How pH and sulfation shape human heparanase structure, stability, and substrate recognition

Human heparanase (HPSE) is an endo-β-d-glucuronidase involved in the degradation of heparan sulfate and in several physiopathological processes, including inflammation, metastasis, and extracellular matrix remodeling. Its activity is strongly influenced by pH and by the sulfation degree of the substrate, yet the structural bases underlying these effects remain only partially understood. In this work, we combine FTIR spectroscopy, temperature-dependent intrinsic fluorescence, and molecular dynamics (MD) simulation experiments to investigate the secondary structure, conformational stability, and structural determinants of substrate recognition in HPSE under different physicochemical conditions. FTIR analyses reveal pH-dependent variations in secondary structure, with acidic environments promoting a more compact and α-helix-rich conformation, consistent with the lysosomal conditions in which HPSE is physiologically active. Fluorescence experiments show a gradual unfolding process at all pH values, with maximal protein stability at pH 5.0. MD simulations of HPSE complexed with heparan-sulfate tetrasaccharides of different sulfation degrees indicate that ionic and hydrogen-bond interactions strongly contribute to binding affinity with highly sulfated ligands forming more stable and persistent interactions. Overall, the integration of spectroscopic analyses and computational simulations suggests that acidic pH and high substrate sulfation favor structural stability and ligand binding in HPSE, providing insights into the physicochemical factors regulating its activity.