Regulation of Heparan Sulfate 6-O-Sulfation Patterns

Sammanfattning: Heparan sulfates (HSs) are linear, negatively charged polysaccharides composed of alternating hexuronic acid (glucuronic acid or iduronic acid) and glucosamine residues that can be substituted to varying degrees with sulfate groups. HS, localized in the extracellular matrix and on the surface of most cells, interacts with a large number of proteins. The actions of HS largely depend on the amount and distribution of its sulfate groups, that provide binding sites for proteins. This thesis focuses on the regulation of the structural diversity in HS, in particular the regulation of its 6-O-sulfation patterns that are generated by the combined action of 6-O-sulfotransferases (6OSTs) during biosynthesis, and 6-O-endosulfatases (Sulfs) after completed biosynthesis. In addition, a new model organism is introduced that offers good prospects for investigating the evolutional aspects of HS structural heterogeneity.Our studies showed that the three mouse 6OSTs (6OST1-3) exhibit similar substrate specificities in vitro, with minor differences in target preferences. Overexpression of the 6OSTs in cells resulted in increased 6-O-sulfation of both N-sulfated and N-acetylated glucosamine residues. The changes were independent of enzyme isoform but positively correlated to the level of enzyme expressed.Quail Sulf1 and Sulf2 enzymes were shown to be cell surface HS 6-O-endosulfatases with preference towards a subset of trisulfated disaccharides within HS chains. The Sulfs contain a “hydrophilic domain” that was shown to be essential for binding of HS, anchorage to the cell surface and endosulfatase activity. QSulf1 was also shown to promote Wnt-Frizzled signaling in cells. An HS-like polysaccharide was isolated from the sea anemone Nematostella vectensis and characterized, and all the enzyme families involved in HS biosynthesis and modification in mammalian model systems were also identified. Our results suggest that Nematostella may be a useful tool for understanding the role of evolution in generating HS structural diversity.