Barnacle cement is an underwater adhesive that is used for permanent settlement, and is an insoluble protein complex. A method for rendering soluble the cement of Megabalanus rosa has been developed, and three major proteins have been identified in a previous study. To survey the M. rosa cement proteins in a lower molecular mass range, the cement proteins were separated by reversed-phase HPLC and a previously unidentified protein named 20kDa M. rosa cement protein (Mrcp-20k) was found. Mrcp-20k cDNA was cloned to reveal its primary structure. This cDNA was 902bp long and encoded a 202 amino acid-long open reading frame, including 19 amino acids of the signal sequence. The molecular mass in the disulphide form was calculated to be 20357Da and the isoelectric point of the mature polypeptide was 4.72. Mrcp-20k was characterized by an abundance of Cys residues and charged amino acids. The most common amino acid was Cys (17.5%), with Asp (11.5%), Glu (10.4%) and His (10.4%) following in order of magnitude. The alignment of the Cys residues indicated the primary structure of this protein to consist of six degenerated repeats, each about 30 residues long. Mrcp-20k has no intermolecular disulphide bonds and no free thiol groups of Cys in the insoluble cement complex. Abundant Cys is thought to play a role in maintaining the topology of charged amino acids on the molecular surface by intramolecular disulphide-bond formation. The possible function of abundant charged amino acids, including the interaction with a variety of surface metals on the substratum, is discussed.
Abbreviations used: Mrcp, Megabalanus rosa cement protein; GSF1 and GSF2, guanidinium chloride-soluble fractions 1 and 2 of M. rosa cement; RP-HPLC, reversed-phase HPLC; GdmCl, guanidinium chloride; MALDI-TOF, matrix-assisted laser-desorption ionization–time of flight; sp185, 185kDa silk protein; Mgfp2, Mytilus galloprovincialis foot protein 2.
The nucleotide sequence data reported have been submitted to the DDBJ, EMBL, GSDB and GenBank Nucleotide Sequence Databases under the accession number AB035415.