Endoglycosidase Heparanase Research Articles

Heparanase Increases Podocyte Survival and Autophagic Flux after Adriamycin-Induced Injury

The kidney glomerular filtration barrier (GFB) is enriched with heparan sulfate (HS) proteoglycans, which contribute to its permselectivity. The endoglycosidase heparanase cleaves HS and hence appears to be involved in the pathogenesis of kidney injury and glomerulonephritis. We have recently reported, nonetheless, that heparanase overexpression preserved glomerular structure and kidney function in an experimental model of Adriamycin-induced nephropathy. To elucidate mechanisms underlying heparanase function in podocytes—key GFB cells, we utilized a human podocyte cell line and transgenic mice overexpressing heparanase. Notably, podocytes overexpressing heparanase (H) demonstrated significantly higher survival rates and viability after exposure to Adriamycin or hydrogen peroxide, compared with mock-infected (V) podocytes. Immunofluorescence staining of kidney cryo-sections and cultured H and V podocytes as well as immunoblotting of proteins extracted from cultured cells, revealed that exposure to toxic injury resulted in a significant increase in autophagic flux in H podocytes, which was reversed by the heparanase inhibitor, Roneparstat (SST0001). Heparanase overexpression was also associated with substantial transcriptional upregulation of autophagy genes BCN1, ATG5, and ATG12, following Adriamycin treatment. Moreover, cleaved caspase-3 was attenuated in H podocytes exposed to Adriamycin, indicating lower apoptotic cell death in H vs. V podocytes. Collectively, these findings suggest that in podocytes, elevated levels of heparanase promote cytoprotection.

Parabacteroides produces acetate to alleviate heparanase-exacerbated acute pancreatitis through reducing neutrophil infiltration

BackgroundThe endoglycosidase heparanase which degrades heparan sulfate proteoglycans, exerts a pro-inflammatory mediator in various inflammatory disorders. However, the function and underlying mechanism of heparanase in acute pancreatitis remain poorly understood. Here, we investigated the interplay between heparanase and the gut microbiota in the development of acute pancreatitis.MethodsAcute pancreatitis was induced in wild-type and heparanase-transgenic mice by administration of caerulein. The differences in gut microbiota were analyzed by 16S ribosomal RNA sequencing. Antibiotic cocktail experiment, fecal microbiota transplantation, and cohousing experiments were used to assess the role of gut microbiota.ResultsAs compared with wild-type mice, acute pancreatitis was exacerbated in heparanase-transgenic mice. Moreover, the gut microbiota differed between heparanase-transgenic and wild-type mice. Heparanase exacerbated acute pancreatitis in a gut microbiota-dependent manner. Specially, the commensal Parabacteroides contributed most to distinguish the differences between wild-type and heparanase-transgenic mice. Administration of Parabacteroides alleviated acute pancreatitis in wild-type and heparanase-transgenic mice. In addition, Parabacteroides produced acetate to alleviate heparanase-exacerbated acute pancreatitis through reducing neutrophil infiltration.ConclusionsThe gut–pancreas axis played an important role in the development of acute pancreatitis and the acetate produced by Parabacteroides may be beneficial for acute pancreatitis treatment.AXqtS6bvN-oakcGDurjdFQVideo abstract

Dissociation of DNA damage sensing by endoglycosidase HPSE

SummaryBalance between cell proliferation and elimination is critical in handling threats both exogenous and of internal dysfunction. Recent work has implicated a conserved but poorly understood endoglycosidase heparanase (HPSE) in the restriction of innate defense responses, yet biochemical mediators of these key functions remained unclear. Here, an unbiased immunopurification proteomics strategy is employed to identify and rank uncharacterized interactions between HPSE and mediators of canonical signaling pathways linking cell cycle and stress responses. We demonstrate with models of genotoxic stress including herpes simplex virus infection and chemotherapeutic treatment that HPSE dampens innate responses to double-stranded DNA breakage by interfering with signal transduction between initial sensors and downstream mediators. Given the long-standing recognition of HPSE in driving late-stage inflammatory disease exemplified by tissue destruction and cancer metastasis, modulation of this protein with control over the DNA damage response imparts a unique strategy in the development of unconventional multivalent therapy.

Heparanase in Kidney Disease.

The primary filtration of blood occurs in the glomerulus in the kidney. Destruction of any of the layers of the glomerular filtration barrier might result in proteinuric disease. The glomerular endothelial cells and especially its covering layer, the glycocalyx, play a pivotal role in development of albuminuria. One of the main sulfated glycosaminoglycans in the glomerular endothelial glycocalyx is heparan sulfate. The endoglycosidase heparanase degrades heparan sulfate, thereby affecting glomerular barrier function, immune reactivity and inflammation. Increased expression of glomerular heparanase correlates with loss of glomerular heparan sulfate in many glomerular diseases. Most importantly, heparanase knockout in mice prevented the development of albuminuria after induction of experimental diabetic nephropathy and experimental glomerulonephritis. Therefore, heparanase could serve as a pharmacological target for glomerular diseases. Several factors that regulate heparanase expression and activity have been identified and compounds aiming to inhibit heparanase activity are currently explored.

The heparan sulfate degrading enzyme heparanase is up‐regulated by the EWS‐Fli1 fusion protein in Ewing sarcoma

Ewing sarcoma is an aggressive form of bone and soft tissue cancer that predominantly affects adolescents. It is commonly caused by the partial translocation of the Ewing sarcoma protein (EWS) gene to the gene encoding the Ets family transcription factor Fli1, resulting in the formation of the EWS‐Fli1 fusion protein. Ewing sarcoma is often highly metastatic, resulting in a poor survival rate of approximately 20%. Expression of the endoglycosidase heparanase (HPSE), a heparan sulfate‐degrading enzyme that contributes to extracellular matrix remodelling, is up‐regulated in approximately 90% of all cancers, including Ewing sarcoma, and possesses pro‐metastatic and ‐angiogenic functions. It is therefore not surprising that it is also a marker of poor prognosis for cancer patients. While increased expression of HPSE in Ewing sarcoma has been reported to correlate with increased tumour size, disease severity and a poor patient outcome, the mechanism by which HPSE expression is increased in Ewing sarcoma has not been defined. We have identified a novel link between the EWS‐Fli1 fusion protein and the expression of HPSE. Using a combination of patient‐derived samples and cell lines, we have been able to show that an increase in expression of EWS‐Fli1 correlates with increased HPSE expression and activity. We also demonstrated that increased EWS‐Fli1 expression is responsible for increased HPSE expression and enzymatic activity by binding to the HPSE gene promoter and driving its expression. These findings highlight a possible role for HPSE in the progression and prognosis of Ewing sarcoma that may contribute to the aggressive nature of the disease.Support or Funding InformationAustralian National Health and Medical Research CouncilThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Heparanase and macrophage interplay in the onset of liver fibrosis

The heparan sulfate endoglycosidase heparanase (HPSE) is involved in tumor growth, chronic inflammation and fibrosis. Since a role for HPSE in chronic liver disease has not been demonstrated to date, the current study was aimed at investigating the involvement of HPSE in the pathogenesis of chronic liver injury. Herein, we revealed that HPSE expression increased in mouse livers after carbon tetrachloride (CCl4)-mediated chronic induction of fibrosis, but with a trend to decline during progression of the disease. In mouse fibrotic liver tissues HPSE immunostaining was restricted in necro-inflammatory areas, co-localizing with F4/80 macrophage marker and TNF-α. TNF-α treatment induced HPSE expression as well as HPSE secretion in U937 macrophages. Moreover, macrophage-secreted HPSE regulated the expression of α-SMA and fibronectin in hepatic stellate LX-2 cells. Finally, HPSE activity increased in the plasma of patients with liver fibrosis but it inversely correlated with liver stiffness. Our results suggest the involvement of HPSE in early phases of reaction to liver damage and inflammatory macrophages as an important source of HPSE. HPSE seems to play a key role in the macrophage-mediated activation of hepatic stellate cells (HSCs), thus suggesting that HPSE targeting could be a new therapeutic option in the treatment of liver fibrosis.

Heparanase 2 Attenuates Head and Neck Tumor Vascularity and Growth.

The endoglycosidase heparanase specifically cleaves the heparan sulfate (HS) side chains on proteoglycans, an activity that has been implicated strongly in tumor metastasis and angiogenesis. Heparanase-2 (Hpa2) is a close homolog of heparanase that lacks intrinsic HS-degrading activity but retains the capacity to bind HS with high affinity. In head and neck cancer patients, Hpa2 expression was markedly elevated, correlating with prolonged time to disease recurrence and inversely correlating with tumor cell dissemination to regional lymph nodes, suggesting that Hpa2 functions as a tumor suppressor. The molecular mechanism associated with favorable prognosis following Hpa2 induction is unclear. Here we provide evidence that Hpa2 overexpression in head and neck cancer cells markedly reduces tumor growth. Restrained tumor growth was associated with a prominent decrease in tumor vascularity (blood and lymph vessels), likely due to reduced Id1 expression, a transcription factor highly implicated in VEGF-A and VEGF-C gene regulation. We also noted that tumors produced by Hpa2-overexpressing cells are abundantly decorated with stromal cells and collagen deposition, correlating with a marked increase in lysyl oxidase expression. Notably, heparanase enzymatic activity was unimpaired in cells overexpressing Hpa2, suggesting that reduced tumor growth is not caused by heparanase regulation. Moreover, growth of tumor xenografts by Hpa2-overexpressing cells was unaffected by administration of a mAb that targets the heparin-binding domain of Hpa2, implying that Hpa2 function does not rely on heparanase or heparan sulfate. Cancer Res; 76(9); 2791-801. ©2016 AACR.

The Matricellular Receptor LRP1 Forms an Interface for Signaling and Endocytosis in Modulation of the Extracellular Tumor Environment.

The membrane protein low-density lipoprotein receptor related-protein 1 (LRP1) has been attributed a role in cancer. However, its presumably often indirect involvement is far from understood. LRP1 has both endocytic and signaling activities. As a matricellular receptor it is involved in regulation, mostly by clearing, of various extracellular matrix degrading enzymes including matrix metalloproteinases, serine proteases, protease inhibitor complexes, and the endoglycosidase heparanase. Furthermore, by binding extracellular ligands including growth factors and subsequent intracellular interaction with scaffolding and adaptor proteins it is involved in regulation of various signaling cascades. LRP1 expression levels are often downregulated in cancer and some studies consider low LRP1 levels a poor prognostic factor. On the contrary, upregulation in brain cancers has been noted and clinical trials explore the use of LRP1 as cargo receptor to deliver cytotoxic agents. This mini-review focuses on LRP1’s role in tumor growth and metastasis especially by modulation of the extracellular tumor environment. In relation to this role its diagnostic, prognostic and therapeutic potential will be discussed.

Syntenin and syndecan in the biogenesis of exosomes.

Cells communicate with their environment in various ways, including by secreting vesicles. Secreted vesicles are loaded with proteins, lipids and RNAs that compose 'a signature' of the cell of origin and potentially can reprogram recipient cells. Secreted vesicles recently gained in interest for medicine. They represent potential sources of biomarkers that can be collected from body fluids and, by disseminating pathogenic proteins, might also participate in systemic diseases like cancer, atherosclerosis and neurodegeneration. The mechanisms controlling the biogenesis and the uptake of secreted vesicles are poorly understood. Some of these vesicles originate from endosomes and are called 'exosomes'. In this review, we recapitulate recent insight on the role of the syndecan (SDC) heparan sulphate proteoglycans, the small intracellular adaptor syntenin and associated regulators in the biogenesis and loading of exosomes with cargo. SDC-syntenin-associated regulators include the endosomal sorting complex required for transport accessory component ALG-2-interacting protein X, the small GTPase adenosine 5'-diphosphate-ribosylation factor 6, the lipid-modifying enzyme phospholipase D2 and the endoglycosidase heparanase. All these molecules appear to support the budding of SDC-syntenin and associated cargo into the lumen of endosomes. This highlights a major mechanism for the formation of intraluminal vesicles that will be released as exosomes.

Heparanase Promotes Exosome Release

The increased abundance of the endoglycosidase heparanase in malignant cells enhances their release of exosomes.

The endoglycosidase heparanase enters the nucleus of T lymphocytes and modulates H3 methylation at actively transcribed genes via the interplay with key chromatin modifying enzymes

The methylation of histones is a fundamental epigenetic process regulating gene expression programs in mammalian cells. Dysregulated patterns of histone methylation are directly implicated in malignant transformation. Here, we report the unexpected finding that the invasive extracellular matrix degrading endoglycosidase heparanase enters the nucleus of activated human T lymphocytes and regulates the transcription of a cohort of inducible immune response genes by controlling histone H3 methylation patterns. It was found that nuclear heparanase preferentially associates with euchromatin. Genome-wide ChIP-on-chip analyses showed that heparanase is recruited to both the promoter and transcribed regions of a distinct cohort of transcriptionally active genes. Knockdown and overexpression of the heparanase gene also showed that chromatin-bound heparanase is a prerequisite for the transcription of a subset of inducible immune response genes in activated T cells. Furthermore, the actions of heparanase seem to influence gene transcription by associating with the demethylase LSD1, preventing recruitment of the methylase MLL and thereby modifying histone H3 methylation patterns. These data indicate that heparanase belongs to an emerging class of proteins that play an important role in regulating transcription in addition to their well-recognized extra-nuclear functions.

Heparanase induces Akt phosphorylation via a lipid raft receptor

The endoglycosidase heparanase is the predominant enzyme that degrades heparan sulfate side chains of heparan sulfate proteoglycans, activity that is strongly implicated in tumor metastasis. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Among these is the induction of Akt/PKB phosphorylation noted in endothelial- and tumor-derived cells. Protein domains of heparanase required for signaling were not identified to date, nor were identified heparanase binding proteins/receptors capable of transmitting heparanase signals. Here, we examined the possible function of mannose 6-phosphate receptor (MPR) and low-density lipoprotein-receptor related protein (LRP), recently implicated in cellular uptake of heparanase, as heparanase receptors mediating Akt phosphorylation. We found that heparanase addition to MPR- and LRP-deficient fibroblasts elicited Akt activation indistinguishable from control fibroblasts. In contrast, disruption of lipid rafts abrogated Akt/PKB phosphorylation following heparanase addition. These results suggest that lipid raft-resident receptor mediates heparanase signaling.

Identification and Characterization of Heparin/Heparan Sulfate Binding Domains of the Endoglycosidase Heparanase

The endo-beta-glucuronidase, heparanase, is an enzyme that cleaves heparan sulfate at specific intra-chain sites, yielding heparan sulfate fragments with appreciable size and biological activities. Heparanase activity has been traditionally correlated with cell invasion associated with cancer metastasis, angiogenesis, and inflammation. In addition, heparanase up-regulation has been documented in a variety of primary human tumors, correlating with increased vascular density and poor postoperative survival, suggesting that heparanase may be considered as a target for anticancer drugs. In an attempt to identify the protein motif that would serve as a target for the development of heparanase inhibitors, we looked for protein domains that mediate the interaction of heparanase with its heparan sulfate substrate. We have identified three potential heparin binding domains and provided evidence that one of these is mapped at the N terminus of the 50-kDa active heparanase subunit. A peptide corresponding to this region (Lys(158)-Asp(171)) physically associates with heparin and heparan sulfate. Moreover, the peptide inhibited heparanase enzymatic activity in a dose-responsive manner, presumably through competition with the heparan sulfate substrate. Furthermore, antibodies directed to this region inhibited heparanase activity, and a deletion construct lacking this domain exhibited no enzymatic activity. NMR titration experiments confirmed residues Lys(158)-Asn(162) as amino acids that firmly bound heparin. Deletion of a second heparin binding domain sequence (Gln(270)-Lys(280)) yielded an inactive enzyme that failed to interact with cell surface heparan sulfate and hence accumulated in the culture medium of transfected HEK 293 cells to exceptionally high levels. The two heparin/heparan sulfate recognition domains are potentially attractive targets for the development of heparanase inhibitors.

Decreased expression of heparanase in glioblastoma multiforme

The authors investigated the presence of endoglycosidase heparanase in human glioblastoma multiforme (GBM) and metastatic brain tumors as well as in healthy brain tissue to explore the relationship between the biological characteristics of GBM and the role of heparanase. Heparanase messenger (m)RNA was almost undetectable in GBMs in vivo, whereas it was frequently seen in metastatic brain tumors according to results of reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical analysis of paraffin-embedded tissue sections showed that neoplastic cells in metastatic brain tumors, especially in cells that invaded blood vessels, exhibit intense heparanase immunoreactivity. Heparanase was present in two highly invasive glioma cell lines, U87MG and U251MG, in vitro. These cell lines did not have metastatic capability, which was tested in an experimental pulmonary metastases model in mice. The activity of heparanase in these cell lines was almost the same as that in the highly metastatic melanoma cell line B16-F1. After nude mice were inoculated with U87MG cells, however, heparanase was no longer detected in subcutaneous or intracerebral experimental glioma in vivo based on results of immunohistochemical analysis. According to results of real-time quantitative PCR, there was a 10-fold increase in heparanase mRNA in U87MG glioma cells in vitro compared with that in experimental U87MG glioma tissue in vivo in nude mice. These results indicate that the expression of heparanase was downregulated in GBM in vivo, which rarely metastasizes to distant organs outside the central nervous system. Heparanase is not implicated in the invasiveness of GBM to surrounding healthy brain tissue in vivo.

Heparanase expression in hepatocellular carcinoma and the cirrhotic liver

The endoglycosidase heparanase plays an important role in tumor cell invasion, angiogenesis and metastasis. In this study, we analyzed heparanase expression in primary hepatocellular carcinoma (HCC), liver cirrhosis and normal liver tissues to further evaluate its role in tumor pathogenesis. Heparanase mRNA expression was measured by real-time quantitative RT-PCR and localized at the cellular level by in situ hybridization. Heparanase protein levels and its localization were determined by Western blotting and immunohistochemistry. Expression of heparanase mRNA in primary HCC was increased 2-fold compared with liver cirrhosis and 2.2-fold in comparison with normal liver tissues, and this overexpression was even more pronounced in advanced stage HCC. In contrast, heparanase expression levels between cirrhotic tissues and normal liver tissues were not significantly different. In HCC the increased heparanase expression was localized in hepatic tumor cells and was only weakly present or absent in normal hepatocytes, bile duct epithelial cells and the connective tissues. These results suggest that increased heparanase expression is involved in the pathogenesis and progression of HCC. Its specific up-regulation in HCC but not in liver cirrhosis indicates that it might be used as a molecular marker for the differentiation of these disorders.

Purification and Characterization of the Endoglycosidase Heparanase 1 from Human Plantar Stratum Corneum: a Key Enzyme in Epidermal Physiology?

A protein exhibiting endoglycosidase activity was purified from plantar stratum corneum to apparent homogeneity in two sequential column chromatographic steps. Protein sequencing revealed its identity with the recently cloned human heparanase 1, an enzyme, the expression of which is reported to be related to the metastasic potential of tumor cells. By using a heparanase 1 specific antibody we were able to demonstrate that, in the plantar stratum corneum, heparanase 1 exists in two forms, the active 50 kDa protein and the inactive 63 kDa form, probably a proform of the enzyme. The antibody also decorated numerous degradation fragments. Reverse transcription polymerase chain reaction studies as well as immunohistochemical analysis using reconstructed and normal human epidermis demonstrated clearly a keratinocyte differentiation related expression of heparanase 1. Interestingly, the antibody also strongly decorated dendritic cells, which after double labeling could be identified to be a subpopulation of the epidermal Langerhans cells. Based on our findings and the known history of this enzyme, we advanced the hypothesis that heparanase 1 has multiple physiologic functions in the epidermis: (i) it plays an important role in epidermal differentiation, possibly by modulating the liberation of heparan sulfate bound (growth) factors; (ii) in the stratum corneum, the endoglycosidase activity of heparanase 1 might be indispensable and represent the first step in the desquamation process; and (iii) in Langerhans cells, its catalytic activity is required for the trans-tissue migration of these cells.

The human endo-glycosidase heparanase is over-expressed in primary and metastatic pancreatic cancer

The human endo-glycosidase heparanase is over-expressed in primary and metastatic pancreatic cancer

The human endo-glycosidase heparanase is over-expressed in primary and metastatic pancreatic cancer

The human endo-glycosidase heparanase is over-expressed in primary and metastatic pancreatic cancer

Molecular properties and involvement of heparanase in cancer progression and mammary gland morphogenesis.

Tumor spread involves degradation of various components of the extracellular matrix and blood vessel wall. Among these is heparan sulfate proteoglycan, which plays a key role in the self-assembly, insolubility and barrier properties of basement membranes and extracellular matrices. Expression of an endoglycosidase (heparanase) which degrades heparan sulfate correlates with the metastatic potential of tumor cells, and treatment with heparanase inhibitors markedly reduces the incidence of metastasis in experimental animals. Heparin-binding angiogenic proteins are stored as a complex with heparan sulfate in the microenvironment of tumors. These proteins are released and can induce new capillary growth when heparan sulfate is degraded by heparanase. Here, we describe the molecular properties, expression and involvement in tumor progression of a human heparanase. The enzyme is synthesized as a latent approximately 65 kDa protein that is processed at the N-terminus into a highly active approximately 50 kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic human cell lines and in tumor biopsy specimens, including breast carcinoma. Overexpression of the heparanase cDNA in low-metastatic tumor cells conferred a high metastatic potential in experimental animals, resulting in an increased rate of mortality. The heparanase enzyme also released ECM-resident bFGF in vitro, and its overexpression elicited an angiogenic response in vivo. Heparanase may thus facilitate both tumor cell invasion and neovascularization, two critical steps in tumor progression. Mammary glands of transgenic mice overexpressing the heparanase enzyme exhibit precocious branching of ducts and alveolar development, suggesting that the enzyme promotes normal morphogenesis and possibly pre-malignant changes in the mammary gland.

Identification of active-site residues of the pro-metastatic endoglycosidase heparanase.

Heparanase is a beta-D-endoglucuronidase that cleaves heparan sulfate (HS) and has been implicated in many important physiological and pathological processes, including tumor cell metastasis, angiogenesis, and leukocyte migration. We report herein the identification of active-site residues of human heparanase. Using PSI-BLAST and PHI-BLAST searches of sequence databases, similarities were identified between heparanase and members of several of the glycosyl hydrolase families (10, 39, and 51) from glycosyl hydrolase clan A (GH-A), including strong local identities to regions containing the critical active-site catalytic proton donor and nucleophile residues that are conserved in this clan of enzymes. Furthermore, secondary structure predictions suggested that heparanase is likely to contain an (alpha/beta)(8) TIM-barrel fold, which is common to the GH-A families. On the basis of sequence alignments with a number of glycosyl hydrolases from GH-A, Glu(225) and Glu(343) of human heparanase were identified as the likely proton donor and nucleophile residues, respectively. The substitution of these residues with alanine and the subsequent expression of the mutant heparanases in COS-7 cells demonstrated that the HS-degrading capacity of both was abolished. In contrast, the alanine substitution of two other glutamic acid residues (Glu(378) and Glu(396)), both predicted to be outside the active site, did not affect heparanase activity. These data suggest that heparanase is a member of the clan A glycosyl hydrolases and has a common catalytic mechanism that involves two conserved acidic residues, a putative proton donor at Glu(225) and a nucleophile at Glu(343).