Proteoglycan-degrading Enzymes Research Articles

Expression of ADAMTS 1-4-8 and placental growth factor in ovary and oviduct during pregnancy in the first trimester.

A Disintegrin and Metalloprotease Domains with Thrombospondins Motifs (ADAMTS), proteinases responsible for the destruction of extracellular matrix structures, have essential roles in the physiological and pathological processes of the female reproductive system, which is a dynamic structure. This study aimed to evaluate the immunoreactivity of placental growth factor (PLGF) and ADAMTS' (1, -4, and -8) in the ovary and oviduct during pregnancy in the first trimester. Our findings suggest a predominant role of ADAMTS-4 and ADAMTS-8 as proteoglycan-degrading enzymes from the ADAMTS-1 in the first trimester. As an angiogenic factor, PLGF showed more immunoreactivity than ADAMTS-1 in the ovary. This study provides the first evidence that ADAMTS-4 and ADAMTS-8 are more expressed in ovarian cells and follicles at different developmental stages during the first trimester of pregnancy than ADAMTS-1. Consequently, we suggest that ADAMTSs and PLGF act together and may exert specific effects on the formation, stabilisation, and function (or a combination thereof) of the matrix surrounding and protecting the follicles.

Matrix metalloproteinase-13 is fully activated by neutrophil elastase and inactivates its serpin inhibitor, alpha-1 antitrypsin: Implications for osteoarthritis.

Matrix metalloproteinase-13 (MMP-13) is a uniquely important collagenase that promotes the irreversible destruction of cartilage collagen in osteoarthritis (OA). Collagenase activation is a key control point for cartilage breakdown to occur, yet our understanding of the proteinases involved in this process is limited. Neutrophil elastase (NE) is a well-described proteoglycan-degrading enzyme which is historically associated with inflammatory arthritis, but more recent evidence suggests a potential role in OA. In this study, we investigated the effect of neutrophil elastase on OA cartilage collagen destruction and collagenase activation. Neutrophil elastase induced significant collagen destruction from human OA cartilage exvivo, in an MMP-dependent manner. Invitro, neutrophil elastase directly and robustly activated pro-MMP-13, and N-terminal sequencing identified cleavage close to the cysteine switch at 72 MKKPR, ultimately resulting in the fully active form with the neo-N terminus of 85 YNVFP. Mole-per-mole, activation was more potent than by MMP-3, a classical collagenase activator. Elastase was detectable in human OA synovial fluid and OA synovia which displayed histologically graded evidence of synovitis. Bioinformatic analyses demonstrated that, compared with other tissues, control cartilage exhibited remarkably high transcript levels of the major elastase inhibitor, (AAT) alpha-1 antitrypsin (gene name SERPINA1), but these were reduced in OA. AAT was located predominantly in superficial cartilage zones, and staining enhanced in regions of cartilage damage. Finally, active MMP-13 specifically inactivated AAT by removal of the serine proteinase cleavage/inhibition site. Taken together, this study identifies elastase as a novel activator of pro-MMP-13 that has relevance for cartilage collagen destruction in OA patients with synovitis.

Intracerebral chondroitinase ABC and heparan sulfate proteoglycan glypican improve outcome from chronic stroke in rats

Physical and chemical constraints imposed by the periinfarct glial scar may contribute to the limited clinical improvement often observed after ischemic brain injury. To investigate the role of some of these mediators in outcome from cerebral ischemia, we treated rats with the growth-inhibitory chondroitin sulfate proteoglycan neurocan, the growth-stimulating heparan sulfate proteoglycan glypican, or the chondroitin sulfate proteoglycan-degrading enzyme chondroitinase ABC. Neurocan, glypican, or chondroitinase ABC was infused directly into the infarct cavity for 7 d, beginning 7 d after middle cerebral artery occlusion. Glypican and chondroitinase ABC reduced glial fibrillary acidic protein immunoreactivity and increased microtubule-associated protein-2 immunoreactivity in the periinfarct region, and glypican- and chondroitinase ABC-treated rats showed behavioral improvement compared with neurocan- or saline-treated rats. Glypican and chondroitinase ABC also increased neurite extension in cortical neuron cultures. Glypican increased fibroblast growth factor-2 expression and chondroitinase ABC increased brain-derived neurotrophic factor expression in these cultures, whereas no such effects were seen following neurocan treatment. Thus, treatment with glypican or enzymatic disruption of neurocan with chondroitinase ABC improves gross anatomical, histological, and functional outcome in the chronic phase of experimental stroke in rats. Changes in growth factor expression and neuritogenesis may help to mediate these effects.

The endogenous proteoglycan-degrading enzyme ADAMTS-4 promotes functional recovery after spinal cord injury

BackgroundChondroitin sulfate proteoglycans are major inhibitory molecules for neural plasticity under both physiological and pathological conditions. The chondroitin sulfate degrading enzyme chondroitinase ABC promotes functional recovery after spinal cord injury, and restores experience-dependent plasticity, such as ocular dominance plasticity and fear erasure plasticity, in adult rodents. These data suggest that the sugar chain in a proteoglycan moiety is essential for the inhibitory activity of proteoglycans. However, the significance of the core protein has not been studied extensively. Furthermore, considering that chondroitinase ABC is derived from bacteria, a mammalian endogenous enzyme which can inactivate the proteoglycans' activity is desirable for clinical use.MethodsThe degradation activity of ADAMTS-4 was estimated for the core proteins of chondroitin sulfate proteoglycans, that is, brevican, neurocan and phosphacan. To evaluate the biological significance of ADMATS-4 activity, an in vitro neurite growth assay and an in vivo neuronal injury model, spinal cord contusion injury, were employed.ResultsADAMTS-4 digested proteoglycans, and reversed their inhibition of neurite outgrowth. Local administration of ADAMTS-4 significantly promoted motor function recovery after spinal cord injury. Supporting these findings, the ADAMTS-4-treated spinal cord exhibited enhanced axonal regeneration/sprouting after spinal cord injury.ConclusionsOur data suggest that the core protein in a proteoglycan moiety is also important for the inhibition of neural plasticity, and provides a potentially safer tool for the treatment of neuronal injuries.

Double-Valve Replacement for Scheie's Syndrome Subtype Mucopolysaccaridosis Type 1-S

Scheie's syndrome is a rare hereditary disorder of proteoglycan-degrading enzymes. Deposition of mucopolysaccharide can cause valvular and other tissue abnormalities. Few studies have reported surgical results for Scheie's syndrome, and the perioperative course is complicated. This is a report of a 35-year-old woman with Scheie's syndrome who underwent double-valve replacement with intensive perioperative management.

Chondroitinase ABC-Mediated Plasticity of Spinal Sensory Function

Experimental therapeutics designed to enhance recovery from spinal cord injury (SCI) primarily focus on augmenting the growth of damaged axons by elevating their intrinsic growth potential and/or by nullifying the influence of inhibitory proteins present in the mature CNS. However, these strategies may also influence the wiring of intact pathways. The direct contribution of such effects to functional restoration after injury has been mooted, but as yet not been described. Here, we provide evidence to support the hypothesis that reorganization of intact spinal circuitry enhances function after SCI. Adult rats that underwent unilateral cervical spared-root lesion (rhizotomy of C5, C6, C8, and T1, sparing C7) exhibited profound sensory deficits for 4 weeks after injury. Delivery of a focal intraspinal injection of the chondroitin sulfate proteoglycan-degrading enzyme chondroitinase ABC (ChABC) was sufficient to restore sensory function after lesion. In vivo electrophysiological recordings confirm that behavioral recovery observed in ChABC-treated rats was consequent on reorganization of intact C7 primary afferent terminals and not regeneration of rhizotomized afferents back into the spinal cord within adjacent segments. These data confirm that intact spinal circuits have a profound influence on functional restoration after SCI. Furthermore, comprehensive understanding of these targets may lead to therapeutic interventions that can be spatially tailored to specific circuitry, thereby reducing unwanted maladaptive axon growth of distal pathways.

Activity of hyaluronidase in placental tissues from cows with and without retained fetal membranes.

Hyaluronidase, a proteoglycan-degrading enzyme, may have an influence on collagenolysis in bovine placenta and take part in the separation processes of the placenta in cows. There was, however, no evidence concerning the activity of this enzyme in those tissues. The experiment was performed on cows divided into two groups as follows: A, placenta not retained (n = 16); and B, placenta retained (n = 9). The activity of the enzyme was measured using a spectrophotometric method and zymography. The results showed statistically significant higher activity of hyaluronidase in the maternal and fetal parts of the placenta in cows with a retention of fetal membranes. Further experiments require substrate specificity studies and the presence of regulatory factors of this enzyme in bovine placental tissues.

P2u purinoceptor regulation of mucin secretion in SPOC1 cells, a goblet cell line from the airways.

The SPOC1 cell, a novel goblet cell line derived from rat trachea, was tested for its ability to exhibit regulated mucin secretion in response to purinergic (P2) agonists. High-molecular mass glycoconjugates (HMMGs) purified by CsCl-density-gradient centrifugation had a buoyant density of 1.45 g/ml. The purified HMMG material exhibited a single major band with an apparent molecular mass of greater than 1000 kDa in SDS/ polyacrylamide gels stained with silver or blotted and stained with soya-bean agglutinin. [3H]HMMG was resistant to proteoglycan-degrading enzymes, but was susceptible to neuraminidase. The HMMG was approx. 91% carbohydrate by weight, and the glycosides were O-linked. The HMMG amino acid composition was enriched in Ser and Thr (sum 27%). Thus SPOC1-cell HMMG possess the characteristics of mucin. Mucin secretion by SPOC1 cells, grown on permeable supports and perfused luminally, was stimulated by ATP, UTP and adenosine 5'-[gamma-thio]triphosphate (100 microM) 4-5-fold over a baseline of 4 ng/min. The three dose-effect relations were nearly identical (K0.5 approximately 4 microM). SPOC1 cells grown on plastic and rat tracheal epithelial primary cells responded similarly to ATP and/or UTP. SPOC1 cells failed to respond to other purinergic agonists, either luminally or serosally, and consequently seem to possess an apical membrane P2u purinoceptor. SPOC1-cell total RNA was probed for P2u purinoceptor mRNA. Using conserved primers for both reverse transcriptase and PCR, a single band of the predicted size was observed, which had a nucleotide base sequence identical with the rat P2u purinoceptor mRNA. Thus SPOC1 cells secrete mucin under the control of a P2u purinoceptor; they should prove useful in dissecting the associated cellular regulatory pathways.

Substrate specificity of ‘elastomucoproteinase’: an enzyme which can degrade cartilage aggrecan

The substrate specificity of elastomucoproteinase (EMP), an enzyme which was first isolated from crude pancreatic elastase and described as a proteoglycan-degrading enzyme, determined on tripeptide-p-nitroanilide substrates indicates the existence of a 'new' chymotrypsin-like enzyme. EMP, however, did not cleave any glycosaminoglycans, i.e., its 'mucolytic' effect has been excluded. Activity of EMP on synthetic or protein substrates (e.g., collagen type-II and aggrecan of cartilage) was completely inhibited by serine proteinase inhibitors, which was also found when using cartilage proteoglycan monomers. EMP cleaves the core protein of proteoglycan monomer (aggrecan) into small peptides, some containing glycosaminoglycan chains resulting in an unusual elution profile on Sepharose CL-6B chromatography when compared to the effects of pancreatic and granulocyte elastases, chymotrypsin, cathepsin G and stromelysin. EMP-like activity also was detected in neutrophil granules of bovine leukocytes and polyclonal antibodies were raised against purified bovine EMP to detect the enzyme in both crude elastase preparations and the granule fraction of bovine leukocytes.

Degradation of Cartilage Collagens Type II, IX, X and XI by Enzymes Derived from Human Articular Chondrocytes

Conditioned culture medium derived from Interleukin-I α-activated human articular chondrocytes contained both collagen- and proteoglycan-degrading activities.Preparations of soluble type I collagen and the cartilage collagens type II, IX, X and XI were all degraded when incubated with the conditioned culture medium at 35°C.Fractionation of the enzymic activities using column chromatography with Ultragel AcA 34 and Heparin-Sepharose allowed the separation and identification of neutral proteinase, collagenolytic and proteoglycan-degrading activities.Eluant fractions which contained type I collagenase activity effectively degraded collagen type II, but these fractions did not correspond precisely with those which degraded collagen types IX, X and XI.These observations indicate that chondrocytes have the potential to produce a conventional interstitial type II collagenase together with other enzymes having some specificity for the minor collagens.Thus IL-1-activated chondrocytes produce a range of collagenolytic and proteoglycan-degrading enzymes which can process most of the structural components of the cartilage matrix.

Neutral protein-degrading enzymes in experimental fracture callus: a preliminary report.

The process of endochondral fracture healing is biochemically similar to growth plate calcification. Recent studies have identified potentially important roles for proteoglycan-degrading enzymes in the growth plate. The purpose of the study described herein was to identify, in healing fractures, neutral enzyme activities capable of degrading proteoglycans and other matrix proteins. Two sets of 60 male Sprague-Dawley rats underwent the production of closed femoral fractures. Calluses were retrieved at timed intervals, and cell and matrix vesicle fractions were prepared for electron microscopy, neutral peptidase, and alkaline phosphatase assays. In another group of 10 animals, fractions were prepared from 14-day calluses and examined for proteoglycanase activity. In the cell fractions, alkaline phosphatase, alanyl-beta-naphthylamidase, aminopeptidase, and endopeptidase activities showed somewhat parallel distributions peaking at approximately 14-17 days. In the matrix vesicle fractions, similar relative distributions were observed for alkaline phosphatase and endopeptidase. However, here the peak activities occurred up to 3 days later than they did in the cell fractions. Significant proteoglycanase activity was confirmed in both cell and matrix vesicle fractions. These findings are consistent with the hypotheses that (a) neutral peptidases, by virtue of their temporal expression in parallel with alkaline phosphatase, may be involved in preparing fracture callus matrix for calcification; and (b) matrix vesicles may convey certain of these enzymes to sites of both matrix degradation and calcification, since the same activities found in cells are found in matrix vesicles a few days later. The possibility that some of these enzymes are involved in growth factor activation remains to be investigated.

Proteoglycan- and collagen-degrading enzymes from human interleukin 1-stimulated chondrocytes from several species: proteoglycanase and collagenase inhibitors as potentially new disease-modifying antiarthritic agents.

Human IL-1-stimulated chondrocytes derived from rabbit, bovine, and human articular cartilage produce proteoglycan- and collagen-degrading enzymes. These studies demonstrate that the biological activity of IL-1 is not species specific. Several thiol, carboxyalkyl, and hydroxamic acid peptide inhibitors showed differential effects. The thiols were equipotent inhibitors of both the collagen- and proteoglycan-degrading enzymes whereas the carboxyalkyls appear to inhibit solely the proteoglycan-degrading enzyme(s). The hydroxamic acid peptides, the most potent inhibitors, appear to be more active against the proteoglycan-degrading enzymes. These synthetic inhibitors of proteoglycan- and/or collagen-degrading enzymes may represent a new class of disease-modifying antiarthritic agents.

Neutral proteases in human osteoarthritic synovium.

The activities of neutral collagenolytic enzymes (CE) and neutral proteoglycan-degrading enzymes (PE) in the synovial membranes of osteoarthritis (OA) patients were determined. The total neutral metallo-CE activity showed a significantly higher level of activity when the membranes of OA patients were compared with those of controls. The severely and moderately inflamed synovia had significantly more enzyme activity than did either mildly inflamed or control synovia. Steroids reduced the total metallo-CE activity. In specimens with severe inflammation, the active form of the neutral metallo-CE was significantly elevated over that found in controls. The serine-CE activity was also significantly elevated in OA synovia with severe inflammation and synovial hypertrophy. The total and active neutral metallo-PE was significantly elevated in synovial membranes of OA patients with severe inflammation. Moreover, the serine-PE showed much more activity in OA patients than in controls. The enzyme activity remained at a significantly high level in the OA synovium, regardless of the presence or absence of macroscopic synovial hypertrophy or the histologic grading of the synovium (mild, moderate, severe). Our data indicate that, in OA, an increased level of neutral proteases in the synovia could be involved in the local tissue destruction of the periarticular structures. Because of the very high level of serine proteases, their diffusion may render plausible a degradative action on the cartilage surface.

Secretion of an articular cartilage proteoglycan-degrading enzyme activity by murine T lymphocytes in vitro.

Destruction of articular cartilage is the hallmark of inflammatory arthritides. Enzymes elaborated by mononuclear cells infiltrating the synovium mediate, in part, the degradation of the cartilage extracellular matrix. Since mononuclear cells are the dominant cell type found in chronic inflammatory synovitis, we investigated whether interaction of immune mononuclear cells with antigen initiated the synthesis and secretion of a proteoglycan-degrading enzyme activity. Proteoglycan-degrading enzyme activity was monitored by the capacity of murine spleen cell conditioned medium to release [3H]serine/35SO4 incorporated into rabbit cartilage proteoglycan monomer fraction (A1D1), and by the relative change in specific viscosity of bovine nasal cartilage proteoglycan monomer. The results demonstrated that both virgin and immune mononuclear cells spontaneously generated proteoglycan-degrading enzyme activity and that cellular activation and proliferation induced by the antigen keyhole limpet hemocyanin or the mitogen phytohemagglutinin was not required. Kinetic studies demonstrated stable release of the enzyme activity over 72 h. Cell separation studies showed that T lymphocytes, a thymoma line, and macrophages separately produced proteoglycan-degrading enzyme activity. The enzyme activity has been partially characterized and appears to belong to a class of neutral pH metal-dependent proteinases. These observations, the first to demonstrate that T lymphocytes secrete an enzyme capable of degrading cartilage proteoglycan, raise the possibility that this enzyme activity contributes to cartilage extracellular matrix destruction in vivo. Moreover, these data support the conclusion that production of this enzyme by T lymphocytes is independent of an antigen-specific stimulus.

A Role for Pericellular Proteoglycan in the Binding of Thrombin or Antithrombin III by the Blood Vessel Endothelium? – The Effects of Proteoglycan-Degrading Enzymes and Glycosaminoglycan-Binding Proteins on 125I-Thrombin Binding by the Rabbit Thoracic Aorta In Vitro

Rabbit thoracic aorta segments were treated with either proteoglycan-degrading enzymes or with glycosaminoglycan-binding proteins to examine the nature of the endothelial and subendothelial binding sites of 125I-thrombin. Treatment (5-30 min) with enzymes (heparitinase, chondroitinases AC or ABC) caused a decrease in 125I-thrombin binding by the endothelium (30-70%) and by the subendothelial (intima-media) layer (20-50%); a low-specificity protease destroyed endothelial binding almost entirely and reduced binding to the subendothelium by approximately 60% over a similar period. Of the glycosaminoglycan-binding proteins, pretreatment of the aorta wall with protamine caused a 30% decrease in thrombin binding to the endothelium whereas lipoprotein lipase (present during 125I-thrombin uptake) decreased binding by up to 40%. Pretreatment with antithrombin III did not significantly affect binding of either 125I-thrombin or 125I-FPR-inactivated thrombin. In contrast to thrombin, 125I-antithrombin III was not readily uptaken by the aorta segments. These observations indicate that, whereas the minimal binding by 125I-antithrombin III probably does not involve endothelial proteoglycan, a strong case can be made for endothelial and subendothelial proteoglycan binding sites for thrombin.

Stimulation by human interleukin 1 of cartilage breakdown and production of collagenase and proteoglycanase by human chondrocytes but not by human osteoblasts in vitro

Human articular chondrocytes in culture synthesise collagenase and neutral proteoglycanase in response to addition of a 12–17 kDa protein produced by cultured human monocytes. This factor copurifies with interleukin 1, as assessed by lymphocyte activating factor activity, on gel filtration chromatography and isoelectric focusing. The interleukin 1 and chondrocyte-stimulating activities are destroyed by pretreatment of the material with phenylglyoxal. The same materials also promote the release of glycosaminoglycans from cultures of intact bovine nasal cartilage. The proteoglycanase activity released from chondrocytes appears to be a metalloproteinase because it is inhibited by EDTA and not phenylmethylsulphonyl fluoride (PMSF), and because detection of its activity is dependent on the presence of 4-aminophenylmercuric acetate. Human osteoblast-like cells do not respond to this factor by increased proteinase production, but are stimulated to produced prostaglandins. These results suggest that interleukin 1 has activities upon non-immune cells which promote degradation of connective tissue matrices. Human osteoblasts do not synthesise neutral collagen- and proteoglycan-degrading enzymes and thus are unlikely to be directly responsible for the matrix degradation which occurs during bone resorption.

The properties of the neutral proteinase released by primary chondrocyte cultures and its action on proteoglycan aggregate

The enzymatic mechanism of proteoglycan breakdown is of major interest, since it has been proposed that osteoarthritis involves increased proteolytic breakdown of proteoglycans. This paper describes the properties of the proteoglycan-degrading enzymes released into the extracellular milieu by chondrocyte cultures that produce cartilage-specific type Il collagen but no detectable type I collagen. Attention has been focused on enzymes active at neutral pH, since the pH of the exracellular matrix is around neutrality. Biogel P-60 chromatography of concentrated culture medium showed a major peak of enzyme activity on proteoglycan monomer entrapped in polyacrylamide beads as well as on native proteoglycan aggregates. The enzyme yields a specific limit digestion peptide from the aggregate of approximately 55000 daltons (in the presence of SDS). This limit peptide is probably derived from the hyaluronic acid-binding region of proteoglycan. The proteolytic enzyme is latent but can be activated by aminophenylmercuric acetate or trypsin. The molecular weight of both the active and latent forms, determined by gel filtration, is approximately 33000. The activity is not inhibited by phenylmethylsulfonyl fluoride or pepstatin but is completely inhibited by o-phenanthroline; the activity is restored by Zn or Co ions in the presence of calcium chloride. Removal of calcium by dialysis results in a reversible loss of activty. The release of such a metalloproteinase by chondrocytes into the extracellular milieu, its activity at physiological pH and its ability to degrade native proteoglycans are consistent with a role of the enzyme in proteoglycan metabolism.

Effects of various anti-inflammatory and anti-rheumatic agents on the synthesis, secretion, and activity of a cartilage proteoglycan-degrading enzyme and other macrophage enzymes

Enzymes released from the macrophage have been implicated in the connective tissue loss noted in a number of chronic inflammatory diseases. The effects of 2- and 3-day exposures to glucocorticosteroids, non-steroidal anti-inflammatory drugs and selected anti-rheumatic agents on release of a cartilage proteoglycan-degrading enzyme, and of β-glucuronidase, cathepsin D, and elastase from the thioglycollate-elicited mouse peritoneal macrophage were determined. The synthesis (and/or release) of the cartilage proteoglycan degrading enzyme was markedly inhibited following exposure to dexamethasone, flufenamic acid, chloroquine and gold sodium thiomalate. It was unaffected by exposure to aspirin, naproxen, indomethacin and dl-penicillamine, but was and stimulated by levamisole. Dexamethasone stimulated β-glucuronidase synthesis and release and inhibited elastase synthesis. The synthesis and release of β-glucuronidase, cathepsin D and elastase were not affected by non-toxic concentrations of indomethacin and chloroquine. These results demonstrate that certain anti-rheumatic and anti-inflammatory agents may derive at least a part of their therapeutic effects from the inhibition of enzyme release from the macrophage.

Properties of the proteoglycan-degrading enzyme released by primary cultures of bovine articular chondrocytes

Properties of the proteoglycan-degrading enzyme released by primary cultures of bovine articular chondrocytes

Proteoglycan-degrading enzymes of rabbit fibroblasts and granulocytes

A neutral proteinase secreted by rabbit synovial fibroblasts in parallel with specific collagenase was partially purified by ion-exchange chromatography. At pH 7.6 this proteinase degraded 35S-labelled bovine nasal proteoglycan and azo-casein. The enzymic activity was inhibited by EDTA, 1,10-phenanthroline and serum, whereas di-isopropyl phosphorofluoridate and soya-bean trypsin inhibitor had little effect. By gel filtration the apparent mol.wt. of the enzyme was 25000. The fibroblast neutral proteinase was compared with the proteoglycan-degrading neutral proteinases of rabbit polymorphonuclear-leucocyte granules. Two distinct activities were found in the granules: one was inhibited by soya-bean trypsin inhibitor and the other by EDTA. The proteoglycan-degrading proteinases of rabbit fibroblasts and polymorphonuclear leucocytes at acid pH also were examined. Both cathepsin D and a thiol-dependent proteinase contributed to the degradation of proteoglycan at pH 4.5.