Anticoagulant heparan sulfate: structural specificity and biosynthesis

Background:The Pasteurella heparosan synthase isozymes are highly homologous yet have different catalytic properties. Results: Chimeric enzymes bearing combinations of desirable traits were produced, and structure/function relationships were studied. Conclusion: Distinct regions of the isozymes are important for high specific activity, sugar analog usage, and chain initiation and elongation. Significance: New catalysts with utility for production of defined heparan sulfate polysaccharides were created.

Section: Journal Of Biological Chemistrymentioning

confidence: 99%

Structure/Function Analysis of Pasteurella multocida Heparosan Synthases

Otto

Green

Masuko

et al. 2012

“…In vivo, the GlcNS residue is synthesized by N-deacetylase/N-sulfotransferase (29). However, it is unclear how to selectively convert a GlcNAc residue to a GlcNS residue when multiple GlcNAc residues are present in an oligosaccharide using N-deacetylase/N-sulfotransferase in vitro.…”

Section: Substratesmentioning

confidence: 99%

Chemoenzymatic Design of Heparan Sulfate Oligosaccharides*

Liu

Chen

et al. 2010

Self Cite

146

193

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 ...

“…HS interacts with a variety of proteins (1,2) and is involved in numerous essential physiological and pathophysiological activities. These include developmental processes, nutritional metabolism, wound repair, blood coagulation and tumor metastasis, and assisting viral infections (3)(4)(5)(6). Heparin, a widely used anticoagulant drug, is a special form of HS with higher iduronic acid content and sulfation levels.…”

Section: Heparan Sulfate (Hs)mentioning

confidence: 99%

Using Engineered 2-O-Sulfotransferase to Determine the Activity of Heparan Sulfate C5-epimerase and Its Mutants

Bethea

Liu

2010

Self Cite

Heparan sulfate (HS) is involved in essential physiological and pathophysiological functions. HS is a highly sulfated polysaccharide consisting of glucuronic acid (or iduronic acid) linked to glucosamine carrying various sulfo groups. Biosynthesis of HS involves sulfotransferases and an epimerase. The HS C 5 -epimerase converts glucuronic acid to iduronic acid. The method for determining the activity has been cumbersome due to the use of a site-specifically 3 H-labeled polysaccharide substrate. Here, we report a two-enzyme coupling assay to determine the activity of C 5 -epimerase. HS 2-O-sulfotransferase (2OST) transfers the sulfo group to the 2-OH-position of glucuronic or iduronic acid. Unlike the wild type protein, 2-O-sulfotransferase mutant (2OST Y94I) transfers sulfate to the iduronic acid but not to the glucuronic acid. Thus, 2OST Y94I cannot sulfate N-sulfated heparosan, a polysaccharide containing glucuronic acid. Incubating N-sulfated heparosan with C 5 -epimerase converts some of the glucuronic acid to iduronic acid, thus becoming a substrate for 2OST Y94I. The susceptibility of the C 5 -epimerasetreated N-sulfated heparosan to 2OST Y94I modification directly correlates to the amount of the activity of C 5 -epimerase, proving that this two-enzyme coupling system can be used to assay for C 5 -epimerase. The method was further used to determine the activities of various C 5 -epimerase mutants. Our approach will significantly reduce the complexity for assaying the activity of C 5 -epimerase and facilitate the structural and functional analysis of C 5 -epimerase.