Exploring the Schistosoma mansoni SmE16 Protein: A Biophysical Study of Its Interactions and Functions

Schistosoma mansoni is a helminth parasite that causes schistosomiasis, a neglected tropical disease affecting thousands globally. Praziquantel, the primary treatment, disrupts calcium homeostasis in adult worms. However, high reinfection rates could  led to drug resistance in endemic regions, highlighting the urgent need for new treatment strategies. The SmE16 is an immunoreactive secreted protein, highly expressed in the esophagus of adult worms, and predicted to bind calcium due to its EF-hand domains, though its functions remains unknown. This study aims to understand SmE16's function by its biophysical characterization and interactions with other proteins. The SmE16 protein was expressed in E. coli Rosetta (DE3) and purified by affinity chromatography followed by size exclusion chromatography. Circular dichroism (CD) spectroscopy was performed with 5 μM of the protein in Tris buffer (25 mM, pH 8.0) and NaCl (150 mM), incubated with 2 mM CaCl₂. Spectra were obtained from 190-260 nm and 260-320 nm and compared with those of the protein without calcium. SDS-PAGE analysis was conducted after trypsin digestion in the presence and absence of Ca²⁺. Fluorescence spectroscopy, using tryptophan and ANS probes, measured emission between 300-450 nm following excitation at 295 nm. The oligomeric state of the protein was evaluated through crosslinking with DSS and size exclusion chromatography, in the presence of calcium and magnesium ions. CD results indicated that SmE16 showed minimal structural changes in the presence of calcium in the Far UV region, which assesses secondary structure. In the Near UV region, however, calcium binding led to more pronounced conformational changes, also observed in the fluorescence experiments. Calcium had a great effect on protein stability, as assessed  by DSC (Differential scanning calorimetry). Trypsin digestion revealed a distinct pattern of bands in the apo and Ca²⁺ bound forms, suggesting a conformational change. Crosslinking experiments showed the formation of dimers with calcium, while magnesium did not interact with SmE16. Our biophysical analyses revealed that SmE16 undergoes significant structural changes in the presence of calcium ions. These changes are characteristic of calcium sensor proteins, indicating that SmE16 might play an active role in calcium-mediated signaling pathways.