New Functional Aspects of Cathepsin D and Cathepsin E

Cathepsin D (CD) and cathepsin E are representative lysosomal and nonlysosomal aspartic proteinases, respectively, and play an important role in the degradation of proteins, the generation of bioactive proteins, antigen processing, etc. Recenty, several lines of evidence have suggested the involvement of these two enzymes in the execution of neuronal death pathways induced by aging, transient forebrain ischemia, and excessive stimulation of glutamate receptors with excitotoxins. CD has also been shown to mediate apoptosis induced by various stimuli and p53-dependent tumor suppression. To gain more insight into in vivo functions of CD, mice deficient in this enzyme were generated. The mutant animals showed a progressive atrophy of the intestinal mucosa, a massive destruction of lymphoid organs, and a profound accumulation of ceroid lipofuscin, and developed a phenotype resembling neuronal ceroid lipofucinosis, suggesting that CD is essential for proteolysis of proteins regulating cell growth and tissue homeostasis. It has also been shown that CD molecules secreted from human prostate carcinoma cells are responsible for the generation of angiostatin, a potent endogenous inhibitor of angiogenesis, suggesting its contribution to the prevention of tumor growth and angiogenesis-dependent growth of metastases. Interestingly, pro-CD from human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that from prostate carcinoma cells. Since deglycosylated CD molecules from both carcinoma cells showed a low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in the carbohydrate structures of CD molecules between the two cell types and to contribute to their potency to prevent tumor growth and metastases.     

New Functional Aspects of Cathepsin D and Cathepsin E

Cathepsin D (CD) and cathepsin E are representative lysosomal and nonlysosomal aspartic proteinases, respectively, and play an important role in the degradation of proteins, the generation of bioactive proteins, antigen processing, etc. Recenty, several lines of evidence have suggested the involvement of these two enzymes in the execution of neuronal death pathways induced by aging, transient forebrain ischemia, and excessive stimulation of glutamate receptors with excitotoxins. CD has also been shown to mediate apoptosis induced by various stimuli and p53-dependent tumor suppression. To gain more insight into in vivo functions of CD, mice deficient in this enzyme were generated. The mutant animals showed a progressive atrophy of the intestinal mucosa, a massive destruction of lymphoid organs, and a profound accumulation of ceroid lipofuscin, and developed a phenotype resembling neuronal ceroid lipofucinosis, suggesting that CD is essential for proteolysis of proteins regulating cell growth and tissue homeostasis. It has also been shown that CD molecules secreted from human prostate carcinoma cells are responsible for the generation of angiostatin, a potent endogenous inhibitor of angiogenesis, suggesting its contribution to the prevention of tumor growth and angiogenesis-dependent growth of metastases. Interestingly, pro-CD from human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that from prostate carcinoma cells. Since deglycosylated CD molecules from both carcinoma cells showed a low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in the carbohydrate structures of CD molecules between the two cell types and to contribute to their potency to prevent tumor growth and metastases.     

Isolation and characterization of a stable activation intermediate of the lysosomal aspartyl protease cathepsin D.

Procathepsin D is the intracellular aspartyl protease precursor of cathepsin D, a major lysosomal enzyme. Procathepsin D is rapidly processed inside the cell, and, thus, examination of its proteolyic activation and structure has been difficult. To study this proenzyme, a nonglycosylated form of the human fibroblast procathepsin D was expressed in Escherichia coli, refold in vitro, and purified by affinity chromatography on pepstatinyl agarose. Sequence analysis of the refolded, autoactivated enzyme allowed determination of the autoproteolytic cleavage site. The sequence surrounding this cleavage site between residues LeuP26 and IleP27 (in the "pro" region) resembled the first cleavage site found during activation of other aspartyl proteases. Thus, the autoactivated procathepsin D is analogous to the pepsin activation intermediate, which has been termed pseudopepsin. The enzymatic activity, thermal and pH stability, and fluorescence spectra of pseudocathepsin D were compared to mature, predominantly two-chain, cathepsin D isolated from human placenta. The results indicated that pseudocathepsin D and mature enzyme have a similar Km toward a peptide substrate and cleave a protein substrate at identical sites. Temperature stability of the recombinant enzyme was similar to that of the tissue-derived enzyme. However, the recombinant enzyme had increased stability at low pH when compared to the glycosylated tissue-derived two-chain cathepsin D. Fluorescence spectra of the recombinant and tissue-derived enzymes were identical. Thus, the absence of asparagine-linked oligosaccharides and the presence of the remaining segment of propeptide did not significantly alter the structural and enzymatic properties of the enzyme.     

Mechanisms and kinetics of procathepsin D activation.

In vitro, procathepsin D is activated to pseudocathepsin D by incubation at low pH. To investigate the mechanism of this activation, recombinant human procathepsin D and two mutants were generated in a baculovirus expression system. One mutant carried a point mutation within the catalytic domain, which resulted in a catalytically inactive enzyme form (D77A). The other carried a point mutation within the propeptide, which prevented activation by processing at the 'autoproteolysis-site' (L26P). Neither mutant is capable of processing itself to form pseudocathepsin D, and L26P is not able to process D77A. Despite the inability of L26P to cleave either its own or a wild-type prosequence, it did exhibit activity against a synthetic peptide substrate. The ability of intact precursor (zymogen) to cleave a peptide, but not a protein substrate, offers new insights into the mechanism of inhibition by the propeptide. Mature cathepsin D can process the inactive D77A mutant to the pseudoform, demonstrating that processed species are capable of cleaving zymogen molecules in an intermolecular interaction. In addition, kinetic studies provide evidence for a two-phase mechanism for the conversion of procathepsin D to pseudocathepsin D, one phase where the first molecules of pseudocathepsin D are formed at a low rate and a second phase where the process is autocatalytically accelerated by newly formed pseudocathepsin D molecules. Finally, with the help of the mutants L26P and D77A it was observed that at least two additional proteinase activities, found in conditioned media from insect cell culture, are capable of activating procathepsin D by cleaving it within the proregion. This observation suggests that there are likely to be multiple proteinases in the extracellular matrix that are capable of activating procathepsin D, thereby triggering the second autocatalytic phase. This may also be important for solid tumors, where the presence of cathepsin D has been correlated with tumor growth and invasion.     

Interaction of human breast fibroblasts with collagen I increases secretion of procathepsin B

Interactions of stromal and tumor cells with the extracellular matrix may regulate expression of proteases including the lysosomal proteases cathepsins B and D. In the present study, we determined whether the expression of these two proteases in human breast fibroblasts was modulated by interactions with the extracellular matrix component, collagen I. Breast fibroblasts were isolated from non-malignant breast tissue as well as from tissue surrounding malignant human breast tumors. Growth of these fibroblasts on collagen I gels affected cell morphology, but not the intracellular localization of vesicles staining for cathepsin B or D. Cathepsins B and D levels (mRNA or intracellular protein) were not affected in fibroblasts growing on collagen I gels or plastic, nor was cathepsin D secreted from these cells. In contrast, protein expression and secretion of cathepsin B, primarily procathepsin B, was induced by growth on collagen I gels. The induced secretion appeared to be mediated by integrins binding to collagen I, as inhibitory antibodies against alpha(1), alpha(2), and beta(1) integrin subunits prevented procathepsin B secretion from fibroblasts grown on collagen. In addition, procathepsin B secretion was induced when cells were plated on beta(1) integrin antibodies. To our knowledge, this is the first examination of cathepsin B and D expression and localization in human breast fibroblasts and their regulation by a matrix protein. Secretion of the cysteine protease procathepsin B from breast fibroblasts may have physiological and pathological consequences, as proteases are required for normal development and for lactation of the mammary gland, yet can also initiate and accelerate the progression of breast cancer.     

Lysosomal cysteine protease, cathepsin B, is targeted to lysosomes by the mannose 6-phosphate-independent pathway in rat hepatocytes: site-specific phosphorylation in oligosaccharides of the proregion

Cathepsin B, a lysosomal cysteine protease, is synthesized as a glycoprotein with two N-linked oligosaccharide chains, one of which is in the propeptide region while the other is in the mature region. When cultured rat hepatocytes were labeled with [(32)P]phosphate, (32)P-labeled cathepsin B was immunoprecipitated only in the proform from cell lysates and medium. Either Endo H or alkaline phosphatase treatment of (32)P-labeled procathepsin B demonstrated the acquisition of a mannose 6-phosphate (Man 6-P) residue on high mannose type oligosaccharides. To identify the site of phosphorylation, immunoisolated (35)S- or (32)P-labeled procathepsin B was incubated with purified lysosomal cathepsin D, since cathepsin D cleaves 48 amino acid residues from the N-terminus of procathepsin B, in which one N-linked oligosaccharide chain was also included [Kawabata, T. et al. (1993) J. Biochem. 113, 389-394]. Treatment of intracellular (35)S-labeled procathepsin B with a molecular mass of 39-kDa with cathepsin D resulted in the production of the 31-kDa intermediate form, but the (32)P-label incorporated into procathepsin B disappeared after treatment with cathepsin D. These results indicate that the phosphorylation of procathepsin B is restricted to an oligosaccharide chain present in the propeptide region. Interestingly, cathepsin B sorting to lysosomes was not inhibited by NH(4)Cl treatment and about 90% of the intracellular procathepsin B initially phosphorylated was secreted into the medium without being dephosphorylated intracellularly, and did not bind significantly to cation-independent-Man 6-P receptor, suggesting the failure of Man 6-P-dependent transport of procathepsin B to lysosomes. Additionally, about 50% of the newly synthesized (35)S-labeled cathepsin B was retained in the cells in mature forms consisting of a 29-kDa single chain form and a 24-kDa two chain form, while part of the procathepsin B was associated with membranes in a Man 6-P-independent manner. Taken together, these results show that in rat hepatocytes, cathepsin B is targeted to lysosomes by an alternative mechanism(s) other than the Man 6-P-dependent pathway.     

Rat procathepsin B. Proteolytic processing to the mature form in vitro.

Expression of rat procathepsin B in yeast led to the secretion of both the latent and mature forms of the enzyme. Culture in the presence of a cysteine proteinase inhibitor prevented this processing. We have expressed and purified a mutant form of rat procathepsin B whose active-site cysteine residue has been changed to a serine, and which also lacks the glycosylation site in the mature region of the protein. This non-active mutant protein was secreted essentially in an unprocessed form. The purified protein has been incubated with a variety of proteinases, and results indicate that cathepsins D and L, as well as mature cathepsin B itself, can produce a processed (single-chain) form of cathepsin B from this precursor. Amino-terminal sequencing of these processed forms has revealed that they are all elongated by a few residues with respect to the mature form found in vivo. The action of a combination of cathepsin B with dipeptidylpeptidase I produced a single-chain form of cathepsin B with the correct amino terminus. This work has also shown that the processing of procathepsin B to a single-chain form can be an autocatalytic process, in at least an intermolecular manner.     

Cathepsin D antigenic epitopes identified by the humoral responses of ovarian cancer patients

 Previous studies implicated cathepsin D as one commonly recognized target of tumor-reactive immunoglobulins from ovarian cancer patients. These immunoglobulins are shown to be immunoreactive with both the 52-kDa procathepsin D and the 32-kDa mature cathepsin D derived from the UL-1 ovarian cancer cell line. Whether the carbohydrate domains or the core protein were associated with its immunogenicity was analyzed with cathepsin D isolated from tunicamycin-treated UL-1 cells. No significant difference was detected in the immunoreactivity of patient serum with the glycosylated and deglycosylated forms of the cathepsin D, suggesting that patient humoral responses are directed primarily against the core protein. To define the antigenic epitopes of cathepsin D, tryptic fragments were prepared from UL-1-derived procathepsin D. The epitopes of the core protein recognized by sera from more than one patient were identified using a peptide-specific enzyme-linked immunosorbent assay and microsequencing of positive immunoreactive peptides. This protocol identified four epitopes: two peptides within the pro-peptide, a third at the carboxy terminus and the fourth at the glycosylation site of the mature enzyme. This approach to the identification of specific antigenic epitopes may be useful in defining effective targets for directed active immunotherapy against cancer. Received: 8 September 1997 / Accepted: 21 October 1997     

Mannose 6-phosphate-independent targeting of cathepsin D to lysosomes in HepG2 cells.

We have studied the role of N-linked oligosaccharides and proteolytic processing on the targeting of cathepsin D to the lysosomes in the human hepatoma cell line HepG2. In the presence of tunicamycin cathepsin D was synthesized as an unglycosylated 43-kDa proenzyme which was proteolytically processed via a 39-kDa intermediate to a 28-kDa mature form. Only a small portion was secreted into the culture medium. During intracellular transport the 43-kDa procathepsin D transiently became membrane-associated independently of binding to the mannose 6-phosphate receptor. Subcellular fractionation showed that unglycosylated cathepsin D was efficiently targeted to the lysosomes via intermediate compartments similar to the enzyme in control cells. The results show that in HepG2 cells processing and transport of cathepsin D to the lysosomes is independent of mannose 6-phosphate residues. Inhibition of the proteolytic processing of 53-kDa procathepsin D by protease inhibitors caused this form to accumulate intracellularly. Subcellular fractionation revealed that the procathepsin D was transported to lysosomes, thereby losing its membrane association. Procathepsin D taken up by the mannose 6-phosphate receptor also transiently became membrane-associated, probably in the same compartment. We conclude that the mannose 6-phosphate-independent membrane-association is a transient and compartment-specific event in the transport of procathepsin D.     

Bafilomycin A1 inhibits the targeting of lysosomal acid hydrolases in cultured hepatocytes

Effects of bafilomycin A1, an inhibitor of vacuolar H(+)-ATPase, on the synthesis and processing of cathepsin D and cathepsin H were investigated in primary cultured rat hepatocytes. Pulse-chase experiments showed that after being synthesized as procathepsin D and procathepsin H the precursors were converted into mature forms in the control cells as the chase time elapsed. However, in the presence of 5 x 10(-7) M of bafilomycin A1, both precursors were largely secreted into the medium and no mature forms were found within the cells. Thus bafilomycin A1 mimics lysosomotropic amines with regard to perturbation of the targeting of lysosomal acid hydrolases. In contrast, bafilomycin A1 was found not to inhibit processings of proalbumin and procomplement component 3, which are thought to occur at the acidic trans-Golgi, implying that the proteolytic event of the proproteins is not sensitive to an increase of intra-Golgi pH. The results suggest that bafilomycin A1 is useful as a pH-perturbant to study the role of acidity in living cells.     

Identification of a precursor form of cathepsin D in microsomal lumen: characterization of enzymatic activation and proteolytic

A precursor form of cathepsin D with 45 kDa was demonstrated in the rat liver microsomal lumen by immunoblotting analysis. The microsomal fraction containing procathepsin D which passed through a pepstatin-Sepharose resin showed no appreciable activity of cathepsin D. The in vitro incubation of this fraction at pH 3.0 resulted in a gradual increase of proteolytic activity toward hemoglobin as substrate and also, the proteolytic conversion of procathepsin D to the mature form was concomitantly observed. The proteolytic processing step was sensitive to pepstatin. These results suggest that procathepsin D is inactive in the endoplasmic reticulum and may be converted to the active forms by autoproteolytic processing mechanism at acidic pH during biosynthesis.     

Human and hamster procathepsin D, although equally tagged with mannose-6-phosphate, are differentially targeted to lysosomes in transfected BHK cells

BHK cells transfected with human cathepsin D (CD) cDNA normally segregate the autologous hamster cathepsin D while secreting a large proportion of the human proenzyme. In the present work, we have utilized these transfectants to examine to what extent the mannose-6-phosphate-dependent pathway for lysosomal enzyme segregation contributes to the differential sorting of human and hamster CD. We report that, in recipient control BHK cells, the rate of mannose-6-phosphate-dependent endocytosis of human procathepsin D secreted by transfected BHK cells is lower than that of hamster procathepsin D and much lower than that of human arylsulphatase A. The missorted human enzyme bears phosphorylated oligosaccharides and most of its phosphate residues are “uncovered”, like the autologous enzyme. Thus, despite both the Golgi-associated modifications of oligosaccharides, i.e. the phosphorylation of mannose and the uncovering of mannose-6-phosphate residues, which proceed on human and hamster procathepsin D with comparable efficiency, only the latter is accurately packaged into lysosomes. Ammonium chloride partially affects the lysosomal targeting of cathepsin D in control BHK cells, whereas in transfected cells, this drug strongly inhibits the maturation of human procathepsin D and slightly enhances its secretion. These data indicate that: (1) over-expression of a lysosomal protein does not saturate the Golgi-associated reactions leading to the synthesis of mannose-6-phosphate; (2) a portion of cathepsin D is targeted independently of mannose-6-phosphate receptors in the transfected BHK cells; and (3) whichever mechanism for lysosomal delivery of autologous procathepsin D is involved, this is not saturated by the high rate of expression of human cathepsin D.     

Pro-cathepsin D can activate in vitro pro-cathepsin B secreted by ovarian cancers

A precursor form of cathepsin B (Mr 45-47 kd) was purified from ascitic fluids of patients with ovarian adenocarcinomas. Following pepsin activation, this precursor produced a 33 kd cathepsin B-like proteinase closely related to lysosomal cathepsin B. A similar activation was found using the 52 kd pro-cathepsin D secreted by the MCF7 human breast cancer cells. This activation was a time, dose and pH dependent process. These results suggest that the 52 kd pro-cathepsin D may be involved in the early steps of the "metastatic cascade", activating pro-cathepsin B in an acidic environment.     

Chondroitin sulfate proteoglycan is a potent enhancer in the processing of procathepsin L

The acceleration effect of chondroitin-4-sulfate(CS-) proteoglycan on the processing of procathepsin L in vitro was investigated using enzyme purified from the culture medium of MLC cells. Procathepsin L was slightly processed even when it was incubated without CS-proteoglycan for 60 min in 50 mm acetate buffer, pH 5.5, and trace amounts of the 31 kDa mature form and 35-38 kDa intermediates of cathepsin L were formed. On the other hand, in the presence of CS-proteoglycan, procathepsin L was completely converted to the mature form within the same 60 minute time period. Moreover, Z-Phe-Arg-MCA hydrolyzing activity was increased significantly by the incubation with CS-proteoglycan, while no considerable increase in the activity was observed during the incubation without CS-proteoglycan. Since the specific cathepsin L inhibitor, CLIK-195, inhibited the processing of procathepsin L accelerated by CS-proteoglycan, the trace amount of cathepsin L activity may participate in the processing. These results suggest that CS-proteoglycan may play a role in accelerating the processing of procathepsin L as an endogenous enhancer in the extracellular environment in vivo.     

Expression and refolding of recombinant human fibroblast procathepsin D

Procathepsin D is a precursor of the human lysosomal protease cathepsin D. Due to its short half-life, procathepsin D is difficult to obtain in quantities sufficient to allow structural and enzymatic studies. To obtain large quantities of this precursor, procathepsin D was expressed using the T7 promoter vector pET3a in bacteria that carry a chromosomal copy of the T7 RNA polymerase gene under the control of the lac promoter. At high cell density in rich medium, basal levels of T7 RNA polymerase were sufficient to express recombinant procathepsin D without addition of an exogenous inducer of the lac promoter. The recombinant protein, constituting almost half of the total cell protein, accumulated in intracytoplasmic inclusion bodies and was isolated from the insoluble fraction of lysed cells. Antibodies prepared against the purified recombinant protein were shown to crossreact with native human placental and porcine spleen cathepsin D. Recombinant procathepsin D was solubilized in denaturants and was refolded. After extended preincubation of the denatured protein at acid pH to allow folding and activation of the zymogen, pepstatin inhibitable catalytic proteolysis was detected. These data demonstrated that the glycosylated aspartic protease, procathepsin D can be refolded and activated in an unglycosylated form and thus provides a system for the study of procathepsin D structure and function.     

Cynomolgus Monkey (Macaca fascicularis) Cathepsin K: Cloning, Expression, Purification, and Activation

Methodology for the production of recombinant active cynomolgus monkey (Macaca fascicularis) cathepsin K (EC 3.4.22.38) was elucidated. The cDNA encoding the cathepsin K was cloned from femaleM. cynomolgusmonkey mRNA. The deduced amino acid sequence ofM. cynomolguspreprocathepsin K from the cDNA sequence showed 94.2% identity to human preprocathepsin K. Sequence differences occurred only in the prepro- domains; the mature domains were identical. The recombinantM. cynomolguscathepsin K was expressed as a secreted proenzyme using baculovirus-infected SF21 insect cells having the predicted N-terminus (LYPEEILDTH … ), indicating proper cleavage of the secretion sequence. Purified monkey procathepsin K was activated under autocatalytic conditions at pH 4.0. The mature enzyme was composed of mixture of enzymes having N-termini of Gly¹¹³and Arg¹¹⁴. The molecular weight was determined to be 23,668.3 Da by MALDI-TOF-MS which is consistent with the absence of carbohydrate on the mature enzyme. These results indicate that monkey procathepsin K is able to autoactivate and produces a mature enzyme which is identical to that of human cathepsin K. Since the sequence of monkey and human mature cathepsin K are identical and thein vitroactivation mechanisms appear to be indistinguishable, monkeys are predicted to be a good animal model for evaluating cathepsin K inhibitorsin vivoas therapeutic agents for diseases characterized by excessive bone loss, such as osteoporosis.     

Matrix metalloproteinase 2 is involved in the regulation of the antimicrobial peptide parasin I production in catfish skin mucosa

A 19-residue antimicrobial peptide parasin I is generated from histone H2A in the skin mucus of catfish by the action of cathepsin D activated by a procathepsin D-processing enzyme induced upon epidermal injury. Here we report the isolation and characterization of the procathepsin D-processing enzyme in the mucus of wounded catfish. Sequence analysis of the cDNA identified the purified procathepsin D-processing enzyme as matrix metalloproteinase 2 (MMP 2). By acting as a procathepsin D convertase upon epidermal injury, MMP 2 is involved in the regulation of parasin I production in catfish skin mucosa.     

Autocatalytic processing of procathepsin E to cathepsin E and their structural differences

The processing of human gastric procathepsin E to its mature form, cathepsin E, was studied at pH 3.5. The results revealed the autocatalytic and apparently one-step conversion of procathepsin E to cathepsin E within 10 min of incubation at 14 degrees C under the conditions used. Analyses of the amino acid sequences of both procathepsin E and cathepsin E showed that cleavage occurred at the Met36-Ile37 bond to produce the mature form, cathepsin E. The NH2-terminal amino acid sequence of procathepsin E thus determined was identical with that predicted from the cDNA sequence by Azuma et al. except that the NH2-terminal glutamine residue in the latter was converted into a pyroglutamic acid residue in the former and that the glycine residue at position 2 in the latter sequence was deleted in the former. On the other hand, the NH2-terminal amino acid sequence of cathepsin E was identical with that reported previously by us.     

Human procathepsin B interacts with the annexin II tetramer on the surface of tumor cells

To study potential roles of plasma membrane-associated extracellular cathepsin B in tumor cell invasion and metastasis, we used the yeast two-hybrid system to screen for proteins that interact with human procathepsin B. The annexin II light chain (p11), one of the two subunits of the annexin II tetramer, was one of the proteins identified. We have confirmed that recombinant human procathepsin B interacts with p11 as well as with the annexin II tetramer in vitro. Furthermore, procathepsin B could interact with the annexin II tetramer in vivo as demonstrated by coimmunoprecipitation. Cathepsin B and the annexin II tetramer were shown by immunofluorescent staining to colocalize on the surface of human breast carcinoma and glioma cells. Taken together, our results indicate that the annexin II tetramer can serve as a binding protein for procathepsin B on the surface of tumor cells, an interaction that may facilitate tumor invasion and metastasis.     

Identification of latent procathepsins B and L in microsomal lumen: characterization of enzymatic activation and proteolytic processing in vitro

Procathepsins B and L in the hepatic endoplasmic lumen were identified as having a molecular weight of 39,000 by immunoblot analysis. The proenzymes were then purified to remove the mature enzymes by concanavalin A-Sepharose chromatography. The concanavalin A-adsorbed fractions containing the proenzymes showed no appreciable activities of cathepsins B and L. When those fractions were incubated at pH 3.0, the enzymatic activities markedly increased: the activities of cathepsins B and L after 36 h incubation were 60 and 210 times those of the controls, respectively. Immunoblot analysis showed that after 36 h incubation the proenzymes disappeared and the mature enzymes increased. Thus the proenzymes were processed to the mature enzymes under acidic conditions of pH 3.0. The marked increases of enzymatic activities and the conversion of the proenzymes to the mature forms were completely blocked with pepstatin, which is a potent inhibitor of aspartic proteases. The results strongly suggested that a processing protease for procathepsins B and L might be cathepsin D, a major lysosomal aspartic protease. Indeed, lysosomal cathepsin D could convert microsomal procathepsin B to the mature enzyme in vitro. Therefore, procathepsins B and L seem first to be synthesized as enzymatically inactive forms in endoplasmic reticulum and successively may be converted into active forms by cathepsin D in lysosomal compartments.