Purification and tissue distribution of rat cathepsin L

Cathepsin L was purified to apparent homogeneity from rat kidney. The molecular weight of the enzyme was estimated to be 30,000, but part of the enzyme was found to consist of two polypeptide chains of Mr 25,000 and 5,000. Antibody against rat kidney cathepsin L did not cross-react with rat cathepsin B or H and detected only cathepsin L in crude rat tissue preparations on immunoblotted sheets. The concentrations of cathepsin L in various rat tissues and peripheral blood cells of rats were determined by a sensitive immunoassay, in which the minimum detectable amount of cathepsin L was 20 pg/assay. The concentration of cathepsin L was found to be highest in the kidneys, where it was more than 3 times higher than in the liver, spleen, lungs, and brain. Nervous tissues, especially the cerebellar cortex, also contained fairly high concentrations of cathepsin L, but the heart, skeletal muscle, and gastrointestinal tract contained low concentrations, as did peripheral blood cells. The cathepsin L content of macrophages was 20% of that of cathepsin B. The concentrations of cathepsin L in lymphocytes, neutrophils, and erythrocytes were 10%, 20%, and less than 0.2%, respectively, of those in resident macrophages.     

Cysteine proteinase inhibitors from rabbit skeletal muscle

1. Two cysteine proteinase inhibitors, I-T (Mr = 29,000) and I-S (Mr = 10,700), were isolated from rabbit skeletal muscle by means of succesive extraction with a neutral buffer solution, precipitation at pH 3.7, acetone fractionation and gel permeation on Sephadex G-75. 2. I-T is a formed trimer of a monomeric inhibitor, I-M (Mr = 10,500), through disulfide bonds. 3. I-S is almost completely stable between pH 3 and 8, while I-M is unstable in the same pH range. 4. I-M acts most effectively towards cathepsins H and L, showing moderate activity towards cathepsin B and only weak activity towards papain. I-S acts most effectively towards cathepsin L, followed by, in decreasing order, cathepsin H, cathepsin B and papain.     

Evidence for a defective thiol protease inhibitor in skeletal muscle of mice with hereditary muscular dystrophy

The thiol protease inhibitor (TPI-d) from hind-limb skeletal muscle of dystrophic 60-day-old male mice (strain 129/ReJ/dy) has been purified to apparent homogeneity and compared with the thiol protease inhibitor (TPI-n) from hind-limb skeletal muscle of normal 60-day-old male littermates. While both TPI-d and TPI-n displayed identical properties on sodium dodecyl sulfate-polyacrylamide gels (14,800 relative mass), analytical isoelectric focusing gels (pI 4.5), and high performance liquid chromatography columns, TPI-d was unable to inhibit papain and cathepsin B after purification by isoelectric focusing. However, a component in the purified TPI-d preparation with an isoelectric point of 4.9 initially masked the functional state of TPI-d, using papain when assayed with the test proteases papain and cathepsins H and L. This inhibitory component was absent from TPI-n preparations. Pure TPI-d was also unable to inhibit in vitro myosin hydrolysis by cathepsin B, whereas TPI-n completely blocked cathepsin B catalyzed myosin hydrolysis. Given the central role of the thiol proteases, especially cathepsin B, in intracellular protein metabolism and the possibility that uncontrolled thiol protease activity in muscle leads to muscle protein breakdown and dystrophy, our data suggest that a modified (defective) thiol protease inhibitor (TPI-d) may be (one of) the end product(s) of the dystrophy gene in mice with the hereditary form of the disease.     

Studies on Proteolysis in Stored Muscle

The amounts of proteolytic products derived from each fraction of rabbit muscle proteins by purified cathepsin D from rabbit muscle, and the effects of cathepsin D and pepsin treatments on ATPase activity of myosin B were studied.

Water-soluble protein was most rapidly hydrolyzed, followed by myosin A, actin, and myosin B.

In comparison with the control, the decrease of the Mg-enhanced ATPase activity of myosin B treated with cathepsin D was observed, but no difference was observed in the Ca-enhanced ATPase activity of myosin B by this treatment.

The removal of native tropomyosin in myosin B was not recognized by cathepsin D treatment, which was different from trypsin treatment.

The effect of pepsin treatment on the Mg-enhanced ATPase activity of myosin B was fairly similar to that of cathepsin D treatment.     

Nucleo-cytoplasmic relationships of oestrogen receptors in rat liver during the oestrous cycle and in response to administered natural and synthetic oestrogen.

The mechanism of degradation of fructose-1,6-bisphosphate aldolase from rabbit muscle by the lysosomal proteinase cathepsin B was determined. Treatment of aldolase with cathepsin B destroys up to 90% of activity with fructose 1,6-bisphosphate as substrate, but activity with fructose 1-phosphate is slightly increased. Cathepsin L, another lysosomal thiol proteinase, and papain are also potent inactivators of aldolase, whereas inactivation is not caused by cathepsins D or H even at high concentrations, or by cathepsin B inhibited by leupeptin or iodoacetate. The cathepsin-B-treated aldolase shows no detectable change in subunit molecular weight, oligomer molecular weight or subunit interactions. Cathepsin B cleaves dipeptides from the C-terminus of th aldolase subunits. Four dipeptides are released sequentially: Ala-Tyr, Asn-His, Ile-Ser and Leu-Phe, and a maximum of five additional dipeptides may be released. There are indications that this peptidyldipeptidase activity of cathepsin B may be an important aspect of its action on protein substrates generally.     

A cathepsin B-like enzyme from mackerel white muscle is a precursor of cathepsin B

A cathepsin B-like enzyme from the white muscle of common mackerel Scomber japonicus was a cysteine protease that hydrolyzed Z-Arg-Arg-MCA, the substrate for cathepsin B. In a partial purified cathepsin B-like enzyme preparation at 4 degrees C left over time, a converted enzyme that hydrolyzes Z-Arg-Arg-MCA and Z-Phe-Arg-MCA appeared in the preparation. The converted enzyme was purified from the cathepsin B-like enzyme, characterized and was identified as mackerel cathepsin B. These results suggested that the mackerel cathepsin B-like enzyme was a precursor of cathepsin B. Mackerel cathepsin B formed in the purified cathepsin B-like enzyme preparation by adding of a small amount of the purified cathepsin B to the preparation. Therefore, mackerel cathepsin B-like enzyme was converted to the mature form of cathepsin B by autoactivation. The conversion of the cathepsin B-like enzyme (molecular mass 60 kDa) to cathepsin B (molecular mass 23 kDa) was detected by immunoblotting by using human anti-(cathepsin B) antibody. The intermediate forms of 40 kDa and 38 kDa were also detected during the conversion.     

Simultaneous isolation of human kidney cathepsins B, H, L and C and their characterisation

A procedure for the simultaneous isolation of four cysteine proteinases, cathepsins B, H, L and C, from human kidney is described. The method includes concentration of the acidified homogenate by ammonium sulphate precipitation. The resuspended and dialysed precipitate was chromatographed on DEAE-cellulose DE-32, to allow separation of cathepsins H and C from cathepsins B and L. The main isoform of cathepsin H was separated from cathepsin C by cation-exchange chromatography on CM-Sephadex C-50. These two enzymes were further purified by covalent chromatography on thiopropyl Sepharose and gel permeation on Sephacryl S-200. The last step allowed separation of cathepsin C and the minor isoform of cathepsin H. Purification of the other two enzymes, cathepsins B and L, was carried out on thiol Sepharose, followed by chromatography on CM-Sepharose C-50. In this step, pure cathepsin L was obtained, while two isoforms of cathepsin B had to be finally purified on Sephacryl S-200 columns. The purity of each enzyme was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, isoelectric focusing on polyacrylamide gels and N-terminal sequencing. The activities of the purified cathepsins B, H and L were determined in terms of k_cat/K_M for three substrates, Z-Phe-Arg-MCA, Z-Arg-Arg-MCA and Arg-MCA. The method produced 25 mg of cathepsin B, 6.5 mg of cathepsin H, 1.5 mg of cathepsin L and 3.8 mg of cathepsin C from 3.5 kg of human kidney.     

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.     

Purification and characterization of cathepsin L from skeletal muscle of the lizard Agama stellio stellio

Cathepsin L from skeletal muscle of the lizard Agama stellio stellio was purified to homogeneity by ion-exchange and gel-permeation chromatography. The molecular weight of the cathepsin L is estimated to be 34 kD, and its isoelectric point is 5.5. The cathepsin L has a pH optimum of 6.1, requires a thiol-reducing reagent for activation, and is inhibited by cysteine protease inhibitors. The Km and kcat values for Z-Phe-Arg-MCA as substrate are 1.4 microM and 6.2 sec-1, respectively. This enzyme readily hydrolyzes proteins such as insulin B chain, hemoglobin, and serum albumin.     

Down-regulation of genes in the lysosomal and ubiquitin-proteasome proteolytic pathways in calpain-3-deficient muscle

Calpain-3 deficiency leads to muscular dystrophy in humans and mice and to perturbation of the NFkappaB/IkappaB pathway. As this phenotype is mainly atrophic, this study was performed to determine whether protein turnover and/or proteolytic gene expression was altered in muscles following calpain-3 deficiency. In vitro rates of protein turnover and of substrate ubiquitination, cathepsin B and B+L activities, and mRNA levels for several proteolytic genes were measured in skeletal muscles from 4-5 month-old control and calpain-3 knockout mice. Rates of protein synthesis and breakdown, cathepsin activities, and rates of substrate ubiquitination remained stable in muscles from calpain-3 deficient mice. However, and surprisingly, mRNA levels for cathepsin L, the 14-kDa ubiquitin-conjugating enzyme E2, and the C2 subunit of the 20S proteasome decreased by approximately 47% (P<0.005) in the gastrocnemius muscle from calpain-3 deficient mice. In contrast, muscle mRNA levels for ubiquitin and subunit S5a of the 26S proteasome were unaffected by calpain-3 deficiency. Taken together these data demonstrate that the expression of some genes that are involved in distinct proteolytic pathways is selectively and coordinately down-regulated without any effect on proteolysis. This suggests new pathophysiological hypotheses, e.g. a lack of maturation of NFkappaB precursor and/or a defect in specific substrate targeting.     

Cathepsin B Is Inhibited in Mutant Cells Selected during Persistent Reovirus Infection

Persistent reovirus infections of murine L929 (L) fibroblast cells select mutant (LX) cells that do not support proteolytic disassembly of reovirus virions within the endocytic pathway. To better understand the function and regulation of endocytic proteases, we conducted experiments to define the block to reovirus disassembly displayed by LX cells. In contrast to parental L cells, mutant LX cells harbor defects that interfere with the maturation and activity of cathepsin B and cathepsin L but not cathepsin H. The cDNAs encoding cathepsin B and cathepsin L in L cells are identical to those in LX cells, indicating that LX cells manifest an extrinsic block to the function of these enzymes. Mixed lysates of L cells and LX cells lack activity of both cathepsin B and cathepsin L, suggesting the presence of an inhibitor of cathepsin function in LX cells. A cathepsin B-green fluorescent protein (GFP) fusion protein expressed in L cells and purified by immunoprecipitation retains cathepsin B activity, whereas cathepsin B-GFP expressed in LX cells does not. However, activity of cathepsin B-GFP expressed in LX cells can be recovered by incubating the immunoprecipitate with L cell lysate followed by immunoprecipitation, providing further evidence that LX cells express a cathepsin inhibitor. Native-gel electrophoresis and gel filtration chromatography demonstrate that, in both cell lines, the double-chain form of cathepsin B is sequestered in a large molecular weight complex that renders this form of the enzyme inactive. Alteration of this sequestration complex appears to be responsible for inhibition of cathepsin B in LX cells. These findings suggest that cathepsins can be regulated within the endocytic pathway. Moreover, this regulation influences host cell susceptibility to intracellular pathogens.     

Selective cleavage of peptide bonds by cathepsins L and B from rat liver

The selective cleavage of peptide bonds by cathepsin L from rat liver was examined with a hexapeptide, luteinizing hormone releasing hormone, neurotensin and oxidized insulin A chain as model substrates. The specificity of cathepsin L was compared with that of cathepsin B. Cathepsin L cleaved peptide bonds that have a hydrophobic amino acid, such as Phe, Leu, Val, and Trp or Tyr, in position P2. A polar amino acid, such as Tyr, Ser, Gly, Glu, Asp, Gln, or Asn, in position P1. enhanced the susceptibility of the peptide bond to cathepsin L, though the importance of the amino acid residue in position P1' was not as great as that of the amino acid in position P2 for the action of cathepsin L. These results suggest that, in contrast to cathepsin B, cathepsin L shows very clear specificity.     

Matrigel® invasion by the prostate cancer cell lines,PC3 and DU145, and cathepsin L+B activity

Cathepsins L and B are lysosomal cysteine proteinases whose activities and cellular location are altered in many types of cancers and cancer cell lines. Cathepsins L and B play an unspecified role in cancer invasion and metastasis. The purpose of our study was to determine whether cathepsins L and B are important for the ability of two prostate cancer cell lines, PC3 and DU 145, to invade the basement membrane-like preparation, Matrigel®. Exposure of PC3 and DU145 to the irreversible cysteine proteinase inhibitor, E64, decreases the invasive ability of DU145, but not PC3. PC3 and DU145 were treated with the phorbol ester analogue, phorbol 12-myristate 13-acetate (PMA), a known tumor promoter that activates protein kinase C and contributes to the metastatic phenotype. PMA increased secreted cathepsin L+B activity and the invasive ability of PC3 and DU145; co-exposure to E64 and PMA decreased both cathepsin L+B activity and invasion. We conclude that DU145 requires cathepsin L+B activity more than PC3 for the invasion of the Matrigel®. When the amount of secreted cathepsin L+B activity is increased by PMA treatment, however, PC3 becomes dependent on cathepsin L+B for invasion. Our study demonstrates that modulation of the amount of secreted cathepsin L+B activity influences the invasive phenotype of PC3 and DU145.     

Shrimp cathepsin L encoded by an intronless gene has predominant expression in hepatopancreas, and occurs in the nucleus of oocyte

We have cloned the cDNA and genomic DNA of an active intronless cathepsin L from Metapenaeus ensis. The encoded enzyme has the shortest prosequence among cathepsin L subgroup. It was predominantly expressed in hepatopancreas with an expression level of at least 10 times higher than in any other tissues. It also has expression in stomach, intestine, eye, testis, ovary and muscle. Western blots visualized the mature enzyme in hepatopancreas and a procathepsin L in ovary, intestine and stomach. Metapenaeus cathepsin L (MeCatL) is localized in the large digestive vacuole of the digestive B cell of hepatopancreas. MeCatL has a role in food digestion. An interesting finding is that it exists in the nucleus of oocyte. MeCatL might have a specified physiological role in the nucleus of oocyte. MeCatL might also have a house-keeping function as is suggested for mammalian cathepsin L.     

ADP-ribosylation factor 1 protein regulates trypsinogen activation via organellar trafficking of procathepsin B protein and autophagic maturation in acute pancreatitis

Several studies have suggested that autophagy might play a deleterious role in acute pancreatitis via intra-acinar activation of digestive enzymes. The prototype for this phenomenon is cathepsin B-mediated trypsin generation. To determine the organellar basis of this process, we investigated the subcellular distribution of the cathepsin B precursor, procathepsin B. We found that procathepsin B is enriched in Golgi-containing microsomes, suggesting a role for the ADP-ribosylation (ARF)-dependent trafficking of cathepsin B. Indeed, caerulein treatment increased processing of procathepsin B, whereas a known ARF inhibitor brefeldin A (BFA) prevented this. Similar treatment did not affect processing of procathepsin L. BFA-mediated ARF1 inhibition resulted in reduced cathepsin B activity and consequently reduced trypsinogen activation. However, formation of light chain 3 (LC3-II) was not affected, suggesting that BFA did not prevent autophagy induction. Instead, sucrose density gradient centrifugation and electron microscopy showed that BFA arrested caerulein-induced autophagosomal maturation. Therefore, ARF1-dependent trafficking of procathepsin B and the maturation of autophagosomes results in cathepsin B-mediated trypsinogen activation induced by caerulein.     

Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane.

Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.     

Matrigel® invasion by the prostate cancer cell lines, PC3 and DU145, and cathepsin L+B activity

Cathepsins L and B are lysosomal cysteine proteinases whose activities and cellular location are altered in many types of cancers and cancer cell lines. Cathepsins L and B play an unspecified role in cancer invasion and metastasis. The purpose of our study was to determine whether cathepsins L and B are important for the ability of two prostate cancer cell lines, PC3 and DU 145, to invade the basement membrane-like preparation, Matrigel®. Exposure of PC3 and DU145 to the irreversible cysteine proteinase inhibitor, E64, decreases the invasive ability of DU145, but not PC3. PC3 and DU145 were treated with the phorbol ester analogue, phorbol 12-myristate 13-acetate (PMA), a known tumor promoter that activates protein kinase C and contributes to the metastatic phenotype. PMA increased secreted cathepsin L+B activity and the invasive ability of PC3 and DU145; co-exposure to E64 and PMA decreased both cathepsin L+B activity and invasion. We conclude that DU145 requires cathepsin L+B activity more than PC3 for the invasion of the Matrigel®. When the amount of secreted cathepsin L+B activity is increased by PMA treatment, however, PC3 becomes dependent on cathepsin L+B for invasion. Our study demonstrates that modulation of the amount of secreted cathepsin L+B activity influences the invasive phenotype of PC3 and DU145.     

Sequence analysis, tissue distribution, and expression of rat cathepsin S.

Cysteine proteases are involved in many diverse cellular processes ranging from processing of precursor proteins to intracellular degradation. In an effort to identify novel cysteine proteases, we used the polymerase chain reaction and primers directed against the catalytic sites of previously cloned cysteine proteases. From rat brain mRNA, a 600-base pair band was amplified; cloning and partial sequence analysis of this band resulted in the identification of cathepsins B and L and five novel sequences. The novel cDNAs contained a number of residues conserved in lysosomal cysteine proteases, including the active site residue His159 (papain numbering). In addition, the amino acid homology between the novel sequences and either cathepsins B, L, or H, ranged from 63 to 32%. The insert with highest homology was used to screen a rat brain cDNA library; a 1334-base pair cDNA was isolated and the nucleotide sequence determined. This sequence encodes an open reading frame of 330 amino acids which is 82% homologous to human cathepsin S, suggesting that this sequence represents rat cathepsin S. Northern blot analysis for rat cathepsin S revealed tissue-specific expression distinct from the distribution of cathepsin B and L. The regulation of expression of rat cathepsin S mRNA in response to thyroid-stimulating hormone was studied in a rat thyroid cell line FRTL-5. The level of cathepsin S mRNA was substantially increased in response to thyroid-stimulating hormone, whereas cathepsin B and cathepsin L mRNA levels were not altered by this treatment. A portion of cDNA encoding the predicted mature protein of rat cathepsin S was expressed as a glutathione S-transferase-fusion protein. The affinity-purified protein exhibited proteolytic activity with properties similar to bovine cathepsin S. Taken together, these results imply highly specific functions for cathepsin S.