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Heparan sulfate biosynthesis: PAPS regulates NDST-mediated formation of N-sulfated domains
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
(English)Manuscript (Other academic)
URN: urn:nbn:se:uu:diva-94912OAI: oai:DiVA.org:uu-94912DiVA: diva2:168931
Available from: 2006-10-05 Created: 2006-10-05 Last updated: 2011-06-28Bibliographically approved
In thesis
1. N-Sulfation and Polymerization in Heparan Sulfate Biosynthesis
Open this publication in new window or tab >>N-Sulfation and Polymerization in Heparan Sulfate Biosynthesis
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Heparan sulfate (HS) is a glycosaminoglycan present in all cell types covalently attached to core proteins forming proteoglycans. HS interacts with different proteins and thereby affects a variety of processes. The biosynthesis of HS takes place in the Golgi network where a complex of the enzymes EXT1 and EXT2 adds N-acetyl glucosamine and glucuronic acid units to the growing chain. The HS chain is N-sulfated by the enzyme N-deacetylase N-sulfotransferase (NDST). N-Sulfation occurs in domains where further modifications (including O-sulfations) take place, giving the chain a complex sulfation pattern.

In this thesis, new data about the regulation of NDST enzyme activity is presented. By studying NDST1 with active site mutations overexpressed in HEK 293 cells we show that N-deacetylation is the rate-limiting step in HS N-sulfation and that two different NDST molecules can work on the same GlcN unit.

By analyzing recombinant forms of NDST1 and NDST2 we determined the smallest substrate for N-deacetylation to be an octasaccharide. Importantly, the sulfate donor PAPS was shown to regulate the NDST enzymes to modify the HS chain in domains and that binding of PAPS had a stimulating effect on N-deacetylase activity.

We could also show that increased levels of NDST1 were obtained when NDST1 was coexpressed with EXT2, while coexpression with EXT1 had the opposite effect. We suggest that EXT2 binds to NDST1, promoting the transport of functional NDST1 to the Golgi network and that EXT1 competes for binding to EXT2.

Using cell lines overexpressing EXT proteins, it was demonstrated that overexpression of EXT1 increases HS chain length and coexpression of EXT2 results in even longer chains. The enhancing effect of EXT2 was lost when EXT2 was carrying mutations identical to those found in patients with hereditary multiple exostoses, a syndrome characterized by cartilage-capped bony outgrowths at the long bones.


Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 47 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 175
Cell and molecular biology, Heparan sulfate, N-deacetylase / N-sulfotransferase, NDST, EXT1, EXT2, Biosynthesis, Cell- och molekylärbiologi
urn:nbn:se:uu:diva-7162 (URN)91-554-6666-4 (ISBN)
Public defence
2006-10-27, B22, BMC, Husargatan 3, Uppsala, 09:15
Available from: 2006-10-05 Created: 2006-10-05Bibliographically approved

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Spillmann, DorotheKjellén, Lena
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