Cathepsin B-like Protease Research Articles

Human osteoclastomas contain multiple forms of cathepsin B

During bone resorption, the osteoclast secretes hydrolytic enzymes into the sealing zone which it creates between itself and the bone surface. Since this environment is acidic; proteinases active at low pH must therefore be responsible for degrading the bone matrix, which is largely composed of type I collagen. To investigate these enzymes, we have used human osteoclastomas as the starting material. Sequential chromatography on S-Sepharose, phenyl-Sepharose, heparin-Sepharose and Sephacryl S-200HR resulted in the separation of six cysteine proteinase activities. These proteinases have M r values ranging from 20 000 to 42 000. The pH profiles of activity showed optima between 3.5–6.0 for both synthetic substrates and type I collagen. All the proteinases were able to degrade soluble and insoluble type I collagen. The kinetics of hydrolysis using Z-Phe-Arg-NHMec and Bz-Phe-Val-Arg-NHMec as substrates resulted in values within the range expected for cathepsin B. The six activities were all inhibited by the cystein proteinase inhibitors antipain, chymostatin, leupeptin and E-64. The rate constants of inactivation using Z-Phe-Tyr-( O- t-Bu)CHN 2 were also similar to the published rates for cathepsin B. Antibodies to cathepsin B reacted with all activities. These antibodies localised the enzyme activities to the osteoclast within the tumour. Northern blotting using a cDNA probe to cathepsin B revealed three species of mRNA transcripts. These results suggest that multiple forms of cathepsin B-like proteinases are involved in osteoclastic bone resorption.

Chromogranin A-processing proteinases in purified chromaffin granules: contaminants or endogenous enzymes?

It was the purpose of this study to define the chromogranin A-processing proteinases present in highly purified preparations of bovine chromaffin granules. The most active enzyme had a pH optimum of 5.0 and was inhibited by pepstatin. It could be identified immunologically as a cathepsin D-like enzyme and subcellular fractionation established its lysosomal origin. After removal of this enzyme the remaining activity at pH 5.0 was mainly due to a cathepsin B-like proteinase. The presence of this enzyme could also be attributed to lysosomal contamination. In the presence of calcium, a further proteolytic activity became apparent at pH 5.0. This enzyme which was inhibited by p-chloromercuriphenylsulfonic acid was localized in chromaffin granules. A trypsin-like peptidase, most active at pH 8.2, was enriched in a membrane wash of chromaffin granules. Subcellular fractionation indicated that this enzyme is preferentially bound to the membranes of very dense particles probably representing a subpopulation of chromaffin granules. This study establishes that the most active chromogranin A-degrading proteinases present in highly purified chromaffin granules are attributable to lysosomal contamination. Two enzymes with low activity (a Ca 2+ activated proteinase and a trypsin-like enzyme) are, apparently, true constituents of chromaffin granules.

The glycosylation state of the precursors of the cathepsin B-like proteinase from human malignant ascitic fluid: Possible implication in the secretory pathway of these proenzymes

We have investigated the structure of the carbohydrate moiety of the precursors of the cathepsin B-like proteinase (PCBt). The largest precursor has an apparent molecular size (Mr) of 45–47 Kd and it contains 3 N-linked oligosaccharide chains which were Endo β N acetylglucosaminidase H (Endo H)-resistant forms. On the other hand, these chains were sequentially removed by Endo β N acetylglucosaminidase F (Endo F). Both results indicate a complex type structure for these oligosaccharides. As usual in these cases, sialic acids were also found. In the deglycosylated state, PCBt has a Mr value of 36 Kd closely related to the 35.9 Kd Mr for human Pro CB, deduced from the cDNa sequence. These findings may explain the extracellular location of PCBt in malignancy: complex oligosaccharides are in most cases associated with secretory or membrane bound glycoproteins. On the contrary, mannose-rich types are involved in the lysosomal routage.

Drosophila cathepsin B-like proteinase: A suggested role in yolk degradation

A cysteine, cathepsin B-like proteinase activity has been found in Drosophila embryos. It appears associated with yolk granules and its activity during embryogenesis correlates well with the degradation of these organelles. In mature oocytes, the enzyme is found in an inactive form which may be activated by limited proteolysis by a serine proteinase also present in oocytes. In early embryos, when solubilized in vitro, the cathepsin B-like proteinase is found in a form of high molecular mass (approx 1000 kDa). This decreases with development down to about 39 kDa, likely the mass of the free proteinase. The heavy form apparently results from the tight association with a yolk protein complex. The proteinase has been found in vitro to degrade readily the yolk polypeptides. The proteinase activity increases during early embryogenesis in parallel with the decrease in molecular weight of the heavy form, and decreases to low values in late embryos. We have also found that ammonium chloride can inhibit in vivo the degradation of yolk and, in parallel, the developmental inactivation of the proteinase. The results altogether suggest that the cathepsin B-like proteinase is implicated in yolk degradation in Drosophila.

Properties of midgut hydrolases from nymphs and adults of the hematophagous bug Rhodnius prolixus

1. 1. The properties of β-acetylglucosaminidase, acid phosphatase, aminopeptidase, cathepsin B-like proteinase, α-galactosidase, α-glucosidase, detergent-solubilized membrane-bound α-glucosidase, β-glucosidase, α-mannosidase and detergent-solubilized membrane-bound α-mannosidase from posterior midgut of Rhodnius prolixus adults were determined by density-gradient centrifugation and kinetic methods. 2. 2. The posterior midgut hydrolases from R. prolixus adults and nymphs were found to be very similar. 3. 3. The results lend support to the hypothesis that digestive enzymes are, in contrast to holometabolous insects, identical in young and adult forms of hemimetabolous insects.

Cathepsin B-like thiol proteases and collagenolytic proteases in middle ear effusion from acute and chronic otitis media with effusion.

Hydrolytic activity of cathepsin B-like thiol proteases and collagenolytic proteases was measured in middle ear effusions (MEE) from pediatric patients with acute and chronic otitis media with effusion (OME). Both activities were significantly higher in MEEs from acute OME than those from chronic OME (p less than 0.01). The ratio of hydrolytic activity in the extracellular portion to the total activity in MEEs from chronic OME was also analysed in both proteases. The ratio ranged from 20 to 100% in individual cases, indicating that the degree of the release of lysosomal thiol proteases including collagenolytic proteases from leukocytes is variable in MEEs with chronic OME. The kinetics of lysosomal thiol proteases including collagenolytic proteases in acute OME seems to be much more active than that in chronic OME, and the presence of these thiol proteases appears to be an important factor leading to chronic OME.

Plasma membrane-associated cysteine proteinases in human and animal tumors.

The ability of tumor cells to invade into and through normal tissue during the metastatic cascade has been attributed to tumor-associated degradative enzymes including proteinases of the metallo, serine and cysteine classes. Work from several laboratories has established that the cysteine proteinases cathepsins L and B are released from tumor cells, primarily as latent precursor forms. In addition, a cathepsin B-like cysteine proteinase has been shown to be associated with the plasma membrane fraction of several animal and human tumors. This form of the enzyme retains activity under physiologic (or pathologic) conditions including at neutral pH and in the presence of low Mr inhibitors. Since we have established that cathepsin B can degrade the basement membrane attachment glycoprotein laminin, we speculate that plasma membrane-associated cathepsin B may participate in focal dissolution of the basement membrane during tumor cell extravasation.

Lysosomal thiol proteases in middle ear effusions.

Hydrolytic activity of lysosomal cathepsins B and H, and trypsin-like proteases in 115 middle ear effusions (MEEs, 40 serous and 75 mucoid) from chronic otitis media with effusion (OME) patients was measured and compared to that in plasma. The activity of both cathepsins in MEEs was significantly higher than that in plasma (p less than 0.01), and cathepsin B activity in mucoid MEEs was also significantly higher than that in serous MEEs (p less than 0.01). The activity of trypsin-like proteases was very weak in both MEEs and plasma. Profiles of various inhibitors indicated the qualitative difference of proteolytic enzymes between MEEs and plasma. Mucoid MEEs had significantly higher activity of thiol proteases than serous ones (p less than 0.01). Cathepsin B-like lysosomal thiol proteases, derived mainly from macrophages, could become a major proteolytic factor to perpetuate and amplify the inflammatory reaction of chronic OME.

Lysosomal Thiol Proteases (Cathepsin B-like Proteases) in Serous Middle Ear Effusions from Adult Patients

Hydrolytic activity of cathepsins B, H and trypsin-like proteases was measured in 38 serous middle ear effusion (MEE) samples. The concentrations of (alpha 1-AT) and alpha 2-macroglobulin (alpha 2-M) were also quantitated. The mean value of cathepsin B activity was 25.0 +/- 20.7 RFU and that of cathepsin H was 14.3 +/- 3.0--both significantly higher than those in plasma (1.8 +/- 0.4 RFU, 1.2 +/- 0.3 RFU, p less than 0.005). Very low trypsin-like protease activity could be observed. The mean concentrations of alpha 1-AT and alpha 2-M were 368 +/- 94.8 mg/dl and 57.5 +/- 57.3 mg/dl. The bulk of alpha 1-AT in MEEs was occupied by free alpha 1-AT, which can saturate exogenous trypsin. Due to the very low molar concentration of alpha 2-M in MEEs, thiol proteases (mainly cathepsin B) could be a possible major factor inflicting proteolytic injury on the middle ear mucosa and reflecting the severity of the inflammatory process.

Extracellular processing of proapolipoprotein A-II in Hep G2 cell cultures is mediated by a 54-kDa protease immunologically related to cathepsin B.

Apolipoprotein A-II is the second most abundant polypeptide found in human plasma high density lipoprotein particles. The primary translation product of human apo-A-II mRNA is a prepropolypeptide. We have previously reported (Gordon, J. I., Sims, H. F., Edelstein, C., Scanu, A. M., and Strauss, A. W. (1984) J. Biol. Chem. 259, 15556-15563) that the prosegment of apo-A-II was removed following export from a human hepatoma cell line (Hep G2). This represented a novel processing compartment for prosegments terminating with paired basic residues and differed from the processing of proalbumin which occurred with high efficiency prior to export from these cells. We have now characterized the enzyme responsible for this extracellular cleavage. The proapo-A-II converting activity is blocked by the thiol protease inhibitors antipain, E-64, leupeptin, and Ala-Lys-Arg chloromethyl ketone. Incubation of 125I-iodotyrosylated Ala-Lys-Arg chloromethyl ketone with serum-free media harvested from cell cultures over a 12-h period revealed a time-dependent accumulation of a 54-kDa protease. Although small quantities of the 54-kDa protease were detected in cell lysates, the major intracellular sequences labeled by the affinity probe had masses of 31.5 and 6 kDa. The 54-kDa extracellular, as well as 31.5- and 6-kDa intracellular, species were all immunoprecipitated by monospecific anti-human liver cathepsin B IgG. Addition of this antibody to media inhibited extracellular conversion of proapo-A-II to the mature protein. Based on these observations, we conclude that a "pro" cathepsin B-like protease exported by Hep G2 cells is responsible for proapo-A-II prosegment removal. It appears that cathepsin B-like proteases exhibit a complex pattern of segregation within the secretory pathway and that larger molecular weight forms of cathepsin B-like proteases are capable of accurately processing propolypeptides.

Proteinase activities of Entamoeba histolytica cytotoxin

The proteinase activity of the low molecular weight cytotoxin of Entamoeba histolytica was correlated with its cytotoxicity. Gel-filtered amebal toxin (mol wt 10–30,000) proteinase activities could be assayed on azocasein at pH 6 or on hemoglobin at pH 4.5. Proteinase activity was inhibited by serum fractions, thiol reagents, heavy metals, leupeptin, and antipain. The cytotoxic activity of gel-filtered amebal toxin was inhibited by serum fractions, leupeptin, and antipain. Increased proteinase and cytotoxic activity was produced by treatment with cysteine. These data support the action of a thiol proteinase in the production of cytopathic effects by gel-filtered amebal toxin in vitro. The cytotoxic and proteinase activities were further purified using a combination of column chromatography and preparative isoelectric focusing. Two low molecular weight cytotoxins with proteinase activity on both substrates were isolated. The major cytotoxin had an isoelectric point of 4.5 and a molecular weight of 22,000; the other cytotoxin had a basic isoelectric point. These substances may be cathepsin B-like proteinase and elastase or cathepsin G-like proteinases of E. histolytica. The major proteinase activity in the high molecular weight fraction was not cytotoxic. The isoelectric points of the high molecular weight proteinase activities corresponded to that of mammalian cathepsin D. The major cell rounding cytotoxic activity of E. histolytica extracts in vitro is probably due to the activity of a thiol-containing cathepsin B-like proteinase.

Cathepsin B-like proteinase as a marker for metastatic tumor cell variants.

Serial transplantation of a spontaneous BDX rat tumor, classified as an anaplastic sarcoma, gives rise to two variants; a rapidly growing nonmetastatic line (AS) and a slowly growing, invasive, and highly metastatic variant (ASML). The availability of two cell lines of the same origin but with markedly differing metastatic potential offers an ideal model for the identification of the cellular properties involved in invasive and/or metastatic behavior. The present work focuses on the pattern of various proteinases in the two tumor cell variants. The findings disclosed one major consistent difference which relates to a cathepsin B-like cysteine proteinase. The metastatic ASML variant manifests exceedingly high intracellular cathepsin B-like activity; in the nonmetastatic AS variant, the activity of this proteinase is significantly lower. Other proteinases, in particular elastase-like, chymotrypsin-like, collagenase-like enzymes and plasminogen activator, showed low, essentially comparable activity patterns. Thus, cathepsin B-like proteinase is a marker enzyme of the metastatic ASML tumor cell variant.

Golgi/granule processing of peptide hormone and neuropeptide precursors: a minireview.

Proteolytic processing of precursor proteins is a phylogenetically ancient and widely used mechanism for producing biologically active peptides. Proteolytic cleavage of proproteins begins only after transport to the Golgi apparatus has been completed and in most systems may continue for many hours within newly formed secretory vesicles as these are stored in the cytosol or transported along axons to more peripheral sites of release. Paired basic residues are required for efficient proteolysis in most precursors, suggesting that a small number of specialized tryptic proteases exist that have great site selectivity but can process many sites within the same precursor or in different precursors within the same cell, or in different cells or tissues. Cleavage-site choice may be strongly influenced by other factors, such as secondary and tertiary structure, but definitive structural information on precursor proteins is lacking. Modifications such as glycosylation, phosphorylation, and sulfation also are Golgi associated but are not known to influence proteolytic processing patterns. Golgi/granule processing also rarely occurs at sites other than pairs of basic amino acids, including single basic residues ( trypsinlike ), Leu-Ala, Leu-Ser, or Tyr-Ala bonds ( chymotrysinlike ) as well as other specialized nontryptic cleavages, suggesting that mixtures of proteases coexist in the Golgi/granule system. Cathepsin B-like thiol proteases, or their precursors, have been implicated as the major processing endopeptidases in several systems. Carboxypeptidase B-like enzymes also have been identified in secretion granules in several tissues and appear to be metalloenzymes similar in mechanism to the pancreatic carboxypeptidases, but with a lower pH optimum. The role of the Golgi apparatus in sorting newly formed secreted products from lysosomal hydrolases may have permitted the development in evolution of an intimate relationship between certain of the lysosomal degradative enzymes, such as cathepsin B or its precursors, and the Golgi/granule processing systems. The sequestration of the proteolytic products of precursors within secretion granules leads to the coordinate discharge of highly complex mixtures of peptides having related or overlapping biological activities. The cosecretion of nonfunctional peptide " leftovers ," such as the proinsulin C-peptide, can serve as useful markers of secretion or cellular localization, as well as of evolutionary relation ships. Errors in cleavage due to point mutations in precursors have been identified in several systems, leading to the accumulation of incorrectly processed materials in the circulation. These and/or defects (ABSTRACT TRUNCATED AT 400 WORDS)

Identification of a cathepsin b-like protease in the crevicular fluid of gingivitis patients.

Crevicular fluid from gingivitis patients contains significant levels of a cysteine protease which was characterized as the lysosomal protease cathepsin B, as judged by substrate specificity, thiol dependence, pH optimum, kinetic parameters, pH stability, and inhibitor sensitivities. A highly-sensitive fluorometric assay procedure was used to establish the mean level of cathepsin B activity for 25 gingivitis patients.

Involvement of a cathepsin B-like cysteine proteinase in platelet aggregation induced by tumor cells and their shed membrane vesicles.

Murine 15091A mammary adenocarcinoma cells and membrane vesicles spontaneously shed from these tumor cells in culture can induce aggregation of washed human platelets. A spectrum of proteinase inhibitors was tested for their ability to inhibit 15091A induced platelet aggregation. Of the inhibitors tested the most effective were those selective for cysteine proteinases. The effect of the spectrum of proteinase inhibitors on 15091A induced platelet aggregation was compared to the effect on cathepsin B-like cysteine proteinase activity in homogenates of 15091A tumor cells and their spontaneously shed vesicles. The results suggest that there is a correlation between activity of a cathepsin B-like proteinase in 15091A cells and vesicles and the ability of these cells and vesicles to induce aggregation of washed human platelets.

Tunicamycin treated fibroblasts secrete a cathepsin B-like protease

We have investigated the effect of tunicamycin on the localization of lysosomal hydrolases in chicken embryo fibroblast cultures. We showed that treatment with tunicamycin (0.05 μg/ml) resulted in a 7–10 fold increase in the cathepsin B-like activity in the culture medium compared to untreated cultures. The protease activity was identified as cathepsin B-like based on 1) substrate specificity (benzoylpro-phe-arg[14C]anilide is rapidly hydrolyzed), 2) the pH optimum for activity of 5.5, 3) inhibition by thiol reactive compounds, 4) inhibition of the activity by leupeptin but not by pepstatin or phenylmethylsulfonyl fluoride, and 5) by the demonstration of a protease with similar properties in the lysosomal fraction of untreated cultures. The secretion of the cathepsin B-like protease was specific and not due to leakage from damaged cells.

Tumor Cell-Platelet Aggregation: Induced by Cathepsin B-Like Proteinase and Inhibited by Prostacyclin

The ability of tumor cells to metastasize may be related to their ability to promote aggregation of host platelets. The use of inhibitors of cysteine proteinases resulted in parallel inhibition of B16 amelanotic melanoma-induced platelet aggregation and of a cathepsin B activity. The antimetastatic agent prostacyclin inhibited platelet aggregation induced by the tumor cells and by papain, a cathepsin B-mimicking agent.

A latent thiol proteinase from ascitic fluid of patients with neoplasia

Pepsin treatment of ascitic fluid from patients with neoplasia generated a cysteine (thiol) proteinase activity which resembles cathepsin B (EC 3.4.22.1) in its requirements for thiol activators, susceptibility to inhibitors and specificity for synthetic substrates. As judged by gel filtration, pepsin reduced the molecular size of the latent enzyme from an M r of 41 000 to 33 000 after activation. Both forms are larger than human liver cathepsin B. In addition to its presence in ascitic fluid, the pepsin-activated species was found in the medium of ascites cells maintained in culture. The latent enzyme may be an enzyme-inhibitor complex or an inactive precursor of a cathepsin B-like proteinase.

Biochemical and immunological studies of lysosomal and related proteinases in health and disease.

A review of continuing studies on the physiological and pathological roles of the lysosomal proteinases, cathepsin D and cathepsin B is presented. Intracellular release of cathepsin D from lysosomes has been demonstrated during myocardial ischemia. Both this proteinase and cathepsin B have been found, by organ culture in the presence of the appropriate antiserum, to be released into the extracellular space in rheumatoid synovium, where they may play a part in cartilage destruction. Also reviewed is the finding of a cathepsin B-like proteinase that, in organ culture, is secreted in markedly increased amounts from human malignant breast carcinomas in comparison to amounts secreted from benign tumors or normal tissue.

Exoproteinase activity in the posterior midgut of Rhodnius prolixus stål (Hemiptera: Reduviidae)

Results of this study suggest that two exoproteinases, lysosomal carboxypeptidase B and aminopeptidase, are present in the posterior midgut of Rhodnius prolixus Stål. Digestive midgut lysosomal carboxypeptidase B hydrolysed hippuryl- dl-phenyllactic acid (HPLA) and N- carbobenzoxy- l-glutamyl- l-tyrosine with maximal activities at pH 4.0 and 3.9 respectively. Activity was higher in the presence of thiol compounds (cysteine, glutathione, dithiothreitol and mercaptoethanol) and was inhibited by iodoacetamide (IAA) and tosyl- l-lysine chloromethyl ketone (TLCK). Digestive midgut aminopeptidase hydrolysed leucine- p-nitroanilide (LPNA) with maximal activity at pH 8.0. Magnesium activated LPNA hydrolysis whereas calcium, thiol compounds and EDTA inhibited activity. IAA and TLCK had no effect. Aminopeptidase was located in the gut wall and lysosomal carboxypeptidase B was located in the gut lumen. The presence of lysosomal carboxypeptidase B and the previously described cathepsin B-like proteinase demonstrate that the digestive proteinases in R. prolixus are unique among those described for blood feeding insects.