In Vitro Calcite Crystal Morphology Is Modulated by Otoconial Proteins Otolin-1 and Otoconin-90

Otoconia are formed embryonically and are instrumental in detecting linear acceleration and gravity. Degeneration and fragmentation of otoconia in elderly patients leads to imbalance resulting in higher frequency of falls that are positively correlated with the incidence of bone fractures and death. In this work we investigate the roles otoconial proteins Otolin-1 and Otoconin 90 (OC90) perform in the formation of otoconia. We demonstrate by rotary shadowing and atomic force microscopy (AFM) experiments that Otolin-1 forms homomeric protein complexes and self-assembled networks supporting the hypothesis that Otolin-1 serves as a scaffold protein of otoconia. Our calcium carbonate crystal growth data demonstrate that Otolin-1 and OC90 modulate in vitro calcite crystal morphology but neither protein is sufficient to produce the shape of otoconia. Coadministration of these proteins produces synergistic effects on crystal morphology that contribute to morphology resembling otoconia.     

Studies of otoconia in the developing chick by polarized light microscopy

Using polarized light microscopy we were able to observe the mineralization patterns of embryonic and neonatal chick otoconia. We compared preparations of freshly dissected material spread under mineral oil to material that had been treated with various fixatives and dehydration agents. We found that the standard fixation agent, glutaraldehyde, and some immersion oils etched embryonic chick otoconia but that fixation with 70% acetone or 70% alcohol followed by dehydration to 100% acetone or 100% alcohol left the otoconia intact. The size and shape of freshly dissected chick otoconia observed with polarized light microscopy were similar to those of acetone-fixed, critical-point-dried material examined by SEM. Embryonic forms of otoconia were found to have a fluted pattern that was different in morphology from otoconia found in hatched chicks and adults. Embryonic chick otoconia did not exhibit the multifaceted surface morphology seen in embryonic rat otoconia. Comparisons of the same fields of otoconia under phase contrast microscopy and polarized light microscopy indicated that the freshly dissected otoconia of embryos exhibit little or no unmineralized (non-birefringent) material but that glutaraldehyde-fixed otoconia exhibited unmineralized areas where etching had occurred. Size frequency distributions of freshly dissected embryonic and mature otoconia in five ages of embryos and hatched chicks were consistent with a hypothesized developmental sequence of otoconia.     

Evolution of Otolithic Membrane. Structure of Otolithic Membrane in Amphibians and Reptilians

Otolithic membrane of utricles, saccules, and lagena of amphibians (Bufo bufo, Xenopus laevis, Rana temporaria) and reptiles (Teratoscincus scincus, Agama sanguinolenta, Ophisaurus apodus, Caiman crocodilus) were studied using light and scanning electron microscopy. Otolithic membrane in various otolithic organs in all studied animals was found to differ by shape, size, structure, and composition of otoconia. Otolithic membrane of utricle of amphibians and reptiles represents a thin plate of non-uniform structure. Otolithic apparatus in saccule represents a large cobble-stone-like conglomerate of otoconia. Otolithic membrane of lagena looks like a bent plate and is poorly differentiated in amphibians, but well differentiated in reptiles. Thus, transition of vertebrates to the earth surface was accompanied by a fundamental reorganization of otolithic membrane structure. Otolithic membrane containing constantly growing large otolith (in fish) was replaced by a thin structurally differentiated otolithic membrane that ceases its growth at early stages of ontogenesis. However, this replacement did not occur simultaneously in all otolithic organs. The changes initially involved otolithic membrane of utricle. Saccule of amphibians and reptiles has a typical compositional otolith. In the course of further phylogenetic development of tetrapods the process of structural differentiation of otolithic membrane was enhanced and otoliths were completely lost. In parallel, there proceeded a process of replacement of prismatic and spindle-shaped aragonitic otoconia by calcitic barrel-shaped otoconia. The data obtained confirm our hypothesis put forward earlier about two directions of evolution of otolithic membrane.     

Fusion of Otoconia: a Stage in the Development of the Otolith in the Evolution of Fishes

Abstract The rigid, polycrystalline otolith of teleosts is a side-branch of the general evolution of endolymphatic otoconia that extends from the sharks and rays to the higher vertebrates including man. The transition from the otoconial mass found in the endolymphatic sac of sharks and rays to the rigid polycrystalline otolith of teleosts probably occurred by progressive fusion of otoconia from a loose aggregate to a semi-rigid mass. Traces of the primitive fused otoconia type of otolith still occur in the otherwise polycrystalline otoliths of some teleosts, and a few species of fish retain otoliths that are probably similar to the primitive fused otoconia type of otolith. The morphology of the fusion of otoconia varies according to the polymorph of calcium carbonate that is involved, as well as the particular crystal habit of the polymorph. Analyses of the size distributions of the polymorph-specific morphologies and crystal structure of otoconia suggest that three physical chemical processes, Ostwald ripening, Keith-Padden spherulitic growth and carbonate cementation are significant in the chemistry of fusion of otoconia in the evolution of the aragonite teleost otolith. Predictions of otoconia growth rate from the theory of Ostwald ripening can be compared with predictions from the Keith-Padden theory of spherulitic growth.     

Principles of Calcite Dissolution in Human and Artificial Otoconia

Human otoconia provide mechanical stimuli to deflect hair cells of the vestibular sensory epithelium for purposes of detecting linear acceleration and head tilts. During lifetime, the volume and number of otoconia are gradually reduced. In a process of degeneration morphological changes occur. Structural changes in human otoconia are assumed to cause vertigo and balance disorders such as benign paroxysmal positional vertigo (BPPV). The aim of this study was to investigate the main principles of morphological changes in human otoconia in dissolution experiments by exposure to hydrochloric acid, EDTA, demineralized water and completely purified water respectively. For comparison reasons artificial (biomimetic) otoconia (calcite gelatin nanocomposits) and natural calcite were used. Morphological changes were detected in time steps by the use of environmental scanning electron microscopy (ESEM). Under in vitro conditions three main dissolution mechanisms were identified as causing characteristic morphological changes of the specimen under consideration: pH drops in the acidic range, complex formation with calcium ions and changes of ion concentrations in the vicinity of otoconia. Shifts in pH cause a more uniform reduction of otoconia size (isotropic dissolution) whereas complexation reactions and changes of the ionic concentrations within the surrounding medium bring about preferred attacks at specific areas (anisotropic dissolution) of human and artificial otoconia. Owing to successive reduction of material, all the dissolution mechanisms finally produce fragments and remnants of otoconia. It can be assumed that the organic component of otoconia is not significantly attacked under the given conditions. Artificial otoconia serve as a suitable model system mimicking chemical attacks on biogenic specimens. The underlying principles of calcite dissolution under in vitro conditions may play a role in otoconia degeneration processes such as BPPV.     

Mutated <Emphasis Type="Italic">otopetrin 1</Emphasis> affects the genesis of otoliths and the localization of Starmaker in zebrafish

Otoliths in bony fishes and otoconia in mammals are composite crystals consisting of calcium carbonate and proteins. These biominerals are part of the gravity and linear acceleration detection system of the inner ear. Mutations in otopetrin 1 have been shown to result in lack of otoconia in tilted and mergulhador mutant mice. The molecular function of Otopetrin 1, a novel protein that contains ten predicted transmembrane domains, however, has remained elusive. Here we show that a mutation in the orthologous gene in zebrafish is responsible for the complete absence of otoliths in backstroke mutants. We examined the localization of Starmaker, a secreted protein that is highly abundant in otoliths in backstroke mutants. Starmaker protein accumulated within cells of the otic epithelium, indicating a possible defect in secretion. Our data suggest that Otopetrin 1 in zebrafish may be involved in the protein trafficking of components required for formation of biominerals in the ear.     

Inactivation of NADPH oxidase organizer 1 results in severe imbalance

Otoconia are biominerals of the vestibular system that are indispensable for the perception of gravity. Despite their importance, the process of otoconia genesis is largely unknown. Reactive oxygen species (ROS) have been recognized for their toxic effects in antimicrobial host defense as well as in aging and carcinogenesis. Enzymes evolved for ROS production belong to the recently discovered NADPH oxidase (Nox) enzyme family . Here we show that the inactivation of a regulatory subunit, NADPH oxidase organizer 1 (Noxo1), resulted in the severe balance deficit seen in the spontaneous mutant "head slant" (hslt) mice whose phenotype was rescued by Noxo1 transgenes. Wild-type Noxo1 was expressed in the vestibular and cochlear epithelia and was required for ROS production by an oxidase complex. In contrast, the hslt mutation of Noxo1 was biochemically inactive and led to an arrest of otoconia genesis, characterized by a complete lack of calcium carbonate mineralization and an accumulation of otoconial protein, otoconin-90/95 (OC-90/95). These results suggest that ROS generated by a Noxo1-dependent vestibular oxidase are critical for otoconia formation and may be required for interactions among otoconial components. Noxo1 mutants implicate a constructive developmental role for ROS, in contrast to their previously described toxic effects.     

The otoconia of the guinea pig utricle: internal structure, surface exposure, and interactions with the filament matrix

A unique feature of the vertebrate gravity receptor organs, the saccule and utricle, is the mass of biomineral structures, the otoconia, overlying a gelatinous matrix also called "otoconial membrane" on the surface of the sensory epithelium. In mammals, otoconia are deposits of calcium carbonate in the form of composite calcite crystals. We used quick-freezing, deep etching to examine the otoconial mass of the guinea pig utricle. The deep-etching step exposed large expanses of intact and fractured otoconia, showing the fine structure and relationship between their internal crystal structure, their surface components, and the filament matrix in which they are embedded. Each otoconium has a compact central core meshwork of filaments and a composite outer shell of ordered crystallites and macromolecular aggregates. A distinct network of 20-nm beaded filaments covers the surface of the otoconia. The otoconia are interconnected and secured to the gelatinous matrix by surface adhesion and by confinement within a loose interotoconial filament matrix. The gelatinous matrix is a dense network made of yet another type of filament, 22 nm in diameter, which are cross-linked by shorter filaments, characteristically 11 nm in diameter. Our freeze-etching data provide a structural framework for considering the molecular nature of the components of the otoconial complex, their mechanical properties, and the degree of biological versus chemical control of otoconia biosynthesis.     

Gene targeting reveals the role of Oc90 as the essential organizer of the otoconial organic matrix

A critical part of the functional development of our peripheral balance system is the embryonic formation of otoconia, composite crystals that overlie and provide optimal stimulus input to the sensory epithelium of the gravity receptor in the inner ear. To date neither the functions of otoconial proteins nor the processes of crystal formation are clearly defined. Using gene targeting and protein analysis strategies, we demonstrate that the predominant mammalian otoconin, otoconin-90/95 (Oc90), is essential for formation of the organic matrix of otoconia by specifically recruiting other matrix components, which includes otolin, a novel mammalian otoconin that we identified to be in wildtype murine otoconia. We show that this matrix controls otoconia growth and morphology by embedding the crystallites during seeding and growth. During otoconia development, the organic matrix forms prior to CaCO3 deposition and provides optimal calcification efficiency. Histological and ultrastructural examinations show normal inner ear epithelial morphology but reduced acellular matrices, including otoconial, cupular and tectorial membranes, in Oc90 null mice, likely due to an absence of Oc90 and a profound reduction of otolin. Our data demonstrate the critical roles of otoconins in otoconia seeding, growth and anchoring and suggest mechanistic similarities and differences between otoconia and bone calcification.     

Immunocytochemical localization of secretory phospholipase A(2)-like protein in the pituitary gland and surrounding tissue of the bullfrog, Rana catesbeiana.

Previously, we obtained a protein that has considerable amino acid sequence homology with secretory phospholipase A(2) (PLA(2)) from a bullfrog pituitary fraction obtained during the purification of thyrotropin (TSH). Subsequently, partial amino acid sequence (N-terminal 45 amino acid residues) analysis revealed this protein to be identical to the N-terminal amino acid sequence of otoconin-22, the major protein of aragonitic otoconia in the Xenopus saccule. In this study we developed an antibody against the N-terminal peptide of the bullfrog protein and applied it for immunocytochemical study of the pituitary and its surrounding tissue. Western blotting analysis showed that this antibody recognizes a 20.4-kD protein that has a molecular mass close to that of otoconin-22. Immunohistochemical reaction with the antibody was not found in any anterior pituitary cells but was intense in the monolayer epithelial cells of the endolymphatic sac surrounding the pituitary gland, which is a major storage site of calcium carbonate in amphibians. An electron microscopic study revealed that the cuboidal cells in the endolymphatic sac contained large, polymorphic secretory granules in their apical cytoplasm. Immunogold particles indicating the presence of a PLA(2)-like protein were observed predominately in these secretory granules. These findings support the view that this PLA(2)-like protein obtained during purification of TSH was derived from the endolymphatic sac adhering to the pituitary and that this protein is a bullfrog otoconin. (J Histochem Cytochem 49:631-637, 2001)     

Influence of substrate mineralogy on bacterial mineralization of calcium carbonate: implications for stone conservation

The influence of mineral substrate composition and structure on bacterial calcium carbonate productivity and polymorph selection was studied. Bacterial calcium carbonate precipitation occurred on calcitic (Iceland spar single crystals, marble, and porous limestone) and silicate (glass coverslips, porous sintered glass, and quartz sandstone) substrates following culturing in liquid medium (M-3P) inoculated with different types of bacteria (Myxococcus xanthus, Brevundimonas diminuta, and a carbonatogenic bacterial community isolated from porous calcarenite stone in a historical building) and direct application of sterile M-3P medium to limestone and sandstone with their own bacterial communities. Field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), powder X-ray diffraction (XRD), and 2-dimensional XRD (2D-XRD) analyses revealed that abundant highly oriented calcite crystals formed homoepitaxially on the calcitic substrates, irrespective of the bacterial type. Conversely, scattered spheroidal vaterite entombing bacterial cells formed on the silicate substrates. These results show that carbonate phase selection is not strain specific and that under equal culture conditions, the substrate type is the overruling factor for calcium carbonate polymorph selection. Furthermore, carbonate productivity is strongly dependent on the mineralogy of the substrate. Calcitic substrates offer a higher affinity for bacterial attachment than silicate substrates, thereby fostering bacterial growth and metabolic activity, resulting in higher production of calcium carbonate cement. Bacterial calcite grows coherently over the calcitic substrate and is therefore more chemically and mechanically stable than metastable vaterite, which formed incoherently on the silicate substrates. The implications of these results for technological applications of bacterial carbonatogenesis, including building stone conservation, are discussed.     

Proteomics Analysis of Avian Eggshell Matrix Proteins: Toward New Advances on Biomineralization

Eggs are widely consumed all over the world. The eggshell is its protective barrier whose original function is to protect the embryo during development. Avian eggshells are made of calcium carbonate with a small amount of organic matrix (proteins and proteoglycans). During eggshell formation, the mineral precursors interact with matrix proteins to regulate the calcification of this highly resistant biomineral. In order to better characterize the functions of matrix proteins in eggshell biominerals, many proteomics studies have been performed during the last 15 years. The chicken eggshell is the main model studied in birds, but there is a need for comparative approaches in order to determine whether there is a general protein toolkits associated with calcitic biomineralization, and to determine its components. The study by Zhu et al., reported in article number 1900011, volume 19, issue 11, is a major step forward as it is the first shell proteomics survey performed on duck. Thus, it will contribute to improved knowledge of the eggshell mineralization process and will provide new insight for shell quality improvement and to guide biomimetic efforts in material sciences.     

Caspartin and calprismin, two proteins of the shell calcitic prisms of the Mediterranean fan mussel Pinna nobilis

We used the combination of preparative electrophoresis and immunological detection to isolate two new proteins from the shell calcitic prisms of Pinna nobilis, the Mediterranean fan mussel. The amino acid composition of these proteins was determined. Both proteins are soluble, intracrystalline, and acidic. The 38-kDa protein is glycosylated; the 17-kDa one is not. Ala, Asx, Thr, and Pro represent the dominant residues of the 38-kDa protein, named calprismin. An N-terminal sequence was obtained from calprismin. This sequence, which comprises a pattern of 4 cysteine residues, is not related to any known protein. The second protein, named caspartin, exhibits an unusual amino acid composition, since Asx constitutes by far the main amino acid residue. Preliminary sequencing surprisingly suggests that the first 75 N-terminal residues are all Asp. Caspartin self-aggregates spontaneously into multimers. In vitro tests show that it inhibits the precipitation of calcium carbonate. Furthermore, it strongly interferes with the growth of calcite crystals. A polyclonal antiserum raised against caspartin was used to localize this protein in the shell by immunogold. The immunolocalization demonstrates that caspartin is distributed within the prisms and makes a continuous film at the interface between the prisms and the surrounding insoluble sheets. Our finding emphasizes the prominent role of aspartic acid-rich proteins for the building of calcitic prisms among molluscs.     

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.     

Elongation factor 2 as the major substrate for Ca2+/calmodulin-dependent protein kinase in rat adrenal glomerulosa cells

We have previously shown the existence of the major substrate protein of Mr 100,000 (substrate 100 K protein) for Ca2+/calmodulin (CaM)-dependent protein kinase in rat adrenal glomerulosa cells. In the present study, the identity of the substrate 100 K protein to elongation factor 2 (EF-2) was investigated. In a 105,000 g-supernatant fraction (cytosol), the protein of Mr 100,000 with the pI (isoelectric point) value of 6.7 was phosphorylated in the presence of calcium and CaM. The optical densities of this phosphorylated band were greatly enhanced in the presence of the EF-2 purified from pig liver (1 microgram) [20-23-fold, n = 5] when compared with those in the absence of the component. In the presence of the purified EF-2, the phosphorylation of Mr 100,000 was detected only in the presence of calcium alone or calcium plus CaM. This phosphorylation in the presence of calcium alone was completely inhibited in the presence of the CaM antagonist pimozide (500 microM), showing the existence of endogenous CaM in the cytosol. In the same fraction, the ADP-ribosylated protein of Mr 100,000 was detected in the presence of diphtheria toxin (fragment A) and (adenylate-32P) NAD, indicating the presence of EF-2 in the cytosol from rat adrenal glomerulosa cells. These results suggest that the substrate 100 K protein may be identical to EF-2 in rat adrenal glomerulosa cells.     

Isolation and sequence analysis of a cDNA clone for a carrot calcium-dependent protein kinase: homology to calcium/calmodulin-dependent protein kinases and to calmodulin

Recently, a novel type of calcium-dependent protein kinase (CDPK) that requires neither calmodulin nor phospholipids for activation, has been described in plants. We have isolated a cDNA clone for carrot CDPK by probing a library of somatic embryo cDNAs with oligonucleotides corresponding to highly conserved regions of protein kinases. The product of this gene overexpressed in Escherichia coli reacted strongly with monoclonal antibodies to soybean CDPK. The deduced amino acid sequence of carrot CDPK reveals two major functional domains. An N-terminal catalytic domain with greatest homology to calcium/calmodulin-dependent protein kinase type II from rat brain is coupled to a C-terminal calcium-binding domain resembling calmodulin. These features of the primary sequence explain how CDPK binds calcium and suggest a model for CDPK regulation based on similarities to animal calcium/calmodulin-dependent protein kinases.     

Amino acid sequence analysis of two mouse calbindin-D9k isoforms by tandem mass spectrometry. Protein modification by internal insertion of a single amino acid

Two forms of calbindin-D9k have sometimes been observed within a single tissue. Sequencing of these proteins has been complicated by the presence of blocked amino termini. Tandem mass spectrometry is a powerful tool for comparing related proteins, and its use does not depend upon an unblocked amino terminus. In the present studies, calbindin-D9k was purified from the intestines of mice (270 animals per purification) by use of gel permeation chromatography and two preparative electrophoresis steps in the presence and absence of EDTA. The purified protein appeared to be homogeneous following electrophoresis under nondenaturing conditions, but two components were identified by sodium dodecyl sulfate-gel electrophoresis and immunoblotting. Two forms of the protein were isolated by reverse-phase high performance liquid chromatography. In each of three preparations, the average ratio of the major:minor isoforms was 2:1. The major form contained 77 amino acids and lacked the amino-terminal serine found in 78-amino acid calbindins from rat and pig. The amino acid sequence was identical with the deduced sequence reported for rat intestinal calbindin-D9k in 73 of 77 positions. In the minor form, a glutamine was found in a location between Lys-43 and Ala-44 of the major form and between the two calcium binding sites of the protein. The minor form was otherwise identical with the major form, including the presence of a blocked amino terminus. The inserted glutamine was located at the site of an intron in the rat calbindin gene, suggesting the possibility that alternative splicing produced the two forms of calbindin-D9k. The functional significance of an inserted amino acid between the two calcium binding sites remains to be explored.     

Anisotropic lattice distortions in biogenic calcite induced by intra-crystalline organic molecules

We have performed precise structural measurements on five different calcitic seashells by high-resolution X-ray powder diffraction on a synchrotron beam line and by laboratory single crystal X-ray diffraction. The unit cell parameters a and c of biogenic calcite were found to be systematically larger than those measured in the non-biogenic calcite. The maximum lattice distortion (about 2.10(-3)) was detected along the c-axis. Under heat treatment above 200 degrees C, a pronounced lattice relaxation was observed, which allowed us to conclude that anisotropic lattice swelling in biogenic calcite is induced by organic macromolecules incorporated within the single crystal calcitic prisms during biomineralization. This conclusion is supported by the results of crystallization experiments in the presence of specific protein extracted from one of the shells.     

Expression in Escherichia coli of full-length and mutant rat brain calbindin D28. Comparison with the purified native protein

Studies of vitamin D-dependent 28-kilodalton calcium binding protein (calbindin D28) have been hindered by difficulties in purifying large amounts of the protein. In order to overcome this problem, we cloned and expressed a full-length rat brain calbindin D28 cDNA. In addition, we isolated and purified to homogeneity, native rat brain calbindin D28. The isolated native protein has an apparent molecular mass of 27 kDa and properties similar to those of the well-characterized chicken calbindin D28. It has an acidic isoelectric point (approximately 4.5), a high affinity for calcium, and an amino terminus blocked to Edman degradation. The properties of the native and the recombinant proteins were examined by gel electrophoresis, isoelectric focusing, protein sequencing, amino acid composition analysis, and calcium binding assays. We demonstrated that: (i) the authentic and the full-length recombinant proteins have similar molecular weights and isoelectric points; (ii) the proteins have the same amino acid composition; (iii) the proteins bind calcium in a similar manner; (iv) the absence of a blocking NH2-terminal group in the recombinant protein does not appreciably influence the binding of calcium. To further examine the calcium binding properties of this protein, we constructed deletion mutants lacking one or both of the two putative degenerated calcium binding sites (EF hand regions). These deletions resulted in smaller proteins that still bound calcium. The ability to express and purify calbindin D28 and mutants thereof should allow the systematic elucidation of structure-function relationships in this class of calcium binding proteins.