Calcite‐specific coupling protein in barnacle underwater cement

Abstract

Sessile organisms are destined for attachment to various materials in water. Because gregariousness is essential for them, the opportunity to attach to a calcific exoskeleton of the same kind is necessarily favored. Thus, calcific material is one of the frequent foreign materials for attachment in the molecular system of the holdfast.The barnacle is a unique sessile crustacean. Once the larva has settled on the foreign substratum, it metamorphoses, calcifying the outer shell at the periphery and base, and permanently attaches to the foreign substratum by a multiprotein complex called cement [1]. This cement is secreted through the calcareous base to an acellular milieu, and joins two different materials, the The barnacle relies for its attachment to underwater foreign substrata on the formation of a multiprotein complex called cement. The 20 kDa cement protein is a component of Megabalanus rosa cement, although its specific function in underwater attachment has not, until now, been known. The recombinant form of the protein expressed in bacteria was purified in soluble form under physiological conditions, and confirmed to retain almost the same structure as that of the native protein. Both the protein from the adhesive layer of the barnacle and the recombinant protein were characterized. This revealed that abundant Cys residues, which accounted for 17% of the total residues, were in the intramolecular disulfide form, and were essential for the proper folding of the monomeric protein structure. The recombinant protein was adsorbed to calcite and metal oxides in seawater, but not to glass and synthetic polymers. The adsorption isotherm for adsorption to calcite fitted the Langmuir model well, indicating that the protein is a calcite-specific adsorbent. An evaluation of the distribution of the molecular size in solution by analytical ultracentrifugation indicated that the recombinant protein exists as a monomer in 100 mm to 1 m NaCl solution; thus, the protein acts as a monomer when interacting with the calcite surface. cDNA encoding a homologous protein was isolated from Balanus albicostatus, and its derived amino acid sequence was compared with that from M. rosa. Calcite is the major constituent in both the shell of barnacle base and the periphery, which is also a possible target for the cement, due to the gregarious nature of the organisms. The specificity of the protein for calcite may be related to the fact that calcite is the most frequent material attached by the cement.Abbreviations ASW, artificial seawater; C eq , equilibrium protein concentration; C I , initial protein concentration; cp, cement protein; fp, mussel foot protein; GSF1 and GSF2, cement fractions separated by their solubility in a guanidine hydrochloride solution; HRP, horseradish peroxidase; Mrcp, Megabalanus rosa cement protein; nMrcp-20k, protein extracted from the secondary cement in pure water; rMrcp-20k, recombinant form of Mrcp-20k expressed in Escherichia coli.

Barnacle biology before, during and after settlement and metamorphosis: a study of the interface

“…Additionally, phosphoproteins are important to the development of extracellular matrices and the transition to calcification (Borbas et al, 1991;Arias et al, 1993;George et al, 1993;Fernández et al, 2002;Johnstone et al, 2015a). Barnacle baseplate and shell biomineralization involve an initial organic matrix in which calcite crystals form (Fernández et al, 2002;Mori et al, 2007;Lewis et al, 2014), with the potential for involvement of calcite-carrying hemocytes, as seen in oyster calcification (Mount et al, 2004;Zhang et al, 2013;Johnstone et al, 2015;Li et al, 2016). Estimates of barnacle hemocyte concentrations are 1.7×10 2 cells μl −1 hemolymph, mostly composed of hyaline cells and some semigranular cells (Waite and Walker, 1988;Dickinson et al, 2009).…”

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.…”

“…Barnacle cement is primarily composed of proteins ranging from 3kDa to upwards of 160kDa with proteins between 50kDa and 100kDa being the most prominent in terms of volume (Kamino et al 1996;Naldrett and Kaplan 1997). Mori et al (2007) identified a calcium coupling protein (20kDa) only found within the primary cement of Megabalanus rosa suggesting cement-specific proteins such as this may relate to the adhesive's incredible ability to cure underwater. Changes in concentration or appearance of these proteins may affect cement tenacity.…”

“…. (Smith et al 1999;Pawlicki et al 2004;Mori et al 2007) 2.4.6 Coating/temperature interaction Substrate modulus has also shown to affect CRS of barnacles ). An experiment conducted by found the glass transition temperature (T g ) of the PDMSe used, to be as high as 18°C.…”

“…Here, liquid cement collection was employed in attempt to analyze cement protein content using 2D SDS PAGE followed by Mass Spectrometry (MALDI/TOFTOF). Only one dimensional SDS PAGE has been conducted on adhesive proteins in previous research (Kamino et al 1996;Naldrett and Kaplan 1997;Kamino et al 2000;Kamino 2001;Mori et al 2007). …”