Cathepsin E from rat neutrophils: its properties and possible relations to cathepsin D-like and cathepsin E-like acid proteinases

An extract of rat neutrophils was found to contain a high hemoglobin-hydrolyzing activity at pH 3.2, about 70% of which does not cross-react with anti-rat liver cathepsin D antibody. A neutrophil non-cathepsin D acid proteinase was successfully isolated from cathepsin D and characterized in comparison with the properties of rat liver cathepsin D. The neutrophil enzyme differed from cathepsin D in chromatographic and electrophoretic behaviors as well as immunological cross-reactivity, and its molecular weight was estimated to be 98,000 by gel filtration on Toyopearl HW 55. These findings strongly suggest that the neutrophil enzyme could be classified as cathepsin E. The enzyme, now designated rat cathepsin E, had an optimal pH at 3.0-3.2, preferred hemoglobin to albumin as substrate, and was markedly resistant to urea denaturation. Rat cathepsins D and E cleaved the insulin B-chain at six and eight sites, respectively; five sites were common for both enzymes. Possible relations among cathepsin E and cathepsin D-like or E-like acid proteinases reported so far were discussed.     

Complex modulation of peptidolytic activity of cathepsin D by sphingolipids

Cathepsin D is an aspartic peptidase involved in cellular processes including proliferation and apoptosis and implicated in human pathologies such as cancer and neurodegeneration. Our knowledge about the relationship between proteolysis and bioactive sphingolipids is still very limited. Here, we describe a complex pattern of modulation of the peptidolytic activity of cathepsin D by sphingolipids. A panel of sphingolipid derivatives was screened in a FRET-based assay; these molecules demonstrated negative or positive modulation of cathepsin D peptidolytic activity, depending on the sphingolipid structure. Certain sphingosines and ceramides inhibited cathepsin D in the submicromolar range, and structural requirements for this inhibitory effect were evaluated. The interaction of cathepsin D with sphingolipids was also demonstrated by fluorescence polarization measurements and determined to follow a competitive inhibition mode. In contrast, monoester phosphosphingolipids, especially ceramide-1-phosphate, were identified as activators of cathepsin D peptidolytic activity at submicromolar concentrations. Thus, sphingolipids and phosphosphingolipids, known to be antagonistic in their cell-signaling functions, displayed opposite modulation of cathepsin D. Sphingolipid-based modulators of cathepsin D are potentially involved in the control of cathepsin D-dependent processes and might serve as a scaffold for the development of novel regulators of this therapeutic target.     

Characterization of endothelin converting enzyme activities in soluble fraction of bovine cultured endothelial cells

Endothelin converting enzyme activities in the soluble fraction of cultured bovine aortic endothelial cells were characterized. The two major endothelin converting enzyme activities were eluted from a hydrophobic chromatography column and the elution profile of the endothelin converting enzyme activities was the same as that of cathepsin D activities. These activities had a same pH optimum at pH 3.5 and were effectively inhibited by pepstatin A. Furthermore, anti-cathepsin D antiserum absorbed these activities as well as cathepsin D activity. Immunoblotting analysis using the antiserum showed the major active fractions have immunostainable components of identical molecular weights with cathepsin D. From these results, we concluded that the major endothelin converting activities in the soluble fraction of endothelial cells are due to cathepsin D. In addition to these cathepsin D activities, a minor endothelin converting enzyme activity with an optimum pH at 3.5 was found, which does not have angiotensin I generating (cathepsin D) activity from renin substrate and needs much higher concentrations of pepstatin A to inhibit the activity than cathepsin D.     

Endocytosis and the recycling of plasma membrane

For study of the time order of glycosylation, formation of complex oligosaccharides and proteolytic maturation as well as the site of proteolytic maturation of cathepsin D, fibroblasts were subjected to pulse-chase labeling, and cathepsin D was isolated from either total cell extracts or subcellular fractions by immune precipitation and analyzed for its molecular forms and sensitivity to endo-beta-N- acetylglucosaminidase H. After a 10-min pulse, cathepsin D was detected in its glycosylated precursor form, indicating an early, probably a cotranslational, N-glycosylation of cathepsin D. Conversion of the high- mannose oligosaccharide side chains into forms resistant to endo-beta-N- acetylglucosaminidase H started after approximately 40 min, indicating that transport of cathepsin D from the endoplasmic reticulum to the trans-Golgi apparatus requires approximately 40 min. Processing of the 53-kdalton precursor polypeptide of cathepsin D to a 47-kdalton intermediate followed about 20 min after the formation of complex oligosaccharides, and, another 30 min later, 31-kdalton mature forms of cathepsin D were detected. Processing of cathepsin D was first observed in light membranes as a partial conversion of the 53-kdalton precursor into the 47-kdalton intermediate. Both the precursor and the intermediate are transferred into the high density-class lysosomes. After 8 h, the processing to the mature 31-kdalton form of cathepsin D is mostly completed.     

Cathepsin D is membrane-associated in macrophage endosomes

Previously we identified an acid protease activity which was located in the endosomes of rabbit alveolar macrophages (Diment, S., and Stahl, P.D. (1985) J. Biol. Chem. 260, 15311-15317). In this study, the endosomal protease is identified as cathepsin D by immunoprecipitation with polyclonal antibodies raised against rabbit cathepsin D and by NH2-terminal sequence. In order to elucidate the mechanism for targeting of cathepsin D to endosomes, we first examined the membrane association of cathepsin D with light (rho = 1.05 g/ml) and heavy density (rho = 1.1 g/ml) vesicles from Percoll density gradients. After sequential washes, 8.4 and 21.9% of cathepsin D activity remained associated with heavy and light density vesicles, respectively. This membrane-associated cathepsin D could not be solubilized in either buffer at pH 5.0 containing mannose 6-phosphate and EDTA or in buffer at pH 10.6. Solubilization required the detergent Triton X-100. To determine whether membrane-associated cathepsin D was found in endosomes, the enzyme was radioiodinated within endosomes and lysosomes with internalized lactoperoxidase. The membrane-associated form was detected in endosomes, but much less in lysosomes. Biosynthetic studies combined with the same extraction procedure revealed that macrophage cathepsin D is first synthesized as an inactive membrane-associated precursor. The precursor is processed to an active, membrane-associated form and then to the active soluble form found in lysosomes. Our studies provide evidence that 1) cathepsin D is in endosomes of macrophages; 2) cathepsin D is transported to endosomes as a membrane-associated form; and 3) the membrane-associated form is a biosynthetic precursor for the soluble form found in endosomes and lysosomes.     

Identificating cathepsin D as a biomarker for differentiation and prognosis of nasopharyngeal carcinoma by laser capture microdissection and proteomic analysis

In this study, we applied laser capture microdissection and a proteomic approach to identify novel nasopharyngeal carcinoma (NPC) biomarkers. Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of the differential protein cathepsin D in the above two tissues as well as four NPC cell lines was determined by Western blotting. Next, siRNA was used to inhibit the expression of cathepsin D in highly metastatic NPC cell line 5-8F to examine whether it associates with NPC metastasis. Immunohistochemistry was also performed to detect the expression of cathepsin D in 72 cases of primary NPC, 28 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of its expression level with clinicopathologic features and clinical outcomes were evaluated. Thirty-six differential proteins between the NPC and NNET were identified. The expression level of cathepsin D in the two types of tissues was confirmed by Western blotting and related to differentiation degree and metastatic potential of the NPC cell lines. Down-regulated cathepsin D expression by siRNA significantly decreased in vitro invasive ability of 5-8F cells. Significant cathepsin D down-regulation was observed in NPC versus NNET, whereas significant cathepsin D up-regulation was observed in lymph node metastasis versus primary NPC. In addition, cathepsin D down-regulation was significantly correlated with poor histological differentiation, whereas cathepsin D up-regulation was significantly correlated with advanced clinical stage, recurrence, and lymph node and distant metastasis. Furthermore, survival curves showed that patients with cathepsin D up-regulation had a poor prognosis. Multivariate analysis confirmed that cathepsin D expression was an independent prognostic indicator. The data suggest that cathepsin D is a potential biomarker for the differentiation and prognosis of NPC, and its dysregulation might play an important role in the pathogenesis of NPC.     

Biosynthesis of the multiple forms of rabbit cardiac cathepsin D

Rabbit cardiac cathepsin D exists as multiple isomeric forms of Mr = 48,000 within cardiac tissue. Their mechanism of formation and their functional role in cardiac protein degradation are unknown. We have previously demonstrated that cathepsin D is initially synthesized as an Mr = 53,000 precursor that is processed by limited proteolysis within cardiac lysosomes to the Mr = 48,000 active forms of the enzyme. To determine if the multiple forms of active cathepsin D originate from a common precursor, isolated perfused Langendorff rabbit hearts were labeled in pulse (15 or 30 min) and pulse-chase (30 or 150 min) experiments with [35S]methionine. Newly synthesized cathepsin D was isolated by butanol/Triton X-100 extraction and immunoadsorption with anti-cathepsin D IgG-Sepharose, and the isomeric forms were separated by two-dimensional electrophoresis and fluorography. After 15- and 30-min pulse perfusions, 35S-labeled cathepsin D appeared as a single precursor form (Mr = 53,000, pI = 6.6). After 30-min pulse and 30-min chase, the precursor was modified to yield multiple precursor forms, all with molecular weight 53,000, but with differing pI values (6.6-6.0). After 30-min pulse and 150-min chase perfusion, multiple forms of both precursor and proteolytically processed active cathepsin D (Mr = 48,000, pI = 6.2-5.6) were detected. The 35S-labeled, proteolytically processed forms of active cathepsin D co-migrated with the major cathepsin D forms present in cardiac tissue. Subcellular fractionation and perfusions in the presence of chloroquine demonstrated that the multiple precursor forms of cathepsin D originated in a nonlysosomal intracellular compartment. Thus, the multiple forms of active cathepsin D originate from a common high molecular weight precursor, and their synthesis occurs prior to the limited proteolysis of the precursor in cardiac lysosomes.     

Low temperature blocks transport and sorting of cathepsin D in fibroblasts

The transport of newly synthesized cathepsin D in fibroblasts at 16-28 degrees C was compared to that at 37 degrees C. At 37 degrees C newly synthesized cathepsin D passes the trans Golgi within 30-60 min, becomes segregated from the secretory route into prelysosomal organelles within 1-2 h and processed to mature forms in dense lysosomes within 1.5-3 h after synthesis. The small fraction of cathepsin D that escapes transport into lysosomes is secreted within less than 2 h. At 16-28 degrees C the transport of cathepsin D to lysosomes is inhibited in a temperature-dependent manner. At 16-28 degrees C cathepsin D precursors are slowly transported to the trans Golgi. The cathepsin D precursors accumulate at a site that is in continuity with the secretory pathway and located within or distal of the trans Golgi and proximal to the site where cathepsin D precursors leave the secretory pathway as complexes with mannose 6-phosphate receptors. The arrest at this site is not complete. The receptor-dependent segregation of the cathepsin D precursors released from the block is impaired at less than or equal to 26 degrees C. The inhibition of segregation results in an increased, albeit retarded secretion of cathepsin D. The fraction of cathepsin D precursors that is segregated from the secretory pathway encounters a further low temperature block in prelysosomal organelles. There cathepsin D precursors are proteolytically processed to an intermediate form, which accumulates transiently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of subcellular compartments involved in biosynthetic processing of cathepsin D.

We have assigned the biosynthetic processing steps of cathepsin D to intracellular compartments which are involved in its transport to lysosomes in HepG2 cells. Cathepsin D was synthesized as a 51-kDa proenzyme. After formation of 51-55-kDa intermediates due to processing of N-linked oligosaccharides, procathepsin D was proteolytically processed to an intermediate 44-kDa and the mature 31-kDa enzyme. The intersection of the biosynthetic pathway of cathepsin D with the endocytic pathway was labeled with horseradish peroxidase and monitored biochemically by 3,3'-diaminobenzidine cytochemistry. Horseradish peroxidase was used either as a fluid-phase marker to label the entire endocytic pathway or conjugated to transferrin (Tf) to label endosomes only. Directly after biosynthesis cathepsin D was accessible neither to horseradish peroxidase nor Tf-horseradish peroxidase. Newly synthesized 51-55-kDa species of cathepsin D present in the trans-Golgi reticulum were accessible to both horseradish peroxidase and Tf-horseradish peroxidase. The accessibility of trans-Golgi reticulum to both endocytosed horseradish peroxidase and Tf-horseradish peroxidase was monitored by colocalization with a secretory protein, alpha 1anti-trypsin. The proteolytic processing of 51-55-kDa to 44-kDa cathepsin D occurred in compartments which were fully accessible to fluid-phase horseradish peroxidase. Tf-horseradish peroxidase had access to only 20% of 44-kDa cathepsin D while it had no access to 31-kDa cathepsin D. In contrast, the 31-kDa species was completely accessible to fluid-phase horseradish peroxidase. We conclude that proteolytic processing of 51-55-kDa to 44-kDa cathepsin D occurs in endosomes, whereas the processing of 44-31-kDa cathepsin D takes place in lysosomes.     

An immunohistochemical study on cathepsin D in human hippocampus

Cathepsin D is a lysosomal enzyme involved in neuronal degeneration. In this study, the immunohistochemistry of cathepsin D was studied in hippocampal CA1 neurons that are vulnerable to ischemia, and parahippocampal glial cells. CA1 neurons from the majority of cases showed cathepsin D immunoreactivity in the cytoplasm, whereas shrunk neurons were unstained in only one case. There was no statistically significant correlation between the postmortem interval between death and autopsy, and cathepsin D immunoreactivity in CA1 neurons. These observations indicate that cathepsin D immunoreactivity is not a sensitive marker for neuronal degeneration or postmortem changes. On the other hand, there was a statistically significant correlation between age and cathepsin D immunoreactivity in the cytoplasm of parahippocampal glial cells. This shows that senescence is correlated with cathepsin D expression in humans as has been reported previously in an animal study.     

Cathepsin D targeted by acid sphingomyelinase-derived ceramide.

Ceramide has been recognized as a common intracellular second messenger for various cytokines, growth factors and other stimuli, such as CD95, chemotherapeutic drugs and stress factors. To understand the role of ceramide during apoptosis and other cellular responses, it is critically important to characterize direct targets of ceramide action. In this paper, we show that ceramide specifically binds to and activates the endosomal acidic aspartate protease cathepsin D. Direct interaction of ceramide with cathepsin D results in autocatalytic proteolysis of the 52 kDa pre-pro cathepsin D to form the enzymatically active 48/32 kDa isoforms of cathepsin D. Acid sphingomyelinase (A-SMase)-deficient cells show decreased cathepsin D activity, which could be reconstituted by transfection with A-SMase cDNA. The results of our study identify cathepsin D as the first endosomal ceramide target that colocalizes with and may mediate downstream signaling effects of A-SMase.     

Tumour budding intensity in relation to cathepsin D expression and some clinicopathological features of colorectal cancer

Although it has been suggested that tumour budding at the invasive edge of colorectal cancer is an important prognostic factor its biological significance for tumour progression is still to be evaluated. The aim of the study was to correlate tumour budding intensity with cathepsin D expression and some other clinicopathological variables of presumed or established prognostic value. 48 patients with colorectal cancer at pT3 stage, G2 grade of histological differentiation and tumour budding at the invasive edge were evaluated. Colorectal tumours were investigated for cathepsin D expression by immunohistochemistry of formalin-fixed and paraffin embedded tissues. There was no statistically significant relationships between tumour budding intensity grade and primary tumour cathepsin D expression, stromal cell cathepsin D expression and histochemical immunostaining of cathepsin D in rumour budding at its invasive edge. The tumour budding intensity was not associated with lymph node status, tumour site, peritumoral inflammatory response as well as the patient's age and sex. The results of this study suggest that intensity of tumour budds formation at the invasive margin of colorectal cancer is not associated with presumed or established prognostic factors such as lymph node metastases, and peritumoural inflammatory reaction as well as cathepsin D expression.     

Structures at the proteolytic processing region of cathepsin D

The amino acid sequences at the "proteolytic processing regions" of cathepsin Ds have been determined for the enzymes from cows, pigs, and rats in order to deduce the sites of cleavage as well as the function of the proteolytic processing of cathepsin D. For bovine cathepsin D, the "processing region" sequence was determined from a peptide isolated from the single-chain enzyme. The COOH-terminal sequence of the light chain and the NH2-terminal sequence of the heavy chain were also determined. The processing region sequence of porcine cathepsin D was determined from its cDNA structure, and the same structure from rat cathepsin D was determined from the peptide sequence of the single-chain rat enzyme. From sequence homology to other aspartic proteases whose x-ray crystallographic structures are known, such as pepsinogen and penicillopepsin, it is clear that the processing regions are insertions to form an extended beta-hairpin loop between residues 91 and 92 (porcine pepsin numbers). However, the sizes of the processing regions of cathepsin Ds from different species are considerably different. For the enzymes from rats, cows, pigs, and human, the sizes of the processing regions are 6, 9, 9, and 11 amino acid residues, respectively. The amino acid sequences within the processing regions are considerably different. In addition, the proteolytic processing sites were found to be completely different in the bovine and porcine cathepsin Ds. While in the porcine enzyme, an Asn-Ser bond and a Gly-Val bond are cleaved to release 5 residues as a consequence of the processing; in the bovine enzyme, two Ser-Ser bonds are cleaved to release 2 serine residues. These findings would argue that the in vivo proteolytic processing of the cathepsin D single chain is probably not carried out by a specific "processing protease." Model building of the cathepsin D processing region conformation was conducted utilizing the homology between procathepsin D and porcine pepsinogen. The beta-hairpin structure of the processing region was found to (i) interact with the activation peptide of the procathepsin D in a beta-structure and (ii) place the Cys residue in the processing region within disulfide linkage distance to Cys-27 of cathepsin D light chain. These observations support the view that the processing region of cathepsin D may function to stabilize the conformation of procathepsin D and may play a role in its activation.     

Molecular forms of cathepsin D in coated vesicle preparations

We have studied the polypeptide pattern of cathepsin D associated with coated vesicle fractions prepared from human placenta. In these fractions cathepsin D was about 35-fold enriched in the precursor polypeptides as compared to the unfractionated tissue extract. The enrichment was more prominent if the vesicles were fractionated in the presence of Triton X-100. Adsorption of exogenously added metabolically labelled cathepsin D precursor to the fractionated material was negligible. It is likely that the precursor and may be also the mature cathepsin D polypeptides are present in the matrix of the coated vesicles. This finding substantiates the idea that coated vesicles participate in the transport of newly synthesized cathepsin D into the lysosomes.     

Characteristic distribution of cathepsin E which immunologically cross-reacts with the 86-kDa acid proteinase from rat gastric mucosa

The antiserum raised against the high-molecular-weight acid proteinase from rat gastric mucosa, termed 86-kDa acid proteinase, has been shown to recognize rat cathepsin E, but not cathepsin D (Muto, N. et al. (1987) J. Biochem. 101, 1069-1075). Using this specific antiserum, characteristic distribution of cathepsin E in rats was demonstrated. The enzyme was detected in a limited number of tissues, such as stomach, thymus, spleen, bladder, and erythrocyte membranes. Among them, the highest activity was observed in the stomach. In contrast, cathepsin D immunoreactive with the antiserum specific to rat gastric cathepsin D was demonstrated in all the tissues examined. Cathepsin E-type enzymes partially purified from these five tissues were precipitated in the same manner by the specific antiserum, and they had the same molecular weight, electrophoretic mobility, and resistance against denaturation by 4 M urea. These results indicate that they could be exactly classified as cathepsin E. This type of enzyme was also detectable in mice and guinea pigs, but they showed relatively weak immunoreactivities with the antiserum. Thus, it is concluded that the distribution of cathepsin E is intrinsically different from ordinary cathepsin D, suggesting that it has a different physiological role from cathepsin D.     

Transformation of leukotriene D4 catalyzed by lysosomal cathepsin H of rat liver

Among several intracellular protease tested, cathepsin H transformed leukotriene D4 to E4 with a release of glycine in a stoichiometric quantity. Under the optimal conditions the rate of leukotriene D4 transformation by cathepsin H was about 3% of the hydrolysis rate of alpha-N-benzoyl-DL-arginine-2-naphthylamide which is commonly utilized as a very efficient substrate to test the peptidase activity of the enzyme. Leukotriene C4 was not transformed to leukotriene D4 by cathepsin H. Neither cathepsin B nor C was active with leukotrienes C4 and D4.     

Degradation of rod outer segment proteins by cathepsin D.

The degradation of proteins of the rod outer segment (ROS) fraction by partially purified cathepsin D [EC 3.4.23.5] from the retinal pigment epithelium was studied. The ROS fraction, prepared from bovine eyes by sucrose density gradient centrifugation, had little cathepsin D activity. Partially purified cathepsin D, obtained from crude extract of bovine retinal pigment epithelium using bovine serum albumin as a substrate, hydrolyzed the porteine of the ROS fraction. The rate of degradation of ROS proteins was proportional to both the enzyme concentration and the incubation time. With ROS proteins as substrate, the optimal pH of cathepsin D was about 3.5. The degradation of ROS proteins was inhibited by pepstatin.     

Comparative studies of two types of acid proteases from rat gastric mucosa and spleen

Two types of acid proteases, cathepsin D and cathepsin E-like enzyme, from rat gastric mucosa and spleen were compared in their biochemical and immunochemical properties. The enzymes were partially purified by employing the same chromatographic procedures and they showed a single proteolytically active band in polyacrylamide gel electrophoresis. Two low molecular weight enzymes, cathepsins D, from both tissues showed the same molecular weight and the same sensitivities to various inhibitors, but slightly different electrophoretic mobilities. The rabbit antiserum raised against gastric mucosa cathepsin D precipitated both enzymes. On the other hand, high molecular weight enzymes, gastric mucosa cathepsin D-like acid proteinase and spleen cathepsin E-like acid proteinase, were similar to each other as judged by their chromatographic profiles, electrophoretic mobilities, and high stabilities in urea solution. Furthermore, the antiserum specific to gastric mucosa cathepsin D-like acid proteinase inhibited both enzyme activities in a similar manner. However, the antiserum specific to one type of enzyme did not react with the other type. These results indicate that: gastric mucosa cathepsin D is immunologically identical with spleen cathepsin D; gastric mucosa cathepsin D-like acid proteinase has biochemical and immunological properties quite similar to spleen cathepsin E-like enzyme; these two types of acid proteases are quite different proteins existing in the individual tissues.