Structure Function Group
Heparan sulfates (HS) are highly sulfated polysaccharides found on the surface of mammalian cells attached to core proteins and can also be secreted into the extracellular matrix. HS can influence a wide range of physiological and pathophysiological functions such as viral/bacterial infection, angiogenesis, embryonic development and blood coagulation through interactions with proteins such as growth factors, protease inhibitors, proteases, cytokines, chemokines and morphogens. Many of these interactions depend on specific sulfation patterns along the HS chain. HS is mainly composed of repeating disaccharide units of glucuronic (GlcA) or iduronic acid (IdoA) along with glucosamine (GlcN). These HS chains are modified in the Golgi by modifying enzymes including a C5-epimerase that converts GlcA to IdoA and a N-deacetylase/N-sulfotransferase which can deacetylate then sulfate the N-acetyl group of GlcN. In addition to these enzymes, there is a sulfotransferase (2-OST) that can sulfate the 2 position of the GlcA or Ido and sulfotransferases that can sulfate the 3- and 6- positions (3-OSTs and 6-OSTs respectively) of the GlcN saccharides. Depending on the cell type and isoforms of the sulfotransferases being expressed different sulfation patterns can occur generating HS that interact with different proteins.
The most characterized example of the HS specificity is a highly sulfated form of HS known as heparin which has a rare 3-O sulfation that allows it to bind anti-thrombin with high specificity inducing a conformational change that then allows anti-throbmin to inhibit the blood clotting enzymes thrombin and factor X. Heparin purified from pig is widely used in the clinic as a potent anti-coagulant whose activity can be reversed by administering protamine. One down side of heparin is that it is heterogenous in nature and on rare occasions it can induce thrombocytopenia that can lead to lose of limb, pulmonary embolisms and death.
Current technology being developed by Jian Liu at the University of North Carolina allows for the production of homogeneous HS for therapeutic purposes using a technique called chemoenzymatic synthesis that utilizes the specificity of the sulfotransferases to generate the desired sulfation pattern. This technique can be used to generate HS that is a potent/reversible anti-coagulant that should have fewer side effects than heparin. In addition, it is conceivable that the synthesis may be tailored to treat other ailments such as inflammation or cancer. In collaboration with Dr. Liu, our lab has solved crystal structures of a number of sulfotransferases with bound HS. From these structures we learn what dictates specificity within these enzymes and we are using this knowledge to change their specificity by site-directed mutatgenesis. It is our hope that these altered enzymes will improve the homogeneity of the desired HS as well as allow for the generation of unique HS sequences which may benefit treatment.