Secretion of Heparanase Protein Is Regulated by Glycosylation in Human Tumor Cell Lines

Simizu, Siro; Ishida, Keisuke; Wierzba, Michal K; Osada, Hiroyuki

doi:10.1074/jbc.m300541200

Cited by 64 publications

(50 citation statements)

References 48 publications

(53 reference statements)

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“…Our results suggest that differences in molecular weights between Spalax and human heparanases are primarily due to a lower glycosylation of the Spalax protein, which lacks three of the six N-glycosylation sites of the human heparanase. Glycosylation of heparanase is not required for its enzymatic activity but may function in heparanase trafficking and secretion (43). Secretion of heparanase appears to be a cardinal step in its processing and activation (34 between the proton donor (Glu-256) and nucleophile (Glu-374) sites.…”

Section: Discussionmentioning

confidence: 99%

Adaptive evolution of heparanase in hypoxia-tolerantSpalax: Gene cloning and identification of a unique splice variant

Nasser

Nevo

Shafat

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Heparan sulfate (HS) side chains of HS proteoglycans bind to and assemble extracellular matrix proteins and play important roles in cell-cell and cell-extracellular matrix interactions. HS chains bind a multitude of bioactive molecules and thereby function in the control of multiple normal and pathological processes. Enzymatic degradation of HS by heparanase, a mammalian endoglycosidase, affects the integrity and functional state of tissues and is involved in, among other processes, inflammation, angiogenesis, and cancer metastasis. Here, we report the cloning of heparanase from four Israeli species of the blind subterranean mole rat (Spalax ehrenbergi superspecies), 85% homologous to the human enzyme. Unlike its limited expression in human tissues, heparanase is highly expressed in diverse Spalax tissues. Moreover, we have identified a unique splice variant of the Spalax enzyme lacking 16 aa encoded by exon 7. This deletion resulted in a major defect in trafficking and processing of the heparanase protein, leading to a loss of its enzymatic activity. Interspecies variation was noted in the sequence and in the expression of the splice variant of the heparanase gene in blind mole rats living under different ecogeographical stresses, indicating a possible role in adaptation to stress in Spalax evolution.alternative splicing ͉ heparan sulfate ͉ blind mole rat ͉ angiogenesis ͉ cancer H eparan sulfate (HS) proteoglycans are macromolecules associated with the cell surface and the extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues (1-3). HS binds to and assembles ECM proteins and plays important roles in the structural integrity of the ECM and in cell-cell and cell-ECM interactions. HS chains sequester a multitude of proteins and bioactive molecules and thereby function in the control of a large number of normal and pathological processes (1-4). Apart from sequestration of bioactive molecules, HS proteoglycans have a coreceptor role in which the proteoglycan, in concert with the other cell surface molecule, comprises a functional receptor complex that binds the ligand and mediates its action (3-5).Enzymatic degradation of HS by heparanase, a mammalian endoglucuronidase, affects the integrity and functional state of tissues and is involved in fundamental biological phenomena, ranging from pregnancy, morphogenesis, and development to inf lammation, angiogenesis, and cancer metastasis (6 -10). Heparanase elicits an indirect angiogenic response by releasing HS-bound angiogenic growth factors [e.g., basic fibroblast growth factor and vascular endothelial growth factor (VEGF)] from the ECM and by generating HS fragments that potentiate basic fibroblast growth factor receptor binding, dimerization, and signaling (5, 8). Despite earlier reports on the existence of several distinct mammalian HS-degrading endoglycosidases (heparanases), the cloning of the same single gene by several groups (6,7,11,12) suggests that mammalian cells express primarily a single dominant functional heparanase e...

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Section: Discussionmentioning

confidence: 99%

Adaptive evolution of heparanase in hypoxia-tolerantSpalax: Gene cloning and identification of a unique splice variant

Nasser

Nevo

Shafat

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…The remaining 6% charged glycans were not identified, as they were not cleaved by either enzyme, presumably due to glycans inaccessible to the enzymes. The existence of six glycosylation sites in the heparanase molecule (Simizu et al, 2004), together with the homogenous appearance of the protein in a native polyacrylamide gel and the finding that 16% of the molecules carry phosphorylated high mannose led us to estimate that each heparanase molecule bears one phosphorylated glycan.…”

Section: Analysis Of Heparanase Glycan Compositionmentioning

confidence: 99%

“…Heparanase has long been characterized as a glycoprotein and including Concanavalin A affinity chromatography in the purification scheme brought this feature into practice (Toyoshima and Nakajima, 1999;Zcharia et al, 2005). Six glycosylation sites were identified in the 50 kDa heparanase subunit (Hulett et al, 1999), and their role in protein secretion was established (Simizu et al, 2004), yet glycan biochemical analysis was not performed to date.…”

Section: Introductionmentioning

confidence: 99%

Low and high affinity receptors mediate cellular uptake of heparanase

Ben-Zaken

Shafat

Gingis-Velitski

et al. 2008

The International Journal of Biochemistry & Cell Biology

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Heparanase is an endoglycosidase which cleaves heparan sulfate and hence participates in degradation and remodeling of the extracellular matrix. Importantly, heparanase activity correlated with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of heparan sulfate cleavage and remodeling of the extracellular matrix barrier. Heparanase has been characterized as a glycoprotein, yet glycan biochemical analysis was not performed to date. Here, we applied the Qproteome ™ GlycoArray kit to perform glycan analysis of heparanase, and compared the kit results with the more commonly used biochemical analyses. We employed fibroblasts isolated from patients with I-cell disease (mucolipidosis II), fibroblasts deficient of low density lipoprotein receptor-related protein and fibroblasts lacking mannose 6-phosphate receptor, to explore the role of mannose 6-phosphate in heparanase uptake. Iodinated heparanase has been utilized to calculate binding affinity. We provide evidence for hierarchy of binding to cellular receptors as a function of heparanase concentration. We report the existence of a high affinity, low abundant (i.e., low density lipoprotein receptor-related protein, mannose 6-phosphate receptor), as well as a low affinity, high abundant (i.e., heparan sulfate proteoglycan) receptors that mediate heparanase binding, and suggest that these receptors cooperate to establish high affinity binding sites for heparanase, thus maintaining extracellular retention of the enzyme tightly regulated.

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“…We previously demonstrated that these six putative N-glycosylation sites were glycosylated in cultured cells. 24) Although glycosylation was not required for enzyme activity, the secretion of heparanase was regulated by glycosylation. 24) The sequence also contains a 35-amino-acid N-terminal signal sequence (Met 1 -Ala 35 ), which represents the most prominent difference between the chicken and human enzymes, and which apparently accounts for the fact that chicken heparanase is readily secreted.…”

Section: Heparanase Structurementioning

confidence: 99%

“…24) Although glycosylation was not required for enzyme activity, the secretion of heparanase was regulated by glycosylation. 24) The sequence also contains a 35-amino-acid N-terminal signal sequence (Met 1 -Ala 35 ), which represents the most prominent difference between the chicken and human enzymes, and which apparently accounts for the fact that chicken heparanase is readily secreted. 25) Use of a chimeric construct composed of the human enzyme and the chicken heparanase signal peptide revealed that cell surface localization and the secretion of heparanase markedly stimulated tumor angiogenesis.…”

Section: Heparanase Structurementioning

confidence: 99%

Heparanase as a molecular target of cancer chemotherapy

2004

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Cancer cells require the ability to degrade the extracellular matrix (ECM) in order to turn into invasive and metastatic cancer cells. Many proteases and glycosidases are essential in the process of dissolving the components of the ECM. An endo-β β β β-D-glucuronidase, heparanase, is capable of specifically degrading one of the ECM components, heparan sulfate, and this activity is associated with the metastatic potential of tumor cells. Since heparanase mRNA is overexpressed in many human tumors (e.g., hepatomas, head and neck tumors, and esophageal carcinomas), the mechanisms regulating the activity of heparanase should be clarified; considering the possible role of heparanase in cancer, the development of heparanase inhibitors would appear to be advantageous. This review will focus on recent findings that have contributed to the characterization of heparanase and to the elucidation of the transcriptional regulation of heparanase mRNA expression, as well as the development of heparanase inhibitors. tudies of the biological function of extracellular matrix (ECM) molecules have revealed the central role of heparan sulfate proteoglycans (HSPGs) in many biological events, such as early embryogenesis and angiogenesis.1, 2) Heparan sulfate (HS) chains, consisting of strongly anionic linear polysaccharides, are structurally heterogeneous and bind a diverse repertoire of proteins under physiological conditions. Cell surface HS provides cells with a mechanism to snare a wide variety of extracellular effectors. These cell surface HSPGs modulate the activity of a wide variety of HS-binding proteins, acting as coreceptors with signaling receptors and directly as endocytosis receptors.3) As co-receptors, they potentiate the activity of sparse ligands by enhancing the formation of ligand-receptor complexes. In addition, intracellular signaling and endocytosis are augmented when HSPGs interact with soluble ligands. 1-3)HS/HSPG is known to interact with various molecules, 3) such as growth factors (e.g., fibroblast growth factors), cytokines (e.g., interleukin-2), ECM proteins (e.g., collagens), factors involved in blood coagulation (e.g., heparin cofactor II), and other proteins such as β-amyloid proteins and matrix metalloproteinase-7.1, 2, 4) Since HS chains are very similar among organisms as diverse as insects, mollusks, and mammals, the conservation of HS binding to extracellular proteins suggests that the association of these proteins with HS is conserved and functionally relevant. Because of the important and pleiotropic roles of HSPGs in cell physiology, the cleavage of HSPGs is likely to alter the integrity and functional state of tissues and to provide a mechanism by which cells can respond rapidly to changes in the extracellular environment. HS/HSPG carbohydrate chains are cleaved either by lyases (eliminative cleavage) or hydrolases (hydrolytic cleavage). These cleaving enzymes are therefore in part responsible for the modulation of the biological functions of HS/HSPGs-binding proteins. The enzymatic degradation o...

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Secretion of Heparanase Protein Is Regulated by Glycosylation in Human Tumor Cell Lines

Cited by 64 publications

References 48 publications

Adaptive evolution of heparanase in hypoxia-tolerantSpalax: Gene cloning and identification of a unique splice variant

Adaptive evolution of heparanase in hypoxia-tolerantSpalax: Gene cloning and identification of a unique splice variant

Low and high affinity receptors mediate cellular uptake of heparanase

Heparanase as a molecular target of cancer chemotherapy

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