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First published online April 28, 2005
doi: 10.1242/10.1242/jcs.02333

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A role for cathepsin E in the processing of mast-cell carboxypeptidase A

¹ Swedish University of Agricultural Sciences, Department of Molecular Biosciences, The Biomedical Centre, Box 575, 751 23 Uppsala, Sweden
² Department of Pharmacology, Graduate School of Dental Science, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan
³ Biochemisches Institut, Christian-Albrechts-Universität Kiel, Olshausenstr. 40, 24098 Kiel, Germany
⁴ Institut fur Molekulare Medizin und Zellforschung, Albert Ludwigs Universitat Freiburg, 79106 Freiburg, Germany
⁵ Swedish University of Agricultural Sciences, Department of Molecular Biology, Uppsala Biomedical Center, Box 590, SE-753 24 Uppsala, Sweden

View larger version (38K): [in a new window]   Fig. 1. Western-blot analysis of CPA and pro-CPA in peritoneal cells from cathepsin-deficient mice. Peritoneal-cell extracts from mice deficient in cathepsin B (A), cathepsin L (B), cathepsin D (C) or cathepsin E (D), and extracts from corresponding wild-type or heterozygous mice, were subjected to western-blot analysis using a an antiserum that recognizes both pro-CPA (55 kDa) and processed CPA (35 kDa). Cat, cathepsin.

View larger version (52K): [in a new window]   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).

View larger version (50K): [in a new window]   Fig. 3. In vitro processing of recombinant pro-CPA by purified cathepsins. Activated recombinant cathepsin E (1.5 ng or 15 ng; A) or cathepsin D (300 ng or 2 µg; B) was added to 1 µl recombinant 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 subjected to western-blot analysis using specific antiserum for CPA 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.

View larger version (102K): [in a new window]   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)

View larger version (35K): [in a new window]   Fig. 5. Release of cathepsin E and CPA after mast-cell degranulation. BMMCs from C57BL/6 mice were degranulated using the calcium ionophore A23187. Cell extracts and 50x concentrated conditioned media were subjected to western-blot analysis using antibodies specific for cathepsin E and CPA.

View larger version (28K): [in a new window]   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.

View larger version (88K): [in a new window]   Fig. 7. Three-dimensional model of mouse cathepsin E. The modelled structure of mouse cathepsin E was based on the known structure of human cathepsin E. His residues are indicated in light blue, other positively charged residues are indicated in dark blue and negatively charged residues are indicated in red. A potential heparin-binding site on the N-terminal domain is outlined by a green border, with contributing side chains labelled. Four different views of the molecule, 90° apart, are shown. (A) Front view. (B) Top view looking down into the active-site cleft. (C) Back view. (D) View from the bottom of the molecule, opposite to the active-site cleft.