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Biomedical subjects

J S Mort

Publications and source records attributed to J S Mort.

At least 91 records · Page 5Linked to original sources
PubMed1990

Cartilage proteoglycan aggregate is degraded more extensively by cathepsin L than by cathepsin B.

The degradative actions of cathepsins L and B on human articular-cartilage proteoglycan aggregates were examined. Cathepsin L was found to be much more extensive than cathepsin B in degrading proteoglycan aggregates. It released products with size similar to that of single chondroitin sulphate chains, and a series of degraded link-protein fragments in the digestion mixtures. These proteolytically modified link-protein components (Mr 25,000 and 33,000) have similar Mr values to those of fragments observed in adult human cartilage. In contrast, cathepsin B exhibited a much more limited degradation on both proteoglycan subunits and link-protein components. Both cathepsins L and B generate multiple but distinct cleavage sites on human link proteins, and the hydrolysed bonds have been identified in the region between residues 18 and 29. Protein sequencing analysis of these modified link-protein components also provided evidence for the location of a second N-linked glycosylation site at residue 41 in human link proteins, in addition to that previously described at residue 6 on a proportion of the link proteins. Furthermore, it allows us to report the sequence of human link protein up to residue 65.

Amino Acid Sequence↗
PubMed1989

Degradation of human proteoglycan aggregate induced by hydrogen peroxide. Protein fragmentation, amino acid modification and hyaluronic acid cleavage.

We have previously shown that treatment of neonatal human articular-cartilage proteoglycan aggregates with H2O2 results in loss of the ability of the proteoglycan subunits to interact with hyaluronic acid and in fragmentation of the link proteins [Roberts, Mort & Roughley (1987) Biochem. J. 247, 349-357]. We now show the following. (1) Hyaluronic acid in proteoglycan aggregates is also fragmented by treatment with H2O2. (2) Although H2O2 treatment results in loss of the ability of the proteoglycan subunits to interact with hyaluronic acid, the loss of this function is not attributable to substantial cleavage of the hyaluronic acid-binding region of the proteoglycan subunits. (3) In contrast, link proteins retain the ability to bind to hyaluronic acid following treatment with H2O2. (4) The interaction between the proteoglycan subunit and link protein is, however, abolished. (5) N-Terminal sequence analysis of the first eight residues of the major product of link protein resulting from H2O2 treatment revealed that cleavage occurred between residues 13 and 14, so that the new N-terminal amino acid is alanine. (6) In addition, a histidine (residue 16) is converted into alanine and an asparagine (residue 21) is converted into aspartate by the action of H2O2. (7) Rat link protein showed no cleavage or modifications in similar positions under identical conditions. (8) This species variation may be related to the different availability of histidine residues required for the co-ordination of the transition metal ion involved in hydroxyl-radical generation from H2O2. (9) Changes in function of these structural macromolecules as a result of the action of H2O2 may be consequences of both fragmentation and chemical modification.

Amino Acid Sequence↗
PubMed1989

Degradation of proteoglycan aggregate by a cartilage metalloproteinase. Evidence for the involvement of stromelysin in the generation of link protein heterogeneity in situ.

Cartilage proteoglycan aggregates were subjected to degradation by a metalloproteinase, capable of degrading proteoglycan, released from cartilage in culture. This proteinase was demonstrated to be immunologically identical with fibroblast stromelysin. An early release of hyaluronic acid-binding region and large glycosaminoglycan-attachment regions was observed. With increasing time the glycosaminoglycan-attachment regions were digested into smaller fragments and the hyaluronic acid-binding regions accumulated. The degradation of link proteins also occurred concomitantly with these events. Link proteins were converted into a component of similar size to that of the smallest native link protein component. N-Terminal sequence analysis of the three human link protein components indicated that they are all derived from the same protein core, which is closely homologous to that of the rat chondrosarcoma link protein. The two larger link proteins (Mr 48,000 and 44,000) contain the same N-terminal sequence, but they differ by the apparent presence of an N-linked oligosaccharide at residue 6 of the largest link protein component. The smallest link protein (Mr 41,000), however, has an N-terminal sequence equivalent to that commencing at residue 17 in the larger link proteins. It was found that the cartilage metalloproteinase cleaves link proteins in human neonatal cartilage proteoglycan aggregates at the His-16-Ile-17 bond, the same position at which the smallest link protein component appears to be derived naturally from the two larger link protein components. These results suggest that stromelysin secreted by chondrocytes can account for the increased accumulation of hyaluronic acid-binding regions and much of the degradation of link protein observed during aging within human articular cartilage.

Amino Acid Sequence↗
PubMed1989

Effect of age on the abundance and fragmentation of link protein of the human intervertebral disc.

The link proteins of the human intervertebral disc were studied in tissue extracts by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), followed by immunoblotting, using a specific monoclonal antibody. Three link proteins were detected, corresponding in electrophoretic mobility to those present in articular cartilage. As with articular cartilage, the largest link protein predominates in the young, whereas in the adult the smallest link protein is equally abundant and internal fragmentation of the link proteins occurs. Only in the newborn is the quantity of extractable link protein comparable to that from articular cartilage. In the adult, the disc contains much less link protein than is present in autologous articular cartilage. Neither the amount nor heterogeneity of the link protein differs among different levels within the lumbar spine, although the proportions of the three proteins can differ between the anulus fibrosus and nucleus pulposus. The anulus always contained more extractable link protein relative to tissue wet weight than the nucleus, and the nuclear link protein, at least in adolescents, contained a greater proportion of the smallest link protein. Such changes in the quantity and structure of the disc link proteins may affect the properties of the proteoglycan aggregates and, thus, could influence disc function.

Aging↗
PubMed1989

Variation in the effects of mononuclear cell products from different individuals on metalloproteinase secretion from human articular cartilage.

Human articular cartilage in organ culture shows a variable degree of endogenous metalloproteinase secretion depending on the individual from whom it was obtained. Cartilage with low endogenous levels are stimulated by interleukin 1 (IL-1) to levels comparable to the high endogenous group. Total blood mononuclear cell products obtained from different individuals either behave in a manner similar to that seen with IL-1, which results in sustained high levels of enzyme secretion, or show an initial stimulation followed by a subsequent dropoff in enzyme secretion even though incubation is continued in the presence of mononuclear cell products. The factor(s) causing this dropoff can be distinguished from IL-1. Production and regulation of such factors may reflect a mechanism whereby the action of IL-1 can be controlled during the inflammatory response.

Adult↗
PubMed1988

Expression of rat and mouse cathepsin B precursors in Escherichia coli.

Vectors for the expression of mouse preprocathepsin B and rat procathepsin B, under the control of a modified lac operon regulatory region, in plasmid pKK233-2 were constructed. In rapidly growing cells induction of preprocathepsin B was cytotoxic. Immune precipitable cathepsin B, compatible in size with that expected for the unglycosylated proenzyme, was produced in both constructs. However, pepsin digestion of extracts did not produce any cathepsin B activity, indicating either that the recombinant forms are not correctly folded or were produced at such low levels that activity could not be detected.

Animals↗
PubMed1987

Treatment of cartilage proteoglycan aggregate with hydrogen peroxide. Relationship between observed degradation products and those that occur naturally during aging.

The effects of treatment of purified neonatal human articular-cartilage proteoglycan aggregate with H2O2 were studied. (1) Exposure of proteoglycan aggregate to H2O2 resulted in depolymerization of the aggregate and modification of the core protein of both the proteoglycan subunits and the link proteins. (2) Treatment of the proteoglycan aggregate with H2O2 rendered the proteoglycan subunits unable to interact with hyaluronic acid, with minimal change in their hydrodynamic size. (3) Specific cleavages of the neonatal link proteins occurred. The order in which the major products were generated and their electrophoretic mobilities resembled the pattern observed during human aging. (4) The proteolytic changes in the link proteins were inhibited in the presence of transition-metal-ion chelators, thiourea or tetramethylurea, suggesting that generation of hydroxyl radicals from H2O2 by trace transition-metal ions via a site-specific Fenton reaction may be responsible for the selective cleavages observed. (5) Cleavage of the link proteins in proteoglycan aggregates by H2O2 was shown to have a limited effect on the susceptibility of these proteins to cleavage by trypsin. (6) The relationship between these changes and those observed in cartilage during human aging suggests that some of the age-related changes in the structure of human cartilage proteoglycan aggregate may be the result of radical-mediated damage.

Aging↗
PubMed1987

The role of mononuclear cell products as modulators of cartilage degradation.

Human articular cartilage cultured in the presence of interleukin 1 (IL-1) produced from activated peripheral blood mononuclear cells, exhibits the concomitant release of a latent metalloproteinase and fragments of proteoglycan. The manner in which the activated form of the metalloproteinase degrades proteoglycan subunit and link protein suggests a role for this enzyme in cartilage degradation in vivo. While cartilage cultured in the continuous presence of purified IL-1 shows sustained release of metalloproteinase, tissue cultured in the presence of the total products released by activated mononuclear cells from some individuals showed initial stimulation followed by a decline, suggesting the presence of a modulator of IL-1 action.

Cartilage, Articular↗
PubMed1986

The action of human articular-cartilage metalloproteinase on proteoglycan and link protein. Similarities between products of degradation in situ and in vitro.

Interleukin 1 stimulation of human articular cartilage in organ culture produced the concomitant release of proteoglycan fragments and latent metalloproteinase. The released fragments ranged in size from that of almost intact proteoglycan subunits to the product of limiting digestion generated by the activated metalloproteinase. None of the fragments possessed the ability to interact with hyaluronic acid. Analysis of proteoglycan aggregate digested with the activated metalloproteinase showed that isolated hyaluronic acid-binding regions were produced from the proteoglycan subunits, and that the two higher-Mr link-protein components (Mr 48,000 and 44,000) were converted into the lowest-Mr component (Mr 41,000). Link protein extracted from cartilage under stimulation with interleukin 1 showed a similar conversion. These results suggest that interleukin 1 stimulates the release of latent metalloproteinase from chondrocytes and that a proportion of the enzyme is activated in situ in the cartilage matrix. The mode of action of the activated enzyme is compatible with a role in the changes in proteoglycan structure seen in aging.

Cartilage, Articular↗
PubMed1986

Interrelationship of active and latent secreted human cathepsin B precursors.

Two high-Mr forms of cathepsin B have been described previously, both of which are stable at alkaline pH, in contrast with the lysosomal proteinase. One form is latent and activated by pepsin treatment; the other form is active as measured with synthetic substrates. In the present study it was shown that the two forms are indistinguishable on the basis of molecular size as determined by gel-filtration chromatography or sodium dodecyl sulphate/polyacrylamide-gel electrophoresis followed by immunoblotting. Both forms lose their alkali-stability upon exposure to Hg2+, and after Hg2+ treatment the latent form becomes immuneprecipitable by an antiserum that reacts only with denatured cathepsin B. Lysosomal cathepsin B is bound by the plasma proteinase inhibitor alpha 2-macroglobulin, a process that requires proteolytic cleavage of the inhibitor. In contrast, the stable active form of cathepsin B is not bound by this inhibitor unless this enzyme is first destabilized by Hg2+ treatment. These results indicate that cathepsin B exists in three different states of activity, completely latent, partially active and fully proteolytically active. To exhibit true endopeptidase activity it seems that the enzyme must be in an alkali-unstable form.

Breast Neoplasms↗
PubMed1986

Human articular cartilage secretes characteristic metal dependent proteinases upon stimulation by mononuclear cell factor.

Human articular cartilage stimulated during organ culture by the addition of human blood mononuclear cell factor concomitantly released latent proteinases and proteoglycan. Gel chromatography at pH 7.4 separated 2 distinct latent enzymes: a general proteinase which could degrade proteoglycan, casein and gelatin, and a collagenolytic activity of smaller molecular size. The general proteinase was calcium dependent and could be activated by either trypsin or 4-aminophenylmercuric acetate; the former procedure invariably generating greatest activity. The enzyme exhibited a broad bimodal pH profile against proteoglycan, with optimum activity at pH 5.5.

Adult↗
PubMed1985

The origin of human cartilage proteoglycan link-protein heterogeneity and fragmentation during aging.

Human articular-cartilage link proteins are resolved into three components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicative of three different structures. The action of the proteinase clostripain yields a single link-protein component with electrophoretic properties analogous to those of the smallest (most mobile) native link protein, suggesting that this link protein may be derived naturally from one or both of the larger molecules by proteolytic cleavage in situ. Upon chemical deglycosylation of native link protein two components are resolved, suggesting that two of the link proteins differ only in their degree and/or type of oligosaccharide substitution. This pattern is compatible with a proteolytic origin for the smallest link protein. During aging further proteolytic fragmentation occurs, though it is only apparent on reduction of disulphide bonds. This fragmentation occurs at identical sites in all three native link proteins, indicating the existence of a large region common to all the link proteins, which appears to consist predominantly of the C-terminal half of the molecules. These observations are compatible with the variation in oligosaccharide and proteolytic heterogeneity occurring at the N-terminus of the link proteins.

Aging↗
PubMed1985

Rat mammary gland in culture secretes a stable high molecular weight form of cathepsin L.

Culture medium from rat mammary gland explants was analyzed for the presence of cysteine proteinases. In addition to a putative precursor of the lysosomal enzyme cathepsin B, a cysteine proteinase with enzymatic properties similar to those reported for cathepsin L was found. Further evidence of the cathepsin L-like nature of this activity was provided by its high sensitivity towards the diazomethane inhibitors Z-Phe-Phe-CHN2 and Z-Phe-Ala-CHN2 and towards leupeptin. The secreted form of cathepsin L is distinguished from the lysosomal form by its increased stability at alkaline pH and by its larger molecular size. It may thus represent an incompletely processed precursor form of the lysosomal enzyme.

Animals↗
PubMed1985

Resolution of cartilage proteoglycan and its proteolytic degradation products by high-performance liquid chromatography using a gel filtration system.

Cartilage proteoglycan subunits are resolved from their various-size proteolytic degradation products by a gel filtration high-performance liquid chromatography system using a Bio-Gel TSK-60 column in tandem with a Bio-Gel TSK-50 column. Molecules ranging in size from the intact proteoglycan to single chondroitin sulfate chains are eluted in the included volume. Each analysis takes less than 30 min to complete, and with purified samples as little as 20 micrograms of proteoglycan is required. The method can be applied to the measurement of proteoglycan in mixtures, such as tissue culture media, by monitoring effluent fractions using the dimethylmethylene blue dye-binding assay.

Adult↗
PubMed1985

Characterization of a cysteine proteinase secreted by mouse mammary gland.

The cysteine proteinase activity secreted by cultured mammary gland was characterized to determine its relationship to a similar enzyme secreted by explants of mouse mammary tumors. Enzymic characterization showed that the secreted enzyme was similar to the lysosomal cysteine proteinase cathepsin B, and physical characterization showed properties identical to a stable cysteine proteinase secreted from mammary tumors reported previously. The secreted enzyme cross-reacted with mouse cathepsin B isolated from liver in a radioimmunoassay. The secreted enzymes are stable at alkaline pH, but irreversible conformational changes can be induced in vitro which render them unstable and thus similar to lysosomal cathepsin B, which is also unstable at alkaline pH. Tissue homogenates from fresh and cultured mammary gland contain mainly pH-unstable cathepsin B; however, the molecular size for the tissue cathepsin B, while smaller than that of the secreted enzymes, was found to be larger than that reported for mouse liver cathepsin B. Isoelectric focusing profiles were also slightly different as compared to those of mouse liver. These data suggest that there might be differences in the processing of cathepsin B between different tissues and organs, and the high degree of similarity between the forms of cathepsin B secreted from mouse mammary tumors, mouse mammary gland, and human malignant breast tumors suggests a similar mechanism for their extracellular release in these tissues.

Animals↗