Jian Liu

University of North Carolina at Chapel Hill, North Carolina, United States

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Publications (90)455.66 Total impact

  • Jian Liu, Robert J Linhardt
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    ABSTRACT: Covering: up to May 2014Heparan 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.
    Natural Product Reports 09/2014; · 10.18 Impact Factor
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    ABSTRACT: The exquisite control of growth factor function by heparan sulfate (HS) is dictated by tremendous structural heterogeneity of sulfated modifications. How specific HS structures control growth factor-dependent progenitor expansion during organogenesis is unknown. We isolated KIT+ progenitors from fetal salivary glands during a stage of rapid progenitor expansion and profiled HS biosynthetic enzyme expression. Enzymes generating a specific type of 3-O-sulfated-HS (3-O-HS) are enriched, and fibroblast growth factor 10 (FGF10)/FGF receptor 2b (FGFR2b) signaling directly regulates their expression. Bioengineered 3-O-HS binds FGFR2b and stabilizes FGF10/FGFR2b complexes in a receptor- and growth factor-specific manner. Rapid autocrine feedback increases 3-O-HS, KIT, and progenitor expansion. Knockdown of multiple Hs3st isoforms limits fetal progenitor expansion but is rescued with bioengineered 3-O-HS, which also increases adult progenitor expansion. Altering specific 3-O-sulfated epitopes provides a mechanism to rapidly respond to FGFR2b signaling and control progenitor expansion. 3-O-HS may expand KIT+ progenitors in vitro for regenerative therapy.
    Developmental cell. 06/2014; 29(6):662-673.
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    ABSTRACT: Heparan sulfate (HS) is a linear polysaccharide expressed on cell surfaces, in extracellular matrices and cellular granules in metazoan cells. Through non-covalent binding to growth factors, morphogens, chemokines and other protein families, HS is involved in all multicellular physiological activities. Its biological activities depend on the fine structures of its protein-binding domains, the determination of which remains a daunting task. Methods have advanced to the point that information-rich product ion mass spectra may be produced on purified HS saccharides. However, the interpretation of these complex product ion patterns has emerged as the bottleneck to the dissemination of these HS sequencing methods. To solve this problem, we designed HS-SEQ, the first comprehensive algorithm for HS de novo sequencing using high-resolution tandem mass spectra. We tested HS-SEQ using negative electron transfer dissociation (NETD) tandem mass spectra generated from a set of pure synthetic saccharide standards with diverse sulfation patterns. The results showed that HS-SEQ rapidly and accurately determined the correct HS structures from large candidate pools.
    Molecular & cellular proteomics : MCP. 06/2014;
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    ABSTRACT: Heparan sulfate and heparin are highly sulfated polysaccharides that consist of a repeating disaccharide unit of glucosamine and glucuronic or iduronic acid. The 2-O-sulfated iduronic acid (IdoA2S) residue is commonly found in heparan sulfate and heparin; however, 2-O-sulfated glucuronic acid (GlcA2S) is a less abundant monosaccharide (∼<5% of total saccharides). Here, we report the synthesis of three GlcA2S-containing hexasaccharides using a chemoenzymatic approach. For comparison purposes, additional IdoA2S-containing hexasaccharides were synthesized. NMR analyses were performed to obtain full chemical shift assignments for the GlcA2S- and IdoA2S-hexasaccharides. These data show that GlcA2S is a more structurally rigid saccharide residue than IdoA2S. The antithrombin binding affinities of a GlcA2S- hexasaccharide and an IdoA2S-hexasaccharide were determined by affinity co-electrophoresis. In contrast to IdoA2S-hexasaccharides, the GlcA2S-hexasaccharide does not bind to antithrombin, confirming that the presence of IdoA2S is critically important for the anticoagulant activity. The availability of pure synthetic GlcA2S-containing oligosaccharides will allow the investigation of the structure and activity relationships of individual sites in heparin or heparan sulfate.
    Glycobiology 04/2014; · 3.54 Impact Factor
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    ABSTRACT: The chemoenzymatic synthesis of heparan sulfate tetrasaccharide (1) and hexasaccharide (2) with a fluorous tag attached at the reducing end is reported. The fluorous tert-butyl dicarbonate ((F)Boc) tag did not interfere with enzymatic recognition for both elongation and specific sulfation, and flash purification was performed by standard fluorous solid-phase extraction (FSPE). Based on an (F)Boc attached disaccharide as acceptor, a series of partial N-sulfated, 6-O-sulfated heparan sulfate oligosaccharides were successfully synthesized employing fluorous techniques.
    Organic Letters 04/2014; · 6.14 Impact Factor
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    ABSTRACT: Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essential physiological functions. HS is composed of sulfated saccharides that are biosynthesized through a complex pathway involving multiple enzymes. In vivo regulation of this process remains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway. Here, we report the crystal structure of the ternary complex of 2OST, 3-phosphoadenosine 5-phosphate, and a heptasaccharide. Utilizing site-directed mutagenesis and specific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OST position in the ordered HS biosynthesis pathway. These studies revealed that Arg80, Lys350, and Arg190 of 2OST interact with the N-sulfo groups near the modification site, consistent with the dependence of 2OST on N-sulfation. In contrast, 6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion within the active site supporting the hypothesis that 2-O-sulfation occurs prior to 6-O-sulfation. Our results provide the structural evidence for understanding the sequence of enzymatic events in this pathway.
    Journal of Biological Chemistry 03/2014; · 4.65 Impact Factor
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    ABSTRACT: Heparin, a commonly used anticoagulant drug, is a mixture of highly sulfated polysaccharides with various molecular weights. The unique sulfation pattern dictates the anticoagulant activity of heparin. Commercial heparins are categorized into three forms according to their average molecular weight (MW): unfractionated heparin (UFH, MWavg 14,000), low molecular weight heparin (LMWH, MWavg 3,500-6,500) and the synthetic pentasaccharide (fondaparinux, MW 1508.3). UFH is isolated from porcine intestine while LMWH is derived from UFH by various methods of depolymerization, which generate a wide range of oligosaccharide chain lengths. Different degradation methods result in structurally distinct LMWH products, displaying different pharmacological and pharmacokinetic properties. In this report, we utilized a chemoenzymatic method to synthesize LMWH with the emphasis on controlling the size distribution of the oligosaccharides. A tetrasaccharide primer and a controlled enzyme-based polymerization were employed to build a narrow size oligosaccharide backbone. The oligosaccharide backbones were further modified by a series of sulfation and epimerization steps in order to obtain a full anticoagulation activity. Determination of the anticoagulation activity in vitro and ex vivo indicated that the synthetic LMWH has higher potency than enoxaparin, a commercial LMWH drug in clinical usage.
    Glycobiology 03/2014; · 3.54 Impact Factor
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    ABSTRACT: Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders, but impurities, structural heterogeneity or functional irreversibility can limit treatment options. We report a series of synthetic LMWHs prepared by cost-effective chemoenzymatic methods. The high activity of one defined synthetic LMWH against human factor Xa (FXa) was reversible in vitro and in vivo using protamine, demonstrating that synthetically accessible constructs can have a critical role in the next generation of LMWHs.
    Nature Chemical Biology 02/2014; · 12.95 Impact Factor
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    ABSTRACT: Four well-defined heparan sulfate (HS) block copolymers, containing S-domains (high sulfo group content) placed adjacent to N-domains (low sulfo group content) were chemoenzymatically synthesized and characterized. The domain lengths in these HS block co-polymers were ~40 saccharide units. Microtiter 96-well and 3D cell-based microarray assays utilizing murine immortalized bone marrow (BaF3) cells were developed to evaluate the activity of these HS block co-polymers. Each recombinant BaF3 cell line expresses only a single type of fibroblast growth factor receptor (FGFR), but produces neither HS nor fibroblast growth factors (FGFs). In the presence of different FGFs, BaF3 cell-proliferation showed clear differences for the four HS block co-polymers examined. These data were used to examine the two proposed signaling models, the symmetric FGF2:HS2:FGFR2 ternary complex model and the asymmetric FGF2:HS1:FGFR2 ternary complex model. In the symmetric FGF2:HS2:FGFR2 model, two acidic HS chains bind in a basic canyon located on the top face of the FGF2-FGFR2 protein complex. In this model, the S-domains at the non-reducing ends of the two HS proteoglycan chains are proposed to interact with the FGF2-FGFR2 protein complex. In contrast, in the asymmetric FGF2:HS1:FGFR2 model, a single HS chain interacts with the FGF2-FGFR2 protein complex through a single S-domain that can be located at any position within an HS chain. Our data comparing a series of synthetically prepared HS block copolymers support a preference for the symmetric FGF2:HS2:FGFR2 ternary complex model.
    Journal of Biological Chemistry 02/2014; · 4.65 Impact Factor
  • Nature Chemical Biology 02/2014; · 12.95 Impact Factor
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    ABSTRACT: O-sulfotransferases (OSTs) are critical enzymes in the cellular biosynthesis of the biologically and pharmacologically important heparan sulfate and heparin. Recently, these enzymes have been cloned and expressed in bacteria for application in the chemoenzymatic synthesis of glycosaminoglycan-based drugs. OST activity assays have largely relied on the use of radioisotopic methods using [(35)S] 3'-phosphoadenosine-5'-phosphosulfate and scintillation counting. Herein, we examine alternative assays that are more compatible with a biomanufacturing environment. A high throughput microtiter-based approach is reported that relies on a coupled bienzymic colorimetric assay for heparan sulfate and heparin OSTs acting on polysaccharide substrates using arylsulfotransferase-IV and p-nitrophenylsulfate as a sacrificial sulfogroup donor. A second liquid chromatography-mass spectrometric assay, for heparan sulfate and heparin OSTs acting on structurally defined oligosaccharide substrates, is also reported that provides additional information on the number and positions of the transferred sulfo groups within the product. Together, these assays allow quantitative and mechanistic information to be obtained on OSTs that act on heparan sulfate and heparin precursors.
    Analytical and Bioanalytical Chemistry 11/2013; · 3.66 Impact Factor
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    ABSTRACT: We have developed an efficient chemoenzymatic synthesis of heparan sulfate oligosaccharides employing the para-nitrophenyl (p-NP) β-glucuronide as an acceptor compatible with enzymatic elongation and one that significantly simplifies oligosaccharide purification on C-18 resin. Employing ceric ammonium nitrate as oxidative reagent to remove the p-NP group unexpectedly also removed the glucuronic acid residue at the reducing-end, affording a smaller oligosaccharide. The application of ceric ammonium sulfate allowed the removal of the p-NP without concomitant loss of the adjacent glucuronic acid offering a route to longer heparin sulfate oligosaccharide products.
    Tetrahedron Letters 08/2013; 54(33):4471-4474. · 2.40 Impact Factor
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    ABSTRACT: Testosteronan, an unusual glycosaminoglycan first isolated from the microbe Comamonas testosteroni, was enzymatically synthesized in vitro by transferring uridine diphosphate sugars on β-p-nitrophenyl glucuronide acceptor. After chemically converting testosteronan to N-sulfotestosteronan it was tested as a substrate for sulfotransferases involved in the biosynthesis of the glycosaminoglycan, heparan sulfate. Studies using (35) S-labeled 3'-phosphodenosine-5'-phosphosulfate (PAPS) showed that only 6-O-sulfotransferases acted on N-sulfotestosteronan. An oxidative depolymerization reaction was explored to generate oligosaccharides from (34) S-labeled 6-O-sulfo-N-sulfotestosteroran using (34) S-labeled PAPS because testosteronan was resistant to all of the tested glycosaminoglycan-degrading enzymes. Liquid chromotography-mass spectrometric analysis of the oxidatively depolymerized polysaccharides confirmed the incorporation of (34) S into ~14% of the glucosamine residues. Nuclear magnetic resonance spectroscopy also showed that the sulfo groups were transferred to ~20% of the 6-hydroxyl groups in the glucosamine residue of N-sulfotestosteronan. The bioactivity of 6-O-sulfo-N-sulfotestosteronan was examined by performing protein-binding studies with fibroblast growth factors and antithrombin III using a surface plasmon resonance competition assay. The introduction of 6-O-sulfo groups enhanced N-sulfotestosteronan binding to the fibroblast growth factors, but not to antithrombin III.
    Biopolymers 04/2013; · 2.88 Impact Factor
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    ABSTRACT: The aim of this study was to evaluate the effect of percutaneous one-channel double stent implantation on hilar biliary obstruction involving both hepatic ducts and its clinical value. A total of 8 patients with hilar biliary obstruction involving the left and right hepatic ducts were enrolled. A percutaneous unilateral approach was adopted. Two stents were implanted, one between the left and right hepatic ducts and the other between the hepatic ducts and the common bile duct for biliary drainage. Interventional therapies such as arterial chemoembolization were performed for antitumor treatment. All surgical procedures were successfully accomplished. At 2 weeks after stenting, total bilirubin decreased to 61.2±13.4 μmol/l (the preoperative value was 267.1±154.7 μmol/l). No severe complications or mortalities occurred. Single-channel double stent implantation should be the preferred method of treatment for patients with hilar biliary obstruction involving both hepatic ducts. Drainage and antitumor treatment should also be used when necessary.
    Experimental and therapeutic medicine 04/2013; 5(4):1179-1183. · 0.34 Impact Factor
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    ABSTRACT: Glycosaminoglycans (GAGs) are complex polysaccharides composed of hexosamine-containing disaccharide repeating units. The three most studied classes of GAGs, heparin/heparan sulfate, hyaluronan, and chondroitin/dermatan sulfate, are essential macromolecules. GAGs isolated from animal and microbial sources have been utilized therapeutically, but naturally occurring GAGs are extremely heterogeneous limiting further development of these agents. These molecules pose difficult targets to construct by classical organic syntheses due to the long chain lengths and complex patterns of modification by sulfation and epimerization. Chemoenzymatic synthesis, a process that employs exquisite enzyme catalysts and various defined precursors (e.g., uridine 5'-diphosphosphate-sugar donors, sulfate donors, acceptors, and oxazoline precursors), promises to deliver homogeneous GAGs. This review covers both theoretical and practical issues of GAG oligosaccharide and polysaccharide preparation as single molecular entities and in library formats. Even at this early stage of technology development, nearly monodisperse GAGs can be made with either natural or artificial structures.
    Glycobiology 03/2013; · 3.54 Impact Factor
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    Sherket B Peterson, Jian Liu
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    ABSTRACT: Heparan sulfate is a highly sulfated polysaccharide abundantly present in the extracellular matrix. Heparan sulfate consists of a disaccharide repeating unit of glucosamine and glucuronic and iduronic acid residues. The functions of heparan sulfate are largely dictated by its size as well as the sulfation patterns. Heparanase is an enzyme that cleaves heparan sulfate polysaccharide into smaller fragments, regulating the functions of heparan sulfate. Understanding the substrate specificity plays a critical role in dissecting the biological functions of heparanase and heparan sulfate. The prevailing view is that heparanase recognizes specific sulfation patterns in heparan sulfate. However, emerging evidence suggests that heparanase is capable of varying its substrate specificities depending on the saccharide structures around the cleavage site. The plastic substrate specificity suggests a complex role of heparanase in regulating the structures of heparan sulfate in matrix biology.
    Matrix biology: journal of the International Society for Matrix Biology 03/2013; · 3.56 Impact Factor
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    ABSTRACT: A 'clickable' disaccharide was prepared by treating the aldehyde precursor with hydroxylamine, followed by the catalytic hydrogenation and diazotransfer reaction. This disaccharide was successfully applied to the elongation of the backbone construction of ultralow molecular weight (ULMW) heparins using two bacterial glycosyl transferases, N-acetyl glucosaminyl transferase from Escherichia coli K5 (KfiA) and heparosan synthase-2 (pmHS2) from Pasteurella multocida.
    Carbohydrate research 03/2013; 372C:30-34. · 2.03 Impact Factor
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    ABSTRACT: 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 many methods of ion-activation, this class of molecules generally exhibit SO3 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 (2 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 mM 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 twelve saccharide subunits and up to eleven sulfo groups.
    Molecular &amp Cellular Proteomics 02/2013; · 7.25 Impact Factor
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    ABSTRACT: Heparin has been the most commonly used anticoagulant drug for nearly a century. The drug heparin is generally categorized into three forms according to its molecular weight (MW), unfractionated (UF, average MW 13,000), low molecular weight (LMW, average MW 5,000), and ultra-low molecular weight heparin (ULMWH, average MW 2,000). Overdose of anticoagulant heparin can lead to very dangerous bleeding in patients. Protamine sulfate can be administered as an antidote to reverse heparin's anticoagulant effect. There is not an effective antidote for ULMWH. In the current study, we examine human N-acetylglucosamine 6-sulfatase (NG6S), expressed in Chinese hamster ovary cells as a reversal agent for ULMWH. NG6S removes a single 6-O-sulfo group at the non-reducing end of the ULMWH Arixtra® (fondaparinux) effectively removing its ability to bind to antithrombin and preventing its inhibition of coagulation factor Xa. These results pave the way to develop human NG6S as an antidote for neutralizing the anticoagulant activity of ULMWHs. © 2013 The Authors Journal compilation © 2013 FEBS.
    FEBS Journal 02/2013; · 4.25 Impact Factor
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    ABSTRACT: K5 lyase A (KflA) is a tailspike protein from the K5A phage that catalyzes the degradation of the capsule polysaccharide of K5 strains of Escherichia coli. The K5 E. coli capsule polysaccharide, also known as heparosan, is composed of the disaccharide repeating unit of [-4)-GlcA-β(1,4)-GlcNAc-α(1-] and therefore identical to the biological precursor of heparin and heparan sulfate. KflA could supplement the heparin lyases for heparin and heparan sulfate analysis. The first part of this study aimed to clarify ambiguity resulting from the revision of the KflA amino acid sequence in 2010 from that published in 2000. We found only the expression of the updated sequence gave a soluble active enzyme, which produced heparosan degradation products similar to those of previous studies. Next, we examined the specificity of KflA towards heparosan oligosaccharides of varying sizes, all containing a single N-sulfated glucosamine (GlcNS) residue. The presence of GlcNS in an octasaccharide and nonasaccharide chain directed cleavage by KflA to a single position at the reducing end of the substrate. However, an N-sulfated decasaccharide exhibited extensive cleavage at the non-reducing end of the chain, illustrating a distinct change in the cleavage pattern of KflA toward substrates of differing sizes. Because KflA is able to cleave substrate containing isolated GlcNS residues, this enzyme could be used for analysis of low sulfate content heparan sulfate domains.
    Glycobiology 09/2012; · 3.54 Impact Factor

Publication Stats

1k Citations
455.66 Total Impact Points

Institutions

  • 2002–2014
    • University of North Carolina at Chapel Hill
      • Division of Chemical Biology and Medicinal Chemistry
      North Carolina, United States
    • Lund University
      • Biomedical Center
      Lund, Skane, Sweden
  • 2013
    • University of Oklahoma Health Sciences Center
      • Department of Biochemistry and Molecular Biology
      Oklahoma City, OK, United States
    • Hefei University of Technology
      Luchow, Anhui Sheng, China
  • 2007–2013
    • Rensselaer Polytechnic Institute
      • • Department of Chemistry and Chemical Biology
      • • Department of Biology
      • • Center for Biotechnology and Interdisciplinary Studies
      Troy, NY, United States
    • University of North Carolina at Pembroke
      North Carolina, United States
  • 2012
    • National Institutes of Health
      • Structural Biophysics Laboratory
      Bethesda, MD, United States
    • Julphar School of Pharmacy
      Charlotte, North Carolina, United States
    • Broad Institute of MIT and Harvard
      Cambridge, Massachusetts, United States
  • 2004–2011
    • University of Illinois at Chicago
      • Department of Ophthalmology and Visual Sciences (Chicago)
      Chicago, IL, United States
  • 2005–2007
    • Michigan Technological University
      • Department of Chemistry
      Michigan City, Indiana, United States