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Robert J. Linhardt - One of the best experts on this subject based on the ideXlab platform.
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filter entrapment enrichment pull down assay for glycosaminoglycan structural characterization and protein interaction
Carbohydrate Polymers, 2020Co-Authors: Fuming Zhang, Gina Renoispredelus, Jonathan I Amster, Robert J. LinhardtAbstract:Abstract Heparins are the most pharmaceutically important polysaccharides. These Heparin-based anticoagulant/antithrombotic agents include unfractionated Heparins, low molecular weight Heparins (LMWHs) and ultralow molecular weight Heparins (ULMWHs). Heparins exhibit their pharmacological and biological activities through interaction with Heparin-binding proteins. The prototypical Heparin-binding protein is antithrombin III (AT), responsible for Heparin’s anticoagulant/antithrombotic activity. This study describes a filter-trapping method to isolate the chains in enoxaparin, a LMWH, which bind to AT. We demonstrate this method using the ULMWH, fondaparinux, which consists of a single well defined AT binding site. The interacting chains of enoxaparin are then characterized by activity assays, top-down liquid chromatography-mass spectrometry, and capillary zone electrophoresis mass spectrometry. This filter-trapping assay is an improvement over affinity chromatography for isolating Heparin chains interacting with Heparin binding proteins.
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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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Heparin-protein interactions.
Angewandte Chemie (International ed. in English), 2002Co-Authors: Ishan Capila, Robert J. LinhardtAbstract:Heparin, a sulfated polysaccharide belonging to the family of glycosaminoglycans, has numerous important biological activities, associated with its interaction with diverse proteins. Heparin is widely used as an anticoagulant drug based on its ability to accelerate the rate at which antithrombin inhibits serine proteases in the blood coagulation cascade. Heparin and the structurally related heparan sulfate are complex linear polymers comprised of a mixture of chains of different length, having variable sequences. Heparan sulfate is ubiquitously distributed on the surfaces of animal cells and in the extracellular matrix. It also mediates various physiologic and pathophysiologic processes. Difficulties in evaluating the role of Heparin and heparan sulfate in vivo may be partly ascribed to ignorance of the detailed structure and sequence of these polysaccharides. In addition, the understanding of carbohydrate-protein interactions has lagged behind that of the more thoroughly studied protein-protein and protein-nucleic acid interactions. The recent extensive studies on the structural, kinetic, and thermodynamic aspects of the protein binding of Heparin and heparan sulfate have led to an improved understanding of Heparin-protein interactions. A high degree of specificity could be identified in many of these interactions. An understanding of these interactions at the molecular level is of fundamental importance in the design of new highly specific therapeutic agents. This review focuses on aspects of Heparin structure and conformation, which are important for its interactions with proteins. It also describes the interaction of Heparin and heparan sulfate with selected families of Heparin-binding proteins.
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measurement of the antithrombin iii binding sites in low molecular weight Heparins by 13c nmr and capillary electrophoresis
Pharmacy and Pharmacology Communications, 1995Co-Authors: Umesh R Desai, Jawed Fareed, Debra Hoppensteadt, Robert J. LinhardtAbstract:Low molecular weight Heparins, prepared from the controlled chemical or enzymatic depolymerization of the Heparin polysaccharide, are currently replacing Heparin as the clinical anticoagulant/antithrombotic agent of choice. A principal mechanism of action of these agents is through the binding of antithrombin III, a plasma serine protease inhibitor, to specific pentasaccharide sequences in these polysaccharides. The content of antithrombin III-binding pentasaccharide sequences within low molecular weight Heparins vary, making these drugs bio-inequivalent. Currently, the only way to examine the content of these pentasaccharide sequences within a low molecular weight Heparin preparation relies on bioassay. This study examined both 13C NMR spectroscopy and oligosaccharide analysis by capillary electrophoresis as alternative measures of the content of antithrombin III binding sites within a variety of low molecular weight Heparins. The number of antithrombin III binding sites per chain, measured by 13C NMR spectroscopy, correlated with the antithrombin III mediated anti-factor Xa activity of various low molecular weight Heparins.
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.
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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.
Daniel R Sandoval - One of the best experts on this subject based on the ideXlab platform.
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sars cov 2 infection depends on cellular heparan sulfate and ace2
Cell, 2020Co-Authors: Daniel R Sandoval, Thomas Mandel Clausen, Charlotte B Spliid, Jessica PihlAbstract:Summary 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.
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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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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.
Fuming Zhang - One of the best experts on this subject based on the ideXlab platform.
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filter entrapment enrichment pull down assay for glycosaminoglycan structural characterization and protein interaction
Carbohydrate Polymers, 2020Co-Authors: Fuming Zhang, Gina Renoispredelus, Jonathan I Amster, Robert J. LinhardtAbstract:Abstract Heparins are the most pharmaceutically important polysaccharides. These Heparin-based anticoagulant/antithrombotic agents include unfractionated Heparins, low molecular weight Heparins (LMWHs) and ultralow molecular weight Heparins (ULMWHs). Heparins exhibit their pharmacological and biological activities through interaction with Heparin-binding proteins. The prototypical Heparin-binding protein is antithrombin III (AT), responsible for Heparin’s anticoagulant/antithrombotic activity. This study describes a filter-trapping method to isolate the chains in enoxaparin, a LMWH, which bind to AT. We demonstrate this method using the ULMWH, fondaparinux, which consists of a single well defined AT binding site. The interacting chains of enoxaparin are then characterized by activity assays, top-down liquid chromatography-mass spectrometry, and capillary zone electrophoresis mass spectrometry. This filter-trapping assay is an improvement over affinity chromatography for isolating Heparin chains interacting with Heparin binding proteins.
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analysis of Heparins derived from bovine tissues and comparison to porcine intestinal Heparins
Clinical and Applied Thrombosis-Hemostasis, 2016Co-Authors: Kalib St Ange, Jawed Fareed, Fuming Zhang, Akihiro Onishi, Jonathan S. Dordick, Xiaojun Sun, Lei Lin, Daisuke Mori, Debra Hoppensteadt, Walter JeskeAbstract:Heparin is a widely used clinical anticoagulant. It is also a linear glycosaminoglycan with an average mass between 10 and 20 kDa and is primarily made up of trisulfated disaccharides comprised of 1,4-linked iduronic acid and glucosamine residues containing some glucuronic acid residues. Heparin is biosynthesized in the Golgi of mast cells commonly found in the liver, intestines, and lungs. Pharmaceutical Heparin currently used in the United States is primarily extracted from porcine intestines. Other sources of Heparin including bovine intestine and bovine lung are being examined as potential substitutes for porcine intestinal Heparin. These additional sources are intended to serve to diversify the Heparin supply, making this lifesaving drug more secure. The current study examines bovine Heparins prepared from both intestines and lung and compares these to porcine intestinal Heparin. The structural properties of these Heparins are examined using nuclear magnetic resonance, gel permeation chromatography, ...
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Analysis of 3-O-sulfo group-containing Heparin tetrasaccharides in Heparin by liquid chromatography-mass spectrometry.
Analytical biochemistry, 2014Co-Authors: Bo Yang, Fuming Zhang, Changhu XueAbstract:Abstract Complete Heparin digestion with Heparin lyase 2 affords a mixture of disaccharides and resistant tetrasaccharides with 3- O -sulfo group-containing glucosamine residues at their reducing ends. Quantitative online liquid chromatography–mass spectrometric analysis of these resistant tetrasaccharides is described in this article. The disaccharide and tetrasaccharide compositions of seven porcine intestinal Heparins and five low-molecular-weight Heparins were analyzed by this method. These resistant tetrasaccharides account for from 5.3 to 7.3 wt% of Heparin and from 6.2 to 8.3 wt% of low-molecular-weight Heparin. Because these tetrasaccharides are derived from Heparin’s antithrombin III-binding sites, we examined whether this method could be applied to estimate the anticoagulant activity of Heparin. The content of 3- O -sulfo group-containing tetrasaccharides in a Heparin correlated positively ( r = 0.8294) to Heparin’s anticoagulant activity.
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structural characterization of pharmaceutical Heparins prepared from different animal tissues
Journal of Pharmaceutical Sciences, 2013Co-Authors: Bo Yang, Akihiro Onishi, Peilong Sun, Fuming ZhangAbstract:Although most pharmaceutical Heparin used today is obtained from porcine intestine, Heparin has historically been prepared from bovine lung and ovine intestine. There is some regulatory concern about establishing the species origin of Heparin. This concern began with the outbreak of mad cow disease in the 1990s and was exacerbated during the Heparin shortage in the 2000s and the Heparin contamination crisis of 2007-2008. Three Heparins from porcine, ovine, and bovine were characterized through state-of-the-art carbohydrate analysis methods with a view profiling their physicochemical properties. Differences in molecular weight, monosaccharide and disaccharide composition, oligosaccharide sequence, and antithrombin III-binding affinity were observed. These data provide some insight into the variability of Heparins obtained from these three species and suggest some analytical approaches that may be useful in confirming the species origin of a Heparin active pharmaceutical ingredient.
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structural characterization of Heparins from different commercial sources
Analytical and Bioanalytical Chemistry, 2011Co-Authors: Fuming Zhang, Bo Yang, Zhongping Xiao, Kemal Solakyildirim, Zhenyu Wang, Julie M Beaudet, Amanda Y Torelli, Jonathan S. DordickAbstract:Seven commercial Heparin active pharmaceutical ingredients and one commercial low molecular weight from different manufacturers were characterized with a view profiling their physicochemical properties. All Heparins had similar molecular weight properties as determined by polyacrylamide gel electrophoresis (MN, 10–11 kDa; MW, 13–14 kDa; polydispersity (PD), 1.3–1.4) and by size exclusion chromatography (MN, 14–16 kDa; MW, 21–25 kDa; PD, 1.4–1.6). one-dimensional 1H- and 13C-nuclear magnetic resonance (NMR) evaluation of the Heparin samples was performed, and peaks were fully assigned using two-dimensional NMR. The percentage of glucosamine residues with 3-O-sulfo groups and the percentage of N-sulfo groups and N-acetyl groups ranged from 5.8–7.9%, 78–82%, to 13–14%, respectively. There was substantial variability observed in the disaccharide composition, as determined by high performance liquid chromatography (HPLC)-mass spectral analysis of Heparin lyase I–III digested Heparins. Heparin oligosaccharide mapping was performed using HPLC following separate treatments with Heparin lyase I, II, and III. These maps were useful in qualitatively and quantitatively identifying structural differences between these Heparins. The binding affinities of these Heparins to antithrombin III and thrombin were evaluated by using a surface plasmon resonance competitive binding assay. This study provides the physicochemical and activity characterization necessary for the appropriate design and synthesis of a generic bioengineered Heparin.
Alejandro Gomez Toledo - One of the best experts on this subject based on the ideXlab platform.
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znf263 is a transcriptional regulator of Heparin and heparan sulfate biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Ryan J. Weiss, Alejandro Gomez Toledo, Philipp Spahn, Austin W T Chiang, Benjamin P Kellman, Christopher Benner, Philip L S M Gordts, Nathan E LewisAbstract: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.
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znf263 is a transcriptional regulator of Heparin and heparan sulfate biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Ryan J. Weiss, Alejandro Gomez Toledo, Philipp Spahn, Austin W T Chiang, Benjamin P Kellman, Christopher Benner, Philip L S M Gordts, Jing Li, Christopher K Glass, Nathan E LewisAbstract: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.
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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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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.