Barnacle cement proteins - Importance of disulfide bonds in their insolubility

Barnacles produce a cement that is a proteinaceous underwater adhesive for their secure attachment to the substratum, The biochemical properties of th e cement have not previously been elucidated, because the insolubility of t he cement proteins hampers their purification and characterization. We deve loped a non-hydrolytic method to render soluble most of the cement componen ts, thereby allowing the proteins to be analyzed. Megabalanus rosa cement c ould be almost completely rendered soluble by its reduction with 0.5 M dith iothreitol at 60 degrees C in a 7 M guanidine hydrochloride solution, the h igh concentration of dithiothreitol being indispensable to achieve this. Th e effectiveness of this reduction treatment was confirmed by the detachment of the barnacle from the substratum. Three proteins comprising up to 94% o f the whole cement were identified as the major cement components. The cDNA clone of one of these major proteins was isolated, and the site-specific e xpression of the gene in the basal portion of the adult barnacle, where the cement glands are located, was demonstrated. A sequence analysis revealed this cement component to be a novel protein of 993 amino acid residues, inc luding a signal peptide. This is the first report of the major component of the barnacle cement protein complex.