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Target selection of heparan sulfate hexuronic acid 2-O-sulfotransferase
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2010 (English)In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 20, no 10, 1274-1282 p.Article in journal (Refereed) Published
Abstract [en]

The signaling of various molecules involved in development and regulation of cell growth are regulated by heparan sulfate (HS). Specific binding of HS to ligand proteins depends on the HS sulfation pattern, where the spacing and number of O-sulfate groups are of special importance. HS 2-O-sulfotransferase catalyzes 2-O-sulfation of glucuronic and iduronic acid residues with a 5-fold higher preference for iduronic acid, as inferred from previously determined kinetic parameters. To study in more detail the regulation of HS hexuronic acid 2-O-sulfation, we tested the ability of the enzyme to catalyze glucuronic acid 2-O-sulfation in polysaccharide mixtures with different glucuronic acid/iduronic acid ratios, using 3'-phosphoadenosine 5'-phospho[S-35]sulfate as sulfate donor. The 2-O-sulfotransferase revealed a more pronounced preference for 2-O-sulfation of iduronic acid than predicted. Even incubations with a 99:1 ratio of glucuronic acid to iduronic acid resulted in almost exclusive iduronic acid 2-O-sulfation. Unexpectedly, when the 2-O-sulfotransferase was co-immunoprecipitated with the glucuronyl C5-epimerase (that converts glucuronic acid to iduronic acid), both glucuronic acid and iduronic acid residues were sulfated to the same extent when a polysaccharide containing only glucuronic acid was used as a substrate. Attempting to understand the mechanism by which extended regions of iduronic acid 2-O-sulfation are formed during HS biosynthesis, a H-3-labeled N-sulfated iduronic acid containing octasaccharide substrate was incubated with the 2-O-sulfotransferase and 3'-phosphoadenosine 5'-phosphosulfate. The 2-O-sulfotransferase showed a preference for mono-2-O-sulfated substrates as compared with octasaccharides with no 2-O-sulfate group.

Place, publisher, year, edition, pages
2010. Vol. 20, no 10, 1274-1282 p.
Keyword [en]
2-O-sulfotransferase, C5-epimerase, glucuronic acid 2-O-sulfation, heparan sulfate
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-92508DOI: 10.1093/glycob/cwq089ISI: 000281718300009OAI: oai:DiVA.org:uu-92508DiVA: diva2:165616
Available from: 2004-12-30 Created: 2004-12-30 Last updated: 2010-12-02Bibliographically approved
In thesis
1. In Vitro Studies of the Substrate Specificities of Heparan Sulfate 2-O- and 6-O-sulfotransferases
Open this publication in new window or tab >>In Vitro Studies of the Substrate Specificities of Heparan Sulfate 2-O- and 6-O-sulfotransferases
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Heparan sulfate (HS), a linear negatively charged polysaccharide located at the cell surface and in the extracellular matrix, interacts with, and thereby regulates the functions of numerous proteins. HS-protein interactions depend on the fine structure of HS, especially its sulfation pattern. This thesis aimed to understand how differently sulfated domains in HS are generated. Specifically, the substrate specificities of HS hexuronic acid 2-O-sulfotransferase (2OST) and HS glucosaminyl 6-O-sulfotransferases (6OSTs) were investigated.

Three different 6OSTs (6OST1-3) have been cloned and characterized. To study the mechanisms controlling 6-O-sulfation we incubated the recombinant purified 6-OST isoforms with different 6-O-desulfated poly- and oligosaccharide substrates and the active sulfate donor 3'-phosphoadenosine 5'-phospho[35S]sulfate (35S-labeled PAPS). All three enzymes catalyzed 6-O-sulfation of both N-acetylated (GlcNAc) as well as N-sulfated (GlcNS) glucosamines next to a nonreducing iduronic acid (IdoA) or glucuronic acid (GlcA). Similar specificities were demonstrated, although some differences in substrate preferences were noted.

To understand how pre-existing 2-O-sulfates affects 6-O-sulfation, 6OST2 and 6OST3 were incubated with pair-wise mixed octasaccharide substrates with different contents of 2-O-sulfates. The specificities for substrates with two or three 2-O-sulfates were higher compared to octasaccharides with no or one 2-O-sulfate indicating that 2-O-sulfate groups substantially promote the subsequent 6-O-sulfation.

Overexpression of the 6OSTs in a mammalian cell line resulted in increased 6-O-sulfation of -GlcA-GlcNS- and -GlcA-GlcNAc- sequences. The results were not isoform specific, but affected by the overexpression level.

The 2OST catalyzes 2-O-sulfation of both IdoA and GlcA residues, with high preference for IdoA units. To study how 2-O-sulfation of GlcA and IdoA is regulated, we incubated the enzyme with different substrates and 35S-labeled PAPS. Our findings revealed that the 2OST almost exclusively sulfated IdoA also with a ratio of GlcA to IdoA of 99:1, suggesting that 2-O-sulfation of GlcA occurs before IdoA is formed.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 59 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 2
Biochemistry, heparan sulfate, heparin, sulfotransferase, 2-O-sulfotransferase, 6-O-sulfotransferase, polysaccharide biosynthesis, glycosaminoglycan, O-sulfotransferase, proteoglycan, Biokemi
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
urn:nbn:se:uu:diva-4738 (URN)91-554-6128-X (ISBN)
Public defence
2005-01-21, C10:301, BMC, Husargatan 3, Uppsala, 09:15
Available from: 2004-12-30 Created: 2004-12-30Bibliographically approved

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