Novel chemical and enzymatic routes to the generation of heparin- related polysaccarides

Abstract: Heparin and heparan sulphate (HS) are structurally related polysaccharides influencing various biological processes. The biological functions of heparin/HS largely depend on interactions between the negatively charged polysaccharide chains and a variety of proteins. Heparin/HS is generated through a complex biosynthetic process, which apparently requires a coordinated action of several enzymes. This thesis aimed to address two questions: 1) how 2-O-sulphation of L-iduronic acid (IdoA) and D-glucuronic acid (GlcA) residues is accomplished in the biosynthesis of heparin/HS; 2) how different saccharide domains must be organised in order to generate biologically active polysaccharides.Using a DNA probe generated from a Chinese hamster ovary (CHO) cell HS 2-OST clone, we isolated a highly similar 2-OST from a mouse mastocytoma cDNA library. Northern-blotting analysis showed a predominant mRNA transcript of ~3 kb in adult mouse tissues, and in addition, ~5 kb and ~7 kb transcripts in some tissues.Overexpression of 2-OST id a mammalian cell line resulted in GlcA 2-O-sulphation. Incubation of different polysaccharide acceptors with cell extract of either 2-OST transfected cells or control cells in the presence of the active sulphate donor adenosine 3'-phosphate 5'-phospho[35S]sulphate revealed that 2-OST prefers to act on IdoA residues. However, the enzyme is capable of sulphating GlcA residues in polysaccharides lacking IdoA residues. Moreover, the activity of 2-OST requires the presence of N-sulphated glucosamine residues. Our data suggest that 2-O-sulphation of GlcA and IdoA residues is catalysed by the same 2-OST.To facilitate the studies on the domain organisation and its biological importance, we developed a novel strategy to generate biologically active neo-glycosaminoglycan (neo-GAG) conjugates by chemical conjugation of two heparin-oligosaccharides. Heparin prevents blood coagulation by binding to, and thus activating antithrombin (AT). An in vitro assay of thrombin inactivation by AT was performed in the presence of heparin with high affinity (HA) to AT or neo-GAG conjugates. Strikingly, the neo-GAG conjugates with the low affinity (LA)-HA (non-reducing end-reducing end) arrangement showed better efficacy to promote the inactivation of thrombin by AT than that of HA-heparin whereas the conjugates with the LA-LA arrangement were virtually inactive.