Control of Polymorphism and Morphology of Calcium Carbonate

Oct 19, 2009 - Faculty of Fisheries Sciences, Hokkaido University, 3-1-1 Minato, Hakodate, Hokkaido 041-8611,. Japan, and §Department of Earth and ...
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DOI: 10.1021/cg9006857

Control of Polymorphism and Morphology of Calcium Carbonate Crystals by a Matrix Protein Aggregate in Fish Otoliths

2009, Vol. 9 4897–4901

Hidekazu Tohse,†,‡ Kazuko Saruwatari,§ Toshihiro Kogure,§ Hiromichi Nagasawa,† and Yasuaki Takagi*,‡ †

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan, ‡Division of Marine Biosciences, Faculty of Fisheries Sciences, Hokkaido University, 3-1-1 Minato, Hakodate, Hokkaido 041-8611, Japan, and §Department of Earth and Planetary Science, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan Received June 19, 2009; Revised Manuscript Received August 27, 2009

ABSTRACT: We show that the high-molecular weight (HMW) protein aggregate of fish otolith matrix is involved in the regulation of crystal polymorphs during otolith biomineralization. Teleost fish otoliths are biominerals that contain calcium carbonate aragonite or vaterite crystals. In a previous study, we identified a novel protein named otolith matrix macromolecule64 (OMM-64) within the otolith organic matrix. In addition, we revealed that the HMW aggregate of otolith matrix architecture was comprised of OMM-64, in which inner-ear-specific short-chain collagen otolin-1 was also contained. By using an in vitro crystallization system in the absence of magnesium ions here, we show that native OMM-64 induced vaterite crystals, whereas native otolion-1 induced calcite crystals. However, the aggregate complex induced the aragonite polymorph in the same condition. The present data suggest that separation and structural and functional analyses of each matrix protein in the aggregate are absolutely imperative, but functional examination of the protein complex itself is equally important in clarifying polymorph control of biomineralization. Introduction Organisms can design and shape minerals to a desired conformation and orientation. Such mineral structures are called biominerals and cannot be formed in abiological environments. Calcium carbonate, formed mainly by invertebrates, is one of the most common biominerals and has three crystal phases: calcite, aragonite, and vaterite. Many organisms form aragonite crystals, but it is unknown why or how they form these thermodynamically metastable crystals. This so-called “calcite-aragonite problem” is one of the most important and difficult unsolved problems in biomineralogy, in spite of extensive research.1 Mollusk shells, especially pearl oysters, have been a target of study because they are composed of two layers: namely, calcite prisms and aragonite nacre. Although many reports suggested that water-soluble macromolecules that comprised nacre induced aragonite,2,3 most results have not identified molecules which can induce aragonite without either magnesium ions or supporting organic substances. Therefore, it may be valid to assume that aragonite crystals are easier to form in seawater because the high concentration of magnesium ions (approximately 50 mM, Mg/Ca ∼ 5) in the present “aragonite sea” prevents calcite crystallization. In fact, the principal components of the abiotic precipitates in shallow seawater are aragonite and Mg-calcite.4 Therefore, discussions on aragonite formation in abiotic environments and induction of calcite by organic molecules may not be contradictory.5 The fish otolith represents another useful biomineral for investigation of the calcite-aragonite problem. Otoliths exist universally in the vertebrate inner ear and their crystal polymorphs have diverged during evolution to include amorphous calcium phosphates in lamprey, aragonites in fishes and

amphibians, and calcites in higher vertebrates. In addition, teleosts have two types of calcium carbonate otoliths: sagittae (saccular otoliths) and lapilli (utricular otoliths) of aragonite, and asterisci (lagenar otoliths) of vaterite. These different polymorphs are formed in the endolymph, which has low Mg2þ concentrations (approximately 0.3 mM, Mg/Ca ∼ 0.2) and is stable in both freshwater and marine fishes,6,7 indicating that these polymorphs are regulated by organic molecules, but not by the ionic environment. Within the past decade, many proteins have been separated from various calcium carbonate biominerals. In the majority of the biomineral matrices, high-molecular-weight (HMW, >100 kDa) proteins are separated by gel electrophoresis. These substances may be aggregates of proteins and polysaccharides, and may play important roles in formation of the phases and/or morphologies of the crystals, because they consist of acidic glycoproteins and may construct waterinsoluble gel-like structures in the biomineral matrices. However, identification of proteins in the aggregates is extremely difficult, because these proteins are not separable by gel electrophoresis or liquid chromatography. In our previous study, we screened an expressed inner ear cDNA library using antibody raised against whole otolith matrix and identified a novel protein that constructs these aggregates in fish otoliths, designated otolith matrix macromolecule-64 (OMM-64).8 Characterization of this protein revealed that the aggregate interacts with the inner ear-specific collagen otolin-1.8 In the present study, we test the effects of natural OMM-64, otolin-1 and a protein aggregate which contains both proteins on calcium carbonate crystallization in vitro. Experimental Section

*Corresponding author. Tel./Fax: 81-138-40-5550. E-mail: takagi@fish. hokudai.ac.jp.

Preparation of Protein-Bound Beads. Preparation of proteinbound beads were precisely described in the previous report.8 In brief, an antiserum against OMM-64 was prepared using recombinant

r 2009 American Chemical Society

Published on Web 10/19/2009

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Tohse et al. Tokyo, Japan). The crystal phases were determined by Raman microscopy according to Dandeu et al.11 The experiments were carried out five times, and the crystals formed on the randomly selected 100 beads were counted in each experiment. Since identities between shapes and polymorphisms of calcite, vaterite, and aragonite crystals were confirmed by Raman spectrometry, the crystal polymorphisms for the counting were estimated from the shapes of the crystals. Statistical differences of the crystal numbers were evaluated by one-way analyses of variance and subsequent posthoc tests (TukeyKramer test). Significance was accepted at P < 0.05.

Results

Figure 1. Schematic representation of the beads used for in vitro crystallization experiments. Details were described in our previous study.8 In brief, single forms of OMM-64 and otolin-1 bound to the anti-OMM-64 beads (O64) and antiotolin-1 beads (O1), respectively, when incubated with the saccular extract (SE). These beads were designated O64 þ SE and O1 þ SE. HMW aggregates which contain both OMM-64 and otolin-1 were bound to both types of beads when incubated with the otolith matrix (OSM). These were designated O64 þ OSM and O1 þ OSM. Control beads were prepared using nProtein-A-bound Sepharose beads (ProA beads) incubated with PBS, SE or OSM, and O64 beads and O1 beads were incubated with PBS. These were designated ProA þ PBS, ProA þ SE, ProA þ OSM, O64 þ PBS, and O1 þ PBS. C-terminal peptide of OMM-64 as an antigen. Anti-otolin-1 antiserum was the one raised by Murayama et al.9 To obtain protein extract of inner ear cells, inner ear sacculi were dissected from the rainbow trout Oncorhynchus mykiss, washed, and homogenized in phosphate buffer saline (PBS). After centrifugation, the supernatant was obtained as the saccular extract. To obtain protein extract of the otolith, trout otoliths were decalcified using 0.5 M ethylenediaminetetraacetic acid (EDTA) solution and centrifuged. The supernatant was used as the extract of the otolith matrix. Aniti-otolin-1- and anti-OMM-64-beads were prepared by incubating nProtein-A-Sepharose beads (GE Healthcare Biosciences) with respective antisera described above. Then, the protein-bound beads were prepared by incubating antisera-bound beads with the protein extracts. In our previous study,8 we showed that single forms of OMM-64 and otolin-1 were bound to the anti-OMM-64 beads and anti-otolin-1 beads, respectively, when incubated with the saccular extract. In contrast, the HMW-aggregate which contains both OMM-64 and otolin-1 (and probably also other components) bound to both types of beads when incubated in the otolith matrix extract (Figure 1). In the present study, we used these beads to examine effects of otolin-1, OMM-64, or the HMW-aggregate on the crystallization of calcium carbonate on the surface of a solid substance. The anti-otolin-1 and anti-OMM-64 beads incubated with PBS were used as respective control beads. nProtein-A-bound Sepharose beads incubated with PBS, saccular extract, or otolith matrix, were also used to examine the effects of both nProtein-ASepharose beads and nonspecific protein binding. In Vitro Crystallization. Calcium carbonate crystals were grown on glass plates in each well of a 96-well plate. A 200-μL aliquot of 25 mM CaCl2 solution was put into each well, and a 2-μL bed of the affinity beads described above was added. Crystallization was allowed to proceed overnight under an ammonium carbonate atmosphere.10 The crystals produced were gently rinsed with distilled water and air-dried, and were observed under light and scanning electron microscopes (SEM, S2300 or S4500, Hitachi,

When control beads (nProtein-A-Sepharose beads incubated with PBS, saccular extract or otolith matrix, or antiOMM-64 or anti-otolin-1 beads incubated with PBS) were tested in the in vitro crystallization system, most of the crystals formed were rhombohedral crystals of ca. 50 μm (Figure 2A). Raman spectra showed that these rhombohedral crystals were calcite (Figure 3). When otolin-1-bound beads (anti-otolin-1 beads incubated with the saccular extract) were used, rhombohedral calcite crystals were also found but they were smaller (ca. 30 μm) than control beads (Figures 2B and 3). In contrast, the HMW-aggregate-bound beads (anti-otolin-1 beads incubated with otolith matrix extract) induced mainly needle-like aragonite crystals, although calcite crystals were also formed (Figures 2C, 3, and 4). Conversely, particulate crystals were formed on the surface of OMM-64-bound beads (anti-OMM64 beads incubated with the saccular extract) (Figure 2D). Rarely (∼10% frequency), spherical-shaped crystals with bubble-like structures were also found on these beads (Figure 2E). The polymorphs of both types of crystal were vaterite (Figure 3). On the surface of the HMW-aggregatebound beads made by incubating anti-OMM-64 beads with otolith matrix extract, needle-like crystals were observed (Figure 2F), like those on the HMW aggregate made by incubating anti-otolin-1 beads with otolith matrix extract. Dumbbell-shaped structures of ca. 5-10 μm (Figure 2G) and the putative central domain of the dumbbell-like crystals were also found (Figure 2H). All of these crystals were aragonite (Figure 3). The crystals shown in Figure 2F,G account for ∼80% and ∼20% of the aragonite crystals. The aragonite crystals shown in Figure 2H were rarely observed. In all experiments, crystals formed at locations other than on the beads were rhombohedral calcite of ca. 50 μm (data not shown). From these results, the numbers of crystals, with respect to each polymorph, on the protein-bound beads were statistically compared with those of respective control beads that were incubated with PBS (Figure 4). It is indicated that (1) the beads that bound otolin-1 alone (anti-otolin-1 beads incubated with saccular extract) induced significantly higher number of calcite crystals, (2) the beads that bound OMM64 alone (anti-OMM-64 beads incubated with saccular extract) induced significantly more vaterite crystals, and (3) the beads that bound HMW-aggregate (anti-otolin-1 and antiOMM-64 beads incubated with otolith matrix extract) induced a significantly higher number of aragonite crystals. The HMW-aggregate bound to the anti-OMM-64 beads also induced significantly more calcite crystals. On the other hand, (4) no significant differences were observed on the numbers of crystals, with respect to each polymorph, among nProtein-ASepharose beads incubated with PBS, saccular extract, and otolith matrix extract.

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Figure 3. Raman spectra of the crystals formed on control beads (ProA þ PBS), otolin-1-bound beads (O1 þ SE), HMW-aggregatebound beads (O1 þ OSM and O64 þ OSM), and OMM-64-bound beads (O64 þ SE). Spectra of crystals on control and otolin-1bound beads represented calcite, which was characterized by peaks at 156, 281, 713, and 1086 cm-1. The HMW aggregates induced aragonite crystallization, marked by 154, 207, 702, and 1085 cm-1 peaks. OMM-64 induced vaterite crystals that were determined by peaks at 750, 1074, and 1089 cm-1.

Figure 2. SEM observation of calcium carbonate crystals formed on nProtein-A-Sepharose beads and on the beads that bind otolin-1, OMM-64, or HMW aggregate. The beads are indicated by “b”. (A) Rhombohedral crystals of approximately 50 μm were formed on the nProtein-A-Sepharose beads incubated with PBS (ProA þ PBS). (B) When otolin-1-bound beads (O1 þ SE) were used, the shape of crystals was also rhombohedral, but crystals were smaller (∼30 μm) than those observed on ProA þ PBS. (C) Needle-like crystals were observed on the surfaces of antiotolin-1 beads that bind HMWaggregate (O1 þ OSM). (D, E) Particulate and bubble-like crystals, respectively, on the OMM-64-bound beads (O64 þ SE). The crystals shown in D and E accounted for about 90% and 10%, respectively. (F-H) Crystals on anti-OMM-64 beads that bind HMW aggregate (O64 þ OSM). These crystal formations accounted for ∼20% (F) and ∼80% (G). The crystals shown in H were rarely observed.

Discussion Most calcium carbonate biominerals contain aspartateand/or glutamate-rich acidic proteins in the water-soluble matrix, and framework macromolecules such as collagen and chitin in the water-insoluble matrix. Among these organic matrices, water-soluble, acidic proteins have been assumed to interact with calcium and regulate crystal formation.12 However, past studies using purified water-soluble proteins of

Figure 4. Species and numbers of crystals formed on nProtein-ASepharose beads (ProA), antiotolin-1 beads (O1), and anti-OMM64 beads (O64), incubated with PBS (PBS), saccular extract (SE), and otolith matrix (OSM). Data are the mean ( standard errors of five experiments. *Significantly different (P < 0.05) compared to numbers of calcite crystals induced on the antiotolin-1 beads incubated in PBS. **Significantly different (P < 0.05) compared to numbers of aragonite crystals induced on the antiotolin-1 beads incubated in PBS. †Significantly different (P < 0.05) compared to numbers of vaterite crystals induced on the anti-OMM-64 beads incubated in PBS. ††Significantly different (P < 0.05) compared to numbers of calcite crystals induced on the anti-OMM-64 beads incubated in PBS. †††Significantly different (P < 0.05) compared to numbers of aragonite crystals induced on the anti-OMM-64 beads incubated in PBS.

calcium carbonate biominerals suggest that none of the purified proteins can induce aragonite crystals, while aggregates of multiple proteins do induce aragonite crystals.13-16 A recent report showed that AP7, a protein obtained from nacre of Haliotis rufescens, could induce aragonite crystals, but this induction required an extremely high concentration of protein.17 Our results also indicate that the HMW aggregate in the otolith matrix, which contained both OMM-64 and otolin-1, induced aragonite, while neither OMM-64 nor otolin-1 itself induced aragonite. Our data suggest two possibilities:

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(1) the HMW aggregate contains an aragonite inducer other than OMM-64 or otolin-1, and (2) each matrix protein in the otolith cannot induce aragonite in a single form, but aggregation of the matrix proteins makes it possible to induce aragonite. At present, both hypotheses seem equally possible, and thus, further identification of components in the HMW aggregate is very important. However, components of the HMW aggregates in the otolith matrix except OMM-64 and otolin-1 are unknown now. Although we have tried to identify component molecules of the HMW aggregates, the components could not be separated by the strong reducing agents for SDS- and 2D-PAGE.8 In addition, we could not identify the component proteins, except OMM-64 and otolin-1, by MS/ MS analyses of the HMW band in the gels after SDS-PAGE using the aggregate-bound beads. The problem may be caused by an insufficient amount of the HMW-aggregate obtained by the present technique.8 The present data along with the previous results also suggest that separation and structural and functional analyses of each matrix protein are absolutely imperative. Further, their biological functions should be examined by considering the native conditions of the molecules. It is important to examine the function of the matrix molecule complexes, because these molecules are able to function as complexes within the biominerals. Indeed, Levi et al.18 succeeded in synthesizing aragonite crystals by combining poly aspartic acid peptides, fibroin and chitin, mimicking the composition of biominerals. OMM-64 has three distinctive domains: namely, two tandem repeat sequences and a glutamate-rich region.8 Because Repeat 1 may be highly phosphorylated, and the glutamaterich region and Repeat 2 contain many acidic residues, OMM64 may be very acidic overall. In addition, the glutamate-rich region of the protein has calcium binding activity.8 We also found starmaker19 as a candidate for an ortholog to OMM64. Starmaker is a matrix protein contained in zebrafish otolith and knocking down starmaker expression induces a variation in the polymorphisms of otolith crystals from aragonite to calcite.19 Structural similarities found between OMM-64 and starmaker may lead to similar functions in these proteins. These data suggest that OMM-64 is a molecule categorized as a water-soluble, acidic matrix protein in the otolith. On the other hand, otolin-1 is a short-chain collagen categorized as a water-insoluble, framework matrix protein in the otolith.9 Therefore, the data support the concept that a complex of structural proteins and acidic calcium-binding proteins function as crystallization regulators in the biominerals. Many aragonite crystals were formed on the beads that bound the HMW aggregate. The number of calcite crystals formed here was also greater than that in control experiments. These results suggest that the HMW aggregate in vitro has a stimulatory activity on crystal nucleation. However, the finding that the number of crystals on the beads binding otolin-1 alone also increased indicates, at least in vitro, that otolin-1 can accelerate crystal nuclei formation. We could not determine the structural conformations of the proteins that comprise the aggregate, and thus, it is unclear whether stimulated crystal nucleation by the HMW aggregate could be attributed to the otolin-1. The precise mechanism of aragonite induction and stimulated nucleation induced by HMW aggregate remains unclear. In zebrafish, the nuclei of sagitta are formed at about 24 h post fertilization (hpf) by aggregating proteins and polysaccharides secreted by otosac epithelial cells.20 Otolin-1, which is

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necessary for aragonite crystal formation in vitro, is expressed at the early stage of the zebrafish inner ear development (48 hpf) and is involved in the seeding and/or nucleation of the sagitta and lapillus.21 Starmaker is also expressed early (24 hpf).19 Therefore, in zebrafish, otolin-1 and starmaker are likely contained in the nuclei of sagitta as an aggregate and contribute to formation of the aragonite polymorph. Similarly, in the trout otolin-1 was immunohistochemically localized in the very early primordia (nuclei) of the sagitta.22 Although expression of OMM-64 during inner ear development of the trout has not been studied, it may be expressed at earlier stages because omm-64 mRNA expression was also found in the trout embryo at the 50% epiboly stage.8 Therefore, it seems likely that the aggregate containing otolin-1 and OMM-64 may also function in the nucleation of sagitta in trout. It is unknown, however, whether these proteins contribute to nucleation of the vaterite otolith asteriscus. During otolith development in zebrafish, both the aragonite sagitta (saccular otolith) and lapillus (utricular otolith) are formed in the same environment of the single otic vesicle, at early developmental stages (24-30 hpf).23 By contrast, the vaterite asteriscus develops in the lagena, which differentiates from the otosac after initiation of the formation of sagitta and lapillus (15 dpf).24 Therefore, it is possible that the developmental process that underlies aragonite otoliths and vaterite otoliths is different. Although otolin-1 is also present in the trout asterisci (unpublished data), it is unknown whether otolin-1 contributes to nucleation of the vaterite crystals. Polymorphism of crystals is thought to be determined at the stage of nucleation of the crystal, and the polymorphism of the subsequently formed crystals is determined by learning the lattices of the already-formed crystals. Therefore, it will be important to study whether the saccular collagen otolin-1 is involved in nucleation of the vaterite otolith asteriscus. In summary, we have clarified that the protein aggregate in the otolith, which contains OMM-64 and otolin-1, is involved in the formation of polymorphs and morphologies of calcium carbonate crystals. The aggregate may function in the formation of otolith morphologies as a crystal nucleator, an inhibitor of crystal growth and an inducer of the aragonite polymorph. Acknowledgment. The authors sincerely appreciate Dr. Motohiro Shimizu, Hokkaido University, for his technical advice on SEM observation, Prof. Hiroyuki Kagi, University of Tokyo, for his kind approval to use Raman microscopy in his laboratory, and Dr. Hirotoshi Endo, Hokkaido University, for his useful advice on the in vitro crystallization experiments using affinity beads. Fish used in this study were kindly supplied by Nikko Station, Freshwater Research Division, National Research Institute of Fisheries Science, and Nanae Freshwater Station, Field Science Center for Northern Biosphere, Hokkaido University. This study was financially supported in part by Grants-in-Aid for Creative Basic Research (Nos. 12NP0201, 17GS0311), for 21st century COE Program, and for Young Scientists (Start-up, No. 18880001). H.T. was supported by the Research Fellowship of the Japan Society for Promotion of Science for Young Scientists (Nos. 15-10657) and the Akiyama Memorial Life Science Foundation (No. 18-6).

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