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)

Heparan sulfate proteoglycans (HSPG) are involved in the binding and uptake of apolipoprotein (apo) E-enriched remnant lipoproteins by cultured cells in vitro. To define the role of hepatic HSPG in remnant lipoprotein clearance in vivo, heparinase (30 units) was infused intravenously into mice to hydrolyze the liver HSPG and determine the effect of HSPG hydrolysis on remnant clearance by the liver. Liver HSPG were prelabeled by peritoneal injection of [35S]Na2SO4. Injection of heparinase decreased the amount of 35S-labeled liver HSPG by approximately 20-40% within 10-15 min. Heparinase infusion significantly inhibited the clearance of chylomicrons, chylomicron remnants, chylomicron remnants + apoE, rabbit beta-very low density lipoproteins (beta-VLDL), and beta-VLDL + apoE. Compared with saline injection in control mice, heparinase injection retarded the plasma clearance of the remnants by 1.5- to 2-fold and decreased liver uptake by 1.3- to 1.6-fold. Confocal fluorescence microscopy of thick slices of liver from mice injected with 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine-labeled beta-VLDL + apoE revealed markedly less intense fluorescence from hepatocytes in heparinase-treated animals compared with those in saline-treated control animals. Intravenous heparinase infusion did not inhibit the clearance of mouse low density lipoproteins (LDL), a ligand for the LDL receptor, and did not affect the clearance of alpha 2-macroglobulin, a ligand for the LDL receptor-related protein. The results suggest an important role of the liver HSPG in remnant clearance in vivo.
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PMID:Intravenous heparinase inhibits remnant lipoprotein clearance from the plasma and uptake by the liver: in vivo role of heparan sulfate proteoglycans. 753 27

Heparan sulfate proteoglycans on the cell surface act as low affinity binding sites for acidic and basic fibroblast growth factor (FGF) [Moscatelli (1987): J Cell Physiol 131:123-130] and play an important role in the interaction of FGF with the FGF receptor (FGFR). In this study, several aspects of the interaction of FGFs with cell surface heparan sulfate proteoglycans were examined. Reciprocal cross blocking studies demonstrated that acidic FGF (aFGF) and basic FGF (bFGF) bind to identical or closely associated heparan sulfate motifs on BALB/c 3T3 cell surface heparan sulfate proteoglycans. However, the binding affinity of the two growth factors for these heparan sulfate proteoglycans differs considerably, competition binding data indicating that aFGF has a 4.7-fold lower affinity than bFGF for 3T3 heparan sulfate proteoglycan. Subsequent studies of dissociation kinetics demonstrated that bFGF dissociates from the FGFR at least 10-fold slower than aFGF, whereas, following removal of cell surface heparan sulfate proteoglycans by heparinase treatment, the dissociation rate of both FGFs is similar and rapid. These results support the concept that cell surface heparan sulfate proteoglycans stabilize the interaction of FGF with FGFR, possibly by the formation of a ternary complex.
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PMID:Acidic and basic fibroblast growth factor bind with differing affinity to the same heparan sulfate proteoglycan on BALB/c 3T3 cells: implications for potentiation of growth factor action by heparin. 754 3

Lipoprotein lipase (LPL) is rapidly and efficiently cleared from the circulation by the liver through an as yet unclear mechanism. In the present study, we determined the nature of LPL interactions with the liver parenchimal cell line HepG2 as compared to other cells in culture. Binding, cell association and degradation of 125I-labelled bovine milk LPL by HepG2 cells, normal and low density lipoprotein (LDL) receptor-negative human fibroblasts and Chinese hamster ovary (CHO) cells show similar values irrespective of source and origin. LPL metabolism in HepG2 cells was characterized by a high capacity to degrade the enzyme, an extremely high sensitivity to heparin and was inhibited by 60%-70% after treatment of the cells with sodium chlorate and heparinase (but not chondroitinase). These findings suggested an important role for heparan sulfate in the process of cell interaction and metabolism of LPL. To further clarify the role of heparan sulfate in determining the LPL-cell interactions, we compared the metabolism of LPL in wild type and mutant heparan sulfate-deficient CHO cells. Heparan sulfate-deficient CHO cells show a low capacity to bind and degrade LPL, about 10%-20% that of the wild type cells. In another set of experiments, we sought to determine whether LPL interactions with HepG2 cells are affected by triglyceride-rich lipoproteins. The results clearly show that whereas unlabeled LPL dramatically enhanced the metabolism of radioiodinated very low density lipoprotein (VLDL), unlabeled VLDL had no effect on radioiodinated LPL metabolism in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Binding to heparan sulfate is a major event during catabolism of lipoprotein lipase by HepG2 and other cell cultures. 760 68

Heparan sulfate biosynthesis initiates by the transfer of alpha-D-GlcNAc from UDP-GlcNAc to the D-GlcA moiety of the linkage tetrasaccharide, GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-core protein. The enzyme catalyzing this reaction differs from the alpha-GlcNAc transferase involved in chain polymerization based on genetic and enzymatic studies of an animal cell mutant defective in chain polymerization (Fritz, T. A., Gabb, M. M., Wei, G., and Esko, J. D. (1994) J. Biol. Chem. 269, 28809-28814). In this report we show that this mutant also accumulates a pentasaccharide intermediate containing alpha-GlcNAc. A fusion protein was made from the IgG-binding domain of protein A and a segment of the proteoglycan, betaglycan. This segment contained one glycosaminoglycan attachment site that primes only chondroitin sulfate and another that primes both heparan sulfate and chondroitin sulfate (Zhang, L., and Esko, J. D. (1994) J. Biol. Chem. 264, 19295-19299). Expression of the chimera in the mutant resulted in the accumulation of an oligosaccharide that labeled with [6-3H]GlcN. The oligosaccharide comigrated with a pentasaccharide standard derived from chondroitin sulfate, but acid hydrolysis gave 98% [3H]GlcN. Heparin lyase III digestion yielded [3H]GlcNAc, suggesting that the GlcNAc residue was alpha-linked to the nonreducing terminus. Enzymatic treatment of [6-3H]Gal-labeled material yielded the tetrasaccharide, delta GlcA-[3H]Gal-[3H]Gal-xylitol. These findings suggest that pentasaccharide had the structure, GlcNAc alpha 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl. Its accumulation in a Chinese hamster ovary cell mutant defective in the polymerizing alpha-GlcNAc transferase provides in vivo evidence that two alpha-GlcNAc transferases catalyze the formation of heparan sulfate.
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PMID:Accumulation of a pentasaccharide terminating in alpha-N-acetylglucosamine in an animal cell mutant defective in heparan sulfate biosynthesis. 775 2

Heparan sulfate proteoglycans (HSPG) are obligatory for receptor binding and mitogenic activity of basic fibroblast growth factor (bFGF). Mutant Chinese hamster ovary cells (pgsA-745) deficient in xylosyltransferase are unable to initiate glycosaminoglycan synthesis and hence can not bind bFGF to low- and high-affinity cell surface receptors. Exposure of pgsA-745 cells to beta-D-xylopyranosides containing hydrophobic aglycones resulted in restoration of bFGF binding in a manner similar to that induced by soluble heparin or by heparan sulfate (HS) normally associated with cell surfaces. Restoration of bF-GF binding correlated with the ability of the beta-D-xylosides to prime the synthesis of heparan sulfate. Thus, both heparan sulfate synthesis and bFGF receptor binding were induced by low concentrations (10-30 microM) of estradiol-beta-D-xyloside and naphthyl-beta-D-xyloside, but not by cis/trans-decahydro-2-naphthyl-beta-D-xyloside, which at low concentration primes mainly chondroitin sulfate. The obligatory involvement of xyloside-primed heparan sulfate in restoration of bFGF-receptor binding was also demonstrated by its sensitivity to heparinase treatment and by the lack of restoration activity in CHO cell mutants that lack enzymatic activities required to form the repeating disaccharide unit characteristic of heparan sulfate. Xyloside-primed heparan sulfate binds to the cell surface. Restoration of bFGF receptor binding was induced by both soluble and cell bound xyloside-primed heparan sulfate and was abolished in cells that were exposed to 0.5-1.0 M NaCl prior to the bFGF binding reaction. These results indicate that heparan sulfate chains produced on xyloside primers behave like heparan sulfate chains attached to cellular core proteins in terms of affinity for bFGF and ability to function as low-affinity sites in a dual receptor mechanism characteristic of bFGF and other heparin-binding growth promoting factors.
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PMID:Heparan sulfate primed on beta-D-xylosides restores binding of basic fibroblast growth factor. 775 55

Heparan sulfate (HS) secreted into the medium of bovine aortic endothelial cell (BAEC) cultures was subjected to chemical and enzymatic degradation followed by analysis using gel-filtration and ion-exchange chromatography. Treatment with HNO2 showed that 41% of the disaccharides were N-sulfated. Degradation by Heparin lyases I (Hep I) showed that 8-9% of the disaccharides contained IdoA(2S) residues. Heparin lyase III (Hep III) degradation produced mainly disaccharides with 67% of the molecules glycosidic linkages susceptible to cleavage. Further degradation of Hep III-resistant fragments with Hep I showed that IdoA(2S) residues were predominantly positioned centrally within the repeating GlcNSO3(+/- 6S)alpha 1-4IdoA containing domains. Digestion with a mixture of Heparin lyases I, II and III degraded the molecule almost entirely to disaccharides, with small amounts of tetrasaccharides containing resistant linkages, suggesting the presence of 3-O sulfated GlcNSO3. Further analysis of the disaccharide products by ion-exchange chromatography and comparison with the data from single enzymatic digestion, allowed an estimate of the disaccharide composition to be made. The results suggest an ordered arrangement of structural domains; however, variations in the structure of these domains results in a heterogeneous population of HS chains. It is suggested that biosynthetic differences in HS structure may act as a regulator of bFGF induced cellular responses.
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PMID:Molecular attributes of bovine aortic endothelial cell heparan sulfate. 776 9

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

Binding of urinary protein C inhibitor (PCI) to cultured human epithelial kidney tumor cells (TCL-598) was studied. Binding was dose-dependent, time-dependent, and saturable. Heparin interfered in a dose-dependent way with PCI binding to TCL-598 as did heparan sulfate and to a lesser degree also dermatan sulfate. Pretreatment of TCL-598 with protamine sulfate inhibited subsequent binding of PCI in a dose-dependent manner and > 100 micrograms/ml protamine sulfate reduced binding of PCI to < 10% of the control. Binding of 125I-PCI was specific, and bound 125I-PCI was recovered from the cells by heparin treatment or detached together with intact cells by EDTA treatment, migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the same mobility (M(r) = 57,000) as unbound 125I-PCI. Furthermore, cell-bound PCI was functionally active as judged from its ability to inhibit the amidolytic activity of urokinase, and its inhibitory activity was stimulated approximately 3-4-fold as compared to fluid-phase PCI. Immunogold electron microscopy revealed that PCI-antigen presented to the cells from the luminal side bound exclusively to that surface in native as well as in prefixed cells. This binding of PCI was abolished in the presence of heparin (50 micrograms/ml) and after pretreatment of the cells either with protamine sulfate (400 micrograms/ml) or with heparinase III (0.5 unit/ml). A slight decrease in PCI binding was seen after pretreatment of the cells with chondroitinase ABC and chondroitinase AC. In contrast, binding of PCI to extracellular matrices of TCL-598 was decreased to approximately 70% after chondroitinase ABC treatment of the extracellular matrices, whereas both heparinase III or chondroitinase AC treatment only reduced matrix-bound PCI to approximately 95%. These data suggest that heparan sulfate-containing proteoglycans are predominantly involved in binding of PCI to the luminal side of TCL-598, while dermatan sulfate-containing proteoglycans, the overall predominant PCI-binding proteoglycans in TCL extracts, are responsible for PCI binding to the extracellular matrix. Heparan sulfate, however, exposed to an environment containing PCI under physiological conditions, might localize PCI and modulate its target enzyme specificity in vivo.
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PMID:Binding of urinary protein C inhibitor to cultured human epithelial kidney tumor cells (TCL-598). The role of glycosaminoglycans present on the luminal cell surface. 818 78

Heparin was extracted and purified from beef intestinal mucosa. The two components, fast moving heparin and slow moving heparin were purified by selective precipitation as barium salts. Heparan sulfate was extracted and purified from beef spleen. Dermatan sulfate was purified from beef intestinal mucosa and chondroitin sulfate from bovine trachea. The purity of the purified glycosaminoglycans was evaluated by agarose-gel and cellulose polyacetate electrophoresis and by specific optical rotation. The relative molecular masses of glycosaminoglycans were estimated by high performance-size exclusion chromatography and the sulfate to carboxyl ratio by titrimetric analysis. The disaccharide pattern of heparin, fast moving and slow moving heparins and heparan sulfate were determined by specific enzymatic cleavage using heparinase I, II and III; the disaccharide composition of dermatan sulfate and chondroitin sulfate was evaluated by cleavage by chondroitinase ABC. The disaccharides obtained by enzymatic cleavage were qualitatively and quantitatively analysed by strong anion exchange-high performance liquid chromatography. The sulfate to carboxyl ratios of glycosaminoglycans were also determined by this technique and compared with the values obtained by titrimetric analysis.
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PMID:Extraction, purification and evaluation of structures and physico-chemical properties of glycosaminoglycans. 835 79

Heparan sulfate and low density lipoprotein receptor related protein (LRP) have been shown to participate in the uptake and degradation of the enzyme lipoprotein lipase (LPL). Yet, the contribution of each of these pathways to LPL metabolism and their possible dependence is unknown. In the present study we examined the metabolism of 125I-labeled LPL in untreated and heparinase-treated primary wild-type mouse embryonic fibroblasts (MEF) and in mouse fibroblasts that express single LRP allele (PEA-10) or are lacking the LRP (PEA-13). The degradation of LPL in PEA-13 cells was 30% lower than in MEF and PEA-10 cells. Heparinase treatment decreased the LPL degradation by 58%, 79% and 92%, whereas heparin reduced such degradation by 87%, 90% and 94% in MEF, PEA-10 and PEA-13 cultures, respectively. Assuming that a) heparinase treatment abolished the heparan-sulfate pathway, and that b) the degradation remaining in heparin-treated cultures represents nonspecific values, it appears that heparan sulfate contributes about 61%, 83% and 95% of total LPL degradation, whereas the LRP pathway contributes 39%, 17% and less than 5% of LPL degradation in MEF, PEA-10 and PEA-13 cells, respectively. In addition, the data indicate that LPL interaction with heparan sulfate and the LRP pathways is independent of each other. The study shows that these cells possess both a heparan sulfate-dependent pathway and an LRP-dependent pathway for LPL metabolism and that the two pathways are independent of each other.
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PMID:Heparan sulfate-dependent and low density lipoprotein receptor-related protein-dependent catabolic pathways for lipoprotein lipase in mouse embryonic fibroblasts. 868 50


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