cDNA cloning and characterization of a novel calmodulin-
like protein from pearl oyster Pinctada fucata
Shuo Li
1
, Liping Xie
1,2
, Zhuojun Ma
1
and Rongqing Zhang
1,2
1 Institute of Marine Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China
2 Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing, China
The shells of bivalve molluscs, especially the internal
lustrous ‘mother of pearl’ layer of the shell, with
exceptional nanoscale architectures and outstanding
mechanical performance, have received a great deal of
attention from many biology and materials scientists
in the past few decades [1]. Shells and pearls are all
products of calcium metabolism which is a very
complicated and highly controlled physiological and
biochemical process. The oyster calcium metabolism
involves calcium ion absorption, transport, accumula-
tion, secretion, deposition and other important steps.
Investigations have mainly focused on purification of
matrix proteins, the end products of oyster calcium
metabolism. However, how calcium is transported into
the cell, is secreted from the mantle epithelium, and
how the calcium carbonate crystals are formed remain
unclear. In particular, what regulatory factors are
involved in these processes is obscure. Recent observa-
tions indicate that hemocytes may be directly involved

is expressed strongly in the outer and inner epithelial cells of the inner
fold, the outer epithelial cells of the middle fold, and the dorsal region of
the mantle. The oyster CaLP protein, with four putative Ca
2+
-binding
domains, is highly heat-stable and has a potentially high affinity for cal-
cium. CaLP also displays typical Ca
2+
-dependent electrophoretic shift,
Ca
2+
-binding activity and significant Ca
2+
-induced conformational chan-
ges. Ca
2+
-dependent affinity chromatography analysis demonstrated that
oyster CaLP was able to interact with some different target proteins from
those of oyster CaM in the mantle and the gill. In summary, our results
have demonstrated that the oyster CaLP is a novel member of the CaM
superfamily, and suggest that the oyster CaLP protein might play a differ-
ent role from CaM in the regulation of oyster calcium metabolism.
Abbreviations
CaM, calmodulin; CaLP, calmodulin-like protein; CD, circular dichroism; EGTA, ethylene glycol-bis-(b-amino-ethyl ether)N,N,N¢,N¢-tetra-acetic
acid; RACE, rapid amplification of cDNA ends; UTR, untranslated region.
FEBS Journal 272 (2005) 4899–4910 ª 2005 FEBS 4899
regulating several crucial processes such as secretion,
cyclic nucleotide metabolism, cellular calcium meta-
bolism, muscle contraction, glycogen metabolism, cell
proliferation and differentiation, and gene expression

expressed in the epithelial cells at the folds and the
outer dorsal region of the mantle. These observations
suggest that CaM may be actively involved in the regu-
lation of calcium transport and secretion in oyster.
The complicated oyster calcium metabolism process
might exist in more factors that also participate in the
many critical steps of calcium metabolism, including
the transport of the extracellular calcium ions to the
mantle epithelium where the calcium is deposited onto
the organic framework formed mainly by matrix pro-
teins. Identification of more regulatory factors involved
in the complicated process will not only provide crit-
ical clues to the understanding of the underlying mech-
anism of calcium metabolism in the process of shell
and pearl formation, but also offer the opportunity to
promote the yield and quality of pearl. In this study,
we isolated a full-length complementary DNA enco-
ding a novel CaLP protein from pearl oyster P. fucata.
Tissue expression and distribution of CaLP mRNA
was examined by RT-PCR and in situ hybridization,
respectively. We also expressed and purified the oyster
CaLP in E.coli, characterized its calcium binding prop-
erties, analyzed its calcium-induced conformational
changes by CD and fluorescence analysis and com-
pared its proteins interaction with oyster CaM in the
mantle and the gill by Ca
2+
-dependent affinity chro-
matography. Our observations described here may
provide important clues to understand the diversity

tide position 719, which is 15 nucleotides upstream
of the poly(A) tail. This cDNA sequence has been
submitted to GenBank with the accession number
AY663847.
Sequence analysis of oyster CaLP protein
The deduced oyster CaLP protein is comprised of 161
amino acids with a calculated molecular mass of
18.3 kDa and an isoelectric point of 4.04. The oyster
CaLP protein shows 67% identity with and 87% simi-
larity with the CaM protein from P. fucata. If the
extra C-terminal end segment of 12 amino acids is
not taken into account, the oyster CaLP shares
93.9% similarity with oyster CaM. The oyster CaLP
and CaM both contain only one Tyr residue, and
each does not contain Cys or Trp residue. The predic-
ted secondary structures for both proteins (Fig. 2A)
are also very similar (helix,  57%; beta-sheet,
A novel calmodulin-like protein from pearl oyster S. Li et al.
4900 FEBS Journal 272 (2005) 4899–4910 ª 2005 FEBS
 3.7% and coil,  38%). All these reveal that the
oyster CaLP protein is closely related to oyster CaM.
A remarkable structural feature of this novel CaLP is
that there are 12 extra hydrophilic amino-acid resi-
dues located at the C-terminal end (Fig. 2B), suggest-
ing that CaLP may have a special function in
Fig. 1. Nucleotide and deduced amino-acid sequence of the P. fucata CaLP cDNA. The stop codon is marked with an asterisk and the pos-
sible polyadenylation signal sequence in the 3¢-untranslated region is underlined. This cDNA sequence has been submitted to GenBank with
accession number AY663847.
B
A

2+
-binding residues (X, Y, Z, -Y, -X, -Z) in the sec-
ond and the fourth EF-hand domains are more con-
served than those in the first and the third EF-hand
domains compared with oyster CaM. Structural varia-
tions in EF-hand domains of oyster CaLP may contrib-
ute significantly to its specific selectivity for substrates
and physiological function. Comparison of the amino-
acid composition of the calcium-binding domains in
canonical EF-hands [30] with that in the EF-hands in
oyster CaLP, reveals a good correlation of Ca
2+
-bind-
ing ligand positions. An exception is the Lys residue in
domain 3 of CaLP at ligand position Z, indicative of a
weaker calcium binding potential in this loop than that
in CaM. However, there are 4 acidic residues (Asp or
Glu) in the ligand positions of domain 2 and 4 in
CaLP, suggesting of a high calcium binding potential.
In the flexible central helix, a region between the sec-
ond and the third EF-hand domain, which contributes
to the functional characteristics of CaM to bind to var-
ious target proteins [31–33], is the most conserved
region of the oyster CaLP. In contrast, the least homol-
ogy region of the oyster CaLP is between the third and
the fourth calcium-binding sites. Finally, oyster CaLP
possesses several putative phosphorylation sites predic-
ted by NetPhos 2.0 Server with high scores [34], which
include five serine, three threonine and one tyrosine res-
idues. Among them, three Ser residues, Ser25, Ser27

cing analyses. As shown in Fig. 3, oyster CaLP mRNA
was expressed in all tissues tested, with the highest
expression levels in the mantle that is a key tissue
responsible for the metabolism of metal ions and parti-
cipates actively in the secretion of calcium and other
ions for mineral growth in the process of the shell and
pearl formation [37,38]. Similar data were obtained
from three independent experiments.
To understand the precise expression site of the oys-
ter CaLP mRNA in the mantle tissue of P. fucata,
in situ hybridization analysis was performed. As can be
seen in Fig. 4, strong hybridization signals were detec-
ted in the outer and inner epithelial cells of the inner
fold and the outer epithelial cells of the middle fold of
the mantle (Fig. 4A), a region for periostracum secre-
tion [39]. However, hybridization signal was weak in
the inner epithelial cells of the outer fold whereas oys-
ter CaM is expressed highly in this place [29]. Strong
hybridization signals were also detected in outer epi-
thelial cells of the dorsal region of the mantle (Fig. 4B)
which is responsible for nacreous layer secretion [39],
but hybridization with the control sense probe yielded
no hybridization signals (data not shown). Calcium is
a major component of oyster shell as well as a key
intracellular second messenger. The shells of oyster
consist of 90% CaCO
3
, products of calcium metabo-
lism, and a few percent of matrix of biological macro-
molecules. This highly controlled process may depend

tography, only a single band with > 95% purity was
observed on 15% SDS ⁄ PAGE stained by Coomassie
Brilliant Blue R-250. The relative molecular mass of
the band is about 18 kDa, which is consistent with the
predicted molecular mass of fusion oyster CaLP, and
the expression level of target protein is 15 mgÆL
)1
in
LB culture. As also can be seen in Fig. 6, the recom-
binant oyster CaLP was homogeneous upon polyacryl-
amide gel electrophoresis with the addition of either
Ca
2+
or EGTA. We have tried to express CaLP
without fusion with His-tag, but failed to purify the
protein by phenyl-sepharose hydrophobic chromatog-
raphy due to the strong hydrophilicity of the 12 extra
Fig. 4. In situ hybridization of oyster CaLP mRNA in the mantle of
pearl oyster P. fucata. To view the distribution of hybridization sig-
nal on the whole tissue, three overlapping pictures of the same
section were taken. Strong hybridization signals were presented in
the outer and inner epithelial cells of the inner fold and the outer
epithelial cells of the middle fold of the mantle (arrow heads) in (A).
Hybridization signals were also shown in the outer epithelial cells
of the dorsal region of the mantle (arrow heads) in (B). OF, outer
fold; MF, middle fold; IF, inner fold. Scale bar, 0.2 mm.
Fig. 5. Expression of recombinant P. fucata CaLP in the culture
supernatant and heat stability profile of CaLP detected by 15%
SDS ⁄ PAGE and stained by Coomassie Brilliant Blue R-250. Arrow
represents the induced proteins after addition of IPTG. M, protein

2+
-dependent
electrophoretic migration analysis was performed to
examine whether the oyster CaLP protein is indeed a
CaM-like protein. Figure 6 shows the electrophoretic
mobility of recombinant oyster CaLP and CaM in the
presence or absence of calcium. Both proteins exhibit
an apparent calcium-dependent mobility, indicating
that there is a close relationship between oyster CaM
and CaLP. In the presence of calcium, oyster CaLP
and CaM appeared as a single band with an apparent
molecular weight of approximately 18 kDa and
14 kDa, respectively, whereas in the absence of cal-
cium, the apparent molecular mass was 25 kDa and
17 kDa, respectively. The shift in the band upon cal-
cium addition could come not only from conforma-
tional changes within CaLP but also from additional
positive charges on the protein upon calcium binding.
The calcium binding ability of CaLP was further stud-
ied using
45
Ca overlay assay. As can be seen in Fig. 7,
CaLP and CaM both exhibit strong ability to bind cal-
cium ion in vitro, suggesting that CaLP may function
as a new Ca
2+
-sensor or play a role for arrest and
temporal storage of calcium ions as CaM.
CD spectroscopy and fluorescence assay
CD is an important method of determining the secon-

blue shift of the peak in 209 nm.
The calcium binding and conformational changes of
oyster CaLP and CaM were further investigated by
monitoring intrinsic phenylalanine and tyrosine fluor-
escence. Intrinsic phenylalanine fluorescence spectra
(with excitation at 250 nm and emission at 280 nm)
were shown in Fig. 9A. The phenylalanine fluorescence
emission of oyster CaM and CaLP upon calcium bind-
ing decreased  32% and  51%, respectively. This
Fig. 7. Identification of calcium binding activity of oyster CaM and
CaLP on nitrocellulose membrane after SDS electrophoresis. A, B
and C are an autoradiograph of the transferred nitrocellulose mem-
brane of oyster CaLP, CaM and BSA (as a negative control),
respectively. The molecular mass in kDa is indicated on the left of
the membrane.
Fig. 8. CD spectra of oyster CaLP and CaM in the presence of
Ca
2+
or EGTA. The spectra of oyster CaM and CaLP were recorded
in 100 m
M KCl, 20 mM Hepes buffer, pH 7.5 in the presence of
2m
M CaCl
2
or EGTA, and corrected using a blank buffer containing
100 m
M KCl, 20 mM Hepes buffer, pH 7.5. The concentration of
both proteins is 10 l
M.
A novel calmodulin-like protein from pearl oyster S. Li et al.

2+
-binding to the third
and fourth EF-hand domains in VanScyoc’s case. Due
to the Phe substitution, The Phe fluorescence reflects
only Ca
2+
-binding to the first, second and third
EF-hand domains in oyster CaM and CaLP, while it
reflected Ca
2+
-binding to the first and second EF-hand
domains in the case of VanScyoc et al.
CaLP and CaM chromatography of extracts from
oyster mantle and gills
Given the high degree of similarity in predicted amino-
acid sequence between oyster CaLP and CaM, it is
possible that these proteins share potential binding
sites or target proteins. Potential CaLP binding and
CaM binding proteins in the extracts of the mantle
and gill tissues, two organs directly involved in oyster
calcium metabolism, were compared by Ca
2+
-depend-
ent affinity chromatography. Figure 10 demonstrates
that more proteins, from the mantle or the gill, were
retained by oyster CaM affinity column than by CaLP
affinity column. Additionally, oyster CaM affinity col-
umn can react with more target proteins in gill than in
mantle, which is in agreement with the previous find-
ing that oyster CaM gene has a higher RNA expres-

min
)] emission fluorescence spec-
tra of oyster CaLP and CaM. The fluorescence of the phenylalanine
(A) and tyrosine residues (B) in oyster CaLP and CaM was meas-
ured using an excitation and emission wavelength pair
(250 ⁄ 280 nm and 277 ⁄ 320 nm, respectively). CaM and CaLP were
diluted in 100 m
M KCl, 20 mM Hepes buffer, pH 7.5 in the pres-
ence of 5 m
M CaCl
2
or EGTA, and the final concentration of both
proteins is 10 l
M.
S. Li et al. A novel calmodulin-like protein from pearl oyster
FEBS Journal 272 (2005) 4899–4910 ª 2005 FEBS 4905
changes. However, the oyster mRNA of CaLP and
CaM is expressed differently in major oyster tissues
and the oyster CaLP protein can interact with target
proteins different from those with oyster CaM, indica-
ting that the oyster CaLP protein may play a different
role in some aspects of oyster calcium metabolism and
calcium signaling pathways.
Experimental procedures
RNA preparation and cDNA synthesis
Adult specimens of P. fucata were purchased from Guofa
Pearl Farm, Beihai, Guangxi Province, China. Tissues
including mantle, gonad, muscle and gill were separated
and kept in RNAlater (Ambion, Austin, TX, USA). Total
RNA was extracted from the tissues by using the TRIzol

(Promega, Madison, WI, USA). The purified PCR products
were then subcloned into pGEM-T Easy vector (Promega)
and sequenced.
The full-length sequence of oyster CaLP cDNA was
obtained by using 5¢- and 3¢-rapid amplification of cDNA
ends technique (RACE). To obtain the 3¢-terminal of
CaLP cDNA ends, the initial round of PCR reaction was
conducted with a gene-specific forward primer LS11
(5¢-TCTCGTGGAAGAAATCGACA-3¢) designed based
on the sequence of fragment CaLP1 obtained above and a
reverse adaptor primer R2 (5¢-TCGAATTCGGATCC
GAGCTC-3¢), using the above first-strand cDNA got as
template. The first round PCR products then were used as
the templates for the second round of PCR reaction. The
fragment named CaLP2 was amplified with a nested for-
ward specific primer LS12 (5¢-CACAGACGGCAATGGA
GAGG-3¢) and adaptor primer R2. The 5¢-RACE was
performed using a SMART
TM
RACE amplification kit
(ClonTech) by two rounds of nested PCR reaction. The
first-strand cDNA was synthesized according to the manu-
facturer’s protocol, and two reverse gene specific primers
LSG1 (5¢-CTACCATCTCTTCTGCTTCTTCGTCGTCG
TCC-3¢) and LSG2 (5¢-CCAAGAACTCGTTGAAAT
CAACC-3¢) prepared based on the sequence of CaLP2 were
used in the nested PCR reactions. The first round of PCR
reaction was performed with a forward primer UPM (a
mixture of primers 5¢-CTAATACGACTCACTATAGGGC
AAGCAGTGGTAACAACGCAGAGT-3¢ and 5¢-CTAAT

of oyster CaLP cDNA obtained by RACE, a PCR
reaction was performed using a pair of specific primers
P3 (5¢-GGAAGAATACAGACACGGACAG-3¢) and P4
(5¢-ATAACAACAGTTTATACATCGCTTC-3¢) correspon-
ding to the 5¢-untranslated and 3¢-untranslated regions of
oyster CaLP mRNA, respectively. The PCR products were
cloned and sequenced as before.
DNA and protein sequence and analyses
All recombinant plasmids were sequenced using an automa-
ted DNA sequencer (Applied Biosystems 377). The nucleo-
tide sequence was blast against GenBank using BlastT
algorithm to identify its coding protein. Multiple align-
ments were created using the clustalx program [41]. The
protein domain was searched on the web site (www.ncbi.
nlm.nih.gov/Structure/cdd/wrpsb.cgi) and the secondary
structure prediction was carried out by the method of
McGuffin et al. [42]. The phosphorylation sites prediction
was carried out by netphos 2.0 Server [34].
Analysis of CaLP expression in oyster tissues
Analysis of CaLP mRNA expression in the different oyster
tissues was performed using RT-PCR analyses. Total RNA
was prepared from tissues including mantle, gonad, muscle
and gill as mentioned above. 1 lg aliquots of total RNA
from different tissues were transcribed into cDNA in 20 lL
reaction mixture using SuperScript II RNase H-Reverse
Transcriptase (Invitrogen). The generated cDNA was used
as template for PCR, which was performed with 1.5 mm
MgCl
2
, 200 lm dNTP, 1.5 U Taq DNA polymerase, and

(TaKaRa). The primers for amplification of oyster CaLP
cDNA were P5 (5¢-GGAT
CCATGGCGGAAGATCTCA
CA-3¢) containing an NcoI site (underlined), and P8
(5¢-CAG
CTCGAGTTTATTTTCTTGTTGCTGTTC-3¢)con-
taining an XhoI site (underlined). The PCR products were
purified with the Wizard PCR Prep DNA Purification Sys-
tem (Promega) and digested with NcoI ⁄ XhoI, then inserted
into a prokaryotic expression vector pET-28b (Novagen,
Madison, WI, USA). The recombinant plasmid named
pET-28b ⁄ CaLP with a His
6
-tag in the C-terminals was con-
firmed by sequencing. The prokaryotic expression vector
pET-28b ⁄ CaLP was fellow transformed into E. coil BL21
(DE3, Novagen). Protein expression was induced with
0.5 mm isopropylthiogalactopyranoside (IPTG) at 37 °C.
IPTG was added when the optical density at 600 nm of the
culture had reached 1.0. After 2.5 h of induction, bacterial
cells were harvested by centrifuging the culture at 8000 g
for 5 min.
The purification of recombinant oyster CaLP protein was
carried out on a precharged HisTrap HP chelating affinity
column (Amersham). The bacterial pellet was washed twice
with binding buffer (20 mm sodium phosphate with 0.5 m
NaCl and 20 mm imidazole, pH 7.5), then was suspended
in the binding buffer, and sonicated on ice. The lysate was
heated 10 min at 90 °C and immediately incubated on ice
for 5 min. The supernatant was collected by centrifuging

ether) N,N,N¢,N¢-tetra-acetic acid (EGTA) in the presence
of SDS. Calcium binding activity was examined by the
method of
45
Ca overlay analysis [45]. The purified recom-
binant oyster CaM and CaLP protein was transferred on
nitrocellulose membrane after electrophoresis, then labeled
with
45
Ca (Amersham) in a 10 mm imidazole ⁄ HCl buffer,
pH 7.5, for 10 min and then washed with Milli-Q water for
5 min. Autoradiography of the
45
Ca labeled proteins on
the nitrocellulose membrane was obtained by Strom 860
scanner (Amersham).
Circular dichroism spectropolarimetry and
fluorescence spectra
Circular dichroism (CD) spectroscopy was carried out at
25 °C with constant N
2
flushing using a CD instrument
(Jasco J-715, Cambs, UK) calibrated with d
10
-camphorsulf-
onic acid. The far-UV CD spectra of CaLP and CaM pro-
teins were measured from 190 to 250 nm in 100 mm KCl,
20 mm Hepes buffer, pH 7.5 in the presence of 2 mm CaCl
2
or EGTA, and corrected using a blank buffer containing

hul et al. [25] with
some modifications. Five grams of oyster mantle and gill
tissues were homogenized in 20 mL extraction buffer
[10 mm Hepes, 150 mm NaCl, 0.1% (w ⁄ v) Trition X-100,
5mm dithiothreitol, 0.5 mm phenylmethanesulfonyl fluor-
ide, 10 lgÆmL
)1
aprotinin, 10 lgÆmL
)1
leupetin, 10 lgÆmL
)1
pepstatin, pH 7.5] at 4 °C, respectively, and centrifuged at
22 000 g for 35 min at 4 °C. The supernatants were passed
over 0.45 lm filters, and adjusted to 2.5 mm CaCl
2
before
chromatographic separation. Then, the supernatants were
loaded onto the rCaLP and rCaM affinity columns pre-
equilibrated with the extraction buffer but added CaCl
2
to
a concentration of 2.5 mm at room temperature. Thereafter,
the columns were washed with 40 column volumes of
extraction buffer containing 2.5 mm CaCl
2
. Elution was
carried out using the extraction buffer containing 5 mm
EGTA. The eluted proteins were analyzed by 12.5%
SDS ⁄ PAGE and silver stained.
Acknowledgements

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