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 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 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 (reviewed in [3–8]). Noticeably, two CaM-regulated calcium metabolism components, Ca 2+ -ATPase and Ca 2+ 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 Ca 2+ -induced Ca 2+ 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 Ca 2+ -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 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 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 indi- 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. S. Li et al. A novel calmodulin-like protein from pearl oyster FEBS Journal 272 (2005) 4899–4910 ª 2005 FEBS 4901 Ca 2+ -mediated cellular process in oyster. Further- more, CaLP contains four putative Ca 2+ -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 Ca 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 and Ser29, are located in the first Ca 2+ 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 Ca 2+ 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 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 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. A novel calmodulin-like protein from pearl oyster S. Li et al. 4902 FEBS Journal 272 (2005) 4899–4910 ª 2005 FEBS cient regulation for the complex process of oyster calcium metabolism, including Ca 2+ 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 90 °C 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 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 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 °Cfor 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 Ca 2+ or EGTA. The sample buffer was added with 2.5 mM CaCl 2 or EGTA. M, protein molecular mass markers. The molecular mass in kDa is indicated on the left of the gel. S. Li et al. A novel calmodulin-like protein from pearl oyster FEBS Journal 272 (2005) 4899–4910 ª 2005 FEBS 4903 amino acids at the C-terminal end of CaLP, absent in oyster CaM (data not shown). Ca 2+ dependent electrophoretic shift and calcium binding properties of oyster CaLP As calcium-induced electrophoretic mobility is a useful method in characterizing CaM, Ca 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- 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 Ca 2+ or EGTA. When 2 mm CaCl 2 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 De 220 increases 112% and 79% in CaM and CaLP, respectively). These data suggest that Ca 2+ can induce reorganization and great changes in the com- position of the secondary structure elements within CaLP. However, when CaCl 2 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 Ca 2+ -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 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. 4904 FEBS Journal 272 (2005) 4899–4910 ª 2005 FEBS fluorescence quenching could be partially due to energy transfer to the nearby tyrosine residues [40]. The high Phe fluorescence of CaLP in the absence of Ca 2+ may due to the fact that CaLP is more unfolded in the Ca 2+ -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 Ca 2+ -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 Ca 2+ - binding to the fourth EF-hand domain in oyster CaM and CaLP, while it reflectes Ca 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- 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 oyster 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, Ca 2+ -dependent electrophoretic shift properties, and Ca 2+ -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 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 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 D 260 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¢-TCGAATTCGGATCCGAGCTCVT 17 -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 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 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. A novel calmodulin-like protein from pearl oyster S. Li et al. 4906 FEBS Journal 272 (2005) 4899–4910 ª 2005 FEBS following conditions: 5 cycles of 50 s at 94 °C; 50 s at 69 °C; 1 min at 72 °C; and 30 cycles of 50 s at 94 °C; 50 s at 61 °C; 1 min at 72 °C, followed by a final extension of 10 min at 72 °C. 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 MgCl 2 , 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 situ hybridization 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 58 °C. 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¢-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 the heated lysate at 22 000 g for 25 min at 4 °C. 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. S. Li et al. A novel calmodulin-like protein from pearl oyster FEBS Journal 272 (2005) 4899–4910 ª 2005 FEBS 4907 Protein yields were measured by BCA assay kit (Pierce, Rockford, IL, USA). Ca 2+ -dependent electrophoretic shift and 45 Ca overlay assay Ca 2+ -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 CaCl 2 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 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 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 25 ° C 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 CaCl 2 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 90 °C) 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, 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. 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(5¢-GGAAGAATACAGACACGGACAG-3¢) and P4 (5¢-ATAACAACAGTTTATACATCGCTTC-3¢) correspon- ding to the 5¢-untranslated and 3¢-untranslated regions of oyster CaLP. 5¢-CTAATACGACTCACTATAGGGC AAGCAGTGGTAACAACGCAGAGT-3¢ and 5¢-CTAAT ACGACTCACTATAGGGC-3¢) and a reverse specific gene primer LSG1. In the second round of PCR reaction,