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Roy Bicknell - One of the best experts on this subject based on the ideXlab platform.
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proteomics based screening of the endothelial Heparan sulfate interactome reveals that c type lectin 14a clec14a is a heparin binding protein
Journal of Biological Chemistry, 2020Co-Authors: Daniel R Sandoval, Chelsea D Painter, Ember M Tota, Osman M Sheikh, Alan Mv West, Alejandro Gomez Toledo, Lance Wells, Ding Xu, Martin Frank, Roy BicknellAbstract:Author(s): Sandoval, Daniel R; Gomez Toledo, Alejandro; Painter, Chelsea D; Tota, Ember M; Sheikh, M Osman; West, Alan MV; Frank, Martin M; Wells, Lance; Xu, Ding; Bicknell, Roy; Corbett, Kevin D; Esko, Jeffrey D | Abstract: Animal cells express Heparan sulfate proteoglycans that perform many important cellular functions by way of Heparan sulfate-protein interactions. The identification of membrane Heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind Heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial Heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.
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proteomics based screening of the endothelial Heparan sulfate interactome reveals that c type lectin 14a clec14a is a heparin binding protein
Journal of Biological Chemistry, 2020Co-Authors: Daniel R Sandoval, Chelsea D Painter, Ember M Tota, Osman M Sheikh, Alan Mv West, Alejandro Gomez Toledo, Lance Wells, Ding Xu, Martin Frank, Roy BicknellAbstract:Animal cells express Heparan sulfate proteoglycans that perform many important cellular functions by way of Heparan sulfate-protein interactions. The identification of membrane Heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind Heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial Heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.
Robert J Linhardt - One of the best experts on this subject based on the ideXlab platform.
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Bioengineered heparins and Heparan sulfates
Advanced Drug Delivery Reviews, 2015Co-Authors: Li Fu, Matthew Suflita, Robert J LinhardtAbstract:Heparin and Heparan sulfates are closely related linear anionic polysaccharides, called glycosaminoglycans, which exhibit a number of important biological and pharmacological activities. These polysaccharides, having complex structures and polydispersity, are biosynthesized in the Golgi of animal cells. While Heparan sulfate is a widely distributed membrane and extracellular glycosaminoglycan, heparin is found primarily intracellularly in the granules of mast cells. While heparin has historically received most of the scientific attention for its anticoagulant activity, interest has steadily grown in the multi-faceted role Heparan sulfate plays in normal and pathophysiology. The chemical synthesis of these glycosaminoglycans is largely precluded by their structural complexity. Today, we depend on livestock animal tissues for the isolation and the annual commercial production of hundred ton quantities of heparin used in the manufacture of anticoagulant drugs and medical device coatings. The variability of animal-sourced heparin and Heparan sulfates, their inherent impurities, the limited availability of source tissues, the poor control of these source materials and their manufacturing processes, suggest a need for new approaches for their production. Over the past decade there have been major efforts in the biotechnological production of these glycosaminoglycans, driven by both therapeutic applications and as probes to study their natural functions. This review focuses on the complex biology of these glycosaminoglycans in human health and disease, and the use of recombinant technology in the chemoenzymatic synthesis and metabolic engineering of heparin and Heparan sulfates.
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chemoenzymatic synthesis of Heparan sulfate and heparin
Natural Product Reports, 2014Co-Authors: Robert J LinhardtAbstract:Covering: up to May 2014 Heparan sulfate is a polysaccharide that plays essential physiological functions in the animal kingdom. Heparin, a highly sulfated form of Heparan sulfate, is a widely prescribed anticoagulant drug worldwide. The Heparan sulfate and heparin isolated from natural sources are highly heterogeneous mixtures differing in their polysaccharide chain lengths and sulfation patterns. The access to structurally defined Heparan sulfate and heparin is critical to probe the contribution of specific sulfated saccharide structures to the biological functions as well as for the development of the next generation of heparin-based anticoagulant drugs. The synthesis of Heparan sulfate and heparin, using a purely chemical approach, has proven extremely difficult, especially for targets larger than octasaccharides having a high degree of site-specific sulfation. A new chemoenzymatic method has emerged as an effective alternative approach. This method uses recombinant Heparan sulfate biosynthetic enzymes combined with unnatural uridine diphosphate-monosaccharide donors. Recent examples demonstrate the successful synthesis of ultra-low molecular weight heparin, low-molecular weight heparin and bioengineered heparin with unprecedented efficiency. The new method provides an opportunity to develop improved heparin-based therapeutics.
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Hyphenated techniques for the analysis of heparin and Heparan sulfate
Analytical and Bioanalytical Chemistry, 2011Co-Authors: Bo Yang, Kemal Solakyildirim, Yuqing Chang, Robert J LinhardtAbstract:The elucidation of the structure of glycosaminoglycan has proven to be challenging for analytical chemists. Molecules of glycosaminoglycan have a high negative charge and are polydisperse and microheterogeneous, thus requiring the application of multiple analytical techniques and methods. Heparin and Heparan sulfate are the most structurally complex of the glycosaminoglycans and are widely distributed in nature. They play critical roles in physiological and pathophysiological processes through their interaction with heparin-binding proteins. Moreover, heparin and low-molecular weight heparin are currently used as pharmaceutical drugs to control blood coagulation. In 2008, the health crisis resulting from the contamination of pharmaceutical heparin led to considerable attention regarding their analysis and structural characterization. Modern analytical techniques, including high-performance liquid chromatography, capillary electrophoresis, mass spectrometry, and nuclear magnetic resonance spectroscopy, played critical roles in this effort. A successful combination of separation and spectral techniques will clearly provide a critical advantage in the future analysis of heparin and Heparan sulfate. This review focuses on recent efforts to develop hyphenated techniques for the analysis of heparin and Heparan sulfate.
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Physiological, Pathophysiological and Therapeutic Roles of Heparin and Heparan Sulfate
Carbohydrate Chemistry Biology and Medical Applications, 2008Co-Authors: Saravanababu Murugesan, Robert J LinhardtAbstract:Publisher Summary Heparin and Heparan sulfate belong to the glycosaminoglycan (GAG) family of carbohydrates. They are linear acidic complex polysaccharides found on the cell surface and in the extracellular matrix. Heparin and Heparan sulfate GAGs are biosynthesized as proteoglycans (PGs) with multiple GAG chains linked to a variety of core proteins. Heparin PGs are found exclusively in the granules of subsets of mast cells, whereas Heparan sulfate PGs have a much greater distribution in the body, being associated with stromal matrices, basement membranes, and almost all cell surfaces. Heparin and Heparan sulfate PGs interact with cell surface binding proteins and are internalized by receptor-mediated endocytosis through their GAG chains. Heparin and Heparan sulfate are the most intensively studied GAGs as a result of their anticoagulant properties. However, it has become obvious that heparin and Heparan sulfate not only have anticoagulant activities but also exhibit a number of diverse biological functions including ones regulating cell growth and differentiation, inflammatory processes, host defense and viral infection mechanisms, cell–cell and cell–matrix interactions, lipid transport, and clearance/metabolism. These functions result from the direct interactions between heparin and Heparan sulfate and heparin-binding proteins.
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Structural characterization of human liver Heparan sulfate.
Biochimica et Biophysica Acta, 2005Co-Authors: Preeyanat Vongchan, Mohamad Warda, Hidenao Toyoda, Toshihiko Toida, Rory M Marks, Robert J LinhardtAbstract:The isolation, purification and structural characterization of human liver Heparan sulfate are described. 1 H-NMR spectroscopy demonstrates the purity of this glycosaminoglycan (GAG) and two-dimensional 1 H-NMR confirmed that it was Heparan sulfate. Enzymatic depolymerization of the isolated Heparan sulfate, followed by gradient polyacrylamide gel, confirmed its heparin lyase sensitivity. The concentration of resulting unsaturated disaccharides was determined using reverse phase ion-pairing (RPIP) HPLC with post column derivatization and fluorescence detection. The results of this analysis clearly demonstrate that the isolated GAG was Heparan sulfate, not heparin. Human liver Heparan sulfate was similar to heparin in that it has a reduced content of unsulfated disaccharide and an elevated average sulfation level. The antithrombin-mediated anti-factor Xa activity of human liver Heparan sulfate, however, was much lower than porcine intestinal (pharmaceutical) heparin but was comparable to standard porcine intestinal Heparan sulfate. Moreover, human liver Heparan sulfate shows higher degree of sulfation than Heparan sulfate isolated from porcine liver or from the human hepatoma Hep 2G cell line. D 2004 Elsevier B.V. All rights reserved.
Ding Xu - One of the best experts on this subject based on the ideXlab platform.
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proteomics based screening of the endothelial Heparan sulfate interactome reveals that c type lectin 14a clec14a is a heparin binding protein
Journal of Biological Chemistry, 2020Co-Authors: Daniel R Sandoval, Chelsea D Painter, Ember M Tota, Osman M Sheikh, Alan Mv West, Alejandro Gomez Toledo, Lance Wells, Ding Xu, Martin Frank, Roy BicknellAbstract:Author(s): Sandoval, Daniel R; Gomez Toledo, Alejandro; Painter, Chelsea D; Tota, Ember M; Sheikh, M Osman; West, Alan MV; Frank, Martin M; Wells, Lance; Xu, Ding; Bicknell, Roy; Corbett, Kevin D; Esko, Jeffrey D | Abstract: Animal cells express Heparan sulfate proteoglycans that perform many important cellular functions by way of Heparan sulfate-protein interactions. The identification of membrane Heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind Heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial Heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.
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proteomics based screening of the endothelial Heparan sulfate interactome reveals that c type lectin 14a clec14a is a heparin binding protein
Journal of Biological Chemistry, 2020Co-Authors: Daniel R Sandoval, Chelsea D Painter, Ember M Tota, Osman M Sheikh, Alan Mv West, Alejandro Gomez Toledo, Lance Wells, Ding Xu, Martin Frank, Roy BicknellAbstract:Animal cells express Heparan sulfate proteoglycans that perform many important cellular functions by way of Heparan sulfate-protein interactions. The identification of membrane Heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind Heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial Heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.
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demystifying Heparan sulfate protein interactions
Annual Review of Biochemistry, 2014Co-Authors: Ding Xu, Jeffrey D EskoAbstract:Numerous proteins, including cytokines and chemokines, enzymes and enzyme inhibitors, extracellular matrix proteins, and membrane receptors, bind heparin. Although they are traditionally classified as heparin-binding proteins, under normal physiological conditions these proteins actually interact with the Heparan sulfate chains of one or more membrane or extracellular proteoglycans. Thus, they are more appropriately classified as Heparan sulfate-binding proteins (HSBPs). This review provides an overview of the various modes of interaction between Heparan sulfate and HSBPs, emphasizing biochemical and structural insights that improve our understanding of the many biological functions of Heparan sulfate.
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Demystifying Heparan Sulfate–Protein Interactions
Annual Review of Biochemistry, 2014Co-Authors: Ding Xu, Jeffrey D EskoAbstract:Numerous proteins, including cytokines and chemokines, enzymes and enzyme inhibitors, extracellular matrix proteins, and membrane receptors, bind heparin. Although they are traditionally classified as heparin-binding proteins, under normal physiological conditions these proteins actually interact with the Heparan sulfate chains of one or more membrane or extracellular proteoglycans. Thus, they are more appropriately classified as Heparan sulfate–binding proteins (HSBPs). This review provides an overview of the various modes of interaction between Heparan sulfate and HSBPs, emphasizing biochemical and structural insights that improve our understanding of the many biological functions of Heparan sulfate.
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Heparan Sulfate Regulates VEGF165- and VEGF121-mediated Vascular Hyperpermeability
Journal of Biological Chemistry, 2010Co-Authors: Ding Xu, Mark M Fuster, Roger Lawrence, Jeffrey D EskoAbstract:Abstract VEGF was first described as vascular permeability factor, a potent inducer of vascular leakage. Genetic evidence indicates that VEGF-stimulated endothelial proliferation in vitro and angiogenesis in vivo depend on Heparan sulfate, but a requirement for Heparan sulfate in vascular hyperpermeability has not been explored. Here we show that altering endothelial cell Heparan sulfate biosynthesis in vivo decreases hyperpermeability induced by both VEGF165 and VEGF121. Since VEGF121 does not bind Heparan sulfate, the requirement for Heparan sulfate suggested that it interacted with VEGF receptors rather than the ligand. By applying proximity ligation assays to primary brain endothelial cells, we show a direct interaction in situ between Heparan sulfate and the VEGF receptor, VEGFR2. Furthermore, the number of Heparan sulfate/VEGFR2 complexes increased in response to both VEGF165 and VEGF121. Genetic or heparin lyase-mediated alteration of endothelial Heparan sulfate attenuated phosphorylation of VEGFR2 in response to VEGF165 and VEGF121, suggesting that the functional VEGF receptor complex contains Heparan sulfate. Pharmacological blockade of Heparan sulfate-protein interactions inhibited hyperpermeability in vivo, suggesting Heparan sulfate as a potential target for treating hyperpermeability associated with ischemic disease.
Yasuo Suda - One of the best experts on this subject based on the ideXlab platform.
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multiple mechanisms for exogenous heparin modulation of vascular endothelial growth factor activity
Journal of Cellular Biochemistry, 2010Co-Authors: Errol S Wijelath, Mayumi Namekata, Jacqueline Murray, Mai Furuyashiki, Siyuan Zhang, Daniel E Coan, Masahiro Wakao, Robert B Harris, Yasuo SudaAbstract:Heparin and heparin-like molecules are known to modulate the cellular responses to vascular endothelial growth factor-A (VEGF-A). In this study, we investigated the likely mechanisms for heparin's influence on the biological activity of VEGF-A. Previous studies have shown that exogenous heparin's effects on the biological activity of VEGF-A are many and varied, in part due to the endogenous cell-surface Heparan sulfates. To circumvent this problem, we used mutant endothelial cells lacking cell-surface Heparan sulfates. We showed that VEGF-induced cellular responses are dependent in part on the presence of the Heparan sulfates, and that exogenous heparin significantly augments VEGF's cellular effects especially when endogenous Heparan sulfates are absent. Exogenous heparin was also found to play a cross-bridging role between VEGF-A165 and putative heparin-binding sites within its cognate receptor, VEGFR2 when they were examined in isolation. The cross-bridging appears to be more dependent on molecular weight than on a specific heparin structure. This was confirmed by surface plasmon resonance binding studies using sugar chips immobilized with defined oligosaccharide structures, which showed that VEGF-A165 binds to a relatively broad range of sulfated glycosaminoglycan structures. Finally, studies of the far-UV circular dichroism spectra of VEGF-A165 showed that heparin can also modulate the conformation and secondary structure of the protein. J. Cell. Biochem. 111: 461–468, 2010. © 2010 Wiley-Liss, Inc.
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sugar chips immobilized with synthetic sulfated disaccharides of heparin Heparan sulfate partial structure
Bioorganic & Medicinal Chemistry Letters, 2008Co-Authors: Masahiro Wakao, Yasuo Suda, Akihiro Saito, Koh Ohishi, Yuko Kishimoto, Tomoaki Nishimura, Michael SobelAbstract:Abstract Carbohydrate chip technology has a great potential for the high-throughput evaluation of carbohydrate–protein interactions. Herein, we report syntheses of novel sulfated oligosaccharides possessing heparin and Heparan sulfate partial disaccharide structures, their immobilization on gold-coated chips to prepare array-type Sugar Chips, and evaluation of binding potencies of proteins by surface plasmon resonance (SPR) imaging technology. Sulfated oligosaccharides were efficiently synthesized from glucosamine and uronic acid moieties. Synthesized sulfated oligosaccharides were then easily immobilized on gold-coated chips using previously reported methods. The effectiveness of this analytical method was confirmed in binding experiments between the chips and heparin binding proteins, fibronectin and recombinant human von Willebrand factor A1 domain (rh-vWf-A1), where specific partial structures of heparin or Heparan sulfate responsible for binding were identified.
Jeffrey D Esko - One of the best experts on this subject based on the ideXlab platform.
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demystifying Heparan sulfate protein interactions
Annual Review of Biochemistry, 2014Co-Authors: Ding Xu, Jeffrey D EskoAbstract:Numerous proteins, including cytokines and chemokines, enzymes and enzyme inhibitors, extracellular matrix proteins, and membrane receptors, bind heparin. Although they are traditionally classified as heparin-binding proteins, under normal physiological conditions these proteins actually interact with the Heparan sulfate chains of one or more membrane or extracellular proteoglycans. Thus, they are more appropriately classified as Heparan sulfate-binding proteins (HSBPs). This review provides an overview of the various modes of interaction between Heparan sulfate and HSBPs, emphasizing biochemical and structural insights that improve our understanding of the many biological functions of Heparan sulfate.
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Demystifying Heparan Sulfate–Protein Interactions
Annual Review of Biochemistry, 2014Co-Authors: Ding Xu, Jeffrey D EskoAbstract:Numerous proteins, including cytokines and chemokines, enzymes and enzyme inhibitors, extracellular matrix proteins, and membrane receptors, bind heparin. Although they are traditionally classified as heparin-binding proteins, under normal physiological conditions these proteins actually interact with the Heparan sulfate chains of one or more membrane or extracellular proteoglycans. Thus, they are more appropriately classified as Heparan sulfate–binding proteins (HSBPs). This review provides an overview of the various modes of interaction between Heparan sulfate and HSBPs, emphasizing biochemical and structural insights that improve our understanding of the many biological functions of Heparan sulfate.
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Heparan Sulfate Regulates VEGF165- and VEGF121-mediated Vascular Hyperpermeability
Journal of Biological Chemistry, 2010Co-Authors: Ding Xu, Mark M Fuster, Roger Lawrence, Jeffrey D EskoAbstract:Abstract VEGF was first described as vascular permeability factor, a potent inducer of vascular leakage. Genetic evidence indicates that VEGF-stimulated endothelial proliferation in vitro and angiogenesis in vivo depend on Heparan sulfate, but a requirement for Heparan sulfate in vascular hyperpermeability has not been explored. Here we show that altering endothelial cell Heparan sulfate biosynthesis in vivo decreases hyperpermeability induced by both VEGF165 and VEGF121. Since VEGF121 does not bind Heparan sulfate, the requirement for Heparan sulfate suggested that it interacted with VEGF receptors rather than the ligand. By applying proximity ligation assays to primary brain endothelial cells, we show a direct interaction in situ between Heparan sulfate and the VEGF receptor, VEGFR2. Furthermore, the number of Heparan sulfate/VEGFR2 complexes increased in response to both VEGF165 and VEGF121. Genetic or heparin lyase-mediated alteration of endothelial Heparan sulfate attenuated phosphorylation of VEGFR2 in response to VEGF165 and VEGF121, suggesting that the functional VEGF receptor complex contains Heparan sulfate. Pharmacological blockade of Heparan sulfate-protein interactions inhibited hyperpermeability in vivo, suggesting Heparan sulfate as a potential target for treating hyperpermeability associated with ischemic disease.
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surfen a small molecule antagonist of Heparan sulfate
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Manuela Schuksz, Mark M Fuster, Jillian R Brown, Brett E Crawford, David Ditto, Roger Lawrence, Charles A Glass, Lianchun Wang, Jeffrey D EskoAbstract:In a search for small molecule antagonists of Heparan sulfate, we examined the activity of bis-2-methyl-4-amino-quinolyl-6-carbamide, also known as surfen. Fluorescence-based titrations indicated that surfen bound to glycosaminoglycans, and the extent of binding increased according to charge density in the order heparin > dermatan sulfate > Heparan sulfate > chondroitin sulfate. All charged groups in heparin (N-sulfates, O-sulfates, and carboxyl groups) contributed to binding, consistent with the idea that surfen interacted electrostatically. Surfen neutralized the anticoagulant activity of both unfractionated and low molecular weight heparins and inhibited enzymatic sulfation and degradation reactions in vitro. Addition of surfen to cultured cells blocked FGF2-binding and signaling that depended on cell surface Heparan sulfate and prevented both FGF2- and VEGF165-mediated sprouting of endothelial cells in Matrigel. Surfen also blocked Heparan sulfate-mediated cell adhesion to the Hep-II domain of fibronectin and prevented infection by HSV-1 that depended on glycoprotein D interaction with Heparan sulfate. These findings demonstrate the feasibility of identifying small molecule antagonists of Heparan sulfate and raise the possibility of developing pharmacological agents to treat disorders that involve glycosaminoglycan–protein interactions.
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Cell Surface Heparan Sulfate Promotes Replication of Toxoplasma gondii
Infection and Immunity, 2005Co-Authors: Joseph R. Bishop, Brett E Crawford, Jeffrey D EskoAbstract:Previous work suggests that cell surface Heparan sulfate acts as a receptor for the Apicomplexan parasite Toxoplasma gondii. Using Chinese hamster ovary cell mutants defective in Heparan sulfate biosynthesis, we show that Heparan sulfate is necessary and sufficient for infectivity. Further, we demonstrate that the parasite requires N sulfation of Heparan sulfate initiated by N-deacetylase/N-sulfotransferase-1, but 2-O sulfation and 6-O sulfation appear to be dispensable. In order to study the role of Heparan sulfate in other cell types, we created a conditional allele for N-deacetylase/N-sulfotransferase-1 by using Cre-loxP technology. Mammary tumor cells lacking N-deacetylase/N-sulfotransferase-1 exhibited reduced toxoplasma infectivity like Chinese hamster ovary cell mutants. Surprisingly, heparin, chemically modified heparinoids, and monoclonal antibodies to Heparan sulfate had no effect on toxoplasma infection. T. gondii attachment and invasion were unchanged in N-deacetylase/N-sulfotransferase-1-inactivated cells as well, but replication was reduced. Thus, Heparan sulfate does not appear to function as a receptor for T. gondii but instead facilitates parasite replication postinvasion.