Calcite-specific coupling protein in barnacle underwater cement Youichi Mori 1 , Youhei Urushida 1 , Masahiro Nakano 1 , Susumu Uchiyama 2 and Kei Kamino 1 1 Marine Biotechnology Institute, Kamaishi, Iwate, Japan 2 Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan Sessile organisms are destined for attachment to vari- ous materials in water. Because gregariousness is essen- tial 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 metamor- phoses, calcifying the outer shell at the periphery and base, and permanently attaches to the foreign substra- tum 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 Keywords adsorption; crustacean; protein complex; sessile organism; underwater adhesive Correspondence K. Kamino, Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001 Japan Fax: +81 193 26 6592 Tel.: +81 193 26 6584 E-mail: kei.kamino@mbio.jp Database The nucleotide sequence data are available in the DNA Data Bank of Japan under the accession number AB329666 (Received 5 July 2007, revised 18 October 2007, accepted 23 October 2007) doi:10.1111/j.1742-4658.2007.06161.x 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 solu- ble 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 character- ized. 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. 6436 FEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBS crustacean’s own calcareous base and the foreign sub- stratum, which can be a metal oxide, synthetic polymer, or the calcareous shell of another animal, in water. Cal- cific material is necessarily the most frequently encoun- tered target for attachment by the barnacle cement. So far, four cement proteins have been identified, with different characteristics [2]. No homologous pro- teins have been found in other organisms. Among the four cement proteins produced by the barnacle, cp-100k and cp-52k are the two major components in terms of amount, and are characterized by their insolu- ble nature [3]. These two components are considered to constitute the bulk region of the cement. A reducing treatment with guanidine hydrochloride was necessary to render the bulk proteins soluble. cp-68k is also a major protein, whose amino acid composition is heav- ily biased towards four amino acids, i.e. Ser, Thr, Ala, and Gly, although the specific function of this protein in underwater attachment is not known at present [3]. cp-20k is a minor cement protein in terms of its amount, and is not post-translationally modified. The amino acid composition of cp-20k is characterized by the unusual abundance of Cys (17%) and charged amino acids (Asp, 11.5%; Glu, 10.4%; His, 10.4%) [4]. Although the high abundance of the Cys residue in the protein has suggested a possible contribution to inter- molecular crosslinking or coupling [5], our previous study has indicated that this is not the case, at least with respect to the latter speculation [4]. Underwater attachment is a multifunctional process, which is different from that of an artificial adhesive in air, and is thus an unachievable technique at present. The process [6] involves such subfunctions as prevent- ing random aggregation during transport via the cement duct, displacing sufficient seawater to prime and spread on the surface without being dispersed in the water, coupling strongly with a variety of material surfaces, and self-assembly to join the calcareous base and the substratum. After the process, it is then neces- sary to cure the cement so that the holdfast remains stiff and tough, and to protect it from microbial degra- dation. The insoluble nature of the complex and the limitations of microanalytical methods for studying each function, however, have hindered elucidation of the specific function of each cement protein [3]. There are two types of sample for studies on barnacle cement: primary cement and secondary cement [1,3]. Primary cement is a natural adhesive of a few microme- ters in thickness between the base and foreign substra- tum, whereas secondary cement is secreted when the animal is free from a substratum. Both forms of cement are similar in their whole amino acid composition [7], and appear to contain the same protein components as determined by peptide mapping with cyanogen bromide treatment [3]. Reattachment of the barnacle to a new substratum by secondary cement has also been reported [1,8], although the adhesive strength was weaker than that of primary cement. The primary cement seemed to be denser and more rigid than the secondary cement. Although these studies indicated that the primary and secondary cements have the same protein composition, it is not clear whether the protein–protein interactions and the topology in the two complexes are the same. Megabalanus rosa (Mr)cp-20k in the secondary cement was chemically characterized in a previous study [4]. However, neither the nature of Mrcp-20k in the primary cement nor the specific function of this protein in underwater attachment has been unraveled. The present study was performed to characterize the nature of the protein in the primary cement. Thereaf- ter, we expressed the recombinant form of the protein in bacteria in a soluble form under physiological con- ditions, and confirmed that the recombinant protein has almost the same structure as that of the native bar- nacle protein. We subsequently showed that the recom- binant protein has a specific affinity for calcite surfaces in water. This is the first report to identify a biotic underwater adhesive protein as a specific adsorbent to calcite, by directly measuring the adsorbing activity of the protein prepared under physiological conditions. Results Confirmation of Mrcp-20k in natural barnacle cement Mrcp-20k was extracted only from guanidine hydro- chloride-soluble fraction 1 (GSF1) of the primary cement, but not from GSF2, which is the guanidine hydrochloride-soluble fraction after reducing treatment (Fig. 1A). This result is consistent with what is found in the secondary cement [4]. Mrcp-20k in GSF1 of the primary cement only gave a band with a monomeric molecular mass on SDS ⁄ PAGE without the reducing treatment (Fig. 1A); this is also consistent with what is found for the secondary cement [4]. This indicates that Mrcp-20k is not covalently crosslinked in the natural cement. Mrcp-20k was not detected in the peripheral shell (Fig. 1B), indicating that Mrcp-20k is not a protein related to calcification of the shell. Preparation of the recombinant form of Mrcp-20k in bacteria The recombinant form of Mrcp-20k in Escherichia coli, rMrcp-20k, was purified in solution under physiologi- Y. Mori et al. Calcite-coupling protein in underwater adhesive FEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBS 6437 cal conditions (Fig. 2A). The elution profiles from both RP-HPLC and ion exchange HPLC were identi- cal to those of native Mrcp-20k in the secondary cement extracted in pure water, nMrcp-20k (supple- mentary Fig. S1A,B). Owing to the vector construc- tion, rMrcp-20k was designed to have an additional tripeptide, Ala-Met-Ala, attached to the N-terminus. The N-terminal sequence and molecular mass of the recombinant protein were determined to be AMAHE- EDGV and 20 629 Da, respectively, which agree well with the deduced sequence and mass (20 629.3 Da). This molecular mass corresponds to the form of the protein in which all Cys residues form disulfide bonds. Alkylation treatment of rMrcp-20k resulted in a same mass, suggesting that no free SH groups are present in rMrcp-20k. The presence of all Cys residues in the intramolecular disulfide form in the recombinant pro- tein is the same as what is found for the protein in the secondary cement [4]. SDS ⁄ PAGE analysis showed that rMrcp-20k without a reduction treatment had a slightly lower mobility than that with the reduction treatment (Fig. 2B); this resembles the behavior of the native Mrcp-20k protein in the secondary cement. The CD spectrum of rMrcp-20k in a 10 mm sodium phos- phate buffer (pH 6.8) was also identical to that of nMrcp-20k; both showed the presence of a mixture of b-turn and random coil structures [9,10]. These spectra were remarkably different from that observed after a reducing treatment, probably due to denaturation of the protein (Fig. 3). Adsorption of rMrcp-20k to underwater material surfaces The adsorption of rMrcp-20k to several underwater material surfaces was investigated, and the findings are summarized in Fig. 4. The protein was adsorbed to calcite in artificial seawater (ASW), whereas it was not adsorbed to glass, gold, polystyrene, or benzo- guanamine-formaldehyde resin, which is a positively charged synthetic polymer. The protein was also adsorbed to a limited extent to metal oxides such as zinc oxide and magnetite. The amount adsorbed to calcite in pure water was almost the same as that in ASW. A B Fig. 2. Purification of rMrcp-20k. (A) Samples were separated by using the 16.5% T Tris ⁄ Tricine buffer system of SDS ⁄ PAGE [30]. Lane 2: crude extract of bacterial cells. Lane 3: rMrcp-20k fused with a tag in the vector construct. Lane 4: rMrcp-20k. Lane 1, low molecular mass markers (Bio-Rad; aldolase, 45.0 kDa; carbonic anhydrase, 31.0 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa). (B) SDS ⁄ PAGE of rMrcp-20k with (left) and without (right) pretreatment with the reducing agent 2-mercaptoethanol. A B Fig. 1. Characterization of Mr cp-20k in the primary cement. (A) Western blotting of fractions rendered soluble from the primary cement by using the antibody to Mrcp-20k. Lane 1: GSF1 with reduction pretreatment in SDS ⁄ PAGE. Lane 2: GSF2 with reduction pretreatment. Lane 3: GSF1 without reduction pretreatment. Num- bers on the left-hand side indicate molecular masses (kDa). (B) Detection of Mrcp-20k in the peripheral shell of the barnacle by using the antibody to Mrcp-20k. Two grams each (dry weight) of the peripheral shell and calcareous base were decalcified and subjected to dot-blotting. Lane 1: 2% acetic acid solution–soluble fraction of the peripheral shell. Lane 2: GSF1 and GSF2 of the peripheral shell. Lane 3: 2% acetic acid solution–soluble fraction of the base. Lane 4: GSF1 and GSF2 of the base. Lane 5: rMrcp-20k as positive control (1 lg). Lane 6: trypsin inhibitor from soybean as negative control (1 lg; Wako Pure Chemical Industries). Calcite-coupling protein in underwater adhesive Y. Mori et al. 6438 FEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBS The relationship between the concentration of the protein at the calcite surface and its solution concen- tration is described by the adsorption isotherm. The linearized forms of the isotherm for the adsorption to calcite were C eq ⁄ Q ¼ 0.3168 · 10 )3 + 4.199C eq [corre- lation coefficient (r 2 ) of 0.97] in ASW and C eq ⁄ Q ¼ 1.7168 · 10 )3 + 3.782C eq (r 2 of 0.98) in the dilute buffer [C eq , equilibrium protein concentration; Q, amount of absorbed protein (lmol) per m 2 of the surface] (Fig. 5). The slope and intercept of the result- ing lines enabled us to estimate the adsorption affinity (K) and the maximum number of adsorption sites (N) to be K ¼ 1.33 · 10 7 m )1 and N ¼ 2.38 · 10 )7 molÆm )2 in ASW, and K ¼ 2.20 · 10 6 m )1 and N ¼ 2.64 · 10 )7 molÆm )2 in the dilute buffer solution. The isotherms for adsorption to zinc oxide and magnetite were not linear (r 2 of 0.75 and 0.58, respectively), so that the adsorption to these surfaces seemed not to be of the typical Langmuir type (supplementary Fig. S2). The adsorption of rMrcp-20k to the barnacle shell was visualized using the antibody to rMrcp-20k with the secondary antibody conjugated by fluorochrome (Fig. 6 and supplementary Fig. S3). A 10 min incuba- tion with rMrcp-20k in ASW gave rise to fluorescence emission at the barnacle shell, demonstrating the Wavelength (nm) [θ] (deg cm -2 dmol -1 ) 200 -30 -20 -10 0 10 [θ] (deg cm -2 dmol -1 ) -30 -20 -10 0 10 [θ] (deg cm -2 dmol -1 ) -30 -20 -10 0 10 A B C 250 300 320 Wavelength (nm) 200 250 300 320 Wavelength (nm) 200 250 300 320 Fig. 3. Comparison of the CD spectra of rMrcp-20k and nMrcp- 20k. The spectra are shown of (A) rMrcp-20k, (B) nMrcp-20k and (C) rMrcp-20k with the reducing pretreatment. A amount of adsorbed protein (ng/cm 2 ) 0 50 100 150 200 250 300 BCDEFGH Fig. 4. Adsorption of rMrcp-20k to various solid surfaces. The adsorption of rMrcp-20k to the particles of several materials in 10 min at 25 °C was evaluated by measuring the decrease in pro- tein amount remaining in the solution. Adsorption to (A) calcite in ASW, (B) glass in ASW, (C) benzoguanamine–formaldehyde resin in ASW, (D) zinc oxide in ASW, (E) magnetite in ASW, (F) gold in ASW, (G) polystyrene in ASW, and (H) calcite in pure water. Error bars indicate the standard deviation. C eq (µmol/mL) C eq /Q (m 2 /mL) -5.2E-18 0 0.01 0.02 0.03 0.04 0.05 C eq /Q (m 2 /mL) 0 0.01 0.02 0.03 0.04 0.05 B A 0.002 0.004 0.006 0.008 0.01 C eq (µmol/mL) -2.08E-1 0.002 0.004 0.006 0.008 0.01 Fig. 5. Linearized adsorption isotherm for adsorption of rMrcp-20k to calcite. (A) Isotherm in ASW. (B) Isotherm in 2.14 m M sodium carbonate (pH 8.2). Y. Mori et al. Calcite-coupling protein in underwater adhesive FEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBS 6439 successful adsorption of the protein to the calcareous shell of the barnacle. The distribution of the molecular size of rMrcp-20k The distribution of the molecular size of the recombi- nant protein was evaluated by analytical ultracentrifu- gation (Table 1). Sedimentation velocity analyses indicated that the protein exists as a single component in 100 mm to 500 mm NaCl solution. The sedimentation coefficient of the component was estimated to be s $ 2.5. The sedimentation equilibrium analyses gave nearly 20 kDa as the molecular mass in 100 mm to 1 m NaCl solution, which is consistent with monomeric molecu- lar mass of the protein. Therefore, the s $ 2.5 species found by sedimentation velocity corresponds to the monomeric form of the protein. The possible change of intramolecular disulfide bonds to intermolecular ones after a longer period of incubation in ASW was evaluated by SDS ⁄ PAGE analysis (Fig. 7). The molecular masses were mono- meric for proteins in both the suspension and the lay- ers adsorbed to calcite, thus confirming that there had been no change of intramolecular disulfide bonds to the intermolecular type in the protein. Isolation of the homologous gene from Balanus albicostatus A PCR investigation of a homologous gene in three barnacle species was attempted with several degener- ated oligonucleotide primers based on the primary structure of Mrcp-20k. All PCR trials with primers designed from the primary structure of Mrcp-20k failed to amplify homologous DNA, except for 3¢-RACE with cDNA of Balanus albicostatus. The sequence of homo- logous cDNA in B. albicostatus determined in this study was 700 bp, and the coding region was deter- mined to encode 125 amino acids (supplementary Fig. S4). The first 20 amino acids are considered to Fig. 6. Demonstration of the adsorption of Mrcp-20k to the barna- cle peripheral shell. The protein adsorbed to the shell was treated with the antibody, and visualized with the secondary antibody linked to fluorochrome Cy3 (GE Healthcare Bio-Science). Images under visible light (left) and those under reflected fluorescence (right) are shown. The image pair was captured from the same angle of the object. In the images under visible light, yellow areas correspond to the shell, and white areas are transparent without any object. Shell was incubated with rMrcp-20k, washed, and trea- ted with the antibody to Mrcp-20k. No fluorescence was observed in the control experiment (supplementary Fig. S3). Table 1. The distribution of the molecular size of rMrcp-20k evalu- ated by analytical ultracentrifugation. The sedimentation coefficients and molecular masses of rMrcp-20k in several solvents were evalu- ated by sedimentation velocity and sedimentation equilibrium, respectively. Sedimentation coefficients were evaluated by sedi- mentation velocity analyses and standardized with the SEDNTERP pro- gram [29]. Molecular masses were determined by sedimentation equilibrium analyses. NaCl concentration ( M) s 20, W (S) Molecular mass (kDa) 0.1 2.6 19.6 0.3 2.5 18.9 0.4 2.5 – 0.5 2.4 – 1.0 – 21.1 Fig. 7. Rearrangement of disulfide bonds in rMrcp-20k during long- term incubation. The molecular masses of rMrcp-20k after several treatments for 1 week at 25 °C were estimated by western blotting with the antibody to Mrcp-20k antibody. rMrcp-20k was incubated in ASW adjusted to pH 8.0 without calcite particles (lane 1), in a dilute buffer adjusted to pH 8.0 without calcite particles (lane 2), or in ASW with calcite particles (lane 3). Calcite-coupling protein in underwater adhesive Y. Mori et al. 6440 FEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBS correspond to the signal peptide, because of its high hydrophobicity and the existence of a predicted signal peptidase cleavage position [11]. The molecular mass and isoelectric point of the mature polypeptide were predicted to be 12 297.0 Da and 8.3, respectively, assuming that all Cys residues were in the disulfide form for prediction of the molecular mass. The amino acid composition deduced from the cDNA indicated that charged amino acids such as His (20%), Lys (10%) and Cys (17%) are the dominant residues; the contents of these residues appear to be significantly higher than in the standard amino acid composition [12]. The charged amino acids Asp, Glu, His, Lys and Arg are estimated to comprise 42% of the total resi- dues. Alignment of the Cys residues indicated that the primary structure of the homologous protein in B. albi- costatus consists of four repeated sequences (Fig. 8). The difference between the B. albicostatus protein and Mrcp-20k in their amino acid lengths depended on the difference in the number of repeats. The similar Cys spacing, the existence of Pro preceding the second Cys, the presence of two amino acids after the second Cys, and the sporadic insertion of clusters of charged amino acids such as HKHHDHGK, HHHDD, RHGKKH and HRKFH, are common characteristics found in both proteins [4]. A BLAST search [13] of the nonredundant database and a sequence profile-based fold-recognition method for three-dimensional struc- tural prediction [14] failed to provide any homologous sequences and meaningful structure from currently available databases. Discussion Although Mrcp-20k was found in the secondary cement in the previous study, neither the presence of this protein in the barnacle natural adhesive layer or pri- mary cement, nor its specific function in underwater attachment, has been characterized so far. The present study was thus conducted to address these questions. The conditions required for extracting the protein from the insoluble primary cement, and its behavior in the SDS ⁄ PAGE analysis, were similar to those of the pro- tein from the secondary cement. The protein exhibited a monomeric molecular mass on SDS ⁄ PAGE even without a reducing pretreatment, a characteristic also found for the protein from the secondary cement. The amino acid composition of Mrcp-20k is characterized by the unusually high contents of Cys (17%) and charged amino acid residues [4], which suggests a possi- ble role of polymerization via intermolecular disulfide bonds for this protein in the process of underwater adhesion [5]. The present study, however, excluded this possibility. This was further supported by the fact that long-term incubation of the bacterial recombinant pro- tein in ASW did not give rise to any polymerized molecular species by the conversion of disulfide bonds to the intermolecular form. The abundance of Cys and charged amino acid residues is reminiscent of proteins involved in biomineralization. As the cement has always been collected from the surface of the barnacle calcareous base, some contamination of the proteins used for calcification may have occurred. However, the fact that Mrcp-20k could not be detected in the periph- eral calcareous shell indicates that the protein is specific to underwater attachment of the base, and does not contribute to the calcification process. The protein con- tains few hydrophobic residues, which would result in a poor hydrophobic core in the structure; this may be a reason for the introduction of abundant intramolecular disulfide bonds to stabilize the structure in molecular evolution. This was confirmed by the marked change in the CD spectrum with the reducing treatment. The limited number of hydrophobic residues may, in turn, suggest the significance of the charged amino acid residues in the function of the protein. Mrcp-20k is a simple protein bearing no post-transla- tional modifications [4]. This allowed us to express this protein in bacteria under physiological conditions, and to compare the characteristics of the recombinant pro- tein with those of the native protein extracted with pure water. Both proteins showed the same elution profiles in column chromatography, the same behaviors as analyzed by SDS ⁄ PAGE, MALDI-TOF MS and CD spectra, and similar resistance to alkylation treatment without any reducing treatment, indicating that both proteins possessed similar molecular structures. We therefore characterized the functional properties of the recombinant protein. This is an unusual case in biotic underwater adhesive studies, as all mussel foot proteins (fps), which represent another model system, are Fig. 8. Alignment of the repetitive sequences in Mr cp-20k and the homologous protein in B. albicostatus. All Cys residues are shown in black, and conserved Pro residues are shown in gray. Y. Mori et al. Calcite-coupling protein in underwater adhesive FEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBS 6441 subjected to heavy post-translational modifications [15], so that the native activity of the simple recombinant protein cannot be obtained. The present study repre- sents the first report based on a recombinant protein retaining almost the same structure as that of the native protein in the study of biotic underwater adhesive. The protein was adsorbed to calcite, a crystalline form of calcium carbonate, but not to glass and syn- thetic polymers. The isotherm for adsorption of the recombinant protein to calcite followed the Langmuir model, which has been extensively applied to the quan- titative evaluation of the interaction between macro- molecules and mineral interfaces [16]. Although the protein was also adsorbed to some metal oxides to a limited extent, this adsorption isotherm did not fit the Langmuir model. These results suggest that the adsorp- tion to calcite is a specific function of Mrcp-20k. This may not be surprising if we consider that half of the material to be attached is the organism’s own calcare- ous base. The barnacle also prefers to attach itself to the peripheral calcite shell of another barnacle, because of the gregarious behavior of this species. It therefore seems that the barnacle arranges a specific protein in the cement to be adsorbed to the most typical target, calcite, although it is not clear whether the target of the protein is specific to the organism’s own base or the foreign calcified shell, or both. The adsorption isotherm for the attachment of rMrcp-20k to calcite determined in the present study indicated that the protein has an affinity for calcite that is one magnitude of order higher than that of the ame- logenin–hydroxyapatite interaction, whose adsorption affinity was 1.97 · 10 6 m )1 [17]. The calculated pI value for rMrcp-20k is 4.7. The points for zero charge of calcite and glass are 9.50 ± 0.50 [18] and 1.80, respec- tively [19], so they are expected to possess positive and negative net charges in seawater (pH 7.8–8.0). This may suggest a simple electrostatic interaction between the protein and calcite. However, the protein was not adsorbed to a positively charged synthetic polymer in seawater. Thus, the adsorption of rMrcp-20k to calcite cannot be explained simply by the electrostatic inter- action, and probably depends on the particular arrange- ment of surface amino acids in the protein structure. Comparison between the sequences of the gene from M. rosa and a homologous gene from B. albicostatus suggests that the abundance of charged amino acids and Cys residues, and the repetitive primary structure, are common features of this protein, whereas the num- ber of repeated sequences was different between differ- ent species. This may indicate that the characteristics of the protein found in this study can also be applied to the cp-20k protein in other barnacle cements. The holdfast system of the barnacle showed no simi- larity to that of the mussel, which is relatively well characterized. There were no sequence similarities among the protein components between the two systems. The mussel holdfast system [15] depends on several protein modifications, typically including 3,4-dihydroxyphenylalanine; however, no involvement of 3,4-dihydroxyphenylalanine in the barnacle cement was found [2]. The mussel attaches to an underwater foreign substratum using a byssal thread as its hold- fast. The tip of the byssus, called the disk, directly attaches to the substratum. At least two proteins, fp-3 and fp-5, have been identified as surface-coupling proteins of this disk [20]. Phosphorylation of the Ser residues in fp-5 has prompted the suggestion that cal- careous material-specific coupling is its functional role [21]. There is a huge quantity of calcareous material in the marine environment. Both the barnacle and mus- sel, at least, seem to provide a specific coupling protein for this frequently encountered material. They have acquired distinct molecular features in the course of evolution: the dependence on common amino acids with a rigid three-dimensional structure in the barna- cle, and the dependence on the function of the amino acid side chains with post-translational modifications in the mussel [15,22]. Moreover, Mrcp-20k may not be covalently linked to other bulk proteins in the barnacle cement; this is also different from the case in the mus- sel, whose surface proteins seem to be covalently linked to other bulk proteins in the disk [23]. Experimental procedures Chemicals The chemicals used were of the highest grade available and purchased from Wako Pure Chemical Industries (Osaka, Japan). ASW was prepared by dissolving Marine Art SF (Senju Seiyaku Co., Osaka, Japan) in ultrapure water that had been ultrafiltered through an MW3000-cutoff mem- brane (YM3; Amicon-Millipore, Billerica, MA, USA). Preparation of the cement samples Specimens of M. rosa attached to a polyethylene substra- tum were collected from Ryou-ishi Bay (Iwate, Japan). The secondary cement was collected as previously reported [3]. The primary cement was prepared from animals that had been carefully dislodged from the substratum by applying vibration, only those specimens without any apparent dam- age being used. The inner soft bodies were physically removed and cleaned. The calcareous base and peripheral shell were separately recovered, and each of them was Calcite-coupling protein in underwater adhesive Y. Mori et al. 6442 FEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBS weighed and decalcified by dialyzing against 2% (v ⁄ v) ace- tic acid at 4 °C. The supernatant was recovered as the ace- tic acid-soluble fraction, and the precipitate was rendered soluble as previously reported [3]. Briefly, the cement was suspended in a solution of 7 m guanidine hydrochloride and 10 mm Hepes at pH 7 and 60 °C for 1 h; the superna- tant of this corresponded to GSF1. The precipitate was ren- dered soluble by reduction in a solution of 0.5 m dithiothreitol, 7 m guanidine hydrochloride, 20 mm EDTA and 1.5 m Tris at pH 8.5 and 60 °C for 1 h in a nitrogen atmosphere; the supernatant was recovered as GSF2. Both fractions were dialyzed against 5% (v ⁄ v) acetic acid at 4 °C and then stored at ) 20 °C until needed. The protein in the secondary cement was partially extracted even in water. Therefore, nMrcp-20k was prepared by suspending the cement in ultrapure water and agitating overnight at 4 °C. The extract was recovered by centrifugation (21 600 g, 4 °C, 15 min), applied to a Mono-Q 5 ⁄ 50GL column (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) that had been equilibrated with 50 mm Tris ⁄ HCl at pH 7.4, and eluted with 50 mm Tris ⁄ HCl at pH 7.4 with a 30 min linear gradient of 1 m NaCl from 30% to 50%. Preparation of rMrcp-20k The Mrcp-20k recombinant system was constructed in bacte- rial cells. cDNA encoding mature Mrcp-20k was first ampli- fied by PCR with M. rosa cDNA [3] and Ex Taq (Takara Bio, Shiga, Japan) as the template and enzyme, respectively. The following oligo-DNA primers were designed from both the N-terminal and C-terminal regions of mature Mrcp-20k to create the NcoI and BamHI restriction sites, respectively: 5¢-AGTTG CCATGGCGCACGAGGAGGA-3¢ and 5¢-TT CTGTTC GGATCCCAAGGCTTA-3¢. The amplified DNA fragment was digested with both NcoI and BamHI, before being inserted into pET32a (Novagen, Darmstadt, Germany) with the same restriction sites. The sequence of the insert was confirmed by using a Prism Dye Deoxy sequencing kit and 3700-DNA analyzer (Applied Biosystems, Foster City, CA, USA). The resulting plasmid was transformed into E. coli OrigamiB (DE3) (Novagen). The transformant was culti- vated in a modified M9 medium [24] with 50 lgÆmL )1 carben- icillin and 0.75% (w ⁄ v) glucose at 37 °C for 16 h to reach the mid-log phase with an attenuance of 0.6–0.9 at 600 nm. Isopropyl thio-b-d-galactoside (0.4 mm) and 0.75% glucose were added to the medium, and the cells were cultivated at 30 °C for 6 h. A crude protein extract was prepared by sonication in 100 mm Tris ⁄ HCl at pH 9.0 on ice, and the supernatant was purified in an Ni-immobilized column (Novagen) with the standard protocol. The protein was eluted with 2 m imidazole, 500 mm NaCl and 50 mm Tris ⁄ HCl at pH 7.9. The rMrcp-20k was dialyzed against a buffer for enterokinase digestion, concentrated with Centri- prep (Amicon-Millipore), and treated with recombinant enterokinase [Novagen; enzyme ⁄ substrate ratio of 1 : 10 (molar ratio)] at 20 °C for 3 days. Final purification was car- ried out in the Mono-Q 5 ⁄ 50GL column as already described. The protein concentration was measured with a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA), with BSA used as a reference [25]. Immunochemical detection of Mrcp-20k The recombinant C-terminal 79 amino acid region was pre- pared as an antigen with a method similar to that used for the whole length protein, except that the vector used was pET30a (Novagen), and a 3.9 mm diameter · 150 mm l-Bondasphere RP-HPLC column (C8, 300 A ˚ ; Waters, Milford, MA, USA) was used for the purification. For PCR amplification of the C-terminal 79 amino acid region, the following oligo-DNA primers were used: 5¢-AATGTA CCATGGAAGCGCCGT-3¢ and 5¢-GCCTTCTGTTCGG ATCCCAAGGCT-3¢. The polyclonal antibody was raised in rabbits by serial subcutaneous injection (Takara Bio). Immunochemical detection was carried out by dot-blotting or electrotransfer to a nitrocellulose membrane (0.45 lm; Bio-Rad, Hercules, CA, USA). Poly(vinylidene difluoride) was not suitable for holding Mrcp-20k in our several trials, probably due to the abnormal characteristics of this pro- tein. A goat anti-rabbit IgG (H + L) horseradish peroxi- dase (HRP) conjugate (Bio-Rad) was used as the secondary antibody, and HRP-100 immunostaining (Konica-Minolta, Tokyo, Japan) was used to develop the signal. Characterization of rMrcp-20k The N-terminal sequence of the recombinant protein was confirmed with a protein sequencer (Procise 494 cLC; Applied Biosystems), and the molecular mass was con- firmed with MALDI-TOF MS. The sample was mixed with synapic acid saturated in 30% (v ⁄ v) acetonitrile and then analyzed with a Voyager-DE STR instrument (Applied Biosystems, Foster City, CA, USA), using Calibration Mixture 3 (Applied Biosystems) as the reference. The Lae- mmli buffer system [26] was used for SDS ⁄ PAGE analysis. The alkylation treatment of the protein was carried out as described in a previous study [4]. A 5 lm amount of rMrcp-20k was suspended in a solution of 7 m guanidine hydrochloride, 20 mm EDTA and 1.5 m Tris ⁄ HCl at pH 8.0. Monoiodo acetic acid (Wako Pure Chemical Indus- tries) was then added to an amount 500 times the number of cysteine residues in rMrcp-20k, and the mixture was incubated in a nitrogen atmosphere in the dark at room temperature for 2 h. The reaction mixture was purified by RP-HPLC and then subjected to MALDI-TOF MS analy- sis. The CD spectra of the protein (32 lgÆmL )1 , dissolved in 10 mm sodium phosphate at pH 6.8) were measured with a J-725 spectropolarimeter (Jasco, Tokyo, Japan). The spec- tra were scanned at 20 ° C from 200 nm to 320 nm, and then integrated 128 times. Prior to the analysis, a reduction Y. Mori et al. Calcite-coupling protein in underwater adhesive FEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBS 6443 treatment was carried out with 100 mm dithiothrei- tol ⁄ 10 mm sodium phosphate at pH 6.8 and 25 °C for 1 h, with subsequent dialysis against 100 mm NaCl and 10 mm sodium phosphate at pH 6.8. Measurement of adsorption to underwater material surfaces The protein adsorption to underwater materials was mea- sured by quantifying the protein amount in soluble fractions after incubating with defined particles. Neither the adsorp- tion of rMrcp-20k to a polypropylene tube nor any precipi- tate formation was apparent. Thus, a polypropylene tube was used to handle the protein solution. The particles used in this study were as follows: calcite (2500 cm 2 surface areaÆg )1 , 8 lm in diameter; Sankyo Seihun Co., Okayama, Japan), glass (50 lm in diameter; Toshinriko Co., Tokyo, Japan), benzoguanamine–formaldehyde resin (3000 cm 2 surface areaÆg )1 , 12.75 lm in diameter; Nippon Shokubai Co., Tokyo, Japan), zinc oxide (20 000 cm 2 surface areaÆg )1 ; 0.70 lm in diameter; Mitsui Mining and Smelting Co., Tokyo, Japan), magnetite (20 000 cm 2 surface areaÆg )1 ; Toda Kogyo Co., Hiroshima, Japan), gold-coated polystyrene (5.0 lm in diameter; Sekisui Chemical Co., Osaka, Japan), and polystyrene (5.0 lm in diameter; Duke Scientific Corpo- ration, Fremont, CA, USA). Each type of particle was suspended in 20 lL of two-fold concentrated ASW or in ultrapure water in a polypropylene tube and then incubated at 25 °C for 10 min. The same volume of protein (0.30 mgÆmL )1 , dissolved in ultrapure water) was preincu- bated at 25 °C, mixed with each type of particle, and incu- bated at 25 °C for 10 min to allow adsorption. A 10 lL aliquot of the supernatant was recovered by centrifugation, and the protein concentration was measured using a bicinch- oninic acid protein assay kit (Pierce) with an ‘enhanced pro- tocol’ according to the manufacturer’s specifications. The incubation time for adsorption was confirmed to be sufficient for maximum adsorption in a preliminary experiment. The adsorption affinity was determined by incubating various concentrations of the protein with each type of par- ticles (total surface area, 12.5 cm 2 each) in ASW, and then evaluating the amount of free protein as described above (N ¼ 3). Calibration curves were constructed as reported elsewhere [17]. The amount of adsorbed protein (lmol) per m 2 of the surface was calculated by the difference between the initial (C I ) and equilibrium (C eq ) protein concentration (lmolÆmL )1 ) according to the following equation: Q ¼½ðC I À C eq ÞV=ðWSÞð1Þ where V is the volume of the solution (0.04 mL), W is the mass of the adsorbent, and S is the specific surface area of the adsorbent. The amount of adsorbed protein reached a plateau under the experimental conditions used. This type of the isotherm can be described by the Langmuir model with the following equation: C eq =Q ¼ 1=NK þ C eq =N ð2Þ where N is the maximum number of adsorption sites per unit of surface area (molÆm )2 ) of the adsorbent, and K is the affinity of the adsorbent molecules (LÆmol )1 ) for the adsorption sites. The protein adsorption to the barnacle peripheral shell was visualized after removing the soft inner body of the animal from the peripheral shell and physically cleaning it. A10lL amount of rMrcp-20k (0.1 mgÆmL )1 ) in ASW was dropped on to the outer surface of the peripheral shell. After incubation at room temperature for 10 min, the shell was immersed in ASW three times for 10 min each and subjected to immunochemical detection with Cy3-labeled anti-rabbit IgG (GE Healthcare Bio-Science Corp.) and fluorescence microscopy. Analyses to evaluate the distribution of the molecular size An Optima XL-I (Beckman Coulter Inc., Fullerton, CA, USA) analytical ultracentrifuge with an AN60-Ti rotor was used in all investigations. Sedimentation velocity experi- ments at 20 °C were conducted at 42 000 r.p.m. The sample cells were double sector charcoal-filled centerpieces equipped with quartz windows. Concentration distributions were acquired by scanning at 215 nm. Protein samples were dia- lyzed against 20 mm NaCl solution, mixed with concentrated NaCl solution in the cell, to form appropriate solutions. The dcdt program in Beckman XLI data analysis soft- ware was used to analyze groups of boundaries to derive sedimentation coefficients. This method is based on the time-derivative method developed by Stafford [27], which fits Gaussian functions to the so-called g(s*) distribution from the time derivative of the concentration distributions (dc ⁄ dt), and the sedimentation coefficient was calculated on the basis of the positions of Gaussian fits to the g(s*) ver- sus s data. Results were confirmed by the method of Van Holde & Weischet [28]. The sedimentation coefficient was corrected to standard solvent conditions (the viscosity, and the density of water at 20 °C) using the same program. The sedimentation equilibrium runs were performed for 15 h before equilibrium absorbance measurements were taken at 215 nm. Protein solutions at three concentrations ranging from 12 to 22 lgÆmL )1 in NaCl solution were cen- trifuged at 21 000 r.p.m. at 20 °C. Molecular weights were obtained using Beckman XLI data analysis software, in which radial position versus absorbance data were fitted to the following equilibrium equation using nonlinear least- squares techniques: AðrÞ¼A 0 ðr 0 Þ exp½HM app ðr 2 À r 2 0 Þ þ B ð3Þ where H ¼ (1 ) mq)x 2 ⁄ 2RT, m is partial specific volume of sample, q is density of solvent, R is gas constant, T is Calcite-coupling protein in underwater adhesive Y. Mori et al. 6444 FEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBS temperature, x is angular velocity, A 0 is absorbance at a ref- erence point r 0 , A(r) is absorbance at a position r cm from the rotor center, and B is baseline correction. In this study, the m of rMrcp-20k (0.6804 mLÆg )1 ) and q of the solvents were calculated from the amino acid composition and solvent composition, respectively, using the program sednterp [29]. In order to confirm whether or not intramolecular disul- fide bonds were rearranged to intermolecular ones, rMrcp- 20k (0.1 mgÆmL )1 )in10mm Tris ⁄ HCl (pH 8.0) or two-fold concentrated ASW with 10 mm Tris ⁄ HCl (pH 8.0) were incubated at 25 °C for 7 days, dialyzed against 10 mm Tris ⁄ HCl at pH 6.8, separated on SDS ⁄ PAGE (15% T) without any reduction treatment, and visualized by western blotting with the rMrcp-20k-C antibody. Confirmation in the adsorbent was carried out in a similar manner. rMrcp- 20k (0.1 mgÆmL )1 ) was incubated in ASW with 60 mg of calcite particles at 25 °C for 7 days. After centrifuging (21 600 g,25°C, 15 min) and washing with ASW, the par- ticles were dialyzed against 5% (v ⁄ v) acetic acid to decal- cify them and to release the adsorbed protein into solution. The protein was then analyzed as described above after evaporation. PCR investigation of the gene homologous to that encoding Mrcp-20k B. albicostatus and Balanus amphitrite were collected from Shimizu Bay (Shizuoka, Japan), and Balanus rostratus was collected from Asamushi Bay (Aomori, Japan). RNA and DNA manipulations were performed as previously described [4]. 3¢-RACE was carried out with a degenerated primer designed from the consensus sequence of the repeti- tive sequences in Mrcp-20k by using a 3¢-RACE core kit (Takara Bio). The degenerated primer used was 5¢- CTG ATCTAGAGGTACCGGATCCTGYAACGANGAKCAY CCTG-3¢, where the underlining corresponds to the three- site adaptor region of the kit. A 336 bp DNA fragment was amplified only from B. albicostatus cDNA. Subsequent 5¢-RACE was carried out using a 5¢-RACE core kit (Taka- ra Bio). The 5¢-RACE primers used were as follows: 5¢-(pG-TG CCA GCA CCG GTG G)-3¢ for reverse tran- scription; 5¢-(AAA CAG TAA GGC CAG CGT AT)-3¢ and 5¢-(GCA TCA TGA TCA CGG AAA GA)-3¢ for the first PCR amplification; and 5¢-(TGA TGG CAA TGT GAT GTT GA)-3¢ and 5¢-(TGC TAC CAC TGC CAC ACC GA)-3¢ for the second PCR amplification. The coding region was finally confirmed by PCR amplification with the primers 5 ¢-(CAA CAC TTC TGT GCT C)-3¢ and 5¢-(GGC GTT CTC TCA GCC G)-3¢. Acknowledgements We thank Professor T. Watanabe of Niigata Univer- sity and Dr T. Shimoyama for their advice on the kinetic analysis and assistance with fluorescence microscopy observations. We also thank Dr S. Kanai and Ms N. Inoue of PharmaDesign, Inc., Japan for bio-informatic analyses. Special thanks are given to Professor J R. Shen of Okayama University for his critical reading of this manuscript. Calcite, benzoguan- amine–formaldehyde resin, zinc oxide, magnetite and gold-coated particles were kindly provided by Sankyo Seihun Co. Ltd, Nippon Shokubai Co. Ltd, Mitsui Mining and Smelting Co. Ltd, Toda Kogyo Co. Ltd, and Sekisui Chemical Co. Ltd, respectively. This work was performed as part of an industrial science and technology project entitled Technological Development for Biomaterials Design Based on Self-organizing Pro- teins, supported by the New Energy and Industrial Technology Development Organization (NEDO). References 1 Saroyan JR, Linder E, Dooley CA & Bleile HR (1970) Repair and reattachment in the Balanidae as related to their cementing mechanism. Ind Eng Chem Prod Res Dev 9, 122–133. 2 Kamino K (2006) Barnacle underwater attachment. In Biological Adhesives (Smith AM & Callow JA, eds), pp. 145–166. Springer-Verlag, Berlin. 3 Kamino K, Inoue K, Maruyama T, Takamatsu N, Harayama S & Shizuri Y (2000) Barnacle cement proteins. 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C eq , equilibrium protein concentration; C I , initial protein concentration; cp, cement protein; fp, mussel foot protein; GSF1 and GSF2, cement fractions. Calcite-specific coupling protein in barnacle underwater cement Youichi Mori 1 , Youhei Urushida 1 , Masahiro Nakano 1 , Susumu Uchiyama 2 and Kei Kamino 1 1