DETERMINATION AND BIOCHEMICAL CHARACTERIZATION OF HEPARAN SULFATE INTERACTION RESIDUES IN THE MORPHOGENS HEDGEHOG, DECAPENTAPLEGIC AND WINGLESS

In cell communication as paracrinal signaling, the morphogen gradients regulate tissue development and homeostasis in animals. Most Drosophila melanogaster morphogens, such as Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) - homologous to Sonic Hedgehog, BMP and WNT in humans, respectively -, interact with heparan sulfate (HS), an essential polysaccharide in cellular signaling. It is known that this is mainly an ionic interaction, and molecular characterization studies are important for understanding how HS regulates paracrinal signalling. The main objective of this project is to map the binding site of these morphogens in order to identify the key residues involved in this interaction. The morphogen protein structures were submitted to molecular docking with heparin and molecular dynamics simulations in order to map the interaction binding site, a helpful approach in recognising the interacting residues. A high-throughput cloning approach enabled the efficient expression and purification of numerous protein constructs. Based on available structural data in research, four variants were designed for each morphogen that was cloned into five plasmids, that add different purification tag combinations into N-terminal and/or C-terminal regions using ligation independent cloning (LIC). We performed a small-scale expression test using different lines of E. coli (BL21) competent. A total of 60 constructs, each with 20 different variants of morphogens, were successfully cloned. The SDS-PAGE gel analysis revealed six high-quality constructs for Hh and six for Dpp. Among these, vector constructions at pNIC-28-Bsa4 and pSUMO-LIC were selected for large-scale protein purification. The purified proteins (Hh and Dpp) were found to interact with Heparin-Sepharose column during affinity chromatography, as demonstrated by their elution with 1M NaCl. To Wg will involve an expression test on the constructs in insect cells. We aim to perform site-directed mutagenesis on the residues identified by molecular dynamics simulations (arginine and lysine) and replace them with glutamic acid, to make the electrostatic potential negative and repulsive to HS. The understanding of the biochemical role of morphogens interaction with HS can allow great insights about regulation mechanisms in paracrine signaling pathways, enabling the development of personalized and modern therapeutic strategies, especially in disease in which paracrine signaling is directly related.