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 metabolism of chylomicron remnants in mice deficient in low density lipoprotein receptor (LDLr) or apolipoprotein E (apoE) was compared with that of control C57BL/6J mice. Mice were injected intravenously with chylomicron-like emulsions labeled with radioactive lipids. Blood samples were taken at fixed time intervals from the retro-orbital sinus, and clearance rates of the lipoproteins were assessed from the decline in plasma radioactivities. To follow the intracellular pathway of remnants in the liver, emulsions labeled with a fluorescent cholesteryl ester (BODIPY) were injected, and liver sections were processed and assayed by laser confocal microscopy. Catabolism of remnant cholesteryl esters was assessed by injecting emulsions labeled with cholesteryl[1-14C]oleate and measuring the expired CO2 from each animal. In apoE-deficient mice, remnant removal from plasma was totally impeded, while the clearance of remnants in LDLr-deficient mice was similar to that in C57BL/6J control mice. The confocal micrographs of livers 20 min after injection of fluorescent chylomicron-like emulsions showed evenly distributed fluorescent particles in the hepatocytes from control mice. In contrast, the fluorescent particles were mainly located in sinusoidal spaces in LDLr-deficient mice. Three hours after injection the livers from control mice showed few fluorescent particles, indicating that remnants have been catabolized, while the sections from LDLr-deficient mice were still highly fluorescent. Micrographs from apoE-deficient mice showed no fluorescent particles in the liver at any time after injection. Measurement of expired radioactive CO2 after injection of emulsions labeled in the fatty acid moiety of cholesteryl oleate indicated that remnant metabolism was slower in the LDLr-deficient mice and essentially nil in the apoE-deficient mice. Control mice had expired 50% of the injected label by 3 h after injection. We conclude that under normal circumstances, chylomicron remnants are rapidly internalized by LDLr and catabolized in hepatocytes, with a critical requirement for apoE. When LDLr is absent, remnants are taken up by a second apoE-dependent pathway, first to the sinusoidal space of the liver, with subsequent slow endocytosis and slow catabolism. Hepatic clearance via this second pathway is increased by heparin, inhibited by lactoferrin, heparinase, and suramin, and down-regulated by feeding a high fat diet.
J Biol Chem 1995 Dec 01
PMID:Intracellular localization and metabolism of chylomicron remnants in the livers of low density lipoprotein receptor-deficient mice and apoE-deficient mice. Evidence for slow metabolism via an alternative apoE-dependent pathway. 749 99

Cytotactin/tenascin is a multidomain extracellular matrix protein that inhibits both cell spreading and intracellular alkalinization. The protein has multiple different domains which are homologous to regions in epidermal growth factor, fibronectin, and fibrinogen. In previous studies, we produced nonoverlapping fusion proteins corresponding to these domains and examined their effects on cell attachment and spreading. Based on their ability either to promote or to inhibit cell attachment, two of these fusion proteins were shown to be adhesive and two were shown to be counteradhesive. To determine how the adhesive and counteradhesive activities of different cytotactin/tenascin domains alter intracellular pH (designated pHi), we have measured pHi, in NIH3T3 and U251MG cells in the presence of the cytotactin/tenascin fusion proteins and intact cytotactin/tenascin, as well as fibronectin. Cells incubated in the presence of intact cytotactin/tenascin or of the counteradhesive fusion proteins had a pHi lower than control cells. In contrast, the presence of the adhesive fusion proteins or of fibronectin caused cells to have higher pHi values than control cells. When two fragments were simultaneously presented, one of which alone increased pHi and the other of which alone decreased pHi, the predominant effect was that of lowered pHi. Incubation with an RGD-containing peptide derived from the cytotactin/tenascin sequence inhibited alkalinization promoted by the adhesive fragment containing the second through sixth fibronectin type III repeats that was known to bind to integrins. Incubation of the cells with heparinase I or III inhibited the intracellular alkalinization of cells plated in the presence of the other adhesive fusion protein containing the fibrinogen domain, suggesting that heparan sulfate proteoglycans were involved in these pHi changes. The activity of protein kinase C appeared to be important for the changes in pHi mediated by all of the proteins. The protein kinase C inhibitor Calphostin C blocked the rise in pHi elicited by the adhesive fusion proteins and by fibronectin. Moreover, activation of protein kinase C by the addition of phorbol esters increased the pHi in cells plated on cytotactin/tenascin or counteradhesive fusion proteins and reversed their effects. The results of this study support the hypothesis that cytotactin/tenascin can bind to multiple cell surface receptors and thereby elicit different physiological responses. Decreases in pHi are correlated with the phenomenon of counteradhesion whereas the ability to increase pHi is associated with cell attachment via at least two different types of cell surface receptors. The data raise the possibility that binding of cytotactin/tenascin may influence primary cellular processes such as migration and proliferation through the differential regulation of pHi.
J Cell Physiol 1994 Dec
PMID:Differential effects of cytotactin/tenascin fusion proteins on intracellular pH and cell morphology. 752 16

Chemokines, a superfamily of 8- to 11-kDa mediators of inflammation, affect the attachment of immune cells to vascular endothelia by binding to cell surface glycosaminoglycans. We analyzed whether chemokines are also involved in interactions between CD4+ T lymphocytes and the subendothelial extracellular matrix (ECM). Soluble mediators, such as MIP-1 beta and RANTES, induced the binding of resting human CD4+ T cells to ECM in an integrin-dependent manner. Both MIP-1 beta and RANTES bound to intact ECM and retained their adhesive properties, and moreover, ECM-bound RANTES and MIP-1 beta prolonged the time course of interactions between the CD4+ T cells and the ECM. Because the adhesive effect of these chemokines was restricted by an inhibitor of GTP-binding proteins, the adhesive effect of ECM-bound RANTES and MIP-1 beta, which requires an intact cytoskeleton, seems to involve activation of a G protein-linked receptor. MIP-1 beta and RANTES exert their pro-adhesive effects through interactions with glycosaminoglycans, because heparinase-treated ECM did not bound chemokines and because the chemokines ability to induce T cell adhesion was abrogated if: 1) either of the chemokines is pretreaed with heparin or heparan-sulfate (HS), 2) HS is removed from intact ECM by heparinase, an HS-specific endoglycosidase, or 3) the ECM-bound chemokines are released by pretreatment with heparinase. Hence, the adhesive effects of immobilized chemokines is not restricted to T cells interacting with endothelial cells, but also affects the migration of immune cells which reside and function in the context of ECM.
J Immunol 1994 Dec 01
PMID:Regulation of adhesion of CD4+ T lymphocytes to intact or heparinase-treated subendothelial extracellular matrix by diffusible or anchored RANTES and MIP-1 beta. 752 18

Bovine lactoferrin inhibits the clearance of remnant lipoproteins from the plasma and competes with the cell-surface binding of apolipoprotein (apo) E-enriched remnants. We established that lactoferrin inhibits remnant binding and uptake by interacting with both heparan sulfate proteoglycans (HSPG) and the low-density lipoprotein receptor-related protein (LRP). The binding of 125I-lactoferrin was inhibited 45% to 60% in HepG2 hepatocytes and wild-type Chinese hamster ovary (CHO) cells treated with heparinase to remove HSPG. In mutant CHO cells (pgsD-677) lacking HSPG, the level of 125I-lactoferrin binding was approximately 50% that seen with wild-type CHO cells; thus, about one half of lactoferrin binding appears to be mediated through cell-surface HSPG. A significant fraction of the residual binding of the lactoferrin appears to be mediated through the LRP. The 39-kd protein known to bind to the LRP and to block ligand interaction inhibited 125I-lactoferrin degradation in wild-type CHO cells by 60% to 65%. The addition of the 39-kd protein plus heparinase treatment reduced the binding by 85% to 90% (this combination blocks direct interaction with both the LRP and HSPG). However, it was also shown that the 39-kd protein bound to HSPG and the LRP. Heparinase treatment of wild-type CHO cells decreased the binding of the 125I-39-kd protein by approximately 40%, and the mutant CHO cells lacking HSPG bound half as much 125I-39-kd protein as wild-type CHO cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Arterioscler Thromb 1994 Dec
PMID:Lactoferrin binding to heparan sulfate proteoglycans and the LDL receptor-related protein. Further evidence supporting the importance of direct binding of remnant lipoproteins to HSPG. 752 99

The fibroblast growth factors (FGFs) act through high affinity tyrosine kinase receptors and, in addition, interact with lower affinity receptors that represent cell- or matrix-associated heparan sulfate proteoglycans. These lower affinity receptors modulate the biological activities of FGFs, but the mechanism by which they exert these effects is rather controversial. We have previously shown (Ron, D., Bottaro, D. P., Finch, P. W., Morris, D., Rubin, J. S., and Aaronson, S. A. (1993) J. Biol. Chem. 268, 2984-2988) that heparin potentiates the mitogenic activity of acidic FGF (aFGF) but inhibits that of the keratinocyte growth factor (KGF) in cells that express the KGF receptor (KGFR). Both growth factors bind the KGFR with high affinity. To gain an insight into the mechanism by which heparin modulates the biological activity of aFGF and KGF, we studied the effect of heparin and cell-associated heparan sulfates on the binding of these two growth factors to the KGFR. To work in a well defined system, we expressed functional KGFR in L6E9 myoblasts that lack detectable high affinity binding sites for FGFs. Low concentrations of heparin inhibited the binding of KGF to the KGFR. By contrast, similar concentrations of heparin enhanced the binding of aFGF to this receptor. The effect of heparin was not unique to L6E9 cells expressing the KGFR; it was also observed in Balb/MK cells that naturally express KGFR. Treatment of cells with sodium chlorate, which blocks sulfation of proteoglycans, reduced the binding of aFGF to its low and high affinity binding sites by 95 and 80%, respectively. In contrast, the binding of KGF to its high affinity binding sites was enhanced about 2-fold. Similar results were obtained after degradation of cell-associated heparan sulfates by heparinase and heparitinase. Heparin restored the high affinity binding of aFGF to chlorate-treated cells and completely abolished the high affinity binding of KGF. Binding competition experiments suggest that aFGF and KGF bind to the same population of cell-associated heparan sulfates. In addition, KGF is apparently interacting with an as yet unidentified type of low affinity binding site that is not affected by chlorate or heparan sulfate-degrading enzymes. An important property of the FGF high affinity receptors is their ability to bind more than one ligand with high affinity. Based on the differential effect of cell-associated heparan sulfates on the binding of KGF and aFGF to the KGFR, we propose a regulatory role for cell-associated heparan sulfates as coordinators of the interaction of aFGF and KGF with the KGFR.(ABSTRACT TRUNCATED AT 400 WORDS)
J Biol Chem 1994 Dec 23
PMID:Differential effect of cell-associated heparan sulfates on the binding of keratinocyte growth factor (KGF) and acidic fibroblast growth factor to the KGF receptor. 752 11

OSF-1/HB-GAM is a member of developmentally regulated growth factors and cytokines. High expression levels of this factor are found in different tissues, e.g., in brain and in bone. We have analyzed the biological function and binding properties of natural OSF-1 to human osteoblasts. Using antibodies raised against the entire OSF-1 molecule or a synthetic carboxy-terminal peptide (amino acids (aa) 110-140) we have investigated the binding sites of rat OSF-1 on human osteoblast-like osteosarcoma cell lines: HOS(TE85) and MG-63. Immunofluorescence microscopic studies and flow cytometric data revealed that OSF-1 is specifically bound to the surface of these cells. Further characterization of the binding sites showed that both osteosarcoma cells express two different kinds of binding sites: Besides binding to a specific OSF-1 receptor, OSF-1 also significantly binds to cell surface heparan sulfates. Using the peptide specific polyclonal antibody we show that the carboxy-terminal domain, aa 110 to 140 of OSF-1, seems to be involved in ligand binding. Studies on the biological function of OSF-1 revealed a strong cell attachment promoting activity in vitro. This activity is not diminished after digestion of cell surface heparan sulfates by heparinase I and heparitinase I, demonstrating that the OSF-1 receptor mediates the cell attachment of osteoblasts. Our results indicate that one biological function of OSF-1 is the promotion of osteoblast attachment to the extracellular bone matrix.
Eur J Cell Biol 1993 Dec
PMID:Receptor binding of osteoblast-specific factor 1 (OSF-1/HB-GAM) to human osteosarcoma cells promotes cell attachment. 792 91

We have previously demonstrated that chemically modified thrombin preparations induce endothelial cell (EC) adhesion, spreading and cytoskeletal reorganization via an Arg-Gly-Asp (RGD) sequence and the alpha v beta 3 integrin. Native thrombin, however, did not exhibit adhesive properties, consistent with crystal structure analysis, showing that Gly-Asp residues of the RGD epitope are buried within the molecule. We have now identified a possible physiological mean of converting thrombin to an adhesive protein. Plasmin, the major end product of the fibrinolytic system, converted thrombin to an adhesive protein for EC in a time and dose-dependent manner. EC adhesion and spreading was also induced by a low molecular weight (approximately 3,000 D) cleavage fragment generated upon incubation of thrombin with plasmin. Cell adhesion mediated by this fragment was completely inhibited by the synthetic peptide GRGDSP. Conversion of thrombin to an adhesive molecule was significantly enhanced in the presence of heparin or heparan sulfate, while other glycosaminoglycans (GAGs) (e.g., dermatan sulfate, keratan sulfate, chondroitin sulfate) had no effect. The role of cell surface heparan sulfate in thrombin conversion to EC adhesive protein was investigated using CHO cell mutants defective in various aspects of GAG synthesis. Incubation of both thrombin and a suboptimal amount of plasmin on the surface of formaldehyde fixed wild-type CHO-KI cells resulted in an efficient conversion of thrombin to an adhesive molecule, as indicated by subsequent induction of EC attachment. In contrast, there was no effect to incubation of thrombin and plasmin with fixed CHO mutant cells lacking both heparan sulfate and chondroitin sulfate, or with cells expressing no heparan sulfate and a three-fold increase in chondroitin sulfate. A similar gain of adhesive properties was obtained upon incubation of thrombin and plasmin in contact with native, but not heparinase-treated extracellular matrix (ECM) produced by cultured ECs. It appears that cell surface and ECM-associated heparan sulfate modulate thrombin adhesive properties through its heparin binding site in a manner that enables suboptimal amounts of plasmin to expose the RGD domain. Our results demonstrate, for the first time, a significant modulation of thrombin molecule by heparin, resulting in its conversion to a potent adhesive protein for ECs. This conversion is most effective in contact with cell surfaces, basement membranes and ECM.
J Cell Biol 1993 Dec
PMID:Thrombin adhesive properties: induction by plasmin and heparan sulfate. 824 31

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.
J Biol Chem 1995 Dec 15
PMID:Lysolecithin-induced alteration of subendothelial heparan sulfate proteoglycans increases monocyte binding to matrix. 853 Mar 67

The versatile biological activities of proteoglycans are mainly mediated by their glycosaminoglycan (GAG) components. Unlike proteins and nucleic acids, no satisfactory method for sequencing GAGs has been developed. This paper describes a strategy to sequence the GAG chains of heparin. Heparin, prepared from animal tissue, and processed by proteinases and endoglucuronidases, is 90% GAG heparin and 10% peptidoglycan heparin (containing small remnants of core protein). Raw porcine mucosal heparin was labelled on the amino termini of these core protein remnants with a hydrophobic, fluorescent tag [N-4-(6-dimethylamino-2-benzofuranyl) phenyl (NDBP)-isothiocyanate]. Enrichment of the NDBP-heparin using phenyl-Sepharose chromatography, followed by treatment with a mixture of heparin lyase I and III, resulted in a single NDBP-linkage region tetrasaccharide, which was characterized as deltaUAp(1-->3)-beta-D-Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp -(1-->O-Ser-NDBP (deltaUAp is 4-deoxy-alpha-L-threo-hex-4-enopyranosyl uronic acid). Several NDBP-octasaccharides were isolated when NDBP-heparin was treated with only heparin lyase I. The structure of one of these NDBP-octasaccharides, deltaUAp2S(1-->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp(1-->3)-beta-D- Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp-(1-->O-Ser NDBP (S is sulphate, Ac is acetate), was determined by 1H-NMR and enzymatic methods. Enriched NDBP-heparin was treated with lithium hydroxide to release heparin, and the GAG chain was then labelled at xylose with 7-amino-1,3-naphthalene disulphonic acid (AGA). The resulting AGA-Xyl-heparin was sequenced on gradient PAGE using heparin lyase I and heparin lyase III. A predominant sequence in heparin at the protein core attachment site was deduced to be -D-GlcNp2S6S(or 6OH)(1-->4)-alpha-L-IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S (or60H) (1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNp2S6S( or 6OH)(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNpAc (1- ->4)-alpha-L-IdoAp(1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-++ +GlcAp(1-->3)-beta-D-Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xyl-AGA.
Glycobiology 1995 Dec
PMID:Strategy for the sequence analysis of heparin. 872 74

We used cationized colloidal gold in order to investigate the distribution of anionic sites in different secretory granules of rat and mouse mast cells. The localization of the anionic sites was performed by post-embedding labeling of thin sections of rat peritoneal cells or mouse skin tissue, fixed in Karnovsky's fixative and OsO4, and embedded in Araldite or LR white, respectively. In all cases anionic sites were demonstrated with a high density variation depending on cell type. In all mast cell secretory granules we have observed the highest density (ca. 500-900 gold particles/microns2), while in other peritoneal cell granules it was about 10 times less (ca. 40-80 gold particles/microns2). Pretreatment of the LR white sections with heparinase I and III resulted in a reduction of 97% and 72%, respectively, in the binding of the gold particles to the granules, indicating that the majority of the gold binding reactivity is due to heparin. Correlation of section profile area with labeling density revealed that the smaller granules were significantly more labeled when compared to the larger profiles. On the basis of these observations it seems that a post-translational change (mainly sulfation of heparin) of secretory content influences the granule anionic charge and thus may affect the intragranule buffer capacity.
Histochem Cell Biol 1995 Dec
PMID:An histochemical approach to characterization of anionic constituents in mast cell secretory granules. 877 31


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