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)

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.
J Virol 1998 Sep
PMID:Adaptation of Sindbis virus to BHK cells selects for use of heparan sulfate as an attachment receptor. 969 32

Heparinase II (no EC number) is one of three lyases isolated from Flavobacterium heparinum that degrade heparin-like complex polysaccharides. Heparinase II is unique among the heparinases in that it has broad substrate requirements and possesses the ability to degrade both heparin and heparan sulfate-like regions of glycosaminoglycans. This study set out to investigate the role of cysteines in heparinase II activity. Through a series of chemical modification experiments, it was found that one of the three cysteines in heparinase II is surface-accessible and possesses unusual chemical reactivity toward cysteine-specific chemical modifying reagents. Substrate protection experiments suggest that this surface-accessible cysteine is proximate to the active site, since addition of substrate shields the cysteine from modifying reagents. The cysteine, present in an ionic environment, was mapped by radiolabeling with N-[3H]ethylmaleimide and identified as cysteine 348. Site-directed mutagenesis of cysteine 348 to an alanine resulted in loss of activity toward heparin but not heparan sulfate, indicating that cysteine 348 is required for heparinase II activity toward heparin but is not essential for the breakdown of heparan sulfate. Furthermore, we show in this study that cysteine 164 and cysteine 189 are functionally unimportant for heparinase II.
J Biol Chem 1998 Sep 04
PMID:Heparinase II from Flavobacterium heparinum. Role of cysteine in enzymatic activity as probed by chemical modification and site- directed mutagenesis. 972 10

The interaction of heparan sulfate (HS) with basic fibroblast growth factor (bFGF) is influential in enabling the growth factor to bind to its cell surface tyrosine kinase receptor. In this study, we have investigated further the structural properties of HS required to mediate the activity of bFGF in a mitogenic assay. We have prepared a library of heparinase III-generated HS oligosaccharides fractionated by both their size (dp6-dp12) and sulfate content. The ability of these oligosaccharides to activate bFGF in a mitogenic assay was then correlated with their length and disaccharide composition. All octa- and hexasaccharide fractions tested were unable to activate bFGF. Dodeca- and decasaccharide fractions were found to contain both activating and non-activating oligosaccharides, and showed a clear correlation between total sulfate content and the level of activatory activity. Disaccharide analysis of a range of dodeca- and decasaccharide fractions showed that both activating and non-activating oligosaccharides were composed mainly of N-sulfated and IdoA(2S)-containing disaccharides. The only significant difference between activating and non-activating oligosaccharides was the content of 6-O-sulfated disaccharides, in particular the disaccharide IdoA(2S)alpha1,4GlcNSO3(6S). These results show that there is a requirement for 6-O-sulfation of N-sulfated glucosamine residues, in addition to the 2-O-sulfation of IdoA, for the promotion of bFGF mitogenic activity by naturally occurring HS oligosaccharides. Analysis of the structure-activity relationships in the dodecasaccharide fractions in particular, suggests that a minimum bFGF activation sequence exists which is dependent on the positioning of at least one 6-O-sulfate group.
J Biol Chem 1998 Sep 04
PMID:Heparan sulfate oligosaccharides require 6-O-sulfation for promotion of basic fibroblast growth factor mitogenic activity. 972 14

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.
J Biol Chem 1998 Sep 04
PMID:In vivo clearance of ternary complexes of vitronectin-thrombin-antithrombin is mediated by hepatic heparan sulfate proteoglycans. 972 80

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.
Histochem J 1998 Sep
PMID:Sulphated glycosaminoglycans in guinea pig eosinophils studied by means of cationic colloidal gold. 987 Jul 69

Patients undergoing extracorporeal membrane oxygenation (ECMO) are at an increased risk for developing coagulopathies due to the adverse effects of extracorporeal circulation on the hemostatic mechanism. Methods of determining causative factors of bleeding diathesis are often inconsistent and non-specific. ECMO patients require aggressive transfusion therapy with autogenic blood products to stabilize and maintain hemostasis. The present study evaluated the coagulation status of newborn patients undergoing ECMO therapy, using a viscoelastic monitor (Thrombelastograph -TEG) that measures functional aspects of clot development and stabilization. Seventeen neonatal patients undergoing ECMO for severe respiratory dysfunction were entered into this study. Serial blood samples were obtained and routine coagulation assessment including fibrinogen concentration, platelet count and ionized calcium was performed. In addition, fibrin(ogen) degradation products (FDP), d-Dimers, antithrombin III and plasma free hemoglobin were measured. Transfusion indicators were established and total transfusion requirements recorded. TEG profiles were determined with the use of heparinase, an enzyme that degrades heparin but has little effect on other coagulation factors. The most commonly encountered complication was hemorrhaging which was diagnosed by laboratory and clinical assessment in 11 of 17 patients. Transfusion requirements (measured in ml/kg/ECMO hour) were the following: packed red blood cells--1.34 +/- 0.5; platelets--0.71 +/- 0.57; fresh frozen plasma--0.09 +/- 0.12; cryoprecipitate 0.05 +/- 0.05. Thrombelastograph profiles reflected hemostatic conditions that ranged from severe coagulopathies (DIC) to hypercoagulability. Interpretation of TEG profiles identified hemostatic abnormalities in 57 of 101 profiles (46.5%), with the most common etiology related to platelet dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)
J Extra Corpor Technol 1995 Sep
PMID:Coagulation monitoring during extracorporeal membrane oxygenation: the role of thrombelastography. 1015 58

Glycosaminoglycans, including heparin, have been demonstrated both in vitro and in vivo to protect the ischemic myocardium against reperfusion injury. In the present study, we sought to determine whether the cardioprotective effects of heparin administration could be reversed by the heparin-degrading enzyme heparinase. New Zealand white rabbits were pretreated with heparin (300 U/kg i.v.) or vehicle (saline). Two hours after treatment, hearts were removed, perfused on a Langendorff apparatus, and subjected to 25 min of global ischemia, followed by 45 min of reperfusion. Hemodynamic variables were obtained before ischemia (baseline) and every 10 min throughout the reperfusion period. Compared with vehicle-treated rabbits, the left ventricular end-diastolic and left ventricular developed pressures were improved significantly (p <.05) in the heparin-treated group. Ex vivo administration of heparinase (5 U/ml) immediately before the onset of global ischemia was associated with a reversal of the heparin-mediated cardioprotection. The uptake of a radiolabeled antibody to the intracellular protein myosin and creatine kinase release were used to determine membrane integrity and discriminate between viable and nonviable myocardial tissue. The uptake of radiolabeled antimyosin antibody and release of creatine kinase after reperfusion were increased in heparin-pretreated hearts exposed to heparinase, indicating a loss of membrane integrity and increased myocyte injury. These results demonstrate that neutralization of heparin by heparinase promotes increased myocardial injury after reperfusion of the ischemic myocardium.
J Pharmacol Exp Ther 1999 Sep
PMID:Ex vivo reversal of heparin-mediated cardioprotection by heparinase after ischemia and reperfusion. 1045 76

Ovarian androgen production is rate limiting for follicular maturation and can induce follicular atresia. Thus, it is important to define the actions of the intraovarian agents, such as apolipoprotein (apo) E, that modulate theca cell androgen production. Theca cell androgen production is stimulated at low concentrations and inhibited at higher concentrations of native apo E. The apo E peptide, acetyl-Y(LRKLRKRLLRDADDL)(2)C or acetyl-Y(141-155)(2)C, has low density lipoprotein (LDL) receptor and LDL receptor-related protein-binding activity, and it mimics the activity of native apo E in the theca-interstitial cell system. To define the role of members of the LDL receptor superfamily in the apo E peptide-mediated responses, we found that receptor-associated protein prevented the stimulation without altering the inhibition of androstenedione production. The apo E peptide (129-162), which has no LDL receptor-binding activity, did not stimulate androstenedione production. The apo E peptide acetyl-Y(141-155)(2)C did not stimulate androstenedione production when cell surface heparan sulfate proteoglycans were degraded with heparinase. The apo E peptide acetyl-Y(141-155)(2)C bound to heparin, a property of LDL receptor ligands, and in this complex the peptide had no effect on androstenedione production. These observations support the conclusion that apo E-mediated stimulation, but not inhibition, of ovarian theca cell androstenedione production was mediated by members of the LDL receptor superfamily.
Biol Reprod 1999 Sep
PMID:Apolipoprotein E peptide stimulation of rat ovarian theca cell androgen synthesis is mediated by members of the low density lipoprotein receptor superfamily. 1045 43

Heparin has been used as a clinical anticoagulant for more than 50 years, making it one of the most effective pharmacological agents known. Much of heparin's activity can be traced to its ability to bind antithrombin III (AT-III). Low molecular weight heparin (LMWH), derived from heparin by its controlled breakdown, maintains much of the antithrombotic activity of heparin without many of the serious side effects. The clinical significance of LMWH has highlighted the need to understand and develop chemical or enzymatic means to generate it. The primary enzymatic tools used for the production of LMWH are the heparinases from Flavobacterium heparinum, specifically heparinases I and II. Using pentasaccharide and hexasaccharide model compounds, we show that heparinases I and II, but not heparinase III, cleave the AT-III binding site, leaving only a partially intact site. Furthermore, we show herein that glucosamine 3-O sulfation at the reducing end of a glycosidic linkage imparts resistance to heparinase I, II, and III cleavage. Finally, we examine the biological and pharmacological consequences of a heparin oligosaccharide that contains only a partial AT-III binding site. We show that such an oligosaccharide lacks some of the functional attributes of heparin- and heparan sulfate-like glycosaminoglycans containing an intact AT-III site.
Proc Natl Acad Sci U S A 2000 Sep 12
PMID:Cleavage of the antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin. 1098 27

Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (alpha-mannose or N-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells.
J Virol 2001 Sep
PMID:Identification of a putative coreceptor on Vero cells that participates in dengue 4 virus infection. 1148 25


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