title: Oligosaccharide library-based assessment of heparan sulfate 6-O-sulfotransferase substrate specificity.
abstract: Heparan sulfate mediates numerous complex biological processes. Its action critically depends on the amount and the positions of O-sulfate groups (iduronyl 2-O-sulfates, glucosaminyl 6-O- and 3-O-sulfates) that form binding sites for proteins. The structures and distribution of these protein-binding domains are influenced by the expression and substrate specificity of heparan sulfate biosynthetic enzymes. We describe a general approach to assess substrate specificities of enzymes involved in glycosaminoglycan metabolism, here applied to 6-O-sulfotransferases involved in heparan sulfate biosynthesis. To understand how 2-O-sulfation affects subsequent 6-O-sulfation reactions, the substrate specificity of 6-O-sulfotransferase 3 was probed using substrates from a heparin-based octasaccharide library. Purified 3H-labeled N-sulfated octasaccharides from a library designed to sample 2-O-sulfated motifs were used as sulfate acceptors, 3'-phosphoadenosine 5'-phosphosulfate as sulfate donor, and cell extract from 6-O-sulfotransferase 3-overexpressing 293 cells as enzyme source in the 6-O-sulfotransferase-catalyzed reactions. The first 6-O-sulfate group was preferentially incorporated at the internal glucosamine unit of the octasaccharide substrate. As the reaction proceeded, the octasaccharides acquired three 6-O-sulfate groups. The specificities toward competing octasaccharide substrates, for 6-O-sulfotransferase 2 and 6-O-sulfotransferase 3, were determined using overexpressing 293 cell extracts and purified octasaccharides. Both 6-O-sulfotransferases showed a preference for 2-O-sulfated substrates. The specificity toward substrates with two to three 2-O-sulfate groups was three to five times higher as compared with octasaccharides with no or one 2-O-sulfate group.
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, Biomedical Center, Box 582, Sweden.
authors.forename: Per
authors.initials: P
authors.lastname: Jemth
authors.forename: Emanuel
authors.initials: E
authors.lastname: Smeds
authors.forename: Anh-Tri
authors.initials: AT
authors.lastname: Do
authors.forename: Hiroko
authors.initials: H
authors.lastname: Habuchi
authors.forename: Koji
authors.initials: K
authors.lastname: Kimata
authors.forename: Ulf
authors.initials: U
authors.lastname: Lindahl
authors.forename: Marion
authors.initials: M
authors.lastname: Kusche-Gullberg
mesh_terms: D000818:Animals; D002460:Cell Line; D020650:Combinatorial Chemistry Techniques; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D051379:Mice; D009844:Oligosaccharides; D013379:Substrate Specificity / Q000235:genetics; D013431:Sulfates; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*
chemical_list: D006497:Heparan Sulfate; D009844:Oligosaccharides; D013431:Sulfates; D015238:Sulfotransferases; C485757:HS6ST2 protein, human; C485756:Hs6st1 protein, mouse; C485758:Hs6st2 protein, mouse; C485759:Hs6st3 protein, mouse; C404189:heparan sulfate 6-O-sulfotransferase