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A novel extracellular EF-hand protein involved in the shell formation of pearl oyster

A novel extracellular EF-hand protein involved in the shell formation of pearl oyster
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  A novel extracellular EF-hand protein involved in the shellformation of pearl oyster  Jing Huang  a  , Cen Zhang  a  , Zhuojun Ma  a  , Liping Xie  a,b, ⁎ , Rongqing Zhang  a,b, ⁎ a   Institute of Marine Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, 100084, China  b  Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing, 100084, China Received 5 December 2006; received in revised form 9 March 2007; accepted 12 March 2007Available online 20 March 2007 Abstract Mollusk shell formation is a complicated and highly controlled calcium metabolism process. Previous studies revealed that several EF-handcalcium-binding proteins actively participate in the regulation of shell mineralization. In this study, we cloned a full-length cDNA encoding anovel extracellular EF-hand calcium-binding protein (named EFCBP) from the pearl oyster,  Pinctada fucata , according to the EF-hand motifs of calmodulin. Although it shares high similarity with the calmodulin family in its EF-hand signatures, EFCBP just has two EF-hand motifs and belongs to a new separate group from the other EF-hand proteins according to a phylogenetic analysis. EFCBP is specifically expressed in shellmineralization-related tissues, viz. the mantle, the gill, and the hemocytes. Moreover, its expression responds quickly only to the shell damage, but not to the damage of other tissues and the infection of the lipopolysaccharides from  Escherichia coli . These results suggest that EFCBP might bean important regulator of shell formation. This finding may help better understand the functions of EF-hand proteins on the regulation of mollusk shell formation.© 2007 Elsevier B.V. All rights reserved.  Keywords:  EF-hand; Biomineralization; Calcium metabolism; Regulator; EFCBP;  Pinctada fucata 1. Introduction The mollusk shell, especially the nacre, is a wonderfulmasterpiece of nature. Although calcium carbonate accounts for more than 95% of the nacre weight, unlike inorganic calciumcarbonate, it is arranged in a highly functional and strictlycontrolled way under the instructions of many organic macro-molecules secreted from the mantle tissues or elsewhere [1 – 3].These characteristics endow nacre with excellent mechanical, physical, and chemical properties [3 – 5], which have attractedthe interest of many biologists, as well as material scientists,nanotechnologists and chemists in recent decades.As for the biologists, unraveling the molecular mechanismunder the macrostructure of the shell and the nacre is not enough. It should not be neglected that from the viewpoint of evolution and physiology, the mollusk shell may be seen as a product of calcium metabolism that releases the stress of calcium accumulated in the body of the marine mollusk and alsoserves as a reservoir of calcium ions. Shell formation involvesthe sequential processes of the calcium absorption, accumula-tion, transportation, and incorporation. And more importantlyincludes the complicated regulation networks which coordinatethe cellular activities with the environmental changes [2].However, these processes, especially the regulation mechanisminvolved in, have not been well investigated. On the other hand,another biominerallization system in vertebrate, namely the bone formation system, has been extensively studied. Its reg-ulation mechanism may give us some clues to our studies onshell formation. In the bone formation process, calcium-binding proteins play a pivotal role in constructing the extracellular  Biochimica et Biophysica Acta 1770 (2007) 1037 –  Abbreviations:  CaM, calmodulin; CaLP, calmodulin-like protein; PFMG1,  Pinctada fucata  mantle gene 1; PFMG6,  Pinctada fucata  mantle gene 6;RACE, rapid amplification of cDNA ends; GAPDH, Glyceraldehyde 3- phosphate dehydrogenase; DEPC, diethypyrocarbonate; LPS, lipopolysacchar-ides; BLAST, basic local alignment search tool ⁎  Corresponding authors. Institute of Marine Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, 100084,China. Tel.: +86 10 62772899; fax: +86 10 62772899.  E-mail addresses: (L. Xie), (R. Zhang).0304-4165/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.bbagen.2007.03.006  matrix for calcium sedimentation and modulating the physio-logical activities of osteoblasts and osteoclasts. The former function is carried out by a group of acidic phosphoproteinswhich are rich in acid amino acids and phosphorylationmodification [6,7]; and the latter is usually performed by anumber of protein families containing the EF-hand domains,such as osteonectin, calcyclin, S100A4, and calbindin [8 – 12].Acidic matrix proteins isolated from the shell act like the phosphoproteins and induce the formation of shell calciumcarbonate crystals [13,14], while as to the functions of EF-hand protein families on shell mineralization, only  Pinctada fucata mantle gene 1 (PFMG1) [15], calmodulin (CaM) [16] and calmodulin-like protein (CaLP) [17,18] cloned from the oyster   P. fucata  and calconectin [19] cloned from the oyster   Pinc-tada margaritifera  have been studied. As the EF-hand proteinsregulate the activities of bone formation-related cells, we postulate that it might function similarly in the mollusk mantlecells. Therefore, we tried to find more EF-hand proteins in themantle cells of the pearl oyster   P. fucata  and reveal their functions on shell formation.In the previous work of our group, we cloned  P. fucata  CaMand CaLP, and studied their functions on calcium metabolismand biomineralization [16 – 18]. Based on these studies, wefirstly tried to find some other calmodulin-like proteins. Wedesigned degenerate primers according to the characteristics of calmodulin EF-hand motifs, viz. the first and the third aminoacids are aspartic acid, the fourth and the sixth amino acid areglycine, the second amino acid is alanine or lysine, and the fifthamino acid is aspartic acid or asparagine. Via rapid amplifica-tion of cDNA ends (RACE), we acquired a full-length cDNAsequence encoding a new kind of extracellular EF-handcalcium-binding protein that was named EFCBP. Using ahomology search with the Basic Local Alignment Search Tool(BLAST) program, we found three other proteins with highsimilarity with EFCBP. A phylogenetic analysis of their EF-hand signatures indicated that they belong to a separate groupfrom the other EF-hand proteins. As described below, EFCBP isexpressed in the specific mantle areas, the gill, and thehemolymph. These tissues and cells have been reported previously to take active part in the shell formation. Moreover,a notching in the shell margin could increase the expression of EFCBP rapidly, while either the damage of other tissues or infection with lipopolysaccharides (LPS) from  Escherichia coli did not alter its expression. Its specific response to shell damagesuggested that EFCBP might be an important regulator of shellformation. 2. Materials and methods 2.1. RNA preparation and RACE  Live individuals of adult oyster   P. fucata  were obtained from the GuofaPearl Farm in Beihai, Guangxi Province, China. Total RNA was extractedseparately from the mantle, viscus, gill, hemocytes, and adductor muscle tissueswith TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to themanufacturer's instructions. The integrity of the RNA was determined byelectrophoresis on a 1.2% formaldehyde-denatured agarose gel stained withethidium bromide. The quantity of RNA was determined by measuring OD 260 with an Ultrospec 3000 UV/Visible Spectrophotometer (Amersham, Piscataway, NJ, USA).5 ′ -RACE and 3 ′ -RACE was performed by using the SMART RACE cDNAAmplification Kit (Clontech, Palo Alto, CA, USA) and Advantage 2 cDNAPolymerase Mix (Clontech) following the manufacturer's instructions. Single-stranded cDNA for all RACE reactions was prepared from the mantle totalRNA, usingPowerScript  ™ (Clontech).Two degenerate primers, EFP5(5 ′ -GAYGCN GAY GGN RAY GG-3 ′ ) and EFP5 ′  (5 ′ -GAYAAR GAY GGN RAY GG-3 ′ ) were designed for 3 ′ -RACE. The total volume of a 3 ′ -RACE reaction was20  μ l. The reaction system contained 1  μ l cDNA template, 2  μ l 10× BDAdvantage 2 PCR Buffer, 2 μ l 10× UPM (Universal Primer A Mix), 1 μ l 50 μ MEFP5 or EFP5 ′ , 0.4  μ l 10 mM dNTP Mix, 0.4  μ l 50× BD Advantage 2Polymerase Mix, and 13.2 μ l PCR-Grade Water. Touchdown PCR program wasadopted, namely within the first 10 cycles, the annealing temperature decreased by 1 °C every PCR cycle till to the calculated Tm values of the degenerate primer, and then the annealing temperature was fixed at (Tm-2) °C for another 25 cycles. The amplified product was cloned into pGEM-T Easy Vector (Promega) and sequenced. Then according to the cDNA sequence acquiredabove, a gene-specific primer EFP3 (5 ′ -TTC TGA CAG GCG TCT ATC AACG-3 ′ ) was synthesized and used for the 5 ′ -RACE. The PCR conditions weresimilar with 3 ′ -RACE except that the concentration of EFP3 was 20  μ M. ThePCR product was then cloned into pGEM-T Easy Vector (Promega) andsequenced.To confirm the accuracy of the cDNA sequence, the full-length cDNA wasamplified with the primer pair of EFgsp5 (5 ′ -CAA AGG AAA AGC TCA ATTAGG AG-3 ′ ) and EFgsp3 (5 ′ -ATA CAG AGA TAA GAT CTG CTT CC-3 ′ ),using the mantle cDNA library of   P. fucata  as a template. The PCR product wasthen cloned into pGEM-T Easy Vector (Promega) and sequenced. 2.2. Analysis of the deduced protein sequence The presence and location of the signal peptide was analyzed with theSignalP v3.0 server (CBS prediction servers; ). Proteindomains were determined using the PROSITE database (  prosite/ ). The secondary structure prediction was carried out according to themethod of McGuffin et al., using the PSIPRED Protein Structure PredictionServer ( Protein sequence simi-larity searches were performed with the BLAST program in GenBank (http:// ). Protein multiple alignments and phylogenetic analysiswere performed by the ClustalX program. 2.3. Gene expression analysis by RT-PCR Total RNA was extracted from different tissues as described above. Equalquantities (2  μ g) of the total RNA were reverse-transcribed into cDNA withSuperscript III RNase H − Reverse Transcriptase and oligo(dT) primers in 20  μ lreaction mixtures (Invitrogen, CA, USA). GAPDH was adopted as the positivecontrol for cDNA preparation and was amplified with the primer pair of GAPDH5 (5 ′ -GCC GAG TAT GTG GTA GAATC-3 ′ ) and GAPDH3 (5 ′ -CACTGT TTT CTG GGTAGC TG-3 ′ ). EFCBP was amplified with the primer pair of EFgsp5 and EFgsp3. Negative control was the RT-PCR in the absence of anycDNA template.First, GAPDH was amplified,and according to the quantities of the PCR products of GAPDH, the amounts of the cDNA templates wereadjusted to be the same. Then EFCBP was amplified. The PCR process of GAPDH and EFCBP consisted of 95 °C for 5 min, 28 cycles of 95 °C for 30 s,50 °C for 30 s, and 72 °C for 45 s, and finally 72 °C for 10 min. The PCR  products were subcloned as described above and verified by sequencing. Theexperiments were repeated using different individuals and the results wereconsistent. 2.4. In situ hybridization The mantle was separated from the adult   P. fucata  and immediately fixed in4% paraformaldehyde and 0.1% DEPC solution overnight.  In situ  hybridizationof EFCBP mRNA was carried out on frozen sections of the mantle (10  μ mthick). Digoxigenin-labeled RNA probes were generated from the pGEM-TEasy Vector with the EFCBP insertion in the multiple cloning sites, using a DIG1038  J. Huang et al. / Biochimica et Biophysica Acta 1770 (2007) 1037   –  1044  RNA Labeling Kit (Roche). The sense probes were synthesized by the T7 RNA polymerase and the antisense ones were by the SP6 RNA polymerase. In situhybridization was performed as described previously with some modification[20]. To avoid false positive signals, the hybridization temperature wasincreased to 50 °C. 2.5. Shell notching experiments and gene expression analysis of   EFCBP  Shell notching experiments were performed according to the method of Mount et al. [21] with some modification. Av-shaped notch was cut on the shellmargin close to the adductor muscle of the oysters, and then they were dividedinto six groups randomly, each of which contained five individuals. The sixgroups were returned to seawater tanks for 3, 6, 9, 12, 24, and 48 h respectively,and then were sacrificed. About 1 cm 2 area of the mantle tissue around the cut was separated. The mantles of the same group were placed together andimmediately in RNA later   (Ambion, Austin, TX, USA), and then stored in theliquid nitrogen. Meanwhile, the mantles of five individuals without shellnotching were separated as described above to act as a control. RNA preparationand RT-PCR were performed as described above except that the PCR cyclenumber of EFCBP was reduced to 20 to ensure that the reactions were within thelinear amplification. The shell notching and succedent RT-PCR experimentshave been repeated, and the results were consistent. 2.6. Injection of LPS and gene expression analysis of EFCBP  Five  μ g LPS solution was injected into the adductor muscle of eachindividual, and then they were divided into six groups, each of which containedten individuals. The six groups were returned to seawater tanks for 3, 6, 9, 12,24, and 48 h respectively. A group of ten individuals without LPS injection wasadopted as a control. The hemolymph was collected with a syringe from theadductor muscle, and the hemolymph of the same group was mixed together.Immediately after extraction, the hemolymph was centrifuged at 1000×  g   for 15 min at 4 °C. The hemocytes were resuspended in TRIzol reagent, and storedat   − 70 °C. RNA preparation and RT-PCR were performed as described above,and the PCR cycle number of EFCBP was 28. 2.7. Tissue damage experiments and gene expression analysis of   EFCBP  A piece of mantle of each individual was cut in the area near the adductor muscle, and a piece of gill of each individual was cut in the middle. Then theoysters were grouped and sacrificed as described in the shell notchingexperiment. About 1 cm 2 area of the mantle and gill tissue around the cut wasseparated and stored. RNA preparation and RT-PCR were performed asdescribed above and the PCR cycle number of EFCBP was 28. 3. Results 3.1. Cloning of the full-length cDNA sequence The 3 ′ -RACE reaction with the EFP5 primer amplified aspecificPCRproductabout167bpinlength,whilethe3 ′ -RACEreaction with the EFP5 ′  primer gave no PCR product. The167-bp PCR product encoded a potential EF-hand motif. Basedon this sequence, the rest of the full-length cDNA sequence wasobtained via the 5 ′ -RACE procedure. To confirm the accuracyof the cDNA sequence, another amplification of the full-lengthcDNA was performed with the primer pair of EFgsp5 andEFgsp3 (Fig. 1), using the mantle cDNA library of   P. fucata  as atemplate. The result was consistent with that of the RACE. Asshown in Fig. 1, the full-length cDNA sequence without the poly(A) tail was 525 bp in length, containing an open readingframe (ORF) encoding 129 amino acids. The putative poly-adenylation signal (AATAAA) began from the 53rd nucleotidedownstream of the stop codon (TGA). The cDNA sequencehas been submitted to GenBank with the accession number DQ494416. 3.2. Analysis of the deduced amino acid sequence The first 22 amino acids of the deduced protein were predicted to be a signal peptide. The mature protein without thesignal peptide was composed of 107 amino acids with anestimated molecular weight of 12.5 kDa and an isoelectric point of 9.78. Analysis of its amino acid composition indicated theexistence of two cysteine residues that might form anintramolecular disulphide bond. A protein domain search of  Fig. 1. The cDNA sequence and the deduced amino acid sequence of EFCBP from the pearl oyster,  Pinctada fucata . The putative signal peptide is underlined. The lowcomplexity region rich in arginine is boxed. The two EF-hand calcium-binding motifs are shadowed. The two cysteine residues that might form a disulphide bond arecircled. The primers used for cloning and RT-PCR are indicated with arrows.1039  J. Huang et al. / Biochimica et Biophysica Acta 1770 (2007) 1037   –  1044  the PROSITE database revealed the existence of two EF-handcalcium-binding motifs located from the 74th to the 85th aminoacid and from the 104th to the 115th amino acid. Moreover, alow complexity region rich in arginine was located from the24th to the 37th amino acid, and lysine was also abundant at the N-terminus (Fig. 1). We named the protein as EFCBP (EF-handcalcium-binding protein).The predicted secondary structures of EFCBP contained fivecoil regions (29.9%), four   α -helix regions (70.1%), and no  β -sheet. As shown in Fig. 2, each EF-hand motif was surrounded by two  α -helixes, which was in accord with the characteristichelix – loop – helix conformation of the canonical EF-handdomain [22].A homology search with the BLAST program against allthe protein sequences in GenBank indicated that EFCBPhad the highest similarity with the calconectin (DQ352042.1)of   P. margaritifera , and was also similar to the PFMG1(DQ104255) and PFMG6 (DQ104260.1) of   P. fucata .Although calconectin shares the highest identity, it lacks thesignal peptide and seems to be intracellular. As for thePFMG1 and PFMG6, they have a predicted signal peptide at the N terminus, and their two EF-hand motifs are located at the C terminus, therefore, from the viewpoint of proteinstructure and subcellular localization, EFCBP might be moresimilar with PFMG1 and PFMG6. Besides, as shown in Fig.3, the EF-hand signatures of EFCBP shared high similaritywith those of calmodulin and calmodulin-like protein family,calbindin, SPARC, and some other calcium-binding proteins.However, a phylogenetic analysis of their EF-hand signaturesrevealed that EFCBP did not belong to any of the abovefamilies. EFCBP, calconectin, PFMG1, and PFMG6 might constitute another new EF-hand protein family with two EF-hand motifs (Fig. 4). Fig. 2. Secondary structure analysis of EFCBP. PRED represents the predictedsecondary structure of EFCBP. The grey barrels indicate the predicted α -helices,the black lines indicate the predicted random coils, and the two EF-hand motifsare boxed.Fig. 3. Protein sequence comparison among the EF-hand signatures of EFCBP, calconectin, PFMG1, PFMG6, calmodulin and calmodulin-like protein family,calbindin, and SPARC. Numbers indicate the positions of the amino acid residues in each sequence. Gaps ( − ) are used to optimize the alignment. The two EF-handcalcium-binding motifs are boxed. Homologous and conservative amino acids are indicated with dots. pf_efcbp:  Pinctada fucata  EFCBP (DQ494416); pm_cn:  Pinctada margaritifera  Calconectin (DQ352042.1); pf_mg1:  Pinctada fucata  PFMG1 (DQ104255); pf_mg6:  Pinctada fucata  PFMG6 (DQ104260.1); ac_cam:  Aplysia californica  Calmodulin (P62145); pf_cam:  Pinctada fucata  Calmodulin (AAQ20043); hs_cam:  Homo sapiens  Calmodulin (CAA36839); ps_cam:  Patino- pecten sp.  Calmodulin (P02595); pf_calp:  Pinctada fucata  Calmodulin-like protein (AAV73912); sc_cam:  Saccharomyces cerevisiae  Calmodulin (P06787);mm_calp5:  Macaca mulatto  Calmodulin-like 5 (XP_001104766); pt_calsp:  Pan troglodytes  Calmodulin-like skin protein (XP_001144681); cb_hp:  Caenorhabditisbriggsae  Hypothetical protein CBG14543 (CAE68649); ce_F43C9.2:  Caenorhabditis elegans  F43C9.2 (NP_508818); bg_cbp1:  Biomphalaria glabrata  Calcium binding protein 1 (AAV91525); bg_cbp2:  Biomphalaria glabrata  Calcium binding protein 2 (AAV91522); hs_calb:  Homo sapiens  Calbindin 1 (NP_004920); pp_calb:  Pongo pygmaeus  Calbindin (Q5R4V1); mm_calb:  Mus musculus  Calbindin-28K (NP_033918); rn_calb:  Rattus norvegicus  Calbindin-d28k (AAA40852);tse_calb:  Trachemys scripta elegans  Calbindin-D28K (AAO49508); af_sparc:  Artemia franciscana  SPARC (BAB20042); dm_sparc:  Drosophila melanogaster   BM-40-SPARC CG6378-PA (NP_651509); ce_sparc:  Caenorhabditis elegans  Osteonectin (sparc) related protein 1(AAB88325); hs_sparc:  Homo sapiens  SPARC(CAG33080); mm_sparc:  Mus musculus  SPARC (CAJ18514).1040  J. Huang et al. / Biochimica et Biophysica Acta 1770 (2007) 1037   –  1044  3.3. Gene expression analysis by RT-PCR and in situhybridization As calconectin, PFMG1 and PFMG6 are mantle-specific proteins, and have been proved to regulate the biomineralization process; we speculated that EFCBP might play a similar role.Therefore, we first analyzed the mRNA expression specificityof EFCBP in different tissues. RT-PCR was performed with thegene-specific primers of EFCBP (EFgsp5 and EFgsp3) andRNA samples from the mantle, viscus, gill, hemocytes, andadductor muscle. The housekeeping gene GAPDH was adoptedas a positive control. As shown in Fig. 5, the mRNA of EFCBPis highly expressed in the mantle, gill, and hemocytes of theoyster. These tissues, especially the mantle, actively participatein the calcium metabolism and shell formation.Then, the precise mRNA expression pattern of EFCBP in themantle tissue was further analyzed via in situ hybridization. Fig.6B shows that the strong hybridization signals appear in the Fig. 4. A phylogenetictree analysis of the EF-hand signaturesof EFCBP, calconectin,PFMG1,PFMG6,calmodulinand calmodulin-like protein family, calbindin, andSPARC. The tree is produced with the ClustalX program by using the method of neighbor-joining analysis. The input alignment is the same as Fig. 3. Bootstrap values(1000 replicates) are indicated at the nodes. The abbreviations of protein names are the same as Fig. 3.1041  J. Huang et al. / Biochimica et Biophysica Acta 1770 (2007) 1037   –  1044
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