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1.
Cell ; 183(4): 1043-1057.e15, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-32970989

RESUMO

We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD). Docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ACE2-binding site. Both ACE2 and heparin can bind independently to spike protein in vitro, and a ternary complex can be generated using heparin as a scaffold. Electron micrographs of spike protein suggests that heparin enhances the open conformation of the RBD that binds ACE2. On cells, spike protein binding depends on both heparan sulfate and ACE2. Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. We suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities.


Assuntos
Betacoronavirus/fisiologia , Heparitina Sulfato/metabolismo , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Linhagem Celular , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Heparina/química , Heparina/metabolismo , Heparitina Sulfato/química , Humanos , Rim/metabolismo , Pulmão/metabolismo , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus
2.
PLoS Pathog ; 16(9): e1008828, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32991636

RESUMO

Field isolates of foot-and-mouth disease viruses (FMDVs) utilize integrin-mediated cell entry but many, including Southern African Territories (SAT) viruses, are difficult to adapt to BHK-21 cells, thus hampering large-scale propagation of vaccine antigen. However, FMDVs acquire the ability to bind to cell surface heparan sulphate proteoglycans, following serial cytolytic infections in cell culture, likely by the selection of rapidly replicating FMDV variants. In this study, fourteen SAT1 and SAT2 viruses, serially passaged in BHK-21 cells, were virulent in CHO-K1 cells and displayed enhanced affinity for heparan, as opposed to their low-passage counterparts. Comparative sequence analysis revealed the fixation of positively charged residues clustered close to the icosahedral 5-fold axes of the virus, at amino acid positions 83-85 in the ßD-ßE loop and 110-112 in the ßF-ßG loop of VP1 upon adaptation to cultured cells. Molecular docking simulations confirmed enhanced binding of heparan sulphate to a model of the adapted SAT1 virus, with the region around VP1 arginine 112 contributing the most to binding. Using this information, eight chimeric field strain mutant viruses were constructed with additional positive charges in repeated clusters on the virion surface. Five of these bound heparan sulphate with expanded cell tropism, which should facilitate large-scale propagation. However, only positively charged residues at position 110-112 of VP1 enhanced infectivity of BHK-21 cells. The symmetrical arrangement of even a single amino acid residue in the FMD virion is a powerful strategy enabling the virus to generate novel receptor binding and alternative host-cell interactions.


Assuntos
Vírus da Febre Aftosa/genética , Febre Aftosa/virologia , Modelos Moleculares , Vírion/metabolismo , Animais , Proteínas do Capsídeo/metabolismo , Cricetinae , Heparitina Sulfato/metabolismo , Simulação de Acoplamento Molecular/métodos , Receptores Virais/metabolismo
3.
Biochim Biophys Acta Gen Subj ; 1864(12): 129707, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32810562

RESUMO

BACKGROUND: Heparan sulfate (HS) is a sulfated linear polysaccharide on cell surfaces that plays an important role in physiological processes. HS is present in skeletal muscles but its detailed role in this tissue remains unclear. METHODS: We examined the role of HS in the differentiation of C2C12 cells, a mouse myoblast cell line. We also phenotyped the impact of HS deletion in mouse skeletal muscles on their functions by using Cre-loxP system. RESULTS: CRISPR-Cas9-dependent HS deletion or pharmacological removal of HS dramatically impaired myoblast differentiation of C2C12 cells. To confirm the importance of HS in vivo, we deleted Ext1, which encodes an enzyme essential for HS biosynthesis, specifically in the mouse skeletal muscles (referred to as mExt1CKO mice). Treadmill and wire hang tests demonstrated that mExt1CKO mice exhibited muscle weakness. The contraction of isolated soleus muscles from mExt1CKO mice was also impaired. Morphological examination of mExt1CKO muscle tissue under light and electron microscopes revealed smaller cross sectional areas and thinner myofibrils. Finally, a model of muscle regeneration following BaCl2 injection into the tibialis anterior muscle of mice demonstrated that mExt1CKO mice had reduced expression of myosin heavy chain and an increased number of centronucleated cells. This indicates that muscle regeneration after injury was attenuated in the absence of HS expression in muscle cells. SIGNIFICANCE: These results demonstrate that HS plays an important role in skeletal muscle function by promoting differentiation.


Assuntos
Heparitina Sulfato/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/fisiologia , Mioblastos/citologia , Animais , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Heparitina Sulfato/antagonistas & inibidores , Heparitina Sulfato/genética , Camundongos , Atividade Motora , Músculo Esquelético/citologia , Mioblastos/metabolismo
4.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1450-1458, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748603

RESUMO

Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.


Assuntos
Carboidratos Epimerases/biossíntese , Carboidratos Epimerases/química , Heparina/metabolismo , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/química , Animais , Carboidratos Epimerases/genética , Escherichia coli , Expressão Gênica , Heparitina Sulfato/metabolismo , Ácido Idurônico/metabolismo , Proteínas de Peixe-Zebra/genética
5.
Proc Natl Acad Sci U S A ; 117(29): 17187-17194, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32636266

RESUMO

Osteoprotegerin (OPG), a secreted decoy receptor for receptor activator of nuclear factor B ligand (RANKL), plays an essential role in regulating bone resorption. While much is known about the function of the N-terminal domains of OPG, which is responsible for binding to RANKL, the exact biological functions of the three C-terminal domains of OPG remain uncertain. We have previously shown that one likely function of the C-terminal domains of OPG is to bind cell surface heparan sulfate (HS), but the in vivo evidence was lacking. To investigate the biological significance of OPG-HS interaction in bone remodeling, we created OPG knock-in mice (opg AAA ). The mutated OPG is incapable of binding to HS but binds RANKL normally. Surprisingly, opg AAA/AAA mice displayed a severe osteoporotic phenotype that is very similar to opg-null mice, suggesting that the antiresorption activity of OPG requires HS. Mechanistically, we propose that the HS immobilizes secreted OPG at the surface of osteoblasts lineage cells, which facilitates binding of OPG to membrane-anchored RANKL. To further support this model, we altered the structure of osteoblast HS genetically to make it incapable of binding to OPG. Interestingly, osteocalcin-Cre;Hs2st f/f mice also displayed osteoporotic phenotype with similar severity to opg AAA/AAA mice. Combined, our data provide strong genetic evidence that OPG-HS interaction is indispensable for normal bone homeostasis.


Assuntos
Conservadores da Densidade Óssea/metabolismo , Conservadores da Densidade Óssea/farmacologia , Heparitina Sulfato/metabolismo , Osteoprotegerina/metabolismo , Osteoprotegerina/farmacologia , Animais , Sítios de Ligação , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Osso e Ossos/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , Osteogênese/fisiologia , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/patologia , Osteoprotegerina/genética , Ligante RANK/metabolismo , Transcriptoma
6.
Arterioscler Thromb Vasc Biol ; 40(9): e240-e255, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32698687

RESUMO

OBJECTIVE: To determine if endothelial dysfunction in a mouse model of diet-induced obesity and in obese humans is mediated by the suppression of endothelial Kir (inwardly rectifying K+) channels. Approach and Results: Endothelial dysfunction, observed as reduced dilations to flow, occurred after feeding mice a high-fat, Western diet for 8 weeks. The functional downregulation of endothelial Kir2.1 using dominant-negative Kir2.1 construct resulted in substantial reductions in the response to flow in mesenteric arteries of lean mice, whereas no effect was observed in arteries of obese mice. Overexpressing wild-type-Kir2.1 in endothelium of arteries from obese mice resulted in full recovery of the flow response. Exposing freshly isolated endothelial cells to fluid shear during patch-clamp electrophysiology revealed that the flow-sensitivity of Kir was virtually abolished in cells from obese mice. Atomic force microscopy revealed that the endothelial glycocalyx was stiffer and the thickness of the glycocalyx layer reduced in arteries from obese mice. We also identified that the length of the glycocalyx is critical to the flow-activation of Kir. Overexpressing Kir2.1 in endothelium of arteries from obese mice restored flow- and heparanase-sensitivity, indicating an important role for heparan sulfates in the flow-activation of Kir. Furthermore, the Kir2.1-dependent component of flow-induced vasodilation was lost in the endothelium of resistance arteries of obese humans obtained from biopsies collected during bariatric surgery. CONCLUSIONS: We conclude that obesity-induced impairment of flow-induced vasodilation is attributed to the loss of flow-sensitivity of endothelial Kir channels and propose that the latter is mediated by the biophysical alterations of the glycocalyx.


Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Glicocálix/metabolismo , Artérias Mesentéricas/metabolismo , Obesidade/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Vasodilatação , Adulto , Animais , Células Cultivadas , Dieta Hiperlipídica , Modelos Animais de Doenças , Endotélio Vascular/fisiopatologia , Feminino , Heparitina Sulfato/metabolismo , Humanos , Masculino , Mecanotransdução Celular , Potenciais da Membrana , Artérias Mesentéricas/fisiopatologia , Camundongos , Pessoa de Meia-Idade , Obesidade/genética , Obesidade/fisiopatologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Fluxo Sanguíneo Regional
7.
J Virol ; 94(17)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32522852

RESUMO

Schmallenberg virus (SBV) is an insect-transmitted orthobunyavirus that can cause abortions and congenital malformations in the offspring of ruminants. Even though the two viral surface glycoproteins Gn and Gc are involved in host cell entry, the specific cellular receptors of SBV are currently unknown. Using genome-wide CRISPR-Cas9 forward screening, we identified 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter 1 (PAPST1) as an essential factor for SBV infection. PAPST1 is a sulfotransferase involved in heparan sulfate proteoglycan synthesis encoded by the solute carrier family 35 member B2 gene (SLC35B2). SBV cell surface attachment and entry were largely reduced upon the knockout of SLC35B2, whereas the reconstitution of SLC35B2 in these cells fully restored their susceptibility to SBV infection. Furthermore, treatment of cells with heparinase diminished infection with SBV, confirming that heparan sulfate plays an important role in cell attachment and entry, although to various degrees, heparan sulfate was also found to be important to initiate infection by two other bunyaviruses, La Crosse virus and Rift Valley fever virus. Thus, PAPST1-triggered synthesis of cell surface heparan sulfate is required for the efficient replication of SBV and other bunyaviruses.IMPORTANCE SBV is a newly emerging orthobunyavirus (family Peribunyaviridae) that has spread rapidly across Europe since 2011, resulting in substantial economic losses in livestock farming. In this study, we performed unbiased genome-wide CRISPR-Cas9 screening and identified PAPST1, a sulfotransferase encoded by SLC35B2, as a host entry factor for SBV. Consistent with its role in the synthesis of heparan sulfate, we show that this activity is required for efficient infection by SBV. A comparable dependency on heparan sulfate was also observed for La Crosse virus and Rift Valley fever virus, highlighting the importance of heparan sulfate for host cell infection by bunyaviruses. Thus, the present work provides crucial insights into virus-host interactions of important animal and human pathogens.


Assuntos
Infecções por Bunyaviridae/genética , Infecções por Bunyaviridae/virologia , Sistemas CRISPR-Cas , Orthobunyavirus/genética , Orthobunyavirus/fisiologia , Animais , Bunyaviridae , Chlorocebus aethiops , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Europa (Continente) , Técnicas de Inativação de Genes , Células HEK293 , Heparitina Sulfato/metabolismo , Humanos , Gado , Glicoproteínas de Membrana/genética , Orthobunyavirus/patogenicidade , Vírus da Febre do Vale do Rift , Transportadores de Sulfato/metabolismo , Sulfotransferases/metabolismo , Células Vero , Ligação Viral
8.
Cell Mol Life Sci ; 77(24): 5059-5077, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32462405

RESUMO

Heparanase (HPSE) is a multifunctional protein endowed with many non-enzymatic functions and a unique enzymatic activity as an endo-ß-D-glucuronidase. The latter allows it to serve as a key modulator of extracellular matrix (ECM) via a well-regulated cleavage of heparan sulfate side chains of proteoglycans at cell surfaces. The cleavage and associated changes at the ECM cause release of multiple signaling molecules with important cellular and pathological functions. New and emerging data suggest that both enzymatic as well as non-enzymatic functions of HPSE are important for health and illnesses including viral infections and virally induced cancers. This review summarizes recent findings on the roles of HPSE in activation, inhibition, or bioavailability of key signaling molecules such as AKT, VEGF, MAPK-ERK, and EGFR, which are known regulators of common viral infections in immune and non-immune cell types. Altogether, our review provides a unique overview of HPSE in cell-survival signaling pathways and how they relate to viral infections.


Assuntos
Glucuronidase/genética , Neoplasias/genética , Viroses/genética , Matriz Extracelular/genética , Glucuronidase/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Imunidade Celular/genética , Neoplasias/patologia , Neoplasias/virologia , Transdução de Sinais/genética , Viroses/imunologia , Viroses/virologia
9.
Adv Exp Med Biol ; 1245: 133-146, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32266656

RESUMO

The tumor microenvironment (TME) is rich in matrix components, growth factors, cytokines, and enzymatic modifiers that respond to changing conditions, to alter the fundamental properties of the tumor bed. Perlecan/HSPG2, a large, multi-domain heparan sulfate proteoglycan, is concentrated in the reactive stroma that surrounds tumors. Depending on its state in the TME, perlecan can either prevent or promote the progression of cancers to metastatic disease. Breast, prostate, lung, and renal cancers all preferentially metastasize to bone, a dense, perlecan-rich environment that is initially a "hostile" niche for cancer cells. Driven by inflammation, production of perlecan and its enzyme modifiers, which include matrix metalloproteinases (MMPs), sulfatases (SULFs), and heparanase (HPSE), increases in the reactive stroma surrounding growing and invading tumors. MMPs act upon the perlecan core protein, releasing bioactive fragments of the protein, primarily from C-terminal domains IV and V. These fragments influence cell adhesion, invasion, and angiogenesis. Sulfatases and heparanases act directly upon the heparan sulfate chains, releasing growth factors from reservoirs to reach receptors on the cancer cell surface. We propose that perlecan modifiers, by promoting the degradation of the perlecan-rich stroma, "flip the molecular switch" and convert the "hostile" stroma into a welcoming one that supports cancer dissemination and metastasis. Targeted therapies that prevent this molecular conversion of the TME should be considered as potential new therapeutics to limit metastasis.


Assuntos
Proteoglicanas de Heparan Sulfato/metabolismo , Neoplasias/metabolismo , Microambiente Tumoral , Proteínas da Matriz Extracelular/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/patologia
10.
Adv Exp Med Biol ; 1245: 147-161, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32266657

RESUMO

The biology of tumor cells strictly depends on their microenvironment architecture and composition, which controls the availability of growth factors and signaling molecules. Thus, the network of glycosaminoglycans, proteoglycans, and proteins known as extracellular matrix (ECM) that surrounds the cells plays a central role in the regulation of tumor fate. Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) are highly versatile ECM components that bind and regulate the activity of growth factors, cell membrane receptors, and other ECM molecules. These HS binding partners modulate cell adhesion, motility, and proliferation that are processes altered during tumor progression. Modification in the expression and activity of HS, HSPGs, and the respective metabolic enzymes results unavoidably in alteration of tumor cell microenvironment. In this light, the targeting of HS structure and metabolism is potentially a new tool in the treatment of different cancer types.


Assuntos
Heparitina Sulfato , Neoplasias , Microambiente Tumoral , Matriz Extracelular/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia
11.
Proc Natl Acad Sci U S A ; 117(17): 9311-9317, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32277030

RESUMO

Heparin is the most widely prescribed biopharmaceutical in production globally. Its potent anticoagulant activity and safety makes it the drug of choice for preventing deep vein thrombosis and pulmonary embolism. In 2008, adulterated material was introduced into the heparin supply chain, resulting in several hundred deaths and demonstrating the need for alternate sources of heparin. Heparin is a fractionated form of heparan sulfate derived from animal sources, predominantly from connective tissue mast cells in pig mucosa. While the enzymes involved in heparin biosynthesis are identical to those for heparan sulfate, the factors regulating these enzymes are not understood. Examination of the promoter regions of all genes involved in heparin/heparan sulfate assembly uncovered a transcription factor-binding motif for ZNF263, a C2H2 zinc finger protein. CRISPR-mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to dramatically increased expression levels of HS3ST1, a key enzyme involved in imparting anticoagulant activity to heparin, and HS3ST3A1, another glucosaminyl 3-O-sulfotransferase expressed in cells. Enhanced 3-O-sulfation increased binding to antithrombin, which enhanced Factor Xa inhibition, and binding of neuropilin-1. Analysis of transcriptomics data showed distinctively low expression of ZNF263 in mast cells compared with other (non-heparin-producing) immune cells. These findings demonstrate a novel regulatory factor in heparan sulfate modification that could further advance the possibility of bioengineering anticoagulant heparin in cultured cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Heparina/metabolismo , Heparitina Sulfato/biossíntese , Animais , Anticoagulantes , Linhagem Celular , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Regulação da Expressão Gênica/genética , Células HeLa , Heparina/biossíntese , Heparina/genética , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Humanos , Mastócitos/metabolismo , Sulfotransferases/metabolismo , Suínos , Fatores de Transcrição
12.
Adv Exp Med Biol ; 1221: 61-69, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274706

RESUMO

Heparanase was discovered during a study of the heparin proteoglycan (serglycin) in mast cells. Newly synthesized polysaccharide chains, kDa 60-100 x 103, were rapidly degraded to fragments similar in size to commercially available heparin (averaging 15 x 103). Analysis of the degradation products identified reducing-terminal glucuronic acid residues, shown by studies of heparin biosynthesis to be of ßD-configuration in the intact polymer. Heparanase, thus identified as an endo-ßD-glucuronidase, was subsequently identified in a variety of tissues and cells. The enzyme was subsequently implicated with a variety of pathophysiological processes, including in particular cancer, inflammatory diseases, and amyloidosis, as detailed in subsequent chapters of this volume. The target for enzyme action in these settings is primarily extracellular heparan sulfate proteoglycans; furthermore, intracellular cleavage initiates degradation of heparan sulfate chains by exolytic hydrolases and sulfatases, as part of normal turnover of the polysaccharide. More unexpectedly, heparanase also influences heparan sulfate biosynthesis, such that overexpression of the enzyme results in generation of highly sulfated, heparin-like oligosaccharides. The mechanism behind this effect remains unclear - along with the overall design of the molecular machinery in control of proteoglycan biosynthesis.


Assuntos
Glucuronidase/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Oligossacarídeos/metabolismo , Especificidade por Substrato
13.
Adv Exp Med Biol ; 1221: 71-96, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274707

RESUMO

Heparanase is an endo-ß-glucuronidase that cleaves at a limited number of internal sites the glycosaminoglycan heparan sulfate (HS). Heparanase enzymatic activity was first reported in 1975 and by 1983 evidence was beginning to emerge that the enzyme was a facilitator of tumor metastasis by cleaving HS chains present in blood vessel basement membranes and, thereby, aiding the passage of tumor cells through blood vessel walls. Due to a range of technical difficulties, it took another 16 years before heparanase was cloned and characterized in 1999 and a further 14 years before the crystal structure of the enzyme was solved. Despite these substantial deficiencies, there was steady progress in our understanding of heparanase long before the enzyme was fully characterized. For example, it was found as early as 1984 that activated T cells upregulate heparanase expression, like metastatic tumor cells, and the enzyme aids the entry of T cells and other leukocytes into inflammatory sites. Furthermore, it was discovered in 1989 that heparanase releases pre-existing growth factors and cytokines associated with HS in the extracellular matrix (ECM), the liberated growth factors/cytokines enhancing angiogenesis and wound healing. There were also the first hints that heparanase may have functions other than enzymatic activity, in 1995 it being reported that under certain conditions the enzyme could act as a cell adhesion molecule. Also, in the same year PI-88 (Muparfostat), the first heparanase inhibitor to reach and successfully complete a Phase III clinical trial was patented.Nevertheless, the cloning of heparanase (also known as heparanase-1) in 1999 gave the field an enormous boost and some surprises. The biggest surprise was that there is only one heparanase encoding gene in the mammalian genome, despite earlier research, based on substrate specificity, suggesting that there are at least three different heparanases. This surprising conclusion has remained unchanged for the last 20 years. It also became evident that heparanase is a family 79 glycoside hydrolase that is initially produced as a pro-enzyme that needs to be processed by proteases to form an enzymatically active heterodimer. A related molecule, heparanase-2, was also discovered that is enzymatically inactive but, remarkably, recently has been shown to inhibit heparanase-1 activity as well as acting as a tumor suppressor that counteracts many of the pro-tumor properties of heparanase-1.The early claim that heparanase plays a key role in tumor metastasis, angiogenesis and inflammation has been confirmed by many studies over the last 20 years. In fact, heparanase expression is enhanced in all major cancer types, namely carcinomas, sarcomas, and hematological malignancies, and correlates with increased metastasis and poor prognosis. Also, there is mounting evidence that heparanase plays a central role in the induction of inflammation-associated cancers. The enzymatic activity of heparanase has also emerged in unexpected situations, such as in the spread of HS-binding viruses and in Type-1 diabetes where the destruction of intracellular HS in pancreatic insulin-producing beta cells precipitates diabetes. But the most extraordinary recent discoveries have been with the realization that heparanase can exert a range of biological activities that are independent of its enzymatic function, most notably activation of several signaling pathways and being a transcription factor that controls methylation of histone tails. Collectively, these data indicate that heparanase is a truly multifunctional protein that has the additional property of cleaving HS chains and releasing from ECM and cell surfaces hundreds of HS-binding proteins with a plethora of functional consequences. Clearly, there are many unique features of this intriguing molecule that still remain to be explored and are highlighted in this Chapter.


Assuntos
Glucuronidase/história , Glucuronidase/metabolismo , Animais , Glucuronidase/genética , Heparitina Sulfato/metabolismo , História do Século XX , História do Século XXI , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/enzimologia , Neoplasias/patologia , Neovascularização Patológica
14.
Adv Exp Med Biol ; 1221: 97-135, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274708

RESUMO

The cell surface heparan sulfate proteoglycan Syndecan-1 acts as an important co-receptor for receptor tyrosine kinases and chemokine receptors, and as an adhesion receptor for structural glycoproteins of the extracellular matrix. It serves as a substrate for heparanase, an endo-ß-glucuronidase that degrades specific domains of heparan sulfate carbohydrate chains and thereby alters the functional status of the proteoglycan and of Syndecan-1-bound ligands. Syndecan-1 and heparanase show multiple levels of functional interactions, resulting in mutual regulation of their expression, processing, and activity. These interactions are of particular relevance in the context of inflammation and malignant disease. Studies in animal models have revealed a mechanistic role of Syndecan-1 and heparanase in the regulation of contact allergies, kidney inflammation, multiple sclerosis, inflammatory bowel disease, and inflammation-associated tumorigenesis. Moreover, functional interactions between Syndecan-1 and heparanase modulate virtually all steps of tumor progression as defined in the Hallmarks of Cancer. Due to their prognostic value in cancer, and their mechanistic involvement in tumor progression, Syndecan-1 and heparanase have emerged as important drug targets. Data in preclinical models and preclinical phase I/II studies have already yielded promising results that provide a translational perspective.


Assuntos
Glucuronidase , Inflamação , Neoplasias , Sindecana-1 , Animais , Heparitina Sulfato/metabolismo , Humanos , Inflamação/enzimologia , Inflamação/metabolismo , Neoplasias/enzimologia , Neoplasias/metabolismo
15.
Adv Exp Med Biol ; 1221: 169-188, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274710

RESUMO

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 ß-1-4 glycosidic bond in -GlcUA-ß(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 et al (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.


Assuntos
Glucuronidase , Glicóis , Heparina , Heparitina Sulfato , Glucuronidase/antagonistas & inibidores , Glucuronidase/química , Glucuronidase/metabolismo , Glicóis/química , Glicóis/metabolismo , Heparina/química , Heparina/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Humanos , Hidrólise , Especificidade por Substrato
16.
Adv Exp Med Biol ; 1221: 365-403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274718

RESUMO

Brain tumors are aggressive and devastating diseases. The most common type of brain tumor, glioblastoma (GBM), is incurable and has one of the worst five-year survival rates of all human cancers. GBMs are invasive and infiltrate healthy brain tissue, which is one main reason they remain fatal despite resection, since cells that have already migrated away lead to rapid regrowth of the tumor. Curative therapy for medulloblastoma (MB), the most common pediatric brain tumor, has improved, but the outcome is still poor for many patients, and treatment causes long-term complications. Recent advances in the classification of pediatric brain tumors reveal distinct subgroups, allowing more targeted therapy for the most aggressive forms, and sparing children with less malignant tumors the side-effects of massive treatment. Heparan sulfate proteoglycans (HSPGs), main components of the neurogenic niche, interact specifically with a large number of physiologically important molecules and vital roles for HS biosynthesis and degradation in neural stem cell differentiation have been presented. HSPGs are composed of a core protein with attached highly charged, sulfated disaccharide chains. The major enzyme that degrades HS is heparanase (HPSE), an important regulator of extracellular matrix (ECM) remodeling which has been suggested to promote the growth and invasion of other types of tumors. This is of clinical interest because GBM are highly invasive and children with metastatic MB at the time of diagnosis exhibit a worse outcome. Here we review the involvement of HS and HPSE in development of the nervous system and some of its most malignant brain tumors, glioblastoma and medulloblastoma.


Assuntos
Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Glucuronidase/metabolismo , Heparitina Sulfato/metabolismo , Glioblastoma/enzimologia , Glioblastoma/patologia , Proteoglicanas de Heparan Sulfato , Humanos , Meduloblastoma/enzimologia , Meduloblastoma/patologia
17.
Adv Exp Med Biol ; 1221: 493-522, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274724

RESUMO

The chapter will review early and more recent seminal contributions to the discovery and characterization of heparanase and non-anticoagulant heparins inhibiting its peculiar enzymatic activity. Indeed, heparanase displays a unique versatility in degrading heparan sulfate chains of several proteoglycans expressed in all mammalian cells. This endo-ß-D-glucuronidase is overexpressed in cancer, inflammation, diabetes, atherosclerosis, nephropathies and other pathologies. Starting from known low- or non-anticoagulant heparins, the search for heparanase inhibitors evolved focusing on structure-activity relationship studies and taking advantage of new chemical-physical analytical methods which have allowed characterization and sequencing of polysaccharide chains. New methods to screen heparanase inhibitors and to evaluate their mechanism of action and in vivo activity in experimental models prompted their development. New non-anticoagulant heparin derivatives endowed with anti-heparanase activity are reported. Some leads are under clinical evaluation in the oncology field (e.g., acute myeloid leukemia, multiple myeloma, pancreatic carcinoma) and in other pathological conditions (e.g., sickle cell disease, malaria, labor arrest).


Assuntos
Glucuronidase/antagonistas & inibidores , Heparina/análogos & derivados , Heparina/farmacologia , Animais , Glucuronidase/metabolismo , Heparina/química , Heparitina Sulfato/metabolismo , Humanos , Neoplasias/tratamento farmacológico
18.
Adv Exp Med Biol ; 1221: 631-645, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274729

RESUMO

Amyloidosis refers to a group of diseases characterized by abnormal deposition of denatured endogenous proteins, termed amyloid, in the affected organs. Analysis of biopsy and autopsy tissues from patients revealed the presence of heparan sulfate proteoglycans (HSPGs) along with amyloid proteins in the deposits. For a long time, HSPGs were believed to occur in the deposits as an innocent bystander. Yet, the consistent presence of HSPGs in various deposits, regardless of the amyloid species, led to the hypothesis that these macromolecular glycoconjugates might play functional roles in the pathological process of amyloidosis. In vitro studies have revealed that HSPGs, or more precisely, the heparan sulfate (HS) side chains interact with amyloid peptides, thus promoting amyloid fibrillization. Although information on the mechanisms of HS participation in amyloid deposition is limited, recent studies involving a transgenic mouse model of Alzheimer's disease point to an active role of HS in amyloid formation. Heparanase cleavage alters the molecular structure of HS, and thus modulates the functional roles of HS in homeostasis, as well as in diseases, including amyloidosis. The heparanase transgenic mice have provided models for unveiling the effects of heparanase, through cleavage of HS, in various amyloidosis conditions.


Assuntos
Amiloidose/metabolismo , Glucuronidase/metabolismo , Heparitina Sulfato/metabolismo , Doença de Alzheimer/enzimologia , Doença de Alzheimer/metabolismo , Amiloidose/enzimologia , Animais , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato/química , Humanos
19.
Adv Exp Med Biol ; 1221: 759-770, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274736

RESUMO

The story of heparanase (HPSE) in viral infection has roots in the longstanding connection between heparan sulfate (HS) and a large number of viruses. As a major viral attachment and entry receptor present on the cell surface, HS serves as the first point of contact between a virus particle and its target host cell. Likewise, direct regulation of HS levels on the cell surface by HPSE enzymatic activity dictates the extent of virus release after replication has occurred. Additionally, virus-induced HPSE activation and nuclear translocation results in higher expression of pro-inflammatory factors and delayed wound healing leading to worsened disease. In this chapter, using herpes simplex virus (HSV) as a prototype virus we provide a brief synopsis of important stages in viral infection, describe how these processes are governed by HS and HPSE, and discuss the recent discoveries that designate HPSE as a major host virulence factor and driver of pathogenesis for several different viruses.


Assuntos
Glucuronidase/metabolismo , Heparitina Sulfato/metabolismo , Simplexvirus/patogenicidade , Viroses/metabolismo , Viroses/virologia , Humanos , Viroses/enzimologia , Liberação de Vírus , Replicação Viral
20.
Adv Exp Med Biol ; 1221: 787-805, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274738

RESUMO

From 1999-2003, Oxford GlycoSciences (OGS) ran a successful drug discovery oncology programme to discover small molecule inhibitors of the Heparanase I enzyme (HPSE1). HPSE1 at the time was widely regarded as being the sole mammalian enzyme capable of cleaving Heparan Sulfate (HS). A second family protein member however called Heparanase 2 (HPSE2) including splice forms was subsequently discovered by PCR analysis based on EST sequences. HPSE2 was found to be expressed mainly in smooth muscle containing tissues, particularly bladder and brain. HPSE2 is poorly expressed in haematopoietic cells and placenta which contrasts with the HPSE1 distribution pattern. HPSE2 binds more strongly to HS than HPSE1 and is believed to out compete for substrate binding and so in effect act as a tumor suppressor. So far, all attempts to show specific HPSE2 endoglycosidase activity against HS have failed suggesting that the enzyme may act as a pseudoenzyme that has evolved to retain only certain non-catalytic heparanase like functions. A breakthrough in the elucidation of functional roles for HPSE2 came about in 2010 with the linkage of HPSE2 gene deletions and mutations to the development of Ochoa/Urofacial Syndrome. Future work into the mechanistic analysis of HPSE2's role in signalling, tumor suppression and bladder/nerve functioning are needed to fully explore the role of this family of proteins.


Assuntos
Clonagem Molecular , Glucuronidase/genética , Animais , Facies , Glucuronidase/classificação , Glucuronidase/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Síndrome , Doenças Urológicas/genética
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