Heparan Sulfate Turnover Research Articles

Heparanase-A single protein with multiple enzymatic and nonenzymatic functions.

Heparanase (Hpa1) is expressed by tumor cells and cells of the tumor microenvironment and functions extracellularly to remodel the extracellular matrix (ECM) and regulate the bioavailability of ECM-bound factors, augmenting, among other effects, gene transcription, autophagy, exosome formation, and heparan sulfate (HS) turnover. Much of the impact of heparanase on tumor progression is related to its function in mediating tumor-host crosstalk, priming the tumor microenvironment to better support tumor growth, metastasis, and chemoresistance. The enzyme appears to fulfill some normal functions associated, for example, with vesicular traffic, lysosomal-based secretion, autophagy, HS turnover, and gene transcription. It activates cells of the innate immune system, promotes the formation of exosomes and autophagosomes, and stimulates signal transduction pathways via enzymatic and nonenzymatic activities. These effects dynamically impact multiple regulatory pathways that together drive tumor growth, dissemination, and drug resistance as well as inflammatory responses. The emerging premise is that heparanase expressed by tumor cells, immune cells, endothelial cells, and other cells of the tumor microenvironment is a key regulator of the aggressive phenotype of cancer, an important contributor to the poor outcome of cancer patients and a valid target for therapy. So far, however, antiheparanase-based therapy has not been implemented in the clinic. Unlike heparanase, heparanase-2 (Hpa2), a close homolog of heparanase (Hpa1), does not undergo proteolytic processing and hence lacks intrinsic HS-degrading activity, the hallmark of heparanase. Hpa2 retains the capacity to bind heparin/HS and exhibits an even higher affinity towards HS than heparanase, thus competing for HS binding and inhibiting heparanase enzymatic activity. It appears that Hpa2 functions as a natural inhibitor of Hpa1 regulates the expression of selected genes that maintain tissue hemostasis and normal function, and plays a protective role against cancer and inflammation, together emphasizing the significance of maintaining a proper balance between Hpa1 and Hpa2.

The Heparanase Regulatory Network in Health and Disease.

The extracellular matrix (ECM) is a structural framework that has many important physiological functions which include maintaining tissue structure and integrity, serving as a barrier to invading pathogens, and acting as a reservoir for bioactive molecules. This cellular scaffold is made up of various types of macromolecules including heparan sulfate proteoglycans (HSPGs). HSPGs comprise a protein core linked to the complex glycosaminoglycan heparan sulfate (HS), the remodeling of which is important for many physiological processes such as wound healing as well as pathological processes including cancer metastasis. Turnover of HS is tightly regulated by a single enzyme capable of cleaving HS side chains: heparanase. Heparanase upregulation has been identified in many inflammatory diseases including atherosclerosis, fibrosis, and cancer, where it has been shown to play multiple roles in processes such as epithelial-mesenchymal transition, angiogenesis, and cancer metastasis. Heparanase expression and activity are tightly regulated. Understanding the regulation of heparanase and its downstream targets is attractive for the development of treatments for these diseases. This review provides a comprehensive overview of the regulators of heparanase as well as the enzyme’s downstream gene and protein targets, and implications for the development of new therapeutic strategies.

An Overview of the Structure, Mechanism and Specificity of Human Heparanase.

The retaining endo-β-D-glucuronidase Heparanase (HPSE) is the primary mammalian enzyme responsible for breakdown of the glycosaminoglycan heparan sulfate (HS). HPSE activity is essential for regulation and turnover of HS in the extracellular matrix, and its activity affects diverse processes such as inflammation, angiogenesis and cell migration. Aberrant heparanase activity is strongly linked to cancer metastasis, due to structural breakdown of extracellular HS networks and concomitant release of sequestered HS-binding growth factors. A full appreciation of HPSE activity in health and disease requires a structural understanding of the enzyme, and how it engages with its HS substrates. This chapter summarizes key findings from the recent crystal structures of human HPSE and its proenzyme. We present details regarding the 3-dimensional protein structure of HPSE and the molecular basis for its interaction with HS substrates of varying sulfation states. We also examine HPSE in a wider context against related β-D-glucuronidases from other species, highlighting the structural features that control exo/endo - glycosidase selectivity in this family of enzymes.

Molecular Aspects of Heparanase Interaction with Heparan Sulfate, Heparin and Glycol Split Heparin.

Heparanase is the principal enzyme that degrades heparan sulfate (HS) in both physiological (HS turnover) and pathological (tumor metastasis, inflammation) cell conditions, catalysing the hydrolysis of the β-1-4 glycosidic bond in -GlcUA-β(1-4)-GlcNX-. Despite efforts to define the minimum trisaccharide sequence that allows glycans to be recognized by heparanase, a rigorous "molecular code" by which the enzyme reads and degrades HS chains has not been identified. The X-ray diffraction model of heparanase, resolved by Wu etal (2015), revealed a complex between the trisaccharide GlcNS6S-GlcUA-GlcNS6S and heparanase. Efforts are ongoing to better understand how HS mimetics longer than three residues are recognized by heparanase before being hydrolyzed or inhibit the enzyme. It is also important to consider the flexibility of the enzyme active site, a feature that opens up the development of heparanase inhibitors with structures significantly different from HS or heparin. This chapter reviews the state-of-the-art knowledge about structural aspects of heparanase activities in terms of substrate recognition, mechanism of hydrolysis, and inhibition.

Heparanase-1 activity and the early postnatal prostate development.

Ventral prostate (VP) morphogenesis starts during embryonic development and continues for the first three postnatal weeks. Heparan sulfate (HS) affects paracrine signaling. Heparanase-1 (HPSE) is the only enzyme capable of cleaving HS. HPSE releases the HS bioactive fragment and mobilizes growth factors. Little is known, however, about HS turnover and HPSE function during VP morphogenesis. In this study, we measured HSPG expression and analyzed the expression and distribution of HPSE in the rat VP. HPSE was predominantly expressed by the VP epithelium. The VP was treated with heparin in ex vivo cultures to interfere with HS and resulted in delayed epithelial growth. Hpse knockdown using siRNA delayed epithelial growth in the first postnatal week ex vivo, which was similar to treating with the lower concentration of heparin. Hpse silencing was related to changes in HS chain length (as determined by size-exclusion chromatography, up-regulation of Mmp9, and down-regulation of Mmp2 expression). It also down-modulated ERK1/2 phosphorylation, suggesting a reduction in signaling, likely due to decreased HS cleavage and growth factor bioavailability. Our results showed that HPSE played a role in early epithelial growth during the first week of VP postnatal development. Developmental Dynamics 248:211-220, 2019. © 2019 Wiley Periodicals, Inc.

Heparanase: From basic research to therapeutic applications in cancer and inflammation.

Heparanase, the sole heparan sulfate degrading endoglycosidase, regulates multiple biological activities that enhance tumor growth, angiogenesis and metastasis. Heparanase expression is enhanced in almost all cancers examined including various carcinomas, sarcomas and hematological malignancies. Numerous clinical association studies have consistently demonstrated that upregulation of heparanase expression correlates with increased tumor size, tumor angiogenesis, enhanced metastasis and poor prognosis. In contrast, knockdown of heparanase or treatments of tumor-bearing mice with heparanase-inhibiting compounds, markedly attenuate tumor progression further underscoring the potential of anti-heparanase therapy for multiple types of cancer. Heparanase neutralizing monoclonal antibodies block myeloma and lymphoma tumor growth and dissemination; this is attributable to a combined effect on the tumor cells and/or cells of the tumor microenvironment. In fact, much of the impact of heparanase on tumor progression is related to its function in mediating tumor-host crosstalk, priming the tumor microenvironment to better support tumor growth, metastasis and chemoresistance. The repertoire of the physio-pathological activities of heparanase is expanding. Specifically, heparanase regulates gene expression, activates cells of the innate immune system, promotes the formation of exosomes and autophagosomes, and stimulates signal transduction pathways via enzymatic and non-enzymatic activities. These effects dynamically impact multiple regulatory pathways that together drive inflammatory responses, tumor survival, growth, dissemination and drug resistance; but in the same time, may fulfill some normal functions associated, for example, with vesicular traffic, lysosomal-based secretion, stress response, and heparan sulfate turnover. Heparanase is upregulated in response to chemotherapy in cancer patients and the surviving cells acquire chemoresistance, attributed, at least in part, to autophagy. Consequently, heparanase inhibitors used in tandem with chemotherapeutic drugs overcome initial chemoresistance, providing a strong rationale for applying anti-heparanase therapy in combination with conventional anti-cancer drugs. Heparin-like compounds that inhibit heparanase activity are being evaluated in clinical trials for various types of cancer. Heparanase neutralizing monoclonal antibodies are being evaluated in pre-clinical studies, and heparanase-inhibiting small molecules are being developed based on the recently resolved crystal structure of the heparanase protein. Collectively, the emerging premise is that heparanase expressed by tumor cells, innate immune cells, activated endothelial cells as well as other cells of the tumor microenvironment is a master regulator of the aggressive phenotype of cancer, an important contributor to the poor outcome of cancer patients and a prime target for therapy.

Distribution of Heparan Sulfate Oligosaccharides in Murine Mucopolysaccharidosis Type IIIA.

Heparan sulfate (HS) catabolism begins with endo-degradation of the polysaccharide to smaller HS oligosaccharides, followed by the sequential action of exo-enzymes to reduce these oligosaccharides to monosaccharides and inorganic sulfate. In mucopolysaccharidosis type IIIA (MPS IIIA) the exo-enzyme, N-sulfoglucosamine sulfohydrolase, is deficient resulting in an inability to hydrolyze non-reducing end glucosamine N-sulfate esters. Consequently, partially degraded HS oligosaccharides with non-reducing end glucosamine sulfate esters accumulate. We investigated the distribution of these HS oligosaccharides in tissues of a mouse model of MPS IIIA using high performance liquid chromatography electrospray ionization-tandem mass spectrometry. Oligosaccharide levels were compared to total uronic acid (UA), which was used as a measure of total glycosaminoglycan. Ten oligosaccharides, ranging in size from di- to hexasaccharides, were present in all the tissues examined including brain, spleen, lung, heart, liver, kidney and urine. However, the relative levels varied up to 10-fold, suggesting different levels of HS turnover and storage. The relationship between the di- and tetrasaccharides and total UA was tissue specific with spleen and kidney showing a different disaccharide:total UA ratio than the other tissues. The hexasaccharides showed a stronger correlation with total UA in all tissue types suggesting that hexasaccharides may more accurately reflect the storage burden in these tissues.

Heparanase expression and glycosaminoglycans profile in renal cell carcinoma

A better understanding of the molecular mechanisms of renal cell carcinogenesis could contribute to a decrease in the mortality rate of this disease. The aim of this study was to evaluate the glycosaminoglycans profile and heparanase expression in renal cell carcinoma. The study included 24 patients submitted to nephrectomy with confirmed pathological diagnosis of renal cell carcinoma. The majority of the samples (87.5%) were classified in the initial stage of renal cell carcinoma (clinical stages I and II). Heparanase messenger ribonucleic acid expression was evaluated by quantitative real-time reverse transcription polymerase chain reaction, and sulfated glycosaminoglycans were identified and quantified by agarose gel electrophoresis of renal cell carcinoma samples or non-neoplastic tissues obtained from the same patients (control group). The sulfated glycosaminoglycans and hyaluronic acid were analyzed in urine samples of the patients before and after surgery. The data showed a significant statistical increase in chondroitin sulfate, and a decrease in heparan sulfate and dermatan sulfate present in neoplastic tissues compared with non-neoplastic tissues. Higher heparanase messenger ribonucleic acid expression in the neoplastic tissues was also shown, compared with the non-neoplastic tissues. The urine glycosaminoglycans profile showed no significant difference between renal cell carcinoma and control samples. Extracellular matrix changes observed in the present study clarify that heparanase is possibly involved with heparan sulfate turnover, and that heparanase and the glycosaminoglycans can modulate initial events of renal cell carcinoma development.

Heparan Sulfate-Dependent Signaling of Fibroblast Growth Factor 18 by Chondrocyte-Derived Perlecan

Perlecan is a large multidomain proteoglycan that is essential for normal cartilage development. In this study, perlecan was localized in the pericellular matrix of hypertrophic chondrocytes in developing human cartilage rudiments. Perlecan immunopurified from medium conditioned by cultured human fetal chondrocytes was found to be substituted with heparan sulfate (HS), chondroitin sulfate (CS), and keratan sulfate (KS). Ligand and carbohydrate engagement (LACE) assays demonstrated that immunopurified chondrocyte-derived perlecan formed HS-dependent ternary complexes with fibroblast growth factor (FGF) 2 and either FGF receptors (FGFRs) 1 or 3; however, these complexes were not biologically active in the BaF32 cell system. Chondrocyte-derived perlecan also formed HS-dependent ternary complexes with FGF18 and FGFR3. The proliferation of BaF32 cells expressing FGFR3 was promoted by chondrocyte-derived perlecan in the presence of FGF18, and this activity was reduced by digestion of the HS with either heparinase III or mammalian heparanase. These data suggest that FGF2 and -18 bind to discrete structures on the HS chains attached to chondrocyte-derived perlecan which modulate the growth factor activities. The presence and activity of mammalian heparanase may be important in the turnover of HS and subsequent signaling required for the establishment and maintenance of functional osteo-chondral junctions in long bone growth.

Heparanase Uptake Is Mediated by Cell Membrane Heparan Sulfate Proteoglycans

Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intrachain sites, an activity that is strongly implicated in cell dissemination associated with metastasis and inflammation. In addition to its structural role in extracellular matrix assembly and integrity, HS sequesters a multitude of polypeptides that reside in the extracellular matrix as a reservoir. A variety of growth factors, cytokines, chemokines, and enzymes can be released by heparanase activity and profoundly affect cell and tissue function. Thus, heparanase bioavailability, accessibility, and activity should be kept tightly regulated. We provide evidence that HS is not only a substrate for, but also a regulator of, heparanase. Addition of heparin or xylosides to cell cultures resulted in a pronounced accumulation of, heparanase in the culture medium, whereas sodium chlorate had no such effect. Moreover, cellular uptake of heparanase was markedly reduced in HS-deficient CHO-745 mutant cells, heparan sulfate proteoglycan-deficient HT-29 colon cancer cells, and heparinase-treated cells. We also studied the heparanase biosynthetic route and found that the half-life of the active enzyme is approximately 30 h. This and previous localization studies suggest that heparanase resides in the endosomal/lysosomal compartment for a relatively long period of time and is likely to play a role in the normal turnover of HS. Co-localization studies and cell fractionation following heparanase addition have identified syndecan family members as candidate molecules responsible for heparanase uptake, providing an efficient mechanism that limits extracellular accumulation and function of heparanase.

Human Heparanase Is Localized within Lysosomes in a Stable Form

Heparanase is an endo-β- d-glucuronidase involved in degradation of heparan sulfate (HS) and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues. The enzymatic activity of heparanase is characterized by specific intrachain cleavage of glycosidic bonds with a hydrolase mechanism. This enzyme facilitates cell invasion and hence plays a role in tumor metastasis, angiogenesis, inflammation, and autoimmunity. Although the expression pattern and molecular properties of heparanase have been characterized, its subcellular localization has not been unequivocally determined. We have previously suggested that heparanase subcellular localization is a major determinant in regulating the enzyme's biological functions. In the present study we examined heparanase localization in three different cell types, utilizing immunofluorescent staining and electron microscopy. Our results indicate that heparanase is localized primarily within lysosomes and the Golgi apparatus. A construct composed of heparanase cDNA fused to green fluorescent protein, utilized in order to visualize the enzyme within living cells, confirmed its localization in acidic vesicles. We suggest that following synthesis, heparanase is transported into the Golgi apparatus and subsequently accumulates in a stable form within the lysosomes, where it functions in HS turnover. The lysosomal compartment may also serve as a site for heparanase confinement within the cells, limiting its secretion and uncontrolled extracellular activities associated with tumor metastasis and angiogenesis.

Human Heparanase Is Localized within Lysosomes in a Stable Form

Heparanase is an endo-β- d -glucuronidase involved in degradation of heparan sulfate (HS) and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues. The enzymatic activity of heparanase is characterized by specific intrachain cleavage of glycosidic bonds with a hydrolase mechanism. This enzyme facilitates cell invasion and hence plays a role in tumor metastasis, angiogenesis, inflammation, and autoimmunity. Although the expression pattern and molecular properties of heparanase have been characterized, its subcellular localization has not been unequivocally determined. We have previously suggested that heparanase subcellular localization is a major determinant in regulating the enzyme's biological functions. In the present study we examined heparanase localization in three different cell types, utilizing immunofluorescent staining and electron microscopy. Our results indicate that heparanase is localized primarily within lysosomes and the Golgi apparatus. A construct composed of heparanase cDNA fused to green fluorescent protein, utilized in order to visualize the enzyme within living cells, confirmed its localization in acidic vesicles. We suggest that following synthesis, heparanase is transported into the Golgi apparatus and subsequently accumulates in a stable form within the lysosomes, where it functions in HS turnover. The lysosomal compartment may also serve as a site for heparanase confinement within the cells, limiting its secretion and uncontrolled extracellular activities associated with tumor metastasis and angiogenesis.