A novel matrix protein participating in the nacre framework formation of pearl oyster,Pinctada fucata

Understanding the molecular composition is of great interest for both nacre formation mechanism and biomineralization in mollusk shell. A cDNA clone encoding an MSI31 relative, termed MSI7 because of its estimated molecular mass of 7.3 kDa, was isolated from the pearl oyster, Pinctada fucata. This novel protein shares similarity with MSI31, a prismatic framework protein of P. fucata. It is peculiar that MSI7 is much shorter in size, harboring only the Gly-rich sequence that has been proposed to be critical for Ca(2+) binding. In situ hybridization result showed that MSI7 mRNA was expressed specifically at the folds and outer epithelia of the mantle, indicating that MSI7 participates in the framework formation of both the nacreous layer and prismatic layer. In vitro experiment on the function of MSI7 suggested that it accelerates the nucleation and precipitation of CaCO(3). Taken together, we have identified a novel matrix protein of the pearl oyster, which may play an important role in determining the texture of nacre.     

Dermatopontin, a shell matrix protein gene from pearl oyster Pinctada martensii, participates in nacre formation

Dermatopontin (DPT) is identified as a major component of the shell matrix protein. However, its exact function in the shell formation remains obscure. In this study, we described the characteristic and function of DPT gene from Pinctada martensii. DPT cDNA was 797bp long, containing an open reading fragment (ORF) of 537bp encoding a polypeptide of 178 amino acids with an estimated molecular mass of 21.4kDa and theoretical isoelectric point of 5.97. The 5' untranslated region (UTR) was 11bp and the 3'UTR was 249 with 18bp poly (A) tail. In the peptide, there was a signal sequence, six potential phosphorylation sites, one glycosylation site and eight cysteine residues. Moreover, a sequence motif (D-R-X-W/F/Y-X-F/Y/I/L/M-X(1-2)-C) was contained and repeated itself three times in the entire sequence. DPT mRNA was constitutively expressed in all studied tissues with the most abundant mRNA in the mantle, which was nacre formation-related tissue. After decreasing DPT expression using RNA interference (RNAi) technology in the mantle, the nacreous layer showed a disordered growth; whereas the prismatic layer of the shells has no significant changes. These results suggested that DPT obtained in this study was a constitutive matrix protein and participated in nacre formation in P. martensii.     

Characterization of MRNP34, a novel methionine-rich nacre protein from the pearl oysters

Nacre of the Pinctada pearl oyster shells is composed of 98% CaCO₃ and 2% organic matrix. The relationship between the organic matrix and the mechanism of nacre formation currently constitutes the main focus regarding the biomineralization process. In this study, we isolated a new nacre matrix protein in P. margaritifera and P. maxima, we called Pmarg- and Pmax-MRNP34 (methionine-rich nacre protein). MRNP34 is a secreted hydrophobic protein, which is remarkably rich in methionine, and which is specifically localised in mineralizing the epithelium cells of the mantle and in the nacre matrix. The structure of this protein is drastically different from those of the other nacre proteins already described. This unusual methionine-rich protein is a new member in the growing list of low complexity domain containing proteins that are associated with biocalcifications. These observations offer new insights to the molecular mechanisms of biomineralization.     

A novel extrapallial fluid protein controls the morphology of nacre lamellae in the pearl oyster, Pinctada fucata

Mollusk shell nacre is known for its superior mechanical properties and precisely controlled biomineralization process. However, the question of how mollusks control the morphology of nacre lamellae remains unresolved. Here, a novel 38-kDa extrapallial fluid (EPF) protein, named amorphous calcium carbonate-binding protein (ACCBP), may partially answer this question. Although sequence analysis indicated ACCBP is a member of the acetylcholine-binding protein family, it is actively involved in the shell mineralization process. In vitro, ACCBP can inhibit the growth of calcite and induce the formation of amorphous calcium carbonate. When ACCBP functions were restrained in vivo, the nacre lamellae grew in a screw-dislocation pattern, and low crystallinity CaCO(3) precipitated from the EPF. Crystal binding experiments further revealed that ACCBP could recognize different CaCO(3) crystal phases and crystal faces. With this capacity, ACCBP could modify the morphology of nacre lamellae by inhibiting the growth of undesired aragonite crystal faces and meanwhile maintain the stability of CaCO(3)-supersaturated body fluid by ceasing the nucleation and growth of calcite. Furthermore, the crystal growth inhibition capacity of ACCBP was proved to be directly related to its acetylcholine-binding site. Our results suggest that a "safeguard mechanism" of undesired crystal growth is necessary for shell microstructure formation.     

The structure-function relationship of MSI7, a matrix protein from pearl oyster Pinctada fucata

We previously identified a matrix protein, MSI7, from pearl oyster Pinctada fucata. According to the structural analysis, the DGD site in the N-terminal of MSI7 is crucial for its role in the shell formation. In this study, we expressed a series of recombinant MSI7 proteins, including the wild-type and several mutants directed at the DGD site, using an Escherichia coli expression system to reveal the structure-function relationship of MSI7. Furthermore, in vitro crystallization, crystallization speed assay, and circular dichroism spectrometry were carried out. Results indicated that wild-type MSI7 could induce the nucleation of aragonite and inhibit the crystallization of calcite. However, none of the mutants could induce the nucleation of aragonite, but all of them could inhibit the crystallization of calcite to some extent. And all the proteins accelerated the crystallization process. Taken together, the results indicated that MSI7 could contribute to aragonite crystallization by inducing the nucleation of aragonite and inhibiting the crystallization of calcite, which agrees with our prediction about its role in the nacreous layer formation of the shell. The DGD site was critical for the induction of the nucleation of aragonite.     

A novel matrix protein family participating in the prismatic layer framework formation of pearl oyster, Pinctada fucata

Understanding the molecular composition and the formation mechanism of shell matrix framework is of great interest for biomineralization in mollusk shell. The cDNAs encoding a novel matrix protein family (KRMP) were cloned from the mantle of pearl oyster, Pinctada fucata. Analysis of the deduced amino acid sequences revealed that KRMP have a high proportion of lysine, glycine, and tyrosine, and their predict isoelectric points are higher than any other identified shell matrix protein to our knowledge. The deduced amino acid sequences of KRMP can be divided into three regions, including an N-terminal signal peptide, a lysine-rich basic region interacting with acidic proteins or CO(3)(2-), and a Gly/Tyr-rich region involved in the protein cross-link via quinone-tanning process. RT-PCR and in situ hybridization demonstrated that KRMP mRNA was specifically expressed in the mantle edge, involved in the prismatic layer formation. Taken together, it seems that KRMP is a matrix protein family participating in the framework formation of prismatic layer.     

cDNA cloning and characterization of a novel calmodulin-like protein from pearl oyster Pinctada fucata

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 identified 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 calcium. CaLP also displays typical Ca(2+)-dependent electrophoretic shift, Ca(2+)-binding activity and significant Ca(2+)-induced conformational changes. 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 different role from CaM in the regulation of oyster calcium metabolism.     

Tissue inhibitor of metalloproteinase gene from pearl oyster Pinctada martensii participates in nacre formation

Tissue inhibitors of metalloproteinases (TIMPs) are nature inhibitors of matrix metalloproteinases and play a vital role in the regulation of extracellular matrix turnover, tissue remodeling and bone formation. In this study, the molecular characterization of TIMP and its potential function in nacre formation was described in pearl oyster Pinctada martensii. The cDNA of TIMP gene in P. martensii (Pm-TIMP) was 901 bp long, containing a 5′ untranslated region (UTR) of 51 bp, a 3′ UTR of 169 bp, and an open reading fragment (ORF) of 681 bp encoding 226 amino acids with an estimated molecular mass of 23.37 kDa and a theoretical isoelectric point of 5.42; The predicted amino acid sequence had a signal peptide, 13 cysteine residues, a N-terminal domain and a C-terminal domain, similar to that from other species. Amino acid multiple alignment showed Pm-TIMP had the highest (41%) identity to that from Crassostrea gigas. Tissue expression analysis indicated Pm-TIMP was highly expressed in nacre formation related-tissues, including mantle and pearl sac. After decreasing Pm-TIMP gene expression by RNA interference (RNAi) technology in the mantle pallium, the inner nacreous layer of the shells showed a disordered growth. These results indicated that the obtained Pm-TIMP in this study participated in nacre formation.     

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

Mollusk shell formation is a complicated and highly controlled calcium metabolism process. Previous studies revealed that several EF-hand calcium-binding proteins actively participate in the regulation of shell mineralization. In this study, we cloned a full-length cDNA encoding a novel 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 shell mineralization-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 be an important regulator of shell formation. This finding may help better understand the functions of EF-hand proteins on the regulation of mollusk shell formation.     

Pf-ALMP, a novel astacin-like metalloproteinase with cysteine arrays, is abundant in hemocytes of pearl oyster Pinctada fucata

The astacin family metalloproteinase is a family of zinc-dependent endopeptidases which play crucial roles in embryonic development, bone growth and morphogenesis. A cDNA clone encoding a putative astacin-like metalloproteinase (pf-ALMP) was isolated from hemocytes of pearl oyster, Pinctada fucata. The novel metalloproteinase presents a molecular organization close to the astacins, but has a novel C-terminal domain with cysteine arrays. RT-PCR analysis revealed that pf-ALMP was expressed dramatically high in hemocytes, which was affected by lipopolysaccharides (LPS) challenge. High expression of pf-ALMP was also found in gill, gonad and digestion gland, and in situ hybridization demonstrated that pf-ALMP was expressed in the epithelia cells of these tissues. Substrate analysis studies indicated that the recombinant pf-ALMP catalytic domain could digest gelatin. Interestingly, the pf-ALMP also could be involved in cell proliferation processes and the cysteine arrays were necessary for the proliferative activity. Taken together, these studies also help to further understand the functions of astacins which may be related to the processes of molluscan inflammatory response, embryo development, proliferation and shell formation.     

A novel carbonic anhydrase from the mantle of the pearl oyster (Pinctada fucata)

A novel carbonic anhydrase (CA) has been purified from the mantle of the pearl oyster, Pinctada fucata, by ammonium sulfate precipitation and affinity chromatography. Its molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be approximately 38 kDa. Native-PAGE shows that the novel CA can bind a fluorescent probe, 5-dimethylamino-1-naphthalenesulfonamide (DNSA), known to specifically bind carbonic anhydrase. Compared to carbonic anhydrase I (CAI) from human erythrocytes, the novel CA migrates faster indicating that it is more acidic. The effect of an inhibitor on the enzyme activity was also examined. The CA from the mantle showed a weak resistance to acetazolamide (AZ), a specific inhibitor of CA. When DNSA was bound to CA, it caused the wavelength of emission maximum intensity to blue shift to 454 nm upon excitation at 326 nm. Histochemical data indicates that the enzyme is distributed widely throughout the mantle tissue, being concentrated at the edge of the mantle. The evidence presented indicates a function for CA in the process of pearl formation and biomineralization.     

A novel ferritin subunit involved in shell formation from the pearl oyster (Pinctada fucata)

Iron is one of the most important minor elements in the shell of bivalves. This study was designed to investigate the involvement of ferritin, the principal protein for iron storage, in shell formation. A novel ferritin cDNA from the pearl oyster (Pinctada fucata) was isolated and characterized. The ferritin cDNA encodes a 206 amino acid polypeptide, which shares high similarity with snail soma ferritin and the H-chains of mammalian ferritins. Oyster ferritin mRNA shows the highest level of expression in the mantle, the organ for shell formation. In situ hybridization analysis revealed that oyster ferritin mRNA is expressed at the highest level at the mantle fold, a region essential for metal accumulation and contributes to metal incorporation into the shell. Taken together, these results suggest that ferritin is involved in shell formation by iron storage. The identification and characterization of oyster ferritin also helps to further understand the structural and functional properties of molluscan ferritins.     

A novel putative tyrosinase involved in periostracum formation from the pearl oyster (Pinctada fucata)

Tyrosinase (monophenol, L-DOPA: oxygen oxidoreductase, EC 1.14.18.1), a kind of copper-containing phenoloxidase, arouses great interests of scientists for its important role in periostracum formation. A cDNA clone encoding a putative tyrosinase, termed OT47 because of its estimated molecular mass of 47kDa, was isolated from the pearl oyster, Pinctada fucata. This novel tyrosinase shares similarity with the cephalopod tyrosinases and other type 3 copper proteins within two conserved copper-binding sites. RT-PCR analysis showed that OT47 mRNA was expressed only in the mantle edge. Further in situ hybridization analysis and tyrosinase activity staining revealed that OT47 was expressed at the outer epithelial cells of the middle fold, different from early histological results in Mercenaria mercenaria, suggesting a different model of periostracum secretion in P. fucata. Taken together, these results suggest that OT47 is most likely involved in periostracum formation. The identification and characterization of oyster tyrosinase also help to further understand the structural and functional properties of molluscan tyrosinase.     

Cloning and characterization of an mRNA encoding a novel G protein alpha-subunit abundant in mantle and gill of pearl oyster Pinctada fucata

Nacre formation is an ideal model to study biomineralization processes. Although much has been done about biomineralization mechanism of nacre, little is known as to how cellular signaling regulates this process. We are interested in whether G protein signaling plays a role in mineralization. Degenerate primers against conserved amino acid regions of G proteins were employed to amplify cDNA from the pearl oyster Pinctada fucata. As a result, the cDNA encoding a novel G(s)alpha (pfG(s)alpha) from the pearl oyster was isolated. The G(s)alpha cDNA encodes a polypeptide of 377 amino acid residues, which shares high similarity to the octopus (Octopus vulgaris) G(s)alpha. The well-conserved A, C, G (switch I), switch II functional domains and the carboxyl terminus that is a critical site for interaction with receptors are completely identical to those from other mollusks. However, pfG(s)alpha has a unique amino acid sequence, which encodes switch III and interaction sites of adenylyl cyclase respectively. In situ hybridization and Northern blotting analysis revealed that the oyster G(s)alpha mRNA is widely expressed in a variety of tissues, with highest levels in the outer fold of mantle and epithelia of gill, the regions essential for biomineralization. We also show that overexpression of the pfG(s)alpha in mammalian MC3T3-E1 cells resulted in increased cAMP levels. Mutant pfG(s)alpha that has impaired CTX substrate diminished its ability to induce cAMP production. Furthermore, the alkaline phosphatase (ALP) activity, an indicator for mineralization, is induced by the G(s)alpha in MC3T3-E1 cells. These results indicated that G(s)alpha may be involved in regulation of physiological function, particularly in biological biomineralization.     

Purification,characterization, and in vitro mineralization studies of a novel goose eggshell matrix protein,ansocalcin

Biomineralization is an important process in which hard tissues are generated through mineral deposition, often assisted by biomacromolecules. Eggshells, because of their rapid formation via mineralization, are chosen as a model for understanding the fundamentals of biomineralization. This report discusses purification and characterization of various proteins and peptides from goose eggshell matrix. A novel 15-kDa protein (ansocalcin) was extracted from the eggshell matrix, purified, and identified and its role in mineralization evaluated using in vitro crystal growth experiments. The complete amino acid sequence of ansocalcin showed high homology to ovocleidin-17, a chicken eggshell protein, and to C-type lectins from snake venom. The amino acid sequence of ansocalcin was characterized by the presence of acidic and basic amino acid multiplets. In vitro crystallization experiments showed that ansocalcin induced pits on the rhombohedral faces at lower concentrations (<50 microg/ml). At higher concentrations, the nucleation of calcite crystal aggregates was observed. Molecular weight determinations by size exclusion chromatography and sodium dodecyl sulfate -polyacrylamide gel electrophoresis showed reversible concentration-dependent aggregation of ansocalcin in solution. We propose that such aggregated structures may act as a template for the nucleation of calcite crystal aggregates. Similar aggregation of calcite crystals was also observed when crystallizations were performed in the presence of whole goose eggshell extract. These results show that ansocalcin plays a significant role in goose eggshell calcification.     

Biomineralization: functions of calmodulin-like protein in the shell formation of pearl oyster

Calmodulin-like protein (CaLP) was believed to be involved in the shell formation of pearl oyster. However, no further study of this protein was ever performed. In this study, the in vitro crystallization experiment showed that CaLP can modify the morphology of calcite. In addition, aragonite crystals can be induced in the mixture of CaLP and a nacre protein (at 16 kDa), which was detected and purified from the EDTA-soluble matrix of nacre. These results agreed with that of immunohistological staining in which CaLP was detected not only in the organic layer sandwiched between nacre (aragonite) and the prismatic layer (calcite), but also around the prisms of the prismatic layer. Take together, we concluded that (1) CaLP, as a component of the organic layer, can induce the nucleation of aragonite through binding with the 16-kDa protein, and (2) CaLP may regulate the growth of calcite in the prismatic layer.     

The extra C-terminal tail is involved in the conformation, stability changes and the N/C-domain interactions of the calmodulin-like protein from pearl oyster Pinctada fucata

Pearl oyster Pinctada fucata calmodulin-like protein (PfCaLP), containing an extra tail (D150-K161) at the C-terminal, is a novel protein involved in the regulation of oyster calcium metabolism. The purpose of this study is to gain insight into the conformational characteristics of the N/C-domain of PfCaLP, especially the detailed contribution of the extra tail to the Ca(2+)/Mg(2+)-induced conformational changes, the stability of the intact PfCaLP molecule and its C-domain, as well as to the interdomain communications in PfCaLP. Our results demonstrate that a strong interaction exists between the hydrophilic tail and the C-domain of PfCaLP. The extra tail, through affecting the C-domain conformational changes, further influences the migration rate, conformational changes, N/C-domain interactions and exposure of the hydrophobic patches of the intact PfCaLP molecule. Furthermore, the tail could actively regulate the stability of PfCaLP and its C-domain. Our studies are helpful to explain our previous finding that the tail plays important roles in PfCaLP-target interaction in the oyster calcium metabolism.