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 thi
Trang 1barnacle cement protein
Youhei Urushida1, Masahiro Nakano1, Satoru Matsuda1, Naoko Inoue2, Satoru Kanai2,
Naho Kitamura3, Takashi Nishino3 and Kei Kamino1
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: kei.kamino@mbio.jp
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.
Trang 2stiff 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 remarkainsolu-ble hydrophobicity, and are
possibly bulk proteins of the cement complex
Reduc-tion treatment with guanidine hydrochloride soluReduc-tion
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 barbar-nacle
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
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.
Trang 3GTVTSSSSHQGSGAGDSIFE 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.
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
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.
Trang 4polypeptides 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 eburneuscement 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 rMrcp-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 VPPP C LG I SK V KQ K V G G SVSTTSA T GSG TTNCVTR TP N SV E KK NVA GN T VTA Bacp19k VPPP C LS I SK L KQ V A A N AVTTTGT T GSG VVKCVVR TP T SV E KK AAV GN T LSA Bicp19k VPPP C FS I SK Q KQ V V A G SVSAKGA T GSG SITCITK TP T SV T KK VAA GN A VSG
70
Mrcp19k TSVS A GD G F NL AA A T LVED T D GLG V T KNG G G FSE G A AIS Q A ANGG ATV K KA Bacp19k VSAS A AN G F NL GK A T EVKT T D GTK V T KTA G G KTG G A TTI Q A ANGG VSE K SL Bicp19k AAAA A GN G F NL VT A T NIST T D ITK V T QTI G G GTG G A TIL Q A ANGG AAL K EV
Mrcp19k KLDLL TD G EDLFDTKKVEK G TV TSSSSH QG SG A DSIFEI LN EA E SKIKKS G
Bacp19k KLDLL TD G LKFVKVTEKKQ G TA TSSSGH KA SG V HSVFKV LN EA E TELELK G
Bicp19k KLDLL PI G TGLGVVKQTKQ G QV TSSSSH KA SG L NSVLKV LN AH E TELKLK G
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.
Trang 5software (supplementary Fig S2) The adsorption
con-stant kaand desorption constant kdwere calculated as
2.17· 105m)1Æs)1and 4.94· 10)4s)1, respectively, for
the formation of the rMrcp-19k–Au complex Using
these data, the equilibrium constant Keq¼ ka⁄ kdcould
be estimated as 4.39· 108m)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 ka,
kd and Keq were calculated to be 9.76· 104m)1Æs)1,
6.67· 10)4s)1 and 1.46· 108m)1, respectively The
amounts adsorbed to the glass and formaldehyde resin
surfaces in 5 min at 25C 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 compocompo-nent 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
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 m M 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
Formaldehyde resin Adsorption amount
(ngÆmm)2)
0.76 (0.83 in dilute buffer)
Surface area per
molecule
(nm2per molecule)
37 (35 in dilute buffer)
Trang 6specifically 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 nm2 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 nm2per 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-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
r19k, 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.
Trang 7repulsion 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,
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
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
atmo-sphere The resulting supernatant was recovered as GSF2
corre-sponding to Mrcp-19k was transferred to a poly(vinylidene difluoride) membrane (ProBlott; Applied Biosystems, Foster
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
Trang 8l-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
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
mass, a Sequazyme peptide mass standard kit (Applied
Biosystems) was used The amino acid composition was
determined using a double-distilled constant-boiling HCl
(Waters) Possible modifications of Mrcp-19k by
glycosyla-tion and phosphorylaglycosyla-tion of Mrcp-19k were also
investi-gated The glycosylation was detected by periodic acid–Schiff
staining [31] with BSA as the positive control
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
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
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
a 3¢-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 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
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
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 +
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
Trang 9recombinant 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
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
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
according to the manufacturer’s instructions The fraction
enterokinase; Novagen) to cleave the fused tag The cleaved
quantitative amino acid analysis, the solvent was changed
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
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,
several protein concentrations and fitted using a Langmuir adsorption isotherm
The SPR measurements were performed with a
polycrystalline gold-coated and octadecanethiol-terminated gold, HPA, were purchased from BIAcore The running
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
minimize the exposure of the protein to any higher salt
evaluated by the relationship
where DRU is the measured change in response units, and
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
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
collected as previously reported [10] The base and peri-pheral shell were separately collected from living M rosa
Trang 10specimens, and physically cleaned to remove all
contamina-tion by the animals’ soft tissue Each shell was decalcified
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 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
Biosciences) The 540 bp DNA encoding the Mrcp-19k
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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