Processing of human cathepsin D is independent of its catalytic function and auto-activation: involvement of cathepsins L and B

Abstract

The current mechanism proposed for the processing and activation of the 52 kDa lysosomal aspartic protease cathepsin D (cath-D) is a combination of partial auto-activation generating a 51 kDa pseudo-cath-D, followed by enzyme-assisted maturation involving cysteine and/or aspartic proteases and yielding successively a 48 kDa intermediate and then 34 + 14 kDa cath-D mature species. Here we have investigated the in vivo processing of human cath-D in a cath-D-deficient fibroblast cell line in order to determine whether its maturation occurs through already active cath-D and/or other proteases. We demonstrate that cellular cath-D is processed in a manner independent of its catalytic function and that auto-activation is not a required step. Moreover, the cysteine protease inhibitor E-64 partially blocks processing, leading to accumulation of 52-48 kDa cath-D intermediates. Furthermore, two inhibitors, CLICK148 and CA-074Met, specific for the lysosomal cath-L and cath-B cysteine proteases induce accumulation of 48 kDa intermediate cath-D. Finally, maturation of endocytosed pro-cath-D is also independent of its catalytic function and requires cysteine proteases. We therefore conclude that the mechanism of cath-D maturation involves a fully-assisted processing similar to that of pro-renin.

Figure 1. Maturation, glycosylation and activation of human wild-type and D231N mutated human cath-D transfected into cath-D-deficient CD55−/− fibroblasts

(a) Maturation of wild-type and D231N mutated human cath-D. Cells were cultured in DMEM medium with FCS and cell lysates were analyzed by immunoblotting with an antibody specific for human cath-D. (b) Effects of tunicamycin on cellular wild-type and D231N mutated cath-D glycosylation. Cells were either untreated or treated with increasing concentrations of tunicamycin for 24 h and cell lysates were analysed for cath-D expression by Western blotting. (c) Effects of endoglycosidase H on the glycosylation of wild-type and D231N secreted pro-cath-D. Conditioned media from CD55−/−cath-D and CD55−/−^D231Ncath-D cells were incubated for different times with or without endoglycosidase H and media were analysed for cath-D expression by Western blotting. (d) Auto-activation of wild-type and D231N secreted pro-cath-D. Conditioned media from CD55−/−cath-D and CD55−/−^D231Ncath-D cells were incubated for different times at pH 3.5 in the absence or presence of pepstatin A, and media were analysed for cath-D expression by Western blotting. Black arrows indicate the migration of the 3 forms of wild-type cath-D; grey arrows indicate the migration of the 3 forms of ^D231Ncath-D; white arrows indicate the migration of non-glycosylated cellular cath-D. K= molecular mass in kDa.

Figure 2. Effects of cysteine protease inhibitors on the maturation of wild-type and D231N mutated cath-D in transfected fibroblasts

(a) Effects of E-64 in fibroblasts. CD55−/−cath-D and CD55−/−^D231Ncath-D cells were either untreated or treated for 48 h with 10 mM E-64 and cellular cath-D was analysed by immunoblotting. (b) Concentration dependency of the E-64 effects. CD55−/−cath-D cells were either untreated or treated for 48 h with increasing concentrations of E-64 and cellular cath-D was analysed by immunoblotting. (c) Effects of E-64 using two-dimensional electrophoresis. CD55−/− cath-D cells were either untreated or treated for 48 h with 10 mM E-64 and cellular cath-D was analysed by two-dimensional electrophoresis and immunoblotting. (d) Effects of CA-074Met. CD55−/−cath-D and CD55−/−^D231Ncath-D cells were untreated or treated for 48 h with 50 μM CA-074Met and cellular cath-D was analysed by immunoblotting. The effect of 10 mM E-64 is shown. (e) Effects of CLICK-148 alone or in combination with CA-074Met. CD55−/−cath-D and CD55−/−^D231Ncath-D cells were either untreated or treated for 48 h with 5 or 20 μM CLICK-148 alone or in combination with 50 μM CA-074Met. Cellular cath-D was analysed by immunoblotting. Brackets indicate the 52-48 kDa processing intermediates detected in CD55−/−cath-D cells and the 51-47 kDa processing intermediates detected in CD55−/−^D231Ncath-D cells.

Figure 3. Schematic representation of human cath-D processing

Cath-D is synthesized on the RER as a pre-pro-enzyme that undergoes several proteolytic cleavages during biosynthesis to produce the mature form. Following the initial co-translational removal of the signal peptide to yield 52 kDa pro-cath-D, this pro-enzyme is then processed into numerous 52-48 kDa processing intermediates. These intermediates are not generated by auto-activation, but by the action of unknown proteases whose activities are only partially inhibited by the cysteine protease inhibitor E64, but are not affected by the cath-B or –L inhibitors. The intermediate 48 kDa single-chain species is then cleaved into a mature two-chain enzyme consisting of a light 14 kDa amino-terminal domain and a heavy 34 kDa carboxyl-terminal domain by the cysteine proteases cath-B or cath-L. Accompanying this conversion to the two-chain species, 7 amino acid residues between the 14 KDa light and 34 kDa heavy chains are removed [15]. Several more amino acids are removed from the carboxyl terminus of the 34 kDa heavy chain [16].

Figure 4. Processing of endocytosed wild-type and D231N mutated human cath-D

(a) Processing of endocytosed human ^D231Npro-cath-D. Parental cath-D-deficient CD55−/− fibroblasts were incubated for 18 h with ³⁵S-labelled conditioned medium containing the secreted wild-type or D231N pro-cath-D. After washing, cell lysates containing endocytosed [S³⁵]methionine-labelled cath-D were analyzed by SDS-PAGE after immunoprecipitation. Immunoprecipitation was performed in triplicate. (b) Effects of E-64. CD55−/− fibroblasts were incubated for 18 h with ³⁵S-labelled conditioned medium containing the wild-type pro-cath-D in the absence or presence of 10 mM E-64. Endocytosed [S³⁵]methionine-labelled cath-D was analyzed by SDS-PAGE, as described in panel a. (c) Effects of CA-074Met. CD55−/− fibroblasts were incubated for 18 h with ³⁵S-labelled conditioned medium containing pro-cath-D in the absence or presence of 25 μM CA-074Met. Endocytosed [S³⁵]methionine-labelled cath-D was analyzed by SDS-PAGE. (d) Effects of CLICK-148 alone or in combination with CA-074Met. CD55−/− fibroblasts were incubated for 18 h with the ³⁵S-labelled conditioned medium containing pro-cath-D in the absence or presence of 20 μM CLICK-148 alone or in combination with 25 μM CA-074Met. Endocytosed [S³⁵]methionine-labelled cath-D was analyzed by SDS-PAGE.