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Calcite-specific coupling protein in barnacle underwatercementYouichi Mori1, Youhei Urushida1, Masahiro Nakano1, Susumu Uchiyama2and Kei Kamino11 Marine Biotechnology Institute, Kamaishi, Iwate, Japan2 Department of Biotechnology, Graduate School of Engineering, Osaka University, JapanSessile organisms are destined for attachment to vari-ous materials in water. Because gregariousness is essen-tial for them, the opportunity to attach to a calcificexoskeleton of the same kind is necessarily favored.Thus, calcific material is one of the frequent foreignmaterials for attachment in the molecular system ofthe holdfast.The barnacle is a unique sessile crustacean. Once thelarva has settled on the foreign substratum, it metamor-phoses, calcifying the outer shell at the periphery andbase, and permanently attaches to the foreign substra-tum by a multiprotein complex called cement [1]. Thiscement is secreted through the calcareous base to anacellular milieu, and joins two different materials, theKeywordsadsorption; crustacean; protein complex;sessile organism; underwater adhesiveCorrespondenceK. Kamino, Marine Biotechnology Institute,3-75-1 Heita, Kamaishi, Iwate 026-0001JapanFax: +81 193 26 6592Tel.: +81 193 26 6584E-mail: kei.kamino@mbio.jpDatabaseThe nucleotide sequence data are availablein the DNA Data Bank of Japan under theaccession number AB329666(Received 5 July 2007, revised 18 October2007, accepted 23 October 2007)doi:10.1111/j.1742-4658.2007.06161.xThe barnacle relies for its attachment to underwater foreign substrata onthe formation of a multiprotein complex called cement. The 20 kDa cementprotein is a component of Megabalanus rosa cement, although its specificfunction in underwater attachment has not, until now, been known. Therecombinant form of the protein expressed in bacteria was purified in solu-ble form under physiological conditions, and confirmed to retain almostthe same structure as that of the native protein. Both the protein from theadhesive 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 wereessential for the proper folding of the monomeric protein structure. Therecombinant protein was adsorbed to calcite and metal oxides in seawater,but not to glass and synthetic polymers. The adsorption isotherm foradsorption to calcite fitted the Langmuir model well, indicating that theprotein is a calcite-specific adsorbent. An evaluation of the distribution ofthe molecular size in solution by analytical ultracentrifugation indicatedthat the recombinant protein exists as a monomer in 100 mm to 1 m NaClsolution; thus, the protein acts as a monomer when interacting with thecalcite surface. cDNA encoding a homologous protein was isolated fromBalanus albicostatus, and its derived amino acid sequence was comparedwith that from M. rosa. Calcite is the major constituent in both the shell ofbarnacle base and the periphery, which is also a possible target for thecement, due to the gregarious nature of the organisms. The specificity ofthe protein for calcite may be related to the fact that calcite is the mostfrequent material attached by the cement.AbbreviationsASW, artificial seawater; Ceq, equilibrium protein concentration; CI, 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 ofMrcp-20k expressed in Escherichia coli.6436 FEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBScrustacean’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 thefour cement proteins produced by the barnacle,cp-100k and cp-52k are the two major components interms of amount, and are characterized by their insolu-ble nature [3]. These two components are consideredto constitute the bulk region of the cement. A reducingtreatment with guanidine hydrochloride was necessaryto render the bulk proteins soluble. cp-68k is also amajor 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 proteinin underwater attachment is not known at present [3].cp-20k is a minor cement protein in terms of itsamount, and is not post-translationally modified. Theamino acid composition of cp-20k is characterized bythe unusual abundance of Cys (17%) and chargedamino acids (Asp, 11.5%; Glu, 10.4%; His, 10.4%) [4].Although the high abundance of the Cys residue in theprotein has suggested a possible contribution to inter-molecular crosslinking or coupling [5], our previousstudy has indicated that this is not the case, at leastwith respect to the latter speculation [4].Underwater attachment is a multifunctional process,which is different from that of an artificial adhesive inair, and is thus an unachievable technique at present.The process [6] involves such subfunctions as prevent-ing random aggregation during transport via thecement duct, displacing sufficient seawater to primeand spread on the surface without being dispersed inthe water, coupling strongly with a variety of materialsurfaces, and self-assembly to join the calcareous baseand the substratum. After the process, it is then neces-sary to cure the cement so that the holdfast remainsstiff and tough, and to protect it from microbial degra-dation. The insoluble nature of the complex and thelimitations of microanalytical methods for studyingeach function, however, have hindered elucidation ofthe specific function of each cement protein [3].There are two types of sample for studies on barnaclecement: 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 theanimal is free from a substratum. Both forms of cementare similar in their whole amino acid composition [7],and appear to contain the same protein components asdetermined by peptide mapping with cyanogen bromidetreatment [3]. Reattachment of the barnacle to a newsubstratum by secondary cement has also been reported[1,8], although the adhesive strength was weaker thanthat of primary cement. The primary cement seemed tobe denser and more rigid than the secondary cement.Although these studies indicated that the primary andsecondary cements have the same protein composition,it is not clear whether the protein–protein interactionsand the topology in the two complexes are the same.Megabalanus rosa (Mr)cp-20k in the secondarycement was chemically characterized in a previousstudy [4]. However, neither the nature of Mrcp-20k inthe primary cement nor the specific function of thisprotein in underwater attachment has been unraveled.The present study was performed to characterize thenature of the protein in the primary cement. Thereaf-ter, we expressed the recombinant form of the proteinin bacteria in a soluble form under physiological con-ditions, and confirmed that the recombinant proteinhas 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 surfacesin water. This is the first report to identify a bioticunderwater adhesive protein as a specific adsorbent tocalcite, by directly measuring the adsorbing activity ofthe protein prepared under physiological conditions.ResultsConfirmation of Mrcp-20k in natural barnaclecementMrcp-20k was extracted only from guanidine hydro-chloride-soluble fraction 1 (GSF1) of the primarycement, but not from GSF2, which is the guanidinehydrochloride-soluble fraction after reducing treatment(Fig. 1A). This result is consistent with what is foundin the secondary cement [4]. Mrcp-20k in GSF1 of theprimary cement only gave a band with a monomericmolecular mass on SDS ⁄ PAGE without the reducingtreatment (Fig. 1A); this is also consistent with what isfound for the secondary cement [4]. This indicates thatMrcp-20k is not covalently crosslinked in the naturalcement. Mrcp-20k was not detected in the peripheralshell (Fig. 1B), indicating that Mrcp-20k is not aprotein related to calcification of the shell.Preparation of the recombinant form of Mrcp-20kin bacteriaThe 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 adhesiveFEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBS 6437cal conditions (Fig. 2A). The elution profiles fromboth RP-HPLC and ion exchange HPLC were identi-cal to those of native Mrcp-20k in the secondarycement extracted in pure water, nMrcp-20k (supple-mentary Fig. S1A,B). Owing to the vector construc-tion, rMrcp-20k was designed to have an additionaltripeptide, Ala-Met-Ala, attached to the N-terminus.The N-terminal sequence and molecular mass of therecombinant protein were determined to be AMAHE-EDGV and 20 629 Da, respectively, which agree wellwith the deduced sequence and mass (20 629.3 Da).This molecular mass corresponds to the form of theprotein in which all Cys residues form disulfide bonds.Alkylation treatment of rMrcp-20k resulted in a samemass, suggesting that no free SH groups are present inrMrcp-20k. The presence of all Cys residues in theintramolecular disulfide form in the recombinant pro-tein is the same as what is found for the protein in thesecondary cement [4]. SDS ⁄ PAGE analysis showedthat rMrcp-20k without a reduction treatment had aslightly lower mobility than that with the reductiontreatment (Fig. 2B); this resembles the behavior of thenative Mrcp-20k protein in the secondary cement. TheCD spectrum of rMrcp-20k in a 10 mm sodium phos-phate buffer (pH 6.8) was also identical to that ofnMrcp-20k; both showed the presence of a mixture ofb-turn and random coil structures [9,10]. These spectrawere remarkably different from that observed after areducing treatment, probably due to denaturation ofthe protein (Fig. 3).Adsorption of rMrcp-20k to underwater materialsurfacesThe adsorption of rMrcp-20k to several underwatermaterial surfaces was investigated, and the findingsare summarized in Fig. 4. The protein was adsorbedto calcite in artificial seawater (ASW), whereas it wasnot adsorbed to glass, gold, polystyrene, or benzo-guanamine-formaldehyde resin, which is a positivelycharged synthetic polymer. The protein was alsoadsorbed to a limited extent to metal oxides such aszinc oxide and magnetite. The amount adsorbed tocalcite in pure water was almost the same as that inASW.ABFig. 2. Purification of rMrcp-20k. (A) Samples were separated byusing the 16.5% T Tris ⁄ Tricine buffer system of SDS ⁄ PAGE [30].Lane 2: crude extract of bacterial cells. Lane 3: rMrcp-20k fusedwith a tag in the vector construct. Lane 4: rMrcp-20k. Lane 1, lowmolecular mass markers (Bio-Rad; aldolase, 45.0 kDa; carbonicanhydrase, 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.ABFig. 1. Characterization of Mr cp-20k in the primary cement. (A)Western blotting of fractions rendered soluble from the primarycement by using the antibody to Mrcp-20k. Lane 1: GSF1 withreduction pretreatment in SDS ⁄ PAGE. Lane 2: GSF2 with reductionpretreatment. 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 byusing the antibody to Mrcp-20k. Two grams each (dry weight) ofthe peripheral shell and calcareous base were decalcified andsubjected to dot-blotting. Lane 1: 2% acetic acid solution–solublefraction of the peripheral shell. Lane 2: GSF1 and GSF2 of theperipheral shell. Lane 3: 2% acetic acid solution–soluble fraction ofthe base. Lane 4: GSF1 and GSF2 of the base. Lane 5: rMrcp-20kas positive control (1 lg). Lane 6: trypsin inhibitor from soybean asnegative 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 FEBSThe relationship between the concentration of theprotein at the calcite surface and its solution concen-tration is described by the adsorption isotherm. Thelinearized forms of the isotherm for the adsorption tocalcite were Ceq⁄ Q ¼ 0.3168 · 10)3+ 4.199Ceq[corre-lation coefficient (r2) of 0.97] in ASW andCeq⁄ Q ¼ 1.7168 · 10)3+ 3.782Ceq(r2of 0.98) in thedilute buffer [Ceq, equilibrium protein concentration;Q, amount of absorbed protein (lmol) per m2of thesurface] (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 · 107m)1and N ¼ 2.38 · 10)7molÆm)2in ASW, and K ¼ 2.20 · 106m)1and N ¼2.64 · 10)7molÆm)2in the dilute buffer solution. Theisotherms for adsorption to zinc oxide and magnetitewere not linear (r2of 0.75 and 0.58, respectively), sothat the adsorption to these surfaces seemed not to beof the typical Langmuir type (supplementary Fig. S2).The adsorption of rMrcp-20k to the barnacle shellwas visualized using the antibody to rMrcp-20k withthe secondary antibody conjugated by fluorochrome(Fig. 6 and supplementary Fig. S3). A 10 min incuba-tion with rMrcp-20k in ASW gave rise to fluorescenceemission at the barnacle shell, demonstrating theWavelength (nm)[θ] (deg cm-2 dmol-1)200-30-20-10010[θ] (deg cm-2 dmol-1)-30-20-10010[θ] (deg cm-2 dmol-1)-30-20-10010ABC250 300 320Wavelength (nm)200 250 300 320Wavelength (nm)200250 300 320Fig. 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.Aamount of adsorbed protein (ng/cm2)050100150200250300BCDEFGHFig. 4. Adsorption of rMrcp-20k to various solid surfaces. Theadsorption of rMrcp-20k to the particles of several materials in10 min at 25 °C was evaluated by measuring the decrease in pro-tein amount remaining in the solution. Adsorption to (A) calcite inASW, (B) glass in ASW, (C) benzoguanamine–formaldehyde resinin ASW, (D) zinc oxide in ASW, (E) magnetite in ASW, (F) gold inASW, (G) polystyrene in ASW, and (H) calcite in pure water. Errorbars indicate the standard deviation.Ceq (µmol/mL)Ceq/Q (m2/mL)-5.2E-18 0 0.01 0.02 0.03 0.04 0.05 Ceq/Q (m2/mL)0 0.01 0.02 0.03 0.04 0.05 B A 0.002 0.004 0.006 0.008 0.01 Ceq (µmol/mL)-2.08E-1 0.002 0.004 0.006 0.008 0.01Fig. 5. Linearized adsorption isotherm for adsorption of rMrcp-20kto calcite. (A) Isotherm in ASW. (B) Isotherm in 2.14 mM sodiumcarbonate (pH 8.2).Y. Mori et al. Calcite-coupling protein in underwater adhesiveFEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBS 6439successful adsorption of the protein to the calcareousshell of the barnacle.The distribution of the molecular size ofrMrcp-20kThe distribution of the molecular size of the recombi-nant protein was evaluated by analytical ultracentrifu-gation (Table 1).Sedimentation velocity analyses indicated that theprotein exists as a single component in 100 mm to500 mm NaCl solution. The sedimentation coefficientof the component was estimated to be s $ 2.5.The sedimentation equilibrium analyses gave nearly20 kDa as the molecular mass in 100 mm to 1 m NaClsolution, which is consistent with monomeric molecu-lar mass of the protein. Therefore, the s $ 2.5 speciesfound by sedimentation velocity corresponds to themonomeric form of the protein.The possible change of intramolecular disulfidebonds to intermolecular ones after a longer period ofincubation in ASW was evaluated by SDS ⁄ PAGEanalysis (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 hadbeen no change of intramolecular disulfide bonds tothe intermolecular type in the protein.Isolation of the homologous gene from BalanusalbicostatusA PCR investigation of a homologous gene in threebarnacle species was attempted with several degener-ated oligonucleotide primers based on the primarystructure of Mrcp-20k. All PCR trials with primersdesigned from the primary structure of Mrcp-20k failedto amplify homologous DNA, except for 3¢-RACE withcDNA of Balanus albicostatus. The sequence of homo-logous cDNA in B. albicostatus determined in thisstudy was 700 bp, and the coding region was deter-mined to encode 125 amino acids (supplementaryFig. S4). The first 20 amino acids are considered toFig. 6. Demonstration of the adsorption of Mrcp-20k to the barna-cle peripheral shell. The protein adsorbed to the shell was treatedwith the antibody, and visualized with the secondary antibodylinked to fluorochrome Cy3 (GE Healthcare Bio-Science). Imagesunder visible light (left) and those under reflected fluorescence(right) are shown. The image pair was captured from the sameangle of the object. In the images under visible light, yellow areascorrespond to the shell, and white areas are transparent withoutany object. Shell was incubated with rMrcp-20k, washed, and trea-ted with the antibody to Mrcp-20k. No fluorescence was observedin the control experiment (supplementary Fig. S3).Table 1. The distribution of the molecular size of rMrcp-20k evalu-ated by analytical ultracentrifugation. The sedimentation coefficientsand 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 theSEDNTERP pro-gram [29]. Molecular masses were determined by sedimentationequilibrium analyses.NaCl concentration (M)s20, W(S)Molecularmass (kDa)0.1 2.6 19.60.3 2.5 18.90.4 2.5 –0.5 2.4 –1.0 – 21.1Fig. 7. Rearrangement of disulfide bonds in rMrcp-20k during long-term incubation. The molecular masses of rMrcp-20k after severaltreatments for 1 week at 25 °C were estimated by western blottingwith the antibody to Mrcp-20k antibody. rMrcp-20k was incubatedin ASW adjusted to pH 8.0 without calcite particles (lane 1), in adilute buffer adjusted to pH 8.0 without calcite particles (lane 2), orin 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 FEBScorrespond to the signal peptide, because of its highhydrophobicity and the existence of a predicted signalpeptidase cleavage position [11]. The molecular massand isoelectric point of the mature polypeptide werepredicted to be 12 297.0 Da and 8.3, respectively,assuming that all Cys residues were in the disulfideform for prediction of the molecular mass. The aminoacid composition deduced from the cDNA indicatedthat charged amino acids such as His (20%), Lys(10%) and Cys (17%) are the dominant residues; thecontents of these residues appear to be significantlyhigher than in the standard amino acid composition[12]. The charged amino acids Asp, Glu, His, Lys andArg are estimated to comprise 42% of the total resi-dues. Alignment of the Cys residues indicated that theprimary structure of the homologous protein in B. albi-costatus consists of four repeated sequences (Fig. 8).The difference between the B. albicostatus protein andMrcp-20k in their amino acid lengths depended on thedifference in the number of repeats. The similar Cysspacing, the existence of Pro preceding the secondCys, the presence of two amino acids after the secondCys, and the sporadic insertion of clusters of chargedamino acids such as HKHHDHGK, HHHDD,RHGKKH and HRKFH, are common characteristicsfound in both proteins [4]. A BLAST search [13] of thenonredundant database and a sequence profile-basedfold-recognition method for three-dimensional struc-tural prediction [14] failed to provide any homologoussequences and meaningful structure from currentlyavailable databases.DiscussionAlthough Mrcp-20k was found in the secondary cementin the previous study, neither the presence of thisprotein in the barnacle natural adhesive layer or pri-mary cement, nor its specific function in underwaterattachment, has been characterized so far. The presentstudy was thus conducted to address these questions.The conditions required for extracting the protein fromthe insoluble primary cement, and its behavior in theSDS ⁄ PAGE analysis, were similar to those of the pro-tein from the secondary cement. The protein exhibiteda monomeric molecular mass on SDS ⁄ PAGE evenwithout a reducing pretreatment, a characteristic alsofound for the protein from the secondary cement. Theamino acid composition of Mrcp-20k is characterizedby the unusually high contents of Cys (17%) andcharged amino acid residues [4], which suggests a possi-ble role of polymerization via intermolecular disulfidebonds for this protein in the process of underwateradhesion [5]. The present study, however, excluded thispossibility. This was further supported by the fact thatlong-term incubation of the bacterial recombinant pro-tein in ASW did not give rise to any polymerizedmolecular species by the conversion of disulfide bondsto the intermolecular form. The abundance of Cys andcharged amino acid residues is reminiscent of proteinsinvolved in biomineralization. As the cement hasalways been collected from the surface of the barnaclecalcareous base, some contamination of the proteinsused for calcification may have occurred. However, thefact that Mrcp-20k could not be detected in the periph-eral calcareous shell indicates that the protein is specificto underwater attachment of the base, and does notcontribute to the calcification process. The protein con-tains few hydrophobic residues, which would result in apoor hydrophobic core in the structure; this may be areason for the introduction of abundant intramoleculardisulfide bonds to stabilize the structure in molecularevolution. This was confirmed by the marked change inthe CD spectrum with the reducing treatment. Thelimited number of hydrophobic residues may, inturn, suggest the significance of the charged aminoacid 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 thisprotein in bacteria under physiological conditions, andto compare the characteristics of the recombinant pro-tein with those of the native protein extracted with purewater. Both proteins showed the same elution profilesin column chromatography, the same behaviors asanalyzed by SDS ⁄ PAGE, MALDI-TOF MS and CDspectra, and similar resistance to alkylation treatmentwithout any reducing treatment, indicating that bothproteins possessed similar molecular structures. Wetherefore characterized the functional properties of therecombinant protein. This is an unusual case in bioticunderwater adhesive studies, as all mussel foot proteins(fps), which represent another model system, areFig. 8. Alignment of the repetitive sequences in Mr cp-20k and thehomologous protein in B. albicostatus. All Cys residues are shownin black, and conserved Pro residues are shown in gray.Y. Mori et al. Calcite-coupling protein in underwater adhesiveFEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBS 6441subjected to heavy post-translational modifications [15],so that the native activity of the simple recombinantprotein cannot be obtained. The present study repre-sents the first report based on a recombinant proteinretaining almost the same structure as that of the nativeprotein in the study of biotic underwater adhesive.The protein was adsorbed to calcite, a crystallineform of calcium carbonate, but not to glass and syn-thetic polymers. The isotherm for adsorption of therecombinant protein to calcite followed the Langmuirmodel, which has been extensively applied to the quan-titative evaluation of the interaction between macro-molecules and mineral interfaces [16]. Although theprotein was also adsorbed to some metal oxides to alimited extent, this adsorption isotherm did not fit theLangmuir model. These results suggest that the adsorp-tion to calcite is a specific function of Mrcp-20k. Thismay not be surprising if we consider that half of thematerial to be attached is the organism’s own calcare-ous base. The barnacle also prefers to attach itself tothe peripheral calcite shell of another barnacle, becauseof the gregarious behavior of this species. It thereforeseems that the barnacle arranges a specific protein inthe cement to be adsorbed to the most typical target,calcite, although it is not clear whether the target of theprotein is specific to the organism’s own base or theforeign calcified shell, or both.The adsorption isotherm for the attachment ofrMrcp-20k to calcite determined in the present studyindicated that the protein has an affinity for calcite thatis one magnitude of order higher than that of the ame-logenin–hydroxyapatite interaction, whose adsorptionaffinity was 1.97 · 106m)1[17]. The calculated pI valuefor rMrcp-20k is 4.7. The points for zero charge ofcalcite and glass are 9.50 ± 0.50 [18] and 1.80, respec-tively [19], so they are expected to possess positive andnegative net charges in seawater (pH 7.8–8.0). This maysuggest a simple electrostatic interaction between theprotein and calcite. However, the protein was notadsorbed to a positively charged synthetic polymer inseawater. Thus, the adsorption of rMrcp-20k to calcitecannot 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 fromM. rosa and a homologous gene from B. albicostatussuggests that the abundance of charged amino acidsand 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 characteristicsof the protein found in this study can also be appliedto 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 wellcharacterized. There were no sequence similaritiesamong the protein components between the twosystems. The mussel holdfast system [15] dependson several protein modifications, typically including3,4-dihydroxyphenylalanine; however, no involvementof 3,4-dihydroxyphenylalanine in the barnacle cementwas found [2]. The mussel attaches to an underwaterforeign substratum using a byssal thread as its hold-fast. The tip of the byssus, called the disk, directlyattaches to the substratum. At least two proteins, fp-3and fp-5, have been identified as surface-couplingproteins of this disk [20]. Phosphorylation of the Serresidues 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 inthe marine environment. Both the barnacle and mus-sel, at least, seem to provide a specific coupling proteinfor this frequently encountered material. They haveacquired distinct molecular features in the course ofevolution: the dependence on common amino acidswith a rigid three-dimensional structure in the barna-cle, and the dependence on the function of the aminoacid side chains with post-translational modificationsin the mussel [15,22]. Moreover, Mrcp-20k may not becovalently linked to other bulk proteins in the barnaclecement; this is also different from the case in the mus-sel, whose surface proteins seem to be covalentlylinked to other bulk proteins in the disk [23].Experimental proceduresChemicalsThe chemicals used were of the highest grade available andpurchased from Wako Pure Chemical Industries (Osaka,Japan). ASW was prepared by dissolving Marine Art SF(Senju Seiyaku Co., Osaka, Japan) in ultrapure water thathad been ultrafiltered through an MW3000-cutoff mem-brane (YM3; Amicon-Millipore, Billerica, MA, USA).Preparation of the cement samplesSpecimens of M. rosa attached to a polyethylene substra-tum were collected from Ryou-ishi Bay (Iwate, Japan). Thesecondary cement was collected as previously reported [3].The primary cement was prepared from animals that hadbeen carefully dislodged from the substratum by applyingvibration, only those specimens without any apparent dam-age being used. The inner soft bodies were physicallyremoved and cleaned. The calcareous base and peripheralshell were separately recovered, and each of them wasCalcite-coupling protein in underwater adhesive Y. Mori et al.6442 FEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBSweighed 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 renderedsoluble as previously reported [3]. Briefly, the cement wassuspended in a solution of 7 m guanidine hydrochlorideand 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 mdithiothreitol, 7 m guanidine hydrochloride, 20 mm EDTAand 1.5 m Tris at pH 8.5 and 60 °C for 1 h in a nitrogenatmosphere; the supernatant was recovered as GSF2. Bothfractions were dialyzed against 5% (v ⁄ v) acetic acid at 4 °Cand then stored at ) 20 °C until needed. The protein in thesecondary cement was partially extracted even in water.Therefore, nMrcp-20k was prepared by suspending thecement 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 (GEHealthcare Bio-Sciences Corp., Piscataway, NJ, USA) thathad been equilibrated with 50 mm Tris ⁄ HCl at pH 7.4, andeluted with 50 mm Tris ⁄ HCl at pH 7.4 with a 30 min lineargradient of 1 m NaCl from 30% to 50%.Preparation of rMrcp-20kThe 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 (TakaraBio, Shiga, Japan) as the template and enzyme, respectively.The following oligo-DNA primers were designed from boththe N-terminal and C-terminal regions of mature Mrcp-20kto create the NcoI and BamHI restriction sites, respectively:5¢-AGTTGCCATGGCGCACGAGGAGGA-3¢ and 5¢-TTCTGTTCGGATCCCAAGGCTTA-3¢. The amplified DNAfragment was digested with both NcoI and BamHI, beforebeing inserted into pET32a (Novagen, Darmstadt, Germany)with the same restriction sites. The sequence of the insert wasconfirmed by using a Prism Dye Deoxy sequencing kit and3700-DNA analyzer (Applied Biosystems, Foster City, CA,USA). The resulting plasmid was transformed into E. coliOrigamiB (DE3) (Novagen). The transformant was culti-vated in a modified M9 medium [24] with 50 lgÆmL)1carben-icillin and 0.75% (w ⁄ v) glucose at 37 °C for 16 h to reachthe 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% glucosewere added to the medium, and the cells were cultivatedat 30 °C for 6 h. A crude protein extract was prepared bysonication in 100 mm Tris ⁄ HCl at pH 9.0 on ice, andthe supernatant was purified in an Ni-immobilized column(Novagen) with the standard protocol. The protein waseluted with 2 m imidazole, 500 mm NaCl and 50 mmTris ⁄ HCl at pH 7.9. The rMrcp-20k was dialyzed against abuffer for enterokinase digestion, concentrated with Centri-prep (Amicon-Millipore), and treated with recombinantenterokinase [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 alreadydescribed. The protein concentration was measured with abicinchonic acid protein assay kit (Pierce, Rockford, IL,USA), with BSA used as a reference [25].Immunochemical detection of Mrcp-20kThe recombinant C-terminal 79 amino acid region was pre-pared as an antigen with a method similar to that used forthe whole length protein, except that the vector used waspET30a (Novagen), and a 3.9 mm diameter · 150 mml-Bondasphere RP-HPLC column (C8, 300 A˚; Waters,Milford, MA, USA) was used for the purification. ForPCR amplification of the C-terminal 79 amino acid region,the following oligo-DNA primers were used: 5¢-AATGTACCATGGAAGCGCCGT-3¢ and 5¢-GCCTTCTGTTCGGATCCCAAGGCT-3¢. The polyclonal antibody was raisedin rabbits by serial subcutaneous injection (Takara Bio).Immunochemical detection was carried out by dot-blottingor 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 secondaryantibody, and HRP-100 immunostaining (Konica-Minolta,Tokyo, Japan) was used to develop the signal.Characterization of rMrcp-20kThe N-terminal sequence of the recombinant protein wasconfirmed with a protein sequencer (Procise 494 cLC;Applied Biosystems), and the molecular mass was con-firmed with MALDI-TOF MS. The sample was mixed withsynapic acid saturated in 30% (v ⁄ v) acetonitrile and thenanalyzed with a Voyager-DE STR instrument (AppliedBiosystems, Foster City, CA, USA), using CalibrationMixture 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 asdescribed in a previous study [4]. A 5 lm amount ofrMrcp-20k was suspended in a solution of 7 m guanidinehydrochloride, 20 mm EDTA and 1.5 m Tris ⁄ HCl atpH 8.0. Monoiodo acetic acid (Wako Pure Chemical Indus-tries) was then added to an amount 500 times the numberof cysteine residues in rMrcp-20k, and the mixture wasincubated in a nitrogen atmosphere in the dark at roomtemperature for 2 h. The reaction mixture was purified byRP-HPLC and then subjected to MALDI-TOF MS analy-sis. The CD spectra of the protein (32 lgÆmL)1, dissolvedin 10 mm sodium phosphate at pH 6.8) were measured witha J-725 spectropolarimeter (Jasco, Tokyo, Japan). The spec-tra were scanned at 20 ° C from 200 nm to 320 nm, andthen integrated 128 times. Prior to the analysis, a reductionY. Mori et al. Calcite-coupling protein in underwater adhesiveFEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBS 6443treatment 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 mmsodium phosphate at pH 6.8.Measurement of adsorption to underwatermaterial surfacesThe protein adsorption to underwater materials was mea-sured by quantifying the protein amount in soluble fractionsafter 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 tubewas used to handle the protein solution. The particles used inthis study were as follows: calcite (2500 cm2surface areaÆg)1,8 lm in diameter; Sankyo Seihun Co., Okayama, Japan),glass (50 lm in diameter; Toshinriko Co., Tokyo, Japan),benzoguanamine–formaldehyde resin (3000 cm2surfaceareaÆg)1, 12.75 lm in diameter; Nippon Shokubai Co.,Tokyo, Japan), zinc oxide (20 000 cm2surface areaÆg)1;0.70 lm in diameter; Mitsui Mining and Smelting Co.,Tokyo, Japan), magnetite (20 000 cm2surface areaÆg)1; TodaKogyo 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 wassuspended in 20 lL of two-fold concentrated ASW or inultrapure water in a polypropylene tube and then incubatedat 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 lLaliquot 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. Theincubation time for adsorption was confirmed to be sufficientfor maximum adsorption in a preliminary experiment.The adsorption affinity was determined by incubatingvarious concentrations of the protein with each type of par-ticles (total surface area, 12.5 cm2each) in ASW, and thenevaluating the amount of free protein as described above(N ¼ 3). Calibration curves were constructed as reportedelsewhere [17]. The amount of adsorbed protein (lmol) perm2of the surface was calculated by the difference betweenthe initial (CI) and equilibrium (Ceq) protein concentration(lmolÆmL)1) according to the following equation:Q ¼½ðCIÀ CeqÞV=ðWSÞð1Þwhere V is the volume of the solution (0.04 mL), W is themass of the adsorbent, and S is the specific surface area ofthe adsorbent. The amount of adsorbed protein reached aplateau under the experimental conditions used. This typeof the isotherm can be described by the Langmuir modelwith the following equation:Ceq=Q ¼ 1=NK þ Ceq=N ð2Þwhere N is the maximum number of adsorption sites perunit of surface area (molÆm)2) of the adsorbent, and K isthe affinity of the adsorbent molecules (LÆmol)1) for theadsorption sites.The protein adsorption to the barnacle peripheral shellwas visualized after removing the soft inner body of theanimal from the peripheral shell and physically cleaning it.A10lL amount of rMrcp-20k (0.1 mgÆmL)1) in ASW wasdropped on to the outer surface of the peripheral shell.After incubation at room temperature for 10 min, the shellwas immersed in ASW three times for 10 min each andsubjected to immunochemical detection with Cy3-labeledanti-rabbit IgG (GE Healthcare Bio-Science Corp.) andfluorescence microscopy.Analyses to evaluate the distribution of themolecular sizeAn Optima XL-I (Beckman Coulter Inc., Fullerton, CA,USA) analytical ultracentrifuge with an AN60-Ti rotor wasused in all investigations. Sedimentation velocity experi-ments at 20 °C were conducted at 42 000 r.p.m. The samplecells were double sector charcoal-filled centerpieces equippedwith quartz windows. Concentration distributions wereacquired by scanning at 215 nm. Protein samples were dia-lyzed against 20 mm NaCl solution, mixed with concentratedNaCl 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 derivesedimentation coefficients. This method is based on thetime-derivative method developed by Stafford [27], whichfits Gaussian functions to the so-called g(s*) distributionfrom the time derivative of the concentration distributions(dc ⁄ dt), and the sedimentation coefficient was calculated onthe basis of the positions of Gaussian fits to the g(s*) ver-sus s data. Results were confirmed by the method of VanHolde & Weischet [28].The sedimentation coefficient was corrected to standardsolvent conditions (the viscosity, and the density of waterat 20 °C) using the same program.The sedimentation equilibrium runs were performed for15 h before equilibrium absorbance measurements weretaken at 215 nm. Protein solutions at three concentrationsranging from 12 to 22 lgÆmL)1in NaCl solution were cen-trifuged at 21 000 r.p.m. at 20 °C. Molecular weights wereobtained using Beckman XLI data analysis software, inwhich radial position versus absorbance data were fitted tothe following equilibrium equation using nonlinear least-squares techniques:AðrÞ¼A0ðr0Þ exp½HMappðr2À r20Þ þ B ð3Þwhere H ¼ (1 ) mq)x2⁄ 2RT, m is partial specific volume ofsample, q is density of solvent, R is gas constant, T isCalcite-coupling protein in underwater adhesive Y. Mori et al.6444 FEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBStemperature, x is angular velocity, A0is absorbance at a ref-erence point r0, A(r) is absorbance at a position r cm fromthe rotor center, and B is baseline correction. In this study,the m of rMrcp-20k (0.6804 mLÆg)1) and q of the solventswere calculated from the amino acid composition and solventcomposition, 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-foldconcentrated ASW with 10 mm Tris ⁄ HCl (pH 8.0) wereincubated at 25 °C for 7 days, dialyzed against 10 mmTris ⁄ HCl at pH 6.8, separated on SDS ⁄ PAGE (15% T)without any reduction treatment, and visualized by westernblotting with the rMrcp-20k-C antibody. Confirmation inthe adsorbent was carried out in a similar manner. rMrcp-20k (0.1 mgÆmL)1) was incubated in ASW with 60 mg ofcalcite 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 afterevaporation.PCR investigation of the gene homologous tothat encoding Mrcp-20kB. albicostatus and Balanus amphitrite were collected fromShimizu Bay (Shizuoka, Japan), and Balanus rostratus wascollected from Asamushi Bay (Aomori, Japan). RNA andDNA manipulations were performed as previouslydescribed [4]. 3¢-RACE was carried out with a degeneratedprimer 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¢-CTGATCTAGAGGTACCGGATCCTGYAACGANGAKCAYCCTG-3¢, where the underlining corresponds to the three-site adaptor region of the kit. A 336 bp DNA fragmentwas amplified only from B. albicostatus cDNA. Subsequent5¢-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 thefirst PCR amplification; and 5¢-(TGA TGG CAA TGTGAT GTT GA)-3¢ and 5¢-(TGC TAC CAC TGC CACACC GA)-3¢ for the second PCR amplification. The codingregion was finally confirmed by PCR amplification with theprimers 5 ¢-(CAA CAC TTC TGT GCT C)-3¢ and 5¢-(GGCGTT CTC TCA GCC G)-3¢.AcknowledgementsWe thank Professor T. Watanabe of Niigata Univer-sity and Dr T. Shimoyama for their advice on thekinetic analysis and assistance with fluorescencemicroscopy observations. We also thank Dr S. Kanaiand Ms N. Inoue of PharmaDesign, Inc., Japan forbio-informatic analyses. Special thanks are given toProfessor J R. Shen of Okayama University for hiscritical reading of this manuscript. Calcite, benzoguan-amine–formaldehyde resin, zinc oxide, magnetite andgold-coated particles were kindly provided by SankyoSeihun Co. Ltd, Nippon Shokubai Co. Ltd, MitsuiMining and Smelting Co. Ltd, Toda Kogyo Co. Ltd,and Sekisui Chemical Co. Ltd, respectively. This workwas performed as part of an industrial science andtechnology project entitled Technological Developmentfor Biomaterials Design Based on Self-organizing Pro-teins, supported by the New Energy and IndustrialTechnology Development Organization (NEDO).References1 Saroyan JR, Linder E, Dooley CA & Bleile HR (1970)Repair and reattachment in the Balanidae as related totheir cementing mechanism. Ind Eng Chem Prod ResDev 9, 122–133.2 Kamino K (2006) Barnacle underwater attachment. InBiological 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 cementproteins. J Biol Chem 275, 27360–27365.4 Kamino K (2001) Novel barnacle underwater adhesiveprotein is a charged amino acid-rich protein constitutedby a Cys-rich repetitive sequence. Biochem J 356, 503–507.5 Weigemann M & Watermann B (2003) Peculiaritiesof barnacle adhesive cured on non-stick surfaces.J Adhesion Sci Technol 17, 1957–1977.6 Waite JH (1987) Nature’s underwater adhesive special-ist. Int J Adhes 7, 9–14.7 Naldrett MJ (1993) The importance of sulphur cross-links and hydrophobic interactions in the polymerizationof barnacle cement. J Mar Bio Assoc UK 73, 689–702.8 Dougherty WJ (1990) Barnacle adhesion: reattachmentof the adult barnacle Chthamalus fragilis Darwin topolystyrene surfaces followed by centrifugational shear-ing,. J Crustacean Biol 10, 469–478.9 Greenfield N & Fasman GD (1969) Computed circulardichroism spectra for the evaluation of protein confor-mation. Biochemistry 8, 4108–4116.10 Brahms S & Brahms J (1980) Determination of proteinsecondary structure in solution by vacuum ultravioletcircular dichroism. J Mol Biol 138, 149–178.11 von Heijne G (1986) A new method for predictingsignal sequence cleavage sites. Nucleic Acids Res 14,4683–4690.Y. Mori et al. Calcite-coupling protein in underwater adhesiveFEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBS 6445[...]... BJ & Klenk DC (1985) Measurement of protein using bicinchroninic acid Anal Biochem 150, 76–85 26 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 27 Stafford WF (1992) Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration... for genomic fold recognition Bioinformatics 19, 874–881 15 Sagert J, Sun C & Waite JH (2006) Chemical subtleties of mussel and polychaete holdfasts In Biological Adhesives (Smith AM & Callow JA, eds), pp 125–140 Springer-Verlag, Berlin 16 Wallwork M, Kirkham J, Zhang J, Brookes S, Shore R, Wood S, Ryu O, Robinson C & Smith DA (2001) Binding of matrix proteins to developing enamel crystals: an atomic... footproteins of Mytilus californianus byssus J Biol Chem 281, 11090–11096 21 Waite JH & Qin XX (2001) Polyphosphoprotein from the adhesive pads of Mytilus edulis Biochemistry 40, 2887–2893 22 Lin Q, Gourdon D, Sun C, Holten-Anderson T, Waite JH & Israelachvili JN (2007) Adhesion mechanisms of the mussel foot proteins mfp-1 and mfp-3 Proc Natl Acad Sci USA 104, 3782–3786 23 Zhao H & Waite JH (2006) Linking... Proc Natl Acad Sci USA 104, 3782–3786 23 Zhao H & Waite JH (2006) Linking adhesive and structural proteins in the attachment plaque of Mytilus californianus J Biol Chem 281, 26150–26158 24 Cai M, Huang Y, Sakaguchi K, Clore GM, Gronenborn AM & Craigie R (1998) An efficient and costeffective isotope labeling protocol for protein expressed in Escherichia coli J Biomol NMR 11, 97–102 6446 25 Smith PK, Krohn...Calcite -coupling protein in underwater adhesive Y Mori et al 12 Jones DT, Taylor WR & Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences J CABIOS 8, 275–282 13 Altschul SF, Gish W, Miller W, Myers EW & Lipman DJ (1990) Basic local alignment search... Pelletier SL (1992) In Analytical Ultracentrifugation in Biochemistry and Polymer Science (Harding AJR & Horton JC, eds), pp 90–125 Royal Society of Chemistry, Cambridge 30 Schager H & Jagow G (1987) Tricine–sodium dodecyl ¨ sulfate-polyacrylamide gel electrophorcsis for the separation of proteins in the range from 1 to 100 kDa Anal Biochem 166, 368–379 Supplementary material The following supplementary... amelogenin nanospheres following the Langmuir model for protein adsorption Calcif Tissue Int 72, 599–603 18 Huang CP (1975) Adsorption of tryptophan onto calcium carbonate surface Environ Lett 9, 7–17 19 Lokar WJ & Ducker WA (2004) Proximal adsorption at glass surfaces: ionic strength, pH, chain length effects, Langmuir 20, 378–388 20 Zhao H, Robertson NB, Jewhurst SA & Waite JH (2006) Probing the... is available online: Fig S1 Comparison of rMrcp-20k and nMrcp-20k by liquid chromatography Fig S2 Adsorption isotherm for adsorption of rMrcp20k to a metal oxide surface Fig S3 Control for Fig 5 Fig S4 cDNA sequence of the homologous protein in B albicostatus This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible... http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 6436–6446 ª 2007 The Authors Journal compilation ª 2007 FEBS . Ceq, equilibrium protein concentration; CI, initial protein concentration; cp, cement protein; fp, mussel foot protein; GSF1 and GSF2, cement fractions. Calcite-specific coupling protein in barnacle underwater cement Youichi Mori1, Youhei Urushida1, Masahiro Nakano1, Susumu Uchiyama2and Kei Kamino11
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