title: Biosynthesis of 3-O-sulfated heparan sulfate: unique substrate specificity of heparan sulfate 3-O-sulfotransferase isoform 5.
abstract: Heparan sulfate 3-O-sulfotransferase transfers sulfate to the 3-OH position of a glucosamine to generate 3-O-sulfated heparan sulfate (HS), which is a rare component in HS from natural sources. We previously reported that 3-O- sulfotransferase isoform 5 (3-OST-5) generates both an antithrombin-binding site to exhibit anticoagulant activity and a binding site for herpes simplex virus 1 glycoprotein D to serve as an entry receptor for herpes simplex virus. In this study, we characterize the substrate specificity of 3-OST-5 using the purified enzyme. The enzyme was expressed in insect cells using the baculovirus expression approach and was purified by using heparin-Sepharose and 3',5'-ADP- agarose chromatographies. As expected, the purified enzyme generates both an antithrombin binding site and a glycoprotein D binding site. We isolated IdoUA-AnMan3S and IdoUA-AnMan3S6S from nitrous acid-degraded 3-OST-5-modified HS (pH 1.5), suggesting that 3-OST-5 enzyme sulfates the glucosamine residue that is linked to an iduronic acid residue at the nonreducing end. We also isolated a disaccharide with a structure of DeltaUA2S-GlcNS3S and a tetrasaccharide with a structure of DeltaUA2S-GlcNS-IdoUA2S-GlcNH23S6S from heparin lyases-digested 3-OST-5-modified HS. Our results suggest that 3-OST-5 enzyme sulfates both N-sulfated glucosamine and N-unsubstituted glucosamine residues. Taken together, the results indicate that 3-OST-5 has broader substrate specificity than those of 3-OST-1 and 3-OST-3. The unique substrate specificity of 3-OST-5 serves as an additional tool to study the mechanism for the biosynthesis of biologically active HS.
authors.affiliation: Division of Medicinal Chemistry and Natural Products, School of Pharmacy, CB#7360, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Jinghua
authors.initials: J
authors.lastname: Chen
authors.forename: Michael B
authors.initials: MB
authors.lastname: Duncan
authors.forename: Kevin
authors.initials: K
authors.lastname: Carrick
authors.forename: R Marshall
authors.initials: RM
authors.lastname: Pope
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
mesh_terms: D000818:Animals; D000991:Antithrombins / Q000378:metabolism; D002460:Cell Line; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000737:chemistry; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000991:Antithrombins; D006497:Heparan Sulfate; D007527:Isoenzymes; D011994:Recombinant Proteins; D015238:Sulfotransferases; C021350:heparitin sulfotransferase