A Novel Acidic Matrix Protein,PfN44, Stabilizes Magnesium Calcite to Inhibit the Crystallization of Aragonite

Magnesium is widely used to control calcium carbonate deposition in the shell of pearl oysters. Matrix proteins in the shell are responsible for nucleation and growth of calcium carbonate crystals. However, there is no direct evidence supporting a connection between matrix proteins and magnesium. Here, we identified a novel acidic matrix protein named PfN44 that affected aragonite formation in the shell of the pearl oyster Pinctada fucata. Using immunogold labeling assays, we found PfN44 in both the nacreous and prismatic layers. In shell repair, PfN44 was repressed, whereas other matrix proteins were up-regulated. Disturbing the function of PfN44 by RNAi led to the deposition of porous nacreous tablets with overgrowth of crystals in the nacreous layer. By in vitro circular dichroism spectra and fluorescence quenching, we found that PfN44 bound to both calcium and magnesium with a stronger affinity for magnesium. During in vitro calcium carbonate crystallization and calcification of amorphous calcium carbonate, PfN44 regulated the magnesium content of crystalline carbonate polymorphs and stabilized magnesium calcite to inhibit aragonite deposition. Taken together, our results suggested that by stabilizing magnesium calcite to inhibit aragonite deposition, PfN44 participated in P. fucata shell formation. These observations extend our understanding of the connections between matrix proteins and magnesium.     

Direct observation of the transition from calcite to aragonite growth as induced by abalone shell proteins

The mixture of EDTA-soluble proteins found in abalone nacre are known to cause the nucleation and growth of aragonite on calcite seed crystals in supersaturated solutions of calcium carbonate. Past atomic force microscope studies of the interaction of these proteins with calcite crystals did not observe this transition because no information about the crystal polymorph on the surface was obtained. Here we have used the atomic force microscope to directly observe changes in the atomic lattice on a calcite seed crystal after the introduction of abalone shell proteins. The observed changes are consistent with a transition to (001) aragonite growth on a (1014) calcite surface.     

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.     

A new matrix protein family related to the nacreous layer formation of Pinctada fucata

We have isolated a new matrix protein family (N16) which is specific to the nacreous layer of the Japanese pearl oyster, Pinctada fucata, and have cloned and characterized the cDNAs coding for the components. Analysis of the deduced amino acid sequence revealed that N16 showed no definitive homology with other proteins. The in vitro studies of the crystallization clarified that N16 induced aragonite crystals when fixed on the substrate but inhibited crystal formation without it. The aragonite crystals showed platy morphology different from those formed inorganically, and long intervals of incubation resulted in crystalline layers highly similar to the nacreous layer.     

A scanning electron microscopic study of the inorganic and organic matrices comprising the mature shell of Amblema, a fresh-water mollusc

The scanning electron microscope has been used to describe the morphology of the mature shell in a fresh-water bivalve. The structure of the organic and inorganic components within the nacre, the myostracum, and the prismatic layer is described. A transitional or intermediate zone, interposed between the prismatic layer and the nacre, was identified. In demineralized samples, the organic component of the nacre was found to consist of parallel matricial sheets interconnected by irregular transverse bridges. The structure of the mineral component of the nacre was found to vary with the method of specimen preparation. With polished-etched samples, brick-like units were seen. When shells were simply broken and fixed in osmium, the layers of nacreous material consisted of fusing rhomboidal crystals of aragonite which demonstrated subconchoidal fractures. On the inner surface of the shell, the rhomboidal crystals showed an apparent spiral growth pattern. The myostracum was characterized by regions of modified nacreous structure consisting of enlarged aragonite crystals with a pyramidal morphology. The peripheral aspect of the muscle scars was characterized by rhomboidal crystals, the latter fusing to form the typical nacreous laminae. The uniqueness of the anterior adductor scar is exemplified by the presence of pores, each pore walled by pyramidal units, for the insertion of adductor fibres. In most regions of the shell, the prismatic layer consisted of one prism unit thickness with a height of approximately 225–250 μm. However, in two specialized regions of the shell, this layer was seen to consist of multiple layers of stacked prisms. The organic matrices of the prismatic layer are arranged in a honeycomb-like arrangement and packed with mineralized spherical subunits.     

CaCO3 biomineralization: acidic 8-kDa proteins isolated from aragonitic abalone shell nacre can specifically modify calcite crystal morphology

Acidic proteins from many biogenic minerals are implicated in directing the formation of crystal polymorphs and morphologies. We characterize the first extremely acidic proteins purified from biomineralized aragonite. These abalone nacre proteins are two variants of 8.7 and 7.8 kDa designated AP8 (for aragonite proteins of approximately 8 kDa). The AP8 proteins have compositions dominated by Asx ( approximately 35 mol %) and Gly ( approximately 40 mol %) residues, suggesting that their structures have high Ca(2+)-binding capacity and backbone flexibility. The growth of asymmetrically rounded CaCO(3) crystals in the presence of AP8 reveals that both proteins preferentially interact with specific locations on the crystal surface. In contrast, CaCO(3) crystals grown with nacre proteins depleted of AP8 retain the morphology of unmodified calcite rhombohedra. Our observations thus identify sites of protein-mineral interaction and provide evidence to support the long-standing theory that acidic proteins are more effective crystal-modulators than other proteins from the same biomineralized material.     

Electron diffraction of mollusc shell organic matrices and their relationship to the mineral phase

Electron diffraction patterns showing orientation of the chitin and protein constituents of the insoluble organic matrix of mollusc shell nacreous layers have been obtained, using low dose conditions and samples cooled to −100°C. Diffraction patterns of the aragonite crystals were also observed. In a gastropod and a bivalve the spatial relationship between the organic matrix constituents and the aragonite crystallographic axes were shown to be the same as was previously observed for a cephalopod using X-ray diffraction, supporting the notion that mineral crystal growth occurs epitaxially upon a matrix template.     

Perlwapin, an abalone nacre protein with three four-disulfide core (whey acidic protein) domains, inhibits the growth of calcium carbonate crystals

We have isolated a new protein from the nacreous layer of the shell of the sea snail Haliotis laevigata (abalone). Amino acid sequence analysis showed the protein to consist of 134 amino acids and to contain three sequence repeats of approximately 40 amino acids which were very similar to the well-known whey acidic protein domains of other proteins. The new protein was therefore named perlwapin. In addition to the major sequence, we identified several minor variants. Atomic force microscopy was used to explore the interaction of perlwapin with calcite crystals. Monomolecular layers of calcite crystals dissolve very slowly in deionized water and recrystallize in supersaturated calcium carbonate solution. When perlwapin was dissolved in the supersaturated calcium carbonate solution, growth of the crystal was inhibited immediately. Perlwapin molecules bound tightly to distinct step edges, preventing the crystal layers from growing. Using lower concentrations of perlwapin in a saturated calcium carbonate solution, we could distinguish native, active perlwapin molecules from denaturated ones. These observations showed that perlwapin can act as a growth inhibitor for calcium carbonate crystals in saturated calcium carbonate solution. The function of perlwapin in nacre growth may be to inhibit the growth of certain crystallographic planes in the mineral phase of the polymer/mineral composite nacre.     

Characterization of two molluscan crystal-modulating biomineralization proteins and identification of putative mineral binding domains

Ethylenediamine-tetraacetic acid extracted water-soluble matrix proteins in molluscan shells secreted from the mantle epithelia are believed to control crystal nucleation, morphology, orientation, and phase of the deposited mineral. Previously, atomic force microscopy demonstrated that abalone nacre proteins bind to growing step edges and to specific crystallographic faces of calcite, suggesting that inhibition of calcite growth may be one of the molecular processes required for growth of the less thermodynamically stable aragonite phase. Previous experiments were done with protein mixtures. To elucidate the role of single proteins, we have characterized two proteins isolated from the aragonitic component of nacre of the red abalone, Haliotis rufescens. These proteins, purified by hydrophobic interaction chromatography, are designated AP7 and AP24 (aragonitic protein of molecular weight 7 kDa and 24 kDa, respectively). Degenerate oligonucleotide primers corresponding to N-terminal and internal peptide sequences were used to amplify cDNA clones by a polymerase chain reaction from a mantle cDNA library; the deduced primary amino acid sequences are presented. Preliminary crystal growth experiments demonstrate that protein fractions enriched in AP7 and AP24 produced CaCO(3) crystals with morphology distinct from crystals grown in the presence of the total mixture of soluble aragonite-specific proteins. Peptides corresponding to the first 30 residues of the N-terminal sequences of both AP7 and AP24 were generated. The synthetic peptides frustrate the progression of step edges of a growing calcite surface, indicating that sequence features within the N-termini of AP7 and AP24 include domains that interact with CaCO(3). CD analyses demonstrate that the N-terminal peptide sequences do not possess significant percentages of alpha-helix or beta-strand secondary structure in solution. Instead, in both the presence and absence of Ca(II), the peptides retain unfolded conformations that may facilitate protein-mineral interaction.     

N40, a novel nonacidic matrix protein from pearl oyster nacre, facilitates nucleation of aragonite in vitro

A novel nonacidic matrix protein from pearl oyster nacre has been purified by cation-exchange chromatography. It was designated N40 for the nacreous protein of approximately 40 kDa. On the basis of the extraction method (with Tris-buffered Milli-Q water) and amino acid compositions (Gly- and Ala-rich), N40 was inferred to be a conventional "insoluble matrix protein". Crystallization experiments showed that N40 could facilitate the nucleation of aragonite drastically. So far, among the macromolecules that have been purified from the shell, N40 is an exclusive protein that can nucleate aragonite by itself, without the need for adsorption to a substrate. Thus, the present study has proposed the possibility that the nonacidic shell protein (maybe a conventional "insoluble framework protein") can also directly participate in aragonite nucleation and even act as a nucleation site. It is a valuable supplement to the classic biomineralization theory, in which the soluble acidic proteins of the shell are generally believed to function as a nucleation site.     

Organization pattern of nacre in Pteriidae (Bivalvia: Mollusca) explained by crystal competition

Bivalve nacre is a brick-wall-patterned biocomposite of aragonite platelets surrounded by organic matter. SEM-electron back scatter diffraction analysis of nacre of the bivalve family Pteriidae reveals that early aragonite crystals grow with their c-axes oriented perpendicular to the growth surface but have their a- and b-axes disoriented. With the accumulation of successive lamellae, crystals progressively orient themselves with their b-axes mutually parallel and towards the growth direction. We propose that progressive orientation is a result of competition between nacre crystals at the growth front of lamellae, which favours selection of crystals whose fastest growth axis (b-axis) is oriented parallel to the direction of propagation of the lamella. A theoretical model has been developed, which simulates competition of rhombic plates at the lamellar growth front as well as epitaxial growth of crystals onto those of the preceding lamella. The model predicts that disordered nacre progressively produces bivalve-like oriented nacre. As growth fronts become diffuse (as is the common case in bivalves) it takes longer for nacre to become organized. Formation of microdomains of nacre platelets with different orientations is also reproduced. In conclusion, not only the organic matrix component, but also the mineral phase plays an active role in organizing the final microstructure.     

Identification of genes associated with shell color in the black-lipped pearl oyster,Pinctada margaritifera

Background

Color polymorphism in the nacre of pteriomorphian bivalves is of great interest for the pearl culture industry. The nacreous layer of the Polynesian black-lipped pearl oyster Pinctada margaritifera exhibits a large array of color variation among individuals including reflections of blue, green, yellow and pink in all possible gradients. Although the heritability of nacre color variation patterns has been demonstrated by experimental crossing, little is known about the genes involved in these patterns. In this study, we identify a set of genes differentially expressed among extreme color phenotypes of P. margaritifera using a suppressive and subtractive hybridization (SSH) method comparing black phenotypes with full and half albino individuals.

Results

Out of the 358 and 346 expressed sequence tags (ESTs) obtained by conducting two SSH libraries respectively, the expression patterns of 37 genes were tested with a real-time quantitative PCR (RT-qPCR) approach by pooling five individuals of each phenotype. The expression of 11 genes was subsequently estimated for each individual in order to detect inter-individual variation. Our results suggest that the color of the nacre is partially under the influence of genes involved in the biomineralization of the calcitic layer. A few genes involved in the formation of the aragonite tablets of the nacre layer and in the biosynthesis chain of melanin also showed differential expression patterns. Finally, high variability in gene expression levels were observed within the black phenotypes.

Conclusions

Our results revealed that three main genetic processes were involved in color polymorphisms: the biomineralization of the nacreous and calcitic layers and the synthesis of pigments such as melanin, suggesting that color polymorphism takes place at different levels in the shell structure. The high variability of gene expression found within black phenotypes suggests that the present work should serve as a basis for future studies exploring more thoroughly the expression patterns of candidate genes within black phenotypes with different dominant iridescent colors.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1776-x) contains supplementary material, which is available to authorized users.     

Mollusk shell formation: mapping the distribution of organic matrix components underlying a single aragonitic tablet in nacre

Control over mineral formation in mollusk shells is exerted by the macromolecules of the organic matrix. Using histochemical methods, we mapped the carboxylates and sulfates of proteins and polysaccharides on the surfaces of decalcified interlamellar matrices from the nacreous shell layer of the cephalopod Nautilus pompilius, expanding upon an earlier study by Crenshaw and Ristedt [Crenshaw, M.A., Ristedt, H., 1976. The histochemical localization of reactive groups in septal nacre from Nautilus pompilius. In: Watabe, N., Wilbur, K.M. (Ed.), The Mechanisms of Mineralization in the Invertebrates and Plants. University of South Carolina Press, Colombia, pp. 355-367]. We observed four different zones underlying a single crystal: (1) a central spot rich in carboxylates; (2) a central ring-shaped area rich in sulfates; (3) an area between the central nucleation region and the imprint periphery containing carboxylates, and (4) the intertabular matrix, rich in carboxylates and sulfates. We also mapped matrix functional groups on the nacreous matrix surfaces of the bivalve Atrina rigida, but did not identify well-defined zones. Immuno-mapping of the constituents of the aragonite-nucleating protein fraction from Atrina nacre showed that these macromolecules are located both in the intertabular matrix and in the center of the crystal imprints for both Atrina and Nautilus matrix surfaces. Their presence at the latter location is consistent with their purported role in aragonite nucleation. The observed differentiation in the distribution of matrix components and their functional groups shows that the different stages of single crystal growth are highly controlled by the matrix.     

A Novel Matrix Protein p10 from the Nacre of Pearl Oyster (Pinctada fucata) and Its Effects on Both CaCO3 Crystal Formation and Mineralogenic Cells

A novel matrix protein, designated as p10 because of its apparent molecular mass of 10 kDa, was isolated from the nacreous layer of pearl oyster (Pinctada fucata) by reverse-phase high-performance liquid chromatography. In vitro crystallization experiments showed that p10 could accelerate the nucleation of calcium carbonate crystals and induce aragonite formation, suggesting that it might play a key role in nacre biomineralization. As nacre is known to contain osteogenic factors, two mineralogenic cell lines, MRC-5 fibroblasts and MC3T3-E1 preosteoblasts, were used to investigate the biological activity of p10. The results showed that p10 could increase alkaline phosphatase activity, an early marker of osteoblast differentiation, while the viability of MRC-5 and MC3T3-E1 remained unchanged after treatment of p10. Taken together, the findings led to identification of a novel matrix protein from the nacre of P. fucata that plays a role in both the mineral phase and in the differentiation of the cells involved in biomineralization.     

Ultrastructural Characteristics of the Nacre in Some Gastropods

The nacreous layer in Gibbula, Calliostoma, Trochus and Haliotis is described on the basis of scanning electron microscopic studies. The central part of each nacreous tablet contains a significant amount of calcified organic matrix which is insoluble in a chromium sulphate and a 25% glutaraldehyde solution. In most cases, the tablet is subdivided by radial vertical organic membranes into a varying number (2 to 50) of crystalline sectors. These sectors represent polysynthetically twinned crystal individuals which form cyclic or interpenetrant twins. The nacreous tablets in gastropods are compared with those in bivalves, and with the non-biogenic aragonite. The mechanical properties of the nacre, and the effects of the interlamellar conchiolin membranes upon the nucleation of the tablets, are discussed.     

Abalone nacre insoluble matrix induces growth of flat and oriented aragonite crystals

Mollusc shell formation takes place in a preformed extracellular matrix, composed of insoluble chitin, coated with proteins and dissolved macromolecules. The water-soluble matrix is known to have a strong influence on the growth of CaCO(3), whereas the role of the insoluble matrix on mineralization is unclear. Therefore, we mineralized the EDTA (ethylenediaminetetraacetic acid) insoluble organic matrix of abalone nacre with a modified double-diffusion set-up, where the diffusing solutions were constantly renewed. Control experiments were performed with cellulose and chitosan foils. The mineralized matrices/foils were analyzed with SEM. We show that the insoluble matrix of abalone nacre induces the growth of flat and roughly polygonal CaCO(3) crystals. In some of the experiments with the insoluble matrix, the growth of three-dimensional parallel sheets of densely packed platelets inside the insoluble matrix was observed. XRD on these samples revealed that they consist of oriented aragonite.     

Expression of genes responsible for biomineralization of Pinctada fucata during development

This study compares the expression levels of nacrein, N16, MSI60, Prismalin-14, aspein and MSI31 genes during the ontogeny of Pinctada fucata. Several novel findings were obtained: 1) The early calcitic prismatic layer was distinguished as a thin membrane-like structure. 2) Initial formation of the nacreous layer started from the mantle pallial region at the age of 31 days. 3) 18S rRNA of P. fucata was determined to be more suitable as a real-time PCR reference gene compared with GAPDH and β-actin genes. 4) A relationship was recognized between the expression levels of the above six organic matrix genes and biomineralization of the larval shell. The lack of calcite in the shells of the veliger and pediveliger stages, when MSI31 and Prismalin-14 genes were expressed, makes a role of polymorph control by these genes less likely. The hypothetical involvement of N16 and MSI60 proteins in aragonitic nacreous layer formation was corroborated by the expression levels of N16 and MSI60 genes during ontogeny. Our results are important with respect to the control of CaCO₃ crystal polymorphism and shell microstructures in P. fucata.     

Novel basic protein, PfN23, functions as key macromolecule during nacre formation

The fine microstructure of nacre (mother of pearl) illustrates the beauty of nature. Proteins found in nacre were believed to be "natural hands" that control nacre formation. In the classical view of nacre formation, nucleation of the main minerals, calcium carbonate, is induced on and by the acidic proteins in nacre. However, the basic proteins were not expected to be components of nacre. Here, we reported that a novel basic protein, PfN23, was a key accelerator in the control over crystal growth in nacre. The expression profile, in situ immunostaining, and in vitro immunodetection assays showed that PfN23 was localized within calcium carbonate crystals in the nacre. Knocking down the expression of PfN23 in adults via double-stranded RNA injection led to a disordered nacre surface in adults. Blocking the translation of PfN23 in embryos using morpholino oligomers led to the arrest of larval development. The in vitro crystallization assay showed that PfN23 increases the rate of calcium carbonate deposition and induced the formation of aragonite crystals with characteristics close to nacre. In addition, we constructed the peptides and truncations of different regions of this protein and found that the positively charged C-terminal region was a key region for the function of PfN23 Taken together, the basic protein PfN23 may be a key accelerator in the control of crystal growth in nacre. This provides a valuable balance to the classic view that acidic proteins control calcium carbonate deposition in nacre.     

几种生物CaCO_3霰石结晶的取向性

ＣａＣＯ３结晶广泛分布于生物界,其主要结晶形式为方解石、霰石及球霰石。用Ｘ－射线衍射法对三角帆蚌及合浦珍珠母贝的珍珠层、墨鱼骨和大黄鱼耳石的ＣａＣＯ３结晶进行测定,发现各样品均有一定取向性,以三角帆蚌和合浦珍珠母贝珍珠层的取向性为最强,墨鱼骨的取向性次之,大黄鱼耳石的取向性最小,以上材料粉末样的衍射分析表明,各样品对应ｄ值间差异极小,均为Ｘ射线衍射卡（５—０４５３）所表征的ＣａＣＯ３霰石结构。