Barnacle cement: a polymerization model based on evolutionary concepts

Abstract

Enzymes 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.

Fig. 1.

Barnacle cement release and polymerization. (A) X-ray microtomograph of a live barnacle (Amphibalanus amphitrite) showing the junction (J) of the base plate (BP) and lateral plates (LP), where cement is released through ducts during growth. (B) Manual removal of polymerized cement from cement ducts resulted in the release of unpolymerized cement, which could be collected in microliter quantities. The barnacle had been gently removed from a silicone substrate. (C) Polymerized cement following release.

Fig. 2.

Atomic force microscopy (AFM) images of polymerized barnacle cement. (A) Cement was imaged in situ, directly on a barnacle base. (B) Unpolymerized cement, collected as shown in Fig. 1, was cured under seawater while sandwiched between glass slides. In both cases, AFM imaging revealed a fibrous ultrastructure.

Fig. 3.

Trypsin-like serine protease in unpolymerized barnacle cement. (A) Left: SDS-PAGE (reducing conditions) of unpolymerized barnacle cement showing multiple doublet bands (Coomassie stained). Prominent bands are labeled with molecular mass in kDa. Right: a mammalian trypsin-like protein in unpolymerized barnacle cement as shown by western blot (WB) using polyclonal bovine pancreatic trypsin antibody. Staining bands occur at 90 and 80 kDa and are indicated by arrows. (B) Verification of trypsin activity in unpolymerized barnacle cement, using BAPNA. Mean optical density at 405 nm (OD₄₀₅) (and s.e.m.) for unpolymerized cement (N=20 individuals), substrate only (no enzyme present) and trypsin control (4.6E^–3 BAPNA units per ml porcine trypsin) is shown. ^*Significant difference between the substrate only and cement groups (rank sum test: P=0.006). (C) Barnacle larval settlement in the presence of polymerizing barnacle cement. A positive settlement response is evidence of trypsin-like serine protease activity, because peptides generated by trypsin-like protease activity induce barnacle settlement. Settlement assays were conducted for 24 h with newly metamorphosed cyprids. Means and s.e.m. are shown. Groups marked with different letters are significantly different as shown by SNK pairwise comparison. N=5 assays per cement volume.

Fig. 4.

Inhibition of barnacle cement polymerization with soybean trypsin inhibitor. Unpolymerized cement was incubated with soybean trypsin inhibitor or deionized water for 2 min and then run on SDS-PAGE. Mean intensity (+s.e.m.) for the protein bands labeled in Fig. 3A is shown (N=4 replicate lanes). ^*Significant difference in band intensity between the trypsin inhibitor and deionized water groups (t-tests: P<0.05).

Fig. 5.

Transglutaminase in unpolymerized barnacle cement. (A,B) Tandem mass spectrometry (MS/MS) identification of the catalytic subunit of human fibrin stabilizing factor, a transglutaminase (factor XIII A1 subunit precursor, accession number NP_000120.1). Identification was made by comparing the MS/MS spectra with the human database. (A) Identified peptides. XC score, cross-correlation score; deltaCn, difference between XC scores for the best and second best match. (B) Mass spectrometry (MS; inset) and MS/MS spectra for the factor XIII A1 peptide shown in bold. Arrow indicates the precursor mass in the MS spectra. b and y ions obtained upon fragmentation of the precursor ion by collision-induced dissociation are shown. For peptide fragmentation and details of the tandem mass spectrometry method see supplementary material Fig. S1. (C) Validation by western blot analysis using anti-human factor XIII A subunit antibody. Markers in kDa. (D) Verification of transglutaminase enzyme activity in unpolymerized barnacle cement. Mean OD₄₅₀ (and s.e.m.) for unpolymerized cement (N=19 individuals), substrate only (no enzyme present) and transglutaminase control (2 mU ml^–1 guinea pig transglutaminase) is shown. ^*Significant difference between the substrate only and cement groups (t-test: P=0.006). (E) Hyaline cells in unpolymerized barnacle cement (phase contrast optics), the principal source of transglutaminase in crustaceans.

Fig. 6.

ε-(γ-Glutamyl)lysine cross-link, which results from transglutaminase activity; 5% of Lys residues in barnacle cement were shown to be bound in ε-(γ-glutamyl)lysine cross-links.

Fig. 7.

The effect of EDTA (left) and EGTA (right) on barnacle cement polymerization. Unpolymerized cement was incubated with EDTA, EGTA or deionized water for 2 min and then run on SDS-PAGE. Mean intensity (+s.e.m.) for the protein bands labeled in Fig. 3A is shown (N=4 replicate lanes for deionized water, N=3 replicate lanes for EDTA and EGTA). ^*Significant difference in band intensity between the EDTA and deionized water groups (t-tests: P<0.05).