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)

We have investigated the proposal that the receptor-associated protein (RAP) of the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor binds to heparan sulfate proteoglycans (HSP). 125I-RAP binds to two sites on the surface of fibroblasts as follows: a high affinity site with a Kd of 1.4 nM and a low affinity site (Kd = 188 nM) with a capacity of more than 1000-fold the maximum amount of lipoprotein receptor-related protein/alpha 2-macroglobulin receptor on the cell surface. 125I-RAP binding to the low affinity site was abolished by heparin or Suramin. However, maximal digestion of the glycosaminoglycan chains of HSP with heparinase or culturing the cells in chlorate, an inhibitor of proteoglycan sulfation, did not affect the binding of 125I-RAP or of 125I-labeled, methylamine-activated alpha 2-macroglobulin. Comparison of 125I-RAP degradation at two different concentrations suggests that the low affinity, high capacity site on the surface of human fibroblasts participates in the endocytosis of 125I-RAP. The nature of the low affinity site remains to be elucidated, but we can exclude the glycosaminoglycan chains of HSP.
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PMID:Exogenous receptor-associated protein binds to two distinct sites on human fibroblasts but does not bind to the glycosaminoglycan residues of heparan sulfate proteoglycans. 751 52

Lipoprotein lipase (LPL) binds to the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor and induces catabolism of normal human very low density lipoproteins (VLDL) via LRP in vitro. Recent studies showed that the C-terminal domain of LPL can bind LRP in solid phase assays and inhibit cellular catabolism of two LRP ligands, activated alpha 2-macroglobulin and the 39-kDa receptor-associated protein (Williams, S.E., Inoue, I., Tran, H., Fry, G. L., Pladet, M.W., Iverius, P.-H., Lalouel, J.-M., Chappell, D.A., and Strickland, D.K. (1994) J. Biol. Chem. 269, 8653-8658). The current study investigated the potential for this region of LPL to promote cellular catabolism of VLDL via LRP. A fragment comprising the C-terminal domain of LPL (designated LPLC) was expressed in bacteria and found to promote cellular binding, uptake, and degradation of normal human VLDL in a dose-dependent manner. These effects were present whether LPLC was added simultaneously with 125I-VLDL or was prebound to cell surfaces prior to the assay. Mutations involving Lys407, Trp393, Trp394, or deletion of the C-terminal 14 residues reduced the effects of LPLC. Three LRP-binding proteins, the receptor-associated protein, lactoferrin, and a polyclonal antibody against LRP, competed for 125I-VLDL degradation induced by LPLC. Heparin or heparinase treatment of cells prevented LPLC-induced 125I-VLDL catabolism. Thus, cell-surface proteoglycans play an important role in this pathway. Interestingly, either LPLC or LPL when added in excess could block LPL-induced 125I-VLDL degradation presumably by interacting directly with LRP. However, unlabeled VLDL could not prevent catabolism of 125I-labeled LPLC or LPL. These data show that cellular fates for VLDL versus LPLC or LPL are divergent. This is probably due to independent catabolism of the latter via cell-surface proteoglycans. In summary, these in vitro studies indicate that a fragment of LPL corresponding to the C-terminal domain mimics the native enzyme with respect to induction of VLDL catabolism via LRP. Because LPLC lacks the catalytic site of native LPL, these studies establish that lipase activity is not required for LRP-mediated lipoprotein catabolism.
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PMID:Cellular catabolism of normal very low density lipoproteins via the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor is induced by the C-terminal domain of lipoprotein lipase. 751 36

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)
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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

Previously, we demonstrated in cultured dorsal root ganglion neurons that, in the presence of beta-migrating very low density lipoproteins (beta-VLDL), apolipoprotein (apo) E4, but not apoE3, suppresses neurite outgrowth. In the current studies, murine neuroblastoma cells (Neuro-2a) were stably transfected with human apoE3 or apoE4 cDNA, and the effect on neurite outgrowth was examined. The stably transfected cells secreted nanogram quantities of apoE (44-89 ng/mg of cell protein in 48 h). In the absence of lipoproteins, neurite outgrowth was similar in the apoE3- and apoE4-secreting cells. The apoE4-secreting cells, when incubated with beta-VLDL, VLDL, cerebrospinal fluid lipoproteins (d < 1.21 g/ml), or with triglyceride/phospholipid (2.7:1 (w/w)) emulsions, showed a reduction in the number of neurites/cell, a decrease in neurite branching, and an inhibition of neurite extension, whereas in the apoE3-secreting cells in the presence of a lipid source, neurite extension was increased. Uptake of beta-VLDL occurred to a similar extent in both the apoE3- and apoE4-secreting cells. With low density lipoproteins or with dimyristoylphosphatidylcholine emulsions, either alone or complexed with cholesterol, no differential effect on neurite outgrowth was observed. A slight differential effect was observed with apoE-containing high density lipoproteins. The differential effect of apoE3 and apoE4 in the presence of beta-VLDL was blocked by incubation of the cells with heparinase and chlorate, with lactoferrin, or with receptor-associated protein, all of which prevent the uptake of lipoproteins by the low density lipoprotein receptor-related protein (LRP). The data suggest that the secreted and/or cell surface-bound apoE interact with the lipoproteins and facilitate their internalization via the heparan sulfate proteoglycan-LRP pathway. The mechanism by which apoE3 and apoE4 exert differential effects on neurite outgrowth remains speculative. However, the data suggest that apoE4, which has been shown to be associated with late onset familial and sporadic Alzheimer's disease, may inhibit neuronal remodeling and contribute to the progression of the disease.
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PMID:Stable expression and secretion of apolipoproteins E3 and E4 in mouse neuroblastoma cells produces differential effects on neurite outgrowth. 759 57

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

We have studied the binding, uptake, and degradation of a recombinant form of apolipoprotein[a] (r-apo[a]) using a cultured cell model. In HepG2 cells and in human fibroblasts, r-apo[a] complexed with low density lipoprotein(LDL) is bound and internalized via high affinity (Kd = 10 nM) receptors; in both cell types, low affinity (Kd = 200-300 nM) sites also mediate free apo[a] uptake. Using competition studies, we found that the high affinity binding component corresponds to the LDL receptor. Involvement of the LDL receptor in r-apo[a] uptake by fibroblasts was confirmed using fibroblasts derived from an individual homozygous for familial hypercholesterolemia; in contrast to normal fibroblasts, these cells lacked the high affinity r-apo[a] binding component. Cell association of 125I-labeled r-apo[a] was increased and decreased concomitantly with the up- and down-regulation of the LDL receptor in response to a number of compounds. The addition of alpha 2-macroglobulin as well as treatment with heparinase, chondroitinase ABC, and sodium chlorate did not decrease total specific binding of r-apo[a], suggesting that neither the low density lipoprotein receptor-related protein nor cell surface proteoglycans are involved in r-apo[a] clearance. The low affinity binding component present in both fibroblasts and HepG2 cells likely corresponds to the plasminogen receptor, as binding of r-apo[a] to these sites was specifically decreased by the addition of plasminogen or the lysine analogue epsilon-aminocaproic acid, but not by the addition of tissue-type plasminogen activator. Heparin abolished uptake of r-apo[a] by the LDL receptor component only; this indicates that apo[a] must be associated with LDL to be cleared by this receptor. In contrast, free apo[a] can be effectively cleared by the plasminogen receptor which may represent a significant route of clearance for free apo[a] in vivo.
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PMID:Interaction of a recombinant form of apolipoprotein[a] with human fibroblasts and with the human hepatoma cell line HepG2. 872 15

Speed and selectivity of hepatocyte invasion by malaria sporozoites have suggested a receptor-mediated mechanism and the specific interaction of the circumsporozoite (CS) protein with liver-specific heparan sulfate proteoglycans (HSPGs) has been implicated in the targeting to the liver. Here we show that the CS protein interacts not only with cell surface heparan sulfate, but also with the low density lipoprotein receptor-related protein (LRP). Binding of 125I-CS protein to purified LRP occurs with a Kd of 4.9 nM and can be inhibited by the receptor-associated protein (RAP). Blockage of LRP by RAP or anti-LRP antibodies on heparan sulfate-deficient CHO cells results in more than 90% inhibition of binding and endocytosis of recombinant CS protein. Conversely, blockage or enzymatic removal of the cell surface heparan sulfate from LRP-deficient embryonic mouse fibroblasts yields the same degree of inhibition. Heparinase-pretreatment of LRP-deficient fibroblasts or blockage of LRP on heparan sulfate-deficient CHO cells by RAP, lactoferrin, or anti-LRP antibodies reduces Plasmodium berghei invasion by 60-70%. Parasite development in heparinase-pretreated HepG2 cells is inhibited by 65% when RAP is present during sporozoite invasion. These findings suggest that malaria sporozoites utilize the interaction of the CS protein with HSPGs and LRP as the major mechanism for host cell invasion.
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PMID:Dual interaction of the malaria circumsporozoite protein with the low density lipoprotein receptor-related protein (LRP) and heparan sulfate proteoglycans. 892 Aug 59

High density lipoprotein (HDL) particles and HDL cholesteryl esters are taken up by both receptor-mediated and non-receptor-mediated pathways. Here we show that cell surface heparan sulfate proteoglycans (HSPG) participate in hepatic lipase (HL)- and apolipoprotein (apo) E-mediated binding and uptake of mouse and human HDL by cultured hepatocytes. The HL secreted by HL-transfected McA-RH7777 cells enhanced both HDL binding at 4 degrees C (approximately 2-4-fold) and HDL uptake at 37 degrees C (approximately 2-5-fold). The enhanced binding and uptake of HDL were partially inhibited by the 39-kDa protein, an inhibitor of low density lipoprotein receptor-related protein (LRP), but were almost totally blocked by heparinase, which removes the sulfated glycosaminoglycan chains from HSPG. Therefore, HL may mediate the uptake of HDL by two pathways: an HSPG-dependent LRP pathway and an HSPG-dependent but LRP-independent pathway. The HL-mediated binding and uptake of HDL were only minimally reduced when catalytically inactive HL or LRP binding-defective HL was substituted for wild-type HL, indicating that much of the HDL uptake required neither HL binding to the LRP nor lipolytic processing. To study the role of HL in facilitating the selective uptake of cholesteryl esters, we used HDL into which radiolabeled cholesteryl ether had been incorporated. HL increased the selective uptake of HDL cholesteryl ether; this enhanced uptake was reduced by more than 80% by heparinase but was unaffected by the 39-kDa protein. Like HL, apoE enhanced the binding and uptake of HDL (approximately 2-fold) but had little effect on the selective uptake of HDL cholesteryl ether. In the presence of HL, apoE did not further increase the uptake of HDL, and at a high concentration apoE impaired or decreased the HL-mediated uptake of HDL. Therefore, HL and apoE may utilize similar (but not identical) binding sites to mediate HDL uptake. Although the relative importance of cell surface HSPG in the overall metabolism of HDL in vivo remains to be determined, cultured hepatocytes clearly displayed an HSPG-dependent pathway that mediates the binding and uptake of HDL. This study also demonstrates the importance of HL in enhancing the binding and uptake of remnant and low density lipoproteins via an HSPG-dependent pathway.
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PMID:Heparan sulfate proteoglycans participate in hepatic lipaseand apolipoprotein E-mediated binding and uptake of plasma lipoproteins, including high density lipoproteins. 939 55

Isoform-specific effects of apolipoprotein E (apoE) on neurite outgrowth and the cytoskeleton are associated with higher intracellular levels of apoE3 than apoE4 in cultured neurons. The current studies, designed to determine the mechanism for the differential intracellular accumulation or retention of apoE, demonstrate that apoE3- and apoE4-containing beta-very low density lipoproteins (beta-VLDL) possess similar cell binding and internalization and delivery of cholesterol to the cells. However, as assessed by immunocytochemistry, analysis of extracted cellular proteins, or quantitation of 125I-apoE-enriched beta-VLDL, there was a 2-3-fold greater accumulation of apoE3 than apoE4 in Neuro-2a cells, fibroblasts, and hepatocytes (HepG2) after 1-2 h, and this differential was maintained for up to 48 h. ApoE2 also accumulated in Neuro-2a cells to a greater extent than apoE4. The differential effect was mediated by the apoE-enriched beta-VLDL and not by free apoE. Neither the low density lipoprotein receptor nor the low density lipoprotein receptor-related protein was responsible for the differential accumulation of apoE3 and apoE4, since cells deficient in either or both of these receptors also displayed the differential accumulation. The effect appears to be mediated primarily by cell surface heparan sulfate proteoglycans (HSPG). The retention of both apoE3 and apoE4 was markedly reduced, and the differential accumulation of apoE3 and apoE4 was eliminated both in mutant Chinese hamster ovary cells that did not express HSPG and in HSPG-expressing cells treated with heparinase. The data suggest that cell surface HSPG directly mediate the uptake of apoE-containing lipoproteins, that the differential accumulation/retention of apoE by cells is mediated via HSPG, and that there is a differential intracellular handling of the specific apoE isoforms.
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PMID:Differential cellular accumulation/retention of apolipoprotein E mediated by cell surface heparan sulfate proteoglycans. Apolipoproteins E3 and E2 greater than e4. 959 78

Thrombin is inhibited by its cognate plasma inhibitor antithrombin, through the formation of covalent thrombin-antithrombin (TAT) complexes that are found as ternary complexes with vitronectin (VN-TAT). To determine whether the metabolism of VN-TAT ternary complexes is different from that previously reported for binary TAT complexes, plasma clearance studies were done in rabbits using human VN-TAT. 125I-VN-TAT was shown to be cleared rapidly from the circulation (t1/2alpha = 3.8 min) in a biphasic manner mainly by the liver. 125I-TAT had a similar initial clearance (t1/2alpha = 5.3 min) but had a significantly faster beta-phase clearance (t1/2beta = 42.8 min versus 85.4 min for VN-TAT; p = 0.005). Protamine sulfate and heparin abolished the rapid initial alpha-phase of 125I-VN-TAT clearance and reduced its liver-specific association and in vivo degradation. Heparin also reduced the alpha-phase clearance of 125I-TAT and was associated with the appearance of high molecular weight complexes, suggesting enhanced complex formation between VN and TAT. 125I-VN-TAT binding to HepG2 cells was reduced by competition with VN-TAT or heparin but to a much lesser extent in the presence of TAT. The binding of VN-TAT to HepG2 cells was not inhibited by competition with the low density lipoprotein receptor-related protein ligand, methylamine-alpha2-macroglobulin. 125I-VN-TAT binding was also inhibited by treating HepG2 cells with heparinase or by growing the cells in the presence of beta-D-xyloside. Finally, both heparin and chloroquine, but not methylamine-alpha2-macroglobulin, reduced the internalization and degradation of VN-TAT by HepG2 cells. Taken together, these data indicate the importance of VN in TAT metabolism and demonstrate that VN-TAT binds to liver-associated heparan sulfate proteoglycans, which mediate its internalization and subsequent intracellular degradation.
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PMID:In vivo clearance of ternary complexes of vitronectin-thrombin-antithrombin is mediated by hepatic heparan sulfate proteoglycans. 972 80


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