Independently our lab is focused on the development of specific sulfotransferases to be used in enzymatic production of therapeutic heparan sulfate. Heparan sulfates (HS) are linear sulfated polysaccharides present on the cell surface and in the extra cellular matrix that play important roles in blood coagulation, inflammation response, cell differentiation and assist in bacterial and viral infection. The specific sulfation pattern of HS determines its functional selectivity. Different sulfotransferases are required for sulfation of specific hydroxyl groups or amines along the polysaccharide. Heparin is a highly sulfated form of HS. Theraputic heparin is a 3 billion dollar a year industry as an anticoagulant. In addition low molecular weight heparin/heparan sulfate mimics show promise as potential anti-cancer/anti-metastasis drugs possibly due to their role in growth factor regulation and as heparanase inhibitors. Currently therapeutic heparin is purified from mast cells of mammalian sources. This can lead to contamination problems as well as and perhaps more importantly heterogeneity problems. One of the major side-effects of administration of heparin is thrombocytopenia due to interaction of the heparin with platelet factor 4 (PF4). Chemical synthesis of homogeneous polysaccharides larger than a hexasaccharide is extremely difficult and currently too challenging for mass production. In collaboration with the Liu lab in the School of Pharmacy at UNC we have been designing mutant forms of HS sulfotransferases for use in enzymatic production of heparan sulfates with specific functions. To accomplish this, we first solve the structures of the sulfotransferases then use structural guided alanine scanning mutagenesis of the active site to try to alter the selectivity of the enzyme. Our most exciting results so far have been mutants of the 2-O-sulfotransferase that allow us to selectively sulfate either iduronic acid or glucuronic acid. In vivo iduronic acid is typically the substrate of choice as it is important for initiating FGF/FGF dimerization and further interaction with the FGF receptor. However as an anti-coagulant this sulfation can play a deleterious role in PF4 binding thus initiating thrombocytopenia. Use of 2-O glucuronic acid containing HS could potentially be utilized for anti-coagulation therapy with reduced side effects while the 2-O iduronic acid containing HS could potentially be used for anti-cancer therapy. Thus the ability to enzymatically design homogenous HS could potentially lead to safer more specific heparin/HS therapies. Another focus of the Structure Function Group is to use X-ray crystallography to support research interest of principal investigators within the intramural community. One of our more recent collaborations is with Geoffrey Mueller in the London group studying crystal structures of dust mite allergens. Recently(unpublished), we have solved the crystal structure of Der p 7, an allergen from the house dust mite Dermatophagoides pteronyssinus. Skin tests have shown reactivity of Der p 7 in 53% of allergic patients yet very little is known about this protein. The crystal structure has revealed a BPI domain-like fold suggesting a possible role as a lipid transporter for lipids such as LPS. Current studies are now underway to investigate this hypothesis. In theory, structural information with regards to Der p 7 could allow for the design of recombinant hypoallergenic Der p 7 for immunotherapy. We are currently working on the crystal structure of Der p 5 another allergen from the house duct mite. In addition, we have extensive collaborations with the Wilson and Kunkel laboratories studying crystal structures of polymerases involved in DNA repair process. In collaboration we continue to help determine structures of human X-family polymerases beta and lambda to better understand the role of these enzymes in base excision and non-homologous end-joining repair processes. As well, we published a paper in collaboration with the Armstrong group in the laboratory of Neurobiology on the crystal structure of RACK1 from Arabidopsis thaliana. This structure provides a structural basis for understanding RACK-1-mediated cellular signaling pathways in both plants and animals.
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