Crystal Structure and Mutational Analysis of Heparan Sulfate 3-O-Sulfotransferase Isoform 1

Edavettal, Suzanne C.; Lee, Karen A.; Negishi, Masahiko; Linhardt, Robert J.; Liu, Jian; Pedersen, Lars C.

doi:10.1074/jbc.m401089200

Cited by 67 publications

(80 citation statements)

References 39 publications

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“…In addition, a ternary complex of 3-OST-3/PAP/tetrasaccharide substrate was solved at 1.9Å (46,47). We have found that 3-OST-1 and 3-OST-3 have very similar overall structure.…”

Section: Crystal Structures Of 3-ost-1 and 3-ost-3mentioning

confidence: 94%

“…Because 3-OSTs play a critical role in controlling the biosynthesis of anticoagulant HS and unique substrate specificities among the isoforms, we investigated the mechanism of 3-OSTs using a structural biology approach coupled with site-directed mutagenesis (46,47). From these studies, several key amino acid residues have been identified that contribute to the interactions with the sulfo donor PAPS, as well as with polysaccharide substrates.…”

Section: Crystal Structures Of 3-ost-1 and 3-ost-3mentioning

confidence: 99%

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Anticoagulant heparan sulfate: structural specificity and biosynthesis

Liu

Pedersen

2007

Appl Microbiol Biotechnol

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123

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SummaryHeparan sulfate (HS) is present on the surface of endothelial and surrounding tissues in large quantities. It plays important roles in regulating numerous functions of the blood vessel wall, including blood coagulation, inflammation response and cell differentiation. HS is a highly sulfated polysaccharide containing glucosamine and glucuronic/iduronic acid repeating disaccharide units. The unique sulfated saccharide sequences of HS determine its specific functions. Heparin, an analogue of heparan sulfate, is the most commonly used anticoagulant drug. Because of its wide range of biological functions, HS has become an interesting molecule to biochemists, medicinal chemists and developmental biologists. Here, we summarize recent progress towards understanding the interaction between heparan sulfate and blood coagulating factors, the biosynthesis of anticoagulant heparan sulfate and the mechanism of action of heparan sulfate biosynthetic enzymes. Further, knowledge of the biosynthesis of HS facilitates the development of novel enzymatic approaches to synthesize HS from bacterial capsular polysaccharides and to produce polysaccharide end products with high specificity for the biological target. These advancements provide the foundation for the development of polysaccharide-based therapeutic agents.

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“…In addition, a ternary complex of 3-OST-3/PAP/tetrasaccharide substrate was solved at 1.9Å (46,47). We have found that 3-OST-1 and 3-OST-3 have very similar overall structure.…”

Section: Crystal Structures Of 3-ost-1 and 3-ost-3mentioning

confidence: 94%

Section: Crystal Structures Of 3-ost-1 and 3-ost-3mentioning

confidence: 99%

Anticoagulant heparan sulfate: structural specificity and biosynthesis

Liu

Pedersen

2007

Appl Microbiol Biotechnol

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123

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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%

Chemoenzymatic Design of Heparan Sulfate Oligosaccharides*

Liu

Chen

et al. 2010

Journal of Biological Chemistry

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148

193

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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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“…AF019385.1; amino acids sequence from G48 to H311) [8] was codon optimized and synthesized by Integrated Device Technology, Inc. (USA) and cloned into the pIDTSMART plasmid with a 5′ overhang containing an XbaI site, a 3′ overhang containing an SmaI site, and a 6× His-tag fused at the N-terminus of expressed protein. The pIDTSMART-3-OST-1 plasmid was double digested using XbaI and SmaI restriction enzymes (Fermentas, USA), and the digested 3-ost-1 gene was ligated into the E. coli-B.…”

Section: Methodsmentioning

confidence: 99%

“…2c). To compare the sulfotransferase activity to that in E. coli-expressed 3-OST-1 constructed previously in another lab [8], we negated the background sulfotransferase activity by subtracting out the measurements from non-induced samples. The measurements for B. megaterium are not in units of micromolar PNP/ milligram protein because no protein concentration measurement was taken as we were only looking for activity qualitatively.…”

Section: Activity Of Expressed Enzymementioning

confidence: 99%

Expression of Low Endotoxin 3-O-Sulfotransferase in Bacillus subtilis and Bacillus megaterium

Wang

Englaender

et al. 2013

Appl Biochem Biotechnol

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A key enzyme for the biosynthesis and bioengineering of heparin, 3-O-sulfotransferase-1 (3-OST-1), was expressed and purified in Gram-positive Bacillus subtilis and Bacillus megaterium. Western blotting, protein sequence analysis, and enzyme activity measurement confirmed the expression. The enzymatic activity of 3-OST-1 expressed in Bacillus species were found to be similar to those found when expressed in Escherichia coli. The endotoxin level in 3-OST-1 from B. subtilis and B. megaterium -fold lower than that of the E. coli-expressed 3-OST-1, which makes the Bacillus expression system of particular interest for the generation of pharmaceutical grade raw heparin from nonanimal sources.

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Crystal Structure and Mutational Analysis of Heparan Sulfate 3-O-Sulfotransferase Isoform 1

Cited by 67 publications

References 39 publications

Anticoagulant heparan sulfate: structural specificity and biosynthesis

Anticoagulant heparan sulfate: structural specificity and biosynthesis

Chemoenzymatic Design of Heparan Sulfate Oligosaccharides*

Expression of Low Endotoxin 3-O-Sulfotransferase in Bacillus subtilis and Bacillus megaterium

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