Oligosaccharide library-based assessment of heparan sulfate 6-O-sulfotransferase substrate specificity
(2003) In The Journal of biological chemistry 278(27). p.6-24371- 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... (More)
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.
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- author
- Jemth, Per ; Smeds, Emanuel LU ; Do, Anh-Tri ; Habuchi, Hiroko ; Kimata, Koji ; Lindahl, Ulf and Kusche-Gullberg, Marion
- publishing date
- 2003
- type
- Contribution to journal
- publication status
- published
- keywords
- Animals, Cell Line, Combinatorial Chemistry Techniques, Heparitin Sulfate/metabolism, Humans, Mice, Oligosaccharides, Substrate Specificity/genetics, Sulfates, Sulfotransferases/genetics
- in
- The Journal of biological chemistry
- volume
- 278
- issue
- 27
- pages
- 6 - 24371
- publisher
- American Society for Biochemistry and Molecular Biology
- external identifiers
-
- pmid:12702732
- scopus:0043092177
- ISSN
- 0021-9258
- DOI
- 10.1074/jbc.M212155200
- language
- English
- LU publication?
- no
- id
- 61708450-a04a-40f3-8050-15a033528015
- date added to LUP
- 2021-07-01 16:43:32
- date last changed
- 2024-12-30 09:59:06
@article{61708450-a04a-40f3-8050-15a033528015,
abstract = {{<p>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.</p>}},
author = {{Jemth, Per and Smeds, Emanuel and Do, Anh-Tri and Habuchi, Hiroko and Kimata, Koji and Lindahl, Ulf and Kusche-Gullberg, Marion}},
issn = {{0021-9258}},
keywords = {{Animals; Cell Line; Combinatorial Chemistry Techniques; Heparitin Sulfate/metabolism; Humans; Mice; Oligosaccharides; Substrate Specificity/genetics; Sulfates; Sulfotransferases/genetics}},
language = {{eng}},
number = {{27}},
pages = {{6--24371}},
publisher = {{American Society for Biochemistry and Molecular Biology}},
series = {{The Journal of biological chemistry}},
title = {{Oligosaccharide library-based assessment of heparan sulfate 6-O-sulfotransferase substrate specificity}},
url = {{http://dx.doi.org/10.1074/jbc.M212155200}},
doi = {{10.1074/jbc.M212155200}},
volume = {{278}},
year = {{2003}},
}