Báo cáo khoa học: Calcite-specific coupling protein in barnacle underwater cement docx

Abbreviations ASW, artificial seawater; Ceq, equilibrium protein concentration; CI, initial protein concentration; cp, cement protein; fp, mussel foot protein; GSF1 and GSF2, cement frac

Youichi Mori1, Youhei Urushida1, Masahiro Nakano1, Susumu Uchiyama2and Kei Kamino1

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 gregarivari-ousness 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; 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 of Mrcp-20k expressed in Escherichia coli.

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 durprevent-ing 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-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 propro-tein 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 Mrcp-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).

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 Ceq⁄ Q ¼ 0.3168 · 10)3+ 4.199Ceq

[corre-lation coefficient (r2) of 0.97] in ASW and

Ceq⁄ Q ¼ 1.7168 · 10)3+ 3.782Ceq (r2 of 0.98) in the

dilute buffer [Ceq, equilibrium protein concentration;

Q, amount of absorbed protein (lmol) per m2 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 K¼ 1.33 · 107m)1 and N¼ 2.38 · 10)7

molÆm)2 in ASW, and K¼ 2.20 · 106m)1 and N¼

2.64· 10)7molÆm)2 in the dilute buffer solution The

isotherms for adsorption to zinc oxide and magnetite

were not linear (r2 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)

-1)

200

-30

-20

-10

0

10

-1)

-30

-20

-10

0

10

-1)

-30

-20

-10

0

10

A

B

C

Wavelength (nm)

Wavelength (nm)

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

2 )

0 50 100 150 200 250 300

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.

Ceq

2 /mL)

0.01 0.02 0.03 0.04 0.05

Ceq

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

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).

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 )

s20, W (S)

Molecular mass (kDa)

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).

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 propro-tein 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 propro-tein 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 Mrcp-20k and the

homologous protein in B albicostatus All Cys residues are shown

in black, and conserved Pro residues are shown in gray.

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 funcadsorp-tion 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· 106 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

weighed and decalcified by dialyzing against 2% (v⁄ v)

ace-tic acid at 4C 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 60C 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 60C 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 4C

The extract was recovered by centrifugation (21 600 g,

4C, 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¢-AGTTGCCATGGCGCACGAGGAGGA-3¢ and 5¢-TT

CTGTTCGGATCCCAAGGCTTA-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 30C 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 20C 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 20C from 200 nm to 320 nm, and then integrated 128 times Prior to the analysis, a reduction

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 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 cm2 surface

areaÆg)1, 12.75 lm in diameter; Nippon Shokubai Co.,

Tokyo, Japan), zinc oxide (20 000 cm2 surface areaÆg)1;

0.70 lm in diameter; Mitsui Mining and Smelting Co.,

Tokyo, Japan), magnetite (20 000 cm2surface 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 25C for 10 min The same volume of protein

(0.30 mgÆmL)1, dissolved in ultrapure water) was

preincu-bated at 25C, mixed with each type of particle, and

incu-bated at 25C 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 cm2each) in ASW, and then

evaluating the amount of free protein as described above

(N¼ 3) Calibration curves were constructed as reported

elsewhere [17] The amount of adsorbed protein (lmol) per

m2of the surface was calculated by the difference between

the initial (CI) and equilibrium (Ceq) protein concentration

(lmolÆmL)1) according to the following equation:

Q¼ ½ðCI CeqÞV=ðWSÞ ð1Þ 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:

Ceq=Q¼ 1=NK þ Ceq=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

A 10 lL 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 20C 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 20C) 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)1in NaCl solution were cen-trifuged at 21 000 r.p.m at 20C 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:

AðrÞ ¼ A0ðr0Þ exp½HMappðr2 r2Þ þ B ð3Þ where H¼ (1) mq)x2⁄ 2RT, m is partial specific volume of sample, q is density of solvent, R is gas constant, T is

temperature, x is angular velocity, A0is absorbance at a

ref-erence point r0, 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) in 10 mm Tris⁄ HCl (pH 8.0) or two-fold

concentrated ASW with 10 mm Tris⁄ HCl (pH 8.0) were

incubated at 25C 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 25C for 7 days After centrifuging

(21 600 g, 25C, 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)

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