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)

It has been postulated that lipoprotein lipase, an enzyme important in the uptake of fatty acids into tissues, is bound to the vascular endothelial cell surface and that this binding occurs through attachment to heparinlike glycosaminoglycans. Furthermore, it is thought that heparin releases the enzyme from its attachment to the endothelium into the circulation. These hypotheses have never been tested directly in cell systems in vitro. In the present study we have directly evaluated the interaction of lipoprotein lipase, purified from bovine skim milk with monolayer cultures of endothelial cells, isolated from bovine pulmonary artery. Endothelial cells in primary culture had no intrinsic lipoprotein lipase activity but were able to bind lipoprotein lipase quantitatively. The binding reached equilibrium and was saturable at 0.24 nmol of lipoprotein lipase/mg of cell protein. The concentration of lipoprotein lipase at half-maximal binding was 0.52 microM. Bound lipoprotein lipase could be detached from cultured cells by increasing concentrations of heparin, and at and above 0.6 microgram/ml of heparin, 90% of the cell-bound lipoprotein lipase activity was released. Heparan sulfate and dermatan sulfate released the enzyme to a lesser extent and chondroitin sulfate caused little, if any, release of lipoprotein lipase. The release of lipoprotein lipase with heparin was not associated with a release of [3S]glycosaminoglycans from 35S-prelabeled cells. Reductions of lipoprotein lipase binding to endothelial cells and of cell surface-associated [3S]glycosaminoglycans in 35S-prelabeled cells occurred in parallel both when cells were pretreated with crude Flavobacterium heparinum enzyme before lipoprotein lipase binding and when cells were treated with this enzyme after lipoprotein lipase binding. The removal of heparan sulfate from the cell surface by purified heparinase totally inhibited the binding of lipoprotein lipase by endothelial cells, but the removal of chondroitin sulfate by chondroitin ABC lyase had no effect on this binding. These results provide direct evidence for lipoprotein lipase attachment to endothelial cells through heparan sulfate on the cell surface, and provide evidence for the release of lipoprotein lipase by heparin through a detachment from this binding site.
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PMID:Involvement of cell surface heparin sulfate in the binding of lipoprotein lipase to cultured bovine endothelial cells. 645 61

Lipoprotein lipase, purified from bovine milk, lost 90% of its activity when incubated in Hanks' balanced salt solution for 5 min at 37 degrees C. Bovine pulmonary artery endothelial cells, maintained in culture, markedly stabilized this enzyme. The stabilizing factor of endothelial cells was non-dialyzable, resistant to heating at 100 degrees C and to changes in pH, and unaffected by treatments of cells with proteolytic enzymes or with heparinase (Flavobacterium heparinum enzyme). However, the stabilizing effect on lipoprotein lipase was reduced by 60-70% by the extraction of cells with chloroform/methanol (2:1). The lipid extract of the cells stabilized the enzyme, suggesting that lipid component(s) of the endothelial cells account for their stabilizing effect. Since the endothelial cell is thought to be the site of action of lipoprotein lipase, stabilization of the enzyme by this cell may play a role in its preservation and function in vivo.
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PMID:Stabilization of lipoprotein lipase by endothelial cells. 706 49

An apolipoprotein (apo) E- and lipoprotein lipase-independent, high affinity, saturable and specific binding site and pathway for uptake of certain triglyceride-rich lipoproteins (TGRLP) by human monocyte-macrophages that leads to lipid accumulation and foam cell formation in vitro has been reported; two membrane binding activities were identified as receptor candidates with apparent molecular masses of 200 and 235 kDa [Gianturco et al. (1994) J. Lipid Res. 35, 1674-1687]. Here we present new evidence that these activities are TGRLP receptors with unique biochemical properties which distinguish them from other lipoprotein receptors. Protease and heparinase susceptibility studies demonstrate that (1) these activities have essential protein, but not heparan sulfate proteoglycan (HSPG) components; (2) the membrane binding proteins (MBPs) are located on the cell surface; (3) HSPGs do not facilitate TGRLP binding to this specific cellular site. Upon reduction, MBP 200 and 235 are both converted into a single, new binding activity of intermediate mobility (MBP 200R); all MBP forms displayed high affinity, saturable TGRLP binding with similar Kds (1.4-2.2 micrograms/mL). Notably, MBP 200R retained the combined ligand binding capacity of MBP 200 and 235 prior to reduction, demonstrating that, unlike members of the LDL receptor or the scavenger receptor families, disulfide bonds are not critical for activity. At 65 degrees C, MBP 235 was converted into MBP 200 without loss of total binding activity, suggesting heat dissociates a small subunit not required for binding from a common large protein subunit that binds TGRLP. Since the MBPs are found on the cell surface, are themselves functionally and structurally related, have distinctly different biochemical properties from members of the LDL receptor and scavenger receptor families, and share all critical characteristics with the cellular binding site, we hypothesize that they represent a new and unique receptor family for apoE- and lipoprotein lipase-independent uptake of TGRLP by human monocyte-macrophages.
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PMID:Human THP-1 monocyte-macrophage membrane binding proteins: distinct receptor(s) for triglyceride-rich lipoproteins. 761 11

The low-density lipoprotein (LDL) receptor plays a crucial role in cholesterol metabolism. A related protein, designated the very low density lipoprotein (VLDL) receptor, that specifically binds apolipoprotein (apo) E has recently been characterized and shown to be expressed in heart, muscle and adipose tissue and the human monocyte-macrophage cell line THP-1. The VLDL receptor binds and internalizes VLDL and intermediate density lipoprotein from Watanabe heritable hyperlipidemic (WHHL) rabbits as well as beta-migrating VLDL from cholesterol-fed rabbits but not LDL from WHHL rabbits. Chinese hamster ovary (CHO) cells transfected with the rabbit VLDL receptor cDNA have now been shown to bind or internalize VLDL (d < 1.006 g/ml) isolated from fasted normolipidemic human subjects with lower affinity than WHHL-VLDL or rabbit beta-VLDL. However, binding and internalization were markedly enhanced when fasted human VLDL was preincubated with either recombinant human apoE (3/3) or lipoprotein lipase (LPL) in CHO cells overexpressing the rabbit or human VLDL receptor. CHO cells transfected with both the rabbit VLDL receptor cDNA and the human LPL cDNA effectively bound, internalized, and degraded fasted human VLDL without pretreatment. Treatment of heparinase reduced the effect of LPL-mediated binding at 4 degrees C, but the inhibitory effect was lower at 37 degrees C. Pseudomonas LPL also enhanced the binding of human fasted VLDL to the VLDL receptor at 37 degrees C in CHO cells overexpressing the human VLDL receptor. Taken together, LPL causes the enhancement of triglyceride-rich lipoproteins binding to the VLDL receptor via both the formation of bridge between lipoproteins and heparan sulfate proteoglycans and its lipolytic effect. Ligand blot analysis showed that the apparent molecular mass of the VLDL receptor is 118 kDa, which is smaller than that of the LDL receptor. These results indicate that the VLDL receptor recognizes both triglyceride-rich lipoproteins that are also relatively rich in apoE, as well as the remnants of triglyceride-rich lipoproteins after catabolism and the interaction with heparan sulfate proteoglycans by LPL. The VLDL receptor may thus function as a receptor for remnants of triglyceride-rich lipoproteins in extrahepatic tissues.
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PMID:Enhancement of the binding of triglyceride-rich lipoproteins to the very low density lipoprotein receptor by apolipoprotein E and lipoprotein lipase. 779 76

We have recently demonstrated that macrophage conditioned medium (MP medium) and beta VLDL enhance cholesterol esterification in cultured aortic smooth muscle cells by LDL receptor mediated and other pathways (Stein, O. et al. (1993) Arteroscl. Thromb. 13, 1350-1358). In view of the presence of extracellular non-lipoprotein cholesteryl ester (in the form of lipid droplets) in the atheroma, the effect of MP medium on the cellular uptake of liposomal cholesteryl linoleyl ether (CLE) or cholesteryl ester (CE) was studied. After 4 h incubation in MP medium, the uptake of liposomal [3H]CLE was up to 10-fold higher than in the presence of control medium of the same composition but not conditioned with macrophages (DV medium). Similar results were seen also with HSF derived from LDL receptor negative donors. The MP medium-stimulated uptake of liposomal [3H]CE resulted also in hydrolysis of 70-90% of the labeled compound, indicating that the [3H]CE was intracellular. While the MP medium effect on liposomal [3H]CLE uptake was evident after 4 h, its effect on [3H]cholesterol esterification by SMC in the presence of beta VLDL could be demonstrated only after 24 h. Addition of apoE to MP medium resulted in a small (30-40%) increase in the uptake of liposomal [3H]CLE; however, it was augmented more than 4-fold when apoE was added to DV medium. The MP medium effect on the uptake of liposomal [3H]CLE was interfered with by heparin, anti-LPL antibody or heparinase, while these treatments did not affect [3H]cholesterol esterification in the presence of beta VLDL. These results suggest that the interaction between SMC and two potential sources of lipids in atheroma, i.e., lipoproteins and non-lipoprotein lipid droplets, could be governed by different components of the MP medium. In the case of the lipid droplets, as modeled here in the form of liposomes, macrophage-derived lipoprotein lipase could play a major role in cholesteryl ester transfer into SMC.
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PMID:Murine macrophages secrete factors that enhance uptake of non-lipoprotein [3H]cholesteryl ester by aortic smooth muscle cells. 819 1

Bovine milk lipoprotein lipase (LPL) induced binding, uptake, and degradation of 125I-labeled normal human triglyceride-rich lipoproteins by cultured mutant fibroblasts lacking LDL receptors. The induction was dose-dependent and occurred whether LPL and 125I-lipoproteins were added to incubation media simultaneously or LPL was allowed to bind to cell surfaces, and unbound LPL was removed by washing prior to the assay. Lipolytic modification of lipoproteins did not appear to be necessary for increased catabolism because the effect of LPL was not prevented by inhibitors of LPL's enzymatic activity, p-nitrophenyl N-dodecylcarbamate or phenylmethylsulfonyl fluoride. However, the effect was abolished by boiling LPL prior to the assay suggesting that major structural features of LPL were required. Also, LPL-induced binding to cells was blocked by an anti-LPL monoclonal antibody but not by antibodies that are known to block apolipoprotein E- or B-100-mediated binding to low density lipoprotein (LDL) receptors. This indicates that LPL itself mediated 125I-lipoprotein binding to cells. Cellular degradation of 125I-lipoproteins was partially or completely blocked by two previously described ligands for the LDL receptor-related protein/alpha 2-macroglobulin receptor (LRP): activated alpha 2-macroglobulin (alpha 2M*), and the 39-kDa receptor-associated protein. These data implicated LRP as mediating LPL-induced lipoprotein degradation and were confirmed by showing that LPL's effects were prevented by an immunoaffinity-isolated polyclonal antibody against LRP. Furthermore, LPL promoted binding of 125I-lipoproteins to highly purified LRP in a solid-phase assay. Heparin or heparinase treatment of cells markedly decreased LPL-induced binding, uptake, and degradation of lipoproteins, but had no effect on catabolism of alpha 2M*. Thus, cell-surface proteoglycans were obligatory participants in the effects of LPL but were not required for LRP-mediated catabolism of alpha 2M*. Taken together, these in vitro findings establish that through interaction with cell-surface proteoglycans, LPL induces catabolism of normal human triglyceride-rich lipoproteins via LRP.
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PMID:Lipoprotein lipase induces catabolism of normal triglyceride-rich lipoproteins via the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor in vitro. A process facilitated by cell-surface proteoglycans. 831 83

Low-density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor is a member of the low-density lipoprotein receptor family. It is known to bind a wide variety of unrelated ligands including alpha 2-macroglobulin-proteinase complexes, tissue plasminogen activator, apolipoprotein E-enriched very low density lipoprotein, lipoprotein lipase, and Pseudomonas exotoxin A. Receptor-associated protein (RAP), a protein which copurifies with LRP, can inhibit the binding and internalization of all known ligands to LRP. Recent studies have shown that some ligands can bind to more than one receptor in this family. However, the ability of low-density lipoprotein (LDL) to bind to LRP in addition to the LDL receptor has not been demonstrated consistently. In this study we demonstrate that LDL binds with high affinity to macrophage cell surface receptors at 4 degrees C (Kd = 1.8 nM) and competes for the binding of a receptor-recognized form of alpha 2-macroglobulin (alpha 2M*) (Ki = 3 nM). alpha 2M* and RAP can inhibit the binding of LDL to macrophages completely (96 and 100% inhibition, respectively), after cell surface heparin has been removed by treatment with heparinase. Using a solid-phase assay, we show that LDL binds specifically, saturably, and with high affinity to purified LRP (Kd = 5 nM). LDL can also completely inhibit the binding of alpha 2M* to purified LRP. These results indicate that LDL binds directly to LRP. The ability of LDL to cross-compete with alpha 2M* for binding to LRP suggests that LDL binds to a similar or overlapping site as alpha 2M*. In addition, the ability of alpha 2M* to inhibit most of the receptor-mediated binding of LDL to macrophages suggests that LDL receptors on murine peritoneal macrophages are predominantly LRP.
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PMID:Low-density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor on murine peritoneal macrophages mediates the binding and catabolism of low-density lipoprotein. 857 70

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

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

Pigeon and rabbit beta-migrating very low density lipoprotein (beta-VLDL) are similar in size and composition, yet rabbit beta-VLDL consistently stimulates greater cholesteryl ester accumulation in pigeon peritoneal macrophages than does pigeon beta-VLDL. The purpose of this study was to determine the mechanism of this difference. Pigeon beta-VLDL bound to both a high and low affinity site while rabbit beta-VLDL bound primarily to a low affinity site. The high affinity site had the characteristics of the LDL receptor. Most rabbit beta-VLDL and some pigeon beta-VLDL bound to the low affinity site that was not down-regulated by cholesterol loading. beta-VLDL binding to the low affinity site and subsequent internalization and degradation were mediated by cell surface heparan sulfate proteoglycans (HSPG). Evidence for this includes inhibition of binding and uptake by chlorate, which prevents sulfation of proteoglycans, and by treatment with heparinase but not chondroitinase ABC. beta-VLDL uptake was stimulated by lipoprotein lipase (LpL) and apolipoprotein E (apoE), both known to bind HSPGs. Uptake and degradation of beta-VLDL were not mediated by the LDL receptor or the alpha(2)MR/LRP. Thus, binding of beta-VLDL to low affinity, high capacity HSPG binding sites on pigeon macrophages appears to directly promote internalization and degradation and is largely responsible for the greater ability of rabbit beta-VLDL to stimulate cholesterol accumulation.
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PMID:Heparan sulfate proteoglycans mediate internalization and degradation of beta-VLDL and promote cholesterol accumulation by pigeon macrophages. 914 91


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