Enzymes involved in biosynthesis and degradation of heparin-related polysaccharides

Sammanfattning: Heparin and heparan sulphate (HS) are structurally related polysaccharides of the glycosaminoglycan type. As the glycan moiety of proteoglycans, they exist covalently linked to various core proteins. Highly sulphated, the polysaccharide chains are prone to interact with proteins at the cell surface and in the extracellular matrix, affecting various biological processes. This thesis focuses on two enzymes involved in biosynthesis and degradation of heparin/HS, with the long-term goal to further understand the physiological functions of these polysaccharides.Heparanases are endoglycosidases that cleave HS at a few sites, generating polysaccharide fragments. The substrate specificity of heparanases from human hepatoma and platelets was characterised. We used a bacterial polysaccharide from E. coli K5, identical to the HS "back-bone" structure, as starting material to generatespecifically modified substrates that were tested for heparanase susceptibility. It is concluded that the heparanases from both sources cleave the polysaccharide at the reducing end of a glucuronic acid (GlcA) residue, provided that a 2-O-sulphate group is located two monosaccharide units from the cleavage site.The specific structures recognised by HS interacting proteins, such as the heparanase, are generated by the concerted action of a number of biosynthetic enzymes. The N-acetylglucosamine N-deacetylase/N-sulphotransferase (NDST) holds a key role during biosynthesis. This enzyme N-sulphates certain regions of the polysaccharide, which can then undergo subsequent modifications (GlcA C5-epimerization and O-sul-phation), resulting in highly heterogenous structures. However, the mechanisms for regulation of HS structure are poorly known.Two isoforms of the enzyme, NDST-1 and -2, were cloned from mouse. They are encoded by two separate genes, which are both expressed in a number of tissues. Functional studies of purified NDST-2 indicated its dependence on a polycationic factor to express N-deacetylase activity. The two catalytic activities of this bifunctionalprotein are tightly coupled to each other. Overexpression of the NDST isoforms in a mammalian cell line illustrated their different characteristics, resulting in different HSN-sulphation patterns. These results suggest specific roles for the NDST isoforms in heparin/HS biosynthesis.