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)

The human breast cancer cell lines MCF-7 and MDA-MB-231 differ in their responsiveness to fibroblast growth factor-2 (FGF-2). This growth factor stimulates proliferation in well-differentiated MCF-7 cells, whereas the less well-differentiated MDA-MB-231 cells are insensitive to this molecule. To investigate the potential regulation of FGF-2 mitogenic activity by heparan sulfate proteoglycans (HSPG), we have treated human breast cancer cells by glycosaminoglycan degrading enzymes or a metabolic inhibitor of proteoglycan sulfation: sodium chlorate. The interaction between FGF-2 and proteoglycans was assayed by examining the binding of 125I-FGF-2 to breast cancer cell cultures as well as to cationic membranes loaded with HSPG. Using MCF-7 cells, we showed that heparinase treatment inhibited FGF-2 binding to HSPG and completely abolished FGF-2 induced growth; chlorate treatment of MCF-7 cells decreased FGF-2 binding to HSPG and cell responsiveness in a dose-dependent manner. This demonstrates a requirement of adequately sulfated HSPG for FGF-2 growth-promoting activity on MCF-7 cells. In highly invasive MDA-MB-231 cells which produce twice as much HSPG as MCF-7 cells and which are not normally responsive to exogenously added FGF-2, chlorate treatment decreased FGF-2 binding to HSPG and induced FGF-2 mitogenic effect. This chlorate effect was dose dependent and observed at concentrations of 10-30 mM; higher chlorate concentrations completely abolished the FGF-2 effect. This shows that the HSPG level of sulfation can also negatively regulate the biological activity of FGF-2. Taken together, these results demonstrate a crucial role for HSPG in both positive and negative control of FGF-2 mitogenic activity in breast cancer cell proliferation.
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PMID:Heparan sulfate proteoglycans play a dual role in regulating fibroblast growth factor-2 mitogenic activity in human breast cancer cells. 898 23

To define the molecular regulation of mast cell phenotype and function optimized procedures must be available to study mRNA from mast cells freshly isolated from tissues. However, rat peritoneal mast cells (PMC) contain large amounts of the proteoglycan heparin, and unfortunately, this molecule which is a potent inhibitor of reverse transcriptase (RT) and Taq polymerase and thus RT-PCR, copurifies with RNA. Here we describe an optimized protocol for extracting and amplifying RNA from rat PMC. Mast cells were isolated from rat peritoneum and a method modified from that of Chomczynski and Sacchi (1987) was used to extract the RNA. Following the removal of heparin by heparinase digestion, first strand cDNA synthesis was primed with oligo-dT and the resulting cDNA was quantified by rapid paper chromatography. The use of a detection system for the reverse transcription reaction ensured that the production of cDNA had occurred and allowed subsequent PCR testing to be optimal. cDNA thus produced can be used to detect relatively specific (histidine decarboxylase) and non-specific (beta-actin) mast cell products. Our PCR studies have shown a 300-fold increase in sensitivity over RNA processed by other methods.
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PMID:Optimization of the isolation and effective use of mRNA from rat mast cells. 905 Sep 42

Skin fibroblasts treated with brefeldin A produce a recycling variant of glypican (a glycosylphosphatidylinositolanchored heparan-sulfate proteoglycan) that is resistant to inositol-specific phospholipase C and incorporates sulfate and glucosamine into heparan sulfate chains (Fransson, L.-A. et al., Glycobiology, 5, 407-415, 1995). We have now investigated structural modifications of recycling glypican, such as fatty acylation from [3H]palmitate, and degradation and assembly of heparan sulfate side chains. Most of the 3H-radioactivity was recovered as lipid-like material after de-esterification. To distinguish between formation of heparan sulfate at vacant sites, elongation of existing chains or degradation followed by re-elongation of chain remnants, cells were pulse-labeled with [3H]glucosamine and then chase-labeled with [14C]glucosamine. Material isolated from the cells during the chase consisted of proteoglycan and mostly [3H]-labeled heparan-sulfate degradation products (molecular mass, 20-80 kDa) showing that the side chains were degraded during recycling. The degradation products were initially glucuronate-rich, but became more iduronate-rich with time. The glypican proteoglycan formed during the chase was degraded either with alkali to release intact side chains or with heparinase to generate distally located chain fragments that were separated from the core protein, containing the proximally located, covalently attached chain remnants. All of the [14C]-radioactivity incorporated during the pulse was found in peripheral chain fragments, and the chains formed were not significantly longer than the original ones. We therefore conclude that newly made heparan-sulfate chains were neither made on vacant sites, nor by extension of existing chains but rather by re-elongation of degraded chain remnants. The remodeled chains made during recycling appeared to be more extensively modified than the original ones.
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PMID:Glypican (heparan sulfate proteoglycan) is palmitoylated, deglycanated and reglycanated during recycling in skin fibroblasts. 906 69

Heparan sulfate moieties of cell-surface proteoglycans modulate the biological responses to fibroblast growth factors (FGFs). We have reported previously that cell-associated heparan sulfates inhibit the binding of the keratinocyte growth factor (KGF), but enhance the binding of acidic FGF to the KGF receptor, both in keratinocytes, which naturally express this receptor, and in rat myoblasts, which ectopically express it (Reich-Slotky, R., Bonneh-Barkay, D., Shaoul, E., Berman, B., Svahn, C. M., and Ron, D. (1994) J. Biol. Chem. 269, 32279-32285). The proteoglycan bearing these modulatory heparan sulfates was purified to homogeneity from salt extracts of rat myoblasts by anion-exchange and FGF affinity chromatography and was identified as rat glypican. Affinity-purified glypican augmented the binding of acidic FGF and basic FGF to human FGF receptor-1 in a cell-free system. This effect was abolished following digestion of glypican by heparinase. Addition of purified soluble glypican effectively replaced heparin in supporting basic FGF-induced cellular proliferation of heparan sulfate-negative cells expressing recombinant FGF receptor-1. In keratinocytes, glypican strongly inhibited the mitogenic response to KGF while enhancing the response to acidic FGF. Taken together, these findings demonstrate that glypican plays an important role in regulating the biological activity of fibroblast growth factors and that, for different growth factors, glypican can either enhance or suppress cellular responsiveness.
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PMID:Identification of glypican as a dual modulator of the biological activity of fibroblast growth factors. 913 88

Vitronectin, a 75-kDa plasma protein is also found in the extracellular matrix, where it is believed to promote cell adhesion and migration. In addition to its role in adhesion, matrix vitronectin is also believed to function as an opsonin promoting the clearance of thrombin-serpin complexes from the matrix. Vitronectin is cleared from the matrix by receptor-mediated endocytosis followed by lysosomal degradation, suggesting that cells can regulate the levels of vitronectin present in the matrix. However, the mechanism by which plasma vitronectin associates with the extracellular matrix remains unclear. Studies were conducted to define the binding site(s) for vitronectin in fibroblast cell layers. Sodium chlorate, a competitive inhibitor of proteoglycan sulfation, produced a dose-dependent decrease in both binding and degradation of vitronectin. This inhibition was reversible in that removal of chlorate returned both binding and degradation of vitronectin to near control levels within 24 h. The binding of vitronectin to cell layers was not dependent on cells because vitronectin bound directly to isolated matrix. Isolated matrices prepared from cell layers treated with sodium chlorate also exhibited a dose-dependent decrease in vitronectin binding, consistent with the binding site for vitronectin in the matrix being sulfated proteoglycans. Binding and degradation of vitronectin were also sensitive to the addition of exogenous heparin, suggesting that the heparin binding domain of vitronectin was mediating binding to the matrix. Incubating fibroblast monolayers with heparinase III resulted in a 40% decrease in binding and degradation of vitronectin. Taken together, the above findings suggest that vitronectin's binding to the matrix and its subsequent degradation are dependent on heparan sulfate proteoglycans.
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PMID:Heparan sulfate proteoglycans function in the binding and degradation of vitronectin by fibroblast monolayers. 916 57

Contact activation occurs when plasma comes in contact with negatively charged manmade surfaces but no substance that initiates contact activation in vivo has been identified. We have isolated a mast cell heparin proteoglycan (MC-HepPG) from a Furth mouse mastocytoma-derived cell line that is analogous to human tissue-type mast cell HepPG. This material and other glycosaminoglycans (GAGs) were tested for their ability to accelerate the reciprocal activation of factor XII and prekallikrein and the autoactivation of factor XII. Quantitative analysis showed the MC-HepPG to be as active as dextran sulfate on a weight basis; hog intestine heparin, dermatan sulfate, keratan polysulfate and chondroitin sulfate C were less active, other sulfated polysaccharides were essentially inactive. Incubation of MC-HepPG in 1:4 diluted plasma resulted in complete cleavage of high molecular weight kininogen in a factor XII-dependent reaction. All of the MC-HepPG dependent reactions described above were inhibited by preincubation of MC-HepPG with heparinase I and II but not by pretreatment with heparitinase, chondroitinase ABC or the serine protease inhibitor aPMSF thus indicating that heparin proteoglycan is indeed acting as an initiating 'surface'. We analysed the proteoglycan preparation by HPLC gel filtration. Fractions spanning a molecular weight range of > 400000-8000 were active initiators. Comparison of the chromatograms obtained before and after cleavage of GAG side chains from the protein core suggested that dissociated GAGs in the MW range 69000-17000 are the most active species rather than the complete proteoglycan. MC-HepPG GAGs therefore represent a physiologic macromolecule with activity comparable to non-physiological surfaces in a purified system and with the capability to induce activation of the contact system in diluted plasma. Its ability to promote kinin generation links cellular and humoral inflammatory responses in the perivasculature and provides a possible explanation for the elevated kinin levels observed after allergen exposure.
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PMID:Mast cell derived heparin activates the contact system: a link to kinin generation in allergic reactions. 920 86

The Lyme disease spirochete, Borrelia burgdorferi, infects multiple tissues, such as the heart, joint, skin, and nervous system and has been shown to recognize heparan sulfate and dermatan sulfate proteoglycans. In this study, we examined the contribution of different classes of proteoglycans to the attachment of the infectious B. burgdorferi strain N40 to several immortalized cell lines and primary cultured cells, including endothelial cells and brain cells. Bacterial attachment was inhibited by exogenous proteoglycans or by treatment of host cells with inhibitors of proteoglycan synthesis or sulfation, indicating that proteoglycans play a critical role in bacterial binding to diverse cell types. Binding to primary bovine capillary endothelial cells or a human endothelial cell line was also inhibited by digestion with heparinase or heparitinase but not with chondroitinase ABC. In contrast, binding to glial cell-enriched brain cell cultures or to a neuronal cell line was inhibited by all three lyases. Binding of strain N40 to immobilized heparin could be completely inhibited by dermatan sulfate, and conversely, binding to dermatan sulfate could be completely blocked by heparin. As measured by 50% inhibitory dose, heparin was a better inhibitor of binding than dermatan sulfate, regardless of whether the substrate was heparin or dermatan sulfate. These results are consistent with the hypotheses that the species of proteoglycans recognized by B. burgdorferi vary with cell type and that bacterial recognition of different proteoglycans is mediated by the same bacterial molecule(s).
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PMID:Different classes of proteoglycans contribute to the attachment of Borrelia burgdorferi to cultured endothelial and brain cells. 948 87

Macrophage binding sites for oxidized LDL (Ox-LDL) include class A scavenger receptors (SR-As), the CD-36 molecule, and an additional but hitherto unidentified binding site. Because cell-surface glycosaminoglycans (GAGs) were previously shown to be involved in the cellular uptake of native LDL and lipoprotein(a), several strategies to assess the participation of heparan sulfate (HS) and chondroitin sulfate (CS) in macrophage catabolism of Ox-LDL were used. First, incubation of J-774 A.1 macrophage-like cells with either heparinase or chondroitinase, or with both enzymes together, reduced the binding, uptake, and degradation of 125I-Ox-LDL by 20% to 45%, in comparison with control nontreated cells, while catabolism of 125I-labeled acetylated LDL (Ac-LDL) and native LDL were unaffected. Second, the proteoglycan (PG) cellular content was increased by cell enrichment with exogenous GAGs or by using human monocyte-derived macrophages from two patients with Sanfilippo mucopolysaccharidosis, which are characterized by cellular HS accumulation. In these macrophages, cellular uptake of 125I-Ox-LDL increased, while catabolism of 125I-Ac-LDL and native LDL were unaffected. Experiments using conditioned media from control, heparinase-digested, or chondroitinase-digested macrophages indicated that neither secreted GAGs nor released digestion products played any role in Ox-LDL catabolism. To evaluate potential interactions between cell-surface GAGs and known receptors for Ox-LDL, we used excess unlabeled Ac-LDL to block SR-As or anti-CD-36 antibodies to block CD-36, and then examined the catabolism of 125I-Ox-LDL by GAG-enriched or -depleted macrophages. Both excess unlabeled Ac-LDL and anti-CD-36 antibodies reduced 125I-Ox-LDL catabolism, but only excess unlabeled Ac-LDL completely abolished the increase in 125I-Ox-LDL catabolism on GAG enrichment of the cells, indicating a cooperation between exogenous GAGs and cell-surface SR-As in the catabolism of OX-LDL. Moreover, the addition of GAGases to macrophages that were preincubated with anti-CD-36 antibodies and excess Ac-LDL further reduced macrophage degradation of Ox-LDL in comparison with cells that were pretreated only with anti-CD-36 antibodies and Ac-LDL, indicating a more complex role for endogenous GAGs. Overall, these studies demonstrate a substantial contribution of macrophage-associated GAGs in the catabolism of Ox-LDL, which is mediated in part by a cooperation between GAGs and cell-surface SR-As.
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PMID:Role of macrophage glycosaminoglycans in the cellular catabolism of oxidized LDL by macrophages. 955 59

This study evaluated whether human monocyte-derived macrophages synthesize specific types of proteoglycans with lipoprotein-binding capability that could contribute to lipid retention in the arterial wall. After labeling with either [35S]SO4 or [35S]methionine, macrophages secreted a high molecular mass proteoglycan, with glycosaminoglycan chains of approximately 18 kDa and core protein bands of approximately 100 and 55 kDa. Both core protein bands were recognized by an antibody to PG-100, an antibody that recognizes the proteoglycan form of macrophage colony-stimulating factor (PG-100/PG-MCSF). The interaction between PG-100/PG-MCSF and low density lipoproteins (LDL) was examined by gel mobility shift. In this system, PG-100/PG-MCSF was resolved further into two forms. The two forms had the same core proteins but differed in their overall size and glycosaminoglycan content. The larger form contained glycosaminoglycan chains that were entirely chondroitin ABC lyase-sensitive, whereas the smaller form contained chains that were sensitive to both chondroitin ABC lyase and heparinase. Both forms bound native LDL with high affinity, but the larger form bound LDL with higher affinity than the smaller form. The glycosaminoglycan chains of PG-100/PG-MCSF, but not the core proteins, were responsible for binding to native LDL. Mildly oxidized LDL and methyl-LDL, which have an electrophoretic charge similar to that of native LDL, also bound PG-100/PG-MCSF. In contrast, extensively oxidized LDL and acetyl-LDL, which are more electronegative than native LDL, did not bind to either form of PG-100/PG-MCSF. The demonstration of two forms of human monocyte-derived macrophage PG-100/PG-MCSF which bind LDL may represent an additional role for macrophages in the extracellular trapping of lipoproteins in atherosclerosis.
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PMID:Human monocyte-derived macrophages secrete two forms of proteoglycan-macrophage colony-stimulating factor that differ in their ability to bind low density lipoproteins. 963 47

Small dense low density lipoprotein (LDL) particles have altered apolipoprotein (apo) B conformation and lowered affinity for the LDL receptor (J. Biol. Chem. 1994. 269: 511-519). Herein, we examine the interaction of small dense LDL with cell LDL receptor-independent binding sites. Compared to normal LDL, at low LDL cell media concentrations (<10 microg/ml), small dense LDL had decreased specific binding to the LDL receptor on normal fibroblasts at 4 degrees C, but a 2-fold increased binding to LDL receptor-independent cell sites. At higher LDL concentration (100 microg/ ml), LDL receptor-independent binding of small dense LDL was 4.5-fold that of normal LDL in normal fibroblasts, but greater (2- to 14- fold) in LDL receptor-negative fibroblasts. In LDL receptor-negative fibroblasts at 37 degrees C, small dense LDL had higher (3-fold) cell association than normal size LDL but no effective LDL degradation. At high LDL concentrations (> or =100 microg/ml), LDL binding to normal or LDL receptor-negative fibroblasts was not affected by several anti-apoB monoclonal antibodies or by cell pretreatment with proteases, chondroitinase, or neuraminidase. In contrast, pretreating normal and receptor-negative fibroblasts with heparinase and heparitinase decreased LDL cell binding by 35% and 50%, respectively. Similarly, preincubation of receptor-negative fibroblasts with sodium chlorate, an inhibitor of proteoglycan sulfation, decreased LDL binding by about 45%. We hypothesize that small dense LDL might be more atherogenic than normal size LDL due to decreased hepatic clearance by the LDL receptor, and enhanced anchoring to LDL receptor-independent binding sites in extrahepatic tissues (e.g., the arterial wall), a process mediated, in part, by cell surface proteoglycans.
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PMID:Small dense low density lipoprotein has increased affinity for LDL receptor-independent cell surface binding sites: a potential mechanism for increased atherogenicity. 964 58


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