Degradation of myofibrillar proteins by cathepsins B and D

1. The procedure of Barrett [(1973) Biochem. J.131, 809-822] for isolating cathepsins B and D from human liver was modified for use with rat liver and skeletal muscle. The purified enzymes appeared to be similar to those reported in other species. 2. Sephadex G-75 chromatography of concentrated muscle extract resolved two peaks of cathepsin B inhibitory activity, corresponding to molecular weights of 12500 and 62000. 3. The degradation of purified myofibrillar proteins by cathepsins B and D was clearly demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After incubation with enzyme, the polypeptide bands representing the substrates decreased in intensity and lower molecular weight products appeared. 4. Cathepsins B and D, purified from either rat liver or skeletal muscle, were shown to degrade myosin, purified from either rabbit or rat muscle. Soluble denatured myosin was degraded more extensively than insoluble native myosin. Degradation by cathepsin B was inhibited by lack of reducing agent, or by myoglobin, iodoacetic acid and leupeptin, but not by pepstatin. The same potential modifiers were applied to cathepsin D, and only pepstatin produced inhibition. 5. Rat liver cathepsin B had a pH optimum of 5.2 on native rabbit myosin. The pH optimum of cathepsin D was 4.0, with a shoulder of activity about 1pH unit above the optimum. 6. Rat liver cathepsins B and D were demonstrated to degrade rabbit F-actin at pH5.0, and were inhibited by leupeptin and pepstain, respectively. 7. The degradation of myosin and actin by cathepsin D was more extensive than that by cathepsin B.     

Angiostatin generation by cathepsin D secreted by human prostate carcinoma cells

Angiostatin, a potent endogenous inhibitor of angiogenesis, is generated by cancer-mediated proteolysis of plasminogen. The culture medium of human prostate carcinoma cells, when incubated with plasminogen at a variety of pH values, generated angiostatic peptides and miniplasminogen. The enzyme(s) responsible for this reaction was purified and identified as procathepsin D. The purified procathepsin D, as well as cathepsin D, generated two angiostatic peptides having the same NH(2)-terminal amino acid sequences and comprising kringles 1-4 of plasminogen in the pH range of 3.0-6.8, most strongly at pH 4.0 in vitro. This reaction required the concomitant conversion of procathepsin D to catalytically active pseudocathepsin D. The conversion of pseudocathepsin D to the mature cathepsin D was not observed by the prolonged incubation. The affinity-purified angiostatic peptides inhibited angiogenesis both in vitro and in vivo. Importantly, procathepsin D secreted by human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that by human prostate carcinoma cells. Since deglycosylated procathepsin D from both prostate and breast carcinoma cells exhibited a similar low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in carbohydrate structures of procathepsin D molecules between the two cell types. The seminal vesicle fluid from patients with prostate carcinoma contained the mature cathepsin D and procathepsin D, but not pseudocathepsin D, suggesting that pseudocathepsin D is not a normal intermediate of procathepsin D processing in vivo. The present study provides evidence for the first time that cathepsin D secreted by human prostate carcinoma cells is responsible for angiostatin generation, thereby causing the prevention of tumor growth and angiogenesis-dependent growth of metastases.     

Human prostatic gastricsinogen: The precursor of seminal fluid acid proteinase

A gastricsinogen-like acid proteinase precursor has been purified by DEAE-cellulose, DEAE-Sephadex A-50, poly-l-lysine-Sepharose 4B, and N-acetyl-l-phenylalanyl-l-tyrosine-Sepharose 4B affinity chromatography from human prostates. The active enzyme hydrolyzes acid-denatured hemoglobin at pH 1.0 and 3.0, while two other active fractions only showed the pH 3.0 activity and resembled cathepsin D (EC 3.4.23.5). The pH optimum, milk-clotting activity, specificity toward synthetic substrates, inhibition by pepstatin, and molecular weight strongly suggest that the prostatic-derived enzyme is identical to seminal fluid and to gastric juice gastricsin.     

Cathepsin D from Atlantic cod (Gadus morhua L.) liver. Isolation and comparative studies

The isolated cathepsin D-like enzyme from Atlantic cod (Gadus morhua L.) liver was shown to be a monomer with a molecular mass of approximately 40 kDa. It was inhibited by Pepstatin A and had an optimum for degradation of haemoglobin at pH 3.0. The purified enzyme had lower temperature stability than bovine cathepsin D. Antibodies raised against the purified enzyme and against two C-terminal peptides of cod cathepsin D recognized a 40 kDa protein in immunoblotting of the samples from the purification process. Both antisera showed cross reactivity with a similar sized protein in liver from cod, saithe (Pollachius virens L.), Atlantic herring (Clupea harengus L.) and Atlantic salmon (Salmo salar L.). A protein of same size was detected in wolffish (Anarhichas lupus L.) liver with the antibody directed against the purified enzyme. This antibody also recognized the native enzyme and detected the presence of cathepsin D in muscle of cod, saithe, herring and salmon. These antibodies may be useful in understanding the mechanisms of post mortem muscle degradation in fish by comparing immunohistochemical localization and enzyme activity, in particular in cod with different rate of muscle degradation. They may also be used for comparing muscle degradation in different fish species.     

A comparison of the substrate specificities of cathepsin D and pseudorenin

Cathepsin D, purified from hog spleen, releases angiotensin I from tetradecapeptide renin substrate and from protein renin substrates purified from hog and human plasma. However, the enzyme does not act on the naturally occurring renin substrate as it exists in plasma nor on purified substrate in the presence of plasma. Cathepsin D releases angiotensin I quantitatively from tetradecapeptide renin substrate and does not further degrade the angiotensin I on prolonged incubation. The pH optimum for cathepsin D prolonged incubation. The pH optimum for cathepsin D acting on tetradecapeptide renin substrate is 4.5, and there is very low activity above pH 7. These properties are very similar to those of pseudorenin, an angiotensin-forming enzyme originally isolated from human kidney, indicating that cathepsin D and pseudorenin may be identical.     

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.     

Purification and characterization of a lysosomal aspartic protease with cathepsin D activity from the mosquito

A lysosomal aspartic protease with cathepsin D activity, from the mosquito, Aedes aegypti, was purified and characterized. Its isolation involved ammonium sulfate (30–50%) and acid (pH 2.5) precipitations of protein extracts from whole previtellogenic mosquitoes followed by cation exchange chromatography. Purity of the enzyme was monitored by SDS-PAGE and silver staining of the gels. The native molecular weight of the purified enzyme as determined by polyacrylamide gel electrophoresis under nondenaturing conditions was 80,000. SDS-PAGE resolved the enzyme into a single polypeptide with M_r = 40,000 suggesting that it exists as a homodimer in its non-denatured state. The pI of the purified enzyme was 5.4 as determined by isoelectric focusing gel electrophoresis. The purified enzyme exhibits properties characteristic of cathepsin D. It utilizes hemoglobin as a substrate and its activity is completely inhibited by pepstatin-A and 6M urea but not by 10 mM KCN. Optimal activity of the purified mosquito aspartic protease was obtained at pH 3.0 and 45°C. With hemoglobin as a substrate the enzyme had an apparent K_m of 4.2 μ M. Polyclonal antibodies to the purified enzyme were raised in rabbits. The specificity of the antibodies to the enzyme was verified by immunoblot analysis of crude mosquito extracts and the enzyme separated by both non-denaturing and SDS-PAGE. Density gradient centrifugation of organelles followed by enzymatic and immunoblot analyses demonstrated the lysosomal nature of the purified enzyme. The N-terminal amino acid sequence of the purified mosquito lysosomal protease (19 amino acids) has 74% identity with N-terminal amino acid sequence of porcine and human cathepsins D.     

Purification and properties of cathepsin D from porcine spleen.

Cathepsin D was purified from porcine spleen to near homogeneity as determined by gel electrophoresis. The isolation scheme involved an acid precipitation of tissue extract, DEAE-cellulose and Sephadex G-200 chromatography, and isoelectric focusing. The end product represented about a 1000-fold purification and about a 10% recovery. The purified enzyme was the major isoenzyme, which represented 60% of cathepsin D present in porcine spleen. Two minor isoenzymes of cathepsin D were present in small amounts. The purified enzyme resembled porcine pepsin in molecular weight (35,000), amino acid composition, and inactivation by specific pepsin inactivators. The pH activity curve of the purified enzyme showed two optima near pH 3 and 4. The relative activities at these optimal pH values were affected by salt concentration. Experimental evidence indicated that the two-optima phenomenon is a property of a single enzyme species.     

Vitellogenesis-related ovary cathepsin D from Xenopus laevis: Purification and properties in comparison with liver cathepsin D

Cathepsin D was purified from ovaries of Xenopus laevis by both QAE-cellulose and pepstatin-Sepharose chromatography and then characterized and compared with Xenopus liver cathepsin D. Ovary cathepsin D appeared predominantly as a 43-kilodalton (kDa) molecular mass, as revealed by SDS-polyacrylamide gel electrophoresis, whereas the liver enzyme was obtained exclusively as a 36-kDa protein. The purified 43-kDa ovary enzyme cleaved vitellogenin limitedly to produce yolk proteins at pH 5.6. The specific activity of ovary cathepsin D was five to six times lower than that of the liver enzyme, as measured by hemoglobin-hydrolysis at pH 3, but the ovary enzyme was shown to be superior to the liver enzyme in terms of vitellogenin-cleaving activity, as examined at pH 5.6. Ovarian enzyme preparations contained variable amounts of 36-kDa species; this form was considered to be an autolytic product of the 43-kDa form arising during purification, because it was not detected in oocyte extracts but was generated by incubation of the purified 43-kDa enzyme alone in an acid solution. The conversion of the 43-kDa form by hepatic factors was accompanied by a marked increase in hemoglobin-hydrolytic activity.     

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.     

Characterization of an analog enzyme to cathepsin L produced by tumor cells

The activity of cathepsin L is examined in the culture supernatants of 38 human, murine and hamster tumor cell lines. It is found that all cell lines secrete the enzyme possessing cathepsin L activity. The supernatant of HPC-YP cell cultures is purified and characterized as the enzyme preparation, because this supernatant shows the highest cathepsin L activity. The results indicate that the enzyme produced in HPC-YP cells is different from cathepsin L of normal liver in the several points. The molecular weight of the enzyme is 68 kd, whereas it is 34 kd for the liver cathepsin L. The enzyme is more stable to heat treatment and at the various pH than the liver cathepsin L. Furthermore, the inhibitors, which inhibit the liver cathepsin L activity, do not inhibit the activity of this enzyme. It is concluded that the enzyme showing cathepsin L activity in the culture supernatants of human tumor cells is different from human normal liver cathepsin L.     

Isolation and some properties of an acid protease from Fasciola hepatica.

A new protease, detected in an extract of Fasciola hepatica, was isolated and partly purified. The pH optimum for the cleavage of denaturated haemoglobin by the enzyme is pH 3.0. This proteolytic activity is inhibited by diazoacetylnorleucine methyl ester, pepstatin, the pepsin inhibitor from Ascaris suum, and phenylalanine. The cathepsin D inhibitor from potatoes, EDTA, mercaptoethanol and the inorganic salts tested have no inhibitory effect. The cleavage of the B-chain of oxidized insulin by enzyme was studied and compared with the digestion of the same substrate by chicken and pig pepsin. The protease from Fasciola hepatica belongs to the carboxyl group of proteases and probably plays an important role in helminth nutrition.     

Cathepsin D. Purification of isoenzymes from human and chicken liver

1. The Barrett (1967) assay for cathepsin D was slightly modified. 2. The enzyme was purified from liver of man and chicken by a procedure involving autolysis, acetone fractionation, ion-exchange chromatography and isoelectric focusing. 3. Several isoenzymes of cathepsin D were resolved in the isoelectric-focusing step, and three major forms, alpha,beta and gamma, were distinguished for each species. 4. A modified analytical method of isoelectric focusing in polyacrylamide gel indicated a high degree of homogeneity of the purified beta and gamma isoenzymes from each species, and this was supported by their constant high specific activities. 5. Gel filtration of the isoenzymes in a calibrated column of Sephadex G-100 showed that each had a molecular weight of 45000. 6. Human cathepsin D had a pH optimum of 3.5, and that of chicken enzyme was 3.0, haemoglobin being used as substrate. In each species, the three isoenzymes have the same pH-dependence curve. 7. The purified cathepsin D samples showed very little action on acid-denatured albumin.     

Unique cathepsin D-type proteases in rat thoracic duct lymphocytes and in rat lymphoid tissues.

Two unique cathepsin D-type proteases apparently present only in rat thoracic duct lymphocytes and in rat lymphoid tissues are described. One, termed H enzyme, has an apparent molecular weight of similar to95,000; the other, termed L enzyme, has an apparent molecular weight of similar to45,000, in common with that of most cathepsins D from other tissues and species. Both enzymes differ from cathepsin D, however, by a considerably greater sensitivity to inhibition by pepstatin and by a smaller degree of inhibition by an antiserum which inhibits rat liver cathepsin D. H enzyme is converted to L enzyme by treatment with beta-mercaptoethanol; the relationship between the two enzymes remains unknown. H and L enzyme have been detected in rat lymphoid tissues and in mouse spleen, but they are not present in other rat tissues (liver, kidney, adrenals), rabbit tissues, calf thymus, bovine spleen, or human tonsils. As measured on acid-denatured bovine hemoglobin as substrate, both enzymes have pH activity curves identical with that of rat liver cathepsin D, with optimal activity at pH 3.6. Activity on human serum albumin is much less and also shows an optimum at pH 3.6; hence, neither enzyme has the properties of cathepsin E. Thiol-reactive inhibitiors have no effect on the activity of H and L enzyme; thus they do not belong to the B group of cathepsins. Additional information, discussed in this paper, leads us to conclude that partially purified H and L enzymes are cathepsin D-type proteases.     

The pH dependence of breakdown of various purified brain proteins by cathepsin D preparations

In a continuing study of control processes of cerebral protein catabolism we compared the activity of cathepsin D from three sources (rat brain, bovine brain, and bovine spleen) on purified CNS proteins (tubulin, actin, calmodulin, S-100 and glial fibrillary acidic protein). The pH optimum was 5 for hydrolysis with tubulin as substrate for all three enzyme preparations, and it was pH 4 with the other substrates. The pH dependence curve was somewhat variable, with S-100 breakdown relatively more active at an acidic pH range. The formation of initial breakdown products and the further catabolism of the breakdown products was dependent on pH; hence the pattern of peptides formed from glial fibrillary acidic protein was different in incubations at different pH's. The relative activity of the enzyme preparations differed, depending on the substrate: with tubulin and S-100 as substrates, rat brain cathepsin D was the most active and the bovine spleen enzyme was the least active. With calmodulin and glial fibrillary acidic protein as substrates, rat brain and spleen cathepsin D activities were similar, and bovine brain cathepsin D showed the lowest activity. Actin breakdown fell between these two patterns.The rates of breakdown of the substrates were different; expressed as μg of substrate split per unit enzyme per h, with rat brain cathepsin D activity was 8–9 with calmodulin and S-100, 4 with glial fibrillary acidic protein, 1.8 with actin, and 0.9 with tubulin. The results show that there are differences in the properties of a protease like cathepsin D, depending on its source; furthermore, the rate of breakdown and the characteristics of breakdown are also dependent on the substrate.We recently measured the breakdown of brain tubulin by cerebral cathepsin D in a continuing study of the mechanisms and controls of cerebral protein catabolism (Bracco et al., 1982a). We found that tubulin breakdown is heterogeneous, that membrane-bound tubulin is resistant to cathepsin D but susceptible to thrombin (Bracco et al., 1982b), and that cytoplasmic tubulin was in at least two pools, one with a higher, another with a lower, rate of breakdown. The pH optimum of tubulin breakdown by cerebral cathepsin D differed significantly from the pH optimum of hemoglobin breakdown by the same enzyme.These findings showed that the properties of breakdown by a cerebral protease depend on the substrate. To further examine this dependence of properties of breakdown on the substrate, we now report measurements of pH dependence of breakdown of several purified proteins (tubulin, actin, calmodulin, S-100, glial fibrillary acidic protein [GFA]) from brain by cathepsin D preparations from three sources, rat brain, bovine brain, and bovine spleen. We also compare the rate of breakdown of the various proteins with the rate of hemoglobin breakdown.     

Breakdown of brain tubulin by cerebral cathepsin D

The breakdown of cytoplasmic tubulin from brain (purified by ammonium sulfate fractionation and DEAE cellulose chromatography) by cathepsin D from brain (purified by ammonium sulfate fractionation and pepstatin Sepharose chromatography) was studied; changes in the intensity of tubulin gel bands were determined. The pH optimum of hemoglobin breakdown by cathepsin D was 3.2; the pH optimum for tubulin breakdown was 5.8; at pH 5.8 there was no significant hemoglobin breakdown by the enzyme. Tubulin breakdown had an apparent K_m of 1.8 × 10⁻⁵ M and a V_max of 0.56 μg tubulin (μg enzyme per min). The rate of breakdown was heterogeneous and studied on length of incubation; the major portion of tubulin was rapidly broken down and a smaller portion was more stable. The rate under our experimental conditions was 18%/h in the 1–4 h period and 2%/h after 4 h. This was not due to enzyme instability: after 4 h of inhibition freshly added tubulin was rapidly broken down, whereas freshly added enzyme did not increase the rate of breakdown. Thus breakdown heterogeneity was due to substrate (tubulin) heterogeneity. Pepstatin inhibited cathepsin D breakdown of tubulin at acid pH; at pH 7.6 it had no effect. Leupeptin was not inhibitory. We calculated that the cathepsin D content in brain, if fully active, could break down cytoplasmic tubulin with a half-life of 24 h, but it is likely that under in vivo conditions enzyme activity is greatly modified.     

Purification and characterization of aspartic protease derived from Sf9 insect cells

An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS-PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K(m) of 0.85 μM. The k(cat) and k(cat)/K(m) values were 13 s(-1) and 15 s(-1) μM(-1) respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K(i), of 25 pM.     

Cathepsin B1. A lysosomal enzyme that degrades native collagen

1. Experiments were made to determine whether the purified lysosomal proteinases, cathepsins B1 and D, degrade acid-soluble collagen in solution, reconstituted collagen fibrils, insoluble collagen or gelatin. 2. At acid pH values cathepsin B1 released (14)C-labelled peptides from collagen fibrils reconstituted at neutral pH from soluble collagen. The purified enzyme required activation by cysteine and EDTA and was inhibited by 4-chloromercuribenzoate, by the chloromethyl ketones derived from tosyl-lysine and acetyltetra-alanine and by human alpha(2)-macroglobulin. 3. Cathepsin B1 degraded collagen in solution, the pH optimum being pH4.5-5.0. The initial action was cleavage of the non-helical region containing the cross-link; this was seen as a decrease in viscosity with no change in optical rotation. The enzyme also attacked the helical region of collagen by a mechanism different from that of mammalian neutral collagenase. No discrete intermediate products of a specific size were observed in segment-long-spacing crystalloids (measured as native collagen molecules aligned with N-termini together along the long axis) or as separate peaks on gel filtration chromatography. This suggests that once an alpha-chain was attacked it was rapidly degraded to low-molecular-weight peptides. 4. Cathepsin B1 degraded insoluble collagen with a pH optimum below 4; this value is lower than that found for the soluble substrate, and a possible explanation is given. 5. The lysosomal carboxyl proteinase, cathepsin D, had no action on collagen or gelatin at pH3.0. Neither cathepsin B1 nor D cleaved Pz-Pro-Leu-Gly-Pro-d-Arg. 6. Cathepsin B1 activity was shown to be essential for the degradation of collagen by lysosomal extracts. 7. Cathepsin B1 may provide an alternative route for collagen breakdown in physiological and pathological situations.     

A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Subcellular localization of the protease in porcine skeletal muscle

A study was done to determine whether the Ca2+-activated muscle protease (CAF) that removes Z disks from myofibrils in the presence of Ca2+ is located in a sedimentable subcellular organelle. Porcine skeletal muscle cells were diced finely with a scalpel and were suspended in 0.25 M sucrose, 4 mM EDTA with a VIRTIS homogenizer. Filtration of the suspended muscle through four layers of cheesecloth removed most of the myofibrils and stromal protein. Nuclear (1,000 gavg for 15 min), mitochondrial-microsomal (50,000 gavg for 60 min), and supernatant fractions were assayed for succinic dehydrogenase, acid ribonuclease, cathepsin D, and CAF activities. Approximately 96% of total succinic dehydrogenase activity, 81% of cathepsin D activity, and 45% of acid ribonuclease activity, but only 14% of total CAF activity, were found in the nuclear and mitochondrial-microsomal fractions. Cathepsin D activity in the nuclear and mitochondrial-microsomal fractions was decreased if assays were done without prior treatment to rupture membranous structures; hence, our cell rupture and homogenization procedures preserved some intact lysosomal organelles. The results indicate that the small amount of CAF activity in the nuclear and mitochondrial-microsomal fractions was due to contamination by supernate and that CAF is not located in a membrane-bounded subcellular particle. Because CAF is active at the intracellular pH and temperature of living skeletal muscle cells and is in direct contact with the cytoplasm of muscle cells, its activity must be regulated by intracellular cellular Ca2+ concentration to prevent continuous and indiscriminate degradation of myofibrils.