus unable to provide any further support for a while not being affected as regards transcription of mast-cell- major role for cysteine proteases in the processing of pro-CPA. specific markers. Clearly, the lack of cathepsin-E staining in Importantly, however, we cannot exclude an involvement of the NDST-2–/– peritoneal cells is therefore in strong agreement other cysteine proteases, such as cathepsins K, O, F, V and X. with a predominant location of cathepsin E in the secretory In the present study, we instead present evidence that the granule of mast cells. This is also strongly supported by our aspartic protease cathepsin E is involved in the processing of finding that mast-cell degranulation is accompanied by the pro-CPA. This notion is supported both by in vivo experiments release of cathepsin E. In further support for the possibility that demonstrating that pro-CPA processing was defective in mice cathepsins localize to secretory compartments, previous studies lacking cathepsin E and by in vitro experiments in which have indicated that cathepsin D and cathepsin C might be purified cathepsin E was found to process recombinant pro- present in the secretory granules of RBL cells (Dragonetti et CPA. Interestingly, the knockout of another aspartic protease, al., 2000) and BMMCs (Wolters et al., 2001) respectively. The effect of NDST-2 deficiency on cathepsin-E staining in mast cells indicates that cathepsin-E storage is dependent on heparin. This dependence could be either direct (cathepsin interacts with heparin proteoglycan) or

Fig. 6. Heparin-binding properties of cathepsin E. Recombinant cathepsin E was subjected to affinity chromatography on a heparin-Sepharose column, eluted with stepwise increasing concentrations of NaCl in PBS buffer of pH 6.0 (A) or pH 7.4 (B). For every NaCl concentration, four fractions containing 100 µl were collected and 30 µl from each fraction was subjected to western-blot analysis using an antibody specific for cathepsin E. dim, dimeric cathepsin E; FT, flow-through fraction; mon, monomeric cathepsin E. Processing of mast-cell carboxypeptidase A 2041 indirect (e.g. that cathepsin is bound to a compound that in turn interacts with heparin). To address these possibilities, we therefore investigated whether cathepsin E interacts with heparin. Indeed, cathepsin E bound strongly to heparin-Sepharose, indicating that cathepsin-E storage in mast- cell granules requires that the cathepsin is associated with the proteoglycan. Interestingly, the interaction of cathepsin E with heparin showed a strong pH dependence, with strong binding at low pH and essentially complete loss of affinity for heparin at neutral pH. The reason for this strong pH dependence is not clear, but one possibility is that the high-affinity interaction to heparin involves His residues. His residues are deprotonated and thereby uncharged at neutral pH but are protonated and positively charged below pH ~6.5. To search for potential heparin-binding regions and, in particular, to search for surface- exposed His residues that might be involved in pH-dependent interaction with heparin, we constructed a three-dimensional model of murine cathepsin E based on the refined Fig. 7. Three-dimensional model of mouse cathepsin E. The modelled structure of mouse 2.35 Å X-ray structure for human cathepsin cathepsin E was based on the known structure of human cathepsin E. His residues are E (Ostermann et al., 2004). Because heparin indicated in light blue, other positively charged residues are indicated in dark blue and binding was not dependent on dimerization negatively charged residues are indicated in red. A potential heparin- on the N- and dimerization does not appear to involve terminal domain is outlined by a green border, with contributing side chains labelled. Four an extensive subunit-subunit interface different views of the molecule, 90° apart, are shown. (A) Front view. (B) Top view looking (Ostermann et al., 2004), only the monomer down into the active-site cleft. (C) Back view. (D) View from the bottom of the molecule, was modelled. The model of mouse opposite to the active-site cleft. cathepsin E (Fig. 7) identified five surface- exposed His residues that might be Journal of Cell Science interesting candidates for heparin binding. Two of the His shown that the heparin part of the heparin-chymase complex residues have negatively charged residues nearby that might attracts heparin-binding substrate proteins and thus presents compensate for their negative charge. However, three His them to the chymase for efficient proteolysis (Tchougounova residues (His61, His65 and His78) and two Lys residues (Lys59 and Pejler, 2001). and Lys114) are found within a relatively limited region of the Although cathepsin E has been implicated in several N-terminal domain of the molecule, where they might contribute physiological and pathological conditions, the true biological to a heparin-binding site. It is interesting that the binding of function of cathepsin E is not known (Tsukuba et al., 2000). two other mast-cell secretory-granule compounds, mMCP-6 However, important clues to the function of this protease were (Hallgren et al., 2004) and mMCP-7 (Matsumoto et al., 1995), obtained through the recent genetic targeting of cathepsin E also involves surface-exposed His residues. A functional (Tsukuba et al., 2003). It was found that the knockout of consequence of this mode of interaction would be that the cathepsin E resulted in mice that were markedly prone to binding to heparin is strong within the mast-cell granule (acidic spontaneously developing atopic dermatitis, a condition that is pH), whereas exposure to neutral pH (e.g. following exocytosis) strongly dependent on T-helper-2 cell responses. Notably, mast will cause His deprotonation and dissociation from the heparin cells are strongly implicated in allergic diseases, including proteoglycan. atopic dermatitis (Irani et al., 1989), and the current Similar to cathepsin E, CPA is tightly associated with identification of cathepsin E in the secretory granule of mast heparin proteoglycan within the mast-cell secretory granule cells might thus point to a potential role of mast-cell cathepsin and its storage is severely affected in the NDST-2-null mast E in allergic disease. However, an identification of the exact role cells (Forsberg et al., 1999; Humphries et al., 1999). It is of mast-cell cathepsin E, in comparison to cathepsin E from thus possible that cathepsin E and CPA show a physical other cellular sources, will require substantial further colocalization through the interaction with heparin investigations. proteoglycan. Such colocalization might be functionally important by bringing cathepsin E into close contact with pro- We thank Nils Ostermann for providing us with cathepsin E CPA, thus facilitating the proteolytic action of cathepsin E on coordinates prior to public release. This work was supported by grants pro-CPA. This would be analogous to the effect of heparin from the Swedish Research Council, Vårdalstiftelsen, Formas and proteoglycan on mast-cell chymase, for which it has been King Gustaf V’s 80th Anniversary Fund. 2042 Journal of Cell Science 118 (9)

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