红条毛肤石鳖齿舌主侧齿中铁还原酶分布及与生物矿化的关系

以红条毛肤石鳖Acanthochiton rubrolineatus(Lischke)齿舌为材料,通过切片和酶组织化学技术,在光镜和电镜下对齿舌主侧齿的微结构及高铁还原酶的存在进行观察,从微观角度了解齿舌主侧齿齿尖的矿化机理。结果显示,成熟主侧齿由齿尖和齿基组成。齿尖结构由外至内分为三层,最外层为磁铁矿层,前后齿面磁铁矿层的厚度不等,后齿面约50μm,前齿面约5-10μm。向内依次为棕红色的纤铁矿层,厚约10μm,及略显黄色的有机基质层,有机基质层占据着齿尖内部的大部分结构。高分辨透射电镜下显示磁铁矿由条状四氧化三铁颗粒组成,长约2-3μm,宽约100-150nm。齿舌的矿化是一个连续过程,不同部段处于不同的矿化阶段,齿舌囊上皮细胞沿囊腔分布,并形成齿片。未矿化的新生主侧齿齿尖中存在由有机基质构成的网状结构。随矿化的进行,有机基质内出现矿物颗粒。初始矿化的齿尖外表面有一个细胞微突层,微突的另一端为囊上皮细胞,矿物质经由微突层达齿尖并沉积于有机基质中,齿尖随之矿化并成熟。初始矿化齿尖的外围有大量的三价铁化物颗粒,稍成熟的齿尖外围同时还出现二价铁化物。新生或初始矿化主侧齿齿尖外围的囊上皮细胞中有大量球形类似于铁蛋白聚集体的内容物,直径0.6-0.8μm,球体由膜包围。齿舌囊上皮组织中存在三价高铁还原酶,此酶分布于上皮细胞的膜表面,可能与齿尖表面磁铁矿的生成有一定的关系。       

A strategy to quantitate global phosphorylation of bone matrix proteins

Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured “in bulk” are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials.     

The Microstructure,Proteomics and Crystallization of the Limpet Teeth

Limpets are marine mollusks that use mineralized teeth, one of the hardest and strongest biomaterials, to feed on algae on intertidal rocks. However, most of studies only focus on the ultrastructure and chemical composition of the teeth while the molecular information is largely unknown, limiting our understanding of this unique and fundamental biomineralization process. The study investigates the microstructure, proteomics, and crystallization in the teeth of limpet Cellana toreuma. It is found that the limpets formed alternatively tricuspid teeth and unicuspid teeth. Small nanoneedles are densely packed at the tips or leading regions of the cusps. In contrast, big nanoneedles resembling chemically synthesized goethite are loosely packed in the trailing regions of the cusps. Proteins extracted from the whole radula, such as ferritin, peroxiredoxin, arginine kinase, GTPase‐Rabs, and clathrin, are identified by proteomics. A goethite‐binding experiment coupled with proteomics and RNA‐seq highlights six chitin‐binding proteins (CtCBPs). Furthermore, the extracted proteins from the cusps of radula or the framework chitin induce packing of crystals and possibly affect crystal polymorphs in vitro. This study provides insight into the unique biomineralization process in the limpet teeth at the molecular levels, which may guide biomimetic strategies aimed at designing hard materials at room temperature.     

iTRAQ‐Based Quantitative Proteomic Analysis of Duck Eggshell During Biomineralization

The avian egg is a valuable model for the calcitic biomineralization process as it is the fastest calcification process occurring in nature and is a clear example of biomineralization. In this study, iTRAQ MS/MS is used to detect and study for the first time: 1) the overall duck eggshell proteome; 2) regional differences in the proteome between the inner and outer portions of the duck eggshell. The new reference protein datasets allow us to identify 179 more eggshell proteins than solely using the current release of Ensembl duck annotations. In total, 484 proteins are identified in the entire duck eggshell proteome. Twenty‐eight novel proteins of unknown function that are involved in eggshell formation are also identified. Among the identified eggshell proteins, 54 proteins show differential abundances between the inner, partially mineralized eggshell (obtained 16 h after ovulation) compared to the overall complete eggshell (normally expulsed eggshell). At least 64 of the abundant matrix proteins are common to eggshell of 4 different domesticated bird species (chicken, duck, quail, turkey) and zebra finch. This study provides a new resource for avian eggshell proteomics, and augments the inventory of eggshell matrix proteins that will lead to a deeper understanding of calcitic biomineralization.     

Expression of phosphophoryn is sufficient for the induction of matrix mineralization by mammalian cells

Mineralized tissues such as dentin and bone assemble extracellular matrices uniquely rich in a variety of acidic phosphoproteins. Although these proteins are presumed to play a role in the process of biomineralization, key questions regarding the nature of their contributions remain unanswered. First, it is not known whether highly phosphorylated proteins alone can induce matrix mineralization, or whether this activity requires the involvement of other bone/dentin non-collagenous proteins. Second, it remains to be established whether the protein kinases that phosphorylate these acidic proteins are unique to cells responsible for producing mineralized tissues. To begin to address these questions, we consider the case of phosphophoryn (PP), due to its high content of phosphate, high affinity for Ca(2+), and its potential role in hydroxyapatite nucleation. We have created a model system of biomineralization in a cellular environment by expressing PP in NIH3T3 fibroblasts (which do not produce a mineralized matrix); as a positive control, PP was expressed in MC3T3-E1 osteoblastic cells, which normally mineralize their matrices. We show that expression of PP in NIH3T3 cells is sufficient for the induction of matrix mineralization. In addition, assessment of the phosphorylation status of PP in these cells reveals that the transfected NIH3T3 cells are able to phosphorylate PP. We suggest that the phosphorylation of PP is essential for mineral formation. The principle goal of this study is to enrich the current knowledge of mineralized tissue phosphorylation events by analyzing them in the context of a complete cellular environment.     

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.     

Biomineralization proteins: from vertebrates to bacteria

Biomineralization processes are frequently found in nature. Living organisms use various strategies to create highly ordered and hierarchical mineral structures under physiologic conditions in which the temperatures and pressures are much lower than those required to form the same mineralized structures by chemical synthesis. Although the mechanism of biomineralization remains elusive, proteins have been found responsible for the formation of such mineral structures in many cases. These proteins are active components in the process of biomineralization. The mechanisms by which their function can vary from providing active organic matrices that control the formation of specific mineral structures to being catalysts that facilitate the crystallization of certain metal ions. This review summarizes the current understanding of the functions of several representative biomineralization proteins from vertebrates to bacteria in the hopes of providing useful insight and guidance for further elucidation of mechanisms of biomineralization processes in living organisms.     

Matrix proteins of the teeth of the sea urchin Lytechinus variegatus

The teeth of the sea urchin Lytechinus variegatus grow continuously. The mineral phase, a high magnesium calcite, grows into single crystals within numerous compartments bounded by an organic matrix deposited by the odontoblasts. Electron microscopic examination of glutaraldehyde-fixed Ethylene Diamine Tetra acetic acid (EDTA) demineralized teeth shows the compartment walls to be organized from multiple layers of cell membrane which might contain cytoplasmic protein inclusions. Proteins extracted during demineralization of unfixed teeth were examined by gel electrophoresis, high performance liquid chromatography, and amino acid analysis. The tooth proteins were acidic, they contained phosphoserine, and they were rich in aspartic acid. By contrast, the proteins of similarly extracted mineralized Aristotle's lantern skeletal elements were nonphosphorylated and were rich in glutamic acid. Vertebrate tooth and bone matrix proteins show similar differences. Surprisingly, an antibody to the principle rat incisor phosphoprotein showed a significant cross-reactivity with the urchin tooth protein, by dot-blot and enzyme-linked immunosorbent assay procedures. Thus, the urchin tooth proteins contain epitope regions similar to those which are phenotypic markers of vertebrate odontoblasts. Whether this is an expression of convergent or divergent evolutionary processes, it is likely that the matrix proteins play a similar role in matrix mineralization. The sea urchin tooth may thus be an excellent model for the study of odontoblast-mediated mineral-matrix relationships.     

Ultrastructure of sea urchin calcified tissues after high-pressure freezing and freeze substitution

The improvements brought by high-pressure freezing/freeze substitution fixation methods to the ultrastructural preservation of echinoderm mineralized tissues are investigated in developing pedicellariae and teeth of the echinoid Paracentrotus lividus. Three freeze substitution (FS) protocols were tested: one in the presence of osmium tetroxide, one in the presence of uranyl acetate, and the last in the presence of gallic acid. FS in the presence of osmium tetroxide significantly improved cell ultrastructure preservation and should especially be used for ultrastructural studies involving vesicles and the Golgi apparatus. With all protocols, multivesicular bodies, suggested to contain Ca(2+), were evident for the first time in skeleton-forming cells. FS in the presence of gallic acid allowed us to confirm the structured and insoluble character of a part of the organic matrix of mineralization in the calcification sites of the tooth, an observation which modifies the current understanding of biomineralization control in echinoderms.     

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.     

A New Method to Extract Matrix Proteins Directly from the Secretion of the Mollusk Mantle and the Role of These Proteins in Shell Biomineralization

Considering the continuous and substantive secretory ability of the mantle in vitro, we report a new technique to produce shell-matrix proteins by inducing the mantle, after removal from the organism's body, to secrete soluble-matrix proteins into phosphate buffer. By this method, a large amount of matrix proteins could be obtained in 2 h. Experiments involving in vitro calcium carbonate crystallization and organic framework calcium carbonate crystallization indicated that these proteins retain high bioactivity and play key roles in shell biomineralization. Phosphate buffer-soluble proteins secreted by the margin of the mantles (MSPs) were used to reconstruct the stages in the growth of the prismatic layer of the decalcified organic frameworks. The MSPs were observed to aggregate calcites in vitro, and this ability enabled the mollusk to form big calcites in the prismatic layer. During shell biomineralization, an important stage after the self-assembly of the biomacromolecules and the formation of crystals is the assembly of the two parts to form a firm structure. Moreover, a new type of matrix protein, functioning as the binding factor between the crystals and the organic frameworks, was shown to exist in the phosphate buffer-soluble proteins secreted by the central part of mantles (CSPs). Nanoscale-sized bowl-like aragonites, with heights of ～800 nm, were induced by CSPs in vitro. This method is a successful example of obtaining functional proteins through secretion by animal tissues.     

Electron microscopic studies on radular tooth formation in the snails Helix pomatia L. and Limax flavus L. (Pulmonata,Stylommatophora)

The radular teeth are secreted at the posterior end of the radular gland and move slowly towards the buccal cavity where they start to function. Helix pomatia and Limax flavus were examined to determine whether the newly formed teeth already show their definite species specific shape, or whether they are gradually finished and moulded in the radular gland. Scanning electron micrographs of Helix pomatia show that teeth are secreted in the odontoblast region in their final form. Their surface is still uneven at the outset; the same is true for the newest teeth of Limax flavus. Older teeth ready for use have a smooth surface. This change seems to be brought about by secretory activity of the superior epithelium of the radular sac. Air-dried radulae, previously isolated by KOH maceration, show considerable artefacts at their posterior end. Maceration leads to shrinking of the newest teeth, but does not change their contours. The newly secreted but as yet unhardened teeth become greatly deformed during the drying process.     

C-terminal Amidation of an Osteocalcin-derived Peptide Promotes Hydroxyapatite Crystallization

Genesis of natural biocomposite-based materials, such as bone, cartilage, and teeth, involves interactions between organic and inorganic systems. Natural biopolymers, such as peptide motif sequences, can be used as a template to direct the nucleation and crystallization of hydroxyapatite (HA). In this study, a natural motif sequence consisting of 13 amino acids present in the first helix of osteocalcin was selected based on its calcium binding ability and used as substrate for nucleation of HA crystals. The acidic (acidic osteocalcin-derived peptide (OSC)) and amidic (amidic osteocalcin-derived peptide (OSN)) forms of this sequence were synthesized to investigate the effects of different C termini on the process of biomineralization. Electron microscopy analyses show the formation of plate-like HA crystals with random size and shape in the presence of OSN. In contrast, spherical amorphous calcium phosphate is formed in the presence of OSC. Circular dichroism experiments indicate conformational changes of amidic peptide to an open and regular structure as a consequence of interaction with calcium and phosphate. There is no conformational change detectable in OSC. It is concluded that HA crystal formation, which only occurred in OSN, is attributable to C-terminal amidation of a natural peptide derived from osteocalcin. It is also proposed that natural peptides with the ability to promote biomineralization have the potential to be utilized in hard tissue regeneration.     

Starmaker exhibits properties of an intrinsically disordered protein

Fish otoliths composed of calcium carbonate and an organic matrix play a primary role in gravity sensing and the perception of sound. Starmaker (Stm) was the first protein found to be capable of influencing the process of biomineralization of otoliths. Stm dictates the shape, size, and selection of calcium carbonate polymorphs in a concentration-dependent manner. To facilitate exploration of the molecular basis of Stm function, we have developed and optimized a protocol for efficient expression and purification of the homogeneous nontagged Stm. The homogeneous nontagged Stm corresponds to its functional form, which is devoid of a signal peptide. A comprehensive biochemical and biophysical analysis of recombinant Stm, along with in silico examinations, indicate for the first time that Stm exhibits the properties of intrinsically disordered proteins. The functional significance of Stm having intrinsically disordered protein properties and its possible role in controlling the formation of otoliths is discussed.     

MEPE-Derived ASARM Peptide Inhibits Odontogenic Differentiation of Dental Pulp Stem Cells and Impairs Mineralization in Tooth Models of X-Linked Hypophosphatemia

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) cause X-linked familial hypophosphatemic rickets (XLH), a disorder having severe bone and tooth dentin mineralization defects. The absence of functional PHEX leads to abnormal accumulation of ASARM (acidic serine- and aspartate-rich motif) peptide − a substrate for PHEX and a strong inhibitor of mineralization − derived from MEPE (matrix extracellular phosphoglycoprotein) and other matrix proteins. MEPE-derived ASARM peptide accumulates in tooth dentin of XLH patients where it may impair dentinogenesis. Here, we investigated the effects of ASARM peptides in vitro and in vivo on odontoblast differentiation and matrix mineralization. Dental pulp stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into a 3D collagen scaffold, and induced towards odontogenic differentiation. Cultures were treated with synthetic ASARM peptides (phosphorylated and nonphosphorylated) derived from the human MEPE sequence. Phosphorylated ASARM peptide inhibited SHED differentiation in vitro, with no mineralized nodule formation, decreased odontoblast marker expression, and upregulated MEPE expression. Phosphorylated ASARM peptide implanted in a rat molar pulp injury model impaired reparative dentin formation and mineralization, with increased MEPE immunohistochemical staining. In conclusion, using complementary models to study tooth dentin defects observed in XLH, we demonstrate that the MEPE-derived ASARM peptide inhibits both odontogenic differentiation and matrix mineralization, while increasing MEPE expression. These results contribute to a partial mechanistic explanation of XLH pathogenesis: direct inhibition of mineralization by ASARM peptide leads to the mineralization defects in XLH teeth. This process appears to be positively reinforced by the increased MEPE expression induced by ASARM. The MEPE-ASARM system can therefore be considered as a potential therapeutic target.     

两种石磺齿舌形态差异分析

显微观察了瘤背石磺(Onchidiumstruma)和石磺(O. verruculatum)齿舌的形态结构。运用差异系数法对两种石磺齿舌参数进行比较分析。利用SPSS10.0对瘤背石磺、石磺齿舌参数(齿舌长、齿舌头宽、齿舌中宽、齿舌尾宽、横列数、每排最少齿片数和每排最多齿片数)与个体参数(体长、体宽、体高、足长、足宽和体重)作回归分析。结果表明,两种石磺齿舌都很发达,外观呈长统靴状;齿片排成许多横列,每一横列均有中央齿一枚,侧齿若干无缘齿;两种石磺的齿舌头宽、齿舌中宽和齿舌尾宽差异极显著,但差异系数小于1.28,认为两种石磺的齿片形态存在明显的种间差异,但齿舌参数不适合作为石磺属贝类的分类依据;瘤背石磺的体宽和石磺的体重在评估各自齿舌生物学性状方面起到比较重要的作用。     

Differential expression and activity of tissue-nonspecific alkaline phosphatase (TNAP) in rat odontogenic cells in vivo.

Among the four existing isoforms of alkaline phosphatase (AP), the present study is devoted to tissue-nonspecific alkaline phosphatase (TNAP) in mineralized dental tissues. Northern blot analysis and measurements of phosphohydrolase activity on microdissected epithelium and ectomesenchyme, in situ hybridization, and immunolabeling on incisors confirmed that the AP active in rodent teeth is TNAP. Whereas the developmental pattern of TNAP mRNA and protein and the previously described activity were similar in supra-ameloblastic and mesenchymal cells, they differed in enamel-secreting cells, the ameloblasts. As previously shown for other proteins involved in calcium and phosphate handling in ameloblasts, a biphasic pattern of steady-state TNAP mRNA levels was associated with additional variations in ameloblast TNAP protein levels during the cyclic modulation process. Although the association of TNAP upregulation and the initial phase of biomineralization appeared to be a basic feature of all mineralized tissues, ameloblasts (and to a lesser extent, odontoblasts) showed a second selectively prominent upregulation of TNAP mRNA/protein/activity during terminal growth of large enamel crystals only, i.e., the maturation stage. This differential expression/activity for TNAP in teeth vs bone may explain the striking dental phenotype vs bone reported in hypophosphatasia, a hereditary disorder related to TNAP mutation. (J Histochem Cytochem 47:1541-1552, 1999)     

The NH2-terminal and COOH-terminal fragments of dentin matrix protein 1 (DMP1) localize differently in the compartments of dentin and growth plate of bone

Multiple studies have shown that dentin matrix protein 1 (DMP1) is essential for bone and dentin mineralization. After post-translational proteolytic cleavage, DMP1 exists within the extracellular matrix of bone and dentin as an NH2-terminal fragment, a COOH-terminal fragment, and the proteoglycan form of the NH2-terminal fragment (DMP1-PG). To begin to assess the biological function of each fragment, we evaluated the distribution of both fragments in the rat tooth and bone using antibodies specific to the NH2-terminal and COOH-terminal regions of DMP1 and confocal microscopy. In rat first molar organs, the NH2-terminal fragment localized to predentin, whereas the COOH-terminal fragment was mainly restricted to mineralized dentin. In the growth plate of bone, the NH2-terminal fragment appeared in the proliferation and hypertrophic zones, whereas the COOH-terminal fragment occupied the ossification zone. Forster resonance energy transfer analysis showed colocalization of both fragments of DMP1 in odontoblasts and predentin, as well as hypertrophic chondrocytes within the growth plates of bone. The biochemical analysis of bovine teeth showed that predentin is rich in DMP1-PG, whereas mineralized dentin primarily contains the COOH-terminal fragment. We conclude that the differential patterns of expression of NH2-terminal and COOH-terminal fragments of DMP1 reflect their potentially distinct roles in the biomineralization of dentin and bone matrices.     

Historical and biomechanical analysis of integration and dissociation in molluscan feeding,with special emphasis on the true limpets (Patellogastropoda: Gastropoda)

Modifications of the molluscan feeding apparatus have long been recognized as a crucial feature in molluscan diversification, related to the important process of gathering energy from the environment. An ecologically and evolutionarily significant dichotomy in molluscan feeding kinematics is whether radular teeth flex laterally (flexoglossate) or do not (stereoglossate). In this study, we use a combination of phylogenetic inference and biomechanical modeling to understand the transformational and causal basis for flexure or lack thereof. We also determine whether structural subsystems making up the feeding system are structurally, functionally, and evolutionarily integrated or dissociated. Regarding evolutionary dissociation, statistical analysis of state changes revealed by the phylogenetic analysis shows that radular and cartilage subsystems evolved independently. Regarding kinematics, the phylogenetic analysis shows that flexure arose at the base of the Mollusca and lack of flexure is a derived condition in one gastropod clade, the Patellogastropoda. Significantly, radular morphology shows no change at the node where kinematics become stereoglossate. However, acquisition of stereoglossy in the Patellogastropoda is correlated with the structural dissociation of the subradular membrane and underlying cartilages. Correlation is not causality, so we present a biomechanical model explaining the structural conditions necessary for the plesiomorphic kinematic state (flexoglossy). Our model suggests that plesiomorphically the radular teeth must flex laterally as they pass over the bending plane as a result of the mechanical restrictions in the flexible but inelastic subradular membrane and close association between subradular membrane and cartilages. Relating this model to the specific character states of the clades, we conclude that lack of flexure in patellogastropods is caused by the dissociation of the subradular membrane and cartilage supports. J. Morphol. 241:175–195, 1999. © 1999 Wiley‐Liss, Inc.