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)

Proteoglycans (PGs) incorporated into cell layer and secreted into media were characterized during retinoic acid-induced neuronal differentiation of cultured P19 murine embryonal carcinoma cells. Heparan sulfate significantly increased (P < 0.01) in cell layer following neuronal differentiation of P19 cells by 3.9-fold. CL-4B gel chromatography revealed the major PGs present in cell layer of stem cells eluted as a broad peak with a Kav = 0.65, and was susceptible to chondroitin ABC lyase. The chondroitin ABC lyase resistant material eluted as a broad peak between Kav = 0.40 and Kav = 0.60, and was only partially digested with heparitinase/heparinase (with resistant material eluting at Kav = 0.70). Therefore, the cell layer of stem cells contained primarily chondroitin sulfate/dermatan sulfate (CS/DS) PGs, with lesser amounts of heparan sulfate proteoglycans (HSPGs). This was confirmed by SDS-PAGE. The CS/DS PGs in the cell layer of stem cells had an apparent M(r) of approximately > 200 kDa, and the HSPGs had an apparent M(r) of approximately 140-230 kDa. In contrast, the major PGs in the cell layer of neurons consisted primarily of HSPGs, with only a minor proportion of CS/DS PGs. Furthermore, both gel filtration chromatography and SDS-PAGE analysis revealed a larger HSPG in the cell layer of neurons (Kav = 0.3-0.6 on CL-4B following chondroitin ABC lyase digestion; M(r) 170 kDa- > 400 kDa on SDS-PAGE) in comparison to stem cells (Kav = 0.4-0.6 on CL-4B following chondroitin ABC lyase digestion; M(r) 140-230 kDa on SDS-PAGE). Likewise, the major PGs secreted into media of stem cells consisted almost exclusively of CS/DS PGs, with lesser amounts of HSPGs, whereas an increase in HSPGs in the media of neurons was apparent. Western, Northern, and immunocytochemical analysis demonstrated that mRNA transcript and protein levels for a specific HSPG (i.e., perlecan) markedly increased in cell layer following P19 neuronal differentiation. Perlecan core protein was identified by Western blot analysis using specific monoclonal and polyclonal antibodies, as a large HSPG with a core protein of apparent M(r) approximately 370-400 kDa, and was observed primarily in extracts from neurons. Northern blot analysis with a cDNA to perlecan revealed a significant (P < 0.01) 12.7-fold increase in expression of perlecan in neurons (day 9) in comparison to stem cells. The increase in perlecan message during P19 neuronal differentiation was concomitant with a significant (P < 0.01) 26.3-fold increase in message for beta-amyloid precursor protein (beta PP).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of proteoglycans synthesized by murine embryonal carcinoma cells (P19) reveals increased expression of perlecan (heparan sulfate proteoglycan) during neuronal differentiation. 780 83

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

Embryonic data and ultrastructural analyses suggest that the primitive endothelium signals undifferentiated mesenchymal cells to migrate to the forming blood vessel and subsequently regulates mural cell growth and behavior. Upon maturation of the blood vessel, chemotactic and mitogenic signals are apparently diminished and differentiated smooth muscle cells normally remain quiescent. This homeostasis is seemingly upset in conditions which lead to pathologies characterized by smooth muscle cell hyperplasia such as atherosclerosis. By culturing endothelial cells at different densities, we attempted to re-create the various stages of vascular development. Whereas media conditioned by sparse endothelial cells stimulate smooth muscle cells, media conditioned by dense endothelial cell cultures are inhibitory. Culture of sparse smooth muscle cells in media conditioned for 3 days by postconfluent endothelial cell cultures leads to dose-dependent and reversible smooth muscle cell inhibition. Furthermore, in the presence of the endothelial cell-derived inhibitor, smooth muscle cells are rendered refractory to mitogens such as fibroblast growth factor and platelet-derived growth factor. The inhibitory activity is not attributable to the well-characterized inhibitors of smooth muscle cell growth, transforming growth factor type-beta, prostaglandin I2, or heparan sulfate proteoglycan. Partial characterization of the inhibitory conditioned media suggests that the active molecule is smaller than 1,000 da, and stable to boiling as well as proteinase K and heparinase digestion. These findings support the concept that there is intercellular communication between endothelial cells and smooth muscle cells and provide evidence for a novel endothelial cell-derived smooth muscle cell growth inhibitor.
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PMID:Density-dependent endothelial cell production of an inhibitor of smooth muscle cell growth. 822 80

The cause and consequence of altered proteoglycans in atherosclerosis are poorly understood. To determine whether proteoglycans affect monocyte binding, we studied the effects of heparin and proteoglycan degrading enzymes on THP-1 monocyte adhesion to subendothelial matrix (SEM). Monocyte binding increased about 2-fold after SEM was treated with heparinase. In addition, heparin decreased monocyte binding to fibronectin, a known SEM protein, by 60%. These data suggest that SEM heparan sulfate inhibits monocyte binding to SEM proteins. We next examined whether lysolecithin, a constituent of modified lipoproteins, affects endothelial heparan sulfate proteoglycan (HSPG) production and monocyte binding. Lysolecithin (10-200 microM) decreased total 35SO4 in SEM (20-75%). 2-fold more monocytes bound to SEM from lysolecithin treated cells than to control SEM. Heparinase treatment did not further increase monocyte binding to lysolecithin-treated SEM. HSPG degrading activity was found in medium from lysolecithin-treated but not control cells. 35SO4-labeled products obtained from labeled matrix treated with lysolecithin-conditioned medium were similar in size to those generated by heparinase. These data suggest that lysolecithin-treated endothelial cells secrete a heparanase-like activity. We hypothesize that decreased vessel wall HSPG, as occurs in atherogenic conditions, allows increased monocyte retention within the vessel and is due to the actions of an endothelial heparanase.
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PMID:Lysolecithin-induced alteration of subendothelial heparan sulfate proteoglycans increases monocyte binding to matrix. 853 Mar 67

Basic fibroblast growth factor (bFGF) has been identified as an important cytokine for blood cells. To determine whether hematopoietic cells have receptors that recognize bFGF, the ability of human leukemia cell lines to bind 125I-bFGF was investigated. Specific bFGF-binding sites were identified on K562 and HL60 cells, but not on U937 cells. DAMI cells bound low amounts of 125I-bFGF specifically. Binding of 125I-bFGF to K562 cell surfaces was reduced in a dose-dependent manner by unlabeled bFGF or by heparin. Scatchard analysis of binding to K562 cells revealed two classes of binding sites: 1,650 high affinity binding sites per cell with a dissociation constant (kd) of 192 pmol/L, and 36,600 low affinity sites per cell with a kd of 9.3 nmol/L. Chemical crosslinking experiments with K562, HL60, and DAMI cells revealed receptor-growth factor complexes with molecular masses of 140 to 160 kD, similar in size to complexes formed by known receptor species. Binding of 125I-bFGF to K562 cells was sensitive to heparinase treatment but not to chondroitinase treatment, suggesting that heparan sulfate proteoglycans (HSPGs) may be responsible for the low affinity binding sites. To further investigate whether K562 cells make HSPG, the incorporation of 35SO4 into proteoglycans was assessed. Metabolically labeled cell-surface proteoglycans with molecular masses of 180 to 300 kD were identified in K562 cells. These proteoglycans were sensitive to heparinase, demonstrating that K562 cells synthesize bFGF-binding HSPG. Treatment of K562 cells with phorbol-12-myristate-13-acetate (PMA) caused a loss of bFGF-binding capacity. This decreased binding capacity reflected a rapid loss of high affinity receptors. The ability to form bFGF-receptor complexes decreased by 65% to 70% within 1 hour and declined continuously thereafter. The decrease in binding of bFGF was not due to an autocrine downregulation of bFGF receptors, because there was no increase in bFGF after PMA treatment as detected by Western blotting, and suramin, which blocks bFGF binding to receptors, did not prevent the loss of receptors after exposure to PMA. In addition, inhibitors of either protein synthesis or protease activity did not prevent the loss of bFGF receptors in PMA-treated cells. In summary, this work demonstrates that leukemia cell lines have receptors that specifically bind bFGF and supports the hypothesis that bFGF acts directly on certain blood cells to stimulate their proliferation.
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PMID:Human leukemia cell lines bind basic fibroblast growth factor (FGF) on FGF receptors and heparan sulfates: downmodulation of FGF receptors by phorbol ester. 854 48

Previous studies have shown that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) mRNA is synthesized in the mouse uterine luminal epithelium temporally, just prior to implantation, and spatially, only at the site of blastocyst apposition (Das, S. K., Wang, X. N., Paria, B. C., Damm, D., Abraham, J. A., Klagsbrun, M., Andrews, G. K. and Dey, S. K. (1994) Development 120, 1071-1083). HB-EGF is synthesized as a transmembrane protein (HB-EGF TM) that can be processed to release the soluble growth factor. An antibody that cross-reacts only with the transmembrane form detected HB-EGF TM in uterine luminal epithelium in a spatial manner similar to that of HB-EGF mRNA. HB-EGF TM is a juxtacrine growth factor that mediates cell-cell contact. To ascertain if HB-EGF TM could be an adhesion factor for blastocysts, a mouse cell line synthesizing human HB-EGF TM was co-cultured with mouse blastocysts. Cells synthesizing HB-EGF TM adhered to day-4 mouse blastocysts more extensively than parental cells or cells synthesizing a constitutively secreted form of HB-EGF. Adhesion of cells synthesizing HB-EGF TM to blastocysts was inhibited by excess recombinant HB-EGF but less so by TGF-alpha. Adhesion was also inhibited by the synthetic peptide P21 corresponding to the HB-EGF heparin binding domain, and by incubating the blastocysts with heparinase. In addition, adhesion to delayed implanting dormant blastocysts, which lack EGF receptor (EGFR), was diminished relative to normal blastocysts. These results suggested that adhesion between blastocysts and cells synthesizing HB-EGF TM was mediated via interaction with both blastocyst EGFR and heparan sulfate proteoglycan (HSPG). It was concluded that HB-EGF TM, which is synthesized exclusively in the luminal epithelium at the site of blastocyst apposition, and which is a juxtacrine adhesion factor for blastocysts, could be one of the mediators of blastocyst adhesion to the uterus in the process of implantation.
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PMID:Mouse preimplantation blastocysts adhere to cells expressing the transmembrane form of heparin-binding EGF-like growth factor. 862 15

Perlecan is a modular heparan sulfate proteoglycan that is localized to cell surfaces and within basement membranes. Its ability to interact with basic fibroblast growth factor (bFGF) suggests a central role in angiogenesis during development, wound healing, and tumor invasion. In the present study we investigated, using domain specific anti-perlecan monoclonal antibodies, the binding site of bFGF on human endothelial perlecan and its cleavage by proteolytic and glycolytic enzymes. The heparan sulfate was removed from perlecan by heparitinase treatment, and the approximately 450-kDa protein core was digested with various proteases. Plasmin digestion resulted in a large fragment of approximately 300 kDa, whereas stromelysin and rat collagenase cleaved the protein core into smaller fragments. All three proteases removed immunoreactivity toward the anti-domain I antibody. We showed also that perlecan bound bFGF specifically by the heparan sulfate chains located on the amino-terminal domain I. Once bound, the growth factor was released very efficiently by stromelysin, rat collagenase, plasmin, heparitinase I, platelet extract, and heparin. Interestingly, heparinase I, an enzyme with a substrate specificity for regions of heparan sulfate similar to those that bind bFGF, released only small amounts of bFGF. Our findings provide direct evidence that bFGF binds to heparan sulfate sequences attached to domain I and support the hypothesis that perlecan represents a major storage site for this growth factor in the blood vessel wall. Moreover, the concerted action of proteases that degrade the protein core and heparanases that remove the heparan sulfate may modulate the bioavailability of the growth factor.
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PMID:The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin, and heparanases. 862 65

Epithelial and mesenchymal cells isolated from mouse embryonic lungs synthesized and responded to amphiregulin (AR) in a different fashion. Mesenchymal cells produced and deposited 3- to 4-fold more AR than epithelial cells, proliferated in the presence of exogenous AR, and their spontaneous growth was blocked by up to 85% by anti-AR antibodies. In contrast, epithelial cells exhibited a broad response to this growth regulator factor depending on whether they were supplemented with extracellular matrix (ECM) and whether this ECM was of epithelial or mesenchymal origin. AR-treated epithelial cells proliferated by up to 3-fold in the presence of mesenchymal-deposited ECM, remained unchanged in the presence of epithelial-deposited ECM, and decreased in their proliferation rate below controls in the absence of ECM supplementation. This effect was abolished by treatment with the glycosaminoglycan-degrading enzymes heparinase and heparitinase suggesting the specific involvement of heparan sulfate proteoglycan (HSPG) in AR-mediated cell proliferation. In whole lung explants, branching morphogenesis was inhibited by antibodies against the AR heparan sulfate binding site and stimulated by exogenous AR. Since during development, epithelial cells are in contact with mesenchymal ECM at the tips of the growing buds and alongside the basement membrane, focal variations in the proportion of epithelial and mesenchymal HSPG will focally affect epithelial proliferation rates. Therefore, AR-HSPG interaction may underlie the process of branching morphogenesis by inducing differential cell proliferation.
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PMID:Amphiregulin in lung branching morphogenesis: interaction with heparan sulfate proteoglycan modulates cell proliferation. 867 15

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

Co-infusion of the specific heparan sulfate proteoglycan (HSPG), perlecan, and beta-amyloid protein (A beta) into rodent hippocampus leads to a consistent animal model to study the effects of fibrillar A beta amyloid in brain [Snow, A.D. et al. (1994) Neuron 12, 219-234]. In the present study, we describe our rapid novel method of perlecan isolation. The isolation method does not require cesium chloride centrifugation and exploits a newly discovered aggregating property of a approximately 220 kDa PG observed during gel filtration chromatography, which allowed it to be affectively separated from non-aggregating perlecan. Fifty or 100 g of EHS tumor were routinely extracted using 4 M guanidine-HCl, followed by anion-exchange and gel filtration chromatography. SDS-PAGE (before and after digestion with heparitinase/heparinase or nitrous acid) followed by staining with silver demonstrated no other contaminating proteins in the perlecan preparations. Western blots using a specific perlecan core protein antibody (HK-102) following heparitinase digestion showed a characteristic doublet at 400 and 360 kDa indicative of intact perlecan core protein. Absence of contamination by other basement membrane components produced by the EHS tumor was confirmed by absence of immunoreactive bands on Western blots using antibodies against laminin, fibronectin, or type IV collagen. One week continuous co-infusion of perlecan obtained from this methodology, with A beta (1-40) into rodent hippocampus, led to deposition of fibrillar A beta amyloid in 100% (10 of 10) of animals. The detailed protocol for isolation and characterization of perlecan from EHS tumor ensures perlecan of the highest quality, and maximizes the potential effects of A beta amyloid deposition/persistence in brain using the animal model. High quality perlecan obtained from this novel isolation method will also allow future studies utilizing in vitro assays to determine the potential interactions of this specific HSPG with other macromolecules.
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PMID:Novel purification and detailed characterization of perlecan isolated from the Engelbreth-Holm-Swarm tumor for use in an animal model of fibrillar A beta amyloid persistence in brain. 888 31


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