David J. Buttle

Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue

The dimethylmethylene blue assay for sulphated glycosaminoglycans has found wide acceptance as a quick and simple method of measuring the sulphated glycosaminoglycan content of tissues and fluids. The available assay methods have lacked specificity for sulphated glycosaminoglycans in the presence of other polyanions, however, and have not discriminated between the different sulphated glycosaminoglycans. We now describe a modified form of the dimethylmethylene blue assay that has improved specificity for sulphated glycosaminoglycans, and we show that in conjunction with specific polysaccharidases, the dimethylmethylene blue assay can be used to quantitate individual sulphated glycosaminoglycans.

Matrix synthesis and degradation in human intervertebral disc degeneration.

Degeneration of the intervertebral disc has been implicated in chronic low back pain. Type II collagen and proteoglycan (predominantly aggrecan) content is crucial to proper disc function, particularly in the nucleus pulposus. In degeneration, synthesis of matrix molecules changes, leading to an increase in the synthesis of collagens type I and III and a decreased production of aggrecan. Linked to this is an increased expression of matrix-degrading molecules including MMPs (matrix metalloproteinases) and the aggrecanases, ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) 1, 4, 5, 9 and 15, all of which are produced by native disc cells. Importantly, we have found that there is a net increase in these molecules, over their natural inhibitors [TIMP-1 (tissue inhibitor of metalloproteinases-1), 2 and 3], suggesting a deregulation of the normal homoeostatic mechanism. Growth factors and cytokines [particularly TNFalpha (tumour necrosis factor alpha) and IL-1 (interleukin 1)] have been implicated in the regulation of this catabolic process. Our work has shown that in degenerate discs there is an increase in IL-1, but no corresponding increase in the inhibitor IL-1 receptor antagonist. Furthermore, treatment of human disc cells with IL-1 leads to a decrease in matrix gene expression and increased MMP and ADAMTS expression. Inhibition of IL-1 would therefore be an important therapeutic target for preventing/reversing disc degeneration.

CA074 methyl ester: A proinhibitor for intracellular cathepsin B

The specificity of compound CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-pro line] for the inactivation of cathepsin B was quantified in in vitro measurements with cysteine endopeptidases from cattle, it being found that the compound is a very rapid inactivator of cathepsin B (rate constant 112,000 M-1.s-1), with barely detectable action on cathepsins H, L, and S or m-calpain. Conversion of the proline carboxyl group of the inhibitor to the methyl ester virtually abolished the effect on cathepsin B, and a possible explanation for the importance of the carboxyl is presented on the basis of the tertiary structure of cathepsin B. It was found that CA074 methyl ester (1 microM, 3 h) caused selective inactivation of the intracellular cathepsin B of human gingival fibroblasts in culture, in contrast to other available agents, and we suggest that CA074 methyl ester will be of value in the elucidation of the biological functions of cathepsin B.

Modified expression of the ADAMTS enzymes and tissue inhibitor of metalloproteinases 3 during human intervertebral disc degeneration

OBJECTIVE Intervertebral disc degeneration is linked to loss of extracellular matrix (ECM), particularly the early loss of aggrecan. A group of metalloproteinases called aggrecanases are important mediators of aggrecan turnover. The present study was undertaken to investigate the expression of the recognized aggrecanases and their inhibitor, tissue inhibitor of metalloproteinases 3 (TIMP-3), in human intervertebral disc tissue. METHODS Twenty-four nondegenerated and 30 degenerated disc samples were analyzed for absolute messenger RNA (mRNA) copy number of ADAMTS 1, 4, 5, 8, 9, and 15 and TIMP-3 by real-time reverse transcription-polymerase chain reaction. Thirty-six formalin-fixed embedded intervertebral disc samples of varying grades of degeneration were used for immunohistochemical analyses. In addition, samples from 8 subjects were analyzed for the presence of matrix metalloproteinase (MMP)- and aggrecanase-generated aggrecan products. RESULTS Messenger RNA for all the aggrecanases other than ADAMTS-8 was identified in intervertebral disc tissue, as was mRNA for TIMP-3. Levels of mRNA expression of ADAMTS 1, 4, 5, and 15 were significantly increased in degenerated tissue compared with nondegenerated tissue. All these aggrecanases and TIMP-3 were also detected immunohistochemically in disc tissue, and numbers of nucleus pulposus cells staining positive for ADAMTS 4, 5, 9, and 15 were significantly increased in degenerated tissue compared with nondegenerated tissue. Aggrecan breakdown products generated by MMP and aggrecanase activities were also detected in intervertebral disc tissue. CONCLUSION The aggrecanases ADAMTS 1, 4, 5, 9, and 15 may contribute to the changes occurring in the ECM during intervertebral disc degeneration. Targeting these enzymes may be a possible future therapeutic strategy for the prevention of intervertebral disc degeneration and its associated morbidity.

Stem bromelain: Amino acid sequence and implications for weak binding of cystatin

The amino acid sequence of stem bromelain, the major cysteine proteinase from pineapple stem is described. It shows that the enzyme is a member of the papain superfamily of cysteine proteinases, but is not very closely related to any other known member of this group. The sequence shows mutation or deletion of several residues that have been conserved in cysteine proteinases examined previously, including Asn‐175 (papain). We suggest that some of these changes have the effect of altering the active‐site geometry of stem bromelain, and that this accounts for the resistance of the enzyme to inhibition by cystatins and E‐64 [L‐3‐carboxy‐2,3‐trans‐epoxypropionylleucylamido(4‐guanidino)butane].

Agonists of toll-like receptor (TLR)2 and TLR4 are unable to modulate platelet activation by adenosine diphosphate and platelet activating factor

Inappropriate platelet activation is a feature of acute and chronic diseases such as disseminated intravascular coagulation (DIC) and atherosclerosis. Since proinflammatory microbial-derived agonists can be involved in the pathogenesis of these diseases, we examined the potential role of TLR4 (mediating responses to LPS) and TLR2 (which responds to bacterial lipopeptides) in platelet activation. Our data suggested low-level expression of TLR2 and TLR4 on platelets, determined by flow cytometry, and we also observed expression of TLR4 on a megakaryocytic cell line by both flow cytometry and immunohistochemistry. Stimulation of the platelets with the TLR4 agonist LPS, and the synthetic TLR2 agonist Pam3CSK4, resulted in no platelet aggregation, no increase in CD62P surface expression and no increase in the cytosolic concentration of Ca2+. The TLR agonists were also unable to directly activate platelets primed with epinephrine, or pretreated with a low concentration of ADP or PAF. Pretreatment of platelets with LPS or Pam3CSK4 also failed to modulate the platelet response to submaximal concentrations of the classical platelet agonists ADP and PAF. We conclude that the TLR agonists LPS and Pam3CSK4 have no direct effect on platelet activation and that platelet TLRs may be a remnant from megakaryocytes. TLR2 and TLR4 agonists are thought to have a significant role in diseases such as atherosclerosis and DIC, but our research suggests that this is through a mechanism other than direct platelet activation or by modification of platelet responses to other agonists.

Assessment of the anthelmintic effect of natural plant cysteine proteinases against the gastrointestinal nematode, Heligmosomoides polygyrus, in vitro

We examined the mechanism of action and compared the anthelmintic efficacy of cysteine proteinases from papaya, pineapple, fig, kiwi fruit and Egyptian milkweed in vitro using the rodent gastrointestinal nematode Heligmosomoides polygyrus. Within a 2 h incubation period, all the cysteine proteinases, with the exception of the kiwi fruit extract, caused marked damage to the cuticle of H. polygyrus adult male and female worms, reflected in the loss of surface cuticular layers. Efficacy was comparable for both sexes of worms, was dependent on the presence of cysteine and was completely inhibited by the cysteine proteinase inhibitor, E-64. LD50 values indicated that the purified proteinases were more efficacious than the proteinases in the crude latex, with purified ficin, papain, chymopapain, Egyptian milkweed latex extract and pineapple fruit extract containing fruit bromelain, having the most potent effect. The mechanism of action of these plant enzymes (i.e. an attack on the protective cuticle of the worm) suggests that resistance would be slow to develop in the field. The efficacy and mode of action make plant cysteine proteinases potential candidates for a novel class of anthelmintics urgently required for the treatment of humans and domestic livestock.

Cleavage of cartilage oligomeric matrix protein (thrombospondin-5) by matrix metalloproteinases and a disintegrin and metalloproteinase with thrombospondin motifs

Cartilage oligomeric matrix protein (COMP) is a pentameric glycoprotein present in cartilage, tendon and ligament. Fragments of the molecule are present in the diseased cartilage, synovial fluid and serum of patients with knee injuries, osteoarthritis and rheumatoid arthritis. Although COMP is a substrate for several matrix metalloproteinases (MMPs), the enzymes responsible for COMP degradation in vivo have yet to be identified. In this study we utilised well-established bovine cartilage culture models to examine IL-1alpha-stimulated COMP proteolysis in the presence and absence of MMP inhibitors. COMP was released from bovine nasal cartilage, in response to IL-1alpha, at an intermediate time between proteoglycans and type II collagen, when soluble MMP levels in the culture medium were undetectable. The major fragment of COMP released following IL-1alpha-stimulation migrated with an apparent molecular mass of approximately 110 kDa (Fragment-110) and co-migrated with both the major fragment present in human arthritic synovial fluid samples and the product of COMP cleavage by purified MMP-9. However, the broad-spectrum MMP and ADAM inhibitor BB94 only partially inhibited the formation of Fragment-110 and failed to inhibit COMP release significantly. Therefore the results of these studies indicate a role for proteinases other than MMPs in the degradation of COMP in bovine cartilage. It was further demonstrated that purified COMP was cleaved by ADAMTS-4, but not ADAMTS-1 or -5, to yield a fragment which co-migrated with Fragment-110. Therefore this is the first demonstration of COMP as a substrate for ADAMTS-4, although it remains to be determined whether this enzyme plays a role in COMP degradation in vivo.

The proteolytic activities of chymopapain, papain, and papaya proteinase III

The three proteinases present in papaya latex: papain (EC 3.4.22.2) chymopapain and papaya proteinase III (EC 3.4.22.6), were standardized by active-site titration, and compared in proteolytic activity against azocasein, serum albumin and cartilage proteoglycan. The activities were all of the same order, although there were differences in pH dependence. SDS-polyacrylamide gel electrophoresis of the early products of digestion of albumin and phosphorylase a showed very similar patterns for the three papaya proteinases. Kinetic parameters for hydrolysis of benzyloxycarbonyl-phenylalanyl-arginyl-7(4-methyl)coumarylamide were determined for the three enzymes. Values for kcat/Km varied only within a factor of 2, but the individual constants were much higher for papain than for chymopapain and papaya proteinase III. In contrast to the results obtained with the synthetic substrate, the kinetic parameters for the initial hydrolysis of succinyl-albumin were very similar for the three papaya proteinases. This was consistent with their similar proteolytic activities in other assays.

Levels of neutrophil elastase and cathepsin B activities, and cystatins in human sputum: relationship to inflammation

Sputum samples from 25 patients with bronchiectasis were assayed enzymatically for myeloperoxidase, neutrophil elastase and cathepsin B, and immunologically for cystatin A, cystatin B, cystatin C, cystatin S and kininogen. High myeloperoxidase and neutrophil elastase levels were found in those sputum samples that were assessed visually to be purulent. These samples were also found to contain high levels of cathepsin B activity and cystatin A, but low levels of cystatin S and of the most effective cathepsin B inhibitor, cystatin C. In contrast, sputum samples that were low in myeloperoxidase and neutrophil elastase activities had low levels of cathepsin B and cystatin A, but high cystatin C and S levels. It is concluded that cathepsin B activity in sputum is positively correlated with the degree of inflammation and neutrophil recruitment. Although this may be due in part to reduced amounts of cathepsin B inhibitors, particularly cystatin C, theoretical considerations suggest that factors other than the gross level of inhibitors must be involved in the control of cathepsin B activity.