biostudies-literatureLi JNIA NIH HHSEshelman Institute for Innovation, University of North CarolinaNational Heart, Lung, and Blood InstituteNHLBI NIH HHSNational Institute of General Medical SciencesNIGMS NIH HHSNational Institute on Aging2026-2035https://www.ebi.ac.uk/biostudies/studies/S-EPMC8526403biostudies-literatureUnknown16(10)Heparan sulfate (HS) 3-O-sulfotransferase isoform 4 (3-OST-4) is a specialized carbohydrate sulfotransferase participating in the biosynthesis of heparan sulfate. Here, we report the expression and purification of the recombinant 3-OST-4 enzyme and use it for the synthesis of a library of 3-O-sulfated hexasaccharides and 3-O-sulfated octasaccharides. The unique structural feature of the library is that each oligosaccharide contains a disaccharide domain with a 2-O-sulfated glucuronic acid (GlcA2S) and 3-O-sulfated glucosamine (GlcNS3S). By rearranging the order of the enzymatic modification steps, we demonstrate the synthesis of oligosaccharides with different saccharide sequences. The structural characterization was completed by electrospray ionization mass spectrometry and NMR. These 3-O-sulfated oligosaccharides show weak to very weak anti-Factor Xa activity, a measurement of anticoagulant activity. We discovered that HSoligo 7 (HS oligosaccharide 7), a 3-O-sulfated octasaccharide, binds to high mobility group box 1 protein (HMGB1) and tau protein, both believed to be involved in the process of inflammation. Access to the recombinant 3-OST-4 expands the capability of the chemoenzymatic method to synthesize novel 3-O-sulfated oligosaccharides. The oligosaccharides will become valuable reagents to probe the biological functions of 3-O-sulfated HS and to develop HS-based therapeutic agents.ACS chemical biologySynthesis of 3-O-Sulfated Heparan Sulfate Oligosaccharides Using 3-O-Sulfotransferase Isoform 4.PMC8526403R01 HL094463R44 HL139187R41 HL139187R42 GM128484RF1 AG069039HL144970GM134738HL139187AG069039GM123792R44 GM123792R44 GM134738GM128484R41 GM123792R01 HL144970HL094463R01 HL062244Liu JLi JArnold KWang CPagadala VSu GXu YfalseSynthesis of 3-O-Sulfated Heparan Sulfate Oligosaccharides Using 3-O-Sulfotransferase Isoform 4.Heparan sulfate (HS) 3-O-sulfotransferase isoform 4 (3-OST-4) is a specialized carbohydrate sulfotransferase participating in the biosynthesis of heparan sulfate. Here, we report the expression and purification of the recombinant 3-OST-4 enzyme and use it for the synthesis of a library of 3-O-sulfated hexasaccharides and 3-O-sulfated octasaccharides. The unique structural feature of the library is that each oligosaccharide contains a disaccharide domain with a 2-O-sulfated glucuronic acid (GlcA2S) and 3-O-sulfated glucosamine (GlcNS3S). By rearranging the order of the enzymatic modification steps, we demonstrate the synthesis of oligosaccharides with different saccharide sequences. The structural characterization was completed by electrospray ionization mass spectrometry and NMR. These 3-O-sulfated oligosaccharides show weak to very weak anti-Factor Xa activity, a measurement of anticoagulant activity. We discovered that HSoligo 7 (HS oligosaccharide 7), a 3-O-sulfated octasaccharide, binds to high mobility group box 1 protein (HMGB1) and tau protein, both believed to be involved in the process of inflammation. Access to the recombinant 3-OST-4 expands the capability of the chemoenzymatic method to synthesize novel 3-O-sulfated oligosaccharides. The oligosaccharides will become valuable reagents to probe the biological functions of 3-O-sulfated HS and to develop HS-based therapeutic agents.2021-01-01T00:00:00Z2021 Oct2025-04-04T20:04:13.062Z2025-04-04T20:04:13.062ZS-EPMC85264033435173210.1021/acschembio.1c00474