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PubMed1978

Characterisation of cathepsin B and collagenolytic cathepsin from human placenta.

1. Human placental cathepsin B and collagenolytic cathepsin were separated by chromatography on columns of Amberlite CG-50. Collagenolytic cathepsin was partially purified by chromatography on DEAE-Sephadex (A-50) and Sephadex G-100. Cathepsin B was purified by chromatography on CM-cellulose and Sephadex G-100. 2. Both enzymes required activation by thiol compounds and were bound to organomercurial-Sepharose-4B. Sulphydryl-blocking reagents were inhibitory, which confirmed an essential thiol group to be present. 3. The enzymes degraded soluble calf skin collagen and insoluble bovine tendon collagen in the telopeptide region at pH 3.5 and 28 degrees C to yield mainly alpha-chain components. 4. In contrast to cathepsin B, collagenolytic cathepsin was found not to hydrolyse any of the low-molecular-weight synthetic substrates that were tested. 5. Leupeptin, a structural analogue of arginine-containing synthetic substrates, and antipain, an inhibitor of papain, were strongly inhibitory to both enzymes. 6. The isoelectric points of the enzymes were similar, being 5.4 for cathepsin B and 5.1 for collagenolytic cathepsin. 7. From chromatography on Sephadex G-100 the molecular weight of cathepsin B was calculated to be 24 500 and that of collagenolytic cathepsin to be 34 600.

Cathepsins↗
PubMed2002

Cathepsin-L, a key molecule in the pathogenesis of drug-induced and I-cell disease-mediated gingival overgrowth: a study with cathepsin-L-deficient mice.

Drug-induced gingival overgrowth, the chronic side effect of calcium antagonists, is frequently seen due to the increase in patients with hypertension, although the etiology of the disease is largely unknown. I-cell disease, which accompanies gingival overgrowth, is characterized by a deficiency in UDP-N-acetyl-glucosamine and is classified as one of the lysosomal storage diseases. Here, we hypothesized that a common mechanism may underlie the etiology of gingival overgrowth seen in patients treated with calcium antagonist and in patients with I-cell disease. A calcium antagonist, nifedipine, specifically suppressed cathepsin-L activity and mRNA expression, but not that of cathepsin-B in cultured gingival fibroblasts. The activity of cathepsin-L was suppressed up to 50% at 24 hours after treatment of the cells with the reagent. The selective suppression of cathepsin-L activity appeared not to be dependent on Ca(2+), since treatment of the cells with thapsigargin suppressed both cathepsin-B and -L activity. Mice deficient in the cathepsin-L gene manifested enlarged gingivae. Histological observation of the gingivae demonstrated typical features of acanthosis, a phenotype very similar to that of experimentally induced gingival overgrowth. Since cathepsin-L deficiency was reported to be associated with thickening of the skin, impaired cathepsin-L activity may play a key role in the establishment of skin and gingival abnormalities seen in I-cell disease. In addition, reduced cathepsin-L activity may play an important role in inducing drug-induced gingival overgrowth.

Animals↗
PubMed1980

Inhibitions by E-64 derivatives of rat liver cathepsin B and cathepsin L in vitro and in vivo.

The mechanism of inhibition of cathepsin B [EC 3.4.22.1] and cathepsin L [EC 3.4.22.-] by E-64 was investigated. Kinetic studies indicated that E-64 was an irreversible inhibitor of these enzymes. [3H]E-64 is incorporated into cathepsin B in a one/one molar ratio in parallel with inactivation of the enzyme. Titration of one of the 10 SH groups of native cathepsin B with 2,2'-dithiodipyridine resulted in complete loss of enzyme activity. Decrease of titratable SH groups and activity of cathepsin B was proportional to the concentration of E-64 added, indicating that E-64 binds to an equimolar amount of active SH residues of cathepsin B. The effects of E-64 and its derivatives on lysosomal cathepsin B and cathepsin L in rat liver were studied in vitro and in vivo. The D form of E-64 inhibited the cathepsin both in vitro and in vivo, although its inhibitory effects were less than those of E-64-(L). E-64-b(RR), in which the terminal agmatine of E-64 is replaced by leucine, was as active as E-64-(L) in vitro, but was completely inactive in vivo. Among the E-64 derivatives tested, E-64-c(SS), in which the terminal agmatine of E-64 is replaced by isoarylamide, showed strong inhibitory activity in vivo, like E-64-(L).

Agmatine↗
PubMed1985

Characteristics of activation of cathepsin B by sodium salicylate and comparison of catalytic site properties of cathepsins B and H.

Attempts to gain greater understanding of characteristics of catalytic site properties of two homologous lysosomal cysteine proteinases, cathepsins B and H, were made by using sodium salicylate (SA) and several specific inhibitors, which extends the studies on the structure-activity relationship and kinetics for the activation of cathepsin B by SA in the previous paper (Yamamoto, K., Takeda, M. and Kato, Y.: Japan. J. Pharmacol. 38, 215-218, 1985). The half-maximal activation of cathepsin B by SA was observed at around a molar ratio of 10(4):1 (SA/cathepsin B). No preincubation time was needed for the SA-stimulated reaction, but the rate of activation was more rapid as pH values in the preincubation mixture decreased. The extent of inactivation of cathepsin B by leupeptin and E-64 significantly decreased in the presence of SA. Catalytic site properties of cathepsins B and H were also distinguished by differences in the extent of inhibition by cysteine proteinase inhibitors. Cathepsin B was more sensitive than cathepsin H to inhibition by antipain, chymostatin, iodoacetic acid and mercuric chloride as well as leupeptin and E-64. Despite the similarity in inhibitory effects of iodoacetic acid and E-64, the rat spleen cathepsin H was characterized by insensitivity to mercuric chloride that had a considerable inhibitory effect on the corresponding enzyme from rat liver and the rat spleen cathepsin B.

Animals↗
PubMedDate not supplied

Expression, proliferation activity and clinical significance of cathepsin B and cathepsin L in operated lung cancer.

AIMS: To immunohistochemically investigate the expression of cathepsin B and cathepsin L in various lung cancer cell types in association with the patients' prognosis. MATERIALS AND METHODS: Histological slides obtained from formalin-fixed, paraffin-embedded tissue of 120 potentially curative resected lung cancer specimens (20 cases of each of the following cell types: squamous cell, adeno, large cell anaplastic, small cell anaplastic, intrapulmonary metastases, mesotheliomas) were quantitatively immunohistochemically analysed with an automated image analysing system and correlated with the patients' prognosis and the tumour proliferation rate measured by the expression of Ki-67. RESULTS: The expression of cathepsin B was most frequently present in large cell anaplastic carcinomas and missing in small cell carcinomas. That of cathepsin L was less frequently seen, and mainly expressed in macrophages and adenocarcinoma tumour cells. The structural heterogeneity of cathepsin B expression measured by syntactic structure analysis was greater than that of cathepsin L. The expression of cathepsin B is of prognostic significance in non-small cell lung cancer, and ranges directly after the pN and pT stages in multivariate statistical analysis. CONCLUSION: In concordance with the literature, the expression of cathepsin B in non-small cell lung cancer should be considered as a significant prognostic parameter in contrast to that of cathepsin L, which is not related to the patients' outcome at a statistically significant level.

Carcinoma, Non-Small-Cell Lung↗
PubMed1985

Purification and properties of rabbit liver cathepsin M and cathepsin B.

Cathepsins M and B from rabbit liver lysosomes were separated by chromatography on Ultrogel AcA34 at low ionic strength and purified to homogeneity, and their catalytic and molecular properties were compared. Cathepsin M was relatively inactive with synthetic peptide substrates. Thus, it hydrolyzed benzoyl arginine naphthylamide at only one-fifth the rate observed with cathepsin B, and no activity was detected with Gly-Phe naphthylamide which is a relatively good substrate for cathepsin B. On the other hand, cathepsin M exhibited a preference for protein substrates. It was more active than cathepsin B in catalyzing the inactivation of the following enzymes: rabbit muscle or liver fructose-1,6-bisphosphate aldolases, rabbit liver fructose-1,6-bisphosphatase and pyruvate kinase, yeast glucose-6-phosphate dehydrogenase, and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. With glucagon as substrate, both enzymes showed similar peptidyl dipeptidase activities with some minor differences in peptide bond specificity. Cathepsins M and B are similar in size, with apparent molecular weights of 30,200 for cathepsin M and 28,800 for cathepsin B, and in amino acid composition and carbohydrate content. Each contains approximately 2-3 equivalents/mol glucosamine, 3 equivalents/mol mannose, and no fucose or galactosamine. They also show similar microheterogeneity in sodium dodecylsulfate-gel electrophoresis and isoelectric focusing; this microheterogeneity is probably related to differences in glycosylation. Extensive homology in primary structure for the two proteins was indicated by the similar patterns of peptides formed on digestion with trypsin.

Amino Acids↗
PubMed2004

Comparative substrate specificity analysis of recombinant human cathepsin V and cathepsin L.

Cathepsins V and L have high identity and few structural differences. In this paper, we reported a comparative study of the hydrolytic activities of recombinant human cathepsins V and L using fluorescence resonance energy transfer peptides derived from Abz-KLRSSKQ-EDDnp (Abz = ortho-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine). Five series of peptides were synthesized to map the S3 to S2' subsites. The cathepsin V subsites S1 and S3 present a broad specificity while cathepsin L has preference for positively charged residues. The S2 subsites of both enzymes require hydrophobic residues with preference for Phe and Leu. The S1' and S2' subsites of cathepsins V and L are less specific. Based on these data we designed substrates to explore the electrostatic potential differences of them. Finally, the kininogenase activities of these cathepsins were compared using synthetic human kininogen fragments. Cathepsin V preferentially released Lys-bradykinin while cathepsin L released bradykinin. This kininogenase activity by cathepsins V and L was also observed from human high and low molecular weight kininogens.

Catalysis↗
PubMed1991

Immunocytochemical localization of cathepsin D in rat ventral prostate: evidence for castration-induced expression of cathepsin D in basal cells.

Cathepsin D (EC3.4.23.5) is an aspartyl endopeptidase involved in lysosomal proteolysis. Its functional role is uncertain. This study was undertaken to determine the cellular and subcellular distribution of cathepsin D in the normal rat ventral prostate and its possible role in the castration-induced atrophy of the gland. Cathepsin D was localized immunohistochemically to perinuclear lysosomes in secretory cells, in capillary endothelial cells, and, occasionally, in stromal cells of the untreated animal. Castration resulted in an increased number of cathepsin D-positive cells in the stroma within 24 hr. By 48 hr after castration autophagolysosomes formed in secretory cells and apoptotic bodies appeared in the epithelium. Although apoptotic bodies generally contained immunoreactive cathepsin D, a subpopulation of larger apoptotic bodies, which commonly rested on the basement membrane and contained multiple inclusions, were more variable in cathepsin D expression. The induction of cathepsin D in dendritic cells basally oriented in the epithelium was noted at 4 days of castration. These cells had a phagocytic phenotype, were distributed periodically along the basement membrane, and were not found in ductal epithelia. Treatment with actinomycin D or hydrocortisone to reduce the rate of regression of the ventral prostate blocked the appearance of these cathepsin D-positive, basally oriented epithelial cells. Our data indicate that this cathepsin D-positive, phagocytic cell differentiates from a cell resident in the prostatic epithelium. We suggest that it differentiates from basal cells in the secretory tubuloalveolar portion of the gland and that it is involved in the destruction of regressed secretory cells.

Animals↗
PubMed1995

Human lysosomal beta-galactosidase-cathepsin A complex: definition of the beta-galactosidase-binding interface on cathepsin A.

Human lysosomal beta-galactosidase is organized as a 680-kDa complex with cathepsin A (also named carboxypeptidase L and protective protein), which is necessary to protect beta-galactosidase from intralysosomal proteolysis. To understand the molecular mechanism of beta-galactosidase protection by cathepsin A, we defined the structural organization of their complex including the beta-galactosidase-binding interface on cathepsin A. Radiation inactivation analysis suggested the existence of a 168-kDa structural subunit of the complex containing both beta-galactosidase and cathepsin A. Chemical cross-linking of the complex confirmed the existence of this subunit and showed that it is composed of one cathepsin A dimer and one beta-galactosidase monomer. The modeling of the cathepsin A dimer tertiary structure based on atomic coordinates of a wheat carboxypeptidase suggested a putative beta-galactosidase-binding cavity formed by the association of two cathepsin A monomers. According to this model two exposed loops of cathepsin A bordering the cavity were chosen as part of a putative beta-galactosidase-binding interface. Synthetic peptides corresponding to these loops were found both to dissociate the complex and to inhibit its in vitro reconstitution from purified cathepsin A and beta-galactosidase. The defined location of the GAL monomer in the complex with 35% of its surface covered by the CathA dimer may explain the stabilizing effect of CathA on GAL in lysosome.

Amino Acid Sequence↗
PubMed1995

Expression of rat cathepsin D cDNA in Saccharomyces cerevisiae: implications for intracellular targeting of cathepsin D to vacuoles.

To investigate the intracellular transport mechanisms of lysosomal cathepsin D in yeast cells, we produced cathepsin D in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin D coding sequence produced an intermediate species which had a slightly higher molecular weight than that of the mature cathepsin D. Cell fractionation experiments demonstrated that the cathepsin D polypeptide was colocalized to the yeast vacuoles with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. A biosynthesis study with pulse-chase kinetic analysis revealed that the precursor polypeptide was accurately sorted to the yeast vacuoles as determined by cell fractionation, and that N-linked carbohydrate modifications were not required for vacuolar sorting of this protein. To elucidate the role of the propeptide region of cathepsin D, which might function in the intracellular targeting to the vacuole, a deletion mutant of cathepsin D lacking the propeptide was prepared and its intracellular targeting was examined after transfection into yeast cells. Immunoblotting analysis demonstrated that the propeptide-deleted mutant protein was recovered in a low quantity as compared with that in the case of yeast cells expressing the wild-type protein in the isolated vacuolar fraction. Immunofluorescence analysis revealed that the deletion mutant protein appeared to be accumulated within the intracellular small vesicles but not in the carboxypeptidase Y-positive vacuoles. Overall, these results indicate that the rat cathepsin D precursor polypeptide is recognized by mechanisms similar to those involved in the intracellular sorting of vacuolar proteins through the ER/Golgi/vacuolar sorting pathway in yeast cells, and that the propeptide has an important function in translocation of the cathepsin D polypeptide to the vacuole.

Animals↗
PubMed2000

Mouse cathepsin M, a placenta-specific lysosomal cysteine protease related to cathepsins L and P.

The complete nucleotide sequence of a novel cathepsin cDNA derived from mouse placenta was determined and is termed cathepsin M. The predicted protein of 333 amino acid is a member of the family C1A proteases and is related to mouse cathepsins L and P. Mouse cathepsin M is highly expressed in placenta, whereas no detectable levels were found in lung, spleen, heart, brain, kidney, thymus, testicle, liver, or embryo. Phylogenic analyses of the sequences of human and mouse cathepsins show that cathepsin M is most closely related to cathepsins P and L. However, the differences are sufficiently large to indicate that the enzymes will be found in other species. This is in contrast to human cathepsins L and V, which probably resulted from a gene duplication after divergence of mammalian species.

Amino Acid Sequence↗
PubMed1988

The design of peptidyldiazomethane inhibitors to distinguish between the cysteine proteinases calpain II, cathepsin L and cathepsin B.

A series of peptidyldiazomethanes was synthesized and tested as inactivators of the cysteine proteinases calpain II, cathepsin L and cathepsin B. Inactivators that react rapidly and that show a degree of selectivity between the enzymes were identified. Z-Tyr(I)-Ala-CHN2 (where Z represents benzyloxycarbonyl) reacts rapidly with cathepsin L and more slowly with cathepsin B, but does not inhibit calpain II. Z-Leu-Leu-Tyr-CHN2 reacts rapidly with cathepsin L and calpain II but very slowly with cathepsin B. Boc-Val-Lys(epsilon-Z)Leu-Tyr-CHN2 (where Boc represents t-butyloxycarbonyl) reacts more rapidly with calpain II than with cathepsin L or cathepsin B. The discriminating inhibitory effects of these compounds make them potentially useful for investigation of enzyme functions in vivo. The data presented also provide insights into the subsite specificity of calpain.

Calpain↗
PubMed1983

Use of new synthetic substrates for assays of cathepsin L and cathepsin B.

Efficient methods were developed for synthesizing synthetic substrates for assays of cathepsin B and cathepsin L. Several 2-naphthylamide compounds with a blocked NH2-terminus, Suc-Tyr-Met-NA, beta-Ala-Tyr-Met-NA, and D-Leu-Tyr-Met-NA, were specific and sensitive substrates for cathepsin L and cathepsin B; they were not specific for cathepsin L only, because all of them were also hydrolyzed by cathepsin B. Some kinetic constants for the hydrolyses of these three synthetic substrates by cathepsin B and cathepsin L are given.

Animals↗
PubMed2001

Cathepsin L, but not cathepsin B, is a potential kininogenase.

Although papain-like enzymes are strongly inhibited by their natural tight-binding inhibitors of the cystatin superfamily, cathepsins B and L may still retain some residual proteolytic activity toward Z-Phe-Arg-AMC in the presence of an excess of kininogen. This activity is abolished by adding E-64 or chicken cystatin. Cathepsins B and L show a single band of gelatinolytic activity when subjected to gelatin-SDS-PAGE. Adding high Mr kininogen, low Mr kininogen, T-kininogen, or chicken cystatin to cathepsin L results in additional intense bands of enzyme activity corresponding to the protease-inhibitor complexes. Cathepsin B does not produce these additional bands. This gelatinolytic activity was inhibited by E-64, but not by EDTA, PMSF or Pefabloc. Cathepsin L also specifically generated kinins from high and low molecular weight kininogens in vitro, but cathepsin B did not. T-kininogen did not release any immunoreactive kinins when complexed with cathepsin L, as previously observed using tissue kallikreins. The ability of cathepsin L to generate vasoactive peptides raises the question of the physiological significance of this mechanism during inflammation.

Animals↗
PubMed1999

Structure based development of novel specific inhibitors for cathepsin L and cathepsin S in vitro and in vivo.

Specific inhibitors for cathepsin L and cathepsin S have been developed with the help of computer-graphic modeling based on the stereo-structure. The common fragment, N-(L-trans-carbamoyloxyrane-2-carbonyl)-phenylalanine-dimethyla mide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (CLIK) specifically inhibited cathepsin L at a concentration of 10(-7) M in vitro, while almost no inhibition of cathepsins B, C, S and K was observed. Four of the CLIKs are stable, and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the CLIK inhibitors contains an aldehyde group, and specifically inhibits cathepsin S at 10(-7) M in vitro.

Animals↗
PubMed1996

Human cathepsin O2, a matrix protein-degrading cysteine protease expressed in osteoclasts. Functional expression of human cathepsin O2 in Spodoptera frugiperda and characterization of the enzyme.

Cathepsin O2, a human cysteine protease predominantly present in osteoclasts, has been functionally expressed in Spodoptera frugiperda Sf9 cells using the Autographa californica nuclear polyhedrosis virus. Following in vitro activation at pH 4.0 with pepsin, active enzyme with an apparent molecular weight of 29,000 was obtained. N-terminal sequencing revealed the typical processing site for cysteine proteases of the papain family with a proline in the position adjacent to the N-terminal alanine residue. The S2P2 subsite specificity of human cathepsin O2 is similar to cathepsin S but distinguished from cathepsins L and B. Similar to cathepsin S, cathepsin O2 is characterized by a bellshaped pH activity profile and is stable at pH 6.5 for 30 min at 37 degrees C. Cathepsin O2 is further distinguished by its potent collagenolytic activity against Type I collagen between pH 5 and 6, and elastinolytic activity against insoluble elastin at pH 7.9. Its capacity to efficiently degrade Type I collagen and its high expression in osteoclasts suggest that cathepsin O2 may play a major role in human osteoclastic bone resorption.

Amino Acid Sequence↗
PubMed1991

Release of dog polymorphonuclear leukocyte cathepsin G, normally and in endotoxin and pancreatitic shock. Isolation and partial characterization of dog polymorphonuclear leukocyte cathepsin G.

Dog polymorphonuclear leukocyte cathepsin G was isolated from a granule extract using a two-step procedure including affinity chromatography on a Trasylol-Sepharose gel and ion-exchange chromatography on a CM 52 column. 22 of the first 24 N-terminal amino acids were determined and showed 83% and 71% identity to those of human and rat cathepsin G, respectively. Total amino-acid composition demonstrated the basic nature of the protein. In an SDS/polyacrylamide-gel electrophoresis the protein showed an Mr of 29,400 compared to the Mr of 26,800 calculated from the total amino-acid composition. The enzyme was shown to form complexes with alpha 1 alpha 2-macroglobulin and alpha 1-proteinase inhibitor. A specific enzyme-linked immunosorbent assay was developed for the determination of cathepsin G/alpha 1-proteinase inhibitor complex in dog plasma and tissue fluids. The mean concentration of cathepsin G in normal dog plasma was determined to be 38 micrograms/l, measured as cathepsin G/alpha 1-proteinase inhibitor complex. When active dog cathepsin G was added to normal dog plasma in vitro, approximately 56% could be measured by the assay. Slow intravenous infusion of a lethal dose of endotoxin in dogs was followed by a marked drop in white blood cell count and thrombocytes and a simultaneous rapid increase in plasma cathepsin G concentration, reaching a maximum level of 150 micrograms/l. Bile-induced experimental pancreatitis in dogs was accompanied by successive increase in cathepsin G levels in plasma as well as in peritoneal exudates, reaching a maximum level of about 300 micrograms/l in plasma and 18 mg/l in the exudates during the late stages of disease.

Acute Disease↗
PubMed2000

Cathepsin K and the design of inhibitors of cathepsin K.

Cathepsin K, a cysteine protease of the papain family, was identified by sequencing complementary DNA libraries derived from osteoclasts. Cathepsin K can cleave bone proteins such as Type I collagen, osteopontin, and osteonectin. The localization and maturation of cathepsin K in activated osteoclasts have been characterized. Furthermore, mutation of the gene expressing cathepsin K in humans results in pycnodysostosis, an autosomal recessive condition, resulting in osteoprosis and increased bone fragility. Knockout of cathepsin K in the mouse also results in retarded bone matrix degradation and osteopetrosis. Together, these data demonstrate that inhibition of cathepsin K should result in a dimunition of osteoclast-mediated bone resorption. Several novel classes of cathepsin K inhibitors have been designed from X-ray co-crystal structures of peptide aldehydes bound to papain. The convergence of the design of novel inhibitors and the discovery of cathepsin K has created opportunities to further understand bone and cartilage biology as well as provide new therapeutic agents for the treatment of disease states in man such as osteoporosis.

Animals↗