Calcium is involved in protein cohesion and interfacial adhesion in a marine invasive fouling ascidian.

Protein-mediated underwater adhesion is vital for the survival of many aquatic organisms and plays central roles in biofouling and bioinspired material development. Metal ions are known to influence underwater adhesion by regulating cohesion between adhesive proteins and interactions at the underwater material interface. However, direct mechanistic evidence of Ca2+ involvement in adhesion of marine organisms remains insufficient. In this study, we investigated the role of Ca2+ in permanent underwater adhesion of ascidian adhesive protein 1 (AAP1), an adhesive protein identified from the ascidian Ciona robusta, a model marine invasive fouling species. Using in vitro experiments, we examined AAP1's cohesion and interfacial adhesion under varying Ca2+ concentrations (0, 1.0, 2.5, 5.0, 10.0, and 25.0 mM). Our results indicated that Ca2+ mediated both cohesion and interfacial adhesion in a concentration-dependent manner. Protein aggregation was induced at 10.0 and 25.0 mM, with denser aggregation at higher concentrations. Surface force apparatus measurements showed a peak in cohesion energy at 25.0 mM Ca2+, while interfacial adhesion energy reached a maximum at 10.0 mM. These results suggest that Ca2+ may facilitate cohesion via salt bridge formation and promote interfacial adhesion by mediating electrostatic interactions between AAP1 and material surfaces. Additionally, the cohesion of AAP1 may enhance molecular alignment on surfaces, contributing its interfacial adhesion. Overall, our results provide direct evidence for the involvement of Ca2+ in protein-mediated ascidian underwater adhesion. These findings will deepen our understanding of the mechanisms of underwater adhesion in aquatic organisms and guide the future development of antifouling strategies and bioinspired underwater adhesives.