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.     

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.     

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.     

A Comparative Study of the Shell Matrix Protein Aspein in Pterioid Bivalves

Aspein is one of the unusually acidic shell matrix proteins originally identified from the pearl oyster Pinctada fucata. Aspein is thought to play important roles in the shell formation, especially in calcite precipitation in the prismatic layer. In this study, we identified Aspein homologs from three closely related pterioid species: Pinctada maxima, Isognomon perna, and Pteria penguin. Our immunoassays showed that they are present in the calcitic prismatic layer but not in the aragonitic nacreous layer of the shells. Sequence comparison showed that the Ser-Glu-Pro and the Asp-Ala repeat motifs are conserved among these Aspein homologs, indicating that they are functionally important. All Aspein homologs examined share the Asp-rich D-domain, suggesting that this domain might have a very important function in calcium carbonate formation. However, sequence analyses showed a significantly high level of variation in the arrangement of Asp in the D-domain even among very closely related species. This observation suggests that specific arrangements of Asp are not required for the functions of the D-domain.     

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.     

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.     

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 (CaCO(3)) deposition on the bare nucleus and (ii) CaCO(3) 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 CaCO(3) 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 CaCO(3), which we believe was amorphous CaCO(3), 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 CaCO(3) crystals.     

Identification of genes directly involved in shell formation and their functions in pearl oyster, Pinctada fucata

Mollusk shell formation is a fascinating aspect of biomineralization research. Shell matrix proteins play crucial roles in the control of calcium carbonate crystallization during shell formation in the pearl oyster, Pinctada fucata. Characterization of biomineralization-related genes during larval development could enhance our understanding of shell formation. Genes involved in shell biomineralization were isolated by constructing three suppression subtractive hybridization (SSH) libraries that represented genes expressed at key points during larval shell formation. A total of 2,923 ESTs from these libraries were sequenced and gave 990 unigenes. Unigenes coding for secreted proteins and proteins with tandem-arranged repeat units were screened in the three SSH libraries. A set of sequences coding for genes involved in shell formation was obtained. RT-PCR and in situ hybridization assays were carried out on five genes to investigate their spatial expression in several tissues, especially the mantle tissue. They all showed a different expression pattern from known biomineralization-related genes. Inhibition of the five genes by RNA interference resulted in different defects of the nacreous layer, indicating that they all were involved in aragonite crystallization. Intriguingly, one gene (UD_Cluster94.seq.Singlet1) was restricted to the 'aragonitic line'. The current data has yielded for the first time, to our knowledge, a suite of biomineralization-related genes active during the developmental stages of P. fucata, five of which were responsible for nacreous layer formation. This provides a useful starting point for isolating new genes involved in shell formation. The effects of genes on the formation of the 'aragonitic line', and other areas of the nacreous layer, suggests a different control mechanism for aragonite crystallization initiation from that of mature aragonite growth.     

Identification of chitin in the prismatic layer of the shell and a chitin synthase gene from the Japanese pearl oyster, Pinctada fucata

The shell of the Japanese pearl oyster, Pinctada fucata, consists of two layers, the prismatic layer on the outside and the nacreous layer on the inside, both of which comprise calcium carbonate and organic matrices. Previous studies indicate that the nacreous organic matrix of the central layer of the framework surrounding the aragonite tablet is beta-chitin, but it remains unknown whether organic matrices in the prismatic layer contain chitin or not. In the present study, we identified chitin in the prismatic layer of the Japanese pearl oyster, Pinctada fucata, with a combination of Calcofluor White staining with IR and NMR spectral analyses. Furthermore, we cloned a cDNA encoding chitin synthase (PfCHS1) that produces chitin, contributing to the formation of the framework for calcification in the shell.     

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.     

The Molecular Evolution of the Pif Family Proteins in Various Species of Mollusks

Various novel proteins have been identified from many kinds of mollusk shells. Although such matrix proteins are believed to play important roles in the calcium carbonate crystal formation of shells, no common proteins that interact with calcium carbonate or that are involved in the molecular mechanisms behind shell formation have been identified. Pif consists of two proteins, Pif 80 and Pif 97, which are encoded by a single mRNA. Pif 80 was identified as a key acidic protein that regulates the formation of the nacreous layer in Pinctada fucata, while Pif 97 has von Willebrand factor type A (VWA) and chitin-binding domains. In this study, we identified Pif homologues from Pinctada margaritifera, Pinctada maxima, Pteria penguin, Mytilus galloprovincialis, and in the genome database of Lottia gigantea in order to compare their primary protein sequences. The VWA and chitin-binding domains are conserved in all Pif 97 homologues, whereas the amino acid sequences of the Pif 80 regions differ markedly among the species. Sequence alignment revealed the presence of a novel significantly conserved sequence between the chitin-binding domain and the C-terminus of Pif 97. Further examination of the Pif 80 regions suggested that they share a sequence that is similar to the laminin G domain. These results indicate that all Pif molecules in bivalves and gastropods may be derived from a common ancestral gene. These comparisons may shed light on the correlation between molecular evolution and morphology in mollusk shell microstructure.     

Studies on the egg shell (oviducal and oviposited) of Chelonia mydas L.

The outer calcified surface of the turtle egg shell consists primarily of crystalline aggregates of calcium carbonate in its aragonite form, together with a small amount (< 5 %) of calcitic material. The latter is first deposited to be followed by aragonite deposition.In the first instance, calcification occurs on the rims of discrete pits formed by the lateral deflection of the ends of soft shell membrane fibres. As crystal deposition continues these pits become filled in and eventually occluded.Micro- and X-ray diffraction analyses of the calcified layer indicate the presence of phosphorus and sulphur. The effects of these elements on the type of crystal deposited, (i.e., aragonite or calcite) is discussed.     

Skeletal chemistry of Nautilus and its taxonomic significance

The strontium (Sr) and magnesium (Mg) chemistry of the shell wall and septum as well as the spherulitic-prismatic and nacreous layers of these structures was determined for Nautilus species: N. belauensis, N. macromphalus, N. pompilius and N. scrobiculatus. Each species of Nautilus exhibits greater variability and higher concentrations of Mg in juvenile portions of the shell than in more mature portions of the shell. This decrease in the variability and amount of Mg in the aragonite lattice suggests a physiochemical system which becomes more efficient with time relative to carbonate production. Statistically significant differences in the Sr and Mg content of spherulitic-prismatic and nacreous layers of the shell and septum indicate that these layers were formed from extracellular fluids of different compositions. Concentrations of Sr and Mg in aragonite of the shell wall are characteristic for each species and sufficiently invariant within species to allow species of Nautilus to be distinguished statistically on the basis of either the Sr or Mg content of the shell wall or the Mg content of septa.     

Identification and Characterization of the Lysine-Rich Matrix Protein Family in <Emphasis Type="Italic">Pinctada fucata</Emphasis>: Indicative of Roles in Shell Formation

Mantle can secret matrix proteins playing key roles in regulating the process of shell formation. The genes encoding lysine-rich matrix proteins (KRMPs) are one of the most highly expressed matrix genes in pearl oysters. However, the expression pattern of KRMPs is limited and the functions of them still remain unknown. In this study, we isolated and identified six new members of lysine-rich matrix proteins, rich in lysine, glycine and tyrosine, and all of them are basic matrix proteins. Combined with four members of the KRMPs previously reported, all these proteins can be divided into three subclasses according to the results of phylogenetic analyses: KRMP1–3 belong to subclass KPI, KRMP4–5 belong to KPII, and KRMP6–10 belong to KPIII. Three subcategories of lysine-rich matrix proteins are highly expressed in the D-phase, the larvae and adult mantle. Lysine-rich matrix proteins are involved in the shell repairing process and associated with the formation of the shell and pearl. What’s more, they can cause abnormal shell growth after RNA interference. In detail, KPI subgroup was critical for the beginning formation of the prismatic layer; both KPII and KPIII subgroups participated in the formation of prismatic layer and nacreous layer. Compared with different temperatures and salinity stimulation treatments, the influence of changes in pH on KRMPs gene expression was the greatest. Recombinant KRMP7 significantly inhibited CaCO₃ precipitation, changed the morphology of calcite, and inhibited the growth of aragonite in vitro. Our results are beneficial to understand the functions of the KRMP genes during shell formation.     

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.     

STRUCTURE OF THE CONCHIOLIN CASES OF THE PRISMS IN MYTILUS EDULIS LINNE

The prisms in the shell of Mytilus edulis Linné are calcite needles. Their small size and their thin conchiolin cases distinguish them from the prisms of many other species of mollusks. These Mytilus prisms have been studied with the electron microscope. The material consisted of positive replicas of surfaces of the prismatic layer, etched with chelating agents, and of preparations of tubular cases from decalcified prisms which were compared with the conchiolin from decalcified mother-of-pearl of the same species. In the replicas, the cases appear as thin pellicles in the intervals between the prism crystals. Both the prism cases and the nacreous conchiolin, disintegrated by exposure to ultrasonic waves and sedimented on supporting films, appear in the form of tightly meshed, reticulated sheets, described as "tight pelecypod pattern" in former studies on nacreous conchiolin of Mytilus. The results show that in the shell of this species the same conchiolin structure is associated with aragonite in mother-of-pearl and with calcite in the prismatic layer.