Barnacle cement: a polymerization model based on evolutionary concepts

Abstract

SUMMARYEnzymes and biochemical mechanisms essential to survival are under extreme selective pressure and are highly conserved through evolutionary time. We applied this evolutionary concept to barnacle cement polymerization, a process critical to barnacle fitness that involves aggregation and cross-linking of proteins. The biochemical mechanisms of cement polymerization remain largely unknown. We hypothesized that this process is biochemically similar to blood clotting, a critical physiological response that is also based on aggregation and cross-linking of proteins. Like key elements of vertebrate and invertebrate blood clotting, barnacle cement polymerization was shown to involve proteolytic activation of enzymes and structural precursors, transglutaminase cross-linking and assembly of fibrous proteins. Proteolytic activation of structural proteins maximizes the potential for bonding interactions with other proteins and with the surface. Transglutaminase cross-linking reinforces cement integrity. Remarkably, epitopes and sequences homologous to bovine trypsin and human transglutaminase were identified in barnacle cement with tandem mass spectrometry and/or western blotting. Akin to blood clotting, the peptides generated during proteolytic activation functioned as signal molecules, linking a molecular level event (protein aggregation) to a behavioral response (barnacle larval settlement). Our results draw attention to a highly conserved protein polymerization mechanism and shed light on a long-standing biochemical puzzle. We suggest that barnacle cement polymerization is a specialized form of wound healing. The polymerization mechanism common between barnacle cement and blood may be a theme for many marine animal glues. Supplementary material available online at

Temperature Affects Adhesion of the Acorn Barnacle (Balanus amphitrite)

“…The protein contents of barnacle cement has been the topic of considerable research as of late (Kamino 2001;Mori et al 2007;Urushida et al 2007;Kamino 2008;Dickinson et al 2009) yet few cement proteins have been added to the database (19kDa, 20kDa, and 100kDa as well as insoluble partial peptide fractions). Although many were collected from a different, though similar species, Megabalanus rosa, we were consequently hopeful in identifying similar cement polymers on the basis that partial sequence data may be used.…”

“…This droplet was quickly collected into a 20µl pipette tip and immediately placed into rehydration buffer (described below). Due to the difficulty of this protein collection method, as described by recent fouling research Dickinson et al 2009), samples from one to three barnacles were pooled giving a maximum of 5µl and minimum of 3µl cement volume per gel. I collected cement from barnacles from both temperature treatments to create four replicates within each treatment.…”

“…An old method of cement collection (Cheung et al 1977) has inspired recent studies of barnacle adhesive (Dickinson et al 2009). By extracting the cement as it is produced in liquid form, solubilizing the proteins becomes much easier.…”

Barnacles resist removal by crack trapping

“…How the adhesive cures is still not fully understood. Recent work of Dickinson et al [14] suggested that cement polymerization may be a specialized form of wound healing similar to blood clotting. The structure of the thin (approx.…”

“…The structure of the thin (approx. 1 mm) adhesive layer has been shown to be a fibrillar [14][15][16][17] protein with secondary structure exhibiting characteristics of amyloid-folded (antiparallel b-sheet) proteins in Balanus amphitrite [17]. Above the proteinaceous cement layer in many barnacles is a calcified base plate, imparting further mechanical rigidity to the adhesive interface.…”