Release of cathepsin B precursors from human and murine tumours.
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Animals↗
Biomedical subjects
J S Mort
Publications and source records attributed to J S Mort.
Cartilage link proteins. Biochemical and immunochemical studies of isolation and heterogeneity.
Native and clostripain-derived link proteins from proteoglycan aggregates were separated by gel chromatography in 4 M guanidine HCl from low-buoyant-density proteoglycan and proteoglycan hyaluronic acid-binding region after extraction from adult bovine nasal cartilage and the Swarm rat chondrosarcoma. Separations were monitored with Laurell immunoelectrophoresis using precipitating antibodies to link protein, hyaluronic acid-binding region, and low-buoyant-density proteoglycan. These immunoanalyses, with sodium dodecyl sulfate-polyacrylamide gel analyses, revealed the usefulness of this combined approach when assessing the purities and identities of these molecules. Using these isolated link proteins we provide data suggesting the presence of two immunologically detectable forms of link proteins of the same molecular weight. On immunoelectrophoresis both rat and bovine link proteins of a single molecular weight each produced two precipitin reactions with antibody to link protein. They were of similar intensity for native molecules but of different intensity for clostripain-isolated link protein. Isoelectric focusing in 6 M urea revealed that the 48,000 and 44,000 molecular weight link proteins of bovine nasal cartilage together produce a complex pattern of many bands. Link proteins of a single molecular size produced a much simpler, predominantly five-banded focusing pattern. Immunoelectrophoresis of the electrofocused clostripain rat link protein (42,000) revealed that the three major central bands each produced double precipitin reactions. Mixing of native and clostripain-derived link protein in 4 M guanidine HCl followed by dialysis to 6 M urea prior to isoelectric focusing did not change the focusing position of the individual bands. This suggests that the heterogeneity of focusing forms was due to the existence of different isoforms. The double precipitin reactions may be due to the existence of two different conformations expressing different epitopes of the kind reported previously by Thonar et al. (Thonar, E. J.-M. A., Kimura, J. H., Hascall, V. C., and Poole, A. R. (1982) J. Biol. Chem. 257, 14173-14180).
Animals↗
The inability to prepare high-buoyant-density proteoglycan aggregates from extracts of normal adult human articular cartilage.
High-buoyant-density proteoglycan aggregates could not be prepared from extracts of adult human cartilage by associative CsCl-density-gradient centrifugation with a starting density of 1.68 g/ml, even though proteoglycan subunits, hyaluronic acid and link proteins were all present. In contrast, aggregates could be prepared when extracts of neonatal human cartilage or bovine nasal cartilage were subjected to the same procedure. This phenomenon did not appear to be due to a defect within the hyaluronic acid-binding region of the adult proteoglycan subunit, but rather to an interference in the stability of the interaction between the proteoglycan subunit and hyaluronic acid towards centrifugation. The factor responsible for this instability was shown to reside within the low-density cartilage protein preparation obtained by direct dissociative CsCl-density-gradient centrifugation of the adult cartilage extract.
Adult↗
The combined action of two enzymes in human serum can mimic the activity of cathepsin B.
Synthetic substrates are often used to measure the activity of proteolytic enzymes. We have investigated the activities which cleave synthetic substrates such as alpha-N-benzyloxycarbonyl-Arg-Arg-beta-naphthylamide, for which the lysosomal proteinase cathepsin B has a high affinity, in sera from normal individuals, pregnant women and patients with breast cancer. As reported by other workers, activities against these substrates were elevated during pregnancy. Naphthylamine release, however, was shown to be the result of the combined action of two enzymes. The substrate is first cleaved by an endopeptidase to yield alpha-N-benzyloxycarbonyl-Arg and the aminopeptidase substrate Arg-beta-naphthylamide, which is then cleaved by serum aminopeptidases, particularly oxytocinase. A similar mechanism of cleavage was also found in the sera of breast cancer patients, where the endopeptidase catalyzing the first reaction was characterized as plasma kallikrein and the second reaction was carried out by serum leucine aminopeptidase. In no serum sample was there evidence for true cathepsin B activity.
Aminopeptidases↗
Extracellular presence of the lysosomal proteinase cathepsin B in rheumatoid synovium and its activity at neutral pH.
The presence of the lysosomal proteinases cathepsin B and cathepsin D at extracellular sites in rheumatoid synovium was demonstrated using the antibody capture technique. Unlike cathepsin D, the cysteine proteinase cathepsin B was commonly detected only at the edges of the synovial explants. Radioimmunoassay and enzyme activity assay of these proteinases demonstrated that both were released from rheumatoid synovial cells in comparable amounts. Since lysosomal cathepsin B is unstable and denatured at physiologic pH and the antibody used only recognizes inactivated enzyme, we believe the selective detection of cathepsin B at the edge of the synovium may be due to the proteinase maintaining a native conformation within the explant, where the pH may be low enough to permit this. By use of a fluorescent substrate in a sensitive, continuous enzyme assay, cathepsin B was shown to express significant activity at neutral and alkaline pH before being inactivated. This and earlier work from this laboratory indicate that cathepsin B secreted by rheumatoid synovial cells may possess extracellular activity in vivo and be involved in the degradation of connective tissue macromolecules.
Antigen-Antibody Complex↗
Activation of latent collagenase by serum proteinases that interact with immobilized immunoglobulin G.
It has been previously observed that collagen destruction occurs in the vicinity of immune complexes present in articular cartilages of patients with rheumatoid arthritis. When IgG is covalently linked to Sepharose it behaves as if it has reacted with an antigen to form an immune complex, in that it binds the complement component C1 from human serum. Other serum components also interact with this matrix, though their interaction may not be specific for IgG. Two of these components were shown to possess proteolytic activity, one being kallikrein and the other having the properties of plasmin. Both of the activities could activate latent human collagenase. Whilst the binding of the plasmin activity is probably nonspecific, the binding of the kallikrein activity may be selective for IgG (although it is not certain whether this binding is direct or indirect via another molecule). These results therefore suggest that active proteinases such as plasma kallikrein may be selectively concentrated on immune complexes in vivo, where they may locally activate latent proteinases such as collagenase thereby initiating tissue destruction.
Antigen-Antibody Complex↗
Age-related changes in the structure of proteoglycan link proteins present in normal human articular cartilage.
Link proteins were identified immunologically in human articular-cartilage protein preparations from various individuals. Irrespective of age, all cartilages contained three link proteins of mol.wts. 48000, 44000 and 41000. However, with increasing age, multiple additional components of mol.wts. 26000-30000 were commonly observed under conditions where disulphide bonds were reduced.
Aging↗
Characterization of a latent cysteine proteinase from ascitic fluid as a high molecular weight form of cathepsin B.
The latent cysteine proteinase present in ascitic fluid of patients with neoplasia and released from ascites cells in culture has been partially purified and the enzyme after pepsin activation was shown to be immunologically related to the lysosomal proteinase, cathepsin B. The latent form was characterized as a single chain of Mr 40 000 as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by Western blotting and immune staining with an antiserum to human cathepsin B. Using the same techniques the enzyme after pepsin activation gave a single band of Mr 33 000. Analysis by isoelectric focusing showed that the latent enzyme before and after pepsin treatment is composed of several acidic isoenzymes. These findings suggest that this latent proteinase represents a precursor form of cathepsin B which is released extracellularly rather than being processed and directed to the lysosome.
Ascitic Fluid↗
Biochemical and morphologic studies of cartilage from the adult human sacroiliac joint.
The cartilage of the adult sacroiliac joint contains type II collagen and a high concentration of glycosaminoglycan. Furthermore, large aggregating proteoglycans and link proteins were extracted from the sacral cartilage. In these respects the sacroiliac cartilage is similar to that in the peripheral joints. However, while the organization of the collagen in the sacral cartilage is typical of an articular cartilage, the organization of the ilial cartilage is very different, and throughout its depth it possesses narrow collagen fibrils arranged parallel to the articular surface.
Adult↗
Proteinase inhibitors of human articular cartilage.
Extracts of human articular cartilage contain a variety of inhibitors to serine, cysteine and metallo-proteinases. By gel filtration chromatography, inhibitory activity towards serine proteinases was resolved into two components of apparent molecular weights 62,000 and 12,000 daltons; whereas inhibitory activity towards cysteine proteinases eluted with an apparent molecular weight of 13,000 daltons. In both cases the low molecular weight inhibitors were further resolved into two components by ion-exchange chromatography. Inhibitory activity towards metallo-proteinases resolved into two components of apparent molecular weights 35,000 and 25,000 daltons. No inhibitor of aspartic proteinases was detected. Although most of the inhibitory activities to serine and cysteine proteinases could be extracted from cartilage with 1 M NaCl, the complete removal of metalloproteinase inhibitory activities required extraction with 4 M guanidinium chloride. This suggests that they are more strongly associated with the cartilage.
Aged↗
A cysteine proteinase secreted from human breast tumours is immunologically related to cathepsin B.
The stable cathepsin B-like cysteine (thiol) proteinase secreted from human breast tumours in culture was shown to be destabilized by mercurial compounds. After such treatment the enzyme cross-reacts in a radioimmunoassay with a monospecific antiserum to human liver cathepsin B. It is suggested that the secreted enzyme may be a precursor form of lysosomal cathepsin B.
Adenocarcinoma↗
The heterogeneity of link proteins isolated from human articular cartilage proteoglycan aggregates.
Link proteins, isolated from proteoglycan aggregates prepared from human articular cartilage, were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. When subjected to the former technique the native link proteins resolve into three components of molecular weights 48,000, 44,000, and 41,000. Link protein, isolated following clostripain digestion of proteoglycan aggregate, is present as a single component of molecular weight 41,000. Under all conditions of isoelectric focusing tested, the native link proteins resolve into at least nine subcomponents having pI values between 6.0 and 7.0. The clostripain-treated link protein resolves into at least five subcomponents which have pI values similar to those of the more basic subcomponents observed in the native molecules. One source of heterogeneity contributing to both isoelectric focusing profiles is variation in sialic acid content, since neuraminidase treatment of the link protein preparations produces a shift to subcomponents with more basic pI. The electrophoretic data are consistent with the two larger link protein components representing the same protein core, but being substituted to different degrees with oligosaccharide chains that may have variable sialic acid contents. The smallest link protein component, which may be derived from either of the larger moieties by limited proteolytic cleavage, is also substituted with sialic acid-containing oligosaccharides.
Cartilage, Articular↗
A radioimmunoassay for total human cathepsin B.
A radioimmunoassay for human cathepsin B is described, which demonstrates the usefulness of protein A-bearing Staphylococcus aureus as an immunosorbent in a system where the primary antibody IgG has a low binding affinity for protein A. The removal of bound antigen from the incubation mixture is achieved by the use of a rabbit second step antiserum which confers high binding affinity for protein A to the primary immune complex. This method, as employed in the assay for human cathepsin B, is very reproducible and economical for large numbers of samples. The use of a monospecific antiserum to human cathepsin B and slightly alkaline assay conditions allow the determination of total cathepsin B protein in tissue fluids which is not possible by enzyme activity determination.
Binding, Competitive↗
A simple, economical method for staining gels for cathepsin B-like activity.
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Cathepsin B↗
Secretion of a thiol proteinase from mouse mammary carcinomas and its characterization.
Spontaneous mammary tumors from C3H/HeJ mice and transplants established from mammary tumors were investigated for their capacity to secrete thiol-dependent proteinase activity in organ culture explants. More activity was detected in culture media from spontaneous tumors than from transplanted spontaneous tumors. The accumulation of thiol proteinase in the culture medium was inhibited by cycloheximide, hydrocortisone, and aldosterone, but not by estradiol or the peptide hormones insulin or prolactin. The thiol proteinase is similar in enzymic properties to lysosomal cathepsin B, but its physical properties are different. It is stable to alkaline pH, has a larger molecular size on gel filtration (relative M.W. 39,000) and shows a different isoenzyme pattern to liver cathepsin B on analytical isoelectric focusing. The characteristics of this thiol proteinase are very similar to an enzyme secreted from malignant human breast tumors.
2-Naphthylamine↗
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 Mr 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.
Ascites↗
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.
Arthritis, Rheumatoid↗
Immunofluorescent localization of cathepsins B and D in human fibroblasts.
The proteinase cathepsin B has been localized in human fibroblasts using a monospecific antiserum that reacts in a precipitating manner with the denatured enzyme only. The distribution of staining as revealed by both direct and indirect immunofluorescence indicates a typical lysosomal localization similar to that observed for cathepsin D. Using dual localization methods employing fluorescein- and tetramethylrhodamine-labeled affinity purified antibodies both proteinases were usually observed in the same lysosome, indicating a combined role in intracellular digestion.
Cathepsin B↗