Deciphering protein-mediated underwater adhesion in an invasive biofouling ascidian: Discovery, validation, and functional mechanism of an interfacial protein.

Discovering macromolecules and understanding the associated mechanisms involved in underwater adhesion are essential for both studying the fundamental ecology of benthos in aquatic ecosystems and developing biomimetic adhesive materials in industries. Here, we employed quantitative proteomics to assess protein expression variations during the development of the distinct adhesive structure - stolon in the model fouling ascidian, Ciona robusta. We found 16 adhesive protein candidates with increased expression in the stolon, with ascidian adhesive protein 1 (AAP1) being particularly rich in adhesion-related signal peptides, amino acids, and functional domains. Western blot and immunolocalization analyses confirmed the prominent AAP1 signals in the mantle, tunic, stolon, and adhesive footprints, indicating the interfacial role of this protein. Surface coating and atomic force microscopy experiments verified AAP1's adhesion to diverse materials, likely through the specific electrostatic and hydrophobic amino acid interactions with various substrates. In addition, molecular docking calculations indicated the AAP1's potential for cross-linking via hydrogen bonds and salt bridges among Von Willebrand factor type A domains, enhancing its adhesion capability. Altogether, the newly discovered interfacial protein responsible for permanent underwater adhesion, along with the elucidated adhesion mechanisms, are expected to contribute to the development of biomimetic adhesive materials and anti-fouling strategies. STATEMENT OF SIGNIFICANCE: Discovering macromolecules and studying their associated mechanisms involved in underwater adhesion are essential for understanding the fundamental ecology of benthos in aquatic ecosystems and developing innovative bionic adhesive materials in various industries. Using multidisciplinary analytical methods, we identified an interfacial protein - Ascidian Adhesive Protein 1 (AAP1) from the model marine fouling ascidian, Ciona robusta. The interfacial functions of AAP1 are achieved by electrostatic and hydrophobic interactions, and the Von Willebrand factor type A domain-based cross-linking likely enhances AAP1's interfacial adhesion. The identification and validation of the interfacial functions of AAP1, combined with the elucidation of adhesion mechanisms, present a promising target for the development of biomimetic adhesive materials and the formulation of effective anti-fouling strategies.