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:4.2.2.7 (heparinase)
1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human lung fibroblasts produce heparan sulphate proteoglycans (HSPG) that are associated with the plasma membrane. A monoclonal-antibody (Mab)-secreting hybridoma, S1, was produced by fusion of SP 2/0-AG 14 mouse myeloma cells with spleen cells from mice immunized with partially purified cellular HSPG fractions. The HSPG character of the material carrying the epitope recognized by Mab S1 was demonstrated by: (i) the co-purification of the S1 epitope with the membrane HSPG of human lung fibroblasts; (ii) the decrease in size of the material carrying the S1 epitope upon treatment with heparinase or heparitinase, and the resistance of this material to heparinase treatment after N-desulphation. The S1 epitope appears to be part of the core protein, since it was destroyed by proteinase treatment and by disulphide-bond reduction, but not by treatments that depolymerize the glycosaminoglycan chains and N-linked oligosaccharide chains. Polyacrylamide-gel electrophoresis of non-reduced heparitinase-digested membrane HSPG followed by Western blotting and immunostaining with Mab S1 revealed a single band with apparent molecular mass of 64 kDa. Membrane proteoglycans isolated from detergent extracts or from 4 M-guanidinium chloride extracts of the cells yielded similar results. Additional digestion with N-glycanase lowered the apparent molecular mass of the immunoreactive material to 56 kDa, suggesting that the core protein also carries N-linked oligosaccharides. Fractionation of 125I-labelled membrane HSPG by immuno-affinity chromatography on immobilized Mab S1, followed by heparitinase digestion and polyacrylamide-gel electrophoresis of the bound material, yielded a single labelled band with apparent molecular mass 64 kDa. Treatment with dithiothreitol caused a slight increase in apparent molecular mass, suggesting that the core protein of this membrane proteoglycan of a single subunit containing (an) intrachain disulphide bond(s).
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PMID:Identification of a 64 kDa heparan sulphate proteoglycan core protein from human lung fibroblast plasma membranes with a monoclonal antibody. 244 76

The heparan sulfate proteoglycans present in a deoxycholate extract of rat brain were purified by ion exchange chromatography, affinity chromatography on lipoprotein lipase agarose, and gel filtration. Heparitinase treatment of the heparan sulfate proteoglycan fraction (containing 86% heparan sulfate and 10% chondroitin sulfate) that was eluted from the lipoprotein lipase affinity column with 1 M NaCl led to the appearance of a major protein core with a molecular size of 55,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the effects of heparinase and heparitinase treatment revealed that the heparan sulfate proteoglycans of brain contain a significant proportion of relatively short N-sulfoglucosaminyl 6-O-sulfate [or N-sulfoglucosaminyl](alpha 1-4)iduronosyl 2-O-sulfate(alpha 1-4) repeating units and that the portions of the heparan sulfate chains in the vicinity of the carbohydrate-protein linkage region are characterized by the presence of D-glucuronic acid rather than L-iduronic acid. After chondroitinase treatment of a proteoglycan fraction that contained 62% chondroitin sulfate and 21% heparan sulfate (eluted from lipoprotein lipase with 0.4 M NaCl), the charge and density of a portion of the heparan sulfate-containing proteoglycans decreased significantly. These results indicate that a population of "hybrid" brain proteoglycans exists that contain both chondroitin sulfate and heparan sulfate chains covalently linked to a common protein core.
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PMID:Structural properties of the heparan sulfate proteoglycans of brain. 252 92

Neurons from embryonic (E18) rat hippocampus were chosen to identify and characterize neurite growth-stimulating proteins accumulating in serum-free conditioned media (CM) obtained from primary or secondary cultures of cerebral astrocytes (less than 5% nonglial cells) using a quantitative cell culture bioassay. CM were fractionated by FPLC on an anion exchange column (Mono Q) and by gel filtration (Superose 6). Column fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and enzyme-linked immunosorbent assay (ELISA) using antibodies to laminin (LN) and fibronectin (FN). The neurite-promoting activity (NPA) was tested by incubating aliquots of the eluted fractions with poly-L-lysine precoated glass coverslips prior to addition of neurons suspended in chemically defined medium. We provide evidence that the NPA in astroglial CM could be assigned mainly to a negatively charged, highly sulfated LN complex consisting predominantly of the B-chains of LN and presumably a sulfated proteoglycan that was sensitive for chondroitinase and to a lower degree to heparinase degradation. In addition, a smaller proportion of the NPA was associated with uncomplexed LN and free FN. FN reached approximately 10 times the concentration of LN in astroglial CM. As revealed by immunofluorescence microscopy, both LN and FN are simultaneously expressed by cultured astrocytes; however, only the production of FN, measured by ELISA, increased during the time astrocytes were in culture, whereas the release of LN remained unchanged. We conclude that, besides the most active LN complex, FN bound to a polycationic matrix is able to induce neurite growth in hippocampal neurons in vitro.
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PMID:Astroglia-released neurite growth-inducing activity for embryonic hippocampal neurons is associated with laminin bound in a sulfated complex and free fibronectin. 252 80

Three types (T1, T2, T3) of proteoglycan (PG) filaments, as demonstrated by cuprolinic blue (CB) under critical electrolyte concentration method in the epithelial-stromal interface of the guinea pig lateral prostate, were characterized cytochemically by using a number of glycosaminoglycan(GAG)-degrading enzymes and nitrous acid. The results showed that T1 filaments located in basement membranes of the epithelium, endothelium, and smooth muscle cells, were removed by nitrous acid, heparitinase, and pronase but resistant to chondroitinase (Ch)-ABC and Ch-AC, heparinase, neuraminidase, and Streptomyces (S) hyaluronidase. The T1 filaments, therefore, contain heparan sulfate. The T2 filaments closely linked to collagen fibrils were removed by Ch-ABC, Ch-ABC plus S-hyaluronidase, and pronase but were resistant to nitrous acid, heparitinase, heparinase, neuraminidase, and S-hyaluronidase. These show that T2 filaments are rich in dermatan sulfate. The T3 filaments in the interstitial spaces and on the surface of fibroblasts were removed by Ch-ABC, Ch-AC, and pronase but were resistant to heparitinase, heparinase, hyaluronidase, neuraminidase, and nitrous acid. They are, therefore, rich in chondroitin sulfate.
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PMID:Cytochemical characterization of cuprolinic blue-stained proteoglycans in the epithelial-stromal interface of the guinea pig lateral prostate. 271 Jun 91

The chemical nature of anionic sites located on both fronts of the endothelial cells (ECs) and in the basement membrane (BM) of mouse brain capillaries was studied using tissue sections embedded in Lowicryl K4M and cationic colloidal gold. Before labelling with cationic probe, the sections were digested with the following enzymes: trypsin, papain, pronase E, proteinase K, collagenase, chondroitinase ABC, hyaluronidase, heparinase, heparitinase, neuraminidase and endoglycosidase H. The results indicate that the negatively charged surface layer on the luminal front differs in chemical nature from that on the abluminal front of the EC. Anionic sites located on the luminal surface of the plasmalemma of the ECs are mainly contributed by sialic acid residues of acidic glycoproteins. On the contrary, the anionic domains on the abluminal front of the EC represent mixed proteoglycan and acid glycopeptides containing hydrophobic amino acids, sialic acid residues, and are rich in heparan sulphate-bearing glycosaminoglycans. The anionic sites of the BM are contributed in a substantial degree by chondroitin and heparan sulphate-rich glycosaminoglycans. The effect of endoglycosidase H suggests that glycopeptides containing oligomannosyl residues linked to N-acetylglucosamine contribute in small degree in maintenance of the negative charge in the BM, but not on the surfaces of the EC. These results show that brain endothelium bears surface anionic domains differing chemically from those described for some fenestrated and continuous endothelia. The distribution of anionic sites indicates that the discrimination against various negatively charged molecules takes place on both fronts of the ECs as well as in the BM of brain micro-blood vessels. The exact role of these domains in the function of the blood-brain barrier remains to be established.
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PMID:Ultracytochemical characterization of anionic sites in the wall of brain capillaries. 274 7

Using low magnification Hoffman Modulation Contrast microscopy to rapidly identify precartilage mesenchymal condensations in chick limb bud cultures, we have determined the effect on condensation number of treatments disruptive of the interaction of cell surface components with endogenously produced fibronectin. A monoclonal antibody directed against the amino-terminal heparin-binding domain of fibronectin reduced the number of condensations by more than 50%, as did the oligopeptide gly-arg-gly, which is a repeated motif in that fibronectin domain. In contrast, monoclonal antibodies directed against the collagen- and integrin-binding domains of fibronectin, or oligopeptides containing the fibronectin integrin-recognition sequence arg-gly-asp-ser, had no significant effect on condensation number. Addition of Flavobacterium heparinase to cultures also reduced condensation number by more than 50%. Alcian blue staining of sulfated proteoglycan was greatly reduced in differentiated cultures that had been exposed to treatments that reduced condensation number. Taken together with the accompanying study, which directly demonstrates an adhesive interaction between the amino-terminal domain of extracellular fibronectin and heparin-like molecules on the surfaces of latex bead probes, the data presented here strongly indicate a major role for the corresponding cell-matrix interaction in mediating precartilage condensation in limb mesenchyme.
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PMID:The mechanism of precartilage mesenchymal condensation: a major role for interaction of the cell surface with the amino-terminal heparin-binding domain of fibronectin. 280 26

We have isolated cDNA clones that code for a proteoglycan-related polypeptide with unique properties. A lambda gt11 expression library made from human fibroblast mRNA was screened with an antiserum made against a proteoglycan fraction from human fetal membranes. One group of positive clones revealed an open reading frame coding for 685 amino acids from the COOH terminus of a polypeptide. This amino acid sequence contains a domain that is strongly homologous with the COOH-terminal core protein domain of the large aggregating cartilage proteoglycan. This domain also contains sequences that are homologous with vertebrate lectins that bind terminal galactosyl, N-acetyl-glucosaminyl or mannosyl residues. On the NH2-terminal side of the lectin-like domain the cDNA-derived amino acid sequence contains two epidermal growth factor-related segments. The cDNA clones were shown to belong to a chondroitin sulfate proteoglycan by using antisera made against two peptides predicted from the cDNA sequence. These antisera were reactive with a proteoglycan fraction from fibroblasts after chondroitinase treatment of the fraction but not after treatment with heparinase or no treatment. Among the several polypeptides reactive with the anti-peptide antibodies the largest one, corresponding to a molecular weight of about 400,000, is likely to be the intact core protein, whereas the smaller polypeptides may be processing products or products of artifactual proteolysis. These results show that the amino acid sequence belongs to a proteoglycan core protein, and the sequence, therefore, provides a molecular definition to this proteoglycan. The lectin-related and growth factor-like sequences in the core protein of this proteoglycan suggest that it may play a role in intercellular signaling.
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PMID:A fibroblast chondroitin sulfate proteoglycan core protein contains lectin-like and growth factor-like sequences. 282 Sep 64

The structure of the glycosaminoglycan chain of a heparan sulfate proteoglycan isolated from the conditioned medium of an endothelial cell line has been analyzed by using various degradative enzymes (heparitinase I, heparitinase II, heparinase, glycuronidase, sulfatases) from Flavobacterium heparinum. This proteoglycan inhibits the thromboplastin-activated pathway of coagulation; as a consequence, the catalytic conversion of prothrombin to thrombin is arrested. Heparitinase I (EC 4.2.2.8), an enzyme with specificity restricted to the heparan sulfate portion of the polysaccharide, releases fragments with the electrophoretic mobility and the structure of heparin. Conversely, an assessment of the size and distribution of the heparan sulfate regions has been provided by the use of heparinase (EC 4.2.2.7), which, by degrading the heparin sections of the chain, releases two segments that exhibit the structure of heparan sulfate. One of these segments is attached to the protein core. On the basis of these findings, the heparan sulfate chain can be defined as a copolymer containing heparin regions in its structure. The combined use of these enzymes has made it possible to establish the disaccharide sequence of parts of the glycosaminoglycan moiety of this proteoglycan.
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PMID:Heparin sequences in the heparan sulfate chains of an endothelial cell proteoglycan. 295 57

The binding of iodinated basic fibroblast growth factor (bFGF) to low-density heparan sulfate proteoglycan purified from the Engelbreth Holm Swarm (EHS) sarcoma was investigated using different techniques. The tumor clearly contained bFGF, the level being comparable to that found in other tissues such as human or bovine brain. 125I bFGF strongly bound to the basement membrane-like matrix of EHS frozen sections as revealed by autoradiography. Iodinated bFGF bound to purified heparan sulfate proteoglycan but not to laminin or collagen type IV, three components isolated from the same tumor. In contrast, acidic fibroblast growth factor (aFGF) displayed negligible binding to heparan sulfate proteoglycan. Binding of bFGF to frozen sections and to purified proteoglycan could be strongly inhibited by heparin and was displaced by an excess of unlabeled factor and completely suppressed after heparitinase and heparinase treatments. Binding was a function of the salt concentration and was abolished at 0.6 M NaCl. Scatchard analysis indicated the affinity site had a Kd of about 30 nM, a value 10-15 higher than that recently reported by Moscatelli (J. Cell. Physiol., 131:123-130, 1987) in the case of the low-affinity binding sites present on the surface of baby hamster kidney (BHK) cells.
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PMID:Specific binding of basic fibroblast growth factor to basement membrane-like structures and to purified heparan sulfate proteoglycan of the EHS tumor. 297 66

The ability of mouse IL-3-dependent, bone marrow culture-derived mast cells (BMMC) to generate serosal mast cells (SMC) in vivo after adoptive transfer to mast cell-deficient mice has been defined by chemical and immunochemical criteria. BMMC differentiated and grown from WBB6F1-+/+ mouse progenitor cells in medium containing PWM/splenocyte-conditioned medium synthesized a approximately 350,000 Mr protease-resistant proteoglycan bearing approximately 55,000 Mr glycosaminoglycans, as defined by gel filtration of each. Approximately 85% of the glycosaminoglycans bound to the cell-associated BMMC proteoglycans were chondroitin sulfates based upon their susceptibility to chondroitinase ABC digestion; HPLC of the chondroitinase ABC-generated unsaturated disaccharides revealed these glycosaminoglycans to be chondroitin sulfate E. As determined by heparinase and nitrous acid degradations, approximately 10% of the glycosaminoglycans bound to BMMC proteoglycans were heparin. In contrast, mast cells recovered from the peritoneal cavity of congenitally mast cell-deficient WBB6F1-W/Wv mice 15 wk after intraperitoneal injection of BMMC synthesized approximately 650,000 Mr protease-resistant proteoglycans that contained approximately 80% heparin glycosaminoglycans of approximately 105,000 Mr. Thus, after adoptive transfer, the SMC of the previously mast cell-deficient mice were like those recovered from the normal WBB6F1-+/+ mice that were shown to synthesize approximately 600,000 Mr proteoglycans that contained approximately 80% heparin glycosaminoglycans of approximately 115,000 Mr. As assessed by indirect immunofluorescence staining and flow cytometry using the B1.1 rat mAb (an antibody that recognizes an epitope located on the neutral glycosphingolipid globopentaosylceramide), approximately 5% of BMMC bound the antibody detectably, whereas approximately 72% of the SMC that were harvested from mast cell-deficient mice 15 wk after adoptive transfer of BMMC were B1.1-positive; approximately 82% of SMC from WBB6F1-+/+ mice bound the antibody. These biochemical and immunochemical data are consistent with the results of previous adoptive transfer studies that characterized mast cells primarily on the basis of morphologic and histochemical criteria. Thus, IL-3-dependent BMMC developed in vitro, cells that resemble mucosal mast cells, can give rise in vivo to SMC that express phenotypic characteristics of connective tissue mast cells.
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PMID:Phenotypic changes of bone marrow-derived mast cells after intraperitoneal transfer into W/Wv mice that are genetically deficient in mast cells. 310 74


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