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

Xiao, Zhihui; Zhao, Wenjing; Yang, Bo; Zhang, Zhenqing; Guan, Huashi; Linhardt, Robert J.

doi:10.1093/glycob/cwq123

Cited by 40 publications

(59 citation statements)

References 24 publications

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“…The purity of each of the oligosaccharides prepared from HPLC was determined to be >95% pure by analytical SAX-HPLC and PAGE analysis. The structures of these standards were characterized by HPLC-ESI-MS, and one-dimensional (1D) and two-dimensional (2D) NMR experiments 18. High resolution mass spectra were obtained for all new compounds using ESI-Fourier transform-mass spectrometry (FT-MS) on an LTQ XL Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) 29…”

Section: Methodsmentioning

confidence: 99%

Heparin Mapping Using Heparin Lyases and the Generation of a Novel Low Molecular Weight Heparin

Xiao

Tappen²,

Ly³

et al. 2010

J. Med. Chem.

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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-CH 2 CONHCH 2 COOH, (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 under sulfated 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) was 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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Section: Methodsmentioning

confidence: 99%

Heparin Mapping Using Heparin Lyases and the Generation of a Novel Low Molecular Weight Heparin

Xiao

Tappen²,

Ly³

et al. 2010

J. Med. Chem.

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“…heparin and HS to a barely detectable or undetectable mixture of disaccharide and tetrasaccharide products [16]. The N405G mutant enzyme afforded complete conversion on heparin and HS and N405A enzyme showed a substantial decrease in the molecular weight of both polysaccharides.…”

Section: Activity Of Hepii Mutant Enzymes On Polysaccharide Substratesmentioning

confidence: 96%

“…Purity of each tetrasaccharide was determined to be >95% by analytical HPLC. Structures of these tetrasaccharides were characterized by liquid chromatography (LC)-mass spectrometry (MS) and 1D and 2D NMR [14][15][16]. …”

Section: Preparation Of the Heparin-derived Tetrasaccharide Substratesmentioning

confidence: 99%

“…1B) were next used to evaluate the effect of the Asn405 mutation on catalysis. The tetrasaccharides were prepared through the complete HepI depolymerization of heparin [16]. Each tetrasaccharide contains a 3-O-sulfoglucosamine residue (+2 subsite) next to the glycosidic uronic acid at the reducing end of the cleavage site (À1, +1) making these substrates resistant to WT HepII and all other known Heps.…”

Section: Activity Of Hepii Mutant Enzymes On Defined Tetrasaccharide mentioning

confidence: 99%

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Site-directed Mutagenesis Using Dpn1

Jing¹

2011

BIO-PROTOCOL

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Edited by Stuart Ferguson Keywords:Heparin lyase II Heparinase Site-directed mutagenesis Protein engineering Heparin 3-O-Sulfoglucosamine Substrate specificity a b s t r a c t 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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“…Fractionation of various uniformly sized oligosaccharide mixtures was carried out using a gradient of water and 2 M NaCl, and chromatographic profiles at 232 nm were used to combine fractions from repeated separations. Primary structure and the level of purity were performed using PAGE analysis (47).…”

mentioning

confidence: 99%

Structurally Informative Tandem Mass Spectrometry of Highly Sulfated Natural and Chemoenzymatically Synthesized Heparin and Heparan Sulfate Glycosaminoglycans

Kailemia

et al. 2013

Molecular & Cellular Proteomics

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The highly sulfated glycosaminoglycan oligosaccharides derived from heparin and heparan sulfate have been a highly intractable class of molecules to analyze by tandem mass spectrometry. Under the many methods of ion activation, this class of molecules generally exhibits SO 3 loss as the most significant fragmentation pathway, interfering with the assignment of the location of sulfo groups in glycosaminoglycan chains. We report here a method that stabilizes sulfo groups and facilitates the complete structural analysis of densely sulfated (two or more sulfo groups per disaccharide repeat unit) heparin and heparan sulfate oligomers. This is achieved by complete removal of all ionizable protons, either by charging during electrospray ionization or by Na ؉ /H ؉ exchange. The addition of millimolar levels of NaOH to the sample solution facilitates the production of precursor ions that meet this criterion. This approach is found to work for a variety of heparin sulfate oligosaccharides derived from natural sources or produced by chemoenzymatic synthesis, with up to 12 saccharide subunits and up to 11 sulfo groups. Molecular & Cellular Proteomics 12: 10.1074/mcp.M112.026880, 979 -990, 2013. Heparin (Hp)1 and heparan sulfate (HS) are linear, polydisperse, and highly sulfated glycosaminoglycans (GAGs), with a repeating disaccharide building block composed of a 1-4-linked glucosamine and a uronic acid residue (1). The saccharide residues may have a variety of modifications, and these are usually heterogeneous due to the nontemplate nature of their biosynthesis (2). Glucosamine residues may be substituted with N-sulfo or N-acetyl and 3-and/or 6-O-sulfo groups. Uronic acid residues can be either glucuronic or iduronic acid and substituted with 2-O-sulfo groups (3, 4). These structural features are thought to control Hp and HS biological activity, e.g. their interactions with proteins, and so the structural characterization of GAGs is an important target for chemical analysis (1,5,6). A particularly well known example of a GAG-protein interaction is the role of Hp as an antithrombin III activator. A pentasaccharide unit with a very specific pattern of modification interacts with antithrombin III causing it to undergo a conformational change that increases the anticoagulation activity of antithrombin III by more than 3 orders of magnitude (7,8). Contamination of pharmaceutical Hp was a major issue recently, i.e. associated with over 70 fatalities worldwide (9 -12). This problem highlights the need for rapid, robust, and sensitive analytical methods for the analysis of heparin and for identifying contaminants of similar composition (11). Although nuclear magnetic resonance spectroscopy is often the method of choice for determining the structure of GAGs, such as Hp and HS, it requires substantial sample preparation to obtain pure samples, relatively large amounts of sample, and time-consuming interpretation.Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) offer high sensitivity and specificity and are often used for t...

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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

Cited by 40 publications

References 24 publications

Heparin Mapping Using Heparin Lyases and the Generation of a Novel Low Molecular Weight Heparin

Heparin Mapping Using Heparin Lyases and the Generation of a Novel Low Molecular Weight Heparin

Site-directed Mutagenesis Using Dpn1

Structurally Informative Tandem Mass Spectrometry of Highly Sulfated Natural and Chemoenzymatically Synthesized Heparin and Heparan Sulfate Glycosaminoglycans

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