In this study, a full-length cDNA encoding a novel calmodulin-like protein CaLP with a long C-terminal sequence was identi-fied from pearl oyster Pinctada fucata, expressed in Escherichia
Trang 1like protein from pearl oyster Pinctada fucata
Shuo Li1, Liping Xie1,2, Zhuojun Ma1and Rongqing Zhang1,2
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, secreaccumula-tion, 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
in shell crystal production in oyster [2]
CaM is a ubiquitous eukaryotic calcium sensor pro-tein that mediates many important signaling pathways
Keywords
Calmodulin; calmodulin-like protein; calcium;
oyster; Pinctada fucata
Correspondence
R Zhang, Department of Biological Sciences
and Biotechnology, Tsinghua University,
Beijing 100084, China
Fax: +86 10 62772899
Tel: +86 10 62772899
E-mail: rqzhang@mail.tsinghua.edu.cn
Note
The nucleotide sequence reported in this
paper has been submitted to GenBank with
the accession number AY663847
(Received 26 May 2005, revised 14 July
2005, accepted 3 August 2005)
doi:10.1111/j.1742-4658.2005.04899.x
Calcium metabolism in oysters is a very complicated and highly controlled physiological and biochemical process However, the regulation of calcium metabolism in oyster is poorly understood Our previous study showed that calmodulin (CaM) seemed to play a regulatory role in the process of oyster calcium metabolism In this study, a full-length cDNA encoding a novel calmodulin-like protein (CaLP) with a long C-terminal sequence was identi-fied from pearl oyster Pinctada fucata, expressed in Escherichia coli and characterized in vitro The oyster CaLP mRNA was expressed in all tissues tested, with the highest levels in the mantle that is a key organ involved in calcium secretion In situ hybridization analysis reveals that CaLP mRNA
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 Ca2+-binding domains, is highly heat-stable and has a potentially high affinity for cal-cium CaLP also displays typical Ca2+-dependent electrophoretic shift,
Ca2+-binding activity and significant Ca2+-induced conformational chan-ges Ca2+-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.
Trang 2regulating several crucial processes such as secretion,
cyclic nucleotide metabolism, cellular calcium
meta-bolism, muscle contraction, glycogen metameta-bolism, cell
proliferation and differentiation, and gene expression
(reviewed in [3–8]) Noticeably, two CaM-regulated
calcium metabolism components, Ca2+-ATPase and
Ca2+ channels, have been suggested to be involved in
the calcification process in some marine invertebrates
[9–14] In addition, recent lines of evidence also
demonstrate that CaM plays an important role in
regulating the function of mature osteoclasts and
osteo-clastogenesis, a bone biomineralization related process
[15] CaM-like protein as a multifunctional calcium
sensor belongs to a new member of CaM superfamily,
which has been found in bacteria [16], nematode [17],
Drosophila[18], plant [19], chicken [20,21], rat [22] and
human [23–25] In human beings, CaLP proteins are
involved in epithelial cell differentiation [25,26] Recent
studies showed that CaLP proteins were able to
regu-late the Ca2+-induced Ca2+ release in rat and human
cell lines [27] Sidhu and Guraya have reported that
CaLP might be involved in calcium transport in
buf-falo sperm [28]
Our previous study reveals that oyster CaM mRNA
is expressed highest in gill [29] that is a key organ
involved in calcium ion uptake, and is also strongly
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 Ca2+-dependent affinity
chro-matography Our observations described here may
provide important clues to understand the diversity
of calcium signaling and the complex mechanism of oyster calcium metabolism
Results and Discussion
Cloning of a full length cDNA encoding a calmodulin-like protein from P fucata
A 377 bp PCR product named CaLP1, which shows high similarity with oyster CaM, was amplified from the gill of P fucata using degenerate oligonucleotide primers derived from the conserved regions of CaM nucleotide sequence Based on this sequence, two spe-cific gene primers (LS11 and LS12) were synthesized and were used to amplify the 3¢ nucleotide sequence
of CaLP cDNA by two rounds of nest PCR reaction The5¢ sequence of oyster CaLP cDNA was also isola-ted by two rounds of nest PCR using the two specific gene primers, LSG1 and LSG2, derived from the sequence isolated by 3¢-RACE To confirm the sequence obtained by RACE, two specific primers (P3 and P4) corresponding to the 5¢-UTR and 3¢-UTR
of CaLP mRNA were designed, and RT-PCR was performed The PCR products were cloned and sequenced, which matched well the sequence expected from the results of 5¢- and 3¢-RACE As shown in Fig 1, the complete CaLP cDNA sequence including the poly(A) tail derived from the mRNA of pearl oys-ter is 757 bp It contains a 130 bases 5¢-untranslated sequence, an open reading frame consisting of 483 bp,
a TGA stop, a 146 bp 3¢-untranslated sequence, and a poly(A) tail of 18 nucleotides A putative polyadenyla-tion signals (AATAAA) is recognized at the nucleo-tide position 719, which is 15 nucleonucleo-tides 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,
Trang 33.7% 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
Fig 2 The secondary structure predictions
(A) and alignment of the amino-acid
sequence of oyster P fucata CaLP and CaM
(B) CaMPRED and CaLPPRED represent
the predicted secondary structures of CaM
and CaLP, respectively Green barrels
cate predicted a-helices, yellow arrows
indi-cate predicted b-strands and black lines
indicate predicted random coils The four
Ca 2+ -binding domains were boxed;
homologous and identical amino acids are
indicated by dots and stars, respectively X,
Y, Z, -Y, -X and -Z indicate the Ca 2+ -binding
ligand residues.
Trang 4Ca2+-mediated cellular process in oyster
Further-more, CaLP contains four putative Ca2+-binding
EF-hand domains (Fig 2B) predicted from the Protein
Families database from the Sanger Institute (http://
www.sanger.ac.uk/Software/Pfam) Among them, the
Ca2+-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 Ca2+
-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
and Ser29, are located in the first Ca2+binding domain
of oyster CaLP While, there are only two threonine
residues (Thr27 and Thr29) that can be potentially
phosphorylated by myosin light-chain kinase [35] in the
same domain of oyster CaM Therefore, these potential
phosphorylated residues may affect the interaction of
CaLP with target proteins In addition, Thr80 and
Ser82 located in the central a-helix of CaM, a region
important for its interaction with target
CaM-depend-ent proteins, are conserved in the oyster CaLP Besides,
Tyr139 located in the fourth Ca2+binding domain that
can be phosphorylated by insulin receptor, epidermal
growth factor and Src family kinases [36], is also
con-served in oyster CaLP
Gene expression analysis and in situ
hybridization
To study the expression of CaLP mRNA in oyster
tissues including mantle, gill, gonad and muscle,
RT-PCR analysis was performed RT-PCR reactions
were performed with RNA samples from mantle, gill, muscle and gonad A 486 bp RT-PCR product was obtained with specific primers (G1 and G2), using the total RNA of various tissues as template, while the negative control exhibited no product (data not shown) The PCR products were then inserted into pGEM-T Easy vector and were subjected to sequen-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 situhybridization 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, hybridizasecre-tion 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% CaCO3, products of calcium metabo-lism, and a few percent of matrix of biological macro-molecules This highly controlled process may depend
on presence of different regulatory proteins available
in different tissues, as well as in the same tissue The observations above also imply that CaLP may function
as a modulator-like CaM to provide a fine and
effi-Fig 3 Expression of CaLP mRNA in tissues of the pearl oyster Agarose gel analysis of RT-PCR products obtained with cDNA from the adult tissues of oyster P fucata muscle (1), gonad (2), mantle (3) and gill (4) The PCR product of CaLP (486 bp) is indicated by an arrow The28 S rRNA was used as a control of equal quantities of total RNA used in RT-PCR.
Trang 5cient regulation for the complex process of oyster
calcium metabolism, including Ca2+absorption,
trans-port, accumulation, secretion, deposition and other
important steps
Expression and purification of P fucata
recombinant CaLP
As a first step to understand the function of CaLP
protein in oyster calcium metabolism, we expressed
His-tagged fusion CaLP protein in E coli, and used
nickel metal affinity chromatography for single-step
purification of this fusion protein As shown in Fig 5,
the expressed recombinant CaLP protein demonstrated
high heat stability and reached approximately 21% of
the total bacterial soluble proteins detected by
SDS⁄ PAGE After heating the lysate for 10 min at
90C and purification by nickel metal affinity
chroma-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
Ca2+ 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 molecular mass markers; lane 1, uninduced whole-cell lysate; lane
2, whole-cell lysate induced by 0.5 m M IPTG for 2.5 h; lane 3, un-induced whole-cell lysate heated at 90 C for 10 min; line 4, whole-cell lysate induced by IPTG after heat treatment at 90 C for
10 min; line 5, purified recombinant CaLP by nickel affinity chroma-tography column The molecular mass in kDa is shown on the left
of the gel.
Fig 6 Ca 2+ -dependent electrophoretic migration of the purified recombinant P fucata CaM and CaLP Purified recombinant oyster CaLP and CaM was run on a 15% SDS ⁄ PAGE in the presence of
Ca2+or EGTA The sample buffer was added with 2.5 m M CaCl 2 or EGTA M, protein molecular mass markers The molecular mass in kDa is indicated on the left of the gel.
Trang 6amino acids at the C-terminal end of CaLP, absent in
oyster CaM (data not shown)
Ca2+dependent electrophoretic shift and calcium
binding properties of oyster CaLP
As calcium-induced electrophoretic mobility is a useful
method in characterizing CaM, Ca2+-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 addiconforma-tional
positive charges on the protein upon calcium binding
The calcium binding ability of CaLP was further
stud-ied using45Ca 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 Ca2+-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-dary structure feature of a protein in solution To investigate the secondary structures of oyster CaLP,
CD spectra in the far-UV region (190–250 nm) were measured Figure 8 showed a similar overall change in the secondary structures of oyster CaM and CaLP in the presence of Ca2+ or EGTA When 2 mm CaCl2 was added, double negative peaks appeared at 209 nm and 220 nm in both proteins, associated with an increase in a-helical content upon calcium binding (the value of De220 increases 112% and 79% in CaM and CaLP, respectively) These data suggest that Ca2+can induce reorganization and great changes in the com-position of the secondary structure elements within CaLP However, when CaCl2 was replaced with EGTA, both proteins seemed to undergo partial unfolding, and the a-helical contents of both proteins are decreased Figure 8 also indicates that the oyster CaLP protein is more unfolded in the Ca2+-free state
in comparison to oyster CaM as indicated by a slight 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
Ca2+or EGTA The spectra of oyster CaM and CaLP were recorded
in 100 m M KCl, 20 m M Hepes buffer, pH 7.5 in the presence of
2 m M CaCl2or EGTA, and corrected using a blank buffer containing
100 m M KCl, 20 m M Hepes buffer, pH 7.5 The concentration of both proteins is 10 l M
Trang 7fluorescence quenching could be partially due to
energy transfer to the nearby tyrosine residues [40]
The high Phe fluorescence of CaLP in the absence of
Ca2+may due to the fact that CaLP is more unfolded
in the Ca2+-free state Figure 9B demonstrated that,
tyrosine fluorescence emission (with excitation at
277 nm and emission at 320 nm) for oyster CaM and
CaLP upon calcium binding increased approximately
1.6-fold and 0.28-fold, respectively The different
chan-ges of intrinsic phenylalanine and tyrosine fluorescence
in CaLP may partially due to the extra C-terminal
end segment of CaLP However, we can not measure
Ca2+-binding to only the N- or only the C-terminal
domain of oyster CaM and CaLP by monitoring intrinsic phenylalanine and tyrosine fluorescence by the method described by VanScyoc et al [40] because the Tyr residue in the third EF-hand domain of rat CaM was replaced by Phe in the same place of oyster CaM and CaLP The Tyr fluorescence reflects only Ca2+ -binding to the fourth EF-hand domain in oyster CaM and CaLP, while it reflectes Ca2+-binding to the third and fourth EF-hand domains in VanScyoc’s case Due
to the Phe substitution, The Phe fluorescence reflects only Ca2+-binding to the first, second and third EF-hand domains in oyster CaM and CaLP, while it reflected Ca2+-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 Ca2+ -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-sion level in the gill [29] In contrast, oyster CaLP can bind more proteins in mantle than in gill, suggesting CaLP protein may play an active role in calcium meta-bolism related processes in the mantle Few differences were noted in the overall patterns of proteins retained
by CaLP column compared with the proteins eluted from CaM column in the mantle Next we will examine whether oyster CaLP is able to active CaM-dependent enzymes, carry out two-dimensional electro-phoresis and use proteomic strategy to identify the different affinity purified proteins interacting with oys-ter CaM and CaLP protein in oysoys-ter mantle and gill, respectively, and this will help us to understand the details of oyster CaLP regulated calcium metabolism processes
In summary, we have identified a full-length cDNA encoding a novel CaM-like protein from P fucata The oyster CaLP shares several characteristics with oyster CaM, which include high heat stability, strong calcium binding capacity, Ca2+-dependent electrophoretic shift properties, and Ca2+-induced conformational
Fig 9 Normalized [(f-f min ) ⁄ (f max –f 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 m M Hepes buffer, pH 7.5 in the
pres-ence of 5 m M CaCl2or EGTA, and the final concentration of both
proteins is 10 l M
Trang 8changes 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
regent (Invitrogen, Carlsbad, CA, USA) The integrity of
RNA was determined by fractionation on 1.2%
formal-dehyde-denatured agarose gel and staining with ethidium
bromide The quantity of RNA was determined by
measur-ing D260 with an Utrospec 3000 UV⁄ Visible
spectrophoto-meter (Amersham, Piscataway, NJ, USA) Total RNA (5 lg) extracted from gill tissue of P fucata was used to synthesis single-strand cDNA using SuperScript II RNase
H–Reverse Transcriptase (Invitrogen) and a oligo-dT adap-tor primer (5¢-TCGAATTCGGATCCGAGCTCVT17-3¢) according to the manufacturer’s instructions
Cloning of pearl oyster CaLP cDNA The cDNA fragment CaLP1 of the pearl oyster CaLP gene from gill tissue was amplified by RT-PCR using Ex Taq DNA polymerase (TaKaRa, Kyoto, Japan) The degenerate oligonucleotide primers used for amplification were designed based on the conserved regions of CaM nucleotide sequence There are the forward primer F1, 5¢-ATYGCW GARTTYAARGARGC-3¢ (corresponding to the sequence from nucleotides +28 to +47), and the reverse primer R1, 5¢-CCRTCWCCATCAATRTCHGC-3¢ (corresponding to the sequence from nucleotides +385 to +404) PCR prod-ucts of the expected size (377 bp) were excised and purified with the Wizard PCR Prep DNA Purification System (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 SMARTTM 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 ACGACTCACTATAGGGC-3¢) and a reverse specific gene primer LSG1 In the second round of PCR reaction, the first round of PCR products then were used as the tem-plates, and amplified using the set of primers NUP (nested universal primer, 5¢-AAGCAGTGGTAACAACGCAGA GT-3¢) and LSG2 in a thermocycler (Biometra) under the
Fig 10 Ca 2+ -dependent affinity chromatography of extracts from
the oyster tissues of mantle and gill by CaM and CaLP affinity
col-umns The extracts from oyster tissues of mantle and gill were
loaded on CaM and CaLP affinity columns After washing with
extraction buffer containing 2.5 m M CaCl 2 , bound proteins were
eluted with extraction buffer containing 5 m M EGTA The eluted
proteins were separated by 12.5% SDS ⁄ PAGE and silver stained.
M, protein molecular mass markers; lane 1, mantle proteins eluted
from CaLP affinity column; line 2, mantle proteins eluted from CaM
affinity column; line 3, gill proteins eluted from CaLP affinity
col-umn; line 4, gill proteins eluted from CaM affinity column The
molecular mass in kDa is shown on the left of the gel.
Trang 9following conditions: 5 cycles of 50 s at 94C; 50 s at
69C; 1 min at 72 C; and 30 cycles of 50 s at 94 C; 50 s
at 61C; 1 min at 72 C, followed by a final extension
of 10 min at 72C To confirm the nucleotide sequence
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
MgCl2, 200 lm dNTP, 1.5 U Taq DNA polymerase, and
20 pm of each primer G1 (5¢-ATGGCGGAAGATC
TCACAGAAGAACAAA-3¢) and G2 (5¢-TCATTTATTTT
CTTGTTGCTGTTC-3¢) Preliminary experiments showed
that a total RNA concentration of 1 lg and 25 cycles were
well within the linear of amplification After amplification,
the PCR products were subcloned into pGEM-T Easy
vector and confirmed by sequence Equal volumes of the
PCR products were applied to 2% agarose gel and stained
with ethidium bromide To avoid the samples across
con-tamination, negative controls reactions for RT-PCR were
performed in absence of cDNA template
In situ hybridization
In situhybridization of oyster CaLP mRNA was performed
on frozen section (10 lm thick) The mantle was sectioned
from the adult P fucata and immediately fixed in 100 mm
phosphate buffer (pH 7.4) containing 4%
paraformalde-hyde overnight Digoxigenin-labeled RNA probes were gen-erated from the cDNA clone encoding oyster CaLP in plasmid using a DIG RNA Labeling kit (Roche), with T7 and SP6 RNA polymerase for the sense and antisense probe, respectively RNA in situ hybridization was carried out as described previously with some modifications [43]
To avoid false positive signals, the hybridization tempera-ture was increased to 58C
Expression and purification of the oyster CaM and CaLP in E coli
The recombinant oyster CaM was obtained as described by
Li et al [29] For expression and purification the oyster CaLP protein in E coli, the coding region of oyster CaLP cDNA was amplified by PCR with Pfu DNA polymerase (TaKaRa) The primers for amplification of oyster CaLP cDNA were P5 (5¢-GGATCCATGGCGGAAGATCTCA CA-3¢) containing an NcoI site (underlined), and P8 (5¢-CAGCTCGAGTTTATTTTCTTGTTGCTGTTC-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 His6-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 37C 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 90C and immediately incubated on ice for 5 min The supernatant was collected by centrifuging the heated lysate at 22 000 g for 25 min at 4C The super-natant was then loaded at room temperature into a HisTrap HP affinity column (Amersham) previously equili-brated with the binding buffer The column was washed with binding buffer until absorption at 280 nm reached baseline Finally, CaLP protein was eluted with elution buf-fer (20 mm sodium phosphate with 0.5 m NaCl and 50 mm imidazole, pH 7.5) Fractions with CaLP protein were ana-lyzed on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS⁄ PAGE), and stained with Coomassie Brilliant Blue R-250 CaLP-containing fractions were col-lected and dialyzed against Milli-Q water and frozen dried
Trang 10Protein yields were measured by BCA assay kit (Pierce,
Rockford, IL, USA)
Ca2+-dependent electrophoretic shift and45Ca
overlay assay
Ca2+-dependent electrophoretic shift assay was carried out
according to the method of Burgess et al [44] with a slight
modification Only the sample buffer and gels were added
with 2.5 mm CaCl2 or ethylene glycol-bis-(b-amino-ethyl
ether) N,N,N¢,N¢-tetra-acetic acid (EGTA) in the presence
of SDS Calcium binding activity was examined by the
method of 45Ca overlay analysis [45] The purified
recom-binant oyster CaM and CaLP protein was transferred on
nitrocellulose membrane after electrophoresis, then labeled
with 45Ca (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 45Ca 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
25C with constant N2 flushing using a CD instrument
(Jasco J-715, Cambs, UK) calibrated with d10
-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 CaCl2
or EGTA, and corrected using a blank buffer containing
100 mm KCl, 20 mm Hepes buffer, pH 7.5 All
measure-ments were performed 10 min after sample preparation
with the following instrument settings: response time, 0.5 s;
scan speed, 200 nmÆmin)1; sensitivity, 100 millidegrees;
1 mm spectral band width, and an average of four scans
The fluorescence emission spectra were collected at 25C
using a Hitachi F-2500 (xxxx, xxxx) spectrofluorimeter
according to the method described by VanScyoc et al [40]
with a slight modification All samples were diluted in
100 mm KCl, 20 mm Hepes buffer, pH 7.5 in the presence
of 5 mm CaCl2 or EGTA, and the final concentration of
each protein is 10 lm The fluorescence of the
phenylala-nine and tyrosine residues in oyster CaLP and CaM was
measured using an excitation and emission wavelength pairs
(250⁄ 280 nm and 277 ⁄ 320 nm, respectively)
Preparation of recombinant oyster CaLP and CaM
affinity chromatography columns and affinity
chromatography
Recombinant oyster CaLP (rCaLP; 5 mg) (sample
prepar-ation as mentioned above but not heated at 90C) and
recombinant CaM (rCaM) were coupled to 0.6 g of
CNBr-activated Sepharose 4B (Amersham Biosciences), according
to the manufacturer’s instructions The coupling efficiency was about 1 mg proteins per mL gel Affinity chromatogra-phy was performed as described by Me´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,
5 mm dithiothreitol, 0.5 mm phenylmethanesulfonyl fluor-ide, 10 lgÆmL)1aprotinin, 10 lgÆmL)1leupetin, 10 lgÆmL)1 pepstatin, pH 7.5] at 4C, respectively, and centrifuged at
22 000 g for 35 min at 4C The supernatants were passed over 0.45 lm filters, and adjusted to 2.5 mm CaCl2 before chromatographic separation Then, the supernatants were loaded onto the rCaLP and rCaM affinity columns pre-equilibrated with the extraction buffer but added CaCl2 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 CaCl2 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
We thank Dr Shengcai Lin (Department of Biochemis-try, Hong Kong University of Science and Technol-ogy) for critical reading of this paper This work was financially supported by the National High Technology Research and Development Program of China (2003AA603430) and the National Science Foundation
of China (30371092)
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