Enzymatic Redesigning of Biologically Active Heparan Sulfate

Abstract

Heparan sulfate carries a wide range of biological activities, regulating blood coagulation, cell differentiation, and inflammatory responses. The sulfation patterns of the polysaccharide are essential for the biological activities. In this study, we report an enzymatic method for the sulfation of multimilligram amounts of heparan sulfate with specific functions using immobilized sulfotransferases combined with a 3-phosphoadenosine 5-phosphosulfate regeneration system. By selecting appropriate enzymatic modification steps, an inactive precursor has been converted to the heparan sulfate having three distinct biological activities, associated with binding to antithrombin, fibroblast growth factor-2, and herpes simplex virus envelope glycoprotein D. Because the recombinant sulfotransferases are expressed in bacteria, and the method uses a low cost sulfo donor, it can be readily utilized to synthesize large quantities of anticoagulant heparin drug or other biologically active heparan sulfates. Heparan sulfate (HS)3 is a ubiquitous component of the cell surface and extracellular matrix. It regulates a wide range of physiologic and pathophysiologic functions, including embryonic development and blood coagulation and can facilitate viral infection (1, 2). HS exerts its biological effects by interacting with the specific proteins involved in a given process (3). HS is a highly charged polysaccharide consisting of 1 3 4-linked glucosamine and glucuronic/iduronic acid units that contain both N-and O-sulfo groups. Unique saccharide sequences within HS determine the specificity of the binding of HS to its target proteins (4). Heparin, a specialized form of HS, is a commonly used anticoagulant drug. Thus, new methods for the synthesis of heparin and HS attract considerable interest for those developing anticoagulant drugs having improved pharmacological effects.Chemical synthesis has been the major route to obtain structurally defined heparin and HS oligosaccharides (5). The most important example involves the structure antithrombin-binding pentasaccharide. A synthetic pentasaccharide, based on this structure, has been marketed in the United States under the trade name Arixtra. Arixtra is a specific factor Xa inhibitor that is used clinically to prevent venous thromboembolic incidents during surgery. Unfortunately, the total synthesis of heparin and HS oligosaccharides, larger than pentasaccharides, is extremely difficult. HS analogues with 14 saccharide units inhibit the activity of thrombin, but these synthetic analogues are simplified hybrid molecules of HS oligosaccharides and highly sulfated glucose units (6, 7) and are not the naturally occurring structures. Although our laboratory and others continue to pursue the synthesis of heparin and HS oligosaccharides (8), it has become clear that chemical synthesis alone is currently incapable of generating most larger oligosaccharide structures. The application of HS biosynthetic enzymes for generating large heparin and HS oligosaccharides with desired biological activities o...

Chemoenzymatic Design of Heparan Sulfate Oligosaccharides*

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

“…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).…”

Inhibition or Activation of Apert Syndrome FGFR2 (S252W) Signaling by Specific Glycosaminoglycans

“…We therefore decided to search for structurally defined polysaccharide inhibitors. By a combined chemical and enzymatic approach, we synthesized 2SNS HS and 6S2SNS HS (36). Synthetic 2SNS HS inhibited FGFR2b (S252W) activity in a dose-dependant manner (Fig.…”

Anticoagulant heparan sulfate: structural specificity and biosynthesis

“…To achieve enzymatic synthesis of HS at a practical pharmaceutical level on a large scale, two obstacles need to be overcome; the availability of large amount of HS sulfotransferases and the inhibition effect of 5′-phosphoadenosine 3′-phosphate (PAP), the desulfated product of PAPS. A proof of principle study has been recently published using completely desulfated and N-sulfated heparin (1) as a starting material (Figure 6) to conduct the O-sulfonation reactions (55). First, highly active HS O-sulfotransferases were expressed in E. coli, in order to provide sufficient quantities of sulfotransferases.…”