Molecular Recognition of Structures Is Key in the Polymerization of Patterned Barnacle Adhesive Sequences

Abstract

The permanent adhesive produced by adult barnacles is held together by tightly folded proteins that form amyloid-like materials distinct among marine foulants. In this work, we link stretches of alternating charged and noncharged linear sequences from a family of adhesive proteins to their role in forming fibrillar nanomaterials. Using recombinant proteins and short barnacle cement derived peptides (BCPs), we find a central sequence with charged motifs of the pattern [Gly/ Ser/Val/Thr/Ala-X], where X are charged amino acids, to exert specific control over timing, structure, and morphology of fibril formation. While most BCPs remain dormant, the core segment demonstrates rapid polymerization as well as an ability to template other peptides with no propensity for self-assembly. Patterned charge domains assemble dormant peptides through a specific antiparallel β-sheet structure as measured by FTIR. While charged domains favor an antiparallel structure, BCPs without charged domains switch fibril assembly to favor simpler parallel β-sheet aggregates. In addition to activation, charged domains direct nanofibers to grow into discrete microns long fibrils similar to the natural adhesive, while segments without such domains only form short branched aggregates. The assembly of adhesive sequences through recognition of structured templates outlines a strategy used by barnacles to control physical mechanisms of underwater adhesive delivery, activation, and curing based on molecular recognition between proteins.

Distribution of Select Cement Proteins in the Acorn Barnacle Amphibalanus amphitrite

“…Here, we use antibodies developed against AaCP19-1 and AaCP43-1 to examine FFPE sections. These antibodies show specific binding in western blots of solubilized cement and purified proteins [see Supplementary Figure S1 and refs (So et al, 2017(So et al, , 2019]. Our immunohistochemistry results and protein analysis of FFPE sections provide two independent techniques to demonstrate that these cement proteins are not exclusive to the adhesive interface, but rather are found in select tissues well removed from the basal region of A. amphitrite.…”

“…As a first step toward understanding the distribution of cement proteins, it was critical to confirm that the polyclonal antibodies bind to specific proteins from A. amphitrite adhesive. Previous analysis with immunoblotting has demonstrated both antibodies directed against AaCP19-1 and AaCP43-1 bound to select components of the adhesive of A. amphitrite when barnacles were grown on nitrocellulose membranes (So et al, 2019). Further, anti-AaCP43-1 has been shown to interact with specific bands after SDS-PAGE gel separation of solubilized adhesive (So et al, 2017).…”

“…Historically, the majority of research groups have designated these proteins as "cement proteins" based on their location of isolation. No experimental validation of the adhesive properties of specific barnacle proteins has been demonstrated to date, although several biochemical mechanisms for adhesion have been proposed, including: hydrophobic forces produced by high levels of aliphatic residues (Naldrett, 1993;Naldrett and Kaplan, 1997;Kamino et al, 2012;Kamino, 2013;Jonker et al, 2014); repetitive sequence motifs similar to those found in silk proteins (So et al, 2016) promoting nanofibril formation (Barlow et al, 2010;Burden et al, 2014;Nakano and Kamino, 2015;Fears et al, 2018;So et al, 2019); protein polymerization as a specialized form of wound healing (Dickinson et al, 2009); and post-translational modifications via oxidation (So et al, 2017). In addition to a lack of experimental verification of their adhesive properties, the function of cement proteins beyond a potential role in adhesion at the interface has not been examined.…”

Comparative analysis of stalked and acorn barnacle adhesive proteomes

“…The AaCrCP group has a low number of predicted sites for either type of glycosylation. The minor clusters of P. pollicipes bulk proteins that show enrichment for either proline or threonine and valine also have a high number of predicted O-linked (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27) and a low number of predicted N-linked (less than four) glycosylation sites (data not shown), similar to the GrCP groups.…”

Adhesion of acorn barnacles on surface-active borate glasses

“…While some CPs self-assembled into fibrils during in vitro experiments [10][11][12], the exact mechanisms of fibril formation and surface adhesion are unclear. Moreover, the identification of families of homologous CPs within cement plaques suggest that processes associated with barnacle adhesion are highly complex [9,13], as conserved domains within the CP19 family are amyloidogenic [14].…”