Oligosaccharide library-based assessment of heparan sulfate 6-O-sulfotransferase substrate specificity.
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.
Jemth
Per
P
Department of Medical Biochemistry and Microbiology, Uppsala University, Biomedical Center, Box 582, Sweden.
Smeds
Emanuel
E
Do
Anh-Tri
AT
Habuchi
Hiroko
H
Kimata
Koji
K
Lindahl
Ulf
U
Kusche-Gullberg
Marion
M
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*
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