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.     

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.     

The structure-function relationship analysis of Prismalin-14 from the prismatic layer of the Japanese pearl oyster, Pinctada fucata

The mollusk shell is a hard tissue consisting of calcium carbonate and organic matrices. The organic matrices are considered to play important roles in shell formation. We have previously identified a prismatic layer-specific protein named Prismalin-14, which consists of 105 amino acid residues and includes four structurally characteristic regions; a repeated sequence of Pro-Ile-Tyr-Arg, a Gly/Tyr-rich region and N- and C-terminal Asp-rich regions. Prismalin-14 showed an inhibitory activity on calcium carbonate precipitation and a calcium-binding ability in vitro. In this study, we prepared some molecular species of recombinant proteins including Prismalin-14 and its truncated proteins in an Escherichia coli expression system to reveal a structure-function relationship of Prismalin-14. The results showed that the Gly/Tyr-rich region was responsible for chitin binding and was identified as a novel chitin-binding sequence. On the other hand, both N- and C-terminal Asp-rich regions are related to inhibitory activity on calcium carbonate precipitation in vitro. Immunohistological observation revealed that Prismalin-14 was localized at the acid-insoluble organic framework including chitin. All these results strongly suggest that Prismalin-14 is a framework protein that mediates chitin and calcium carbonate crystals by using its acidic and chitin-binding regions.     

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.     

Molecular Evolution of Mollusc Shell Proteins: Insights from Proteomic Analysis of the Edible Mussel Mytilus

Shell matrix proteins (SMPs) that are embedded within calcified layers of mollusc shells are believed to play an essential role in controlling the biomineral synthesis and in increasing its mechanical properties. Among the wide diversity of mollusc shell textures, nacro-prismatic shells represent a tremendous opportunity for the investigation of the SMP evolution. Indeed, nacro-prismatic texture appears early in Cambrian molluscs and is still present in the shell of some bivalves, gastropods, cephalopods and very likely also, of some monoplacophorans. One key question is to know whether these shells are constructed from similar matrix protein assemblages, i.e. whether they share a common origin. Most of the molecular data published so far are restricted to two genera, the bivalve Pinctada and the gastropod Haliotis. The shell protein content of these two genera are clearly different, suggesting independent origins or considerable genetic drift from a common ancestor. In order to describe putatively conserved mollusc shell proteins, here we have investigated the SMP set of a new bivalve model belonging to another genera, the edible mussel Mytilus, using an up-to-date proteomic approach based on the interrogation of more than 70,000 EST sequences, recently available from NCBI public databases. We describe nine novel SMPs, among which three are completely novel, four are homologues of Pinctada SMPs and two are very likely homologues of Haliotis SMPs. This latter result constitutes the first report of conserved SMPs between bivalves and gastropods. More generally, our data suggest that mollusc SMP set may follow a mosaic pattern within the different mollusc models (Mytilus, Pinctada, Haliotis). We discuss the function of such proteins in calcifying matrices, the molecular evolution of SMP genes and the origin of mollusc nacro-prismatic SMPs.     

The distribution of chitin in larval shells of the bivalve mollusk Mytilus galloprovincialis

The insoluble matrix of larval shells of the marine bivalve mollusk Mytilus galloprovincialis is investigated by confocal laser scanning microscopy using a GFP fusion protein with a chitin-binding domain for labeling of chitinous structures. We show that chitinous material is present in the larval shell, presumably as a chitin-protein complex. We further show that the structure of the chitinous material changes with the development of the larvae. We conclude from the presence of characteristic chitinous structures in certain shell regions that chitin fulfills an important function in the formation and functionality of larval bivalve shells.     

A Bivalve Biomineralization Toolbox

Mollusc shells are a result of the deposition of crystalline and amorphous calcite catalyzed by enzymes and shell matrix proteins (SMP). Developing a detailed understanding of bivalve mollusc biomineralization pathways is complicated not only by the multiplicity of shell forms and microstructures in this class, but also by the evolution of associated proteins by domain co-option and domain shuffling. In spite of this, a minimal biomineralization toolbox comprising proteins and protein domains critical for shell production across species has been identified. Using a matched pair design to reduce experimental noise from inter-individual variation, combined with damage-repair experiments and a database of biomineralization SMPs derived from published works, proteins were identified that are likely to be involved in shell calcification. Eighteen new, shared proteins likely to be involved in the processes related to the calcification of shells were identified by the analysis of genes expressed during repair in Crassostrea gigas, Mytilus edulis, and Pecten maximus. Genes involved in ion transport were also identified as potentially involved in calcification either via the maintenance of cell acid–base balance or transport of critical ions to the extrapallial space, the site of shell assembly. These data expand the number of candidate biomineralization proteins in bivalve molluscs for future functional studies and define a minimal functional protein domain set required to produce solid microstructures from soluble calcium carbonate. This is important for understanding molluscan shell evolution, the likely impacts of environmental change on biomineralization processes, materials science, and biomimicry research.     

In-depth proteomic analysis of a mollusc shell: acid-soluble and acid-insoluble matrix of the limpet Lottia gigantea

Background

Invertebrate biominerals are characterized by their extraordinary functionality and physical properties, such as strength, stiffness and toughness that by far exceed those of the pure mineral component of such composites. This is attributed to the organic matrix, secreted by specialized cells, which pervades and envelops the mineral crystals. Despite the obvious importance of the protein fraction of the organic matrix, only few in-depth proteomic studies have been performed due to the lack of comprehensive protein sequence databases. The recent public release of the gastropod Lottia gigantea genome sequence and the associated protein sequence database provides for the first time the opportunity to do a state-of-the-art proteomic in-depth analysis of the organic matrix of a mollusc shell.

Results

Using three different sodium hypochlorite washing protocols before shell demineralization, a total of 569 proteins were identified in Lottia gigantea shell matrix. Of these, 311 were assembled in a consensus proteome comprising identifications contained in all proteomes irrespective of shell cleaning procedure. Some of these proteins were similar in amino acid sequence, amino acid composition, or domain structure to proteins identified previously in different bivalve or gastropod shells, such as BMSP, dermatopontin, nacrein, perlustrin, perlucin, or Pif. In addition there were dozens of previously uncharacterized proteins, many containing repeated short linear motifs or homorepeats. Such proteins may play a role in shell matrix construction or control of mineralization processes.

Conclusions

The organic matrix of Lottia gigantea shells is a complex mixture of proteins comprising possible homologs of some previously characterized mollusc shell proteins, but also many novel proteins with a possible function in biomineralization as framework building blocks or as regulatory components. We hope that this data set, the most comprehensive available at present, will provide a platform for the further exploration of biomineralization processes in molluscs.     

Breaking the long-standing morphological paradigm: Individual prisms in the pearl oyster shell grow perpendicular to the c-axis of calcite

Cross-sections of calcitic prismatic layers in mollusk shells, cut perpendicular to growth direction, reveal well-defined polygonal shapes of individual “grains” clearly visible by light and electron microscopy. For several kinds of shells, it was shown that the average number of edges in an individual prism approaches six during the growth process. Taking into account the rhombohedral symmetry of calcite, often presented in hexagonal axes, all this led to the long-standing opinion that calcitic prisms grow along the c-axis of calcite. In this paper, using X-ray diffraction and electron backscatter diffraction (EBSD), we unambiguously show that calcitic prisms in pearl oyster Pinctada margaritifera predominantly grow perpendicular to the c-axis. The obtained results imply that the hexagon-like habitus of growing crystallites may be not necessarily connected to calcite crystallography and, therefore, other factors should be taken into consideration. We analyze this phenomenon by comparing the organic contents in Pinctada margaritifera and Pinna nobilis shells, the later revealing regular growth of calcitic prisms along the c-axis.     

Mid-Neolithic Exploitation of Mollusks in the Guanzhong Basin of Northwestern China: Preliminary Results

Mollusk remains are abundant in archaeological sites in the Guanzhong Basin of Northwestern China, providing good opportunities for investigations into the use of mollusks by prehistoric humans. Here we report on freshwater gastropod and bivalve mollusks covering the time interval from about 5600 to 4500 cal. yrs BP from sites of Mid-Late Neolithic age. They are identified as Cipangopaludina chinensis and Unio douglasiae, both of which are currently food for humans. The shells are well preserved and have no signs of abrasion. They are all freshwater gastropods and bivalves found in pits without water-reworked deposits and have modern representatives which can be observed in rivers, reservoirs, and paddy fields in the studied region. Mollusk shells were frequently recovered in association with mammal bones, lithic artifacts, and pottery. These lines of evidence indicate that the mollusks are the remains of prehistoric meals. The mollusk shells were likely discarded into the pits by prehistoric humans after the flesh was eaten. However, these mollusk remains may not have been staple food since they are not found in large quantities. Mollusk shell tools and ornaments are also observed. Shell tools include shell knives, shell reaphooks and arrowheads, whereas shell ornaments are composed of pendants and loops. All the shell tools and ornaments are made of bivalve mollusks and do not occur in large numbers. The finding of these freshwater mollusk remains supports the view that the middle Holocene climate in the Guanzhong Basin may have been warm and moist, which was probably favorable to freshwater mollusks growing and developing in the region.     

Aragonite shells are more ancient than calcite ones in bivalves: new evidence based on omics

Two calcium carbonate crystal polymorphs, aragonite and calcite, are the main inorganic components of mollusk shells. Some fossil evidences suggest that aragonite shell is more ancient than calcite shell for the Bivalvia. But, the molecular biology evidence for the above deduction is absent. In this study, we searched for homologs of bivalve aragonite-related and calcite-related shell proteins in the oyster genome, and found that no homologs of calcite-related shell protein but some homologs of aragonite-related shell proteins in the oyster genome. We explained the results as the new evidence to support that aragonite shells are more ancient than calcite shells in bivalves combined the published biogeological and seawater chemistry data.     

The carbonic anhydrase domain protein nacrein is expressed in the epithelial cells of the mantle and acts as a negative regulator in calcification in the mollusc Pinctada fucata

Signals and organic matrix proteins secreted from the mantle are critical for the development of shells in molluscs. Nacrein, which is composed of a carbonic anhydrase domain and a Gly-X-Asn repeat domain, is one of the organic matrix proteins that accumulates in shells. In situ hybridization revealed that nacrein was expressed in the outer epithelial cells of the mantle of the pearl oyster Pinctada fucata. The recombinant nacrein protein inhibited the precipitation of calcium carbonate from a saturated solution containing CaCl2 and NaHCO3, indicating that it can act as a negative regulator for calcification in the shells of molluscs. Because deletion of the Gly-X-Asn repeat domain of nacrein had a significant effect on the ability of nacrein to inhibit the precipitation of calcium carbonate, it is conceivable that the repeat domain has a primary role in the inhibitory function of nacrein in shell formation. Together these studies suggest that nacrein functions as a negative regulator in calcification in the extrapallial space between the shell and the mantle by inhibiting the precipitation of CaCO3.     

厚壳贻贝一种新型贝壳胶原蛋白的重组表达与功能分析

贝壳是一种具有优异力学性能的生物硬组织,贝壳基质蛋白质对贝壳的形成具有重要意义。厚壳贻贝(Mytilus coruscus)贝壳中发现一种类似胶原蛋白质的新型贝壳基质蛋白质,命名为collagen-like protein 2（CLP-2）。然而,该蛋白质的结构与功能以及对贝壳形成的影响机制尚不清楚。为此,本研究对CLP 2开展了序列分析;进一步采取密码子优化结合原核重组表达策略,开展了CLP-2的重组表达;在此基础上分析了重组CLP-2对酸钙结晶的诱导、结晶速率抑制以及碳酸钙结合能力。对CLP-2的序列分析结果表明,该蛋白质序列中含有信号肽及两个Von Willebrand factor A（VWA）结构域。CLP-2在数据库中尚无高同源性蛋白质存在,表明这是一种较为新颖的贝壳基质蛋白。所获得的重组CLP-2对碳酸钙体外结晶表现出明显的诱导作用,扫描电镜以及傅里叶红外光谱结果表明,重组CLP-2可诱导碳酸钙晶体的形貌由立方体形转化为球形,并在高浓度下进一步转化为哑铃形;同时,重组CLP-2可促使碳酸钙晶体的晶型由方解石型向文石型转化;重组CLP-2在体外具有碳酸钙晶体结合作用;此外,重组CLP-2能显著抑制碳酸钙晶体的结晶速度（P<0.01）,并具有浓度依赖性。上述结果表明,厚壳贻贝贝壳CLP-2蛋白质在贝壳,特别是文石型肌棱柱层的生物矿化过程中具有重要作用。上述研究为深入了解贻贝贝壳的形成机制,以及胶原类蛋白质对生物矿化过程的影响奠定了基础。     

Temperature and Food Influence Shell Growth and Mantle Gene Expression of Shell Matrix Proteins in the Pearl Oyster Pinctada margaritifera

In this study, we analyzed the combined effect of microalgal concentration and temperature on the shell growth of the bivalve Pinctada margaritifera and the molecular mechanisms underlying this biomineralization process. Shell growth was measured after two months of rearing in experimental conditions, using calcein staining of the calcified structures. Molecular mechanisms were studied though the expression of 11 genes encoding proteins implicated in the biomineralization process, which was assessed in the mantle. We showed that shell growth is influenced by both microalgal concentration and temperature, and that these environmental factors also regulate the expression of most of the genes studied. Gene expression measurement of shell matrix protein thereby appears to be an appropriate indicator for the evaluation of the biomineralization activity in the pearl oyster P. margaritifera under varying environmental conditions. This study provides valuable information on the molecular mechanisms of mollusk shell growth and its environmental control.     

The Bacteroides sp. 3_1_23 Pif1 protein is a multifunctional helicase

ScPif1 DNA helicase is the prototypical member of a 5′-to-3′ helicase superfamily conserved from bacteria to human and plays various roles in the maintenance of genomic homeostasis. While many studies have been performed with eukaryotic Pif1 helicases, including yeast and human Pif1 proteins, the potential functions and biochemical properties of prokaryotic Pif1 helicases remain largely unknown. Here, we report the expression, purification and biochemical analysis of Pif1 helicase from Bacteroides sp. 3_1_23 (BsPif1). BsPif1 binds to a large panel of DNA substrates and, in particular, efficiently unwinds partial duplex DNAs with 5′-overhang, fork-like substrates, D-loop and flap-like substrates, suggesting that BsPif1 may act at stalled DNA replication forks and enhance Okazaki fragment maturation. Like its eukaryotic homologues, BsPif1 resolves R-loop structures and unwinds DNA–RNA hybrids. Furthermore, BsPif1 efficiently unfolds G-quadruplexes and disrupts nucleoprotein complexes. Altogether, these results highlight that prokaryotic Pif1 helicases may resolve common issues that arise during DNA transactions. Interestingly, we found that BsPif1 is different from yeast Pif1, but resembles more human Pif1 with regard to substrate specificity, helicase activity and mode of action. These findings are discussed in the context of the possible functions of prokaryotic Pif1 helicases in vivo.     

The Biomineralization Proteome: Protein Complexity for a Complex Bioceramic Assembly Process

There are over 62 different biominerals on Earth and a diverse array of organisms that generate these biominerals for survival. This review will introduce the process of biomineralization and the current understanding of the molecular mechanisms of mineral formation, and then comparatively explore the representative secretomes of two well‐documented skeletal systems: vertebrate bone (calcium phosphate) and invertebrate mollusk shell (calcium carbonate). It is found that both skeletal secretomes have gross similarities and possess proteins that fall into four functional categories: matrix formers, nucleation assisters, communicators, and remodelers. In many cases the mineral‐associated matrix former and nucleation assister sequences in both skeletal systems are unique and possess interactive conserved globular domains, intrinsic disorder, post‐translational modifications, sequence redundancy, and amyloid‐like aggregation‐prone sequences. Together, these molecular features create a protein‐based environment that facilitates mineral formation and organization and argue in favor of conserved features that evolve from the mollusk shell to bone. Interestingly, the mollusk shell secretome appears to be more complex compared to that of bone tissue, in that there are numerous protein subcategories that are required for the nucleation and organization of inner (nacre) and outer (prismatic) calcium carbonate regions of the shell. This may reflect the organizational and material requirements of an exoskeletal protective system.