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Robert J. Linhardt - One of the best experts on this subject based on the ideXlab platform.
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heparosan chain characterization sequential depolymerization of e coli k5 heparosan by a bacterial eliminase Heparin Lyase iii and a bacterial hydrolase heparanase bp to prepare defined oligomers
Biotechnology Journal, 2021Co-Authors: Payel Datta, Jonathan S. Dordick, Lufeng Yan, Adeola Awofiranye, Robert J. LinhardtAbstract:Heparosan is a non-sulfated polysaccharide and potential applications include, chemoenzymatic synthesis of Heparin and heparan sulfates. Heparosan is produced using microbial cells (natural producers or engineered cells). The characterization of heparosan isolated from both natural producers and engineered-cells are critical steps towards the potential applications of heparosan. Heparosan is characterized using 1) analysis of intact chain size and polydispersity, and 2) disaccharide composition. The current paper describes a novel method for heparosan chain characterization, using Heparin Lyase III (Hep-3, an eliminase from Flavobacterium Heparinum) and heparanase Bp (Hep-Bp, a hydrolase from Burkholderia pseudomallei). The partial digestion of E. coli K5 heparosan with purified His-tagged Hep-3 results in oligomers of defined sizes. The oligomers (degree of polymerization from 2 to 8, DP2-DP8) are completely digested with purified GST-tagged Hep-Bp and analyzed using gel permeation chromatography. Hep-Bp specifically cleaves the linkage between d-glucuronic acid (GlcA) and N-acetyl-d-glucosamine (GlcNAc) but not the linkage between 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid (deltaUA) and GlcNAc, and results in the presence of a minor resistant trisaccharide (GlcNAc-GlcA-GlcNAc). This method successfully demonstrated the substrate selectivity of Hep-BP on heparosan oligomers. This analytical tool could be applied towards heparosan chain mapping and analysis of unnatural sugar moieties in the heparosan chain.
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structural analysis of Heparin derived 3 o sulfated tetrasaccharides antithrombin binding site variants
Journal of Pharmaceutical Sciences, 2017Co-Authors: Yin Chen, Fuming Zhang, Lei Lin, Isaac Agyekum, Xing Zhang, Kalib St Ange, Jian Liu, Jonathan I Amster, Robert J. LinhardtAbstract:Abstract Heparin is a polysaccharide that is widely used as an anticoagulant drug. The mechanism for Heparin’s anticoagulant activity is primarily through its interaction with a serine protease inhibitor, antithrombin III (AT), that enhances its ability to inactivate blood coagulation serine proteases, including thrombin (factor IIa) and factor Xa. The AT-binding site in the Heparin is one of the most well-studied carbohydrate-protein binding sites and its structure is the basis for the synthesis of the Heparin pentasaccharide drug, fondaparinux. Despite our understanding of the structural requirements for the Heparin pentasaccharide AT-binding site, there is a lack of data on the natural variability of these binding sites in Heparins extracted from animal tissues. The present work provides a detailed study on the structural variants of the tetrasaccharide fragments of this binding site afforded following treatment of a Heparin with Heparin Lyase II. The 5 most commonly observed tetrasaccharide fragments of the AT-binding site are fully characterized, and a method for their quantification in Heparin and low-molecular-weight Heparin products is described.
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abnormally high content of free glucosamine residues identified in a preparation of commercially available porcine intestinal heparan sulfate
Analytical Chemistry, 2016Co-Authors: Barbara Mulloy, Robert J. Linhardt, Fuming Zhang, Ten Feizi, Frederick Gyaponquast, Lei Lin, Matthew C Pickering, Wengang ChaiAbstract:Heparan sulfate (HS) polysaccharides are ubiquitous in animal tissues as components of proteoglycans, and they participate in many important biological processes. HS carbohydrate chains are complex and can contain rare structural components such as N-unsubstituted glucosamine (GlcN). Commercially available HS preparations have been invaluable in many types of research activities. In the course of preparing microarrays to include probes derived from HS oligosaccharides, we found an unusually high content of GlcN residue in a recently purchased batch of porcine intestinal mucosal HS. Composition and sequence analysis by mass spectrometry of the oligosaccharides obtained after Heparin Lyase III digestion of the polysaccharide indicated two and three GlcN in the tetrasaccharide and hexasaccharide fractions, respectively. 1H NMR of the intact polysaccharide showed that this unusual batch differed strikingly from other HS preparations obtained from bovine kidney and porcine intestine. The very high content of G...
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Abnormally High Content of Free Glucosamine Residues Identified in a Preparation of Commercially Available Porcine Intestinal Heparan Sulfate
2016Co-Authors: Barbara Mulloy, Robert J. Linhardt, Fuming Zhang, Ten Feizi, Lei Lin, Matthew C Pickering, Frederick Gyapon-quast, Wengang ChaiAbstract:Heparan sulfate (HS) polysaccharides are ubiquitous in animal tissues as components of proteoglycans, and they participate in many important biological processes. HS carbohydrate chains are complex and can contain rare structural components such as N-unsubstituted glucosamine (GlcN). Commercially available HS preparations have been invaluable in many types of research activities. In the course of preparing microarrays to include probes derived from HS oligosaccharides, we found an unusually high content of GlcN residue in a recently purchased batch of porcine intestinal mucosal HS. Composition and sequence analysis by mass spectrometry of the oligosaccharides obtained after Heparin Lyase III digestion of the polysaccharide indicated two and three GlcN in the tetrasaccharide and hexasaccharide fractions, respectively. 1H NMR of the intact polysaccharide showed that this unusual batch differed strikingly from other HS preparations obtained from bovine kidney and porcine intestine. The very high content of GlcN (30%) and low content of GlcNAc (4.2%) determined by disaccharide composition analysis indicated that N-deacetylation and/or N-desulfation may have taken place. HS is widely used by the scientific community to investigate HS structures and activities. Great care has to be taken in drawing conclusions from investigations of structural features of HS and specificities of HS interaction with proteins when commercial HS is used without further analysis. Pending the availability of a validated commercial HS reference preparation, our data may be useful to members of the scientific community who have used the present preparation in their studies
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the responses of hyperglycemic dividing mesangial cells to Heparin are mediated by the non reducing terminal trisaccharide
Journal of Biological Chemistry, 2015Co-Authors: Christina P Wang, Robert J. Linhardt, Fuming Zhang, Vincent C Hascall, Amina Abbadi, Aimin WangAbstract:Our previous studies showed: (i) that growth-arrested G0/G1 rat mesangial cells stimulated to divide in hyperglycemic medium initiate intracellular hyaluronan synthesis that induces autophagy and the cyclin D3-induced formation of a monocyte-adhesive extracellular hyaluronan matrix after completing cell division; and (ii) that Heparin inhibits the intracellular hyaluronan and autophagy responses, but after completing division, induces hyaluronan synthesis at the plasma membrane with the formation of a larger monocyte-adhesive hyaluronan matrix. This study shows: (i) that the non-terminal trisaccharide of Heparin is sufficient to initiate the same responses as intact Heparin, (ii) that a fully sulfated tetrasaccharide isolated from bacterial Heparin Lyase 1 digests of Heparin that contains a Δ-2S-iduronate on the non-reducing end does not initiate the same responses as intact Heparin, and (iii) that removal of the Δ-2S-iduronate to expose the fully sulfated trisaccharide (GlcNS(6S)-IdoUA(2S)-GlcNS(6S)) does initiate the same responses as intact Heparin. These results provide evidence that mammalian heparanase digestion of Heparin and heparan sulfate exposes a cryptic motif on the non-reducing termini that is recognized by a receptor on dividing cells.
Zhongping Xiao - One of the best experts on this subject based on the ideXlab platform.
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Heparinase 1 selectivity for the 3,6-di-O-sulfo-2-deoxy-2-sulfamido-R-D-glucopyranose (1,4) 2-O-sulfo-R-L-idopyranosyluronic acid (GlcNS3S6SIdoA2S) linkages. Glycobiology 2011
2015Co-Authors: Zhongping Xiao, Zhenqing Zhang, Bo Yang, Wenjing Zhao, Huashi Guan, Robert J. LinhardtAbstract:Porcine intestinal mucosa Heparin was partially depolymer-ized by recombinant Heparinase 1 (Heparin Lyase 1, orig-inating from Flavobacterium Heparinum and expressed in Escherichia coli) and then fractionated, leading to the iso-lation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel elec-trophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their struc-tures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600 MHz. Five of the characterized oligosaccharides represent new com-pounds. The most prominent oligosaccharide comprises the common repeating unit of Heparin, ΔUA2S-[-GlcNS6S-IdoA2S-]n-GlcNS6S, where ΔUA is 4-deoxy-α-L-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-D-glucopyranose, IdoA is L-idopyranosyluronic acid, S is sulfate and n = 0–7. A second prominent Heparin oligosac-charide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S]n-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n = 0–5 and GlcA is D-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in Heparin. The promi-nence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the-GlcNS3S6S-IdoA2S- linkage is particularly susceptible to Heparinase 1
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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.
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asparagine 405 of Heparin Lyase ii prevents the cleavage of glycosidic linkages proximate to a 3 o sulfoglucosamine residue
FEBS Letters, 2011Co-Authors: Wenjing Zhao, Bo Yang, Zhongping Xiao, Marieline Garron, Jeffrey D Esko, Miroslaw Cygler, Robert J. LinhardtAbstract:Heparin and heparan sulfate contain a rare 3-O-sulfoglucosamine residue critical for anticoagulation and virus recognition, respectively. The glycosidic linkage proximate to this 3-O-sulfoglucosamine is resistant to cleavage by all Heparin Lyases (Heps). HepII has a broad specificity. The crystal structure of the wild type HepII identified its active site and showed a close spatial proximity between Asn405 and the 3-OH group of the bound glucosamine residue. In this study, we mutated Asn405 to the less sterically demanding Ala405 or Gly405, which broadened the substrate specificity of HepII and caused it to cleave the resistant linkage proximate to the 3-O-sulfoglucosamine residue.
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Heparinase 1 selectivity for the 3 6 di o sulfo 2 deoxy 2 sulfamido α d glucopyranose 1 4 2 o sulfo α l idopyranosyluronic acid glcns3s6s idoa2s linkages
Glycobiology, 2011Co-Authors: Zhongping Xiao, Robert J. Linhardt, Zhenqing Zhang, Bo Yang, Wenjing Zhao, Huashi GuanAbstract:Porcine intestinal mucosa Heparin was partially depolymerized by recombinant Heparinase 1 (Heparin Lyase 1, originating from Flavobacterium Heparinum and expressed in Escherichia coli) and then fractionated, leading to the isolation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel electrophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their structures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600 MHz. Five of the characterized oligosaccharides represent new compounds. The most prominent oligosaccharide comprises the common repeating unit of Heparin, ΔUA2S-[-GlcNS6S-IdoA2S-](n)-GlcNS6S, where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-d-glucopyranose, IdoA is l-idopyranosyluronic acid, S is sulfate and n = 0-7. A second prominent Heparin oligosaccharide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S](n)-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n = 0-5 and GlcA is d-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in Heparin. The prominence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the -GlcNS3S6S-IdoA2S- linkage is particularly susceptible to Heparinase 1.
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Heparinase 1 selectivity for the 3,6-di-O-sulfo-2-deoxy-2-sulfamido--Dglucopyranose(1,4)2-O-sulfo--L-idopyranosyluronic acid (GlcNS3S6SIdoA2S) linkages
2011Co-Authors: Zhongping Xiao, Zhenqing Zhang, Bo Yang, Wenjing Zhao, Huashi Guan, Robert J. LinhardtAbstract:Porcine intestinal mucosa Heparin was partially depolymer-ized by recombinant Heparinase 1 (Heparin Lyase 1, orig-inating from Flavobacterium Heparinum and expressed in Escherichia coli) and then fractionated, leading to the iso-lation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel elec-trophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their struc-tures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600 MHz. Five of the characterized oligosaccharides represent new com-pounds. The most prominent oligosaccharide comprises the common repeating unit of Heparin, ΔUA2S-[-GlcNS6S-IdoA2S-]n-GlcNS6S, where ΔUA is 4-deoxy-α-L-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-D-glucopyranose, IdoA is L-idopyranosyluronic acid, S is sulfate and n = 0–7. A second prominent Heparin oligosac-charide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S]n-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n = 0–5 and GlcA is D-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in Heparin. The promi-nence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the-GlcNS3S6S-IdoA2S- linkage is particularly susceptible to Heparinase 1
Bo Yang - One of the best experts on this subject based on the ideXlab platform.
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Heparinase 1 selectivity for the 3,6-di-O-sulfo-2-deoxy-2-sulfamido-R-D-glucopyranose (1,4) 2-O-sulfo-R-L-idopyranosyluronic acid (GlcNS3S6SIdoA2S) linkages. Glycobiology 2011
2015Co-Authors: Zhongping Xiao, Zhenqing Zhang, Bo Yang, Wenjing Zhao, Huashi Guan, Robert J. LinhardtAbstract:Porcine intestinal mucosa Heparin was partially depolymer-ized by recombinant Heparinase 1 (Heparin Lyase 1, orig-inating from Flavobacterium Heparinum and expressed in Escherichia coli) and then fractionated, leading to the iso-lation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel elec-trophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their struc-tures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600 MHz. Five of the characterized oligosaccharides represent new com-pounds. The most prominent oligosaccharide comprises the common repeating unit of Heparin, ΔUA2S-[-GlcNS6S-IdoA2S-]n-GlcNS6S, where ΔUA is 4-deoxy-α-L-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-D-glucopyranose, IdoA is L-idopyranosyluronic acid, S is sulfate and n = 0–7. A second prominent Heparin oligosac-charide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S]n-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n = 0–5 and GlcA is D-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in Heparin. The promi-nence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the-GlcNS3S6S-IdoA2S- linkage is particularly susceptible to Heparinase 1
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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 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.
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asparagine 405 of Heparin Lyase ii prevents the cleavage of glycosidic linkages proximate to a 3 o sulfoglucosamine residue
FEBS Letters, 2011Co-Authors: Wenjing Zhao, Bo Yang, Zhongping Xiao, Marieline Garron, Jeffrey D Esko, Miroslaw Cygler, Robert J. LinhardtAbstract:Heparin and heparan sulfate contain a rare 3-O-sulfoglucosamine residue critical for anticoagulation and virus recognition, respectively. The glycosidic linkage proximate to this 3-O-sulfoglucosamine is resistant to cleavage by all Heparin Lyases (Heps). HepII has a broad specificity. The crystal structure of the wild type HepII identified its active site and showed a close spatial proximity between Asn405 and the 3-OH group of the bound glucosamine residue. In this study, we mutated Asn405 to the less sterically demanding Ala405 or Gly405, which broadened the substrate specificity of HepII and caused it to cleave the resistant linkage proximate to the 3-O-sulfoglucosamine residue.
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Heparinase 1 selectivity for the 3 6 di o sulfo 2 deoxy 2 sulfamido α d glucopyranose 1 4 2 o sulfo α l idopyranosyluronic acid glcns3s6s idoa2s linkages
Glycobiology, 2011Co-Authors: Zhongping Xiao, Robert J. Linhardt, Zhenqing Zhang, Bo Yang, Wenjing Zhao, Huashi GuanAbstract:Porcine intestinal mucosa Heparin was partially depolymerized by recombinant Heparinase 1 (Heparin Lyase 1, originating from Flavobacterium Heparinum and expressed in Escherichia coli) and then fractionated, leading to the isolation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel electrophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their structures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600 MHz. Five of the characterized oligosaccharides represent new compounds. The most prominent oligosaccharide comprises the common repeating unit of Heparin, ΔUA2S-[-GlcNS6S-IdoA2S-](n)-GlcNS6S, where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-d-glucopyranose, IdoA is l-idopyranosyluronic acid, S is sulfate and n = 0-7. A second prominent Heparin oligosaccharide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S](n)-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n = 0-5 and GlcA is d-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in Heparin. The prominence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the -GlcNS3S6S-IdoA2S- linkage is particularly susceptible to Heparinase 1.
Wenjing Zhao - One of the best experts on this subject based on the ideXlab platform.
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Heparinase 1 selectivity for the 3,6-di-O-sulfo-2-deoxy-2-sulfamido-R-D-glucopyranose (1,4) 2-O-sulfo-R-L-idopyranosyluronic acid (GlcNS3S6SIdoA2S) linkages. Glycobiology 2011
2015Co-Authors: Zhongping Xiao, Zhenqing Zhang, Bo Yang, Wenjing Zhao, Huashi Guan, Robert J. LinhardtAbstract:Porcine intestinal mucosa Heparin was partially depolymer-ized by recombinant Heparinase 1 (Heparin Lyase 1, orig-inating from Flavobacterium Heparinum and expressed in Escherichia coli) and then fractionated, leading to the iso-lation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel elec-trophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their struc-tures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600 MHz. Five of the characterized oligosaccharides represent new com-pounds. The most prominent oligosaccharide comprises the common repeating unit of Heparin, ΔUA2S-[-GlcNS6S-IdoA2S-]n-GlcNS6S, where ΔUA is 4-deoxy-α-L-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-D-glucopyranose, IdoA is L-idopyranosyluronic acid, S is sulfate and n = 0–7. A second prominent Heparin oligosac-charide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S]n-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n = 0–5 and GlcA is D-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in Heparin. The promi-nence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the-GlcNS3S6S-IdoA2S- linkage is particularly susceptible to Heparinase 1
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asparagine 405 of Heparin Lyase ii prevents the cleavage of glycosidic linkages proximate to a 3 o sulfoglucosamine residue
FEBS Letters, 2011Co-Authors: Wenjing Zhao, Bo Yang, Zhongping Xiao, Marieline Garron, Jeffrey D Esko, Miroslaw Cygler, Robert J. LinhardtAbstract:Heparin and heparan sulfate contain a rare 3-O-sulfoglucosamine residue critical for anticoagulation and virus recognition, respectively. The glycosidic linkage proximate to this 3-O-sulfoglucosamine is resistant to cleavage by all Heparin Lyases (Heps). HepII has a broad specificity. The crystal structure of the wild type HepII identified its active site and showed a close spatial proximity between Asn405 and the 3-OH group of the bound glucosamine residue. In this study, we mutated Asn405 to the less sterically demanding Ala405 or Gly405, which broadened the substrate specificity of HepII and caused it to cleave the resistant linkage proximate to the 3-O-sulfoglucosamine residue.
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Heparinase 1 selectivity for the 3 6 di o sulfo 2 deoxy 2 sulfamido α d glucopyranose 1 4 2 o sulfo α l idopyranosyluronic acid glcns3s6s idoa2s linkages
Glycobiology, 2011Co-Authors: Zhongping Xiao, Robert J. Linhardt, Zhenqing Zhang, Bo Yang, Wenjing Zhao, Huashi GuanAbstract:Porcine intestinal mucosa Heparin was partially depolymerized by recombinant Heparinase 1 (Heparin Lyase 1, originating from Flavobacterium Heparinum and expressed in Escherichia coli) and then fractionated, leading to the isolation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel electrophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their structures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600 MHz. Five of the characterized oligosaccharides represent new compounds. The most prominent oligosaccharide comprises the common repeating unit of Heparin, ΔUA2S-[-GlcNS6S-IdoA2S-](n)-GlcNS6S, where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-d-glucopyranose, IdoA is l-idopyranosyluronic acid, S is sulfate and n = 0-7. A second prominent Heparin oligosaccharide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S](n)-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n = 0-5 and GlcA is d-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in Heparin. The prominence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the -GlcNS3S6S-IdoA2S- linkage is particularly susceptible to Heparinase 1.
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Heparinase 1 selectivity for the 3,6-di-O-sulfo-2-deoxy-2-sulfamido--Dglucopyranose(1,4)2-O-sulfo--L-idopyranosyluronic acid (GlcNS3S6SIdoA2S) linkages
2011Co-Authors: Zhongping Xiao, Zhenqing Zhang, Bo Yang, Wenjing Zhao, Huashi Guan, Robert J. LinhardtAbstract:Porcine intestinal mucosa Heparin was partially depolymer-ized by recombinant Heparinase 1 (Heparin Lyase 1, orig-inating from Flavobacterium Heparinum and expressed in Escherichia coli) and then fractionated, leading to the iso-lation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel elec-trophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their struc-tures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600 MHz. Five of the characterized oligosaccharides represent new com-pounds. The most prominent oligosaccharide comprises the common repeating unit of Heparin, ΔUA2S-[-GlcNS6S-IdoA2S-]n-GlcNS6S, where ΔUA is 4-deoxy-α-L-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-D-glucopyranose, IdoA is L-idopyranosyluronic acid, S is sulfate and n = 0–7. A second prominent Heparin oligosac-charide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S]n-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n = 0–5 and GlcA is D-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in Heparin. The promi-nence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the-GlcNS3S6S-IdoA2S- linkage is particularly susceptible to Heparinase 1
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Heparin Mapping Using Heparin Lyases and the Generation of a Novel Low Molecular Weight Heparin
Journal of medicinal chemistry, 2010Co-Authors: Zhongping Xiao, Britney R. Tappen, Wenjing Zhao, Lauren P. Canova, Huashi GuanAbstract:Seven pharmaceutical Heparins were investigated by oligosaccharide mapping by digestion with Heparin Lyase 1, 2, or 3, followed by high performance liquid chromatography analysis. The structure of one of the prepared mapping standards, ΔUA-Gal-Gal-Xyl-O-CH2CONHCH2COOH (where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, Gal is β-d-galactpyranose, and Xyl is β-d-xylopyranose) released from the linkage region using either Heparin Lyase 2 or Heparin Lyase 3 digestion, is reported for the first time. A size-dependent susceptibility of site cleaved by Heparin Lyase 3 was also observed. Heparin Lyase 3 acts on the undersulfated domains of the Heparin chain and does not cleave the linkages within Heparin’s antithrombin III binding site. Thus, a novel low molecular weight Heparin (LMWH) is afforded on Heparin Lyase 3 digestion of Heparin due to this unique substrate specificity, which has anticoagulant activity comparable to that of currently available LMWH.
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Ž.Comparative Biochemistry and Physiology Part B 130 2001 513519 Localization and characterization of acharan sulfate in the body of the giant African snail Achatina fulica
2015Co-Authors: Jia Jeonga, Toshihiko Toidab, Yuki Munetab, Ichiro Kosiishib, Toshio Imanarib, Robert Linhardt J. C, Hyung Seok Choia, Song Ji Wua, Yeong Shik KimAbstract:Ž.. ŽAcharan sulfate is a glycosaminoglycan GAG, having the structure 4-2-acetamido-2-deoxy--D-glucopyranose 1. Ž4-2-sulfo--L-idopyranosyluronic acid 1 , isolated from the body of the giant African snail Achatina fulica. This GAG represents 35 % of the dry weight of this snail’s soft body tissues. Frozen sections and polyester wax sections of Ž.the snail’s body were stained by Alcian blue-periodic acid-Schiff’s reagent PAS to localize acharan sulfate. Alcian blue staining indicated that GAG was mainly secreted into the outer surface of the body from internal granules. A highly mucous material was collected and treated and the acharan sulfate was recovered by ethanol and cetyl pyridinium chloride precipitation. Crude acharan sulfate was purified by DEAE-Sephacel ion-exchange chromatography. De-Ž.polymerization of intact mucus and purified acharan sulfate fractions by Heparin Lyase II heparitinase I from Flaobacterium Heparinum produced an unsaturated disaccharide as a major product, establishing the repeating unit of acharan sulfate. These results demonstrate that mucus in the granule and secreted to the outside of the body i
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structural snapshots of Heparin depolymerization by Heparin Lyase i
Journal of Biological Chemistry, 2009Co-Authors: Younghyun Han, Zhenqing Zhang, Zhongping Xiao, Wan Seok Kim, Marieline Garron, Hyeyeon Kim, Kyeongseok Ryu, David Shaya, Chaejoon Cheong, Yeong Shik KimAbstract:Heparin Lyase I (Heparinase I) specifically depolymerizes Heparin, cleaving the glycosidic linkage next to iduronic acid. Here, we show the crystal structures of Heparinase I from Bacteroides thetaiotaomicron at various stages of the reaction with Heparin oligosaccharides before and just after cleavage and product disaccharide. The Heparinase I structure is comprised of a β-jellyroll domain harboring a long and deep substrate binding groove and an unusual thumb-resembling extension. This thumb, decorated with many basic residues, is of particular importance in activity especially on short Heparin oligosaccharides. Unexpected structural similarity of the active site to that of Heparinase II with an (α/α)6 fold is observed. Mutational studies and kinetic analysis of this enzyme provide insights into the catalytic mechanism, the substrate recognition, and processivity.
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purification and characterization of Heparin Lyase i from bacteroides stercoris hj 15
Journal of Biochemistry and Molecular Biology, 2004Co-Authors: Wan Seok Kim, Byung Taek Kim, Donghyun Kim, Yeong Shik KimAbstract:Heparin Lyase I was purified to homogeneity from Bacteroides stercoris HJ-15 isolated from human intestine, by a combination of DEAE-Sepharose, gel-filtration, hydroxyapatite, and CM-Sephadex C-50 column chromatography. This enzyme preferred Heparin to heparan sulfate, but was inactive at cleaving acharan sulfate. The apparent molecular mass of Heparin Lyase I was estimated as 48,000 daltons by SDS-PAGE and its isoelectric point was determined as 9.0 by IEF. The purified enzyme required 500 mM NaCl in the reaction mixture for maximal activity and the optimal activity was obtained at pH 7.0 and 50 degrees C. It was rather stable within the range of 25 to 50 degrees C but lost activity rapidly above 50 degrees C. The enzyme was activated by Co(2+) or EDTA and stabilized by dithiothreitol. The kinetic constants, K(m) and V(max) for Heparin were 1.3 10(-5) M and 8.8 micromol/min.mg. The purified Heparin Lyase I was an eliminase that acted best on porcine intestinal Heparin, and to a lesser extent on porcine intestinal mucosa heparan sulfate. It was inactive in the cleavage of N-desulfated Heparin and acharan sulfate. In conclusion, Heparin Lyase I from Bacteroides stercoris was specific to Heparin rather than heparan sulfate and its biochemical properties showed a substrate specificity similar to that of Flavobacterial Heparin Lyase I.
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localization and characterization of acharan sulfate in the body of the giant african snail achatina fulica
Comparative Biochemistry and Physiology B, 2001Co-Authors: Jia Jeong, Robert J. Linhardt, Toshihiko Toida, Toshio Imanari, Yuki Muneta, Ichiro Kosiishi, Hyung Seok Choi, Yeong Shik KimAbstract:Acharan sulfate is a glycosaminoglycan (GAG), having the structure -->4)-2-acetamido-2-deoxy-alpha-D-glucopyranose(1-->4)-2-sulfo-alpha-L-idopyranosyluronic acid (1-->, isolated from the body of the giant African snail Achatina fulica. This GAG represents 3-5% of the dry weight of this snail's soft body tissues. Frozen sections and polyester wax sections of the snail's body were stained by Alcian blue-periodic acid-Schiff's reagent (PAS) to localize acharan sulfate. Alcian blue staining indicated that GAG was mainly secreted into the outer surface of the body from internal granules. A highly mucous material was collected and treated and the acharan sulfate was recovered by ethanol and cetyl pyridinium chloride precipitation. Crude acharan sulfate was purified by DEAE-Sephacel ion-exchange chromatography. Depolymerization of intact mucus and purified acharan sulfate fractions by Heparin Lyase II (heparitinase I) from Flavobacterium Heparinum produced an unsaturated disaccharide as a major product, establishing the repeating unit of acharan sulfate. These results demonstrate that mucus in the granule and secreted to the outside of the body is composed entirely of acharan sulfate.