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D J Buttle

Publications and source records attributed to D J Buttle.

At least 37 records · Page 2Linked to original sources
PubMed1998

Characterization of helical cleavages in type II collagen generated by matrixins.

Several vertebrate collagenases have been reported to cleave type II collagen, leading to irreversible tissue destruction in osteoarthritis. We have investigated the action of MMP-1 and MMP-13 on type II collagen by use of neoepitope antibodies and N-terminal sequencing. Previous studies have suggested that the initial cleavage of type II collagen by MMP-13 is followed by a second cleavage, three amino acids carboxy-terminal to the primary cleavage site. We show here that this cleavage is also produced by APMA-activated MMP-1 in combination with MMP-3 (i.e. fully activated MMP-1). The use of a selective inhibitor of MMP-3 has shown that it is this enzyme, rather than interstitial collagenase which had been exposed to MMP-3, which makes the second cleavage. In addition we have identified, through N-terminal sequencing, a third cleavage site, three residues carboxy-terminal to the secondary site. Since MMP-2 is thought to be responsible for gelatinolytic action on type II collagen we have investigated the effect of MMP-2 after the initial helical cleavage made by either MMP-1 or MMP-13. A combination of MMPs-1, -2 and -3 results in both the second and third cleavage sites; adding MMP-2 to MMP-13 did not alter the cleavage pattern produced by MMP-13 on its own. We conclude that none of the three cleavage sites will provide information about the specific identity of the collagenolytic enzymes involved in collagen cleavage in situ. Staining of cartilage sections of osteoarthritis patients with the neoepitope antibodies revealed type II collagen degradation starting at or near the articular surface and extending into the mid and deep zones with increasing degeneration of the cartilage.

Animals↗
PubMed1998

Cleavage of native type I collagen by human neutrophil elastase.

The ability of purified human neutrophil elastase (EC 3.4.21.37) to cleave native type I collagen has been investigated. Soluble human, bovine or rat type I collagen was incubated with neutrophil elastase for 16 h at 25 degrees C before catalysis was stopped with 3, 4-dichloroisocoumarin. Analysis by SDS/PAGE of the collagen digests revealed 3/4-length fragments similar in size to those produced by interstitial collagenase. The collagenolytic activity was dose dependent and was not due to a contaminating metalloproteinase or cysteine proteinase, as it was not inhibited by 1,10-phenanthroline, EDTA or L-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane. The identity of the cleavage products was confirmed using a new antibody that recognizes the unwound alpha2(I)-chain. This detected the 3/4-length fragment of type I collagen following neutrophil elastase cleavage. In addition to cleaving soluble collagen, neutrophil elastase also cleaved reconstituted, radiolabelled type I collagen fibrils, at a rate of 16 microg/min per nmol. These results indicate that neutrophil elastase can cleave native type I collagen in the helix, an activity that might contribute to its roles in connective-tissue pathology.

Animals↗
PubMed1998

Participation of intracellular cysteine proteinases, in particular cathepsin B, in degradation of collagen in periosteal tissue explants.

The involvement of cysteine proteinases in the degradation of soft connective tissue collagen was studied in cultured periosteal explants. Using cysteine proteinase inhibitors that were active intracellularly or extracellularly (Ep453 and Ep475, respectively), it was shown that over-all collagen degradation, as measured by the release of hydroxyproline, decreased significantly on inhibition of the intracellular pool of cysteine proteinases by Ep453. This inhibitor also induced an accumulation of intracellular fibrillar collagen in fibroblasts, indicating a decreased degradation of phagocytosed collagen. The extracellular inhibitor, Ep475, had minor or no effects. Histochemical analysis using a substrate for the cysteine proteinases cathepsins B and L revealed a high level of enzyme activity, which was completely blocked in explants preincubated with a selective intracellular inhibitor of cathepsin B, Ca074-Me. Moreover, the cathepsin B inhibitor strongly affected collagen degradation, decreasing the release of hydroxyproline and increasing the accumulation of phagocytosed collagen. These effects were comparable or slightly stronger than those found with the general intracellular inhibitor (Ep453). Taken together, these data strongly suggest that intracellular cysteine proteinases, in particular cathepsin B, play an important role in the digestion of soft connective tissue collagen.

Animals↗
PubMed1998

Stromelysin 1, neutrophil collagenase, and collagenase 3 do not play major roles in a model of chondrocyte mediated cartilage breakdown.

AIMS: To determine the collective roles of stromelysin 1, neutrophil collagenase, and collagenase 3 in chondrocyte mediated cartilage proteoglycan and type II collagen degradation in tissue culture model systems. METHODS: Bovine nasal cartilage explants were cultured with and without recombinant human interleukin 1 alpha (IL-1 alpha), recombinant human tumour necrosis factor alpha, or retinoic acid. Proteoglycan and type II collagen release were determined by colorimetric assay and immunoassay, respectively, in the absence and presence of matrixin inhibitors. Potential toxic effects of the inhibitors were assessed by measuring rates of glycolysis. RESULTS: Loss of proteoglycan and type II collagen from nasal cartilage was inhibited by batimastat, a broad spectrum matrixin inhibitor. BB-3437, a selective inhibitor of stromelysin, neutrophil collagenase, and collagenase 3, at the concentrations used in this study, showed a weak but dose dependent inhibitory effect on the IL-1 stimulated degradation of type II collagen, but had virtually no effect on proteoglycan breakdown. Neither inhibitor affected rates of glycolysis. CONCLUSIONS: Stromelysin 1, neutrophil collagenase, and collagenase 3 are unlikely to contribute to chondrocyte mediated proteoglycan degradation in our model system. The modest effect of a selective inhibitor of these enzymes on IL-1 stimulated collagen breakdown suggests a minor role for one or more of these proteinases; potent inhibition by an inhibitor of interstitial collagenase and the gelatinases suggests that these enzymes play a major role in IL-1 stimulated, chondrocyte mediated type II collagen breakdown from nasal cartilage.

Amides↗
PubMed1997

Proteoglycan breakdown from bovine nasal cartilage is increased, and from articular cartilage is decreased, by extracellular ATP.

The addition of ATP, but not ADP or AMP, to the culture media of bovine nasal cartilage explants caused an acceleration in the rate of proteoglycan loss from the tissue. The ATP-stimulated loss of proteoglycan was not inhibited by the IL1-receptor antagonist protein, but was partially inhibited by the presence of ADP or AMP. The proteolytic events resulting from the presence of ATP were found to be similar to those following treatment with IL1, in that inhibitors of the cysteine-peptidase cathepsin B, serine-proteinases with trypsin-like specificity, and of some of the matrixins, could all prevent proteoglycan loss, which was mediated, at least in part, by the action of 'aggrecanase'. In contrast to its effects on nasal cartilage, ATP inhibited basal and stimulated proteoglycan release from articular cartilage. Both ADP and AMP had no effect on proteoglycan release in articular cartilage but enhanced the response to ATP when added concurrently. We conclude that extracellular ATP, probably acting via P2-purinoceptors, stimulates proteoglycan breakdown from bovine nasal cartilage and thus, may have a role in diseases which primarily involve destruction of non-articular cartilage. Extracellular ATP has, in contrast, a chondroprotective effect on bovine articular cartilage.

Adenosine Diphosphate↗
PubMed1997

Degradation of type II collagen, but not proteoglycan, correlates with matrix metalloproteinase activity in cartilage explant cultures.

OBJECTIVE: To determine the contribution of certain matrix metalloproteinases (MMPs) to the degradation of proteoglycan and type II collagen in cartilage. METHODS: Bovine nasal and articular cartilage explants were cultured with recombinant human interleukin-1 alpha (IL-1 alpha) for up to 4 weeks. Release of proteoglycan and type II collagen into the medium was determined by colorimetric assay and immunoassay, respectively. The activity of MMPs in the medium was assayed using a quenched fluorescent substrate, as well as with a collagen fibril assay, by zymography, and in Western immunoblots. In some experiments, the effects of specific MMP inhibitors on type II collagen degradation were studied. RESULTS: In cultures of nasal cartilage with IL-1 alpha, almost all the proteoglycan was released within the first week, whereas there was no detectable release of type II collagen for the first 2 weeks of culture. A rapid period of almost complete dissolution of the collagen occurred in the third or fourth week. MMP activity measured using a quenched fluorescent substrate was negligible during the first 2 weeks of culture but was substantially increased in the third week of culture, at the time of collagen degradation. Similarly, there was a large increase in collagenolytic activity (by collagen fibril assay) and gelatinolytic activity (by zymography) during the third week of culture. Articular cartilage cultured with IL-1 alpha lost proteoglycan progressively during the 4-week period; however, there was no loss of type II collagen from the matrix in that time and no significant increase in MMP activity. The loss of type II collagen from nasal cartilage stimulated with IL-1 alpha was inhibited by BB87, an inhibitor of both collagenases and gelatinases, and by BB3003, a selective inhibitor of gelatinase A. In Western immunoblots, procollagenase and active interstitial collagenase could be readily detected in nasal cartilage cultures. Some procollagenase 3 and active collagenase 3 was also shown to be present. CONCLUSION: MMP activity correlates with degradation of type II collagen, but not proteoglycan, in cartilage cultures. Interstitial collagenase, collagenase 3, and gelatinases are all likely to contribute to cleavage and removal of collagen from the cartilage matrix. The proteinase(s) responsible for aggrecan breakdown remains unclear.

Animals↗
PubMed1997

Cathepsin B.

Cathepsin B is a lysosomal cysteine protease of the papain family. It functions in intracellular protein catabolism and in certain situations may also be involved in other physiological processes, such as processing of antigens in the immune response, hormone activation and bone turnover. There is also evidence that cathepsin B is implicated in the pathology of chronic inflammatory diseases of airways and joints, and in cancer and pancreatitis. In this short review we outline the major structural features of the enzyme, and describe how these relate to its synthesis, trafficking, processing and function.

Animals↗
PubMed1997

"Aggrecanase" activity is implicated in tumour necrosis factor alpha mediated cartilage aggrecan breakdown but is not detected by an in vitro assay.

AIMS: To develop an in vitro assay for the putative glutamyl endopeptidase, "aggrecanase", which is thought to degrade cartilage aggrecan, and to examine the role of the enzyme in tumour necrosis factor stimulated aggrecan cleavage. METHODS: Aggrecan fragments released by bovine nasal cartilage explants, with and without exposure to tumour necrosis factor alpha, were purified and analysed by western blotting and N-terminal sequencing. Intact bovine aggrecan was incubated with extracts of cartilage, lysed chondrocytes, or cartilage explant conditioned culture medium under a variety of conditions. Deglycosylated aggrecan was incubated with nasal cartilage explants. Proteoglycan breakdown was assessed by metachromatic assay of fragments in culture media, and cleavage of the substrate at the aggrecanase cleavage site was detected and measured using the antibody BC3, which recognises a neoepitope produced by aggrecanase cleavage of aggrecan. RESULTS: Aggrecan fragments generated from explants treated with tumour necrosis factor had N-terminal sequences consistent with cleavage of aggrecan at a restricted number of glutamyl bonds. Aggrecanase generated fragments were found in cartilage explant culture medium and chondrocyte monolayers. However, no aggrecanase activity could be detected in extracts of cartilage, or chondrocytes from which endogenous aggrecan fragments had been removed, under a variety of assay conditions. Deglycosylated aggrecan, added to explant cultures, efficiently inhibited endogenous aggrecan breakdown. CONCLUSIONS: Aggrecanase is active in cartilage and in chondrocyte monolayers, and its action is stimulated by tumour necrosis factor alpha. However, activity due to this enzyme could not be detected in vitro under our assay conditions, although a deglycosylated version of the substrate inhibited aggrecan breakdown in explant cultures.

Aggrecans↗
PubMed1996

Inhibitors of collagenase but not of gelatinase reduce cartilage explant proteoglycan breakdown despite only low levels of matrix metalloproteinase activity.

Aims-To investigate the level of matrix metalloproteinase activity during the time-course of cartilage explant proteoglycan breakdown; to determine the effects of selective small-molecule inhibitors of matrix metalloproteinases on proteoglycan degradation.Methods-The levels of matrix metalloproteinase activity in cartilage explant cultures and conditioned media were monitored by use of a quenched fluorescent substrate. The constants for inhibition of certain matrix metalloproteinases by a series of synthetic inhibitors were determined. Bovine and human cartilage explant cultures were treated with interleukin-1, tumor necrosis factor or retinoic acid and the amount of proteoglycan released into the culture medium in the absence and presence of the inhibitors was quantified. Control experiments, examining the inhibition of other proteinases, and investigating possible toxic or non-specific effects of the inhibitors, were carried out.Results-The profile of inhibition of proteoglycan release suggested the involvement of interstitial collagenase-like, rather than gelatinase- or possibly stromelysin-like, proteinases. No evidence was found for toxic or non-specific mechanisms of inhibition. Very low levels of activity of the known matrix metalloproteinases were present during the time-course of aggrecan breakdown.Conclusions-A novel collagenase-like proteinase(s) may be involved in cartilage proteoglycan breakdown. Gelatinase-type matrix metalloproteinases do not seem to be involved in this process. Specific collagenase inhibitors may be therapeutically efficacious in the treatment of arthritis.

Journal Article↗
PubMed1995

Crystal structure of glycyl endopeptidase from Carica papaya: a cysteine endopeptidase of unusual substrate specificity.

Glycyl endopeptidase is a cysteine endopeptidase of the papain family, characterized by specificity for cleavage C-terminal to glycyl residues only and by resistance to inhibition by members of the cystatin family of cysteine proteinase inhibitors. Glycyl endopeptidase has been crystallized from high salt with a substrate-like inhibitor covalently bound to the catalytic Cys 25. The structure has been solved by molecular replacement with the structure of papain and refined at 2.1 A to an R factor of 0.196 (Rfree = 0.258) with good geometry. The structure of the S1 substrate binding site of glycyl endopeptidase differs from that of papain by the substitution of glycines at residues 23 and 65 in papain, with glutamic acid and arginine, respectively, in glycyl endopeptidase. The side chains of these residues form a barrier across the binding pocket, effectively excluding substrate residues with large side chains from the S1 subsite. The constriction of this subsite in glycyl endopeptidase explains the unique specificity of this enzyme for cleavage after glycyl residues and is a major component of its resistance to inhibition by cystatins.

Amino Acid Sequence↗
PubMed1995

Kinetic constants for the hydrolysis of aggrecan by the papaya proteinases and their relevance for chemonucleolysis.

The four known proteinases from papaya latex, namely papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), caricain (EC 3.4.22.30), and glycyl endopeptidase (EC 3.4.22.25), were purified to homogeneity and fully characterized by single radial immunodiffusion and active-site titration. A modified HPLC gel permeation assay was used to determine the kinetic constants for aggrecan hydrolysis by the papaya proteinases. The disappearance of intact aggrecan monomer was first-order, indicating that for the four enzymes studied the Km was much larger than 0.5 microM and that kcat/Km = 1.2 +/- 0.1 x 10(6) M-1 s-1 for chymopapain, 1.20 +/- 0.08 x 10(6) M-1 s-1 for caricain, 0.90 +/- 0.02 x 10(6) M-1 s-1 for papain, and 0.120 +/- 0.005 x 10(6) M-1 s-1 for glycyl endopeptidase. Chymodiactin, the chymopapain preparation used for chemonucleolysis, consists of a mixture of chymopapain (70%), caricain (20%), and glycyl endopeptidase (4%). The rate constant for the aggrecan hydrolysis by such a mixture was not significantly different from the rate constant for pure chymopapain. As a result of these observations, we predict that pure chymopapain could replace partially purified chymopapain preparations for chemonucleolysis.

Aggrecans↗
PubMed1995

Immunoglobulin E antibodies to papaya proteinases and their relevance to chemonucleolysis.

STUDY DESIGN: Levels of four papaya cysteine proteinases were determined in Chymodiactin, a pharmaceutical preparation of chymopapain (EC 3.4.22.6) used in chemonucleolysis for the treatment of sciatica. Twelve sera known to contain immunoglobulin E antibodies to Chymodiactin were assayed for immunoglobulin E antibodies to these enzymes. OBJECTIVES: The goal of the study was to determine what contribution each of the four proteinases makes to the allergic response that occasionally occurs during injection of a damaged intervertebral disc with chymopapain preparations. SUMMARY OF BACKGROUND DATA: The occurrence of an allergic reaction during chemonucleolysis implies prior sensitization to components of the injected enzyme solution. The latex of the unripe fruit of the papaya plant Carica papaya, from which chymopapain is purified, contains another three immunologically distinct cysteine proteinases: 1) caricain (EC 3.4.22.30), 2) glycyl endopeptidase (EC 3.4.22.25), and 3) papain (EC 3.4.22.2). METHODS: A dot-blot immunoassay was developed to quantify each enzyme in Chymodiactin. Total serum immunoglobulin E levels and specific immunoglobulin E antibody levels to each of the four papaya cysteine proteinases were assayed by an enzyme-linked immunoassay in 12 sera containing immunoglobulin E antibodies to Chymodiactin. RESULTS: Chymodiactin contained 70% chymopapain, 20% caricain, 4% glycyl endopeptidase, and 0.1% papain. Immunoglobulin E antibodies to all four proteinases were found in most of the 12 sera, but in varying proportions. Antibodies to glycyl endopeptidase were predominant in eight sera, and the mean amounts of immunoglobulin E directed against each protein were: glycyl endopeptidase, 4.21 IU/ml; caricain, 2.9 IU/ml; chymopapain, 1.97 IU/ml; and papain, 1.39 IU/ml. Total serum immunoglobulin E levels showed little correlation with immunoglobulin E responses to Chymodiactin. CONCLUSIONS: The results suggested that removal of glycyl endopeptidase and caricain from pharmaceutical preparations of chymopapain may help reduce the incidence of allergic reactions during chemonucleolysis.

Chymopapain↗
PubMed1995

Synthesis and secretion of procathepsin B and cystatin C by human bronchial epithelial cells in vitro: modulation of cathepsin B activity by neutrophil elastase.

Procathepsin B and cystatin C are found in human lung secretions. We investigated the capacity of human bronchial epithelial cells to synthesize and secrete these proteins. Immunoprecipitation of [35S]methionine-labeled proteins from cultured bronchial epithelial cell lysates, followed by denaturing gel electrophoresis and autoradiography, showed the presence of newly synthesized procathepsin B of M(r) 42,000; no mature form was detected. Cathepsin B in conditioned medium from epithelial cells was tagged with benzyloxycarbonyl-125I-tyrosyl-alanine-diazomethane before and after treatment of the medium with neutrophil elastase. Control medium again showed a predominant form of cathepsin B with a M(r) of 42,000, but upon treatment with neutrophil elastase this protein was converted to a M(r) of 38,000, similar to the active form previously found in lung secretions, and cathepsin B activity was generated. The medium also contained the cathepsin B inhibitor, cystatin C, but cystatins A, B, S, SN, SA, and kininogen were not detected. After removal of cystatin C from the medium, elastase was still required to activate procathepsin B. These results suggest that bronchial epithelial cells are a source of procathepsin B and cystatin C in lung secretions. Cleavage both of cystatin C and procathepsin B by neutrophil elastase is essential for the generation of cathepsin B activity in the medium.

Bronchi↗
PubMed1994

Inhibition of bone resorption by selective inactivators of cysteine proteinases.

Inactivators of cysteine proteinases (CPs) were tested as inhibitors of bone resorption in vitro and in vivo. The following four CP inactivators were tested: Ep475, a compound with low membrane permeability which inhibits cathepsins B, L, S, H, and calpain; Ep453, the membrane-permeant prodrug of Ep475; CA074, a compound with low membrane permeability which selectively inactivates cathepsin B; and CA074Me, the membrane-permeant prodrug of CA074. The test systems consisted of 1) monitoring the release of radioisotope from prelabelled mouse calvarial explants and 2) assessing the extent of bone resorption in an isolated osteoclast assay using confocal laser microscopy. Ep453, Ep475, and CA074Me inhibited both stimulated and basal bone resorption in vitro while CA074 was without effect; the inhibition was reversible and dose dependent. None of the inhibitors affected protein synthesis, DNA synthesis, the PTH-enhanced secretion of beta-glucuronidase, and N-acetyl-beta-glucosaminidase, or the spontaneous release of lactate dehydrogenase. Ep453, Ep475, and CA074Me dose-dependently inhibited the resorptive activity of isolated rat osteoclasts cultured on bone slices with a maximal effect at 50 microM. The number of resorption pits and their mean volume was reduced, whilst the mean surface area remained unaffected. Again, CA074 was without effect. Ep453, Ep475, and CA074Me, but not CA074, when administered subcutaneously at a dose of 60 micrograms/g body weight inhibited bone resorption in vivo as measured by an in vivo/in vitro assay, by about 20%. This study demonstrates that cathepsins B, L, and/or S are involved in bone resorption in vitro and in vivo. Whilst cathepsin L and/or S act extracellularly, and possibly intracellularly, cathepsin B mediates its effects intracellularly perhaps through the activation of other proteinases involved in subosteoclastic collagen degradation.

Animals↗