pubs.acs.org/crystal Published on Web 10/19/2009 r 2009 American Chemical Society DOI: 10.1021/cg9006857 2009, Vol. 9 4897–4901 Control of Polymorphism and Morphology of Calcium Carbonate Crystals by a Matrix Protein Aggregate in Fish Otoliths 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 macromolecule- 64 (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 inverte- brates, is one of the most common biominerals and has three crystal phases: calcite, aragonite, and vaterite. Many organ- isms 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 magne- sium 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 poly- morphs 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 Mg 2þ concentrations (approximately 0.3 mM, Mg/Ca ∼ 0.2) and is stable in both freshwater and marine fishes, 6,7 indicat- ing 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 poly- saccharides, 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 water- insoluble gel-like structures in the biomineral matrices. How- ever, 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 Preparation of Protein-Bound Beads. Preparation of protein- bound beads were precisely described in the previous report. 8 In brief, an antiserum against OMM-64 was prepared using recombinant *Corresponding author. Tel./Fax: 81-138-40-5550. E-mail: takagi@fish. hokudai.ac.jp.