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EXT1, EXT2 and EXTL3 siRNA gene silencing result in altered heparan sulfate chain length
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Manuscript (Other academic)
URN: urn:nbn:se:uu:diva-93949OAI: oai:DiVA.org:uu-93949DiVA: diva2:167607
Available from: 2006-01-26 Created: 2006-01-26 Last updated: 2010-01-13Bibliographically approved
In thesis
1. EXT Proteins in Heparan Sulfate Biosynthesis
Open this publication in new window or tab >>EXT Proteins in Heparan Sulfate Biosynthesis
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Heparan sulfate (HS) is a long unbranched polysaccharide composed of alternating glucosamine and hexuronic (glucuronic or iduronic) acid residues. Modification by sulfate groups in various positions generates a highly heterogeneous molecule. HS is synthesized as a proteoglycan by virtually all cells, and play pivotal functions in signaling and developmental patterning, but also in pathogenic events such as tumor metastasis and microbial adhesion.

This thesis deals with the properties of enzymes involved in HS chain elongation. Polymerization of the HS chain is believed to be catalyzed by the EXT family of proteins. In humans, the EXT family consists of five members: EXT1, EXT2, EXTL1, EXTL2 and EXTL3; their respective functions in HS biosynthesis are not fully understood.

In this study, for the first time, successful in vitro HS polymerization on oligosaccharide acceptor substrates was demonstrated, using recombinant EXT1 and EXT1/EXT2 complex. EXT1 formed longer chains than EXT1/EXT2 and their mechanisms of sugar incorporation were different.

Suppression of EXT1 or EXT2 expression by siRNA in a human cell line resulted in reduction of HS chain length. In contrast, cells transfected with EXTL3 siRNA produced longer HS chains. Overexpression of soluble EXT1, alone or co-expressed with EXT2, resulted in increased chain length, whereas overexpression of soluble EXT2 or EXTL3 has no detectable effect on HS chain elongation.

Structural analysis of HS from fibroblasts isolated from mice with a hypomorphic mutation in Ext1 showed that they produced significantly shorter HS chains then the wild-type fibroblasts (20 and 70 kDa, respectively). The disaccharide composition of the HS produced by the mutant cells was virtually indistinguishable from that of the wild-type HS, however, the mutant HS chains contained higher proportions of unmodified regions. Mutant cells responded less efficiently than wild-type cells to low concentrations of FGF2, as analyzed by ERK phosphorylation assay.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 48 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 101
Molecular biology, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-6303 (URN)91-554-6445-9 (ISBN)
Public defence
2006-02-10, Room C10:310, BMC, Uppsala, 13:15
Available from: 2006-01-26 Created: 2006-01-26Bibliographically approved

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