Báo cáo khoa học: Identification and functional characterization of a novel barnacle cement protein pptx

Identification and functional characterization of a novel
barnacle cement protein
Youhei Urushida
1
, Masahiro Nakano
1
, Satoru Matsuda
1
, Naoko Inoue
2
, Satoru Kanai
2
,
Naho Kitamura
3
, Takashi Nishino
3
and Kei Kamino
1
1 Marine Biotechnology Institute, Iwate, Japan
2 Pharma Design, Inc., Tokyo, Japan
3 Department of Chemical Science & Engineering, Faculty of Engineering, Kobe University, Japan
Living on a boundary brings various advantages for
organisms; such organisms therefore have developed a
variety of molecular systems to hold themselves on the
boundary during their evolution. Marine sessile organ-
isms possess underwater attachment capability as an
indispensable physiologic function, enabling them to
live on a liquid–solid boundary during most of their
life cycle. This underwater attachment is closely related
to other biological functions such as metamorphosis,

molting and biomineralization. Recent advances in
underwater holdfast studies on mussel [1–3], and
barnacle [4], which represent two typical organisms
possessing this kind of activity, have indicated that the
biological adhesion is, in general, mediated by an
insoluble multiprotein complex. Each constitutive pro-
tein of the complex has been suggested to have a
special function in a multifunctional process of under-
water attachment. These functions [5] include displace-
ment of the bound-water layer on a foreign substratum
by the adhesive, as well as spreading, coupling of
the adhesive with a variety of material surfaces, self-
assembly of the adhesive, curing to make the holdfast
Keywords
biological adhesive; extracellular protein;
holdfast protein; protein adsorption; sessile
organism
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:
Database
The nucleotide sequence data are available
in the DNA Data Bank of Japan under the
accession numbers AB242294, AB242295,
and AB242296
(Received 20 March 2007, revised 26 June

2007, accepted 29 June 2007)
doi:10.1111/j.1742-4658.2007.05965.x
Barnacle attachment to various foreign materials in water is guided by an
extracellular multiprotein complex. A 19 kDa cement protein was purified
from the Megabalanus rosa cement, and its cDNA was cloned and
sequenced. The gene was expressed only in the basal portion of the animal,
where the histologically identified cement gland is located. The sequence of
the protein showed no homology to other known proteins in the databases,
indicating that it is a novel protein. Agreement between the molecular mass
determined by MS and the molecular weight estimated from the cDNA
indicated that the protein bears no post-translational modifications. The
bacterial recombinant was prepared in soluble form under physiologic con-
ditions, and was demonstrated to have underwater irreversible adsorption
activity to a variety of surface materials, including positively charged, nega-
tively charged and hydrophobic ones. Thus, the function of the protein was
suggested to be coupling to foreign material surfaces during underwater
attachment. Homologous genes were isolated from Balanus albicostatus and
B. improvisus, and their amino acid compositions showed strong resem-
blance to that of M. rosa, with six amino acids, Ser, Thr, Ala, Gly, Val
and Lys, comprising 66–70% of the total, suggesting that such a biased
amino acid composition may be important for the function of this protein.
Abbreviations
ASW, artificial seawater; Balcp-19k, Balanus albicostatus 19 kDa cement protein; Bicp-19k, Balanus improvisus 19 kDa cement protein;
cp, cement protein; Dopa, 3,4-dihydroxyphenylalanine; GSF1 and GSF2, cement fractions separated by their solubility in a guanidine
hydrochloride solution; Mrcp, Megabalanus rosa cement protein; rMrcp-19k, recombinant 19 ka Megabalanus rosa cement protein in
Escherichia coli; RU, response unit; SPR, surface plasmon resonance.
4336 FEBS Journal 274 (2007) 4336–4346 ª 2007 The Authors Journal compilation ª 2007 FEBS
stiff and tough, and protection from microbial degra-
dation. This multifunctionality, together with the
insoluble ⁄ sticky and complex nature of the adhesive,

have hindered any detailed analysis of its function,
especially the direct evaluation of the adhesive process.
Thus, biological underwater attachment remains an
unachievable technology, which is considered to be
based on a completely different approach from that
used in developing artificial adhesives in air.
The barnacle, a unique sessile crustacean, has long
been noted for its underwater adhesive capability [6–8].
This underwater adhesive material, called cement, joins
two different materials, the animal’s own calcareous
base and the foreign substratum, together in water as
a molecular event. Development of a method to render
this barnacle cement soluble [9] has enabled us to iden-
tify its components. Four cement proteins, designated
as Megabalanus rosa cement protein (Mrcp)-100k [9],
Mrcp-52k, Mrcp-68k [10], and Mrcp-20k [11], have so
far been identified; these were shown to be novel pro-
teins that are distinct from each other. The cp-100k
and cp-52k proteins are characterized by their insolu-
ble nature and remarkable hydrophobicity, and are
possibly bulk proteins of the cement complex. Reduc-
tion treatment with guanidine hydrochloride solution
was indispensable to render these proteins soluble.
cp-68k is characterized by its bias toward four-amino
acids, Ser, Thr, Ala and Gly which comprise 57% of
the total residues. cp-20k is characterized by its abun-
dant charged amino acids, with its primary structure
being a repeat of a well-defined segment in which Cys
residues are found in designated positions. Although
both cp-100k and cp-52k seem to constitute the bulk

of the adhesive, no proteins contributing the necessary
surface functions such as priming, spreading and cou-
pling have been identified. Nor have any direct mea-
surement of these activities been reported for the
cement proteins prepared under physiologic conditions,
and such kinds of measurement have never been
achieved in any biotic underwater adhesive protein
studies.
The holdfast system of the barnacle shows no simi-
larity to that of the mussel, a relatively well-character-
ized one. There are no sequence similarities among the
protein components between the two systems. The
mussel holdfast system [1] depends on several protein
modifications, including 3,4-dihydroxyphenylalanine
(Dopa); however, no involvement of Dopa in the bar-
nacle cement was found [10,12]. Thus, the barnacle
system represents a novel biological adhesive system.
The present study identified a novel cement protein,
cp-19k, in the barnacle holdfast system, and demon-
strated its ability to be adsorbed to a foreign material
surface in seawater using a bacterial recombinant pro-
tein prepared under physiologic conditions. We also
show that the function of the protein is reliant upon
common amino acids, with no specific modifications.
Results
Purification and characterization of Mrcp-19k
Mrcp-19k was detected by SDS ⁄ PAGE in both guani-
dine hydrochloride-soluble fractions 1 (GSF1) and 2
(GSF2) of barnacle cement [9] with the same mobility
(Fig. 1). The molecular mass was estimated to be

18 500 Da from SDS ⁄ PAGE. Mrcp-19k was purified
from GSF1 by column chromatography, which gave
rise to a molecular mass of 16 992.34 Da as measured
by MALDI-TOF MS (Table 1). The protein (Table 2)
was rich in Gly (17.3%), Thr (12.3%), Ser (11.3%),
Ala (10.6%), Lys (8.5%), and Val (8.7%). The amino
acid sequence of the mature N-terminus was deter-
mined as VPPPXDLGIASKVKQKGVTGGGASV
STT, where X was most likely to be Cys. The
N-terminal sequences of three internal peptide frag-
ments were determined to be GVTGGGASVSTT
SATQGSG, GFSEGTAAISQTAGANGGATV, and
Fig. 1. Mrcp-19k from M. rosa cement and its bacterial recombi-
nant analyzed by SDS ⁄ PAGE. Lanes 1 and 2: GSF1 and GSF2 pre-
pared from M. rosa cement, respectively. Lane 3: the bacterial
recombinant protein rMrcp-19k. Lane 4: molecular weight markers.
The samples were separated by SDS ⁄ PAGE (a Tris ⁄ Tricine buffer
system, 16.5% T ⁄ 3% C [28]) and stained with Coomassie Brilliant
Blue R-250. The numbers on the right-hand side indicate molecular
masses (kDa). The arrow indicates Mrcp-19k. The bacterial recom-
binant, rMrcp-19k, has an additional dipeptide, Met-Ala, at the
N-terminus of mature Mrcp-19k, due to the vector construction.
Y. Urushida et al. Barnacle surface-cement protein
FEBS Journal 274 (2007) 4336–4346 ª 2007 The Authors Journal compilation ª 2007 FEBS 4337
GTVTSSSSHQGSGAGDSIFE. Specific staining for
detection of either glycosylation or phosphorylation
gave negative results in both cases.
Cloning of cp-19k cDNA from M. rosa, Balanus
albicostatus and B. improvisus
A 53 bp DNA corresponding to the N-terminal part

was first amplified from M. rosa cDNA by PCR. The
deduced amino acid sequence of the 53 bp DNA com-
pletely matched the N-terminal amino acid sequence
of the mature Mrcp-19k. Subsequent 3¢-RACE and
5¢-RACE resulted in a 750 bp and a 102 bp DNA
fragment, respectively. An 852 bp cDNA fragment
encoding the Mrcp-19k protein was finally determined.
Ten randomly selected clones for the coding region of
Mrcp-19k had one nonsynonymous substitution and
several synonymous substitutions, presumably due to
errors introduced by the PCR amplifications (as each
substitution was found only in one randomly selected
clone but not in any other clones). Both B. albicostatus
(Bal)cp-19k (881 bp) and B. improvisus (Bi)cp-19k
(970 bp) cDNAs were also amplified by 3¢-RACE with
the oligonucleotide primers designed from the N-termi-
nal region of Mrcp-19k.
Structural outline of cp-19ks
The coding region of Mrcp-19k encoded 198 amino
acids (supplementary Fig. S1A). The mature N-termi-
nal sequence was found to start at residue number 26;
thus the first 25 amino acids function as the signal
peptide that has been cleaved off in the mature pro-
tein. The amino acid sequences of the N-terminal and
three internal peptide fragments of Mrcp-19k deter-
mined experimentally were found to be contained in
the deduced sequence and are in complete agreement
with those of the deduced sequence. The cDNA frag-
ments of 881 bp and 970 bp encoding 173 amino acids
each were also determined for Balcp-19k and

Bicp-19k, respectively (supplementary Fig. S1A). The
molecular masses and isoelectric points of the mature
Table 1. Predicted and observed molecular masses and predicted
isoelectric points of cp-19ks. Calculated mass (Mass
cDNA
) is based
on a sequence deduced from the cDNA, and m ⁄ z
obs
value corre-
sponds to [M +H]
+
observed with MALDI TOF-MS.
Mrcp-19k Balcp-19k Bicp-19k
m ⁄ z
obs
16 993.34 – –
Mass
cDNA
16 995.52 17 336.27 16 841.99
pI 5.8 10.3 10.3
Table 2. Amino acid compositions of various cp-19ks and their deviations from standard compositions. The amino acid compositions of
mature cp-19ks are presented as the number of residues per protein in columns 1–4. The ratios of each number of residues to the average
contents of the amino acids [13] are shown to indicate the bias in columns 5–7. ND, not determined.
Mrcp19k
a
Mrcp19k
b
Balcp19k
a
Bicp19k

a
Mrcp19k ⁄ standard Balcp19k ⁄ standard Bicp19k ⁄ standard
Asp 10.00 14.8
c
5.00 5.00 1.92 0.96 0.96
Asn 8.00 – 6.00 7.00 1.86 1.40 1.63
Ser 18.00 19.30 15.00 17.00 2.61 2.17 2.46
Glu 9.00 11.2
d
8.00 3.00 1.45 1.29 0.48
Gln 4.00 – 3.00 8.00 0.98 0.73 1.95
Gly 27.00 29.60 22.00 25.00 3.65 2.97 3.38
His 1.00 1.10 2.00 2.00 0.43 0.87 0.87
Arg 1.00 2.30 1.00 0.00 0.20 0.20 0.00
Thr 21.00 21.20 25.00 20.00 3.56 4.24 3.39
Ala 18.00 18.20 18.00 21.00 2.34 2.34 2.73
Pro 4.00 4.90 4.00 5.00 0.78 0.78 0.98
Cys 2.00 ND 2.00 2.00 1.00 1.00 1.00
Tyr 0.00 0.60 0.00 0.00 0.00 0.00 0.00
Val 14.00 14.60 17.00 16.00 2.12 2.58 2.42
Met 0.00 0.00 1.00 0.00 0.00 0.42 0.00
Lys 17.00 14.60 24.00 17.00 2.88 4.07 2.88
Ile 5.00 5.20 3.00 9.00 0.94 0.57 1.70
Leu 10.00 9.40 13.00 14.00 1.10 1.43 1.54
Phe 4.00 4.00 4.00 2.00 1.00 1.00 0.50
Trp 0.00 ND 0.00 0.00 0.00 0.00 0.00
a
The amino acid composition calculated from the deduced sequence.
b
The amino acid composition analyzed by amino acid analysis.

c
Sum
of the numbers of Asp and Asn.
d
Sum of the numbers of Glu and Gln.
Barnacle surface-cement protein Y. Urushida et al.
4338 FEBS Journal 274 (2007) 4336–4346 ª 2007 The Authors Journal compilation ª 2007 FEBS
polypeptides were predicted to be 16 995.52 Da
(Table 1) and 5.8 for Mrcp-19k, 17 336.27 Da and 10.3
for Balcp-19k, and 16 841.99 Da and 10.3 for Bicp-19k,
respectively. The molecular mass of Mrcp-19k estimated
by SDS ⁄ PAGE was slightly higher than that predicted
from the cDNA sequence and that determined by
MALDI-TOF MS. This may be due to unusual migra-
tion on SDS ⁄ PAGE caused by the biased amino acid
composition. The amino acid composition of Mrcp-19k
deduced from the cDNA (Table 2) agreed well with that
of Mrcp-19k determined by the amino acid analysis,
with six amino acids, Gly (15.6%), Thr (12.1%), Ser
(10.4%), Ala (10.4%), Lys (9.8%) and Val (8.1%), as
dominant residues and representing 66.4% of all
residues. This ratio is significantly higher than that
deduced from the standard amino acid composition
[13]. The sequence identity and similarity (Fig. 2) were
as follows: Mrcp-19k versus Balcp-19k, 54% identity
and 65% similarity; Mrcp-19k versus Bicp-19k, 51%
identity and 68% similarity; and Balcp-19k versus
Bicp-19k, 61% identity and 75% similarity. All cp-19ks
contained two Cys residues, whose positions are
conserved. The amino acid compositions among three

cp-19ks agreed well with each other, especially in terms
of the content of the six dominant residues, Gly, Thr,
Ser, Ala, Lys and Val (Table 2).
A blast search of the nonredundant database and
a sequence profile-based fold-recognition method for
three-dimensional structural prediction failed to pro-
vide any homologous sequences and meaningful struc-
ture (supplementary Document S1). In particular, no
sequence similarity between cp-19ks and foot proteins
in the mussel was evident. The primary structures of
cp-19ks also showed no homology with cp-100k and
cp-20k. Naldrett & Kaplan [14] have reported the par-
tial amino acid sequences of peptide fragments from
B. eburneus cement. Among these fragments, WCD-21,
a peptide fragment obtained by cyanogen bromide
treatment of B. eburneus cement, showed homology to
the N-terminal region of cp-19ks (supplementary
Fig. S1B), indicating that the protein homologous to
cp-19k should also be present in B. eburneus cement.
Characterization of the recombinant Mrcp-19k
protein
Recombinant (r)Mrcp-19k was expressed in Escherichia
coli as a soluble cytosolic fraction, and was purified to
homogeneity (Fig. 1). rMrcp-19k had a slightly lower
mobility than that of the native Mrcp-19k isolated
from the cement. This was due to the additional N-ter-
minal dipeptide in the recombinant protein as the
result of the vector design. The N-terminal sequence
and molecular mass were determined to be
AMVPPPXDLG and 17 201 Da (predicted molecular

mass from the cDNA, 17 197.60 Da), respectively.
Digestion of rMrcp-19k by a specific protease gener-
ated a peptide fragment with a molecular mass of
4509.24 Da, which corresponds to two peptides; each
contains one Cys residue, and they are linked by a
disulfide bond (Ala1-Lys14 and Gly19-Lys51, predicted
molecular mass, 4509.17 Da). Treatment with reduc-
tants led to the loss of the MS peak, and alternatively
gave two MS peaks corresponding to each single pep-
tide with molecular masses of 3112.36 Da (Gly19-
Lys51, predicted molecular mass, 3111.47 Da) and
1398.6 Da (Ala1-Lys14, predicted molecular mass,
1397.70 Da). This confirmed that the two Cys residues
in rMrcp-19k form an intramolecular disulfide bond.
The properties of adsorption of rMr cp-19k to under-
water surfaces of glass, formaldehyde resin, alkylated
gold, and bare gold were measured either in artificial
seawater (ASW) or in a dilute buffer solution. Figure 3
shows the mass uptake by the adsorption of rMrcp-
19k on the gold and alkylated gold surfaces versus
time from the surface plasmon resonance (SPR) mea-
surement. The proteins showed rapid adsorption to the
sensor surfaces that corresponded to sharp increases in
the SPR shift. Upon washing, the response units
(RUs) were slightly decreased, probably due to dissoci-
ation of loosely attached protein. The final RUs after
washing were almost the same after repetitive injec-
tions of the protein on each surface. The adsorption
kinetics were estimated by nonlinear curve fitting with
theoretical models described in the biaevaluation

Mrcp19k VPPPCDLGIASKVKQKGVTGGGASVSTTSATQGSGTTNCVTRTPNSVEKKNVAGNTGVTA
Bacp19k VPPPCDLSIKSKLKQVGATAGNAAVTTTGTTSGSGVVKCVVRTPTSVEKKAAVGNTGLSA
Bicp19k VPPPCDFSIKSKQKQVGVTAGGASVSAKGATSGSGSITCITKTPTSVTKKVAAGNAGVSG
70
11020 30 4050 60
80 90 100 110 120
Mrcp19k TSVSAGDGAFGNLAAALTLVEDTEDGLGVKTKNGGKGFSEGTAAISQTAGANGGATVKKA
Bacp19k VSASAANGFFKNLGKATTEVKTTKDGTKVKTKTAGKGKTGGTATTIQIADANGGVSEKSL
Bicp19k AAAAAGNGVFKNLVTALTNISTTDDITKVQTQTIGSGGTGGAATILQLADANGGAALKEV
130 140 150 160 170
Mrcp19k KLDLLTDGEDLFDTKKVEKGTVTSSSSHQGSGAGDSIFEILNEAESKIKKSGD
Bacp19k KLDLLTDGLKFVKVTEKKQGTATSSSGHKASGVGHSVFKVLNEAETELELKGL
Bicp19k KLDLLPIGTGLGVVKQTKQGQVTSSSSHKASGLGNSVLKVLNAHETELKLKGI
Fig. 2. Alignment of the amino acid
sequences of mature cp-19ks.The deduced
amino acid sequences of mature Mrcp-19k,
Balcp-19k and Bicp-19k were aligned by
CLU-
STALW
[34]. The three homologous proteins
have the same amino acid length, and the
two Cys residues are conserved. Identical
amino acids are reversed.
Y. Urushida et al. Barnacle surface-cement protein
FEBS Journal 274 (2007) 4336–4346 ª 2007 The Authors Journal compilation ª 2007 FEBS 4339
software (supplementary Fig. S2). The adsorption con-
stant k
a
and desorption constant k
d

were calculated as
2.17 · 10
5
m
)1
Æs
)1
and 4.94 · 10
)4
s
)1
, respectively, for
the formation of the rMrcp-19k–Au complex. Using
these data, the equilibrium constant K
eq
¼ k
a
⁄ k
d
could
be estimated as 4.39 · 10
8
m
)1
.rMrcp-19k was simi-
larly adsorbed to the hydrophobic alkylated gold sur-
face, although the adsorbed amount was two-thirds of
that adsorbed to bare gold (Table 3). The values of k
a
,

k
d
and K
eq
were calculated to be 9.76 · 10
4
m
)1
Æs
)1
,
6.67 · 10
)4
s
)1
and 1.46 · 10
8
m
)1
, respectively. The
amounts adsorbed to the glass and formaldehyde resin
surfaces in 5 min at 25 °C were estimated, and the
results are shown in Table 3 and supplementary
Fig. S3.
Localization and expression site of Mrcp-19k
M. rosa cement was usually collected by gently scrap-
ing the surface of the calcareous base on the side
attached to the foreign material surface [10], making
the cement proteins vulnerable to contamination by
calcified material during the process of collection. We

therefore attempted to confirm the identified protein
as a cement component. The cement joins the ani-
mal’s own calcareous base to the foreign substratum.
Therefore, the cement should be present on one side
of the barnacle’s calcareous base, whereas the periph-
eral shell should be free from cement. If the protein
is present in the protein fraction from the calcareous
base and not in that from the peripheral shell, this
would confirm that the protein is a cement compo-
nent and not a component involved in calcification.
Western blot analysis of the primary cement, and of
the protein fractions in the calcareous base and
periphery, indicated that Mrcp-19k was present in the
primary cement and protein fraction in the calcareous
base, but not in the peripheral shell (Fig. 4). A wes-
tern blot analysis with the polyclonal antibody raised
against Mrcp-100k gave a similar result to that for
Mrcp-19k.
Northern blot analysis using Mrcp-19k DNA as the
probe indicated that the corresponding mRNA was
–200
200
600
1000
1400
1800
0 100 200 300 400 500 600 700
Response (R.U.)
Au:ASW
Au:Buffer

HPA:ASW
Fig. 3. Typical SPR analyses on polycrystalline gold and alkylated gold. The arrows and thick arrows indicate the starts of sample loading
(2 l
M) and washing by the running buffer, respectively. The processes of sample loading and washing were sequentially repeated three
times. Open circular symbols, squares and triangles indicate changes of resonance after protein adsorption on polycrystalline gold in ASW,
on the same material in a dilute buffer containing 10 m
M Tris (pH 7.4) ⁄ 25 mM NaCl, and on alkylated gold (HPA) in ASW, respectively. DRUs
after each washing process were as follows: first loading on Au in ASW, 1174 RU; second loading on Au in ASW, 1177 RU; third loading on
Au in ASW, 1182 RU; first loading on Au in dilute buffer, 1278 RU; second loading on Au in dilute buffer, 1318 RU; third loading on Au in
dilute buffer, 1345 RU; first loading on alkylated gold in ASW, 768 RU; second loading on alkylated gold in ASW, 827 RU; third loading on
alkylated gold in ASW, 858 RU.
Table 3. Amount of adsorption of rMrcp-19k to several material
surfaces. The adsorbed amount in ASW or dilute buffer solution
was calculated from the change in RU on SPR [36] for gold and
alkylated gold, and from a quantitative amino acid analysis for glass
and the formaldehyde resin (see details in supplementary Fig. S3).
Surface area per molecule was calculated by a assuming full sur-
face monolayer coverage.
Gold
Alkylated
gold Glass
Formaldehyde
resin
Adsorption amount
(ngÆmm
)2
)
0.76
(0.83 in
dilute buffer)

0.5 2.48 4.38
Surface area per
molecule
(nm
2
per molecule)
37
(35 in dilute
buffer)
57 11 7
Barnacle surface-cement protein Y. Urushida et al.
4340 FEBS Journal 274 (2007) 4336–4346 ª 2007 The Authors Journal compilation ª 2007 FEBS
specifically expressed in the basal portion of the barna-
cle where the cement gland was located (Fig. 5).
Discussion
The present study identified a novel protein, cp-19k, in
the cement of the barnacle. Amino acid composition
analysis indicated that this protein is heavily biased
toward six residues, namely, Gly, Thr, Ser, Ala, Lys
and Val, with their total proportion exceeding 66%
in M. rosa. MALDI-TOF MS analysis of Mrcp-19k
isolated from barnacle cement, as well as scrutiny of
the specific staining for glycosylation and phosphoryla-
tion, revealed that the protein is a simple one bearing
no post-translational modifications. As all mussel foot
proteins found so far are subjected to extensive post-
translational modifications [1], mussel underwater
attachment relies heavily on the functionality of modi-
fied amino acids [15]. Among the barnacle cement pro-
teins, at least Mrcp-19k, and another cement protein

Mrcp-20k, which has been identified previously [11],
were shown to be simple proteins. Thus, the barnacle
seems to manage its underwater attachment activity
well with common amino acids.
The bacterial recombinant protein of Mrcp-19k,
rMrcp-19k, was prepared in soluble form under physi-
ologic conditions, enabling us to directly measure its
adsorption to underwater surfaces. Two Cys residues
in the protein formed an intramolecular disulfide bond,
probably with the help of a thioredoxin-tag in the
vector system. rMrcp-19k was adsorbed to various
characteristic surfaces, including negatively charged,
positively charged and hydrophobic surfaces. The bar-
nacle attaches to various foreign material surfaces,
including metal oxide, glass, plastic, wood, and rock.
Naturally occurring surfaces such as rock are not
microscopically homogeneous, and have a patchwork
of different surface characteristics. The cement is there-
fore required to simultaneously adapt the molecular
event to different surfaces. The ability of Mrcp-19k
to be adsorbed to various surfaces suggests that this
protein may be responsible for the surface func-
tions, at least for the ability of the barnacle cement
to adsorb to foreign materials with different surface
characteristics.
Polycrystalline gold and hydrophobic alkylated gold
were used as the representative surfaces in this study
for evaluating the adsorption isotherm. The surface
attachment area of a protein molecule on the gold sur-
face was calculated to be 37 nm

2
per molecule by
assuming full surface coverage. Although no informa-
tion is available on the three-dimensional structure of
Mrcp-19k, this value is higher than the surface contact
area of the well-known globular protein lysozyme (bac-
teriophage lambda; molecular mass 17 700 Da [16]
32 · 32 · 40A
˚
, approximately 8–10 nm
2
per molecule),
which has a similar molecular mass. Thus, the Mrcp-
19k molecule may be flatter to maximize contact with
the material surface. The adsorption to alkylated gold
was two-thirds of that to bare gold. It is not clear
from this study whether this was due to an enlarged
contact area of the protein as a result of conforma-
tional change on the surface, or imperfect surface cov-
erage at some distance as a result of intermolecular
GSF1
A
B
GSF2 rMrcp19k 1stcp peripheral base
GSF2 1stcp peripheral base
Fig. 4. Western blotting analysis to identify the location of Mrcp-
19k and Mrcp-100k in the cement. (A) Antibody to Mrcp-19k was
used for western blotting analysis. Lane 4 shows the primary
cement [10] with the dithiothreitol ⁄ guanidine hydrochloride treat-
ment [9]. Lanes 5 and 6 show the barnacle peripheral shell and

base plate, respectively, which have been decalcified and rendered
soluble by the dithiothreitol ⁄ guanidine hydrochloride treatment.
Lanes 1–3 correspond to GSF1, GSF2 and the recombinant protein
rMrcp-19k, respectively, as positive controls. (B) Antibody to Mrcp-
100k was used for the analysis. Lane 2 shows the primary cement
with the dithiothreitol ⁄ guanidine hydrochloride treatment. Lanes 3
and 4 show the barnacle peripheral shell and base plate, respec-
tively, which have been decalcified and rendered soluble by the
dithiothreitol ⁄ guanidine hydrochloride treatment. Lane 1 corre-
sponds to GSF2 as a positive control.
Basal
Upper
Basal
Upper
Fig. 5. Site specificity of Mrcp-19k gene expression in the basal
portion of the adult barnacle, where the histologically identified
cement gland is located.Twenty micrograms of total RNA extracted
from the basal or upper portion of the adult barnacle was electro-
phoresed in formaldehyde gel, transferred to a nylon membrane,
and hybridized with a probe. The basal portion mainly comprises
the mantle, muscle, ovariole, cement gland [20–22], and hemo-
lymph, whereas the upper portion contains the cirri, thorax, pro-
soma and hemolymph. Left, northern blot; right, 18S rRNA on gel
stained by ethidium bromide.
Y. Urushida et al. Barnacle surface-cement protein
FEBS Journal 274 (2007) 4336–4346 ª 2007 The Authors Journal compilation ª 2007 FEBS 4341
repulsion on the surface. The amounts adsorbed to
both glass and formaldehyde resin were two-fold to
five-fold the amount adsorbed to bare gold. These
data, however, were obtained with a method that

involved a different principle of measurement, making
a direct comparison difficult at this stage.
The fact that the amino acid compositions have been
well conserved in cp-19k from three species, although
the similarity of sequences was by no means high, indi-
cates that the function of the protein may be associ-
ated with the amino acid bias. The four amino acids
Ser, Thr, Lys and Val in the six amino acid-biased
protein would be useful for coupling with various
foreign material surfaces via hydrogen bonding,
electrostatic interactions, hydrophobic interactions, etc.
During the initial process of underwater attachment, a
cement protein is required to approach the solid sub-
stratum to which water molecules are bound, and to
displace this water prior to coupling with the substra-
tum surface. Waite [5] has suggested the significance of
the hydroxyl group on the Ser residue and Thr residue
for the priming process. In a relevant protein, the anti-
freeze protein, which binds to the ice nucleus to inhibit
crystal growth in the cytosolic space of several organ-
isms, including bacteria and fish [17], the Ala and ⁄ or
methyl group of Thr on the molecular surface of the
protein are known to be essential in the process of
binding to the ice nucleus [18,19], although the exact
roles of these amino acids are not yet clearly under-
stood. The requirements of coupling to various
foreign material surfaces and displacing water mole-
cules bound to a solid substratum may result in the
bias of six amino acids in the barnacle cement protein.
Although the content of Mrcp-19k in cement was

not accurately determined in this study, it was by no
means a major component. Cement proteins contribut-
ing to surface functions might be minor constituents,
whereas the proteins for bulk functions [9] would be
present in much higher amounts in the adhesive layer.
Northern blot analysis has indicated that the Mrcp-
19k gene is specifically expressed in the basal portion
of the animal, where the histologically identified
cement gland is located [20–22]. This result is consis-
tent with that for Mrcp-100k [9]. The cement proteins
are probably biosynthesized together in the cement
gland and transported by a duct to the narrow inter-
space outside, between the animal’s base and the for-
eign substratum.
In conclusion, this study has identified a novel pro-
tein, cp-19k, in barnacle cement and demonstrated that
it is able to be adsorbed to various underwater sur-
faces, suggesting that this protein is a surface protein
of the cement complex. Our results also revealed that
the function of cp-19k is dependent on common amino
acid residues on the molecular surface. This is in con-
trast to the underwater adhesive proteins of mussel
and tubeworm studied so far, where modified amino
acids have been found to play major roles [23,24]. The
barnacle cement protein characterized in this study
may therefore represent a new mechanism of biological
adhesion, which is likely to be useful in helping the
interdisciplinary links between biotechnology and
material science, e.g. development of adsorbents for
various material surfaces, of support for protein align-

ment on a solid surface [25–27], and of underwater
adhesives for surgical use [6].
Experimental procedures
Chemicals
All chemicals used were of the highest grade available, with
most being purchased from Wako Pure Chemical Industries
(Osaka, Japan) and Takara Shuzo Co. (Otsu, Japan). Two-
fold-concentrated ASW was prepared by dissolving ASW
(Senju Seiyaku Co., Osaka, Japan) in ultrapure water,
which was ultrafiltered through an M
r
3000-cutoff mem-
brane (YM3; Amicon-Millipore, Billerica, MA, USA).
Purification and characterization of Mrcp-19k
GSF1 and GSF2 were prepared from M. rosa cement basi-
cally as described previously [9]. Briefly, the cement was
suspended in 10 mm sodium phosphate buffer at pH 6.0 con-
taining 6 m guanidine hydrochloride, and the suspension was
centrifuged at 200 000 g for 1 h at 20 °C (CS120 centrifuge
with RP100AT rotor, Hitachi Koki, Tokyo, Japan). The pro-
tein fraction in the supernatant corresponded to GSF1. The
precipitate in the GSF1 preparation was reduced with 0.5 m
dithiothreitol ⁄ 7 m guanidine hydrochloride ⁄ 0.5 m Tris ⁄ HCl
(pH 8.5) ⁄ 20 mm EDTA at 60 °C for 2 h in a nitrogen atmo-
sphere. The resulting supernatant was recovered as GSF2.
Both fractions were dialyzed against 1% acetic acid at 4 °C,
before being evaporated and stored at ) 20 °C until needed.
GSF1 and GSF2 were separated by SDS ⁄ PAGE (a Tris ⁄ Tri-
cine buffer system, 16.5% T ⁄ 3% C [28]). The band corre-
sponding to Mrcp-19k was transferred to a poly(vinylidene

difluoride) membrane (ProBlott; Applied Biosystems, Foster
City, CA, USA) using a Tris ⁄ borate buffer containing 0.1%
SDS [29], and was stained with Coomassie Brilliant Blue
R-250. In order to get peptide fragments of Mrcp-19k, the
band corresponding to Mrcp-19k on the poly(vinylidene di-
fluoride) membrane before Coomassie Brilliant Blue staining
was cut out and subjected to in situ enzymatic digestion [30]
using lysylendopeptidase (Wako Pure Chemical Industries).
The generated peptide fragments were separated and frac-
tionated by RP-HPLC in a 3.9 mm diameter · 150 mm
Barnacle surface-cement protein Y. Urushida et al.
4342 FEBS Journal 274 (2007) 4336–4346 ª 2007 The Authors Journal compilation ª 2007 FEBS
l-Bondasphere column (C18, 100 A
˚
; Waters, Milford, MA,
USA). The amino acid sequence was determined with a
Procise 494 cLC (Applied Biosystems) or PSQ-2 protein
sequencer (Shimadzu, Kyoto, Japan). Mrcp-19k was also
purified from GSF1 by ion exchange chromatography (SP
Sepharose FF; Amersham Biosciences, Uppsala, Sweden).
The column was equilibrated with 50 mm acetic acid, and
eluted with a linear gradient of NaCl from 0 m to 0.6 m in
80 min. The fractions were monitored with a polyclonal anti-
body raised against the bacterial recombinant protein corre-
sponding to the C-terminal 10 kDa portion of Mrcp-19k, as
described in the latter section of recombinant in E. coli except
for using 5¢-TGG CCG CAG
CCA TGG CAT TGG T-3¢
as the 5¢-primer. The fraction containing Mrcp-19k was
concentrated by ultrafiltration (Microcon YM-3; Amicon-

Millipore), and further purified by gel filtration chromatogra-
phy (G3000SWXL; Tosoh, Tokyo, Japan) with 50 mm acetic
acid ⁄ 20 mm NaCl as the eluent. The purified Mrcp-19k was
subjected to MALDI-TOF MS with a Voyager-DE STR
instrument (Applied Biosystems) incorporating a 337 nm
nitrogen laser operated in the linear mode at an acceleration
voltage of 20 kV. For the MALDI matrix, saturated sinapi-
nic acid dissolved in 30% (v ⁄ v) acetonitrile containing 0.3%
(v ⁄ v) trifluoroacetic acid was used, and for calibration of the
mass, a Sequazyme peptide mass standard kit (Applied
Biosystems) was used. The amino acid composition was
determined using a double-distilled constant-boiling HCl
hydrolysate at 110 °C for 24 h, with an AccQ-Tag system
(Waters). Possible modifications of Mrcp-19k by glycosyla-
tion and phosphorylation of Mrcp-19k were also investi-
gated. The glycosylation was detected by periodic acid–Schiff
staining [31] with BSA as the positive control. Phosphory-
ation was detected by staining the SDS ⁄ PAGE gel with
Pro-Q diamond (Invitrogen, Eugene, OR, USA), and then
observed under a UV-transilluminator, with BSA and bovine
milk b-casein as positive controls.
Molecular cloning of cDNAs encoding Mrcp-19k,
Balcp-19k, and Bicp-19k
M. rosa, B. improvisus and B. albicostatus were collected
from Miyako Bay (Iwate), Yodo River (Osaka) and Shi-
mizu Bay (Shizuoka, Japan), respectively. RNA and DNA
manipulation was generally performed as described previ-
ously [9]. Total RNA was extracted from basal tissue of the
barnacle by a Total RNA Separator kit (BD Biosciences
Clontech, Mountain View, CA, USA), and poly(A)

+
RNA
was isolated using Oligo(dT)-Latex Super (Takara Shuzo
Co.). cDNA was prepared from mRNA with a Zap-cDNA
synthesis kit (Stratagene, La Jolla, CA, USA) according to
the instructions of the supplier. DNA fragments of Mrcp-
19k were first amplified by PCR (ExTaq; Takara) with fully
degenerated PCR primers designed from the N-terminal
amino acid sequence of Mrcp-19k: 5¢-GTN
CCN CCN CCN TGY GA-3¢ and 5¢-CAN CCY TTY TGY
TTN ACY TT-3¢. The PCR products were resolved by 3%
NuSieve 3 : 1 agarose (Takara) gel electrophoresis, and a
53 bp DNA fragment from M. rosa was purified from
the gel. The DNA fragment was subcloned in pT7 Blue
T-Vector (Novagen, EMD Biosciences, Madison, WI,
USA), and the insert was sequenced using a Prism Dye
Deoxy sequencing kit and 3700-DNA analyzer (Applied
Biosystems). 3¢-RACE was then carried out with a specific
3¢-RACE primer designed from the 53 bp DNA and using
a3¢-RACE core kit (Takara). The 3¢-RACE primer used
was 5¢-CTG ATC TAG AGG TAC CGG ATC CGT TCC
CCC ACC ATG CGA CCT TGG CAT-3¢. The PCR pro-
duct was subcloned and then sequenced. To obtain the
full-length cDNA, 5¢-RACE was carried out with oligo-
nucleotide primers designed from the sequence of 750 bp
DNA and using a 5¢-RACE core kit (Takara). The 5¢-
RACE primers used were as follows: 5¢-G#CC GTC CCC
GGC CGA C-3¢, where G# is phosphorylated, for reverse
transcription; 5¢-GTG CCG GAG CCC TGC GTG GC-3¢
and 5¢-AAC TCC GTG GAG AAG AAG AA-3¢ for the

first PCR amplification; and 5¢-TGC TGA CCG ACG
CGC CTC CT-3¢ and 5¢-GGC AAC ACG GGC GTC
ACC GC-3¢ for the second PCR amplification. The 102 bp
DNA amplified by 5¢-RACE was purified, subcloned, and
sequenced. Finally, 665 bp DNA for the coding region of
Mrcp-19k was amplified from M. rosa total cDNA using
the primers 5¢-ACC AAC GCA GCA GTT ATG GT-3¢
and 5¢-GCT GCA CAT CTT CGA CCT CA-3¢, and then
subcloned. KOD-plus DNA polymerase (Toyobo, Osaka,
Japan) was used for PCR amplification to achieve high
fidelity. Ten randomly selected clones were sequenced.
DNA fragments encoding Balcp-19k and Bicp-19k were
amplified by 3¢-RACE, respectively, using the degenerated
oligonucleotide primer designed for 3¢-RACE of Mrcp-19k
as already described. The amplified DNA fragments were
subcloned and sequenced.
A homology search was performed with the nonredun-
dant GenBank CDS translations + Protein Data Bank +
swissprot + PIR + PRF database using the blast pro-
gram [32]. A sequence profile-based fold-recognition
method involving the mgenthreader [33] program was
used for further analysis to identify a family of homologous
proteins. The clustalw [34] program was used to identify
the clustered sequence alignment among cp-19ks.
Characterization of the Mrcp-19k recombinant
in E. coli
The Mrcp-19k recombinant in E. coli, designated rMrcp-
19k, was prepared as follows. The cDNA was amplified by
PCR with primers around the mature N-terminal and
C-terminal regions, which, respectively, included the newly

created NcoI and BamHI restriction sites. The primers used
were 5¢-ACCGGCCATGGGCAAGGCCGT-3¢ and 5¢-AT
GGTCACGGGATCCCTCCGGTGGTCTTA, whereby the
Y. Urushida et al. Barnacle surface-cement protein
FEBS Journal 274 (2007) 4336–4346 ª 2007 The Authors Journal compilation ª 2007 FEBS 4343
recombinant was designed to have the N-terminal sequence
of AMGKAVTV, in which the mature N-terminal sequence
of Mrcp-19k with an additional dipeptide sequence, AM,
was created after removing the fused tag by enterokinase
cleavage, and with the original C-terminal end. The ampli-
fied DNA was subcloned in pT7 Blue T-Vector (Novagen),
and the sequence was confirmed. Insert DNA was gene-
rated by digestion with the NcoI and BamHI restriction
enzymes, and then subcloned into pET32b (Novagen) with
the same restriction sites. The pET32 vector system pro-
duces fusion proteins with a thioredoxin-tag, which
enhances disulfide bond formation of the target protein in
the cytoplasm of the host strain. The created vector was
transformed into the expression host strain Oligami (DE3)
(Novagen). The recombinant protein was purified with a
metal-chelating column according to the affinity of the His-
tag fused into Mrcp-19k. The cells were inoculated in LB
medium [35] containing ampicillin at 37 °C for 3 h, and
transferred to freshly prepared medium, and inoculated for
another 3 h; protein expression was induced by 0.2% iso-
propyl-thio-b-d-galactoside for an additional 4 h. The cyto-
solic fraction was prepared by sonicating on ice in 20 mm
Tris (pH 7.4) ⁄ 500 mm NaCl ⁄ 40 mm imidazole, and purified
with an Ni
2+

-immobilized column (His-bind kit; Novagen)
according to the manufacturer’s instructions. The fraction
containing rMrcp-19k was recovered and dialyzed at 4 °C
against 20 mm Tris ⁄ HCl (pH 7.4) ⁄ 50 mm NaCl ⁄ 2mm
CaCl
2
, and then treated with enterokinase (recombinant
enterokinase; Novagen) to cleave the fused tag. The cleaved
protein was purified by Ni
2+
-immobilized column chroma-
tography and gel filtration column chromatography
(TSK-gel G3000 SW
XL
; Tosoh), using 20 mm Tris
(pH 7.4) ⁄ 50 mm NaCl as the eluent. For the following
quantitative amino acid analysis, the solvent was changed
to 20 mm Hepes (pH 7.4) ⁄ 20 mm NaCl by dialysis. The
protein was quickly stored at ) 80 °C in small volumes.
Freeze–thaw cycles and storage for more than 1 month
were avoided, and handling of sample solutions was mini-
mized, because these processes caused loss of the protein in
solution.
Inspection of the chemical forms of two Cys residues in
the recombinant protein was performed as follows. The
protein was digested with lysylendopeptidase [Wako;
enzyme ⁄ substrate, 1 : 100 (molar ratio)] in 20 mm Tris
(pH 7.4) ⁄ 50 mm NaCl at 30 °C for 3 h, and the molecular
masses of the resulting peptide fragments were determined
by LC-ESI-MS (LCQ-Advantage instrument; Thermo Elec-

tron, Waltham, MA, USA), either with or without pretreat-
ment with dithiothreitol.
Adsorption of the recombinant protein to underwater
material surfaces was analyzed by: (a) quantitative amino
acid analysis; and (b) SPR.
Protein adsorption to glass and a positively charged poly-
mer were evaluated by quantification of the bound protein
and unbound protein, respectively, by amino acid analysis
after hydrolysis (see details in supplementary Fig. S3). The
substrates to be analyzed were the inner surface of small
glass test tube (5 mm in diameter and 29 mm in length,
73.6 mm
2
for covering the surface area of a 20 lL solution)
and benzoguanamine ⁄ formaldehyde resin particles (Epostar
L15, 11.6 lm in diameter, 73.6 mm
2
for the surface area
test; Nippon Shokubai, Osaka, Japan). The amount
adsorbed in 5 min at 25 °C in ASW was measured with
several protein concentrations and fitted using a Langmuir
adsorption isotherm.
The SPR measurements were performed with a BIA-
core 3000 system (Biacore AB, Uppsala, Sweden) at 25 °C
and with a flow rate of 10 lLÆmin
)1
. The sensor chips of
polycrystalline gold-coated and octadecanethiol-terminated
gold, HPA, were purchased from BIAcore. The running
buffer was 10 mm Tris (pH 7.4) ⁄ 25 mm NaCl with or with-

out ASW. A baseline was first established by pumping the
buffer, and the port was then switched to the protein solu-
tion. After saturation of the protein, the buffer was
pumped once again to monitor the desorption behavior.
rMrcp-19k at a concentration of 4 lm was adequately
diluted by the buffer, before being mixed with the same vol-
ume of buffer with or without 2 · ASW immediately before
injection. The mixing process with 2 · ASW was used to
minimize the exposure of the protein to any higher salt
concentration. The mass uptake of protein, Dm
SPR
, was
evaluated by the relationship
Dm
SPR
¼ C
SPR
DRU
where DRU is the measured change in response units, and
C
spr
has been calibrated to be 6.5 · 10
)2
ng
)1
Æcm
)2
for
adsorption to a flat surface [36]. The kinetics for adsorption
of rMrcp-19k to gold and alkylated gold were evaluated

using biaevaluation version 3.1 software that was sup-
plied with the instrument.
Localization and expression site of Mrcp-19k
To confirm that cp-19k was a cement component, the local-
ization of Mrcp-19k in the primary cement and in the pro-
tein fractions of both the base shell and peripheral shell of
the animal were investigated by western blotting. Polyclonal
antibodies were raised using bacterial recombinants of the
respective C-terminal regions of approximately 10 kDa in
Mrcp-19k and Mrcp-100k as antigens in rabbits with serial
subcutaneous injections. The recombinants were prepared
as described earlier. The primers used for amplifying the
Mrcp-19k portion were 5¢-TGG CCG CAG CCA TGG
CAT TGG T-3¢ and 5¢-ACC TCA GGA TCC AGG TCG
AGA AAA-3 ¢. The primers used for amplifying the Mrcp-
100k portion were 5¢-AGT GCA GCC CAT GGG GGC
AGC CAT-3 ¢ and 5¢-TTG CCT AGG TGG ATC CTC
AGC ATC TGA A-3¢. M. rosa primary cement was
collected as previously reported [10]. The base and peri-
pheral shell were separately collected from living M. rosa
Barnacle surface-cement protein Y. Urushida et al.
4344 FEBS Journal 274 (2007) 4336–4346 ª 2007 The Authors Journal compilation ª 2007 FEBS
specimens, and physically cleaned to remove all contamina-
tion by the animals’ soft tissue. Each shell was decalcified
by dialyzing against 2% acetic acid at 4 °C, and the precip-
itate was recovered. Although the supernatant was also
analyzed, no signal was detected by western blotting. The
precipitate was evaporated to dryness, denatured, separated
by SDS ⁄ PAGE (a Tris ⁄ Tricine buffer system, 16.5% T ⁄ 3%
C for Mrcp-19 k, and 8% T [37] with 6 m urea for Mrcp-

100k), and finally subjected to western blotting as described
elsewhere [38].
To evaluate the expression site of the Mrcp-19k gene in
the animal, RNAs were separately purified from tissues in
the upper or lower part of the barnacle in the same manner
as described above. The upper part included the cirri, tho-
rax, prosoma and hemolymph, and the lower part included
the mantle, muscle, ovariole, cement gland and hemolymph.
Twenty micrograms of RNA was electrophoresed and
transferred to a Hybond-N
+
nylon membrane (Amersham
Biosciences). The 540 bp DNA encoding the Mrcp-19k
ORF was labeled with [
32
P]dCTP[a
32
P] using a Random
Primer DNA Labeling kit (Takara Shuzo Co.). The labeled
probe thus obtained was used for northern blotting analysis
with the prepared membrane.
Acknowledgements
We thank Ms Futaba Sasaki and Ms Chikako Kajim-
oto for their technical assistance. Special thanks are
given to Professor J R. Shen of Okayama University
for his critical reading of the manuscript. Part of this
work was performed as an industrial science and tech-
nology project entitled Technological Development for
Biomaterials Design Based on Self-organizing Proteins,
which is supported by The New Energy and Industrial

Technology Development Organization (NEDO).
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Supplementary material
The following supplementary material is available
online:
Fig. S1. (A) cDNA and deduced amino acid sequences
of Mrcp-19k, Balcp-19k and Bicp-19k. (B) Alignment of
the mature N-terminal region of cp-19ks with WCD-21,

the peptide fragment generated by the cyanogen bro-
mide treatment of crude B. eburneus cement [11].
Fig. S2. SPR measurement at various concentrations
for the adsorption ⁄ desorption to ⁄ from (A) polycrystal-
line gold and (B) alkylated gold in ASW.
Fig. S3. Quantification of protein adsorption by amino
acid analysis and data fitting with the Langmuir
model.
Doc. S1. Search for homologous proteins.
This material is available as part of the online article
from
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 corre-
sponding author for the article.
Barnacle surface-cement protein Y. Urushida et al.
4346 FEBS Journal 274 (2007) 4336–4346 ª 2007 The Authors Journal compilation ª 2007 FEBS