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 use structural guided mutagenesis to understand the specificity of the heparan sulfate sulfotransferases. If we are able to redesign these enzymes we can tailor the production of task specific heparan sulfates which are more homogenous and have fewer side effects. 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 such collaboration is with Geoffrey Mueller in the London group studying crystal structures of dust mite allergens. This year we published the crystal structures of two dust mite allergens, Der p 7 and Der p 5. 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. The crystal structure of Der p 5 is a three helical bundle and resolved a dispute in the literature with regard to the ordering of the helices for Group 5 allergens. The structure suggests that Der p 5 also has the potential to bind hydrophobic ligands and suggests a potential to form higher ordered oligomers. In theory, structural information on these allergens could allow for the design of recombinant hypoallergenic proteins for immunotherapy. 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. This past year we also published on a technique that we developed which appears to improve the likelihood of obtaining diffraction quality crystals from recalcitrant proteins. Combining the techniques of MBP-fusion/fix-arm crystallization with surface entropy reduction, we have developed a suite of vectors with different mutations on the surface of the MBP that allows one to screen for a specific target using multiple surfaces that can form potential crystal lattice contacts. This technique has allowed us to solve the structure of five proteins in the lab that we were unable to obtain structures of prior to applying this technique.
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