Identification of a novel matrix protein contained in a protein aggregate associated with collagen in fish otoliths Hidekazu Tohse1,2, Yasuaki Takagi2 and Hiromichi Nagasawa1 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan Division of Marine Biosciences, Graduate School of Fisheries Science, Hokkaido University, Japan Keywords biomineralization; calcium binding; calcium carbonate; collagen; otolith matrix Correspondence Y Takagi, Division of Marine Bioscience, Graduate School of Fisheries Science, Hokkaido University, 3-1-1 Minato, Hakodate, Hokkaido 041-8611, Japan Tel ⁄ Fax: +81 138 40 5550 E-mail: takagi@fish.hokudai.ac.jp Database Nucleotide sequence data are available in the DDBJ ⁄ EMBL ⁄ GenBank databases under the accession number AB213022 (Received 31 December 2007, revised 10 March 2008, accepted 13 March 2008) doi:10.1111/j.1742-4658.2008.06400.x In the biomineralization processes, proteins are thought to control the polymorphism and morphology of the crystals by forming complexes of structural and mineral-associated proteins To identify such proteins, we have searched for proteins that may form high-molecular-weight (HMW) aggregates in the matrix of fish otoliths that have aragonite and vaterite as their crystal polymorphs By screening a cDNA library of the trout inner ear using an antiserum raised against whole otolith matrix, a novel protein, named otolith matrix macromolecule-64 (OMM-64), was identified The protein was found to have a molecular mass of 64 kDa, and to contain two tandem repeats and a Glu-rich region The structure of the protein and that of its DNA are similar to those of starmaker, a protein involved in the polymorphism control in the zebrash otoliths [Sollner C, Burghamă mer M, Busch-Nentwich E, Berger J, Schwarz H, Riekel C & Nicolson T (2003) Science 302, 282–286] 45Ca overlay analysis revealed that the Glu-rich region has calcium-binding activity Combined analysis by western blotting and deglycosylation suggested that OMM-64 is present in an HMW aggregate with heparan sulfate chains Histological observations revealed that OMM-64 is expressed specifically in otolith matrix-producing cells and deposited onto the otolith Moreover, the HMW aggregate binds to the inner ear-specific short-chain collagen otolin-1, and the resulting complex forms ring-like structures in the otolith matrix Overall, OMM-64, by forming a calcium-binding aggregate that binds to otolin-1 and forming matrix protein architectures, may be involved in the control of crystal morphology during otolith biomineralization Organisms can design and shape minerals to the desired conformation and orientation Such mineral structures are called biominerals and cannot be formed by any non-biological environments Calcium carbonate is one of the most common biominerals, formed mainly by invertebrates, and has three crystal phases: calcite, aragonite and vaterite Although calcite is the most stable crystal thermodynamically, many organisms can form metastable aragonite crystals with desired morphologies under normal environments of pressure and temperature It is thought that the morphology and polymor- phism of biominerals can be controlled by the proteins, polysaccharides and complexes (organic matrices) within the biominerals themselves [1,2] In the past decade, many proteins have been isolated from various calcium carbonate biominerals, and their roles in the formation of crystal morphologies have been discussed These isolated single proteins have some activity in changing crystal morphologies; however, analyses of the single proteins has not led to insights into how these morphologies and polymorphisms are formed in the biominerals, as the Abbreviations GST, glutathione S-transferase; HMW, high molecular weight; IPTG, isopropyl-b-D-thiogalactopyranoside; OMM-64, otolith matrix macromolecule-64; PVDF, polyvinylidene difluoride; TFMS, trifluoromethanesulfonic acid 2512 FEBS Journal 275 (2008) 2512–2523 ª 2008 The Authors Journal compilation ª 2008 FEBS H Tohse et al organic matrices are thought to be formed from a complex of individual matrix proteins For example, in biomineralization of mollusk shells, which have chitin as the structural molecule in their EDTA-insoluble fraction [3], isolated proteins from the EDTA-insoluble fraction exhibit different actions on the crystal formation when they are applied to crystal induction systems with framework organic substrates [4–7], indicating that such proteins may interact with the framework molecules [8] In biomineral matrices, it is believed that framework molecules construct the basic scaffold and form water- or EDTA-insoluble matrices with other proteins that bind to the frameworks In addition, it is thought that water-soluble proteins and polysaccharides are bound to the frameworks, possess mineral (calcium)-binding activity and enable water-gaining dilatation to form gel-like structure of organic matrices However, the structural and biochemical bases of the biomineralization framework and mineralassociated protein have not been elucidated On the other hand, vertebrates possess collagens as a main component of the structural framework In fish otoliths of the inner ear (a calcium carbonate biomineral of vertebrates), collagen functions as a structural framework substance [9] The structure of fish otoliths comprises a tree-ring-like layered biomatrix [10], and collagen forms the ring structures by periodic deposition onto the otolith matrix [11,12] A gel-like structure containing the framework is observed upon decalcification, suggesting that the otolith matrix is constructed from large aggregates of framework molecules and mineral-associated molecules In the majority of biomineral matrices, not just mollusk shells and otoliths, high-molecular-weight (HMW, > 100 kDa) proteins are observed 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 water-insoluble, gel-like structures in the biomineral matrices Identifying the proteins that construct the aggregates is extremely difficult, however, because these proteins are not separable by gel electrophoresis or liquid chromatography In the present study, we have examined and characterized the proteins that form these aggregates in fish otoliths We had previously raised an antiserum against whole otolith matrix containing mainly HMW (> 100 kDa) proteins [13], and here we used this antiserum to screen an inner ear cDNA library and thereby clone a cDNA encoding a protein, named otolith matrix macromolecule-64 (OMM-64), that is contained in a HMW aggregate in the otolith matrix During characterization of this A novel protein in the otolith matrix framework protein, we revealed that the aggregate also contains the inner ear-specific collagen otolin-1 [9] Results Cloning of cDNA and DNA encoding OMM-64 To obtain cDNA clones encoding proteins contained in the HMW aggregate, immunoscreening was performed using an antiserum that reacts mainly with the aggregate in the otolith matrix [13] After screening, clones containing omm-64 cDNA were obtained, but the sequence of the 5¢ end could not be determined Therefore, 5¢ RACE was performed In addition, genomic DNA encoding OMM-64 was also obtained by genome walking Structures of OMM-64 protein and DNA The cDNA cloned had a length of 2776 bp and encodes a protein of 628 amino acids (Fig and supplementary Fig S1) The open reading frame is followed by a 3¢ UTR containing a putative polyadenylation signal, AATAAA (nt 2747–2752) The relative molecular weights of the precursor including the signal peptide and of OMM-64 without the signal peptide were calculated to be 66 580 and 64 486, respectively, based on the deduced amino acid sequence Sequence analysis showed that OMM-64 has three distinct domains: two tandem-repeat domains of SP(G ⁄ E ⁄ R)- Fig (A) Schematic of omm-64 DNA and protein structure Detailed sequences of the mRNA and amino acids are shown in supplementary Fig S1 (the GenBank accession number for omm64 mRNA is AB213022) The DNA encoding OMM-64 is split into 23 exons (closed boxes), and several transcription factor-binding sites (closed circles) are predicted to occur in the region 5¢ to the gene OMM-64 has two tandem repeats (R1 and R2) and a Glu-rich region (E-rich) SP, signal peptide (B) Expression of omm-64 mRNA examined by RT-PCR Expression of b-actin mRNA was also examined as an endogenous control S.C., semicircular canal; W muscle, white muscle; R muscle, red muscle; B kidney, body kidney; H kidney, head kidney FEBS Journal 275 (2008) 2512–2523 ª 2008 The Authors Journal compilation ª 2008 FEBS 2513 A novel protein in the otolith matrix framework H Tohse et al SDS(T ⁄ A)(E ⁄ D) (·6) and MDK(D ⁄ E)D (·5) and a glutamate-rich region Overall, including these domains, OMM-64 is rich in acidic residues (Asp + Glu, 35%) In silico analysis using netphos (http:// www.cbs.dtu.dk/services/NetPhos/) predicted that most serine residues in tandem repeat are phosphorylated In the whole sequence, 14% of the amino acids are predicted to be phosphorylated (66 serines, 19 threonines and one tyrosine) A blastp search using the amino acid sequence of OMM-64 identified starmaker, a zebrafish otolith matrix protein that contributes to the regulation of otolith crystal polymorphism [14] Although the identity between these proteins was only 25%, some distinctive domains of starmaker are conserved in OMM-64 (supplementary Fig S2): an N-terminal sequence containing signal peptides (Met1–Ala36) is highly conserved, and two (V ⁄ G)TTD sequences found in the tandem repeats of starmaker are also found in OMM-64 By contrast, a distinctive sequence that is rich in serine and aspartic acid in starmaker is not conserved in OMM-64, which has a glutamic acid-rich sequence instead A partial sequence of omm-64 mRNA was found in the GenBank EST database (accession number CX067293) This rainbow trout mRNA had been identified by random sequencing analysis of a cDNA library constructed by suppressive subtraction of whole-embryo mRNA at late neurogenesis stages (hindbrain swelling + heart tube with peristalsis) from that at early neurogenesis stages (neural groove + 50% epiboly), suggesting that omm-64 is expressed in the early neurogenesis stage of the embryo and is involved in inner ear development In omm-64 gene, the sequence encoding OMM-64 is divided into 23 exons, including two large exons in the middle region of the ORF and the 3¢ UTR (Fig 1) This exon ⁄ intron structure is highly similar to that of the starmaker gene (supplementary Fig S2): many small introns are present in the region encoding the Nterminal portion of the protein (including the signal peptide and small tandem repeats), a large exon comprises the middle region of the ORF, which encodes the Glu-rich region in OMM-64 and the Ser ⁄ Asp-rich region in starmaker, and the 3¢ UTR is transcribed from a single large exon In addition, all of the distinctive domains of the proteins are translated from single exons (supplementary Fig 1A) Inner ear-specific expression of omm-64 mRNA Expression of omm-64 mRNA was specific to inner ear tissues (sacculus and semicircular canals), with the exception of the ovary (Fig 1B and supplementary 2514 Fig S3) In the inner ear sacculus, strong hybridization signals were detected, mainly in the cells at the periphery of the macula and in transitional epithelial cells except mitochondria-rich cells (ion-transporting ionocytes, Fig 2A,B), which can be distinguished from Fig Localization of omm-64 mRNA expression in the inner ear sacculus by in situ hybridization (A) Sagittal section of whole sacculus The regions magnified in (B)–(D) are indicated by boxes (B) Macula (M) and transitional epithelial (TE) regions Intense hybridization signals were observed in the cells at the periphery of the macula (arrowhead) and in transitional epithelial cells omm-64 mRNA was not expressed in the mitochondria-rich cells (MRC) (C) No hybridization signal was observed in the ventral region of the sacculus (D) Hybridization signals were barely detected in the distal region of the sacculus (E) Hematoxylin ⁄ eosin staining of the region shown in (B), to differentiate between transitional epithelial cells (TC) and mitochondria-rich cells (ion-transporting ionocytes), which stain positive with eosin Sense-strand probes did not hybridize to any regions of the sacculus (data not shown) CT, connective tissue; EL, endolymph region; SqE, squamous epithelial cells FEBS Journal 275 (2008) 2512–2523 ª 2008 The Authors Journal compilation ª 2008 FEBS H Tohse et al other types of cells owing to their large size and shape and positive eosin staining (Fig 2E), and which, like chloride cells, have Na+ ⁄ K+-ATPase activity [15] Weak expression of omm-64 mRNA was detected in the sensory epithelium (macula) In the ventral, dorsal and distal areas of the sacculus, by contrast, mRNA hybridization signals were barely detectable (Fig 2C,D) Identification of the calcium-binding domain in OMM-64 To determine the regions that have calcium-binding activity, six fusions of GST with recombinant proteins of OMM-64 (rOMM-64) were produced and applied to a 45Ca overlay assay (Fig 3) Of these recombinant Fig 45Ca overlay analysis of fusions of GST and recombinant OMM-64 variants (rOMM-64-I-V and -C), containing different domains of the protein, to determine the calcium-binding domain of the protein (A) Schematic drawing of the recombinant proteins Six GST-fused recombinant proteins containing the three distinctive domains of tandem repeat (R1), the Glu-rich domain (E-rich) and ⁄ or tandem repeat (R2) were synthesized SP, signal peptide of the OMM-64 precursor (B) ‘Stains-all’ staining of the recombinant OMM-64 variants separated by SDS–PAGE to detect negatively charged proteins as blue bands (left) and 45Ca overlay analysis of the proteins (right) I–V, C and G indicate the respective recombinant proteins G, GST Calmodulin (C), used as a positive control, was detected at approximately 17 kDa A novel protein in the otolith matrix framework proteins, rOMM-64-I, III, IV and V, which include the Glu-rich domain, were found to have calcium-binding activity rOMM-64-II and -C and GST were stained red using ‘Stains-all’ and were not detected by 45Ca This result suggests that the Glu-rich domain of OMM-64 has affinity for calcium We cannot conclude, however, that other regions of the protein not have calcium-binding activity, because we used recombinant proteins that were not phosphorylated Characterization of native OMM-64 To characterize the native form of OMM-64, western blotting was performed using anti-rOMM-64-C serum In the sacculus and endolymph, multiple bands were detected but most of these were non-specific, as assessed by comparison with the preimmune serum; however, a 64 kDa band bound specifically to the antiserum (Fig 4) In the EDTA-soluble otolith matrix, a diffuse immunoreaction band was observed at > 100 kDa, but weak non-specific binding around 100 kDa was also detected However, a strong specific reaction in the HMW region was observed in both EDTA-soluble and -insoluble matrices, indicating that OMM-64 may be contained in the aggregate of the HMW proteins described above After digestion of the side chains using deglycosylation enzymes, the intensity of the immunoreactive band in the HMW region was decreased and a new band was detected at 64 kDa, but only after treatment with heparitinase II (Fig 5A) Although the same band was obtained after digestion Fig Detection of OMM-64 in the inner ear tissues by western blotting using anti-rOMM-64-C serum In the saccular extract (S) and endolymph (E), OMM-64 bands were observed by both ‘Stainsall staining’ and western blotting (arrowheads) All proteins in the EDTA-soluble (OS) and -insoluble (OI) otolith matrix were stained blue using ‘Stains-all’ In these matrices, strong immunoreactions were detected in the high-molecular-weight region (arrows) FEBS Journal 275 (2008) 2512–2523 ª 2008 The Authors Journal compilation ª 2008 FEBS 2515 A novel protein in the otolith matrix framework H Tohse et al Fig OMM-64 is contained in the HMW aggregate in the otolith matrix and is excised from the aggregate by deglycosylation using TFMS or heparitinase II (A) Western blotting of EDTA-soluble otolith matrix proteins (OSM) after digestion of polysaccharides by glycopeptidase A (0.5 munits, G), chondroitinase ABC (0.5 units, C), heparitinase II (10 munits, H), hyaluronidase SD (25 munits, Y) and endo-a-N-acethylagalactosaminidase (70 munits, E) The HMW aggregate was digested only by heparitinase II (arrowhead), and a 64 kDa protein band appeared instead (arrow) Some non-specific binding was observed when these enzymes alone were subjected to SDS–PAGE (Enzyme) (B) Time course of the effect of TFMS treatment on the aggregate and free OMM-64 Although the 64 kDa band was observed by western blotting after treatment with TFMS for at least (arrow) (aOMM-64), ‘Stains-all’ staining showed that the HMW aggregate was not digested completely even after 30 of treatment (arrowhead) Silver staining indicated that the other proteins may be damaged by the 30 TFMS treatment (C) Heparitinase II digests the HMW aggregate (arrowhead) and separates free OMM-64 (arrow) in a concentrationdependent manner Bovine serum albumin, which was contained in the enzyme solution, was observed at 66 kDa by both silver and ‘Stains-all’ staining glycosaminoglycans, and can be released from the aggregate by deglycosylation Localization of OMM-64 in the extracellular matrices To determine the in vivo localization of OMM-64, immunohistochemistry was performed using antirOMM-64-C serum Similar to omm-64 mRNA expression, immunoreactivity was detected in cells at the periphery of the macula and in transitional epithelial cells except mitochondria-rich cells (Fig 6A,B) The basement membranes and connective tissues were immunonegative We found that OMM-64 accumulates at the apical membrane in macula (Fig 6A) and in the ring-like structures in otoliths (Fig 6C) of the sugar chains using trifluoromethanesulfonic acid (TMSF), the HMW aggregate was not completely digested even after 30 of treatment (Fig 5B) After 15 min, many protein bands were detected by silver staining, suggesting that several proteins in the otolith matrix are glycosylated and can be separated by electrophoresis However, most of these protein bands disappeared after 30 of treatment, indicating that these proteins are damaged by long incubation with TFMS On the other hand, heparitinase II was able to completely digest the HMW aggregate (Fig 5A), and OMM-64 was separated from the aggregate in a concentration-dependent manner (Fig 5C) These results suggest that OMM-64 is contained in the otolith matrix aggregate consisting of heparan sulfate 2516 Inner ear-specific collagen otolin-1 is contained in the OMM-64-bound HMW aggregate To purify the mature form of OMM-64, anti-rOMM-64 affinity beads were allowed to react with saccular and otolith matrix extracts After incubation with the otolith matrix extract and stringent washing with acidic glycine, the beads were found to bind a HMW protein and two proteins of approximately 95 and 140 kDa (Fig 7) The HMW protein band reacted strongly with anti-rOMM-64 serum, whereas the other two bands of 95 and 140 kDa immunoreacted with anti-recombinant otolin-1 (rOtolin-1) serum, as previously reported [9,11] These results suggest that the HMW aggregate contains OMM-64 and otolin-1 within the otolith FEBS Journal 275 (2008) 2512–2523 ª 2008 The Authors Journal compilation ª 2008 FEBS H Tohse et al A novel protein in the otolith matrix framework Fig Separation of native OMM-64, otolin-1 and their complex by co-immunoprecipitation Anti-rOMM-64 or anti-rOtolin-1 affinity beads were incubated with NaCl ⁄ Pi (N), saccular extract (S) or EDTA-soluble otolith matrix (O), and specifically bound proteins were subjected to electrophoresis and staining using ‘Stains-all’ Western blotting using anti-rOMM-64 and anti-rOtolin-1 antisera was also performed When the affinity beads were incubated with saccular extract, OMM-64 (arrows) and otolin-1 (arrowheads) bound separately to the beads By contrast, incubation with otolith extract resulted in binding of a complex of the HMW aggregate containing OMM-64 and otolin-1 to the beads Fig Localization of OMM-64 in the inner ear sacculus by immunohistochemistry (A) Macular region of the sacculus after removal of the otolith Strongly immunoreactive cells were observed at the periphery of the macula (arrows) OMM-64 was also detected in the apical region of the macula (arrowheads) No immunoreaction was detected in connective tissue (CT) EL, endolymphatic space (B) Transitional epithelium observed by differential interference contrast microscopy Intense signals were observed in the transitional epithelial cells (arrows) but not in the mitochondria-rich cells (arrowheads) [15] (C) Localization of OMM-64 in the otolith region observed by differential interference contrast microscopy OMM64 was localized in the ring-like structures in the otolith (D) No immunoreaction was observed in the negative control section of the otolith region incubated with preimmune serum altered to primary antibody CT, connective tissue; EL, endolymph region; M, macula; O, otolith; TE, transitional epithelium (E) Schematic of the inner ear sacculus containing the otolith, indicating the sections in (A)–(D) matrix We also observed this interaction when antirOtolin-1 affinity beads were used for the same experiments However, because non-specific immunoreactive bands were observed in western blotting using antirOtolin-1 serum, the 95 and 140 kDa bands could not be confirmed to be otolin-1 Therefore, MALDI-TOFTOF tandem mass spectrometry was performed to identify the proteins The tryptic peptide mass fingerprinting spectra of these proteins were highly similar (supplementary Fig S4), and both proteins were identified as otolin- by both peptide mass fingerprinting and MS ⁄ MS ion searches on the mascot server [16] with high scores Although some differences between the spectra were found, structural differences in these proteins could not be identified By contrast, the HMW aggregate and otolin-1 bands were separately detected when the beads were reacted with the saccular extract (Fig 7), suggesting that these factors exist independently in the cells and are not FEBS Journal 275 (2008) 2512–2523 ª 2008 The Authors Journal compilation ª 2008 FEBS 2517 A novel protein in the otolith matrix framework H Tohse et al associated directly In addition, using anti-rOMM-64 beads, a 64 kDa protein, which was not detected by gel staining, was detected by anti-rOMM-64 serum Overall, these data suggest that both OMM-64 and otolin-1 are contained in the HMW protein aggregate in the otolith matrix Discussion Since the proposal of aragonite crystal induction by water-soluble organic matrices from aragonite biominerals [1,2], numerous studies have investigated proteins, mainly in the nacre of mollusk shells, to reveal how aragonite polymorphs are formed in biominerals However, no single protein that induces aragonite formation has been identified, although some reports have presented evidence that multiple matrix proteins induce aragonite crystals [4–7] We therefore examined proteins contained in aggregates within the otolith matrix and identified a protein contained in the HMW protein–glycosaminoglycan aggregate that also contains the otolith structural protein otolin-1 This protein may exist freely in the saccular cells and be incorporated into the HMW aggregate in the otolith In our previous study, an antiserum raised against whole EDTA-soluble otolith matrix, which was used for immunoscreening in the present study, did not bind to a 64 kDa band, but did bind to the HMW aggregate in the otolith matrix [13] This indicates that OMM-64 is not freely localized, but is contained in the HMW aggregate in the otolith matrix However, what kinds of molecules are present in the aggregate in addition to OMM-64, otolin-1 and heparan sulfate, and how these proteins interact, remains unknown The protein identified has three distinctive domains: namely, two tandem repeat sequences and a Glu-rich region Because repeat may be highly phosphorylated, and the Glu-rich region and repeat contain many acidic residues, OMM-64 may be very acidic overall and may function in interactions with calcium and subsequent mineral crystallization Although the putative isoelectric point of the OMM-64 was calculated to be 3.5, the mature form of OMM-64 may be more acidic because it may be highly phosphorylated Although we determined that the Glu-rich region of the protein has calcium-binding activity, we could not confirm whether repeat also has activity because we used non-phosphorylated recombinant proteins for the calcium-binding assay Therefore, the functions of the two tandem repeat domains remain unknown at present We found starmaker and human dentin sialophosphoprotein to be homologous proteins to 2518 OMM-64 by blast search (blastp and tblastn) The relationship between starmaker and dentin sialophosphoprotein has been discussed in detail in a previous report [14] Although some structural similarities in the protein and gene were found between OMM-64 and starmaker (see Results), they may not be orthologs because of their relatively low identity (25%) However, their structural similarities may lead to similar functions In fact, knocking down starmaker expression induces a variation in the polymorphism of otolith crystals, from aragonite to calcite [14] Therefore, OMM-64 and starmaker are thought to be related proteins in terms of both structure and function Although we carried out various blast searches using amino acid, mRNA and genomic DNA sequences as queries, no orthologous gene in any other species was identified Sollner et al [14] also ă reported that they could not nd an ortholog of starmaker These observations suggest two possibilities: (i) omm-64 and starmaker are orthologous genes that are highly diverged, so the identity of their sequences is low and orthologs cannot be found, or (ii) they are different but similar genes that are conserved only in species that can form aragonite otoliths At present, however, we are unable to differentiate between these possibilities We have shown that OMM-64 is contained in the HMW aggregate, which may comprise proteins and heparan sulfate glycosaminoglycan chains, although the structures of the proteins and the glycosaminoglycan chains were not characterized Although it has been suggested that glycosaminoglycan chains are involved in the nanoscale processes of calcium carbonate biomineralization by associating with crystals via their sulfates [17], no proteoglycan has been identified in fish otoliths In mammalian bone, heparan sulfate proteoglycans are localized mainly on the cell surfaces, basement membranes and bone matrix, and are involved in bone formation through regulation of cell differentiation factors such as bone morphogenetic proteins and fibroblast growth factors [18] Similar to the extracellular matrices in bone, it is conceivable that structural proteins such as collagens may also construct the extracellular matrices in the inner ear by binding to glycosaminoglycans, which can hold water and form gels The tissue-specific and proximal side-specific distribution of mRNA expression and immunolocalization suggests the potential function of OMM-64 In the inner ear sacculus, the otolith is close to the proximal side of the sacculus, and calcification of the otolith occurs mainly at the proximal surface [19] In addition, proteins that may be involved in otolith calcification FEBS Journal 275 (2008) 2512–2523 ª 2008 The Authors Journal compilation ª 2008 FEBS H Tohse et al are concentrated in the proximal endolymph [20] Therefore, the proximal region of the sacculus produces the otolith matrix proteins [13] and forms the environment for otolith mineralization In particular, the cells located at the periphery of the macula may be specialized for production of the otolith matrix proteins, because these cells are rich in rough endoplasmic reticulum [13], and two otolith matrix proteins, otolith matrix protein-1 and otolin-1, are also localized in these cells [11] Similar to the other otolith proteins, OMM-64 may contribute to the heterogeneity of the endolymph chemistry and otolith biomineralization In the otolith matrix, OMM-64 was localized in ring-like structures, indicating that OMM-64 is periodically incorporated into the otoliths The manner of incorporation may be regulated by the binding activity of OMM-64 to otolin-1, because periodic expression of omm-64 mRNA was not observed (data not shown), which binds to OMM-64 indirectly, is localized in the ring-like structures [9,11] and its mRNA expression does vary periodically [12] During otolith development in zebrafish, the sagitta (saccular otolith) and lapillus (utricular otolith), both of which composed of aragonite, are formed in the single otosac of the inner ear at early developmental stages [24–30 hours post fertilization (hpf)] [21] By contrast, the vaterite asteriscus develops in the lagena, which differentiates from the otosac after initiation of the formation of sagitta and lapillus [15 days post fertilization (dpf)] [22] Therefore, it is possible that the developmental process that underlies aragonite otoliths and vaterite otoliths is different Otolin-1, which is necessary for aragonite crystal formation in vitro, is expressed in the early stages of development of the inner ear (48 hpf) and is involved in the seeding and ⁄ or nucleation of the sagitta and lapillus [23] On the other hand, omm-64 may be expressed at earlier stages because mRNA expression was found in the trout embryo at the 50% epiboly stage (see Results) If omm-64 mRNA is really expressed at this stage, it represents the earliest known expression of all inner ear-specific marker genes found to date Otolith nuclei are formed at about 24 hpf by aggregating proteins and polysaccharides secreted from epithelial cells [24] Starmaker is also expressed at an early stage (24 hpf) [25] Therefore, OMM-64 and starmaker are likely to be contained in the aggregate and contribute to formation of the aragonite polymorph In summary, we have identified a novel protein, OMM-64, contained in the HMW aggregate in the otolith matrix, and shown that the aggregate also contains ear-specific collagen, otolin-1, and forms framework mineral constructs The two proteins, OMM-64 A novel protein in the otolith matrix framework and otolin-1, are expressed in the same cells in the inner ear sacculus and are secreted into the extracellular matrices of the inner ear In the otoliths, they are both localized in the ring-like structures These findings identify for the first time proteins with these functions that construct matrix aggregates in calcium carbonate biominerals Experimental procedures Animals Rainbow trout (Oncorhynchus mykiss) weighing approximately 1000 g were used They were reared in outdoor ponds at 10–15 °C under natural light for at least 10 days before collection of the samples Cloning of cDNA and DNA encoding OMM-64 As described previously [13], we detected HMW proteins that may be aggregated in the otolith matrix by western blotting using an antiserum raised against whole water-soluble otolith matrix To identify proteins contained in these aggregates, immunoscreening of a cDNA library was performed using this antiserum Approximately 200 000 clones contained in a kZAP II (Stratagene, La Jolla, CA, USA) inner ear cDNA library, constructed according to the method described by Murayama et al [26], were grown on LB agar ⁄ LB top agarose plates Recombinant proteins in each clone were induced and transferred to poly(vinylidene difluoride) (PVDF) membranes (Millipore, Billerica, MA, USA), which had been soaked with 20 mm isopropyl-bd-thiogalactopyranoside (IPTG) at 37 °C for h After blocking with 5% fat-free dried milk in NaCl ⁄ Tris (50 mm NaCl, 20 mm Tris ⁄ HCl pH 8.0) for h, the membranes were incubated with the antiserum at a : 1000 dilution overnight Each immunoreacted membrane was then incubated with secondary antibody (horseradish peroxidase conjugated anti-rabbit IgG; Bio-Rad, Hercules, CA, USA) at a : 1000 dilution for h Immunoreaction was detected by catalysis of the substrate diaminobenzidine diluted in NaCl ⁄ Tris at 10 mm Clones containing immunoreactive proteins were selected, and the pBluescript phagemids were excised using ExAssist helper phage (Stratagene) according to the manufacturer’s protocol To determine the 5¢ end of the cDNA, 5¢ RACE was performed using a SMARTÔ RACE cDNA amplification kit (Clontech, Mountain View, CA, USA) with reverse primer 5¢-GTGACAACATTGTGA TGGGATAGTTT-3¢ (nt 79–54) After cDNA cloning, the sequence of the genomic DNA encoding OMM-64 was determined by PCR-based cloning To determine the internal introns, PCR was performed using gene-specific primers designed according to the sequence of omm-64 cDNA A GenomeWalker universal kit FEBS Journal 275 (2008) 2512–2523 ª 2008 The Authors Journal compilation ª 2008 FEBS 2519 A novel protein in the otolith matrix framework H Tohse et al (Clontech) was used to clone the region 5¢ to the gene PCR products were ligated into pGEM-T Easy vector (Promega, Madison, WI, USA), and the ligated plasmid DNAs were transformed into XL1-blue-competent cells (Stratagene) After growth and harvest of the Escherichia coli cells, the amplified DNAs were recovered using a QIAprep miniprep kit (Qiagen, Hilden, Germany) and sequenced using a DNA sequencer (3130xl Genetic Analyzer; Applied Biosystems, Foster City, CA, USA) Expression analyses of omm-64 mRNA Total RNA was isolated from various organs (see Fig 2) using ISOGEN (Nippon Gene, Tokyo, Japan), and treated with unitsỈlL)1 of DNase I (Takara, Kyoto, Japan) at 37 °C overnight Complete digestion of genomic DNA contamination in the total RNA was confirmed by lack of amplification of a b-actin mRNA fragment by PCR using a pair of primers (5¢-ATCACCATCGGCAACGAGAG-3¢ and 5¢-TGGAGTTGTAGGTGGTCTCGTG-3¢) without reverse transcription After purification using phenol ⁄ chloroform, lg of the total RNA was reverse-transcribed using a first-strand cDNA synthesis kit (Amersham Biosciences, Little Chalfont, UK) Using ⁄ 100 aliquots of the first-strand cDNAs as templates, PCR was performed using primers 5¢-GCTATGTTTCTGCAGGGTTCCTA-3¢ (nt 2385–2407) and 5¢-GCGTCATTAAACGTATGTACACT-3¢ (nt 2600–2578) Expression of b-actin mRNA was verified using the primers described above For in situ hybridization, a 216 bp fragment (nt 2385– 2600) of omm-64 cDNA was amplified by PCR as described above and ligated into pGEM-T vector (Promega) The plasmid DNA was digested with NotI or NcoI, and antisense and sense probes labeled with digoxigenin were produced by in vitro transcription using T7 and SP6 RNA polymerase (Roche, Mannheim, Germany) The specificity of the probes was confirmed by northern hybridization (supplementary Fig S1), and omm-64 mRNA expression in the paraffin sections of the sacculi was detected by in situ hybridization as described previously [27] For northern blotting analysis, total RNA of the inner ear sacculus and ovary (10 lg each) extracted using ISOGEN (Nippon Gene) and the sense- and antisense-strand RNA probes (0.1 lg each) produced as described above were subjected to 1.2% agarose gel electrophoresis After electrophoresis, the RNAs were blotted onto a nitrocellulose membrane (Hybond N+, Amersham Biosciences) and hybridized with digoxigenin-labeled sense- and antisensestrand RNA probes at 68 °C for 30 each for overnight After washing the membrane twice each with 2· SSC and 0.1· SSC at 68 °C for 30 each, hybridization signals were detected by immunodetection using alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (Roche) coupled with CDP-star alkaline phosphatase substrate (Roche) according to the manufacturer’s protocol 2520 Determination of the calcium-binding domain using recombinant OMM-64 variants Six recombinant fusion proteins comprising glutathione S-transferase (GST) and various regions of OMM-64 (rOMM-64-I, Thr141–Ser543; rOMM-64-II, Thr141– Ser233; rOMM-64-III, Ser233–Lys496; rOMM-64-IV, Ala21–Ser628; rOMM-64-V, Arg410–Ser543; rOMM-64-C, Asp544–Ser628) were synthesized The corresponding regions of the omm-64 cDNA were amplified by RT-PCR using six pairs of primers (rOMM-64-I, 5¢-CGCGGATCC ACCGTAGACACTTATGATATA-3¢ and 5¢-CGCCTCCA CCTAAGAGGCATCCTTGTCCAC-3¢; rOMM-64-II, 5¢CGCGGATCCACCGTAGACACTTATGATATA-3¢ and 5¢-CGCCTCGAGCTAAGAGTCAGCTTGCACGTC-3¢; rOMM-64-III, 5¢-CGCGGATCCGCTGATGTGACCAGT GATGAC-3¢ and 5¢-CGCCTCGAGCTATTTGGGCTCTT TCATCAT-3¢; rOMM-64-IV, 5¢-CGCGGATCCGCCCCT GTTAATGATGGAACC-3¢ and 5¢-CGCCTCGAGCTAA GAAGACTGGGCTGCCAG-3¢; rOMM-64-V, 5¢-CGCGG ATCCAGGCAAGATTTTAAGCATCCA-3¢ and 5¢-CGCC TCCACCTAAGAGGCATCCTTGTCCAC-3¢; rOMM-64C, 5¢-CGCGGATCCGACTCAGTGGATGACCAATCC-3¢ and 5¢-CGCCTCGAGCTAAGAAGACTGGGCTGCC AG-3¢) that had 5¢ adapters corresponding to BamHI (GGATCC) and XhoI (CTCGAG) restriction sites, respectively In the reverse primers, stop codons (TAG) before the XhoI sites were also added PCR products were doubly digested by the restriction enzymes, purified using a QIAquick PCR purification kit (Qiagen), and ligated into pGEX 6p-1 vector (Amersham Biosciences), which had been digested and purified in the same way as the PCR products After transformation into XL1-blue cells and confirmation of the sequences, the plasmid DNA was transformed again into BL21 E coli cells (Amersham Biosciences) The cells were grown in LB medium containing 50 lgỈmL)1 ampicillin at 37 °C overnight Subsequently, 200 lL of the cells was transferred to 20 mL of new LB medium without ampicillin and grown at 37 °C for h The GST–recombinant protein fusions were then induced by addition of IPTG at a final concentration of mm After incubation at 37 °C for h, the cells were collected by centrifugation at 3000 g for 10 and lysed in 10 mL of extraction buffer (5 mm EDTA, 0.5% Triton X-100, 40 mm Tris ⁄ HCl, pH 7.5) by sonication The lysate was then centrifuged at 30 000 g for 10 and the supernatant was collected We added a 0.5 mL bed of glutathione– Sepharose beads (Amersham Biosciences) equilibrated with NaCl ⁄ Pi (140 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4, pH 7.4) to the extract, and allowed the GST–recombinant protein fusions to bind to the beads at °C for h The beads were then washed five times with 10 mL of extraction buffer and NaCl ⁄ Pi The beads that bound recombinant proteins were directly applied to SDS–PAGE under reducing conditions FEBS Journal 275 (2008) 2512–2523 ª 2008 The Authors Journal compilation ª 2008 FEBS H Tohse et al Separated proteins were stained with ‘Stains-all’ (Sigma, St Louis, MO, USA) [28] or were blotted onto a PVDF membrane to detect 45Ca2+-binding activity [29] Production of antibody against recombinant OMM-64 The recombinant rOMM-64-C protein that bound to glutathione–Sepharose beads was digested from GST by Prescission protease (80 unitsỈmL)1 gel bed; Amersham Biosciences) at °C for days, eluted with mL of NaCl ⁄ Pi, and concentrated and desalted using Ultrafree cartridges (Millipore, 5000 Da cut-off) The digested rOMM-64-C was completely separated from GST using a Sep-Pak Cartridge C18 column (Waters, Millford, MA, USA) by stepwise elution with acetonitrile After the purity and molecular mass (m ⁄ z 9227) of rOMM-64-C had been confirmed by MALDI-TOF mass spectrometry (4700 Proteomics Analyzer, Applied Biosystems), the buffer of the protein was changed to NaCl ⁄ Pi using Ultrafree cartridges (5000 Da cut-off) Production of rabbit antiserum raised against the recombinant protein and affinity purification of the antibody were performed by Hokkaido System Science (Hokkaido, Japan) Collection of inner ear proteins Dissection of whole inner ear and collection of the endolymph and otoliths were performed as previously described [30] After the endolymph and otoliths had been collected, the sacculi were washed three times with 0.9% NaCl and homogenized in the same solution The homogenate and endolymph were centrifuged at 100 000 g for hr and the supernatants were collected Otoliths were washed vigorously five times each for each with 1% SDS and distilled water, and were immediately decalcified in 0.5 m EDTA at °C with gentle agitation The EDTA solution was changed every day by centrifugation at 25 000 g for 10 min, and the supernatant was collected The supernatants were stored at )30 °C After complete decalcification (approximately days), the stored solutions were concentrated and the solvent was changed to 20 mm Tris ⁄ HCl (pH 8.0) using Ultrafree cartridges (5000 Da cut-off) The EDTA-insoluble matrix (the pellet from the final centrifugation of the EDTA-decalcified solution) was washed five times with 20 mm Tris ⁄ HCl (pH 8.0) and the proteins were extracted by boiling in denaturing solution (8 m urea, 10 mm dithiothreitol, 1% SDS, 10% Chaps, 20 mm Tris ⁄ HCl pH 8.0) for 10 Analyses of protein profiles SDS–PAGE was performed under reducing conditions After separation of the proteins, the gels were stained with A novel protein in the otolith matrix framework ‘Stains-all’ [28] or silver to detect negatively charged proteins and all proteins, respectively To detect OMM-64 and otolin-1 by western blotting, antirOMM-64-C and anti-recombinant otolin-1-C [9] sera were used Ten micrograms of protein extracted from inner ear was separated by SDS–PAGE and blotted onto a PVDF membrane The membrane was incubated first in 5% fat-free dried milk in NaCl ⁄ Tris for h, and then in the same solution containing the antibodies (1 : 1000 dilution) overnight After washing the membrane twice (10 mins each) with NaCl ⁄ Tris containing 0.1% Tween-20 and once with NaCl ⁄ Tris, specific binding of the antibodies was detected by using Supersignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA), and the corresponding secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG, : 5000), according to the manufacturer’s protocol Deglycosylation of proteins Ten micrograms of otolith matrix protein were desalted in an Ultrafree cartridge (5000 Da cut-off) and completely dried in a centrifugal concentrator (VC-96W, Taitec, Saitama, Japan) Chemical deglycosylation of the proteins was performed by incubation with 50 lL of trifluoromethanesulfonic acid (TFMS) at °C for 0, 5, 15 and 30 The solutions were neutralized by adding 500 lL of ice-cold buffer (1 m Tris) The sample solvent was changed to 20 mm Tris ⁄ HCl pH 8.0 using an Ultrafree cartridge (5000 Da cut-off) For enzymatic digestion, 10 lg of watersoluble otolith matrix protein, completely dried in a centrifugal concentrator, was incubated at 37 °C overnight with 10 lL of the following deglycosylation enzymes dissolved in buffers described in the manufacturer’s protocol (Seikagaku, Tokyo, Japan): glycopeptidase A (0.5 munits), chondroitinase ABC (0.5 units), heparitinase II (0, 1, 2, 3, and 10 munits), hyaluronidase SD (25 munits) or endo-a-N-acethylgalactosaminidase (70 munits) The samples were subjected to 10% SDS–PAGE, and OMM-64 was detected by western blotting as described above Immunohistochemistry Paraffin sections (5 lm) of the sacculi containing otoliths were produced as previously described [13] After de-paraffination and rehydration, the sections were incubated at room temperature first with normal rabbit serum (1 : 1000) for h and then with anti-rOMM-64 serum (1 : 1000) in 10 mm NaCl ⁄ Pi (pH 7.4) overnight After immunoreaction with secondary antibody (1 : 2000, peroxidase-conjugated anti-rabbit IgG, Bio-Rad) for h, the sections were washed three times with NaCl ⁄ Pi, and specific binding of the antibody was detected by incubating the sections in a solution of 10 mm diaminobenzidine in 10 mm NaCl ⁄ Pi FEBS Journal 275 (2008) 2512–2523 ª 2008 The Authors Journal compilation ª 2008 FEBS 2521 A novel protein in the otolith matrix framework H Tohse et al Co-immunoprecipitation of OMM-64 and otolin-1 )1 First, mL each of anti-rOMM-64 serum (1.5 mgỈmL ) or anti-rOtolin-1 serum was allowed to bind to a 0.5 mL bed of Protein A–Sepharose beads (Amersham Biosciences) according to the manufacturer’s protocol, and then the antibodies and beads were covalently bound by incubation with 20 mm dimethylpimelimidate (MP Biochemicals, Solon, OH, USA) for h at room temperature After washing the beads with 10 mm NaCl ⁄ Pi (pH 7.4), complete binding of the antibody was confirmed by the detection of no bands in the supernatant SDS–PAGE Subsequently, 100 lL of saccular extract or water-soluble otolith matrix proteins were bound to 10 lL of the affinity beads at °C for h, the beads were then washed twice each by voltexing 10 sec and centrifuged at 5000 g for 30 sec with mL of 0.5 mm NaCl in 10 mm NaCl ⁄ Pi (pH 7.4), 0.1 m glycine (pH 2.5) and 20 mm Tris ⁄ HCl (pH 8.0) Proteins that bound to the affinity beads were analyzed by applying a lL bed of the beads directly to SDS–PAGE Tandem mass spectrometry To identify the two proteins (95 and 130 kDa) bound to recombinant otolin-1-C beads, tryptic peptides of these proteins were applied to MS ⁄ MS analysis as follows After gel electrophoresis, the two protein bands were excised and destained using a solution of 50% acetonitrile, 25 mm NH4HCO3 After reduction and carbamidomethylation of the proteins by incubation with 20 mm dithiothreitol at 56 °C and 20 mm iodoacetamide at room temperature for h each, the proteins were digested with trypsin (10 lgỈmL)1 in 25 mm NH4HCO3, 30% dimethylformamide) The digested peptides were purified using a SepPak C18 column (Waters) and applied to tandem mass spectrometry (MALDI-TOF-TOF, 4700 Proteomics Analyzer, Applied Biosystems) The tryptic peptide mass fingerprinting and MS ⁄ MS spectra were used for a mascot search (Matrix Science, http://www.matrixscience.com) Acknowledgements The authors sincerely appreciate the useful advice of Dr Hirotoshi Endo (Hokkaido University) regarding on the 45Ca overlay assay The 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 the Ministry of Education, Science, Sports and Culture (Grants-in-Aid for Creative Basic Research numbers 12NP0201 and 17GS0311 and a Scientific Research 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