The effects of inorganic and organic additives on the hydration structure on the crystal surface have been discussed in X-ray reflectivity studies and in molecular dynamics simulations. We now demonstrate their effects on the hydration structure by conducting in situ observations of the hydration structure at a growing calcite surface by frequency modulation atomic force microscopy (FM-AFM). We show the atomic scale change of the hydration structure on the calcite surface in a supersaturated solution of CaCO3 by the addition of magnesium ions and a hydrophilic polypeptide. The FM-AFM images of the hydration structure revealed that magnesium ions increase the number of hydration layers on the terrace of the calcite surface from two to four layers. On the other hand, the hydrophilic polypeptide was ineffective for the hydration of the calcite surface. When both the magnesium ions and the hydrophilic polypeptide were added to the CaCO3 solution, the number of hydration layers increased and the magnitude of the oscillation hydration force as well as the long-range electrostatic force became larger than in the case when they were individually added. This is a noteworthy effect on the hydration structure on the calcite surface by cooperation of the magnesium ions and the polypeptide.

Tyrosinase plays an important role in the formation of the shell matrix and melanin synthesis in mollusks shells. A cDNA clone encoding a 47 kDa protein was isolated from the pearl oyster Pinctada fucata. The cDNA was 1,957 base pairs long and encodes a 417 residue protein that has extensive sequence identity with tyrosinase (polyphenol oxidase: EC 1.14.18.1). This tyrosinase-like protein, termed PfTy, contains an N-terminal signal sequence and the two copper-binding domain signatures (CuA and CuB), suggesting that PfTy belongs to the α-subclass of type-3 copper proteins. Enzyme activity of PfTy was examined by a spectrophotometric method using the translation product derived from an S30 T7 high-yield protein expression system. Tyrosinase activity was seen in this recombinant product. RT-PCR analysis showed that PfTy mRNA was expressed in the mantle pallial, but not in the mantle edge. Therefore, PfTy may participate in insoluble shell matrix formation of the nacreous layer. PfTy expression was also observed in the foot, liver, and adductor muscle, suggesting that PfTy participates in the synthesis of melanins, which are effective scavengers of free radicals formed in multiple intracellular oxidative processes. This is the first report of a novel α-class tyrosinase from the pearl oyster P. fucata. © 2014 Ryousuke Takgi and Tomoyuki Miyashita.

In molluscs, shell matrix proteins are associated with biomineralization, a biologically controlled process that involves nucleation and growth of calcium carbonate crystals. Identification and characterization of shell matrix proteins are important for better understanding of the adaptive radiation of a large variety of molluscs. We searched the draft genome sequence of the pearl oyster Pinctada fucata and annotated 30 different kinds of shell matrix proteins. Of these, we could identified Perlucin, ependymin-related protein and SPARC as common genes shared by bivalves and gastropods; however, most gastropod shell matrix proteins were not found in the P. fucata genome. Glycinerich proteins were conserved in the genus Pinctada. Another important finding with regard to these annotated genes was that numerous shell matrix proteins are encoded by more than one gene; e.g., three ACCBP-like proteins, three CaLPs, five chitin synthase-like proteins, two N16 proteins (pearlins), 10 N19 proteins, two nacreins, four Pifs, nine shematrins, two prismalin-14 proteins, and 21 tyrosinases. This diversity of shell matrix proteins may be implicated in the morphological diversity of mollusc shells. The annotated genes reported here can be searched in P. fucata gene models version 1.1 and genome assembly version 1.0 (http://marinegenomics.oist.jp/pinctada_fucata). These genes should provide a useful resource for studies of the genetic basis of biomineralization and evaluation of the role of shell matrix proteins as an evolutionary toolkit among the molluscs.

Bone morphogenetic protein (BMP)-2 plays an important role in morphogenesis in both vertebrates and invertebrates. BMP-2 is one of the most powerful bioactive substances known to induce the osteogenic differentiation of mesenchymal cells. We examined the structural and functional conservation of Pinctada fucata BMP-2 in inducing osteogenesis in the murine mesenchymal stem cells, C3H10T1/2. Exposure of C3H10T1/2 cells to the recombinant mature fragment of Pinctada fucata BMP-2 resulted in osteoblastic differentiation. The sequence, SVPKPCCVPTELSSL, within the C-terminal portion of Pinctada fucata BMP-2, is homologous to the knuckle epitope of human BMP-2. This synthetic polypeptide was able to induce differentiation of C3H10T1/2 along the osteoblastic lineage, as confirmed by an increase in alkaline phosphatase activity, and the accumulation of calcium, as determined by von Kossa staining. Furthermore, using immunohistochemical staining, we observed an increased expression of collagen type I, osteopontin, and osteocalcin, which are known markers of osteogenesis. These results show that BMP-2 is conserved, not only in terms of its homology at the amino acid sequence, but also in terms of driving the formation of hard tissues in vertebrates and invertebrates. © 2013 Akiko Takami et al.

The carbonic anhydrase nacrein participates in the formation of the nacreous or prismatic layer of Pinctada fucata. We isolated a genomic clone containing the nacrein gene and cloned the 5'-flanking region. Within the 1336 bp 5' flanking region, we identified putative cis-acting elements, including the TATA box (TATAAAA) at -82 bp, and AP1 (-819 bp) and Oct-1 (-1244 bp) binding sites. In addition to the mantle, the nacrein gene is also expressed in the adductor muscle, liver, and foot. These results showed that nacrein not only takes part in the formation of the hard tissue but also might be involved in acid-base balance, ion transport, and maintenance of ionic concentration. In vitro transcription experiments showed that the addition of human c-jun activates transcription from the nacrein promoter. This is the first report of a promoter from a gene that controls the formation of the hard tissue of mollusk shells.

The hard tissue of the Japanese pearl oyster, Pinctada fucata, consists of two layers, the outer prismatic layer, bearing calcite, and the inner nacreous layer, bearing aragonite. An EDTA-insoluble fraction of the prismatic layer of P. fucata was extracted with urea. In-vitro crystallization experiments showed that this urea-soluble fraction contained the factor(s) that promoted the growth of calcite crystals. We purified a protein from this fraction and deduced the internal amino acid sequences EYDFDRPDPYDP and EYDFERPD. We performed 3' RACE using primer DPPF1, encoding EYDFDRPDPYDP, and an oligo-dT adapter primer and amplified a fragment of approximately 300 bp. We screened cDNA libraries using the 300 bp fragment and obtained two clones that we named prismin 1 and 2. Both cDNAs encode proteins of 51 amino acids. Homology searches revealed 91% amino acid identity between prismin 1 and 2. The synthetic peptide DFDRPDPYDPY-DRFD, corresponding to the carboxy terminal region of prismin 1, has calcite growing activity and calcium binding capability, showing that the carboxy-terminal region is a functional domain. Prismin 1 is expressed strongly in the outer edge and in the inner part of the mantle tissue. However, immunoblot analysis revealed that prismin protein exists only in the prismatic layer, not in the nacreous layer, despite the presence of the mRNA. Therefore, we conclude that prismin is a novel prismatic layer-specific calcite growth factor.

The bone morphogenetic proteins (BMps) constitute a subfamily of the transforming growth factor type beta (TGF-beta) supergene family. BMP-2 plays an important role not only in osteoblast differentiation but also in pattern formation during development. To determine the function of BMP-2 in Pinctada fucata development and hard tissue formation, we isolated a BMP-2 genomic DNA clone and the BMP-2 cDNA. The deduced BMP-2 sequence consisted of 447 amino acids. The BMP-2 gene was composed of three exons. The C-terminal portion (149 amino acids) had 86% and 66% identity to the Crassostrea gigas and the human BMP-2 sequence respectively. The 5' flanking promoter region contained putative glioma transcription factor (Gli) and retinoic acid receptor (RAR) responding elements. The BMP-2 gene was expressed strongly in the inner part of the mantle tissue, corresponding to the nacreous aragonite shell layer. This finding suggests that BMP-2 has a key role in nacreous layer formation.

We have found a carbonic anhydrase (CA) in the prismatic layer of Pinctada fucata. This CA has the same kinetic properties as Nacrein, which is a CA existing in the nacreous layer of Pinctada fucata. We have examined the effects of inhibitors on the enzyme activity. Sodium sulfide and sulfanilamide are typical inhibitors of various types of CA; however. a CA in the prismatic layer and Nacrein were found to be resistant to sodium Sulfide and to show a weak resistance to sulfanilamide. This is the first report of a carbonic anhydrase with resistance to sodium sulfide, The molecular mass of the prismatic layer CA was estimated by SDS-PAGE to be approximately 60 kDa. Moreover, we have determined the N-terminal amino acid sequence of a CA in the prismatic layer. The sequence of the first 11 amino acids was in agreement with that of Nacrein, as deduced from the cDNA sequence. From these results, we have concluded that the carbonic anhydrase of the prismatic layer is Nacrein. Nacrein contributes to the formation of a prismatic layer as well as a nacreous layer of mollusk shells as a carbonic anhydrase and is a matrix component.

Pearlin is a representative of the EDTA-insoluble proteins and the nacreous layer-specific protein. Effects of pearlin on calcium carbonate crystallization were examined in vitro. The rate of calcium carbonate precipitation was examined by recording the decrease in pH of NaHCO3 solution when CaCl2 solution containing pearlin was added in the presence or absence of magnesium ion. As for the inhibitory effect of calcium carbonate precipitation, it was more remarkable in the presence of magnesium ion. Experiments of inhibition of calcium carbonate crystallization were performed using supersaturated solution of Ca(HCO3)2 in the presence or absence of magnesium ion. Pearlin strongly inhibited the aragonite crystallization of CaCO3, but not calcite crystallization. These results suggest that pearlin regulates negatively the crystal growth of the aragonite calcium carbonate in the processes of nacreous layer formation of Pinctada fucata.

真珠貝における硬組織は炭酸カルシウムの方解石結晶を主成分とする稜柱層とアラレ石結晶を主成分とする真珠層より形成され、両層とも外套膜から分泌された数パーセントの有機質を含んでいる。有機質のEDTA不溶性分画をEDTA-尿素で、高温下抽出して得られた大きさ約16 kDaのタンパク質パーリンの機能解析をin vitro結晶形成系において行った。パーリンはアラレ石結晶の析出を特異的に阻害することが解った。パーリンは真珠層形成を抑制的に制御しているものと推測される。

稜柱層のEDTA不溶性分画を尿素で高温下の条件で抽出して得られたタンパク質をプリズミンと名付け、このタンパク質を用いてIn vitroでの結晶形成実験を行うと同時にその機能を解析した。稜柱層のEDTA不溶性分画から抽出したタンパク質は大きさが約48kDaであり、これをプリズミンと名付けた。プリズミンを用いてIn vitroでの結晶形成実験を行ったところ、方解石の表面に結晶を成長させた。このことから、プリズミンは方解石を成長させる機能を持つと推察される。

アコヤ貝の貝殻は主に稜柱層と真珠層の２層から構成される硬組織であり、どちらも炭酸カルシウムを主成分とし、少量の有機物質を含んでいる。しかし、結晶構造は前者が方解石（三方晶系）であるのに対し、後者はアラレ石構造（斜方晶系）である。そして、貝殻に含まれる少量のタンパク質が、これらの結晶多形を制御し、結晶を成長させると考えられる。昨年度の本学会において、アコヤ貝真珠層のEDTA不溶性分画に含まれるタンパク質複合体は、平板状アラレ石結晶を成長させる機能があると報告した。そこで、本研究では稜柱層における、結晶成長に関与するタンパク質の同定を試みた。 稜柱層のEDTA不溶性分画を尿素で高温下の条件で抽出して得られたタンパク質をプリズミンと名付け、In Vitroでの結晶形成実験を行うと同時にその機能を解析した。 その結果、プリズミンは方解石の表面に結晶を成長させた。

アコヤ貝の真珠層より発見・同定された炭酸脱水酵素ナクレインは外套膜特異的に発現する。TATA boxからなるナクレインのプロモーターの上流、約１kbp内にはCCAAT boxやCREB結合様配列が存在する。また、脊椎動物の骨の代謝に重要な役割を果たしている２型炭酸脱水酵素の遺伝子の転写を活性化する転写調節因子であるAP1の結合様配列も存在している。これらナクレイン遺伝子の発現調節に関与する因子の解析をin vitro転写系を用いて試みたところ、c-junタンパク質を添加したときに転写物の量が増大した。AP1結合様配列を欠失した変異体を用いたときは転写物が確認できなかったことから、c-junはホモダイマーか、c-Fosとヘテロダイマーを形成して、AP1結合様配列を介してナクレインの転写を活性化していると考えられる。