MOLECULAR CHARACTERIZATION OF HEDGEHOG AND DECAPENTAPLEGIC INTERACTIONS WITH HEPARIN

Morphogen gradients play essential roles in tissue development and homeostasis by regulating cell–cell communication through paracrine signaling. In Drosophila melanogaster, Hedgehog (Hh) and Decapentaplegic (Dpp) are key morphogens whose activity is modulated by interactions with heparan sulfate (HS). Although these interactions are critical for morphogen distribution and signaling, the molecular determinants underlying morphogen–HS recognition remain poorly understood. Herein, we investigated the molecular basis of Hh and Dpp interactions with heparin, used as a structural model of HS, combining bioinformatics and biochemical approaches. Bioinformatic analyses identified positively charged regions in the wild-type proteins as potential heparin-binding sites. Based on these predictions, 16 mutants of each morphogen were generated and characterized by fluorescence spectroscopy, circular dichroism (CD), and dynamic light scattering (DLS). Fluorescence measurements revealed significant quenching of the wild-type proteins upon heparin addition, whereas this effect was attenuated in the mutant variants, indicating that the targeted residues contribute to heparin recognition. Consistently, CD analyses showed reduced heparin-binding affinity following residue substitutions compared with the corresponding wild-type proteins. DLS measurements further demonstrated the formation of stable macromolecular complexes in the presence of heparin. Notably, wild-type Hh and Dpp exhibited distinct interaction dynamics and a greater propensity to form large-scale aggregates, suggesting that heparin binding may influence not only morphogen association but also their supramolecular organization. Overall, these results identify molecular determinants involved in Hh– and Dpp–heparin interactions and provide insights into how HS may regulate morphogen properties and paracrine signaling. Given the evolutionary conservation of these pathways, these findings may also contribute to understanding the regulation of mammalian morphogens, including Sonic Hedgehog and Bone Morphogenetic Proteins, and their dysregulation in human disease. Importantly, defining the residues that mediate HS recognition provides a molecular framework for understanding how extracellular matrix components modulate morphogen availability, distribution, and signaling range. Such mechanisms may be particularly relevant to conserved signaling systems in vertebrates, where aberrant regulation of SHH and BMP pathways is associated with developmental disorders and cancer. Thus, the present study establishes an experimental framework for dissecting morphogen–HS interactions and their structural consequences, supporting further investigation of their roles in physiological and pathological processes.