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Kovalszky, Ilona; Dudás, József; Oláh-Nagy, Julia; Pogány, Gábor; Töváry, József; Tı́már, József; Kopper, László; Jeney, A.; Iozzo, Renato V.

doi:10.1023/a:1006898920637

Cited by 51 publications

(29 citation statements)

References 67 publications

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“…fibronectins and collagens), factors involved in blood coagulation (e.g. heparin cofactor II), and other proteins such as lipoproteins, DNA topoisomerases, and ␤-amyloid proteins (5)(6)(7)(8)(9)(10)(11)(12). HS/HSPG carbohydrate chains are depolymerized enzymatically either by eliminative cleavage with lyases or by hydrolytic cleavage with hydrolases, and these enzymes therefore, in part, modulate the biological functions of HS/HSPG-binding proteins (13).…”

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confidence: 99%

Human Heparanase

Toyoshima

Nakajima

1999

Journal of Biological Chemistry

283

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Heparan sulfate and heparan sulfate proteoglycans are present in the extracellular matrix as well as on the external cell surface. They bind various molecules such as growth factors and cytokines and modulate the biological functions of binding proteins. Heparan sulfate proteoglycans are also important structural components of the basement membrane. Heparanase is an endo-␤-D-glucuronidase capable of cleaving heparan sulfate and has been implicated in inflammation and tumor angiogenesis and metastasis. In this study, we report the purification of a human heparanase from an SV40-transformed embryonic fibroblast cell line WI38/VA13 by four sequential column chromatographies. The activity was measured by high speed gel permeation chromatography of the degradation products of fluorescein isothiocyanate-labeled heparan sulfate. The enzyme was purified to homogeneity, yielding a peptide with an apparent molecular mass of 50 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. Using the amino acid sequences of the N-terminal and internal heparanase peptides, a cDNA coding for human heparanase was cloned. NIH3T3 and COS-7 cells stably transfected with pBK-CMV expression vectors containing the heparanase cDNA showed high heparanase activities. The homology search revealed that no homologous protein had been reported. Heparan sulfate (HS)1 and heparan sulfate proteoglycans (HSPG), localized in the extracellular matrix and on the external surface of cell membranes, play a major role in cell-cell and cell-extracellular matrix interactions (1, 2). HSPG are implicated in a number of cellular processes, including cell adhesion, migration, differentiation, and proliferation (3, 4). Various molecules have been reported to interact with HS/HSPG; they are growth factors (e.g. fibroblast growth factors and platelet-derived growth factor), cytokines (e.g. interleukin-2), extracellular matrix proteins (e.g. fibronectins and collagens), factors involved in blood coagulation (e.g. heparin cofactor II), and other proteins such as lipoproteins, DNA topoisomerases, and ␤-amyloid proteins (5-12). HS/HSPG carbohydrate chains are depolymerized enzymatically either by eliminative cleavage with lyases or by hydrolytic cleavage with hydrolases, and these enzymes therefore, in part, modulate the biological functions of HS/HSPG-binding proteins (13).Heparanase, which cleaves HS into characteristic large molecular weight fragments, was identified in murine metastatic melanoma cells by Nakajima et al. (14). Heparanase activity was correlated with the lung colonization potential of murine B16 melanoma sublines (14). Nakajima et al. (15) concluded that the enzyme responsible for HS degrading is an endoglucuronidase, cleaving the linkage between GlcUA and GlcNAc, and named it heparanase. Heparanase is a hydrolase distinct from flavobacterium heparitinase and heparinase, which are eliminases capable of specifically degrading heparan sulfate and heparin, respectively, into di-and tetrasaccharides (15)(16)(17).Various methods for detecting hepara...

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confidence: 99%

Human Heparanase

Toyoshima

Nakajima

1999

Journal of Biological Chemistry

283

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“…We focused on topoisomerase I. Certainly, heparin and liver HS, but not liver cancer HS, bound and inhibited topoisomerase I plasmid relaxation in vitro [21, 44]. Our present work also demonstrates that heparin and HS hinder the in vitro plasmid cleavage effect of TpT.…”

Section: Discussionmentioning

confidence: 54%

“…While heparin is mainly present in mast cells, HS that is structurally strongly related to heparin is present everywhere in the living organisms [37]. Earlier we demonstrated that cells could take up labeled heparin and liver HS and transport them into the nucleus [17, 21]. …”

Section: Discussionmentioning

confidence: 99%

“…Protein concentration of the nuclear extract was determined by the Coomassie protein kit of Pierce Biotechnology, Inc. (Rockford, USA) according to the protocol of the manufacturer. The isolation of GAG and HS, their electrophoresis, and their colorimetry were described previously [17, 21, 28]. …”

Section: Methodsmentioning

confidence: 99%

“…We reported that heparin and liver HS inhibit the plasmid relaxation activity of topoisomerase I enzyme in vitro [21]. Furthermore, we provided evidence for heparin and HS cellular uptake and accumulation in the nucleus [17, 22].…”

Section: Introductionmentioning

confidence: 99%

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Heparin and Liver Heparan Sulfate Can Rescue Hepatoma Cells from Topotecan Action

Dudás

Bocsi

Fullár

et al. 2014

BioMed Research International

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Topotecan (TpT) is a major inhibitory compound of topoisomerase (topo) I that plays important roles in gene transcription and cell division. We have previously reported that heparin and heparan sulfate (HS) might be transported to the cell nucleus and they can interact with topoisomerase I. We hypothesized that heparin and HS might interfere with the action of TpT. To test this hypothesis we isolated topoisomerase I containing cell nuclear protein fractions from normal liver, liver cancer tissues, and hepatoma cell lines. The enzymatic activity of these extracts was measured in the presence of heparin, liver HS, and liver cancer HS. In addition, topo I activity, cell viability, and apoptosis of HepG2 and Hep3B cells were investigated after heparin and TpT treatments. Liver cancer HS inhibited topo I activity in vitro. Heparin treatment abrogated topo I enzyme activity in Hep3B cells, but not in HepG2 cells, where the basal activity was higher. Heparin protected the two hepatoma cell lines from TpT actions and decreased the rate of TpT induced S phase block and cell death. These results suggest that heparin and HS might interfere with the function of TpT in liver and liver cancer.

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Heparin inhibits the interaction of DNA topoisomerase I/anti–topoisomerase I immune complexes with heparan sulfate on dermal fibroblasts

Arcand¹,

Robitaille²,

Koenig³

et al. 2012

Arthritis & Rheumatism

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Objective. Previous studies have demonstrated that the systemic sclerosis (SSc)-associated autoantigen DNA topoisomerase I (topo I) binds specifically to the surface of fibroblasts when released in the extracellular environment and recruits anti-topo I autoantibodies, which subsequently leads to the adhesion and activation of monocytes. This study aimed to characterize the molecular interactions of topo I with fibroblast surfaces in order to elucidate the pathogenic role of topo I/antitopo I immune complexes (ICs) in SSc.Methods. Topo I directly coupled to fluorochromes was used to follow its binding to fibroblast surfaces by flow cytometry and fluorescence microscopy. Purified IgG from normal subjects or SSc patients was added with topo I to the cells; unfractionated heparin (UFH) and low molecular weight heparin (LMWH) were used to determine their effects on the binding of topo I and topo I/anti-topo I IC to fibroblast surfaces.Results. Heparan sulfate (HS) proteoglycans on fibroblast surfaces were found to act as coreceptors for topo I binding. The addition of anti-topo I autoantibodies from SSc sera led to the amplification of topo I binding to HS chains. UFH and LMWH were shown to inhibit topo I and topo I/anti-topo I IC binding to HS chains.Conclusion. This study is the first to show that topo I binds specifically to HS proteoglycans on fibroblast surfaces and that anti-topo I autoantibodies from SSc patients amplify topo I binding to HS chains. The accumulation of topo I on cell surfaces by anti-topo I autoantibodies could contribute to the initiation of an inflammatory cascade stimulating the fibrosis. UFH and LMWH inhibited the binding of topo I/anti-topo I IC to fibroblasts, suggesting a potential therapeutic role in SSc-associated fibrosis.

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Cited by 51 publications

References 67 publications

Human Heparanase

Human Heparanase

Heparin and Liver Heparan Sulfate Can Rescue Hepatoma Cells from Topotecan Action

Heparin inhibits the interaction of DNA topoisomerase I/anti–topoisomerase I immune complexes with heparan sulfate on dermal fibroblasts

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