Using an Enzymatic Combinatorial Approach to Identify Anticoagulant Heparan Sulfate Structures

Chen, Jinghua; Jones, Courtney L.; Liu, Jian

doi:10.1016/j.chembiol.2007.07.015

Cited by 97 publications

(113 citation statements)

References 35 publications

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“…N-sulfotransferase (NST), C 5 -epi, 2-OST, 6-OST-1, 6-OST-3, 3-OST-1, 3-OST-5, and N-acetyl-D-glucosaminyl transferase of E. coli K5 strain (KfiA) were expressed and purified as previously described (6,(13)(14)(15)(16). PmHS2 was expressed as an N-terminal fusion to His 6 using a PET-15b vector (Novagen) (17).…”

Section: Methodsmentioning

confidence: 99%

“…HPLC Analysis-HPLC analysis of oligosaccharides followed the procedures as previously described (6,9).…”

Section: Methodsmentioning

confidence: 99%

“…Heparin, a widely used anticoagulant drug, is a specialized form of highly sulfated HS. The diverse biological functions present considerable opportunities for exploiting HS or HS-protein conjugates for developing new classes of anticancer (4), antiviral (5), and improved anticoagulant drugs (6). Furthermore, a recent worldwide outbreak of contaminated heparin underscores the needs for synthetic heparins to replace those isolated from animal tissues (7).…”

Section: Heparan Sulfate (Hs)mentioning

confidence: 99%

“…With the exception of HS polymerase, all of these biosynthetic enzymes have been expressed at high levels in Escherichia coli (1), permitting easy access to an abundance of enzymes. Using HS sulfotransferases and C 5 -epi, we previously developed a method to synthesize HS from a bacteria capsular polysaccharide with biological activities (6,9,10).…”

Section: Heparan Sulfate (Hs)mentioning

confidence: 99%

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Chemoenzymatic Design of Heparan Sulfate Oligosaccharides*

Liu

Chen

et al. 2010

Journal of Biological Chemistry

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Heparan sulfate is a sulfated glycan that exhibits essential physiological functions. Interrogation of the specificity of heparan sulfate-mediated activities demands a library of structurally defined oligosaccharides. Chemical synthesis of large heparan sulfate oligosaccharides remains challenging. We report the synthesis of oligosaccharides with different sulfation patterns and sizes from a disaccharide building block using glycosyltransferases, heparan sulfate C 5 -epimerase, and sulfotransferases. This method offers a generic approach to prepare heparan sulfate oligosaccharides possessing predictable structures. Heparan sulfate (HS)3 is a unique class of macromolecular natural product that is present in large quantities on the mammalian cell surface and in the extracellular matrix. HS participates in regulating blood coagulation, embryonic development, and the inflammatory response and assists viral/bacterial infections. It consists of a repeating disaccharide unit of glucuronic acid (GlcUA) or iduronic acid (IdoUA) and glucosamine, both capable of carrying sulfo groups (1). The sulfation pattern of HS dictates its biological activity (2, 3). Heparin, a widely used anticoagulant drug, is a specialized form of highly sulfated HS. The diverse biological functions present considerable opportunities for exploiting HS or HS-protein conjugates for developing new classes of anticancer (4), antiviral (5), and improved anticoagulant drugs (6). Furthermore, a recent worldwide outbreak of contaminated heparin underscores the needs for synthetic heparins to replace those isolated from animal tissues (7). Chemical synthesis is a powerful tool to obtain structurally defined heparin/HS oligosaccharides. The most successful example is the total synthesis of an antithrombin-binding pentasaccharide (8). This pentasaccharide is marketed under the trade name Arixtra for the treatment of venous thromboembolic disorders. However, the chemical synthesis of oligosaccharides larger than an octasaccharide is extremely difficult, especially when multiple target structures are required for biological evaluation (8). An enzyme-based method offers a promising alternative approach to synthesize HS.The HS biosynthetic pathway involves multiple enzymes, including HS polymerase, epimerase, and sulfotransferases ( Fig. 1). HS polymerase is responsible for building the polysaccharide backbone, containing the repeating unit of -GlcUA-GlcNAc-. The backbone is then modified by N-deacetylase/N-sulfotransferase (having two separate domains exhibiting the activity of N-deacetylase and N-sulfotransferase, respectively), C 5 -epimerase (C 5 -epi, converting GlcUA to IdoUA), 2-O-sulfotransferase (2-OST), 6-O-sulfotransferase (6-OST) and 3-O-sulfotransferase (3-OST) to produce the fully elaborated HS. With the exception of HS polymerase, all of these biosynthetic enzymes have been expressed at high levels in Escherichia coli (1), permitting easy access to an abundance of enzymes. Using HS sulfotransferases and C 5 -epi, we previously developed a method ...

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Section: Methodsmentioning

confidence: 99%

“…HPLC Analysis-HPLC analysis of oligosaccharides followed the procedures as previously described (6,9).…”

Section: Methodsmentioning

confidence: 99%

Section: Heparan Sulfate (Hs)mentioning

confidence: 99%

Section: Heparan Sulfate (Hs)mentioning

confidence: 99%

See 2 more Smart Citations

Chemoenzymatic Design of Heparan Sulfate Oligosaccharides*

Liu

Chen

et al. 2010

Journal of Biological Chemistry

Self Cite

144

192

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“…However, the synthesis of those molecules larger than hexasaccharides is extremely difficult. Using HS biosynthetic enzymes to prepare biologically active polysaccharides and oligosaccharides has recently gained momentum (8)(9)(10)(11)(12).…”

mentioning

confidence: 99%

Using a 3-O-Sulfated Heparin Octasaccharide To Inhibit the Entry of Herpes Simplex Virus Type 1

et al. 2008

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Heparan sulfate (HS) is a highly sulfated polysaccharide and is present in large quantities on the cell surface and in the extracellular matrix. Herpes simplex virus type 1(HSV-1) utilizes a specialized cell surface HS, known as 3-O-sulfated HS, as an entry receptor to establish infection. Here, we exploit an approach to inhibit HSV-1 infection by using a 3-O-sulfated octasaccharide, mimicking the active domain of the entry receptor. The 3-O-sulfated octasaccharide was synthesized by incubating a heparin octasaccharide (3-OH octasaccharide) with HS 3-O-sulfotransferase isoform 3. The resultant 3-O-sulfated octasaccharide has a structure of ΔUA2S-GlcNS6S-IdoUA2S-GlcNS6S-IdoUA2S-GlcNS3S6S-IdoUA2S-GlcNS6S (where ΔUA is 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid, GlcN is D-glucosamine and IdoUA is L-iduronic acid). Results from cell based assays revealed that the 3-O-sulfated octasaccharide has stronger activity in blocking HSV-1 infection than that of the 3-OH octasaccharide, suggesting that the inhibition of HSV-1 infection requires a unique sulfation moiety. Our results suggest the feasibility of inhibiting HSV-1 infection by blocking viral entry with a specific oligosaccharide. KeywordsHeparin; heparan sulfate; herpes simplex virus; glycoprotein D; sulfotransferase; oligosaccharides Heparan sulfate (HS) is a highly sulfated linear polysaccharide present ubiquitously on the cell surface and in the extracellular matrix. HS plays a role in regulating embryonic development, inflammatory response, blood coagulation and assisting viral/bacterial infections (1). Heparin, a commonly used anticoagulant drug, is a special form of HS containing glucuronic (GlcUA)/ iduronic acid (IdoUA) and glucosamine, each carrying sulfo groups (Fig. 1A). The uniquely distributed sulfation pattern of HS polysaccharide is believed to regulate its functional specificity (2-4). Thus, understanding the structure and function relationship of HS attracts considerable interest in improving the anticoagulant efficacy of heparin and exploiting heparin *Corresponding author: Rm 309, Beard Hall, University of North Carolina, Chapel Hill, NC 27599. Tel.: 919-843-6511; Fax: 919-843-5432; E-mail: jian_liu@unc.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 May 27. Published in final edited form as:Biochemistry. 2008 May 27; 47(21): 5774-5783. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript or heparin-like molecules for the development of anticancer and antiviral drugs (5-7). The major difficulty in dissecting the structure and function relationship of HS is to obtain HS oligosaccharides or polysaccharides with defined structures. Chemical synthesis has been the major route to prepare structurally defined oligosaccharides to mimic the functions of HS. However, the synthesis of those molecules larger than hexasaccharides is extremely difficult. Using HS biosynthetic enzymes to prepare biologically active polysaccharides and oligosaccharides has recently gained momentum...

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