Gene 555 (2015) 217–222
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Silkmapin of Hyriopsis cumingii, a novel silk-like shell matrix protein involved in nacre formation Xiaojun Liu a,1, Shaojian Dong a,1, Can Jin a, Zhiyi Bai a, Guiling Wang a, Jiale Li a,b,c,⁎ a b c
Key Laboratory of Freshwater Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Agriculture, Shanghai 201306, China E-Institute of Shanghai Universities, Shanghai Ocean University, Shanghai 201306, China Mobile postdoctoral center of Agriculture, Shanghai Ocean University, Shanghai 201306, China
a r t i c l e
i n f o
Article history: Received 24 September 2014 Received in revised form 2 November 2014 Accepted 4 November 2014 Available online 7 November 2014 Keywords: Freshwater mussel Hyriopsis cumingii Biomineralization Nacre Matrix protein
a b s t r a c t Understanding the role of matrix proteins in nacre formation and biomineralization in mollusks is important for the pearl industry. In this study, the gene encoding the novel Hyriopsis cumingii shell matrix protein silkmapin was characterized. The gene encodes a protein of 30.89 kDa in which Gly accounts for 34.41% of the amino acid content, and the C-terminal region binds Ca2+. Secondary structure prediction indicated a predominantly β-fold and a structure typical of filamentous proteins. Real-time quantitative PCR and in situ hybridization showed that silkmapin was expressed in epithelial cells at the edge and pallial of mantle tissue, indicated that silkmapin play roles in the shell nacreous and prismatic layer formation. Further real-time PCR results indicated an involvement in pearl formation via nucleation of calcium carbonate prior to formation of the nacre. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Shell biomineralization in molluscs involves crystallization of calcium carbonate under the guidance of matrix proteins. During this process, two major structural shell layers are formed, namely the nacreous layer and the prismatic layer. The nacreous layer in particular protects the softer body parts, and has a high mechanical strength (Smith et al., 1999) due to the dense regular microstructure consisting of 5% organic matrix and 95% calcium carbonate. Although the matrix proteins and other biological macromolecules only account for 5%, the organic matrix is responsible for regulating the various stages of nacreous layer biomineralization including nucleation, crystallization, crystal orientation and crystal morphology (Weiner and Hood, 1975; Wheeler et al., 1981; Belcher et al., 1996; Falini et al., 1996). Recent studies have focused on the proteins present in the organic matrix, and many matrix proteins have been identified and isolated (Miyamoto et al., 1996; Sudo et al., 1997; Samata et al., 1999; Miyashita et al., 2000; Zhang et al., 2003; Yano et al., 2007; Ma et al., 2007; Zhang and Zhang, 2006; Suzuki et al., 2009). While information on tissue distribution and the role in crystallization of calcium carbonate has been obtained, many matrix proteins remain unidentified and uncharacterized, which is a barrier to our understanding of nacre formation.
Hyriopsis cumingii is the most important oyster in the pearl industry in China, which produces 95% of freshwater pearls worldwide, and H. cumingii accounts for 70% of this (Wang et al., 2007; Li and Li, 2009). A better understanding of nacre formation is therefore highly desirable. H. cumingii shell is composed of prismatic and nacreous layers made of aragonite, unlike many other shellfish in which the prismatic and nacreous layers are made of calcite and aragonite, respectively (Liu and Li, in press). The mechanism of this unusual biomineralization is poorly understood, and research on the nacreous layer of H. cumingii largely focused on proteomics and transcriptomics studies on the whole matrix proteome (Ma et al., 2010, 2011, 2012; Bai et al., 2013; Berland et al., 2013). Identification and extraction of individual proteins was limited. Natoli et al. (2010) characterized a 48 kDa protein from the pearl of H. cumingii that induced the formation of vaterite. Additionally, the matrix protein perlucin was studied and found to be involved in H. cumingii nacre formation (Lin et al., 2013). In this study, the gene encoding the novel shell matrix protein silkmapin was isolated from a H. cumingii cDNA library. Silkmapin was found to be expressed in the mantle and is involved in nacre formation. 2. Materials and methods 2.1. Animal samples and RNA extraction
Abbreviations: PCR, Polymerase chain reaction. ⁎ Corresponding author at: Key Laboratory of Freshwater Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Agriculture, Shanghai 201306, China. E-mail address: [email protected] (J. Li). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.gene.2014.11.006 0378-1119/© 2014 Elsevier B.V. All rights reserved.
H. cumingii samples were obtained from the Weiwang pearl farm of Jinhua City, Zhejiang Province, China. Mantle, gill, foot, adductor muscle, intestine and hemocyte tissue were frozen in liquid nitrogen and
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pulverized. On days 1, 2, 4, 7, 14, 21 and 28 following implantation, six oysters were harvested and pearl sacs were carefully separated from mantle tissue and immediately placed in RNAzol (BIOTEX, Inc.) at a ratio of 50–100 mg per mL of RNAzol. Total RNA was extracted according to the manufacturer's instructions. The integrity of the RNA was determined by fractionation on formaldehyde-denaturing 1.2% agarose gels and staining with ethidium bromide. The quantity of RNA was determined by measuring the absorbance at 260 nm using a nanodrop (ThermoScientific, USA). Approximately 2 μg of mantle and pearl sac RNA was used as a template for cDNA synthesis using the BD PowerScript Reverse Transcriptase (BD Biosciences Clontech). 2.2. Rapid amplification of cDNA ends and sequence analysis 3′-RACE and 5′-RACE were conducted with a SMART RACE cDNA Amplication kit (Clontech) and Advantage 2 cDNA polymerase mix (Clontech) according to the manufacturer's instructions. 3′-RACE was performed using the degenerate primer GYR-F1 (5′-GGN TAY GGN GGN GGN TAY GGN GGN-3′, N = A/G/C/T, Y = C/T). This primer was designed based on the shematrin-3 amino acid repeat GYGG to include two repeats (GYGGGYGG). Based on the 3′-RACE sequencing results, the gene-specific primer GYR-R1 (5′-CAA GTC CAC CCC CGT ATA GGC CAG GAC C-3′) was designed for 5′-RACE. Multiple sequence alignment
of homologous proteins was performed using GenBank, SwissProt and the BLAST program. Signal peptide sequence predicted was performed using the SignalP 3.0 program (http://www.cbs.dtu.dk/servies/SignalP. 3.0/). Secondary structure prediction was performed using Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre/).
2.3. Gene expression analysis First-strand cDNA synthesis was conducted using the Clontech RACE kit according to manufacturer's instructions. Based on the silkmapin sequence, quantitative real-time PCR primers GYR-F2 (5′-TTGGCGGTTTTG GCGGACCCTCTTTT-3′) and GYR-R2 (5′-ATGGCATGATGGTCAAGCAACT GGCACACT-3′) were designed, along with primers EF-F (5′-GGAACTTC CCAGGCAGACTGTGC-3′) and EF-R (5′-TCAAAACGGGCCGCAGAGAAT3′) for the reference gene EF-1α. qPCR was carried out on a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, USA), with samples repeated in triplicate. The program used for amplification included 40 cycles of 95 °C for 5 s and 60 °C for 20 s. The threshold cycle (CT) of each sample was measured with Mx3000P software. cDNAs were detected for each sample after seven cycles, and repeated nine times per sample. For data analysis, − ΔΔCT data were standardized using the 5th day as the zero point.
Fig. 1. Nucleotide sequence of the cDNA clone and deduced amino acid sequence of the mussel silkmapin. The cDNA sequence has been submitted to GenBank (Accession No. KM588085).
X. Liu et al. / Gene 555 (2015) 217–222 Table 1 Amino acid compositions (mole percent) of silkmapin. Amino acid
silkmapin
Gly Val Ser Phe Thr Pro Tyr Ala Asp Leu Ile Arg Asn Lys Gln Met
34.41 8.36 7.72 5.79 5.79 5.47 5.47 5.14 4.5 3.22 3.22 2.57 1.93 1.61 0.96 0.96
219
2.4. In situ hybridization (1) Preparation of frozen sections. Fresh mantle tissue from H. cumingii was immediately fixed in 4% paraformaldehyde (freshly prepared using 0.1% DEPC water) for 6 h. After washing with 0.1 M phosphate buffer (freshly prepared using 0.1% DEPC water), 20–25% sucrose buffer (freshly prepared using 0.1 M phosphate buffer) was added and samples incubated at 4 °C until tissues sank to the bottom of the container. Following slicing, 10 μm thick samples were mounted on slides pre-treated with poly-lysine. (2) In situ hybridization. Plasmids containing the silkmapin gene were linearized using restriction enzyme NdeI to generate template. Using the DIG RNA Labeling Kit (Roche), RNA probes were labeled with digoxigenin, and T7 or SP6 RNA polymerase were added to synthesize sense and antisense probes as described previously (Qiu et al., 2008).
Fig. 2. Secondary structure prediction of silkmapin. Based on the protein sequence of Silkmapin, the secondary structure prediction is performed by Phyre2. The amino acids are colored based on the physiochemical properties of the side chains. The regions adopting putative β-sheet conformations are represented as blue arrow. The degrees of confidence 0.9 are also indicated by a rainbow color gradient.
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molecular weight was 29.35 kDa and the isoelectric point was 6.87. Three Gly-rich regions were apparent, and Gly accounted for 34.41% of amino acid content (Table 1). Most Asp residues were in the C-terminal region. 3.2. Protein structure prediction
Fig. 3. Three dimensional structure prediction of silkmapin. The tertiary structure prediction is performed by Phyre2.
Phyre2 was used to predict the secondary structure of silkmapin (excluding the signal peptide). A mainly β-fold structure was predicted, which accounted for 21% of the sequence, and α-helical structure was not present (Fig. 2). Furthermore, a filamentous higher-order structure was predicted (Fig. 3).
3. Results 3.3. Tissue expression and in situ hybridization 3.1. cDNA cloning and sequence analysis An 804 bp fragment was obtained using 3′-RACE using the designed primers, and 5′-RACE generated a further 514 bp fragment. Upon combining the two fragments, the full 1242 bp silkmapin cDNA sequence was determined (Fig. 1). Sequence analysis revealed an open reading frame of 978 bp encoding a protein of 325 amino acids with a theoretical molecular weight of 30.89 kDa, an isoelectric point of 7.39, and a signal peptide from residues 1–15. After the removal of the signal peptide, the theoretical
Quantitative analysis of silkmapin expression was performed on seven H. cumingii tissues (intestine, adductor muscle, foot, gill, blood, mantle edge and pallial) using real time PCR. The results indicated that expression occurred mainly in the mantle (Fig. 4), with higher expression in the pallial portion than the edge of the mantle. In situ hybridization using DIG-labeled silkmapin-specific probes on the frozen mantle sections gave a strong signal in the epithelial cells at the mantle edge and pallial region (Fig. 5). 3.4. Expression of silkmapin during pearl sac formation and early development Samples were taken on days 1, 2, 4, 7, 14, 21 and 28 after implantation, and the analysis of expression of silkmapin during pearl sac formation was performed using real time PCR. Silkmapin expression in the pearl sac increased between days 1–14, then decreased after day 14 (Fig. 6). 4. Discussion
Fig. 4. Tissue-specific expression of silkmapin by real-time PCR.
The shell and pearls of H. cumingii provide new biomineralization models, but the matrix proteins involved in formation of the nacreous layer are poorly understood. In this study, the gene encoding the novel matrix protein silkmapin was isolated from a H. cumingii mantle cDNA library. Like many other shell matrix proteins, silkmapin is rich in Gly, which accounts for 34.41% of the amino acid content (Table 1).
Fig. 5. In situ hybridization analysis of silkmapin gene expression in the mantle of Hyriopsis cumingii.
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day, silkmapin was still expressed at a relatively high level. Previous studies showed that during the formation of the pearl, mineral deposition proceeded through two stages: ordered and disordered deposition. During the ordered deposition stage, before the first glossy nacre layer is formed, the critical process of nucleation takes place, and a brown sediment is generated (Liu et al., 2012). We speculate that silkmapin expression peaked at day 14 because this protein provides nucleation sites for the biomineralization of calcium carbonate prior to formation of the nacre. Acknowledgments
Fig. 6. The relative expression level of silkmapin in the pearl sac during the early stages after implantation of pearl formation.
Tissue expression analysis showed that silkmapin is specifically expressed in the mantle, at the edge and in the center, and in situ hybridization indicated expression in mantle epithelial cells. The above results are consistent with an involvement in the formation of both prismatic and nacreous layers. It should be noted that the region between the mantle edge cells and pallial epithelial cells showed no silkmapin expression. This region of the mantle is closely integrated with the shell, and divides the extrapallial fluid into two parts. Mineralization of H. cumingii shell could be controlled by epithelial cells on both sides of this region. Blast results identified no homology with other shell matrix proteins or indeed any other known proteins. Secondary structure prediction of silkmapin revealed a mainly βfold structure, consistent with other silk-like proteins. According to the current model of the molecular mechanism of nacreous layer mineralization, prior to mineral formation, a large class of silk-like proteins form anamorphous hydrated gel framework (Addadi et al., 2006). This gel, as part of the organic framework, plays a key role in the subsequent mineralization process. These silk-like matrix proteins are generally non-acidic and rich in Gly and Ala residues, have a typical β-fold secondary structure, and form a filamentous higher-order structure. Before mineralization, silk-like proteins fill the chitin framework and inhibit crystallization outside the acidic nucleation sites. After mineralization, these junctions are tightly closed between the crystal plates and become part of the organic layer (Mann, 1988; Addadi et al., 1987). Matrix proteins are secreted into the extrapallial fluid by mantles, and an organic framework of insoluble matrix proteins and other biomolecules is formed that provides nucleation sites upon which calcium and carbonate ions are deposited in an ordered fashion. Insoluble matrix proteins therefore influence nacre biomineralization, and studies on the molecular composition and mechanism of nacre formation will help to clarify our understanding of biomineralization. Acidic Asp residues were found to be mainly located at the Cterminal portion of silkmapin, with Asp-Ile-Asp and Asp-Asp-Asp motifs at the most distal end. Mann (1988) reported that the Asp-X-Asp motif may provide an ideal binding site for Ca2+, therefore Ca2+ binding may occur in this region. During mineralization, the C-terminus of silkmapin may become exposed on the surface of the organic framework, which may provide nucleation sites for the biomineralization of calcium carbonates. Silkmapin also appears to be involved in the formation of pearls, since expression in the pearl sac increased between days 1 and 14 following implantation. According to Lin et al. (2013), after the insertion of the pearl nucleus, the pearl sac of H. cumingii quickly forms and begins to secrete minerals. On the 14th day, brown minerals were observed, and on the 21st day, a shiny nacre secretion was visible (Lin et al., 2013). The results of the current study showed that on the 21st
This work was supported by a grant from the National Science and Technology Support Program (2012BAD26B04), the Ability Improvement Project for Engineering and Technology Research Center of the Shanghai Municipal Science and Technology Commission (13DZ228050031272657), and the National Natural Science Foundation of China (31272657). References Addadi, L., Moradian, J., Shay, E., Maroudas, N.G., Weiner, S., 1987. A chemical model for the cooperation of sulfates and carboxylates in calcite crystal nucleation: relevance to biomineralization. Proc. Natl. Acad. Sci. U. S. A. 84, 2732–2736. Addadi, L., Joester, D., Nudelman, F., Weiner, S., 2006. Mollusk shell formation: a source of new concepts for understanding biomineralization processes. Chem. Eur. J. 12, 981–987. Bai, Z., Zheng, H., Lin, J., Wang, G., Li, J., 2013. Comparative analysis of the transcriptome in tissues secreting purple and white nacre in the pearl mussel Hyriopsis cumingii. PLos One 8, e53617. Belcher, A.M., Wu, X., Christensen, R.J., Hansma, P.K., Stucky, G.D., Morse, D.E., 1996. Control of crystal phase switching and orientation by soluble mollusc-shell proteins. Nature 381, 56–58. Berland, S., Ma, Y., Marie, A., Andrieu, J., Bédouet, L., Feng, Q., 2013. Proteomic and profile analysis of the proteins laced with aragonite and vaterite in the freshwater mussel Hyriopsis cumingii shell biominerals. Protein Pept. Lett. 20, 1170–1180. Falini, G., Albeck, S., Weiner, S., Addadi, L., 1996. Control of aragonite or calcite polymorphism by mollusk shell macromolecules. Science 271, 67–69. Li, J., Li, Y., 2009. Aquaculture in China—freshwater pearl culture. World Aquacult. 40, 60. Lin, J., Ma, K., Bai, Z., Li, J., 2013. Molecular cloning and characterization of perlucin from the freshwater pearl mussel, Hyriopsis cumingii. Gene 520, 210–216. Liu, X., Li, J., 2014. Formation of the prismatic layer in the freshwater bivalve Hyriopsis cumingii: the feedback of crystal growth on organic matrix. Acta Zool. http://dx.doi. org/10.1111/azo.12048 (in press). Liu, X., Li, J., Xiang, L., Sun, J., Zheng, G., Zhang, G., Wang, H., Xie, L., Zhang, R., 2012. The role of matrix proteins in the control of nacreous layer deposition during pearl formation. Proc. R. Soc. B Biol. Sci. 279, 1000–1007. Ma, Z., Huang, J., Sun, J., Wang, G., Li, C., Xie, L., Zhang, R., 2007. A novel extrapallial fluid protein controls the morphology of nacre lamellae in the pearl oyster, Pinctada fucata. J. Biol. Chem. 282, 23253–23263. Ma, Y., Gao, Y., Feng, Q., 2010. Effects of pH and temperature on CaCO3 crystallization in aqueous solution with water soluble matrix of pearls. J. Cryst. Growth 312, 3165–3170. Ma, Y., Gao, Y., Feng, Q., 2011. Characterization of organic matrix extracted from fresh water pearls. Mater. Sci. Eng. C 31, 1338-1134. Ma, Y.F., Qiao, L., Feng, Q.L., 2012. In-vitro study on calcium carbonate crystal growth mediated by organic matrix extracted from fresh water pearls. Mater. Sci. Eng. C 32, 1963–1970. Mann, S., 1988. Molecular recognition in biomineralization. Nature 332, 119–124. Miyamoto, H., Miyashita, T., Okushima, M., Nakano, S., Morita, T., Matsushiro, A., 1996. A carbonic anhydrase from the nacreous layer in oyster pearls. Proc. Natl. Acad. Sci. U. S. A. 93, 9657–9660. Miyashita, T., Takagi, R., Okushima, M., Nakano, S., Miyamoto, H., Nishikawa, E., Matsushiro, A., 2000. Complementary DNA cloning and characterization of pearlin, a new class of matrix protein in the nacreous layer of oyster pearls. Mar. Biotechnol. 2, 409–418. Natoli, A., Wiens, M., Schröder, H.C., Stifanic, M., Batel, R., Soldati, A.L., Jacob, D.E., Müller, W.E., 2010. Bio−vaterite formation by glycoproteins from freshwater pearls. Micron 41 (4), 359–366. Qiu, G., Weber, G.M., Rexroad, C.E., Yao, J., 2008. Identification of RtGST-1, a novel germ cell-specific mRNA-like transcript predominantly expressed in early previtellogenic oocytes in rainbow trout (Oncorhynchus mykiss). Mol. Reprod. Dev. 75, 723–730. Samata, T., Hayashi, N., Kono, M., Hasegawa, K., Horita, C., Akera, S., 1999. A new matrix protein family related to the nacreous layer formation of Pinctada fucata. FEBS Lett. 462, 225–229. Smith, B.L., Schäffer, T.E., Viani, M., Thompson, J.B., Frederick, N.A., Kindt, J., Belcher, A., Stucky, G.D., Morse, D.E., Hansma, P.K., 1999. Molecular mechanistic origin of the toughness of natural adhesives, fibres and composites. Nature 399, 761–763. Sudo, S., Fujikawa, T., Nagakura, T., Ohkubo, T., Sakaguchi, K., Tanaka, M., Nakashima, K., Takahashi, T., 1997. Structures of mollusc shell framework proteins. Nature 387, 563–564. Suzuki, M., Saruwatari, K., Kogure, T., Yamamoto, Y., Nishimura, T., Kato, T., Nagasawa, H., 2009. An acidic matrix protein, Pif, is a key macromolecule for nacre formation. Science 325, 1388–1390.
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Wang, G., Yuan, Y., Li, J., 2007. SSR analysis of genetic diversity and phylogenetic relationships among different populations of Hyriopsis cumingii from the five lakes of China. J. Fish. China 31, 152–158. Weiner, S., Hood, L., 1975. Soluble protein of the organic matrix of mollusk shells: a potential template for shell formation. Science 190, 987–989. Wheeler, A.P., George, J.W., Evans, C.A., 1981. Control of calcium carbonate nucleation and crystal growth by soluble matrix of oyster shell. Science 212, 1397–1398.
Yano, M., Nagai, K., Morimoto, K., Miyamoto, H., 2007. A novel nacre protein N19 in the pearl oyster Pinctada fucata. Biochem. Biophys. Res. Commun. 326, 158–163. Zhang, C., Zhang, R., 2006. Matrix proteins in the outer shells of mollusks. Mar. Biotechnol. 8, 572–586. Zhang, Y., Xie, L., Meng, Q., Jiang, T., Pu, R., Chen, L., Zhang, R., 2003. A novel matrix protein participating in the nacre framework formation of pearl oyster, Pinctada fucata. Comp. Biochem. Physiol. B Mol. Biol. 135, 565–573.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Silkmapin of Hyriopsis cumingii, a novel silk-like shell matrix protein involved in nacre formation Xiaojun Liu a,1, Shaojian Dong a,1, Can Jin a, Zhiyi Bai a, Guiling Wang a, Jiale Li a,b,c,⁎ a b c
Key Laboratory of Freshwater Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Agriculture, Shanghai 201306, China E-Institute of Shanghai Universities, Shanghai Ocean University, Shanghai 201306, China Mobile postdoctoral center of Agriculture, Shanghai Ocean University, Shanghai 201306, China
a r t i c l e
i n f o
Article history: Received 24 September 2014 Received in revised form 2 November 2014 Accepted 4 November 2014 Available online 7 November 2014 Keywords: Freshwater mussel Hyriopsis cumingii Biomineralization Nacre Matrix protein
a b s t r a c t Understanding the role of matrix proteins in nacre formation and biomineralization in mollusks is important for the pearl industry. In this study, the gene encoding the novel Hyriopsis cumingii shell matrix protein silkmapin was characterized. The gene encodes a protein of 30.89 kDa in which Gly accounts for 34.41% of the amino acid content, and the C-terminal region binds Ca2+. Secondary structure prediction indicated a predominantly β-fold and a structure typical of filamentous proteins. Real-time quantitative PCR and in situ hybridization showed that silkmapin was expressed in epithelial cells at the edge and pallial of mantle tissue, indicated that silkmapin play roles in the shell nacreous and prismatic layer formation. Further real-time PCR results indicated an involvement in pearl formation via nucleation of calcium carbonate prior to formation of the nacre. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Shell biomineralization in molluscs involves crystallization of calcium carbonate under the guidance of matrix proteins. During this process, two major structural shell layers are formed, namely the nacreous layer and the prismatic layer. The nacreous layer in particular protects the softer body parts, and has a high mechanical strength (Smith et al., 1999) due to the dense regular microstructure consisting of 5% organic matrix and 95% calcium carbonate. Although the matrix proteins and other biological macromolecules only account for 5%, the organic matrix is responsible for regulating the various stages of nacreous layer biomineralization including nucleation, crystallization, crystal orientation and crystal morphology (Weiner and Hood, 1975; Wheeler et al., 1981; Belcher et al., 1996; Falini et al., 1996). Recent studies have focused on the proteins present in the organic matrix, and many matrix proteins have been identified and isolated (Miyamoto et al., 1996; Sudo et al., 1997; Samata et al., 1999; Miyashita et al., 2000; Zhang et al., 2003; Yano et al., 2007; Ma et al., 2007; Zhang and Zhang, 2006; Suzuki et al., 2009). While information on tissue distribution and the role in crystallization of calcium carbonate has been obtained, many matrix proteins remain unidentified and uncharacterized, which is a barrier to our understanding of nacre formation.
Hyriopsis cumingii is the most important oyster in the pearl industry in China, which produces 95% of freshwater pearls worldwide, and H. cumingii accounts for 70% of this (Wang et al., 2007; Li and Li, 2009). A better understanding of nacre formation is therefore highly desirable. H. cumingii shell is composed of prismatic and nacreous layers made of aragonite, unlike many other shellfish in which the prismatic and nacreous layers are made of calcite and aragonite, respectively (Liu and Li, in press). The mechanism of this unusual biomineralization is poorly understood, and research on the nacreous layer of H. cumingii largely focused on proteomics and transcriptomics studies on the whole matrix proteome (Ma et al., 2010, 2011, 2012; Bai et al., 2013; Berland et al., 2013). Identification and extraction of individual proteins was limited. Natoli et al. (2010) characterized a 48 kDa protein from the pearl of H. cumingii that induced the formation of vaterite. Additionally, the matrix protein perlucin was studied and found to be involved in H. cumingii nacre formation (Lin et al., 2013). In this study, the gene encoding the novel shell matrix protein silkmapin was isolated from a H. cumingii cDNA library. Silkmapin was found to be expressed in the mantle and is involved in nacre formation. 2. Materials and methods 2.1. Animal samples and RNA extraction
Abbreviations: PCR, Polymerase chain reaction. ⁎ Corresponding author at: Key Laboratory of Freshwater Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Agriculture, Shanghai 201306, China. E-mail address: [email protected] (J. Li). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.gene.2014.11.006 0378-1119/© 2014 Elsevier B.V. All rights reserved.
H. cumingii samples were obtained from the Weiwang pearl farm of Jinhua City, Zhejiang Province, China. Mantle, gill, foot, adductor muscle, intestine and hemocyte tissue were frozen in liquid nitrogen and
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pulverized. On days 1, 2, 4, 7, 14, 21 and 28 following implantation, six oysters were harvested and pearl sacs were carefully separated from mantle tissue and immediately placed in RNAzol (BIOTEX, Inc.) at a ratio of 50–100 mg per mL of RNAzol. Total RNA was extracted according to the manufacturer's instructions. The integrity of the RNA was determined by fractionation on formaldehyde-denaturing 1.2% agarose gels and staining with ethidium bromide. The quantity of RNA was determined by measuring the absorbance at 260 nm using a nanodrop (ThermoScientific, USA). Approximately 2 μg of mantle and pearl sac RNA was used as a template for cDNA synthesis using the BD PowerScript Reverse Transcriptase (BD Biosciences Clontech). 2.2. Rapid amplification of cDNA ends and sequence analysis 3′-RACE and 5′-RACE were conducted with a SMART RACE cDNA Amplication kit (Clontech) and Advantage 2 cDNA polymerase mix (Clontech) according to the manufacturer's instructions. 3′-RACE was performed using the degenerate primer GYR-F1 (5′-GGN TAY GGN GGN GGN TAY GGN GGN-3′, N = A/G/C/T, Y = C/T). This primer was designed based on the shematrin-3 amino acid repeat GYGG to include two repeats (GYGGGYGG). Based on the 3′-RACE sequencing results, the gene-specific primer GYR-R1 (5′-CAA GTC CAC CCC CGT ATA GGC CAG GAC C-3′) was designed for 5′-RACE. Multiple sequence alignment
of homologous proteins was performed using GenBank, SwissProt and the BLAST program. Signal peptide sequence predicted was performed using the SignalP 3.0 program (http://www.cbs.dtu.dk/servies/SignalP. 3.0/). Secondary structure prediction was performed using Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre/).
2.3. Gene expression analysis First-strand cDNA synthesis was conducted using the Clontech RACE kit according to manufacturer's instructions. Based on the silkmapin sequence, quantitative real-time PCR primers GYR-F2 (5′-TTGGCGGTTTTG GCGGACCCTCTTTT-3′) and GYR-R2 (5′-ATGGCATGATGGTCAAGCAACT GGCACACT-3′) were designed, along with primers EF-F (5′-GGAACTTC CCAGGCAGACTGTGC-3′) and EF-R (5′-TCAAAACGGGCCGCAGAGAAT3′) for the reference gene EF-1α. qPCR was carried out on a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, USA), with samples repeated in triplicate. The program used for amplification included 40 cycles of 95 °C for 5 s and 60 °C for 20 s. The threshold cycle (CT) of each sample was measured with Mx3000P software. cDNAs were detected for each sample after seven cycles, and repeated nine times per sample. For data analysis, − ΔΔCT data were standardized using the 5th day as the zero point.
Fig. 1. Nucleotide sequence of the cDNA clone and deduced amino acid sequence of the mussel silkmapin. The cDNA sequence has been submitted to GenBank (Accession No. KM588085).
X. Liu et al. / Gene 555 (2015) 217–222 Table 1 Amino acid compositions (mole percent) of silkmapin. Amino acid
silkmapin
Gly Val Ser Phe Thr Pro Tyr Ala Asp Leu Ile Arg Asn Lys Gln Met
34.41 8.36 7.72 5.79 5.79 5.47 5.47 5.14 4.5 3.22 3.22 2.57 1.93 1.61 0.96 0.96
219
2.4. In situ hybridization (1) Preparation of frozen sections. Fresh mantle tissue from H. cumingii was immediately fixed in 4% paraformaldehyde (freshly prepared using 0.1% DEPC water) for 6 h. After washing with 0.1 M phosphate buffer (freshly prepared using 0.1% DEPC water), 20–25% sucrose buffer (freshly prepared using 0.1 M phosphate buffer) was added and samples incubated at 4 °C until tissues sank to the bottom of the container. Following slicing, 10 μm thick samples were mounted on slides pre-treated with poly-lysine. (2) In situ hybridization. Plasmids containing the silkmapin gene were linearized using restriction enzyme NdeI to generate template. Using the DIG RNA Labeling Kit (Roche), RNA probes were labeled with digoxigenin, and T7 or SP6 RNA polymerase were added to synthesize sense and antisense probes as described previously (Qiu et al., 2008).
Fig. 2. Secondary structure prediction of silkmapin. Based on the protein sequence of Silkmapin, the secondary structure prediction is performed by Phyre2. The amino acids are colored based on the physiochemical properties of the side chains. The regions adopting putative β-sheet conformations are represented as blue arrow. The degrees of confidence 0.9 are also indicated by a rainbow color gradient.
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molecular weight was 29.35 kDa and the isoelectric point was 6.87. Three Gly-rich regions were apparent, and Gly accounted for 34.41% of amino acid content (Table 1). Most Asp residues were in the C-terminal region. 3.2. Protein structure prediction
Fig. 3. Three dimensional structure prediction of silkmapin. The tertiary structure prediction is performed by Phyre2.
Phyre2 was used to predict the secondary structure of silkmapin (excluding the signal peptide). A mainly β-fold structure was predicted, which accounted for 21% of the sequence, and α-helical structure was not present (Fig. 2). Furthermore, a filamentous higher-order structure was predicted (Fig. 3).
3. Results 3.3. Tissue expression and in situ hybridization 3.1. cDNA cloning and sequence analysis An 804 bp fragment was obtained using 3′-RACE using the designed primers, and 5′-RACE generated a further 514 bp fragment. Upon combining the two fragments, the full 1242 bp silkmapin cDNA sequence was determined (Fig. 1). Sequence analysis revealed an open reading frame of 978 bp encoding a protein of 325 amino acids with a theoretical molecular weight of 30.89 kDa, an isoelectric point of 7.39, and a signal peptide from residues 1–15. After the removal of the signal peptide, the theoretical
Quantitative analysis of silkmapin expression was performed on seven H. cumingii tissues (intestine, adductor muscle, foot, gill, blood, mantle edge and pallial) using real time PCR. The results indicated that expression occurred mainly in the mantle (Fig. 4), with higher expression in the pallial portion than the edge of the mantle. In situ hybridization using DIG-labeled silkmapin-specific probes on the frozen mantle sections gave a strong signal in the epithelial cells at the mantle edge and pallial region (Fig. 5). 3.4. Expression of silkmapin during pearl sac formation and early development Samples were taken on days 1, 2, 4, 7, 14, 21 and 28 after implantation, and the analysis of expression of silkmapin during pearl sac formation was performed using real time PCR. Silkmapin expression in the pearl sac increased between days 1–14, then decreased after day 14 (Fig. 6). 4. Discussion
Fig. 4. Tissue-specific expression of silkmapin by real-time PCR.
The shell and pearls of H. cumingii provide new biomineralization models, but the matrix proteins involved in formation of the nacreous layer are poorly understood. In this study, the gene encoding the novel matrix protein silkmapin was isolated from a H. cumingii mantle cDNA library. Like many other shell matrix proteins, silkmapin is rich in Gly, which accounts for 34.41% of the amino acid content (Table 1).
Fig. 5. In situ hybridization analysis of silkmapin gene expression in the mantle of Hyriopsis cumingii.
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day, silkmapin was still expressed at a relatively high level. Previous studies showed that during the formation of the pearl, mineral deposition proceeded through two stages: ordered and disordered deposition. During the ordered deposition stage, before the first glossy nacre layer is formed, the critical process of nucleation takes place, and a brown sediment is generated (Liu et al., 2012). We speculate that silkmapin expression peaked at day 14 because this protein provides nucleation sites for the biomineralization of calcium carbonate prior to formation of the nacre. Acknowledgments
Fig. 6. The relative expression level of silkmapin in the pearl sac during the early stages after implantation of pearl formation.
Tissue expression analysis showed that silkmapin is specifically expressed in the mantle, at the edge and in the center, and in situ hybridization indicated expression in mantle epithelial cells. The above results are consistent with an involvement in the formation of both prismatic and nacreous layers. It should be noted that the region between the mantle edge cells and pallial epithelial cells showed no silkmapin expression. This region of the mantle is closely integrated with the shell, and divides the extrapallial fluid into two parts. Mineralization of H. cumingii shell could be controlled by epithelial cells on both sides of this region. Blast results identified no homology with other shell matrix proteins or indeed any other known proteins. Secondary structure prediction of silkmapin revealed a mainly βfold structure, consistent with other silk-like proteins. According to the current model of the molecular mechanism of nacreous layer mineralization, prior to mineral formation, a large class of silk-like proteins form anamorphous hydrated gel framework (Addadi et al., 2006). This gel, as part of the organic framework, plays a key role in the subsequent mineralization process. These silk-like matrix proteins are generally non-acidic and rich in Gly and Ala residues, have a typical β-fold secondary structure, and form a filamentous higher-order structure. Before mineralization, silk-like proteins fill the chitin framework and inhibit crystallization outside the acidic nucleation sites. After mineralization, these junctions are tightly closed between the crystal plates and become part of the organic layer (Mann, 1988; Addadi et al., 1987). Matrix proteins are secreted into the extrapallial fluid by mantles, and an organic framework of insoluble matrix proteins and other biomolecules is formed that provides nucleation sites upon which calcium and carbonate ions are deposited in an ordered fashion. Insoluble matrix proteins therefore influence nacre biomineralization, and studies on the molecular composition and mechanism of nacre formation will help to clarify our understanding of biomineralization. Acidic Asp residues were found to be mainly located at the Cterminal portion of silkmapin, with Asp-Ile-Asp and Asp-Asp-Asp motifs at the most distal end. Mann (1988) reported that the Asp-X-Asp motif may provide an ideal binding site for Ca2+, therefore Ca2+ binding may occur in this region. During mineralization, the C-terminus of silkmapin may become exposed on the surface of the organic framework, which may provide nucleation sites for the biomineralization of calcium carbonates. Silkmapin also appears to be involved in the formation of pearls, since expression in the pearl sac increased between days 1 and 14 following implantation. According to Lin et al. (2013), after the insertion of the pearl nucleus, the pearl sac of H. cumingii quickly forms and begins to secrete minerals. On the 14th day, brown minerals were observed, and on the 21st day, a shiny nacre secretion was visible (Lin et al., 2013). The results of the current study showed that on the 21st
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