Determination and biochemical characterization of heparan sulfate interaction residues in the morphogens Hedgehog, Decapentaplegic, and Wingless

• Grant number: 24/13088-8
• Support Opportunities: Scholarships in Brazil - Master
• Start date: July 01, 2025
• End date: July 31, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Guilherme Oliveira Barbosa
• Grantee: Geovana Almeida Carneiro
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

Most paracrine signaling morphogens are heparan sulfate (HS) ligands. This property of HS is extremely significant for cellular signaling activity during the development and homeostasis of organs and tissues. It is known that the interaction between HS ligands and HS is primarily ionic. Therefore, characterizing this interaction at the molecular level is important for deepening the understanding of how HS regulates paracrine signaling. Thus, the aim of this project is to identify, through bioinformatics, the heparan binding site (HBS) of the morphogens Hh, Dpp, and Wg to map in vitro the most relevant HBS residues for the interaction between the morphogens and HS. Subsequently, through site-directed mutagenesis, arginine and lysine residues will be substituted with glutamate, making the electric potential of the binding site negative and repulsive to HS (”HBS). After the expression and purification of the different morphogen variants, we will verify the effect of each identified residue substitution on morphogen affinity for HS through affinity chromatography assays using a Sepharose-heparin column. Finally, we will determine whether the acidification of the medium can exert any effect on this interaction, mimicking in vitro what happens in the protein export process in biosynthetic pathways. Thus, we will dissect at the amino acid level the biochemical role of the interaction of these morphogens with HS (AU)