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)

The mechanisms of Marek's disease virus (MDV) entry to host cells have not yet been analyzed. Heparan sulfate (HS) on the cell surface serves as a receptor for several herpesviruses in mammalian species. In this study, we demonstrated that plaque formation by cell-free MDV is inhibited by the addition of soluble heparin to the cell culture. Moreover, pretreatment of susceptible cells, chicken embryo fibroblasts, with heparinase, partially reduced infectivity of the cell-free MDV. From these results, it was suggested that the MDV entry, at least in the case of cell-free MDV, is dependent on the presence of cell surface glycosaminoglycans, principally HS.
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PMID:Heparin inhibits plaque formation by cell-free Marek's disease viruses in vitro. 1134 78

Cell surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines (Belting, M., Persson, S., and Fransson, L.-A. (1999) Biochem. J. 338, 317-323; Belting, M., Borsig, L., Fuster, M. M., Brown, J. R., Persson, L., Fransson, L.-A., and Esko, J. D. (2001) Proc. Natl. Acad. Sci. U. S. A., in press). Here, we have analyzed the effect of polyamine deprivation on the structure and polyamine affinity of the heparan sulfate chains in various glypican-1 glycoforms synthesized by a transformed cell line (ECV 304). Heparan sulfate chains of glypican-1 were either cleaved with heparanase at sites embracing the highly modified regions or with nitrite at N-unsubstituted glucosamine residues. The products were separated and further degraded by heparin lyase to identify sulfated iduronic acid. Polyamine affinity was assessed by chromatography on agarose substituted with the polyamine spermine. In heparan sulfate made by cells with undisturbed endogenous polyamine synthesis, free amino groups were restricted to the unmodified, unsulfated segments, especially near the core protein. Spermine high affinity binding sites were located to the modified and highly sulfated segments that were released by heparanase. In cells with up-regulated polyamine uptake, heparan sulfate contained an increased number of clustered N-unsubstituted glucosamines and sulfated iduronic acid residues. This resulted in a greater number of NO/nitrite-sensitive cleavage sites near the potential spermine-binding sites. Endogenous degradation by heparanase and NO-derived nitrite in polyamine-deprived cells generated a separate pool of heparan sulfate oligosaccharides with an exceptionally high affinity for spermine. Spermine uptake in polyamine-deprived cells was reduced when NO/nitrite-generated degradation of heparan sulfate was inhibited. The results suggest a functional interplay between glypican recycling, NO/nitrite-generated heparan sulfate degradation, and polyamine uptake.
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PMID:Modulations of glypican-1 heparan sulfate structure by inhibition of endogenous polyamine synthesis. Mapping of spermine-binding sites and heparanase, heparin lyase, and nitric oxide/nitrite cleavage sites. 1157 85

The purpose of this study was to examine the ability of type I- (porcine pancreas and Naja mocambique mocambique venom), type II- (bothropstoxin-I, bothropstoxin-II, and piratoxin-I), and type III- (Apis mellifera venom) secretory phospholipases A2 (sPLA2s) to induce human neutrophil chemotaxis, and the role of the cell surface proteoglycans, leukotriene B4 (LTB4), and platelet-activating factor (PAF), in mediating this migration. The neutrophil chemotaxis assays were performed by using a 48-well microchemotaxis chamber. Piratoxin-I, bothropstoxin-I, N. m. mocambique venom PLA2 (10-1000 microg/mL each), bothropstoxin-II (30-1000 microg/mL), porcine pancreas PLA2 (0.3-30 microg/mL), and A. mellifera venom PLA2 (30-300 microg/mL) caused concentration-dependent neutrophil chemotaxis. Heparin (10-300 U/mL) concentration-dependently inhibited the neutrophil migration induced by piratoxin-I, bothropstoxin-II, and N. m. mocambique and A. mellifera venom PLA2s (100 microg/mL each), but failed to affect the migration induced by porcine pancreas PLA2. Heparan sulfate (300 and 1000 microg/mL) inhibited neutrophil migration induced by piratoxin-I, whereas dermatan sulfate and chondroitin sulfate (30-1000 microg/mL each) had no effect. Heparitinase I and heparinase (300 mU/mL each) inhibited by 41.5 and 47%, respectively, piratoxin-I-induced chemotaxis, whereas heparitinase II and chondroitinase AC failed to affect the chemotaxis. The PAF receptor antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thienol-[3,2-f] -triazolo-[4,3-a] -diazepine-2-yl]-1-(4-morpholynil)-1-propionate) (0.1-10 microM) and the LTB4 synthesis inhibitor AA-861 [2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone] (0.1-10 microM) significantly inhibited the piratoxin-I-induced chemotaxis. Piratoxin-I (30-300 microg/mL) caused a concentration-dependent release of LTB4. Our results suggest that neutrophil migration in response to sPLA2s is independent of PLA activity, and involves an interaction of sPLA2s with cell surface heparin/heparan binding sites triggering the release of LTB4 and PAF.
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PMID:Human neutrophil migration in vitro induced by secretory phospholipases A2: a role for cell surface glycosaminoglycans. 1175 75

Understanding the process of wound healing will provide valuable insight for the development of new strategies to treat diseases associated with improper regeneration, such as blindness induced by corneal scarring. Heparan sulfate proteoglycans (HSPG) are not normally expressed in the corneal stroma, but their presence at sites of injury suggests their involvement in the wound healing response. Primary cultured corneal stromal fibroblasts constitutively express HSPG and represent an injured phenotype. Recently, nuclear localization of HSPG was shown to increase in corneal stromal fibroblasts plated on fibronectin (FN), an extracellular matrix protein whose appearance in the corneal stroma correlates with injury. One possible role for the nuclear localization of HSPG is to function as a shuttle for the nuclear transport of heparin-binding growth factors, such as basic fibroblast growth factor (FGF-2). Once in the nucleus, these growth factors might directly modulate cellular activities. To investigate this hypothesis, cells were treated with (125)I-labelled FGF-2 under various conditions and fractionated. Our results show that nuclear localization of FGF-2 was increased in cells plated on FN compared to those on collagen type I (CO). Interestingly, FGF-2-stimulated proliferation was increased in cells plated on FN compared to CO and this effect was absent in the presence of heparinase III. Furthermore, pre-treatment with heparinase III decreased nuclear FGF-2, and CHO cells defective in the ability to properly synthesize heparan sulfate chains showed reduced nuclear FGF-2 indicating that the heparan sulfate chains of HSPG are critical for this process. HSPG signaling, particularly through the cytoplasmic tails of syndecans, was investigated as a potential mechanism for the nuclear localization of FGF-2. Treatment with phorbol 12-myristate-13-acetate (PMA), under conditions that caused downregulation of protein kinase Calpha (PKCalpha), decreased nuclear FGF-2. Using pharmacological inhibitors of specific PKC isozymes, we elucidated a potential mode of regulation whereby PKCalpha mediates the nuclear localization of FGF-2 and PKCdelta inhibits it. Our studies suggest a novel mechanism in which FGF-2 translocates to the nucleus in response to injury.
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PMID:Nuclear localization of basic fibroblast growth factor is mediated by heparan sulfate proteoglycans through protein kinase C signaling. 1264 3

The assembly and activation of the kinin forming system components on human umbilical vein endothelial cells (HUVEC) have been studied in great detail. Proteins such as gC1qR, cytokeratin-1 and u-PAR have been identified to be responsible for Zn2+-dependent binding of high molecular weight kininogen (HK) to HUVEC. Heparan sulfate has also been shown to have a major role in Zn2+-dependent binding of HK to the endothelial cell line, Ea.hy 926. In this study, we have analyzed the possible contribution of heparan sulfate to high molecular weight kininogen binding to HUVEC using multiple approaches. The presence of heparan sulfate on HUVEC was analyzed by staining with an antibody specific for heparan sulfate. Incubation of the cells with bacterial heparinases removed the heparan sulfate from the cell surface to the level seen with a control antibody, however, the Zn2+-dependent binding of HK was not affected. Further, blocking of heparan sulfate with a specific antibody to heparan sulfate even after digestion with heparinases did not reduce HK binding whereas antibodies to the proteins gC1qR and cytokeratin-1 consistently reduced the binding of HK to the endothelial cells. The binding intensities of FITC-labeled HK were similar in heparinase-treated and -untreated HUVEC. The rate of kallikrein formation by the assembly of factor XII, HK and PK were similar in both heparinase-treated and non-treated HUVEC. All of these data indicate that heparan sulfate does not contribute significantly to HK binding to HUVEC.
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PMID:Assessment of the role of heparan sulfate in high molecular weight kininogen binding to human umbilical vein endothelial cells. 1462 81

Flow-induced mechanotransduction in vascular endothelial cells has been studied over the years with a major focus on putative connections between disturbed flow and atherosclerosis. Recent studies have brought in a new perspective that the glycocalyx, a structure decorating the luminal surface of vascular endothelium, may play an important role in the mechanotransduction. This study reports that modifying the amount of the glycocalyx affects both short-term and long-term shear responses significantly. It is well established that after 24 h of laminar flow, endothelial cells align in the direction of flow and their proliferation is suppressed. We report here that by removing the glycocalyx by using the specific enzyme heparinase III, endothelial cells no longer align under flow after 24 h and they proliferate as if there were no flow present. In addition, confluent endothelial cells respond rapidly to flow by decreasing their migration speed by 40% and increasing the amount of vascular endothelial cadherin in the cell-cell junctions. These responses are not observed in the cells treated with heparinase III. Heparan sulfate proteoglycans (a major component of the glycocalyx) redistribute after 24 h of flow application from a uniform surface profile to a distinct peripheral pattern with most molecules detected above cell-cell junctions. We conclude that the presence of the glycocalyx is necessary for the endothelial cells to respond to fluid shear, and the glycocalyx itself is modulated by the flow. The redistribution of the glycocalyx also appears to serve as a cell-adaptive mechanism by reducing the shear gradients that the cell surface experiences.
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PMID:Glycocalyx modulates the motility and proliferative response of vascular endothelium to fluid shear stress. 1746 37

Heparan sulfate (HS) within the glomerular basement membrane (GBM) is thought to play a major role in the charge-selective properties of the glomerular capillary wall. Recent data, however, raise questions regarding the direct role of HS in glomerular filtration. For example, in situ studies suggest that HS may prevent plasma macromolecules from clogging the GBM, keeping it in an "open" state. We evaluated this potential role of HS in vivo by studying the passage of protein through the glomerular capillary wall in the presence and absence of HS. Intravenous administration of neuraminidase removed neuraminic acid--but not HS--from the GBM, and this led to albuminuria. Concomitant removal of HS with heparinase III, confirmed by ultrastructural imaging, prevented the development of albuminuria in response to neuraminidase treatment. Taken together, these results suggest that HS keeps the GBM in an open state, facilitating passage of proteins through the glomerular capillary wall.
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PMID:Removal of heparan sulfate from the glomerular basement membrane blocks protein passage. 1830 8

Heparan sulfate moieties of cell surface proteoglycans serve as receptors for several herpesviruses. For herpes simplex virus 1, pseudorabies virus and equine herpesvirus 1, glycoprotein C (gC) homologues have been shown to mediate the binding to cell surface heparan sulfate. However, the role of gC in equine herpesvirus 4 (EHV-4) infection has not yet been analyzed. Using pull-down assay, we first determined that EHV-4 gC as well as gB are heparin-binding glycoproteins. To study the role of gC in EHV-4 infection, we constructed a gC-deletion mutant, WA79DeltagC, where the kanamycin resistant gene was inserted instead of the open reading frame encoding gC. We found that soluble heparin was capable of blocking both wild-type EHV-4 and WA79DeltagC infection of fetal horse kidney. Furthermore, pretreatment of cells with heparinase reduces considerably the ability of both viruses to adsorb to these cells and to form plaques. Similar results were obtained when cellular glycosaminoglycan synthesis was inhibited by chlorate treatment. In addition, we did find that gC protects EHV-4 from complement-mediated neutralization. These results suggest that, like other herpesviruses, EHV-4 gC plays a role in the interaction of the virus with cellular heparan sulfate. Moreover, gC can protect the virus from complement-mediated neutralization.
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PMID:Glycoprotein C of equine herpesvirus 4 plays a role in viral binding to cell surface heparan sulfate. 2023 10

Growth and remodeling of lymphatic vasculature occur during development and during various pathologic states. A major stimulus for this process is the unique lymphatic vascular endothelial growth factor-C (VEGF-C). Other endothelial growth factors, such as fibroblast growth factor-2 (FGF-2) or VEGF-A, may also contribute. Heparan sulfate is a linear sulfated polysaccharide that facilitates binding and action of some vascular growth factors such as FGF-2 and VEGF-A. However, a direct role for heparan sulfate in lymphatic endothelial growth and sprouting responses, including those mediated by VEGF-C, remains to be examined. We demonstrate that VEGF-C binds to heparan sulfate purified from primary lymphatic endothelia, and activation of lymphatic endothelial Erk1/2 in response to VEGF-C is reduced by interference with heparin or pretreatment of cells with heparinase, which destroys heparan sulfate. Such treatment also inhibited phosphorylation of the major VEGF-C receptor VEGFR-3 upon VEGF-C stimulation. Silencing lymphatic heparan sulfate chain biosynthesis inhibited VEGF-C-mediated Erk1/2 activation and abrogated VEGFR-3 receptor-dependent binding of VEGF-C to the lymphatic endothelial surface. These findings prompted targeting of lymphatic N-deacetylase/N-sulfotransferase-1 (Ndst1), a major sulfate-modifying heparan sulfate biosynthetic enzyme. VEGF-C-mediated Erk1/2 phosphorylation was inhibited in Ndst1-silenced lymphatic endothelia, and scratch-assay responses to VEGF-C and FGF-2 were reduced in Ndst1-deficient cells. In addition, lymphatic Ndst1 deficiency abrogated cell-based growth and proliferation responses to VEGF-C. In other studies, lymphatic endothelia cultured ex vivo from Ndst1 gene-targeted mice demonstrated reduced VEGF-C- and FGF-2-mediated sprouting in collagen matrix. Lymphatic heparan sulfate may represent a novel molecular target for therapeutic intervention.
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PMID:Lymphatic endothelial heparan sulfate deficiency results in altered growth responses to vascular endothelial growth factor-C (VEGF-C). 2134 5

Heparan sulfate (HS) glycosaminoglycans (GAGs) regulate a host of biological functions. To better understand their biological roles, it is necessary to gain understanding about the structure of HS, which requires identification of the sulfation pattern as well as the uronic acid epimerization. In order to model HS structure, it is necessary to quantitatively profile depolymerization products. To date, liquid chromatography-mass spectrometry (LC-MS) methods for profiling heparin lyase decomposition products have been shown. These enzymes, however, destroy information about uronic acid epimerization. Deaminative cleavage using nitrous acid (HONO) is a classic method for GAG depolymerization that retains uronic acid epimerization. Several chromatographic methods have been used for analysis of deaminative cleavage products. The chromatographic methods have the disadvantage that there is no direct readout on the structures producing the observed peaks. This report demonstrates a porous graphitized carbon (PGC)-MS method for the quantification of HONO generated disaccharides to obtain information about the sulfation pattern and uronic acid epimerization. Here, we demonstrate the separation and identification of uronic acid epimers as well as geometric sulfation isomers. The results are comparable to those expected for benchmark HS and heparin samples. The data demonstrate the utility of PGC-MS for quantification of HS nitrous acid depolymerization products for structural analysis of HS and heparin.
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PMID:Mass spectrometric method for determining the uronic acid epimerization in heparan sulfate disaccharides generated using nitrous acid. 2287 17


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