Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.36 (hyaluronidase)
4,606 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Extracts of human peripheral blood polymorphonuclear leukocyte granules, and two purified proteases derived from such extracts, an elastase and a chymotrypsin-like enzyme, degrade isolated bovine nasal cartilage proteoglycan at neutral pH. Viscosity studies indicate that the leukocyte granule extracts lack hyaluronidase activity and that their degradative effect on proteoglycan at physiological pH is due entirely to proteolytic action. Sepharose 4B gel chromatography and SDS-polyacrylamide gel electrophoresis of proteoglycan fractions treated with leukocyte granule enzymes at pH 7.0 indicate that they degrade one of the proteoglycan link proteins, release a fragment from the hyaluronic acid-binding portion of the proteoglycan subunit core protein, and break down the remainder of the proteoglycan subunit molecule into peptide fragments with varying numbers of chondroitin sulfate chains. Immunodiffusion studies indicate that the antigenic determinants of the proteoglycan subunit core protein and the link proteins survive treatment with granule proteases. Similar degradation of human articular cartilage proteoglycan by granule neutral proteases can be presumed to occur, in view of the similarity of structure of human articular and bovine nasal cartilage proteoglycans. The release of granule enzymes in the course of neutrophil-mediated inflammation can thus result in the degradation of cartilage matrix proteoglycan, leading to cartilage destruction and joint injury.
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PMID:Degradation of cartilage proteoglycan by human leukocyte granule neutral proteases--a model of joint injury. II. Degradation of isolated bovine nasal cartilage proteoglycan. 12 83

The basic subunit of cartilage proteoglycan consists of multiple glycosaminoglycan chains covalently attached to a core protein. It is unclear as to whether there is a single core protein or multiple different core proteins, since previous studies using either chondroitinase or testicular hyaluronidase to enzymatically remove chondroitin sulfate side chains from the proteoglycan subunit have yielded conflicting results. In the present study, a chondroitinase-produced core protein preparation isolated as a single peak on Sepharose gel chromatography was found to contain at least two immunologically distinct components. Hyaluronidase-produced core protein from the same proteoglycan subunit fraction was found to contain multiple components nearly all of which were smaller than the components in the chondroitinase digest. A possible explanation of these findings is that they resulted from proteolytic degradation of the core protein in the course of the enzymatic removal of its chondroitin sulfate. The presence of small amounts of protease contaminants in several commercial chondroitinase and hyaluronidase preparations was detected by an extremely sensitive radioassay. Until proteases can be rigorously excluded from enzyme preparations used to degrade the proteoglycan subunit, it will not be possible to determine whether it consists of a single or several different core proteins.
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PMID:A comparison of bovine nasal cartilage proteoglycan core protein produced by chondroitinase and hyaluronidase: the possible role of protease contaminants. 14 80

In order to investigate the coordinated synthesis of matrix components by individual chondrocytes, specific antibodies to type I collagen, type II collagen, and chondroitin sulfate proteoglycan core protein were used in simultaneous double immunofluorescence reactions. Extensive accumulation of core protein surrounding chondrocytes and the intracellular accumulation of type II collagen were observed. Extracellular core protein immunofluorescence obscured the intracellular reaction product, but the extracellular immunoreactive material could be removed by digestion with purified testicular hyaluronidase prior to fixation. Subsequent to digestion, core protein and type II collagen were observed in the same chondrocytes within discrete, sometimes identical, cytoplasmic regions, thus demonstrating the simultaneous localization of these two products characteristic of differentiating cartilage.
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PMID:Simultaneous localization of type II collagen and core protein of chondroitin sulfate proteoglycan in individual chondrocytes. 37 34

A large proteoglycan (365 kDa), identified with monoclonal antibodies raised against chondroitin sulfate, was isolated from human brain. The isolation required anion-exchange chromatography followed by gel filtration through a Sephacryl S-500 column. The proteoglycan bound specifically to [3H]hyaluronate (HA). The binding was not reduced by high salt concentrations (up to 4 M) and was inhibited at low pH (< 4.0). The binding was inhibited by the octamer and decamer (but not the hexamer) oligosaccharides of HA. Limited proteolysis of the proteoglycan gave rise to a relatively stable polypeptide (80 kDa). The amino-terminal sequence of the 80-kDa polypeptide was identical to the cDNA-derived amino-terminal sequence of versican, a large human fibroblast proteoglycan. A monoclonal antibody raised against bovine proteoglycans and recognizing the versican core protein reacted by immunoblotting with the proteoglycan isolated from human brain. The antibody was used to localize the proteoglycan in acetone-fixed cryostat sections of bovine spinal cord. The localization of the proteoglycan in the central nervous system was identical to that previously reported for glial hyaluronate-binding protein (GHAP), a 60-kDa glycoprotein of the brain extracellular matrix (ECM). However, a major difference was observed with respect to the sensitivity of the two antigens to hyaluronidase. As previously reported, GHAP was released from the tissue by hyaluronidase digestion, whereas the proteoglycan persisted under these conditions. We conclude that the protein-hyaluronate aggregates in brain ECM contain both GHAP and versican, that GHAP is only retained in the ECM by its interaction with hyaluronate, and that the proteoglycan is anchored in some other manner and probably connects cell surfaces with the ECM since it was not released by hyaluronidase digestion.
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PMID:Isolation of a large aggregating proteoglycan from human brain. 142 26

After immunization of mice with partially-purified heparan sulfate proteoglycan (HSPG) isolated from rat glomeruli, a monoclonal antibody (mAb JM-403) was obtained, which was directed against heparan sulfate (HS), the glycosaminoglycan side chain of HSPG. In ELISA it reacted with isolated human glomerular basement membrane (GBM) HSPG, HS and hyaluronic acid, but not with the core protein of human GBM HSPG, and not with chondroitin sulfate A and C, dermatan sulfate, keratan sulfate and heparin. Furthermore, it did not bind to laminin, collagen type IV or fibronectin. Specificity of JM-403 for HS was also suggested by results of inhibition studies, which found that intact HSPG and HS, but not the core protein, inhibited the binding of JM-403 to HS. In indirect immunofluorescence on cryostat sections of rat kidney, a fine granular to linear staining of the GBM was observed, along with a variable staining of the other renal basement membranes. Pretreatment of the sections with heparitinase completely prevented the binding of mAb JM-403, whereas pretreatment with chondroitinase ABC or hyaluronidase had no effect. The precise binding site of mAb JM-403 was investigated by indirect immunoelectron microscopy. It revealed a diffuse staining of the whole width of the GBM. One hour after intravenous injection of JM-403 into rats, the mAb was detected along the glomerular capillary wall in a fine granular pattern, which shifted towards a more mesangial localization after 24 hours. No binding was observed anymore by day 15. Intravenous injection induced a dose-dependent, transient and selective proteinuria that was maximal immediately after the injection.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A monoclonal antibody against GBM heparan sulfate induces an acute selective proteinuria in rats. 159 46

We have recently shown that the large hyaluronan-aggregating chondroitin sulfate proteoglycan from cartilage (PG-LA) is unfavorable as a substrate for neural crest cell migration in vitro and that this macromolecule inhibits cell dispersion on fibronectin substrates when included in the medium (R. Perris and S. Johansson, 1987, J. Cell Biol. 105, 2511-2521). In this study we present data on the specificity of the migration-repressing activity of PG-LA and data on the molecular mechanisms by which the proteoglycan might impair neural crest cell motility. Soluble PG-LA potently impaired cell migration on substrates of laminin/laminin-nidogen, vitronectin, and collagen types I, III, IV, and VI. When tested in solid-phase binding assays, PG-LA bound avidly to substrates of collagen types I-III and V. Conversely, minimal amounts of the proteoglycan bound to substrates of laminin-nidogen, vitronectin, collagen types IV and VI, and fibronectin or to a proteolytic fragment encompassing its cell-binding domain (105 kDa). Preincubation of these substrates with soluble PG-LA prior to plating of the cells had no effect on their locomotory behavior. These results indicate that PG-LA affects neural crest cell movement primarily through an interaction with the cell surface, rather than by association with the cell motility-promoting substrate molecules. The molecular interaction of soluble PG-LA with neural crest cells was further examined by analyzing the effects of isolated domains of the proteoglycan on cell migration on fibronectin. Addition of chondroitin sulfate chains, the core protein free of glycosaminoglycans, the isolated hyaluronan-binding region (HABr), or a proteolytic fragment corresponding to the keratan sulfate-enriched domain of the PG-LA to neural crest cells migrating on fibronectin or the 105-kDa fibronectin fragment had no significant effect on their motility. After reduction and alkylation, PG-LA was considerably less efficient in perturbing cell movement on fibronectin substrates and virtually ineffective in altering migration on the 105-kDa fragment. In the presence of hyaluronan fragments of 16-30 monosaccharides in length, or an antiserum against the HABr, the migration repressing activity of PG-LA was reduced in a dose-dependent fashion. Furthermore, the inhibitory action of PG-LA was significantly reduced by treatment of the cells with Streptomyces hyaluronidase.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Inhibition of neural crest cell migration by aggregating chondroitin sulfate proteoglycans is mediated by their hyaluronan-binding region. 168 36

Chondrons have recently been extracted from adult articular cartilages and techniques developed to study their structure and composition in isolation. This study introduces methods to immobilize isolated canine chondrons in thin layers of agarose gel for immunohistochemistry and future in vitro studies. An antibody to Type VI collagen which stained the chondron in suspension was used to successfully validate the system and its feasibility for immunoelectron microscopy. Monoclonal and polyclonal antibodies to a variety of epitopes on the proteoglycan molecule were tested on fresh and fixed plugs cored from chondron-agarose gels. Plugs were immunolabeled with peroxidase-diaminobenzidine before or after digestion with testicular hyaluronidase or chondroitinase ABC. Trypsin/chymotrypsin were used to challenge epitopes of the core protein. The results indicate that epitopes to keratan sulfate, chondroitin sulfate, hyaluronate binding region, and core protein are localized in the chondron. Consistent staining was found in the tail and interconnecting segments between chondrons, whereas staining of the pericellular matrix and capsule adjacent to the chondrocyte varied according to the enzyme pre-treatment employed. We conclude that isolated chondrons are rich in proteoglycan monomer, which is particularly concentrated in the tail and interconnecting segments of the chondron where it could function to protect and stabilize the chondrocyte.
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PMID:Chondrons from articular cartilage. (IV). Immunolocalization of proteoglycan epitopes in isolated canine tibial chondrons. 171 45

NG2 is a chondroitin sulfate proteoglycan previously found to be expressed by glial progenitor cells of the O2A lineage. We have examined the expression of NG2 in the developing rat limb by immunohistochemistry and northern blot analysis. Staining of embryonic day 14 (E14) rat limb bud sections with polyclonal and monoclonal anti-NG2 antibodies reveals reactivity in the precartilaginous mesenchymal condensation. The staining intensity increases with the differentiation of chondrocytes until E16. NG2 staining is not detected in the mature hypertrophic chondrocytes of E17 and postnatal day 3 (P3) limbs even after treatment of the sections with hyaluronidase or collagenase. Immuno-precipitations with anti-NG2 antibody using 125I-labeled limb cells in culture showed a 400 to 800 x 10(3) Mr proteoglycan species with a core protein size of 300 x 10(3) Mr, comparable to NG2 from O2A cells and neural cell lines. Northern blot analysis reveals the expression of an 8.9 kb mRNA in E16 limbs and at a lower level in P1 cartilage. The northern blot analyses also show that NG2 is distinct from the large aggregating proteoglycan of the cartilage. Our results indicate that in the developing limb cartilage, as in the differentiating oligodendrocytes, NG2 is present on immature cells in the process of differentiating, but its expression is downregulated as terminal differentiation of chondrocytes takes place.
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PMID:The expression of NG2 proteoglycan in the developing rat limb. 187 62

Cytotactin is an extracellular matrix protein that is involved in neuron-glia adhesion and is found in both neural and nonneural sites. It is synthesized by glia but not by neurons. In this study, we have examined the binding of cytotactin to a variety of extracellular matrix components using uniform microscopic beads (Covaspheres) that could be labeled and then linked to purified molecules. Cytotactin-coated beads bound well to neurons, and this binding was strongly inhibited by anti-cytotactin antibodies but not by anti-neural cell adhesion molecule (anti-N-CAM) antibodies. In contrast, the binding of N-CAM-coated beads to neurons was inhibited by anti-N-CAM antibodies and not by anti-cytotactin antibodies. To identify a neuronal ligand for cytotactin, we tested several molecules for their ability to block the binding of cytotactin-coated beads to cells. A proteoglycan-containing fraction that copurified with cytotactin from brain extracts strongly inhibited binding, whereas neither a heparan sulfate proteoglycan from Engelbreth-Holm-Swarm tumor cells nor soluble cytotactin itself had a significant inhibitory effect. The neural proteoglycan also inhibited the binding of cytotactin-coated beads to fibroblasts. Digestion with chondroitinase, heparitinase, and hyaluronidase as well as immunological analyses suggested that the predominant species in the active fraction was a chondroitin sulfate proteoglycan with a Mr280,000 core protein bearing HNK-1 antigenic determinants and also indicated that hyaluronic acid was present in this fraction. In experiments on in vitro synthesis, it was found that the proteoglycan was synthesized in culture by embryonic chicken brain tissue but not by embryonic chicken glial cells. A series of binding experiments was performed on appropriately derivatized beads to confirm that the proteoglycan is a ligand for cytotactin and to check for the possibility that other extracellular matrix proteins might interact with one or the other member of this binding couple. Proteoglycan-coated beads and cytotactin-coated beads coaggregated readily. The aggregation was inhibitable by anti-cytotactin antibodies, soluble cytotactin, or soluble proteoglycan. Addition of laminin inhibited the binding of cytotactin-coated beads to proteoglycan-coated beads or to cells; this is consistent with data indicating that laminin interacts with a component of the proteoglycan-containing fraction. In contrast, fibronectin bound to cytotactin, but it did not bind to proteoglycan or interfere with the binding of cytotactin to proteoglycan. The results of this study are in accord with the idea that the functions of extracellular matrix components during neural and nonneural development may be modulated both by competition for shared cell surface receptors and by a network of molecular interactions among the matrix components themselves.
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PMID:A proteoglycan with HNK-1 antigenic determinants is a neuron-associated ligand for cytotactin. 243 34

This paper makes three points about how the chick corneal epithelium lays down the primary stroma, an orthogonally arranged array of well-spaced, 20-nm-diameter collagen fibrils. (1) Isolated corneal epithelia will, when cultured, lay down de novo stromas whose fibril-diameter distribution, fibril spacing, and proteoglycan profile are similar to those laid down in vivo. They differ from embryonic stromas in two ways: first, much of the chondroitin sulfate is released to the medium and, second, there is a relatively small amount of orthogonal organization. Epithelia seem only to lay down such stromas if they are separated from their original stromas with dispase, which leaves an intact basal lamina, and spread out, basal lamina downward, on a Nuclepore filter (poresize, 0.1 micron). (2) Chondroitin sulfate (CS), the predominant proteoglycan (greater than 85%), seems to play no significant role in collagen fibrillogenesis in vitro. Stromas laid down in its absence were indistinguishable from controls as assayed by fibril diameter, organization, and spacing and the amount of collagen synthesized. For these experiments, epithelia were cultured in the presence of hyaluronidase, which degrades CS, and p-nitrophenyl beta-D-xyloside, which inhibits the formation of links between the core protein and glycosaminoglycan side chains in the PG; the absence of intact CS was confirmed by gel filtration. We suggest that, in vivo, CS may facilitate the interfibrillar movement that takes place as the cornea grows. We have also found that keratinase, which degrades the very small amount of keratan sulfate present in the primary stroma, has no effect on stromal deposition. (3) There are substantial amounts of unidentified matrix components in primary stromas laid down both in vivo and in vitro. This conclusion was drawn from SEM observations on both types of stroma after they had been freeze-dried, a process which does not condense hydrated macromolecules. Even after being treated with hyaluronidase to remove the CS, substantial amounts of interfibrillar matrix were still present. Until these components are identified and their interactions with collagen are understood, the mechanisms responsible for stromal morphogenesis are unlikely to be understood.
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PMID:Does chondroitin sulfate have a role to play in the morphogenesis of the chick primary corneal stroma? 249 96


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