Biosynthesis of 3-O-sulfated heparan sulfate: Unique substrate specificity of heparan sulfate 3-O-sulfotransferase isoform 5

Jinghua Chen, Michael B Duncan, Kevin Carrick, R. Marshall Pope, Jian Liu

Research output: Contribution to journalArticle

34 Citations (Scopus)

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 ΔUA2S-GlcNS3S and a tetrasaccharide with a structure of ΔUA2S-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.

Original languageEnglish (US)
Pages (from-to)785-794
Number of pages10
JournalGlycobiology
Volume13
Issue number11
DOIs
StatePublished - Nov 1 2003

Fingerprint

Sulfotransferases
Heparitin Sulfate
Biosynthesis
Substrate Specificity
Protein Isoforms
Substrates
Glucosamine
Binding Sites
Enzymes
Antithrombins
Sulfates
Iduronic Acid
Heparin Lyase
Nitrous Acid
Agarose Chromatography
Disaccharides
Baculoviridae
Simplexvirus
Chromatography
Viruses

Keywords

  • Anticoagulation
  • Antithrombin
  • Heparan sulfate
  • Heparan sulfate sulfotransferase
  • Herpes simplex virus

ASJC Scopus subject areas

  • Biochemistry

Cite this

Biosynthesis of 3-O-sulfated heparan sulfate : Unique substrate specificity of heparan sulfate 3-O-sulfotransferase isoform 5. / Chen, Jinghua; Duncan, Michael B; Carrick, Kevin; Pope, R. Marshall; Liu, Jian.

In: Glycobiology, Vol. 13, No. 11, 01.11.2003, p. 785-794.

Research output: Contribution to journalArticle

Chen, Jinghua ; Duncan, Michael B ; Carrick, Kevin ; Pope, R. Marshall ; Liu, Jian. / Biosynthesis of 3-O-sulfated heparan sulfate : Unique substrate specificity of heparan sulfate 3-O-sulfotransferase isoform 5. In: Glycobiology. 2003 ; Vol. 13, No. 11. pp. 785-794.
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AB - 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 ΔUA2S-GlcNS3S and a tetrasaccharide with a structure of ΔUA2S-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.

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