Adhesion in barnacles is still poorly understood. The cement gland secretes an insoluble multi-protein complex, which adheres very strongly to a variety of substrates in the presence of water. This adhesion mechanism is bioinspiring for the engineering of new adhesive materials, but to replicate this adhesive system, the genes coding for the cement constitutive proteins must be identified and elucidated, and their products characterised. Here, the complete sequences of three cement protein (CP) genes (CP-100K, CP-52K, and CP-19K) isolated from the cement gland of the stalked barnacle Pollicipes pollicipes (order Scalpelliformes) were obtained using RACE PCR. The three genes were compared to the 23 other acorn barnacle CP genes so far sequenced (order Sessilia) to determine common and differential patterns and molecular properties, since the adhesives of both orders have visibly different characteristics. A shotgun proteomic analysis was performed on the cement, excreted at the membranous base of specimens, where the products of the three genes sequenced in the gland were identified, validating their function as CPs. A principal component analysis (PCA) was performed, to cluster CPs into groups with similar amino acid composition. This analysis uncovered three CP groups, each characterised by similar residue composition, features in secondary structure, and some biochemical properties, including isoelectric point and residue accessibility to solvents. The similarity among proteins in each defined group was low despite comparable amino acid composition. PCA can identify putative adhesive proteins from NGS transcriptomic data regardless of their low homology. This analysis did not highlight significant differences in residue composition between homologous acorn and stalked barnacle CPs. The characteristics responsible for the structural differences between the cement of stalked and acorn barnacles are described, and the presence of nanostructures, such as repetitive homologous domains and low complexity regions, and repetitive β-sheets are discussed relatively to self-assembly and adhesion.
Keywords: Adhesive proteins; Adult barnacle; Biased residue composition; Cement proteins; Interface surface adhesion; Underwater adhesion.