THE JOURNAL OF BIOLOGICAL CHEMISTRY
27360 –27365, 2000
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Barnacle Cement Proteins
IMPORTANCE OF DISULFIDE BONDS IN THEIR INSOLUBILITY*
Received for publication, December 23, 1999, and in revised form, April 29, 2000
Published, JBC Papers in Press, June 5, 2000, DOI 10.1074/jbc.M910363199
Kei Kamino‡§, Koji Inoue¶储, Tadashi Maruyama¶, Nobuhiko Takamatsu**, Shigeaki Harayama¶,
and Yoshikazu Shizuri‡
From the ‡Shimizu Laboratories, Marine Biotechnology Institute, 1900 Sodeshi, Shimizu, Shizuoka 424-0037, Japan,
¶Kamaishi Laboratories, Marine Biotechnology Institute, 75-1, Heita, Kamaishi, Iwate 026-0001, Japan, and the
**Department of Biosciences, School of Science, Kitasato University, 1-15-1, Kitasato, Sagamihara, Kanagawa 228, Japan
Barnacles produce a cement that is a proteinaceous
underwater adhesive for their secure attachment to the
substratum. The biochemical properties of the cement
have not previously been elucidated, because the insol-
ubility of the cement proteins hampers their purifica-
tion and characterization. We developed a non-hydro-
lytic method to render soluble most of the cement
components, thereby allowing the proteins to be ana-
lyzed. Megabalanus rosa cement could be almost com-
pletely rendered soluble by its reduction with 0.5 M di-
thiothreitol at 60 °C in a 7 M guanidine hydrochloride
solution, the high concentration of dithiothreitol being
indispensable to achieve this. The effectiveness of this
reduction treatment was confirmed by the detachment
of the barnacle from the substratum. Three proteins
comprising up to 94% of the whole cement were identi-
fied as the major cement components. The cDNA clone of
one of these major proteins was isolated, and the site-
specific expression of the gene in the basal portion of
the adult barnacle, where the cement glands are located,
was demonstrated. A sequence analysis revealed this
cement component to be a novel protein of 993 amino
acid residues, including a signal peptide. This is the first
report of the major component of the barnacle cement
protein complex.
The barnacle is a marine organism that attaches firmly to
various substrata in water. The barnacle achieves the underwa-
ter adhesion by secreting proteinaceous cement from the cement
gland into the space between its calcareous base and the substra-
tum (1–3). To adhere effectively, the cement needs to accomplish
several functions such as coagulation, displacement of water
from the substratum, establishment of interfacial contact, and
molecular attraction between dissimilar materials (4, 5). Under-
standing the structures and functions of the cement components
may help to elucidate the mechanisms for the biological adhesive
that is involved in barnacle settlement and to design interesting
biomimetic polymers. This may also lead to the development of a
specific remediation strategy for barnacle fouling.
* This work was performed as part of the Industrial Science and
Technology Frontier Program supported by the New Energy and Indus-
trial Technology Development Organization. The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBankTM/EBI Data Bank with accession number(s) AB033942.
§ To whom correspondence should be addressed: Tel.: 81-543-66-
9215; Fax: 81-543-66-9256; E-mail: keikamino@shimizu.mbio.co.jp.
储 Present address: Ocean Research Institute, The University of
Tokyo, 1-15-1 Minamidai, Nakano-ku, Tokyo 164-8639, Japan.
27360
Vol. 275, No. 35, Issue of September 1, pp.
Printed in U.S.A.
A quantitative amino acid analysis has revealed that the
cement is principally composed of proteinaceous substances (6).
DOPA (peptidyl-3,4-dihydroxyphenylalanine), which is a com-
mon constituent of mussel-foot proteins (7), has not been found
in the cement (8, 9). The partial compositions of the cement
proteins in Megabalanus rosa (9) and Balanus eburneus (10)
have recently been reported. M. rosa cement was shown to
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consist of three groups of proteins, i.e. a formic acid-soluble
fraction (SF1),1 a formic acid-soluble fraction after reduction by
tri-n-butylphosphine (SF2), and an insoluble fraction after re-
duction (IF) (9). SF1 and SF2 contain three similar proteins of
approximately 60 kDa that are rich in Ser, Thr, Gly, and Ala,
and other smaller proteins. IF, which accounts for 47% of the
cement, was not characterized, because it could only be ren-
dered soluble after cyanogen bromide (CNBr) cleavage.
We developed in this study a method to render soluble nearly
all the components of M. rosa cement that enabled all the major
cement proteins to be identified without any cleavage of the
peptide bonds. In addition, the complementary DNA clone cor-
responding to a major M. rosa cement protein was isolated and
sequenced.
EXPERIMENTAL PROCEDURES
Fractionation of the Cement According to the Solubility in a Guani-
dine Hydrochloride Solution—Cement of M. rosa was collected as de-
scribed in our previous study (9) within 1 day of its secretion and stored
at ⫺20 °C until being used. The cement was suspended in a 10 mM
sodium phosphate buffer (pH 6.0) containing 6 M guanidine hydrochlo-
ride (GdnHCl), and the suspension was centrifuged at 200,000 ⫻ g for
1 h at 20 °C. The protein fraction in the supernatant is designated as
GdnHCl-soluble fraction 1 (GSF1). The precipitate was resuspended in
the same solution and again centrifuged. This procedure was conducted
twice more to completely remove GSF1 from the precipitate. The pre-
cipitate was resuspended in a 1.5 M Tris-HCl buffer (pH 8.5) containing
7 M GdnHCl and 20 mM EDTA (3 mg/ml) and then reduced with 0.5 M
dithiothreitol (DTT) for 1 h at 60 °C while continuously agitating. The
sulfhydryl groups of the proteins were carboxymethylated by a 2.5-fold
amount of monoiodoacetic acid (w/w) to DTT in the dark at room
temperature for 20 min, the reaction being terminated by adding 2-mer-
captoethanol. The resulting suspension was centrifuged as already
described. The protein fraction in the supernatant is designated as
GdnHCl-soluble fraction 2 (GSF2), and the precipitate is designated as
the GdnHCl-insoluble fraction (GIF). Each fraction was dialyzed
against 0.1% acetic acid at 4 °C and then flash-evaporated. The meth-
1
The abbreviations used are: SF1, SF2 and IF, Megabalanus rosa
cement fractions separated by their solubility in aqueous formic acid;
GdnHCl, guanidine hydrochloride; GSF1, GSF2 and GIF, Megabalanus
rosa cement fractions separated by their solubility in a GdnHCl solu-
tion; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; CB
peptides, eight major peptide fragments generated by the CNBr treat-
ment of IF, named CB-1 through CB-8; PCR, polymerase chain reaction;
Mrcp, Megabalanus rosa cement protein; HRP, horseradish peroxidase;
bp, base pair(s); PVDF, polyvinylidene difluoride.
This paper is available on line at http://www.jbc.org
 Barnacle Underwater Adhesive Proteins 27361

FIG. 1. Oligonucleotide primers used for PCR amplification of

the Mrcp-100k cDNA fragment, and nucleotide sequence of the
amplified 110-bp DNA. The DNA sequences of the primers are de-
noted according to the IUPAC code, r ⫽ (A/G), Y ⫽ (C/T), K ⫽ (G/T), D ⫽
(A/G/T), and n ⫽ (A/C/G/T). Arrows indicate the sense and antisense
primers. The N-terminal amino acid sequence of CB-8 (9) are shown for
comparison, and the amplified region is underlined. 110-bp DNA was
used for screening the M. rosa cDNA library. FIG. 2. Major constituents in the M. rosa cement identified by
SDS-PAGE. Lanes 2 and 3, GSF1 and GSF2 prepared from M. rosa
ods of Laemmli (11) and of Schäger and Jagow (12) were employed for cement, respectively. Lane 4, M. rosa cement proteins rendered soluble
an SDS-PAGE analysis. A peptide map analysis of the major cement by heat denaturation in 4.2% 2-mercaptoethanol and 2% SDS after
proteins was carried out as follows. Each protein band by SDS-PAGE removing GSF1. Lane 1, high molecular mass standards (Bio-Rad).
was visualized with the Copper Stain kit (Bio-Rad) and then cut out. Numbers on the left side of lane 1 indicate molecular masses (kDa). The

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Each gel piece was destained and treated with CNBr in 70% (v/v) formic names of major proteins are indicated on the right side of lane 4. The
acid (9). After evaporating to remove the CNBr and formic acid, the samples were separated by SDS-PAGE (8% polyacrylamide gel includ-
peptide fragments derived from each protein were separated by SDS- ing 6 M urea and a Tris-Gly buffer system). The gel was stained with
PAGE and visualized by CBB-R250 staining. Electrophoretic transfer of Coomassie Blue R-250 after electrophoresis.
the major cement proteins and peptide fragments to a polyvinylidene
difluoride (PVDF) membrane (Pro Blott, PE-Biosystems) was conducted TABLE I
according to the method of Ikeuchi (13) by adding 0.1% SDS in a Amino acid compositions of the cement protein
blotting buffer. The N-terminal amino acid sequences were determined Each value is given as residues per thousand.
with a PSQ-2 protein sequencer (Shimadzu, Japan). The amino acid
composition of Mrcp-100k, a major protein component of M. rosa ce- Mrcp-100ka Mrcp-100kb 58-kDa
Mrcp-68kc
(deduced) (analyzed) Protein
ment, was determined as follows. GSF2 was separated by SDS-PAGE
and then electrophoretically transferred to a PVDF membrane. After a Asx 77.95 78.78 87.51 65.70
brief CBB-R250 staining, Mrcp-100k was cut out and hydrolyzed in Glx 91.28 102.66 86.50 96.80
vacuo in constantly boiling HCl (5.7 N), including 0.02% phenol at Ser 86.15 90.47 159.81 128.10
110 °C for 24, 48, and 72 h, or in 4 M methane sulfonic acid (Pierce) at Gly 42.05 56.84 147.84 165.30
110 °C for 24 h. The amino acid compositions of the hydrolysates were His 14.36 8.67 2.48 2.90
analyzed by a Pico-Tag amino acid analysis system (Waters, Division of Thr 46.15 61.32 138.06 128.70
Millipore). The glycosylation of Mrcp-100k was investigated as follows. Ala 56.41 63.99 124.10 134.80
After separating GSF2 by SDS-PAGE and electroblotting to a PVDF Arg 68.72 56.09 36.23 29.90
membrane, the sample was treated with periodate to oxidize the oligo- Pro 49.23 56.92 25.45 51.30
saccharide. The generated aldehyde was reacted with a biotinhydrazide Val 70.77 75.94 72.84 61.60
reagent and with horseradish peroxidase (HRP)-labeled avidin by using Met 25.64 11.14 0.00 4.00
a GP-sensor (Honen, Japan). Bound HRP was visualized by its reaction Cys 14.36 6.37 8.91 6.60
Ile 76.92 81.99 17.27 26.00
with an HRP1000 immunostaining kit (Konica, Japan).
Leu 113.85 107.84 17.83 32.00
Test on Barnacles of Their Detachment from the Substratum—Bar-
Trp 1.03 ND 0.00 0.00
nacles of about 1 cm in diameter attached to mussel shells (M. rosa) or Phe 47.18 49.76 6.50 14.00
to a plastic substratum (Balanus amphitrite) were collected, and the Lys 55.38 36.36 51.02 48.00
whole soft tissue within the shell was carefully removed. Each intact Tyr 62.56 55.06 5.27 4.20
barnacle shell attached to the substratum was put into a 50-ml conical a
tube and immersed in a 1.5 M Tris-HCl buffer (pH 8.5) containing 7 M The composition was calculated from the predicted sequence, except
GdnHCl. The barnacle shell was then treated by adding or not 0.2 M or for the signal sequence.
b
0.5 M DTT in a nitrogen atmosphere while gently agitating at 60 °C. The composition was determined by an amino acid analysis of the
hydrolysate of Mrcp-100k. ND, not detected
Isolation of mRNA and cDNA Synthesis—Barnacles (M. rosa) of c
The amino acid compositions of Mrcp-68k and of the 58-kDa protein
about 4 cm in diameter at the calcareous base were collected from
in B. eburneus cement are from Refs. 9 and 10, respectively.
Miyako Bay in Iwate prefecture, Japan. The whole soft tissue of the
barnacle was homogenized, and total RNA was extracted with a total the SmaI site of pUC19. The insert was sequenced with a Prism dye
RNA separator kit (CLONTECH Laboratories). Poly(A)⫹ RNA was iso- terminator cycle sequencing kit and 373A DNA sequencer (PE-Biosys-
lated by using Oligotex-dT30 (Takara Shuzo Co., Japan). cDNA was tems). The insert excised from the pUC19 clone by digestion with EcoRI
prepared from M. rosa mRNA with a Zap-cDNA synthesis kit (Strat- and BamHI was 32P-labeled by a random primer DNA labeling kit
agene) according to the instructions of the supplier. (Takara), apart from using an oligonucleotide primer (TACCTAGAC-
Screening the cDNA Library—The DNA probe for screening the CACGAACTGCCC) complementary to the 110-bp insert. This labeled
cDNA library was generated by the polymerase chain reaction (PCR) probe was used for screening a ␭-phage cDNA library of M. rosa (14).
with two primers designed from the partial amino acid sequence of one Ten positive clones were picked up, and the cDNA inserts were sub-
of the CB peptides, CB-8 (9) (Fig. 1). Primary PCR was performed in cloned into pBluescript SK(II) according to the manufacturer’s specifi-
100 ␮l of a reaction mixture containing 3 ␮g of each primer, 200 ␮M cation for the ExAssist system (Stratagene). The molecular sizes of the
dNTPs, 1⫻ Tth buffer, 4 units of Tth DNA polymerase (Toyobo, Japan), inserts were determined by agarose gel electrophoresis after being
and 0.3 ␮g of the M. rosa cDNA. DNA amplification was carried out digested by the appropriate restriction enzymes. The cDNA clone con-
with 29 thermal cycles, each involving 95 °C for 1 min, 52 °C for 30 s, taining the longest insert was sequenced as already described.
and 70 °C for 2 min. Secondary PCR was performed in the same man- Sequence Analysis by Computer—A homology search analysis was
ner, except that 10 ␮l of the amplified reaction mixture from primary made of the SwissProt and Protein Information Resource (PIR) data
PCR was used as the DNA template. Amplified DNA of the expected bases by using the FASTA or BLAST program. The secondary structure
size (110 bp) was purified by electrophoresis on 3% NuSieve 3:1 Agarose (15), isoelectric points (16), and hydropathic characteristics (17) were
gel (FMC Bio Products). The 110-bp DNA fragment was subcloned into predicted by using GENETYX-MAC, version 7.0.1.
27362 Barnacle Underwater Adhesive Proteins

FIG. 3. Nucleotide sequence of

Mrcp-100k cDNA and the predicted
amino acid sequence of Mrcp-100k.
The nucleotides are numbered on the
right side of the sequence, the termina-

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tion codon is indicated by an asterisk, and
the Met residues are boxed. Underlined
are the partial amino acid sequences of
the CB peptides (CB-2, -3, -5, -6, -7 and -8)
that were determined by a protein se-
quence analysis (9). The N-terminal se-
quence of mature Mrcp-100k is shown by
a dotted underline.

Northern Blot Hybridization—The upper portion of the body, which
contained the cirri, thorax, prosoma, and hemolymph, and the basal
portion mainly comprising the mantle, muscle, ovariole, cement gland
(18), and hemolymph were separated with a surgical knife and col-
lected. Total RNA was prepared from each portion by using a total RNA
separator kit (CLONTECH). 20 ␮g of total RNA was electrophoresed on
1% agarose gel, transferred to a Hybond N⫹ nylon membrane (Amer-
sham Pharmacia Biotech), and hybridized with [␣-32P]dCTP-labeled
110-bp DNA.
RESULTS
Constituents of the Barnacle Cement—The proportions by
weight of GSF1, GSF2, and GIF in the cement were 24%, 70%,
and 6%, respectively. This indicates that more than 90% of the
cement had been rendered soluble by this method. The SDS-
PAGE analysis showed that GSF1 was composed of a protein
with a molecular mass of ca. 68 kDa, which was named M. rosa
cement protein-68k (Mrcp-68k), and some minor proteins
(Fig. 2, lane 2). Proteins with molecular masses of 180 kDa, 40
kDa, and of a little less than 20 kDa were consistently detected
as minor constituents. The N-terminal sequence and amino
acid composition of Mrcp-68k agree with those of SF2-60k from
our previous study (9), which contained high levels of Ser, Thr,
Gly, and Ala. This previously designated protein was therefore
renamed Mrcp-68k.
The SDS-PAGE analysis of the GSF2 fraction obtained after
the treatments with 0.5 M DTT revealed that the fraction con-
tained two major proteins of 100 and 52 kDa, in addition to
Mrcp-68k, which was also found in GSF1. These proteins were
named Mrcp-100k and Mrcp-52k, respectively (Fig. 2, lane 3).
Mrcp-100k and Mrcp-52k were not rendered soluble by a 15 mM
Tris-HCl buffer (pH 6.8) containing 4.2% 2-mercaptoethanol
and 2% SDS with heat denaturation at 100 °C for 3 min (Fig. 2,
lane 4). After the DTT treatment, these two proteins became
soluble in a 0.1% acetic acid solution, but were insoluble in a
neutral pH buffer without SDS. The addition of SDS to the
blotting buffer was required for electrophoretic transfer of the
two major proteins, Mrcp-100k and -52k, to the hydrophobic
PVDF membrane.
In our previous work (9), the eight major CB peptides (CB-1
 Barnacle Underwater Adhesive Proteins
through CB-8) were derived from the formic acid-insoluble
fraction (IF) of M. rosa cement by CNBr cleavage. An SDS-
PAGE analysis of the CNBr-cleaved products of Mrcp-100k and
-52k gave six (CB-2, -3, -5, -6, -7, and -8) and two CB peptides
(CB-1 and -4), respectively (data not shown). The N-terminal
amino acid sequence of Mrcp-100k was HRPSFERRXXGXLR-
SPVAADLDDDEIGM, where X is not determined, but it was
most likely Cys. The amino acid composition of Mrcp-100k
isolated by SDS-PAGE was determined as shown in Table I. No
glycosylation was detected in Mrcp-100k.
The insoluble fraction after this DTT treatment was named
GIF. Although it was a proteinaceous substance, a method for
rendering GIF soluble without hydrolysis was not discovered in
this study.
Effect of the Reduction Treatment on the Detachment of Bar-
nacles from the Substratum—The effect of the DTT treatment
on barnacle detachment from the substratum was the same for
both M. rosa and B. amphitrite. The barnacle shell became
spontaneously detached from the substratum after a 1-h treat-
ment by 0.5 M DTT, and became detached after a 1-day treat-
ment by 0.2 M DTT, whereas the shell remained attached
without any DTT treatment for 2 days.
Molecular Cloning of Mrcp-100k cDNA—cDNA clones were
isolated on the basis of the partial amino acid sequence of the
CB peptides. According to the amino acid sequence of the CB-8
peptide fragment (9) in Mrcp-100k, two PCR primers were
synthesized (Fig. 1), and PCR was performed by using M. rosa
cDNA as the template. The amplified 110-bp-long DNA was
subsequently cloned and sequenced. The predicted amino acid
sequence of the 110-bp DNA fragment completely matched the
corresponding amino acid sequence of the CB-8 peptide (Fig. 1).
About 100,000 clones of an M. rosa cDNA library were screened
by using 32P-labeled 110-bp DNA as a probe, and more than 30
positive clones were obtained. DNA inserts from 10 randomly
selected clones were subcloned into pBluescript SK(⫺) and
were found to carry inserts of about 3.3 kbp. A restriction
endonuclease analysis of these 10 clones indicated them to be
identical (data not shown). A plasmid containing the largest
cDNA fragment was selected for sequencing.
Structures of Mrcp-100k cDNA and the Encoding Polypep-
tide—The DNA insert of the longest plasmid was 3299 bp long
(Fig. 3) and encoded a polypeptide of 993 amino acids. The
molecular mass and isoelectric point were deduced to be
113,639 daltons and 9.86, respectively. The first 18 amino acid
residues were thought to be the signal peptide, because of its
high hydrophobicity, and the N-terminal amino acid sequence
of mature Mrcp-100k was thought to begin at the 19th residue.
The 9th, 10th, and 12th amino acids of the mature N-terminal
sequence of Mrcp-100k were confirmed to be Cys residues by
the deduced sequence from the cDNA. When the putative sig-
nal peptide was omitted from the deduced amino acid sequence,
a discrepancy in molecular mass between the estimated figure
from SDS-PAGE (100 kDa) and the calculated one from the
predicted sequence (112 kDa) was apparent. The amino acid
sequence of mature Mrcp-100k deduced from the cDNA had 23
Met residues and was presumably cleaved into 24 fragments by
the CNBr treatment. The predicted amino acid sequence of
Mrcp-100k contained six of the eight CB peptides (CB-2, -3, -5,
-6, -7, and -8). The only discrepancy between the amino acid
sequences of the CB peptides and the predicted Mrcp-100k
sequence was the second residue of CB-3: It was Thr in the
CB-3 sequence, whereas Ile was predicted in the Mrcp-100k
sequence. Mrcp-100k was confirmed to give the six CB peptides
in the SDS-PAGE peptide map obtained by CNBr cleavage. The
other smaller fragments of Mrcp-100k by CNBr cleavage,
which could not be detected by SDS-PAGE, were thought to
27363
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FIG. 4. Comparison of the fragments divided into 10 segments
(r1 to r10) from the whole Mrcp-100k sequence. A, proportions of
hydrophobic, neutral, and hydrophilic amino acids. B, transition of the
predicted isoelectric point for each region.
have migrated to the front of SDS-PAGE. Two peptide bands
with slightly higher molecular masses than that of CB-1 on a
gel of SDS-PAGE (9) were partial cleavage products of Mrcp-
100k. The most abundant amino acid residue was Leu (111
residues of the 975 total residues; Table I), with Ser (84 of 975)
and Ile (75 of 975) following. The content of Cys was calculated
to be 1.4% of the total residues (14 of 975). The experimentally
determined amino acid composition of Mrcp-100k agrees, in
general, with that deduced from the cDNA sequence (Table I).
No repetitive motif or sequence periodicity was suggested in
the predicted amino acid sequence of Mrcp-100k. The hydro-
pathic profile indicates a short alternating pattern of hydopho-
bic and hydrophilic residues throughout Mrcp-100k. The ar-
rangement of amino acid species in its primary structure was
investigated by comparing the proportions of hydrophobic, neu-
tral, and hydrophilic amino acids in 10 segments of the Mrcp-
100k sequence (r1–r10; Fig. 4A). The isoelectric points were
also calculated in the 10 segments (Fig. 4B). Although the
proportions of the hydrophobic, neutral, and hydrophilic amino
acids were almost the same in all the regions of Mrcp-100k, a
gradient in the isoelectric points from the N-terminal region,
r1, to the C-terminal region, r10, was apparent. Prediction of
the secondary structure (15) suggested that 87% of the total
sequence formed a ␤-sheet structure. No similar sequence has
so far been found by a computer-aided homology search of the
SwissProt and PIR data bases.
RNA Blot Analysis—The RNA blot analysis was performed
by using total RNA, which had been prepared from the upper or
basal portion of the body of adult M. rosa, to confirm the site of
the Mrcp-100k gene expression. The transcript of the Mrcp-
100k gene was only detected in the basal portion where the
cement glands are located (Fig. 5).
DISCUSSION
We have previously shown (9) that, although proteins like
SF2-60k (renamed Mrcp-68k in this study) and those smaller
than 20 kDa, could be rendered soluble in an aqueous formic
acid solution by reduction with tri-n-butylphosphine, a half
27364 Barnacle Underwater Adhesive Proteins
FIG. 5. Site specificity of Mrcp-100k gene expression in the
basal portion of the adult barnacle where the histologically
distinct cement gland is localized. 20 ␮g of total RNA extracted
from the basal or upper portion of the adult barnacle was electrophore-
sed in a formaldehyde gel, transferred to a nylon membrane, and
hybridized with a 110-bp DNA probe. The basal portion mainly com-
prises the mantle, muscle, ovariole, cement gland (18), and hemolymph,
whereas the upper portion contained the cirri, thorax, prosoma, and
hemolymph.
portion (47%) of the cement proteins (IF) remained insoluble.
In this study, we successfully rendered more than 90% of M.
rosa cement soluble by reducing with 0.5 M DTT in a GdnHCl
solution. Peptide mapping by CNBr cleavage indicated that IF
was mainly composed of Mrcp-100k and -52k. Thus, more than
90% of M. rosa cement was composed of the three major pro-
teins, Mrcp-100k, Mrcp-68k, and Mrcp-52k, and some minor
proteins. The similarity between Mrcp-100k and -52k is note-
worthy, i.e. their behavior in rendering the cement soluble,
contents in the cement, and electroblotting characteristics. Mrcp-
68k was different in these respects from Mrcp-100k and -52k in
that it could easily be rendered soluble by conventional reduction
with 2-mercaptoethanol in SDS containing a buffer (pH 6.8). The
amino acid composition of Mrcp-68k was rich in Ser, Thr, Gly,
and Ala, and considerably different from the composition of
Mrcp-100k. Barnacle underwater adhesion thus seems to be
cooperatively achieved by a complex of distinct proteins.
Naldrett et al. (10) have reported that B. eburneus cement
could be rendered partially soluble by a reductive treatment in
2.5% 2-mercaptoethanol and 2% SDS. The 58-kDa protein in B.
eburneus cement resembles Mrcp-68k in its amino acid compo-
sition and molecular mass (9, 10) (Table I). Although nothing
resembling Mrcp-100k and -52k has been reported in B. ebur-
neus cement, the sequence of a short peptide fragment (WCD-
11), which had been derived from whole B. eburneus cement
(10) by the CNBr cleavage, indicates good homology with part
of the Mrcp-100k sequence (Fig. 6). B. eburneus cement thus
appears to have similar constituents to those of M. rosa cement.
Although Mrcp-100k and -52k were both rendered soluble by
reduction with 0.5 M DTT in a GdnHCl solution at pH 8.5, they
were not by reduction with 4.2% 2-mercaptoethanol in 2% SDS
at pH 6.8, nor by reduction with tri-n-butylphosphine (9). With
the latter treatment, tri-n-butylphosphine has poor solubility
in an aqueous solution, so reduction might be insufficient.
Reduction with 2.5% 2-mercaptoethanol in the presence of 2%
SDS at pH 8.45 was also inadequate to render soluble the
corresponding proteins in B. eburneus cement (10). The reli-
ability of the DTT treatment was also confirmed by the detach-
ment test on M. rosa and B. amphitrite from substrata. DTT
has been shown to be at least 1000 times more effective than
2-mercaptoethanol for cleaving disulfide bonds (19). This indi-
cates that disulfide bonds would have contributed to the sta-
bility of the protein complex in the barnacle cement. The low
Cys content of Mrcp-100k seems to be incompatible with the
extensive requirement of a reductant to give solubility. How-
ever, Mrcp-100k was composed of abundant hydrophobic resi-
dues, and a small number of disulfide bonds in Mrcp-100k may
be hidden by the hydrophobic barrier, providing a possible
explanation for the requirement of a high concentration of DTT
and GdnHCl. It is not known whether the disulfide bonds of the
cement proteins are intermolecular or intramolecular.
FIG. 6. Alignment between Mrcp-100k and WCD11, the latter
being the peptide fragment generated by the CNBr treatment of
crude B. eburneus cement (10). Identical amino acids are indicated
with vertical lines, and conserved replacements are indicated by double
dots. The residue before the first amino acid of WCD11, Leu, is likely to
be Met, because WCD11 was prepared by the CNBr treatment.
A discrepancy in the molecular mass estimation for Mrcp-
100k was apparent when comparing the mass calculated from
the predicted sequence with the apparent mass determined by
SDS-PAGE. The addition of SDS to the blotting buffer was
required for the electrophoretic transfer of Mrcp-100k and -52k
to the PVDF membrane. This observation is consistent with the
high content of hydrophobic residues in the protein. The high
hydrophobicity of the polypeptide may have contributed to the
anomalous mobility by SDS-PAGE. Post-translational process-
ing of the C-terminal region is another possible explanation.
Although the N-terminal sequences of Mrcp-100k and of the CB
peptides agree with those of the predicted sequence, determi-
nations at the C-terminal end and of the exact mass by matrix-
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assisted laser desorption ionization-time of flight (MALDI-
TOF) mass spectrometry were unsuccessful. Although the
experimentally determined amino acid composition of Mrcp-
100k was generally in agreement with that deduced from the
cDNA sequence, complete agreement was not apparent. This
may suggest post-translational processing of Mrcp-100k. The
cause of this discrepancy in the molecular mass estimation for
Mrcp-100k was not found in this study.
This is the first report on the complete primary structure of
barnacle underwater adhesive protein. No protein similar to
Mrcp-100k has been found in sequence data bases, suggesting
that the function of Mrcp-100k is unique and it has not previ-
ously been reported. Specific characteristics are generally be-
lieved to be required for underwater adhesion, i.e. coagulation,
displacement of water from the substratum, and establishment
of interfacial contact and molecular attraction between unlike
materials (4, 5). Although the role of Mrcp-100k in underwater
adhesion is not clear, its insoluble behavior is noteworthy. The
results of this work lead us to believe that Mrcp-100k and -52k
are essential for stabilizing the cement complex in seawater.
The hydropathic profile of Mrcp-100k indicates a pattern of
short alternating hydrophobic and hydrophilic residues
throughout the whole region. The proteins involved in the
formation of insoluble amyloid plaque have recently been char-
acterized (20), and the pattern of alternating polar and nonpo-
lar residues in a “cross-␤ ” sheet structure was found to be
essential to form insoluble fibrils. The ␤-sheet structure was
also predicted to be rich in Mrcp-100k. Thus, the molecular
mechanisms for forming an insoluble proteinaceous multimer
may be similar between amyloid plaque and barnacle cement.
Although a similar distribution of hydrophobic, neutral, and
hydrophilic amino acids was found in each region of Mrcp-100k
(Fig. 4A), the predicted isoelectric points indicate a gradient
from the N-terminal region (r1) to the C-terminal region (r10)
(Fig. 4B). The adhesive protein of the mussel, foot protein-2
(fp-2), has been pointed out to contain clusters of amino acids
with acidic charges in the N- and C-terminal regions. It has
been speculated that these clusters of acidic charges play a role
in initiating the assembly of these proteins in seawater by
reducing the basic charges in the central region of fp-2 (7). In
Mrcp-100k, the gradient of charge distribution from the N to C
termini may play a similar role in the initial assembly of the
proteins by reducing the charge at the site for attachment and
may provide the mechanism for assemble with other cement
components in seawater.
 Barnacle Underwater Adhesive Proteins
Some researchers (1, 2, 18, 21) have reported a histologically
distinct cement organ and cement duct in the basal portion,
near the ovariole, of the adult barnacle. The Northern blot
analysis indicates that the Mrcp-100k gene was probably ex-
pressed by the histologically distinct cement gland in the basal
portion of the barnacle. The cement would be transported
through the duct and then secreted into the space between the
calcareous base and the substratum.
Acknowledgments—We thank F. Sasaki, S. Dobashi, I. Hiramatsu, S.
Komukai, D. Miki, S. Ohdo, and S. Kanai for their technical assistance
and advice, and S. Miyachi for encouragement. Special thanks are given
to J. H. Waite for his critical reading of this manuscript.
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 Barnacle Cement Proteins: IMPORTANCE OF DISULFIDE BONDS IN THEIR
INSOLUBILITY
Kei Kamino, Koji Inoue, Tadashi Maruyama, Nobuhiko Takamatsu, Shigeaki Harayama
and Yoshikazu Shizuri
J. Biol. Chem. 2000, 275:27360-27365.
originally published online August 25, 2000

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