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.
