Cement Proteins of the Acorn Barnacle,

Components of the proteinaceous cement secreted by barnacles have yet to be studied because of their insolubility. We solubilized and characterized the proteins of secondary cement, which is produced when the barnacle is detached from the substratum, in Megabalanus rosa. The cement was fractionated, according to its solubility in aqueous formic acid, into a soluble fraction, SF1 (2 1%); a fraction soluble after reduction, SF2 (37%); and a fraction insoluble after reduction, IF (42%). Analysis of the SF 1 and SF2 by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that they contained three polypeptides (SFl-60 k, -57 k, -47 k) and one polypeptide (SF2-60 k), respectively. The amino acid compositions of these polypeptides were similar and their N-terminal amino acid sequences were identical. These polypeptides had an unusual amino acid composition, rich in Set-, Thr, Ala, and Gly, like the tube cement of a marine polychaete, Phragmatopoma cal@rnica. The IF, solubilized in aqueous formic acid after cleavage with cyanogen bromide, was shown by SDS-PAGE to contain eight fragment peptides (CB-peptides). N-terminal amino acid sequences of the CB-peptides were also determined. We conclude that the barnacle cement is composed of at least two types of protein: highly hydroxylated protein in the SF1 and SF2 and insoluble protein in the IF. Received 25 August 1995; accepted 29 December 1995. Ahhr~viutions: SF1 and SF2, soluble fraction 1 and 2 of the cement; IF, formic-acid-insoluble fraction of the cement; CB-peptides, major peptides derived from IF using CNBr treatment; DOPA, 3,4-dihydroxyphenylalanine; CNBr, cyanogen bromide; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; TRICINE, NTris(hydroxymethyl)methylglycine; PVDF, polyvinylidene difluoride; CAPS, 3-cyclohexylaminopropanesulfonic acid; NBD-F, 4-fluoro-7-nitrobenzofurazan; RP-HPLC, reversed-phase-high-performance liquid chromatography; Hyp, hydroxyproline; EDTA, ethylenediaminetetraacetic acid. The SDS-PAGE pattern of CB-peptides from the secondary cement was identical to that of the primary cement produced while the barnacle is attached to a substratum. In addition, immunoblot analysis, using a polyclonal antibody against one of the CB-peptides from the secondary cement, also cross-reacted with a CNBr-fragment peptide of the primary cement. These results indicate that the primary and secondary cements are similar in protein composition.