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.
Highlights
Introduction of PAPS Regeneration SystemPAP inhibits sulfotransferase-catalyzed reactions
Heparin and HS have a wide range of biological activities, including anticoagulation, antiviral, and anticancer activities
Sulfo group-containing saccharide sequences dominate the specificity of the functions of heparin and HS
Summary
Introduction of PAPS Regeneration SystemPAP inhibits sulfotransferase-catalyzed reactions. Complete modification of the substrate could be demonstrated by the low susceptibility of polysaccharide product to undergo additional sulfation using [35S]PAPS with soluble enzymes, as described under “Experimental Procedures.”. 2-OST, 6-OST, and 3-OST-1 afforded 98, 97, and 98% complete modification, respectively, using the PAPS regeneration system. These results demonstrate that the PAPS regeneration system functioned properly, providing sufficient PAPS for the complete sulfotransferase-catalyzed modification of polysaccharide substrates. This conclusion was further supported by characterizing the structures of the newly synthesized polysaccharide products 2, 3, 4a, 4b, and 5 (described below). We used [35S]PAPS as the sulfo donor to sulfate the polysaccharides in order to estimate the num-
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