Novel Matrix Proteins of Pteria penguin Pearl Oyster Shell Nacre Homologous to the Jacalin-Related β-Prism Fold Lectins

Nacreous layers of pearl oyster are one of the major functional biominerals. By participating in organic compound-crystal interactions, they assemble into consecutive mineral lamellae-like photonic crystals. Their biomineralization mechanisms are controlled by macromolecules; however, they are largely unknown. Here, we report two novel lectins termed PPL2A and PPL2B, which were isolated from the mantle and the secreted fluid of Pteria penguin oyster. PPL2A is a hetero-dimer composed of α and γ subunits, and PPL2B is a homo-dimer of β subunit, all of which surprisingly shared sequence homology with the jacalin-related plant lectin. On the basis of knockdown experiments at the larval stage, the identification of PPLs in the shell matrix, and in vitro CaCO₃ crystallization analysis, we conclude that two novel jacalin-related lectins participate in the biomineralization of P. penguin nacre as matrix proteins. Furthermore, it was found that trehalose, which is specific recognizing carbohydrates for PPL2A and is abundant in the secreted fluid of P. penguin mantle, functions as a regulatory factor for biomineralization via PPL2A. These observations highlight the unique functions, diversity and molecular evolution of this lectin family involved in the mollusk shell formation.     

不溶性基质中天冬氨酸丰富的蛋白在珊瑚的钙质骨片形成中的重要作用

一般认为, 酸性蛋白在控制矿物的形成和发展中发挥重要作用。因此, 在不溶性有机基质中鉴定酸性蛋白对于理解珊瑚中个体蛋白的功能是非常重要的一步。在短指多型软珊瑚(Sinularia polydactyla)的可溶性和不溶性基质层中分析蛋白组分表明, 在不溶性基质和可溶性基质层中天冬氨酸的含量分别是61%和29%。利用体外分析法发现, 基质蛋白诱导碳酸钙形成非晶态析出相先于其形成钙质的结晶态。利用X-射线衍射来鉴定骨片上结晶态的碳酸钙, 结果表明钙质的多晶态呈现强反射。傅利叶变换红外光谱分析表明珊瑚基质中富含天冬氨酸的蛋白和多醣的结构。在不溶性基质组分中用钙离子结合分析显示一个分子量为109 kD的蛋白质可以与形成骨片的钙离子结合, 这一过程对骨片形成非常重要。在对生物钙化过程中起重要作用的碳酸酐酶的分析中显示了此酶的新颖的活性。以上结果显示珊瑚中不溶性基质内的富含天冬氨酸的蛋白在生物矿化调控过程中起重要作用。     

Proteomics of CaCO3 biomineral‐associated proteins: How to properly address their analysis

In a recent editorial (Proc. Natl. Acad. Sci., 2013 110, E2144–E2146) and elsewhere, questions have been raised regarding the experimental practices in relation to the proteomic analysis of organic matrices associated to the biomineralized CaCO₃ skeletons of metazoans such as molluscan shells and coral skeletons. Indeed, although the use of new high sensitivity MS technology potentially allows to identify a greater number of proteins, it is also equally (or even more) sensitive to contamination of residual proteins from soft tissues, which are in close contact with the biomineral. Based on our own past and present experimental know‐how—observations that are reproducible and coherent with the current understanding of extracellular biomineralization processes—we are convinced that a careful and appropriate cleaning of biominerals prior to any analysis is crucial for accurate proteomic investigations and subsequent pertinent interpretation of the results. Our goal is to alert the scientific community about the associated bias that definitely should be avoided, and to provide critical recommendations on sample preparation and experimental design, in order to better take advantage of the aptness of proteomic approaches aiming at improving our understanding of the molecular mechanisms in biomineralization.     

An evolutionary fast‐track to biocalcification

The ability to construct mineralized shells, spicules, spines and skeletons is thought to be a key factor that fuelled the expansion of multicellular animal life during the early Cambrian. The genes and molecular mechanisms that control the process of biomineralization in disparate phyla are gradually being revealed, and it is broadly recognized that an insoluble matrix of proteins, carbohydrates and other organic molecules are required for the initiation, regulation and inhibition of crystal growth. Here, we show that Astrosclera willeyana, a living representative of the now largely extinct stromatoporid sponges (a polyphyletic grade of poriferan bauplan), has apparently bypassed the requirement to evolve many of these mineral‐regulating matrix proteins by using the degraded remains of bacteria to seed CaCO₃ crystal growth. Because stromatoporid sponges formed extensive reefs during the Paelozoic and Mesozoic eras (fulfilling the role that stony corals play in modern coral reefs), and fossil evidence suggests that the same process of bacterial skeleton formation occurred in these stromatoporid ancestors, we infer that some ancient reef ecosystems might have been founded on this microbial–metazoan relationship.     

In-depth,high-accuracy proteomics of sea urchin tooth organic matrix

Background  

The organic matrix contained in biominerals plays an important role in regulating mineralization and in determining biomineral properties. However, most components of biomineral matrices remain unknown at present. In sea urchin tooth, which is an important model for developmental biology and biomineralization, only few matrix components have been identified. The recent publication of the Strongylocentrotus purpuratus genome sequence rendered possible not only the identification of genes potentially coding for matrix proteins, but also the direct identification of proteins contained in matrices of skeletal elements by in-depth, high-accuracy proteomic analysis.     

The sea urchin (<Emphasis Type="Italic">Strongylocentrotus purpuratus</Emphasis>) test and spine proteomes

Background  

The organic matrix of biominerals plays an important role in biomineral formation and in determining biomineral properties. However, most components of biomineral matrices remain unknown at present. In sea urchin, which is an important model organism for developmental biology and biomineralization, only few matrix components have been identified and characterized at the protein level. The recent publication of the Strongylocentrotus purpuratus genome sequence rendered possible not only the identification of possible matrix proteins at the gene level, but also the direct identification of proteins contained in matrices of skeletal elements by in-depth, high-accuracy, proteomic analysis.     

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.     

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.     

MMS6 protein regulates crystal morphology during nano-sized magnetite biomineralization in vivo

Biomineralization, the process by which minerals are deposited by organisms, has attracted considerable attention because this mechanism has shown great potential to inspire bottom-up material syntheses. To understand the mechanism for morphological regulation that occurs during biomineralization, many regulatory proteins have been isolated from various biominerals. However, the molecular mechanisms that regulate the morphology of biominerals remain unclear because there is a lack of in vivo evidence. Magnetotactic bacteria synthesize intracellular magnetosomes that comprise membrane-enveloped single crystalline magnetite (Fe(3)O(4)). These nano-sized magnetite crystals (<100 nm) are bacterial species dependent in shape and size. Mms6 is a protein that is tightly associated with magnetite crystals. Based on in vitro experiments, this protein was first implicated in morphological regulation during nano-sized magnetite biomineralization. In this study, we analyzed the mms6 gene deletion mutant (Δmms6) of Magnetospirillum magneticum (M. magneticum) AMB-1. Surprisingly, the Δmms6 strain was found to synthesize the smaller magnetite crystals with uncommon crystal faces, while the wild-type and complementation strains synthesized highly ordered cubo-octahedral crystals. Furthermore, deletion of mms6 gene led to drastic changes in the profiles of the proteins tightly bound to magnetite crystals. It was found that Mms6 plays a role in the in vivo regulation of the crystal structure to impart the cubo-octahedral morphology to the crystals during biomineralization in magnetotactic bacteria. Magnetotactic bacteria synthesize magnetite crystals under ambient conditions via a highly controlled morphological regulation system that uses biological molecules.     

Recombinant production of a shell matrix protein in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its application to the biomimetic synthesis of spherulitic calcite crystals

Objectives

To overcome the limited production capability of shell matrix proteins and efficiently conduct in vitro CaCO₃ biomineralization studies, a putative recombinant shell matrix protein was prepared and characterized.

Results

A glycine-rich protein (GRP_BA) was found in Pinctada fucata as a putative shell matrix protein (NCBI reference sequence; BAA20465). It was genetically redesigned for the production in Escherichia coli. The recombinant protein was obtained in a 400 ml shake-flask culture at approx. 30 mg l^?1 with a purity of >95 %. It efficiently formed a complex with Ca²⁺. Ca²⁺-induced agglomeration was like other calcification-related proteins. Spherulitic calcite micro-particles, 20–30 µm diam. with rosette- and sphere-like structures were synthesized in the presence of the recombinant shell protein, which could be formed by stacking and/or aggregation of calcite nanograins and the bound protein.

Conclusions

Recombinant production of a shell matrix protein could overcome potential difficulties associated with the limited amount of protein available for biomineralization studies and provide opportunities to fabricate biominerals in practical aspects.

     

Insights into the composition,morphology, and formation of the calcareous shell of the serpulid Hydroides dianthus

To date, the calcareous tubes of serpulid marine worms have not been studied extensively in a biomineralization context. The structure and composition of the tube shell and adhesive cement of the marine tubeworm Hydroides dianthus were studied using a variety of characterization techniques, including powder XRD, FTIR, SEM, EDX, and AFM. The tube and cement were determined to be inorganic–organic composite materials, consisting of inorganic aragonite (CaCO₃) and Mg-calcite ((Ca_0.8Mg_0.2)CO₃) crystals, and both soluble and insoluble organic matrices (SOM and IOM). SEM imaging revealed a variety of crystal morphologies. AFM nanoindentation of the inorganic components yielded Young’s moduli of ～20 GPa in the wet state, and ～50 GPa in the dry state. Amino acid analysis of the SOM indicated substantial amounts of acidic and non-polar neutral amino acids. Part of the insoluble organic tube lining was identified as being composed of collagen-containing fibres aligned in a criss-crossed structure. The SOM and organic tube lining were found to contain carboxylated and sulphated polysaccharides. In an artificial seawater solution, the SOM and the organic tube lining mediated CaCO₃ mineralization in vitro.     

Calcium associated with a fibrillar organic matrix in the scleractinian coral Galaxea fascicularis

Summary.  Field emission scanning electron microscopy of frozen-hydrated preparations of the scleractinian coral Galaxea fascicularis revealed organic fibrils which have a diameter of 26 nm and are located between calicoblastic ectodermal cells and the underlying CaCO₃ skeleton. Small (37 nm in diameter) nodular structures observed upon this fibrillar organic material possibly correspond to localised Ca-rich regions detected throughout the calcifying interfacial region of freeze-substituted preparations by X-ray microanalysis. We propose that these Ca-rich regions associated with the organic material are nascent crystals of CaCO₃. Significant amounts of S were also detected throughout the calcifying interfacial region, further verifying the likely presence of organic material. However, the bulk of this S is unlikely to be derived from mucocytes within the calicoblastic ectoderm. It is suggested that in the scleractinian coral G. fascicularis, nodular crystals of CaCO₃ establish upon a fibrillar, S-containing, organic matrix within small but distinct extracellular pockets formed between calicoblastic ectodermal cells and skeleton. This arrangement conforms with the criteria necessary for biomineralisation and with the long-held theory that organic matrices may act as templates for crystal formation and growth in biological mineralising systems. Received April 30, 2002; accepted September 11, 2002; published online March 11, 2003     

Effect of Coccolith Polysaccharides Isolated from the Coccolithophorid, <Emphasis Type="Italic">Emiliania huxleyi</Emphasis>, on Calcite Crystal Formation in In Vitro CaCO<Subscript>3</Subscript> Crystallization

Marine coccolithophorids (Haptophyceae) produce calcified scales “coccoliths” which are composed of CaCO₃ and coccolith polysaccharides (CP) in the coccolith vesicles. CP was previously reported to be composed of uronic acids and sulfated residues, etc. attached to the polymannose main chain. Although anionic polymers are generally known to play key roles in biomineralization process, there is no experimental data how CP contributes to calcite crystal formation in the coccolithophorids. CP used was isolated from the most abundant coccolithophorid, Emiliania huxleyi. CaCO₃ crystallization experiment was performed on agar template layered onto a plastic plate that was dipped in the CaCO₃ crystallization solution. The typical rhombohedral calcite crystals were formed in the absence of CP. CaCO₃ crystals formed on the naked plastic plate were obviously changed to stick-like shapes when CP was present in the solution. EBSD analysis proved that the crystal is calcite of which c-axis was elongated. CP in the solution stimulated the formation of tabular crystals with flat edge in the agarose gel. SEM and FIB-TEM observations showed that the calcite crystals were formed in the gel. The formation of crystals without flat edge was stimulated when CP was preliminarily added in the gel. These observations suggest that CP has two functions: namely, one is to elongate the calcite crystal along c-axis and another is to induce tabular calcite crystal formation in the agarose gel. Thus, CP may function for the formation of highly elaborate species-specific structures of coccoliths in coccolithophorids.     

Proteomic analysis of sea urchin <Emphasis Type="Italic">(Strongylocentrotus purpuratus)</Emphasis> spicule matrix

Background  

The sea urchin embryo has been an important model organism in developmental biology for more than a century. This is due to its relatively simple construction, translucent appearance, and the possibility to follow the fate of individual cells as development to the pluteus larva proceeds. Because the larvae contain tiny calcitic skeletal elements, the spicules, they are also important model organisms for biomineralization research. Similar to other biominerals the spicule contains an organic matrix, which is thought to play an important role in its formation. However, only few spicule matrix proteins were identified previously.     

Distinct macroscopic structures developed from solutions of chemical compounds and periodic proteins

By controlling the growth of inorganic crystals, macro-biomolecules, including proteins, play pivotal roles in modulating biomineralization. Natural proteins that promote biomineralization are often composed of simple repeats of peptide sequences; however, the relationship between these repetitive structures and their functions remains largely unknown. Here we show that an artificial protein containing a repeated peptide sequence allows NaCl, KCl, CuSO₄ and sucrose to form a variety of macroscopic structures, as represented by their dendritic configurations. Mutational analyses revealed that the physicochemical characteristics of the protein, not the peptide sequence per se, were responsible for formation of the dendritic structures. This suggests that proteins that modulate crystal growth may have evolved as repeat-containing forms at a relatively high rate. These observations could serve as the basis for developing new genetic programming systems for creation of artificial proteins able to modulate crystal growth from inorganic compounds, and may thus provide a new tool for nano-biotechnology.     

Soluble proteins control growth of skeleton crystals in three coralline demosponges

Summary Calcified demosponges (coralline sponges, sclero-sponges), the first metazoa producing a carbonate skeleton, used to be important reef building organisms in the past. The relatives of this group investigated here,Spirastrella (Acanthochaetetes) wellsi, Astrosclera willeyana andVaceletia cf.crypta, are restricted to cryptic niches of modern Pacific coral reefs and may be considered as “living fossils’. They are characterized by a basic biologically controlled metazoan biomineralization process. Each of the investigated taxa forms its calcareous basal skeleton in a highly specialized way. Moreover, each taxon secretes distinct Ca²⁺-binding macromolecules which were entrapped within the calcium carbonate crystals during skeleton formation. Therefore these Ca²⁺-binding macromolecules were also described as intracrystalline macromolecules. When isolated and separated by SDS polyacrylamide gel electrophoresis, the organic skeleton matrix of the three species revealed to be composed of a respective distinct array of EDTA-soluble proteins. A single protein of 41 kDa was detected inS. wellsi, two proteins of 38 and 120 kDa inA. willeyana, and four proteins of 18 kDa, 30 kDa, 33 kDa, and 37 kDa inVaceletia sp. When run on IEF gel, the Ca²⁺-binding proteins gave staining bands at pH values between 5.25 and 5.65. As proved by anin vitro mineralization assay, the extracted proteins effectively inhibit CaCO₃ and SrCO₃ precipitation, respectively, in a saturated solution. Biochemical properties and behavior of the extracted proteins strongly suggest that they are involved in crystal nucleation and skeleton carbonate formation within the calcified sponges studied here.     

Otolin-1, an otolith- and otoconia-related protein,controls calcium carbonate bioinspired mineralization

BackgroundOtoliths and otoconia are calcium carbonate biomineral structures that form in the inner ear of fish and humans, respectively. The formation of these structures is tightly linked to the formation of an organic matrix framework with otolin-1, a short collagen-like protein from the C1q family as one of its major constituents.MethodsIn this study, we examined the activity of recombinant otolin-1 originating from Danio rerio and Homo sapiens on calcium carbonate bioinspired mineralization with slow-diffusion method and performed crystals characterization with scanning electron microscopy, two-photon excited fluorescence microscopy, confocal laser scanning microscopy and micro-Raman spectroscopy.ResultsWe show that both proteins are embedded in the core of CaCO₃ crystals that form through the slow-diffusion mineralization method. Both of them influence the morphology but do not change the polymorphic mineral phase. D.rerio otolin-1 also closely adheres to the crystal surface.General significanceThe results suggest, that otolin-1 is not a passive scaffold, but is directly involved in regulating the morphology of the resulting calcium carbonate biocrystals.     

Intrinsically Disordered and Pliable Starmaker-Like Protein from Medaka (Oryzias latipes) Controls the Formation of Calcium Carbonate Crystals

Fish otoliths, biominerals composed of calcium carbonate with a small amount of organic matrix, are involved in the functioning of the inner ear. Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths. Recently, a gene was identified encoding the Starmaker-like (Stm-l) protein from medaka (Oryzias latipes), a putative homologue of Stm and human dentine sialophosphoprotein. Although there is no sequence similarity between Stm-l and Stm, Stm-l was suggested to be involved in the biomineralization of otoliths, as had been observed for Stm even before. The molecular properties and functioning of Stm-l as a putative regulatory protein in otolith formation have not been characterized yet. A comprehensive biochemical and biophysical analysis of recombinant Stm-l, along with in silico examinations, indicated that Stm-l exhibits properties of a coil-like intrinsically disordered protein. Stm-l possesses an elongated and pliable structure that is able to adopt a more ordered and rigid conformation under the influence of different factors. An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals. The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed.     

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.