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)

Schwann cells synthesize both hydrophobic and peripheral cell surface heparan sulfate proteoglycans (HSPGs). Previous analysis of the kinetics of radiolabeling suggested the peripheral HSPGs are derived from the membrane-anchored forms (Carey, D., and D. Evans. 1989. J. Cell Biol. 108:1891-1897). Peripheral cell surface HSPGs were purified from phytic acid extracts of cultured neonatal rat sciatic nerve Schwann cells by anion exchange, gel filtration, and laminin-affinity chromatography. Approximately 250 micrograms of HSPG protein was obtained from 2 X 10(9) cells with an estimated recovery of 23% and an overall purification of approximately 2000-fold. SDS-PAGE analysis indicated the absence of non-HSPG proteins in the purified material. Analysis of heparinase digestion products revealed the presence of at least six core protein species ranging in molecular weight from 57,000 to 185,000. The purified HSPGs were used to produce polyclonal antisera in rabbits. The antisera immunoprecipitated a subpopulation of 35SO4-labeled HSPGs that were released from Schwann cells by incubation in medium containing phosphatidylinositol-specific phospholipase C (PI-PLC); smaller amounts of immunoprecipated HSPGs were also present in phytic acid extracts. In the presence of excess unlabeled PI-PLC-released proteins, immunoprecipitation of phytic acid-solubilized HSPGs was inhibited. SDS-PAGE analysis of proteins immunoprecipitated from extracts of [35S]methionine labeled Schwann cells demonstrated that the antisera precipitated an HSPG species that was present in the pool of proteins released by PI-PLC, with smaller amounts present in phytic acid extracts. Nitrous acid degradation of the immunoprecipitated proteins produced a single 67,000-Mr core protein. When used for indirect immunofluorescence labeling, the antisera stained the external surface of cultured Schwann cells. Preincubation of the cultures in medium containing PI-PLC but not phytic acid significantly reduced the cell surface staining. The antisera stained the outer ring of Schwann cell membrane in sections of adult rat sciatic nerve but did not stain myelin or axonal membranes. This localization suggests the HSPG may play a role in binding the Schwann cell plasma membrane to the adjacent basement membrane surrounding the individual axon-Schwann cell units.
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PMID:Identification of a lipid-anchored heparan sulfate proteoglycan in Schwann cells. 217 60

Intermediate density lipoproteins (IDL) were shown to bind to high- and low-affinity binding sites on rat liver membranes. The low-affinity sites were named lipoprotein binding sites (LBS), since they bind all classes of lipoproteins. This study was undertaken to further characterize the interaction of 125I-labelled IDL with the LBS of rat liver membranes to determine the chemical nature of the LBS. We found that the binding of IDL to the LBS is insensitive to EDTA and sensitive to heparin and that it is present on plasma membranes. Also, membranes were pretreated with various enzymes that have an effect on the membrane constituents, and the activity of the LBS on these treated membranes was determined. Our results reveal that the LBS of rat liver membranes is insensitive to heparinase I, chondroitinase ABC, and phospholipase C, while it is partially sensitive to phospholipase A2 and sensitive to proteases and heat. Rat liver membrane proteins were solubilized with Triton X-100, reconstituted in liposomes, and analyzed for their ability to bind lipoproteins. 125I-labelled IDL were shown to bind to high- and low-affinity sites that are similar, in affinity and specificity, to the ones observed with intact rat liver membranes, indicating that a LBS activity is detectable on these liposomes. We found that the binding capacity of low-affinity sites in liposomes containing either no protein or containing proteins solubilized from Escherichia coli membranes is five times weaker than low-affinity sites in liposomes containing liver membrane proteins. Thus, a protein solubilized from rat liver membranes has LBS activity when reconstituted in liposomes. Taken altogether our results provide new information on the binding of IDL to the LBS and indicate that the LBS activity is in part mediated by a protein. Thus, the LBS appears to be a bona fide receptor.
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PMID:Analysis of the lipoprotein binding site of rat liver membranes. 781 47

Lipoprotein lipase (LPL)-binding heparan sulfate proteoglycans (HSPGs) were isolated from cell extracts and conditioned media of cultured adipocytes treated with phosphatidylinositol-specific phospholipase C (PIPLC). The methodology employed included anion exchange chromatography, affinity chromatography on LPL Affi-Prep 10 and hydrophobic chromatography. HSPGs were resolved into two distinct fractions on the Octyl-Sepharose CL-4B matrix. Treatment of the eluted fractions with heparinase and heparitinase yielded core proteins of 48.4 and 39 kDa. The 39-kDa core protein is anchored to the cell surface by a glycosyl phosphatidylinositol anchor as evidenced by 1) release of the HSPG with the 39-kDa core protein into media by PIPLC treatment and 2) biosynthetic incorporation of [3H]ethanolamine and [32P]orthophosphate into the PIPLC-releasable 39-kDa core protein. PIPLC released 23% of the total heparin-releasable LPL. A similar percentage (24.5%) of the total heparan sulfate chains was released by PIPLC. Over 96% of the total adipocyte heparan sulfate chains bound to LPL Affi-Prep 10 column. The heterogeneity of core proteins of HSPGs with affinity for LPL may provide a structural basis for the multiple fates of LPL on the surface of adipocytes, i.e. internalization, degradation, or recycling to the cell surface and translocation into the medium.
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PMID:Purification and characterization of adipocyte heparan sulfate proteoglycans with affinity for lipoprotein lipase. 808 54

Previous studies (Sivaram, P., Choi, S. Y., Curtiss, L. K., and Goldberg, I. J.(1994) J. Biol. Chem. 269, 9409-9412) from this laboratory showed that the NH2-terminal region of apoB (NTAB) has binding domains for lipoprotein lipase (LPL). LPL binding to endothelial cells, we hypothesize, involves interaction both with heparan sulfate proteoglycans and with a protein that has homology to NTAB. To test whether cell-surface NTAB would increase the amount and affinity of LPL binding to cells, we produced stable Chinese hamster ovary cell lines that have NTAB anchored to the cell surface. A cDNA encoding the amino-terminal 17% of apoB (apoB17) was fused to a cDNA coding for the last 37 amino acids of decay-accelerating factor (DAF), which contains the signal for glycosylphosphatidylinositol anchor attachment. The fused construct was sequence-verified and cloned into expression vector pCMV5. The pCMV5-apoB17-DAF plasmid was cotransfected with a neomycin resistance gene into wild-type (WT) cells and mutant heparan sulfate proteoglycan-deficient Chinese hamster ovary cells (745 cells), and stable cell lines were established. Expression of apoB17 on the cell surface was confirmed by the release of apoB17 by phosphatidylinositol-specific phospholipase C. LPL binding to WT and apoB17-DAF-transfected cells was determined. Using 0.8-6 microg of LPL, 1.3-2.2-fold more LPL associated with apoB17-DAF WT cells compared with WT cells; apoB17-DAF also increased LPL binding to 745 cells. After heparinase treatment, LPL binding to apoB17-DAF cells was still greater than to treated WT cells. This increased binding to apoB17-DAF cells was almost abolished by treatment of cells with phosphatidylinositol-specific phospholipase C or anti-apoB monoclonal antibody. LPL dissociated from WT cells with k-1 = 2.55 x 10(-2) min-1, whereas LPL dissociated more slowly from apoB17-DAF-containing cells with k-1 = 1.08 x 10(-2) min-1. Furthermore, almost 95% of the LPL on WT cells was dissociated by 1 M NaCl, while only 65% of the LPL dissociated from apoB17-DAF cells at the same high salt concentration. Similarly, in high salt, more LPL remained associated with apoB17-DAF cells than with nontransfected 745 cells. These data show that NTAB on cell surfaces can function as a LPL-binding protein. Moreover, they demonstrate that LPL association with cells can be increased by simultaneously binding to both proteoglycan and non-proteoglycan binding sites.
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PMID:Cell-surface expression of an amino-terminal fragment of apolipoprotein B increases lipoprotein lipase binding to cells. 870 44

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

We have previously demonstrated that thrombin possesses an active yet cryptic Arg-Gly-Asp (RGD) site which upon exposure induces endothelial cell (EC) adhesion via alpha nu beta 3 integrin [Bar-Shavit et al. (1991): J Cell Biol 112:335]. This was achieved in the presence of cell surface-associated heparan sulfate proteoglycans (HSPG) and exceedingly low concentrations of plasmin [Bar-Shavit et al. (1993): J Cell Biol 123:1279]. A portion of the cell surface-associated HSPG (glypican) is anchored via a covalently linked glycosyl-phosphatidylinositol (PI) residue, which can be released by treatment with glycosyl-PI-specific phospholipase C (PI-PLC). We report here that exposure of either bovine aortic EC, smooth muscle cells (SMC), or wild-type CHO cells to PI-PLC released HSPG involved in the conversion of thrombin to an adhesive molecule. The adhesion-promoting activity of the released HSPG was abolished following treatment with heparinase but not chondroitinase ABC. Incubation of thrombin with heparan sulfate-deficient CHO cells or cells that were pretreated with PI-PLC failed to induce its conversion to an adhesive molecule, indicating that glypican was playing a major role in this conversion. Moreover, affinity-purified glypican, but not syndecan or fibroglycan, elicited efficient conversion of plasmin-treated thrombin into an adhesive molecule. Antibodies raised against the RGD site in thrombin failed to interact with native thrombin, prothrombin, or the RGD site in other adhesive proteins such as vitronectin, fibrinogen, or fibronectin. Anti-thrombin-RGD antibodies which blocked the adhesion-promoting activity of thrombin were also capable of recognizing thrombin that was first incubated with a suboptimal concentration of plasm in in the presence of PI-PLC-released HSPG. Heparin, heparan sulfate, and PI-PLC-released HSPG had no effect on other cellular properties of thrombin such as receptor binding and growth-promoting activity. Altogether we have demonstrated that the heparin binding domain in thrombin plays a specific role in promoting thrombin adhesive properties and that membrane-associated glypican is likely to be the major physiological inducer of this property.
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PMID:Specific involvement of glypican in thrombin adhesive properties. 917 91

Cellular entry of human T-cell leukaemia virus type 1 (HTLV-1) was studied by a quantitative assay system using vesicular stomatitis virus (VSV) pseudotypes in which a recombinant VSV (VSVDeltaG*) containing the gene for green fluorescent protein instead of the VSV G protein gene was complemented with viral envelope glycoproteins in trans. Most of the cell lines tested showed susceptibility to VSVDeltaG* complemented with either HTLV-1 envelope glycoproteins (VSVDeltaG*-Env) or VSV G protein (VSVDeltaG*-G), but not to VSVDeltaG* alone, indicating that cell-free HTLV-1 could infect many cell types from several species. High concentration pronase treatment of cells reduced their susceptibility to VSVDeltaG*-Env, while trypsin treatment, apparently, did not. Treatment of the cells with sodium periodate, heparinase, heparitinase, phospholipase A2 or phospholipase C reduced the susceptibility of cells to VSVDeltaG*-Env, but not to VSVDeltaG* complemented with measles virus (Edmonston strain) H and F proteins (VSVDeltaG*-EdHF), which was used as a control. Purified phosphatidylcholine also inhibited the infectivity of VSVDeltaG*-Env, but not VSVDeltaG*-G. These findings indicated that, in addition to cell surface proteins, glycosaminoglycans and phospholipids play an important role in the process of cell-free HTLV-1 entry.
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PMID:Analysis of the molecules involved in human T-cell leukaemia virus type 1 entry by a vesicular stomatitis virus pseudotype bearing its envelope glycoproteins. 1125 87

The genome of hepatitis C virus (HCV) encodes two envelope glycoproteins (E1 and E2), which are thought to be responsible for receptor binding and membrane fusion resulting in virus penetration. To investigate cell surface determinants important for HCV infection, we used a recombinant vesicular stomatitis virus (VSV) in which the glycoprotein gene was replaced with a reporter gene encoding green fluorescent protein (GFP) and produced HCV-VSV pseudotypes possessing chimeric HCV E1 or E2 glycoproteins, either individually or together. The infectivity of the pseudotypes was determined by quantifying the number of cells expressing the GFP reporter gene. Pseudotypes that contained both of the chimeric E1 and E2 proteins exhibited 10--20 times higher infectivity on HepG2 cells than the viruses possessing either of the glycoproteins individually. These results indicated that both E1 and E2 envelope proteins are required for maximal infection by HCV. The infectivity of the pseudotype virus was not neutralized by anti-VSV polyclonal antibodies. Bovine lactoferrin specifically inhibited the infection of the pseudotype virus. Treatment of HepG2 cells with Pronase, heparinase, and heparitinase but not with phospholipase C and sodium periodate reduced the infectivity. Therefore, cell surface proteins and some glycosaminoglycans play an important role in binding or entry of HCV into susceptible cells. The pseudotype VSV possessing the chimeric HCV glycoproteins might offer an efficient tool for future research on cellular receptors for HCV and for the development of prophylactics and therapeutics for hepatitis C.
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PMID:Characterization of pseudotype VSV possessing HCV envelope proteins. 1148 95

Human papillomavirus type 33 (HPV-33) pseudovirus infection is a slow process dependent on the initial interaction with cell-surface heparan sulfate (T. Giroglou, L. Florin, F. Schafer, R. E. Streeck, and M. Sapp, 2001a, J. Virol. 75, 1565-1570). We have now further dissected the initial steps of pseudovirus uptake using removal of cell-surface proteoglycans and selective inhibition of entry pathways. Treatment of cells with heparinase I, but not with phosphoinositol-specific phospholipase C (PIPLC), prevented binding of papillomavirus-like particles and infection with HPV-33 pseudovirions, indicating that GPI-linked proteoglycans (glypicans) are not required for productive infection. The slow entry of pseudovirions was inhibited by cytochalasin D and nocodazole in a concentration-dependent manner, suggesting actin polymerization and intact microtubuli be required. Inhibitors of the caveolae-mediated uptake did not significantly affect pseudoinfection. Interestingly, pseudoinfection was blocked by selective inhibitors of endosomal acidification up to 12 h postinfection. Together, our results suggest that binding of HPV pseudovirions to heparan sulfate proteoglycans, most likely syndecans, is followed by delayed internalization via the endosomal pathway.
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PMID:Analysis of the infectious entry pathway of human papillomavirus type 33 pseudovirions. 1220 31

Extracellular RNA has been shown to induce vascular endothelial growth factor (VEGF)-dependent hyperpermeability in vivo as well as in vitro. Studies were performed to investigate the mechanism of these effects. For permeability studies primary cultures of porcine brain-derived microvascular endothelial cells (BMECs) and for all other analytical studies the human brain endothelial cell line HCMEC/D3 or human umbilical vein endothelial cells (HUVECs) were used. RNA, but not DNA, initiated signaling events by binding of VEGF to neuropilin-1, followed by VEGF-R2 phosphorylation, activation of phospholipase C (PLC), and intracellular release of Ca(2+). Activation of these pathways by RNA also resulted in the release of von Willebrand Factor from Weibel-Palade bodies. Pretreatment of cells with heparinase totally abrogated the RNA-induced permeability changes, whereas RNA together with VEGF completely restored VEGF-R2-mediated hyperpermeability. Although poly:IC increased the interleukin-6 release via activation of toll-like receptor-3 (TLR-3), permeability changes mediated by poly:IC or RNA remained unchanged after blocking TLR-3 or NF-kB activation. These results indicate that extracellular RNA serves an important cofactor function to engage VEGF for VEGF-R2-dependent signal transduction, reminiscent of the coreceptor mechanism mediated by proteoglycans, which might be of relevance for the mobilization and cellular activities of RNA-binding cytokines in general.
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PMID:Signaling mechanism of extracellular RNA in endothelial cells. 1924 91


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