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Overexpression of heparan sulfate 6-O-sulfotransferases in human embryonic kidney 293 cells results in increased N-acetyl glucosaminyl 6-O-sulfation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
(English)Manuscript (Other academic)
Identifiers
URN: urn:nbn:se:uu:diva-92507OAI: oai:DiVA.org:uu-92507DiVA: diva2:165615
Available from: 2004-12-30 Created: 2004-12-30 Last updated: 2011-06-28Bibliographically 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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 2
Keyword
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)
Identifiers
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
Opponent
Supervisors
Available from: 2004-12-30 Created: 2004-12-30Bibliographically approved

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Spillmann, Dorothe

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