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)

Human amphiregulin (AR) is a heparin-binding growth factor which functions by binding to and activating the epidermal growth factor (EGF) receptor tyrosine kinase. AR contains an EGF-like domain (residues 44-84) and a Lys/Arg-rich NH2-terminal extension (residues 1-43). Synthetic peptides corresponding to residues 8-26, 26-44, and 68-84 of AR were tested for their ability to compete for the binding of AR to immobilized heparin. AR8-26 and AR68-84 had no significant effect on the binding of AR to heparin, whereas AR26-44 bound to heparin and blocked the binding of AR to heparin. Both soluble heparin and heparan sulfate inhibited AR-induced mitogenesis in MCF-10A human mammary epithelial cells with an IC50 of 5 and 2 micrograms/ml, respectively, whereas soluble chondroitin sulfate had only a slight inhibitory effect. When MCF-10A cells were grown in the presence of chlorate, an inhibitor of sulfation, or exposed to the glycosaminoglycan-degrading enzymes heparitinase or heparinase, the ability of AR to evoke mitogenesis in these cells was lost. Chlorate, heparitinase, or heparinase treatment inhibited AR-induced autophosphorylation of tyrosine residues in the EGF receptor. None of these treatments had any significant effect on EGF-triggered mitogenic signaling by the EGF receptor. These results indicate that extracellular heparan sulfate glycosaminoglycan is essential to AR-induced mitogenic signaling by the EGF receptor tyrosine kinase.
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PMID:Heparan sulfate is essential to amphiregulin-induced mitogenic signaling by the epidermal growth factor receptor. 792 59

Much evidence has suggested that the superoxide generated by xanthine oxidase (XOD) within the endothelial cell triggers characteristic free-radical-mediated tissue injuries. Although it has been reported that XOD exists not only in the cytoplasm, but also on the outside surface of the endothelial cell membrane, it is not clear how XOD localizes on the outside of the plasma membrane. Purified human xanthine oxidase (h-XOD) had an affinity for heparin-Sepharose. The binding was largely independent of the pH over the physiological range, whereas it tended to increase at lower pH and to decrease at higher pH. Exposure of h-XOD to the lysine-specific reagent trinitrobenzenesulphonic acid or the arginine-specific reagent phenylglyoxal caused it to lose its affinity for heparin-Sepharose. The binding of h-XOD to heparin is apparently of electrostatic nature, and both lysine and arginine residues are involved in the binding. h-XOD was found to bind to cultured porcine aortic endothelial cells, and this binding was inhibited by the addition of heparin or pretreatment of the cells with heparinase and/or heparitinase. Intravenous injection of heparin into two healthy persons led to a prompt increase in plasma h-XOD concentration. These results suggest that XOD localizes on the outside surface of endothelial cells by association with polysaccharide chains of heparin-like proteoglycans on the endothelial-cell membranes. Superoxide extracellularly generated by XOD may injure the source-endothelial-cell membrane and also attract and activate closely appositional neutrophils, which themselves actually cause progressive oxidative damage.
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PMID:Binding of human xanthine oxidase to sulphated glycosaminoglycans on the endothelial-cell surface. 842 93

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

Platelet factor 4 is a tetrameric heparin binding chemokine released from the alpha-granules of activated platelets. In this study we show that platelet factor 4 binds with high affinity and specificity to an approximately 9-kDa sequence in heparan sulfate, which it protects from degradation by heparinase enzymes. This protected fragment is enriched in N-sulfated disaccharides and iduronate 2-O-sulfate residues, the latter being important for binding to platelet factor 4. The major structural motif of the fragment appears to consist of a pair of sulfated domains positioned at both ends separated by a central mainly N-acetylated region. On the basis of these findings, we propose a model in which the heparan sulfate fragment wraps around the ring of positive charges on platelet factor 4 with the iduronate 2-O-sulfates within the sulfated domains binding strongly to lysine clusters on opposite faces of the tetramer.
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PMID:Specific binding of the chemokine platelet factor 4 to heparan sulfate. 925 63

Heparinases are bacterial enzymes that are powerful tools to study the physiological roles of heparin-like complex polysaccharides. In addition, heparinases have significant therapeutic applications. We had proposed earlier that cysteine 135 and histidine 203 together form the catalytic domain in heparinase I. We had also identified a heparin binding domain in heparinase I containing two positively charged clusters HB-1 and HB-2 in a primary heparin binding site and other positively charged residues in the vicinity of cysteine 135. In this study, through systematic site-directed mutagenesis studies, we show that the alteration of the positive charge of the HB-1 region has a pronounced effect on heparinase I activity. More specifically, site-directed mutagenesis of K199A (contained in HB-1) results in a 15-fold reduction in catalytic activity, whereas a K198A mutation (also in HB-1) results in only a 2- to 3-fold reduction in heparinase I activity. A K132A mutation, in close proximity to cysteine 135, also resulted in reduced (8-fold) activity. Heparin affinity chromatography experiments indicated moderately lowered binding affinities for the K132A, K198A, and the K199A mutant enzymes. The above results, taken together with our previous observations, lead us to propose that the positively charged heparin binding domain provides the necessary microenvironment for the catalytic domain of heparinase I. The dominant effect of lysine 199 suggests an additional, more direct, role in catalysis for this residue.
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PMID:Heparinase I from Flavobacterium heparinum. Role of positive charge in enzymatic activity. 941 72

The three heparinases derived from Flavobacterium heparinum are powerful tools for studying heparin-like glycosaminoglycans in major biological processes, including angiogenesis and development. Heparinase II is unique among the three enzymes because it is able to catalytically cleave both heparin and heparan sulfate-like regions of heparin-like glycosaminoglycans. Toward understanding the catalytic mechanism of heparin-like glycosaminoglycan degradation by heparinase II, we set out to investigate the role of the histidines of heparinase II in catalysis. We observe concentration-dependent inactivation of heparinase II in the presence of the reversible histidine-modifying reagent diethylpyrocarbonate (DEPC). With heparin as the substrate, the rate constant of inactivation was found to be 0.16 min-1 mM-1; with heparan sulfate as the substrate, the rate constant was determined to be 0.24 min-1 mM-1. Heparinase II activity is restored following hydroxylamine treatment. This, along with other experiments, strongly suggests that the inactivation of heparinase II by DEPC is specific for histidine residues and that three histidines are modified by DEPC. Substrate protection experiments show that heparinase II preincubation with heparin followed by the addition of DEPC resulted in a loss of enzymatic activity toward heparan sulfate but not heparin. However, heparinase II preincubation with heparan sulfate was unable to protect heparinase II from DEPC inactivation for either of the substrates. Proteolytic mapping studies with Lys-C were consistent with the chemical modification experiments and identified histidines 238, 451, and 579 as being important for heparinase II activity. Further mapping studies identified histidine 451 as being essential for heparin degradation. Site-directed mutagenesis experiments on the 13 histidines of heparinase II corroborated the chemical modification and the peptide mapping studies, establishing the importance of histidines 238, 451 and 579 in heparinase II activity.
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PMID:Heparinase II from Flavobacterium heparinum. Role of histidine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis. 955 64

Attachment of Sindbis virus to the cell surface glycosaminoglycan heparan sulfate (HS) and the selection of this phenotype by cell culture adaptation were investigated. Virus (TR339) was derived from a cDNA clone representing the consensus sequence of strain AR339 (K. L. McKnight, D. A. Simpson, S. C. Lin, T. A. Knott, J. M. Polo, D. F. Pence, D. B. Johannsen, H. W. Heidner, N. L. Davis, and R. E. Johnston, J. Virol. 70:1981-1989, 1996) and from mutant clones containing either one or two dominant cell culture adaptations in the E2 structural glycoprotein (Arg instead of Ser at E2 position 1 [designated TRSB]) or this mutation plus Arg for Ser at E2 114 [designated TRSB-R114]). The consensus virus, TR339, bound to baby hamster kidney (BHK) cells very poorly. The mutation in TRSB increased binding 10- to 50-fold, and the additional mutation in TRSB-R114 increased binding 3- to 5-fold over TRSB. The magnitude of binding was positively correlated with the degree of cell culture adaptation and with attenuation of these viruses in neonatal mice. HS was identified as the attachment receptor for the mutant viruses by the following experimental results. (i) Low concentrations of soluble heparin inhibited plaque formation on and binding of mutant viruses to BHK cells by >95%. In contrast, TR339 showed minimal inhibition at high concentrations. (ii) Binding and infectivity of TRSB-R114 was sensitive to digestion of cell surface HS with heparinase III, and TRSB was sensitive to both heparinase I and heparinase III. TR339 infectivity was only slightly affected by either digestion. (iii) Radiolabeled TRSB and TRSB-R114 attached efficiently to heparin-agarose beads in binding assays, while TR339 showed virtually no binding. (iv) Binding and infectivity of TRSB and TRSB-R114, but not TR339, were greatly reduced on Chinese hamster ovary cells deficient in HS specifically or all glycosaminoglycans. (v) High-multiplicity-of-infection passage of TR339 on BHK cell cultures resulted in rapid coselection of high-affinity binding to BHK cells and attachment to heparin-agarose beads. Sequencing of the passaged virus population revealed a mutation from Glu to Lys at E2 70, a mutation common to many laboratory strains of Sindbis virus. These results suggest that TR339, the most virulent virus tested, attaches to cells through a low-affinity, primarily HS-independent mechanism. Adaptive mutations, selected during cell culture growth of Sindbis virus, enhance binding and infectivity by allowing the virus to attach by an alternative mechanism that is dependent on the presence of cell surface HS.
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PMID:Adaptation of Sindbis virus to BHK cells selects for use of heparan sulfate as an attachment receptor. 969 32

The internalization of a basic peptide, 001-C8 [H-MeTyr-Arg-MeArg-D-Leu-NH(CH2)8NH2], into enterocyte-like Caco-2 cells was evaluated. Internalization of 125I-labeled 001-C8 (125I-001-C8) increased time dependently and reached steady state at 60 min. The steady-state internalization of 125I-001-C8 (7.24 +/- 0. 41 microl/mg protein) was temperature and concentration dependent and was significantly decreased by dansylcadaverine (500 microM), protamine (1 mM), poly-L-lysine (1 mM), E-2078 (1 mM), and ebiratide (1 mM), whereas poly-L-glutamic acid (1 mM), tyrosine (1 mM), and glycylglycine (25 mM) were not inhibitory. Predigestion of acid mucopolysaccharides by heparinase I, heparitinase, and chondroitinase ABC also decreased the internalization. The maximal internalization, the half-saturation constant, and the nonsaturable internalization of 125I-001-C8 were 1.13 +/- 0.23 pmol/mg protein, 0. 47 +/- 0.43 microM, and 3.13 +/- 0.19 microl/mg protein, respectively. Confocal microscopy also indicated the internalization of fluorescence-derived 001-C8 [001-C8-4-nitrobenz-2-oxa-1,3-diazole (001-C8-NBD)]. Granular staining seen within the cell, excluding nuclei, indicated the sequestration of 001-C8-NBD within endocytotic vesicles. Dansylcadaverine and protamine strongly decreased the granular distribution of 001-C8-NBD within the cell. These results demonstrate that 001-C8 is taken up by Caco-2 cells via adsorptive-mediated endocytosis.
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PMID:Adsorptive-mediated endocytosis of a basic peptide in enterocyte-like Caco-2 cells. 972 63

Lp(a) is a major inherited risk factor for premature atherosclerosis. The mechanism of Lp(a) atherogenicity has not been elucidated, but likely involves both its ability to interfere with plasminogen activation and its atherogenic potential as a lipoprotein particle after receptor-mediated uptake. We demonstrate that Lp(a) stimulates production of vascular cell adhesion molecule 1 (VCAM-1) and E-selectin in cultured human coronary artery endothelial cells (HCAEC). This effect resulted from a rise in intracellular free calcium induced by Lp(a) and could be inhibited by the intracellular calcium chelator, BAPTA/AM. The involvement of the LDL and VLDL receptors in Lp(a) activation of HCAEC were ruled out since Lp(a) induction of adhesion molecules was not prevented by an antibody (IgGC7) to the LDL receptor or by receptor-activating protein, an antagonist of ligand binding to the VLDL receptor. Addition of alpha2-macroglobulin as well as treatment with heparinase, chondroitinase ABC, and sodium chlorate did not decrease levels of VCAM-1 and E-selectin stimulated by Lp(a), suggesting that neither the low density lipoprotein receptor-related protein nor cell-surface proteoglycans are involved in Lp(a)-induced adhesion molecule production. Neither does the binding site on HCAEC responsible for adhesion molecule production by Lp(a) appear to involve plasminogen receptors, as levels of VCAM-1 and E-selectin were not significantly decreased by the addition of glu-plasminogen, the lysine analog epsilon-aminocaproic acid, or by trans-4-(aminomethyl)-cyclohexanecarboxymethylic acid (tranexamic acid), which acts by binding to the lysine binding sites carried on the kringle structures in plasminogen. In contrast, recombinant apolipoprotein (a) [r-apo(a)] competed with Lp(a) and attenuated the expression of VCAM-1 and E-selectin. In summary, we have identified a calcium-dependent interaction of Lp(a) with HCAEC capable of inducing potent surface expression of VCAM-1 and E-selectin that does not appear to involve any of the known potential Lp(a) binding sites. Because leukocyte recruitment to the vessel wall appears to represent one of the important early events in atherogenesis, this newly described endothelial cell-activating effect of Lp(a) places it at a crucial juncture in the initiation of atherogenic disease and may lead to a better understanding of the role of Lp(a) in the vascular biology of atherosclerosis.
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PMID:Expression of adhesion molecules by lp(a): a potential novel mechanism for its atherogenicity. 983 67

Using bone marrow embedded in hydrophilic resin Lowicryl K4M and cationic colloidal gold pH 1.0 labelling, we studied sites of sulphation and sulphated glycosaminoglycans ultrastructurally in various maturational stages of both eosinophil granulocytes and eosinophil granules of guinea pig. Eosinophil granules reacted positively to cationic gold, the pattern of labelling varying according to the degree of cell maturation. The formation of eosinophil granules takes place throughout the myelocyte stage. Early eosinophil myelocytes contain a large Golgi apparatus with active granulogenesis, while late ones contain a small and less active Golgi apparatus. All the immature granules were labelled positively. However, mature granules with a central crystal bar lost their affinity towards colloidal gold. Interestingly, strong colloidal gold labelling was also observed in the trans to transmost Golgi apparatus, especially in immature eosinophil granulocytes. This indicates that sulphation of glycosaminoglycans occurs in the trans to transmost Golgi apparatus of eosinophil granulocytes. Prior absorption with poly-L-lysine prevented colloidal gold labelling of tissue sections. Methylation of sections at 37 degrees C did not alter the gold labelling, whereas the labelling disappeared after methylation at 60 degrees C. Prior treatment with chondroitinase ABC or heparinase I abolished the majority of colloidal gold labelling in immature eosinophil granules. Taking these results together, we conclude that immature eosinophil granules contain sulphated glycosaminoglycans including chondroitin sulphate or heparan sulphate or both.
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PMID:Sulphated glycosaminoglycans in guinea pig eosinophils studied by means of cationic colloidal gold. 987 Jul 69


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