Guiyou Zhang

The role of matrix proteins in the control of nacreous layer deposition during pearl formation

To study the function of pearl oyster matrix proteins in nacreous layer biomineralization in vivo, we examined the deposition on pearl nuclei and the expression of matrix protein genes in the pearl sac during the early stage of pearl formation. We found that the process of pearl formation involves two consecutive stages: (i) irregular calcium carbonate (CaCO3) deposition on the bare nucleus and (ii) CaCO3 deposition that becomes more and more regular until the mature nacreous layer has formed on the nucleus. The low-expression level of matrix proteins in the pearl sac during periods of irregular CaCO3 deposition suggests that deposition may not be controlled by the organic matrix during this stage of the process. However, significant expression of matrix proteins in the pearl sac was detected by day 30–35 after implantation. On day 30, a thin layer of CaCO3, which we believe was amorphous CaCO3, covered large aragonites. By day 35, the nacreous layer had formed. The whole process is similar to that observed in shells, and the temporal expression of matrix protein genes indicated that their bioactivities were crucial for pearl development. Matrix proteins controlled the crystal phase, shape, size, nucleation and aggregation of CaCO3 crystals.

Novel Basic Protein, PfN23, Functions as Key Macromolecule during Nacre Formation

Background: The microstructure of nacre is controlled by the proteins in them. Results: When PfN23 was knocked down, the shell formation in adults and larvae was suppressed. Conclusion: The basic protein PfN23 is important for the control of crystal growth in nacre. Significance: This might provide a valuable complementary to the classic view that acidic proteins control 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.

Culture of outer epithelial cells from mantle tissue to study shell matrix protein secretion for biomineralization

Mantle tissue plays an important role in shell biomineralization by secreting matrix proteins for shell formation. However, the mechanism by which it regulates matrix protein secretion is poorly understood, largely because of the lack of cellular tools for in vitro study and techniques to evaluate matrix protein secretion. We have isolated the outer epithelial cells of the mantle of the pearl oyster, Pinctada fucata, and evaluated cellular metabolism by measuring the secretion of the matrix protein, nacrein. A novel sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was established to quantify nacrein. Mantle explant culture was demonstrated to provide dissociated tissue cells with high viability. Single dissociated cell types from explant culture were separated by density in a discontinuous Percoll gradient. The outer epithelial cells were isolated from other cell types by their higher density and identified by immunolabeling and ultrastructure analysis. ELISA assays revealed that the outer epithelial cells retained the ability to secrete nacrein in vitro. Moreover, increased nacrein secretion resulted from an increased Ca2+ concentration in the culture media of the outer epithelial cells, in a concentration-dependent manner. These results confirm that outer epithelial cell culture and the ELISA method are useful tools for studying the regulatory mechanisms of shell biomineralization.

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

Background: Thermodynamically unstable magnesium calcite is deposited in the shell of pearl oysters at ambient pressure. Results: The novel acidic matrix protein PfN44 interacts with magnesium to inhibit the deposition of aragonite. Conclusion: PfN44 participates in shell formation by inhibiting aragonite formation. Significance: Results of this study suggest a connection between the matrix protein and magnesium. 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.

Amorphous calcium carbonate precipitation by cellular biomineralization in mantle cell cultures of Pinctada fucata.

The growth of molluscan shell crystals is generally thought to be initiated from the extrapallial fluid by matrix proteins, however, the cellular mechanisms of shell formation pathway remain unknown. Here, we first report amorphous calcium carbonate (ACC) precipitation by cellular biomineralization in primary mantle cell cultures of Pinctada fucata. Through real-time PCR and western blot analyses, we demonstrate that mantle cells retain the ability to synthesize and secrete ACCBP, Pif80 and nacrein in vitro. In addition, the cells also maintained high levels of alkaline phosphatase and carbonic anhydrase activity, enzymes responsible for shell formation. On the basis of polarized light microscopy and scanning electron microscopy, we observed intracellular crystals production by mantle cells in vitro. Fourier transform infrared spectroscopy and X-ray diffraction analyses revealed the crystals to be ACC, and de novo biomineralization was confirmed by following the incorporation of Sr into calcium carbonate. Our results demonstrate the ability of mantle cells to perform fundamental biomineralization processes via amorphous calcium carbonate, and these cells may be directly involved in pearl oyster shell formation.

Patterns of Expression in the Matrix Proteins Responsible for Nucleation and Growth of Aragonite Crystals in Flat Pearls of Pinctada fucata

The initial growth of the nacreous layer is crucial for comprehending the formation of nacreous aragonite. A flat pearl method in the presence of the inner-shell film was conducted to evaluate the role of matrix proteins in the initial stages of nacre biomineralization in vivo. We examined the crystals deposited on a substrate and the expression patterns of the matrix proteins in the mantle facing the substrate. In this study, the aragonite crystals nucleated on the surface at 5 days in the inner-shell film system. In the film-free system, the calcite crystals nucleated at 5 days, a new organic film covered the calcite, and the aragonite nucleated at 10 days. This meant that the nacre lamellae appeared in the inner-shell film system 5 days earlier than that in the film-free system, timing that was consistent with the maximum level of matrix proteins during the first 20 days. In addition, matrix proteins (Nacrein, MSI60, N19, N16 and Pif80) had similar expression patterns in controlling the sequential morphologies of the nacre growth in the inner-film system, while these proteins in the film-free system also had similar patterns of expression. These results suggest that matrix proteins regulate aragonite nucleation and growth with the inner-shell film in vivo.

Identification and Comparison of Amorphous Calcium Carbonate-Binding Protein and Acetylcholine-Binding Protein in the Abalone, Haliotis discus hannai

Nacre has two different microarchitectures: columnar nacre and sheet nacre. We previously identified an important regulator of the morphology of sheet nacre tablets, which was named amorphous calcium carbonate-binding protein (pf-ACCBP). However, little is known about its counterpart in columnar nacre. Moreover, pf-ACCBP shares significant sequence similarity with a group of acetylcholine-binding proteins (AChBP) that participate in neuronal synapses transmission, but the relationships between the two proteins, which are homologous in sequences but disparate in function, have not been studied yet. Here, we identified an amorphous calcium carbonate-binding protein and an acetylcholine-binding protein in the abalone, Haliotis discus hannai, named hdh-ACCBP and hdh-AChBP, respectively. Studies of hdh-ACCBP indicated that it was a counterpart of pf-ACCBP in gastropods that might function similarly in columnar nacre formation and supersaturated extrapallial fluid. Analysis of hdh-AChBP showed that unlike previously identified AChBP, hdh-AChBP was not only expressed in the nervous system but could also be detected in non-nervous system cells, such as the goblet cells of the mantle pallial. Additionally, its expression patterns during embryo and larval development did not accord with ganglion development. These phenomena indicated that AChBP might play more general roles than just in neuronal synapses transmission. Comparison of hdh-ACCBP and hdh-AChBP revealed that they were quite different in their post-translational modification and oligomerization and that they were controlled under different transcriptional regulation systems, consequently obtaining disparate expression profiles. Our results also implied that ACCBP and AChBP might come from a common ancestor through gene duplication and divergence.

Structural characterization of amorphous calcium carbonate-binding protein: an insight into the mechanism of amorphous calcium carbonate formation.

ACC (amorphous calcium carbonate) plays an important role in biomineralization process for its function as a precursor for calcium carbonate biominerals. However, it is unclear how biomacromolecules regulate the formation of ACC precursor in vivo. In the present study, we used biochemical experiments coupled with bioinformatics approaches to explore the mechanisms of ACC formation controlled by ACCBP (ACC-binding protein). Size-exclusion chromatography, chemical cross-linking experiments and negative staining electron microscopy reveal that ACCBP is a decamer composed of two adjacent pentamers. Sequence analyses and fluorescence quenching results indicate that ACCBP contains two Ca²⁺-binding sites. The results of in vitro crystallization experiments suggest that one Ca²⁺-binding site is critical for ACC formation and the other site affects the ACC induction efficiency. Homology modelling demonstrates that the Ca²⁺-binding sites of pentameric ACCBP are arranged in a 5-fold symmetry, which is the structural basis for ACC formation. To the best of our knowledge, this is the first report on the structural basis for protein-induced ACC formation and it will significantly improve our understanding of the amorphous precursor pathway.

Transformation of amorphous calcium carbonate nanoparticles into aragonite controlled by ACCBP

Polymorph selection during shell or pearl formation has been an intriguing issue, especially for those species of interest for human consumption. The polymorph switching of calcium carbonate controlled by amorphous calcium carbonate-binding protein (ACCBP), an extrapallial fluid (EPF) protein from the pearl oyster identified by our group in 2007, is investigated in this research. FTIR and TGA analysis suggest that ACCBP decreases the bound water content of amorphous calcium carbonate (ACC), suggesting that ACCBP may be involved in biogenic anhydrous ACC formation. In vitro crystallization and ACC transformation experiments show that ACCBP induces aragonite formation via an ACC precursor in Mg/Ca = 1 and Mg/Ca = 2 solutions at low temperature. Raman, FTIR, ICP and XPS analyses of the initial-stage ACC nanoparticles in the ACC transformation experiment suggest that this polymorph switching may be controlled by increasing the surface Mg/Ca ratio of the ACC, rather than by regulating the bulk Mg/Ca ratio or the short-range ordered structure. These results suggest that the polymorph selection in nacre or pearl growth may be controlled not only by the nucleating template on the matrix but also by the physicochemical effects of EPF proteins.

Ubiquitylation functions in the calcium carbonate biomineralization in the extracellular matrix.

Mollusks shell formation is mediated by matrix proteins and many of these proteins have been identified and characterized. However, the mechanisms of protein control remain unknown. Here, we report the ubiquitylation of matrix proteins in the prismatic layer of the pearl oyster, Pinctada fucata. The presence of ubiquitylated proteins in the prismatic layer of the shell was detected with a combination of western blot and immunogold assays. The coupled ubiquitins were separated and identified by Edman degradation and liquid chromatography/mass spectrometry (LC/MS). Antibody injection in vivo resulted in large amounts of calcium carbonate randomly accumulating on the surface of the nacreous layer. These ubiquitylated proteins could bind to specific faces of calcite and aragonite, which are the two main mineral components of the shell. In the in vitro calcium carbonate crystallization assay, they could reduce the rate of calcium carbonate precipitation and induce the calcite formation. Furthermore, when the attached ubiquitins were removed, the functions of the EDTA-soluble matrix of the prismatic layer were changed. Their potency to inhibit precipitation of calcium carbonate was decreased and their influence on the morphology of calcium carbonate crystals was changed. Taken together, ubiquitylation is involved in shell formation. Although the ubiquitylation is supposed to be involved in every aspect of biophysical processes, our work connected the biomineralization-related proteins and the ubiquitylation mechanism in the extracellular matrix for the first time. This would promote our understanding of the shell biomineralization and the ubiquitylation processes.