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J. Biol. Chem., Vol. 275, Issue 35, 27360-27365, September 1, 2000

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Barnacle Cement Proteins
IMPORTANCE OF DISULFIDE BONDS IN THEIR INSOLUBILITY^*

Kei Kamino^§, Koji Inoue^¶, Tadashi Maruyama^¶, Nobuhiko Takamatsu^**, Shigeaki Harayama^¶, and Yoshikazu Shizuri

From the  Shimizu Laboratories, Marine Biotechnology Institute, 1900 Sodeshi, Shimizu, Shizuoka 424-0037, Japan, ^¶ Kamaishi Laboratories, Marine Biotechnology Institute, 75-1, Heita, Kamaishi, Iwate 026-0001, Japan, and the ^** Department of Biosciences, School of Science, Kitasato University, 1-15-1, Kitasato, Sagamihara, Kanagawa 228, Japan

Barnacles produce a cement that is a proteinaceous underwater adhesive for their secure attachment to the substratum. The biochemical properties of the cement have not previously been elucidated, because the insolubility of the cement proteins hampers their purification and characterization. We developed a non-hydrolytic method to render soluble most of the cement components, thereby allowing the proteins to be analyzed. Megabalanus rosa cement could be almost completely rendered soluble by its reduction with 0.5 M dithiothreitol at 60 °C in a 7 M guanidine hydrochloride solution, the high concentration of dithiothreitol being indispensable to achieve this. The effectiveness of this reduction treatment was confirmed by the detachment of the barnacle from the substratum. Three proteins comprising up to 94% of 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 expression 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, including a signal peptide. This is the first report of the major component of the barnacle cement protein complex.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AB033942.

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