Tài liệu Báo cáo khoa học: Identification of a novel matrix protein contained in a protein aggregate associated with collagen in fish otoliths pdf

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
1 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan
2 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.

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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. 1 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. 1. (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.

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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 1 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
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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. 2. 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.

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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. 3. 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 1 (R1), the Glu-rich domain (E-rich)
and ⁄ or tandem repeat 2 (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 do
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. 4. 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).

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Fig. 5. 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 5 min (arrow) (aOMM-64), ‘Stains-all’ staining showed that the HMW aggregate was not digested completely
even after 30 min of treatment (arrowhead). Silver staining indicated that the other proteins may be damaged by the 30 min 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 min 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 min 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
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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

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A novel protein in the otolith matrix framework

Fig. 7. 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. 6. 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

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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 1 may be highly phosphorylated, and the Glu-rich region and repeat 2 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 1 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
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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 identied. Sollner et al. [14] also
ă
reported that they could not find 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

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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 4 h. After
blocking with 5% fat-free dried milk in NaCl ⁄ Tris (50 mm
NaCl, 20 mm Tris ⁄ HCl pH 8.0) for 1 h, the membranes
were incubated with the antiserum at a 1 : 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

1 : 1000 dilution for 2 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 SMARTƠ
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

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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 4 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, 1 lg of the total RNA was reverse-transcribed
using a first-strand cDNA synthesis kit (Amersham Biosciences, Little Chalfont, UK). Using 1 ⁄ 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 min each for overnight.
After washing the membrane twice each with 2· SSC and
0.1· SSC at 68 °C for 30 min 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 2 h.
The GST–recombinant protein fusions were then induced
by addition of IPTG at a final concentration of 1 mm.
After incubation at 37 °C for 2 h, the cells were collected
by centrifugation at 3000 g for 10 min 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 min 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
4 °C for 1 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.

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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 4 °C for 2 days, eluted with 1 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 1 hr and the
supernatants were collected. Otoliths were washed vigorously five times each for 1 min each with 1% SDS and distilled water, and were immediately decalcified in 0.5 m
EDTA at 4 °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 5 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 min.

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 1 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, 1 : 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 0 °C for 0, 5, 15 and 30 min.
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, 5 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 1 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 2 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.

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2521


A novel protein in the otolith matrix framework

H. Tohse et al.

Co-immunoprecipitation of OMM-64 and otolin-1
)1

First, 1 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 1 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 4 °C
for 2 h, the beads were then washed twice each by voltexing
10 sec and centrifuged at 5000 g for 30 sec with 1 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
5 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
1 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, ).

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 for
Young Scientists Start-Up Grant number 18880001).

2522

H. T. was supported by research fellowships from the

Japan Society for Promotion of Science for Young Scientists (number 15-10657) and Akiyama Memorial Life
Science Foundation (number 18-6).

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Supplementary material
The following supplementary material is available
online:
Fig. S1. Sequences of the omm-64 cDNA and its
deduced protein.
Fig. S2. Alignment of amino acid sequences of OMM64 and starmaker.
Fig. S3. Northern hybridization analysis of omm-64
transcripts.
Fig. S4. Tryptic peptide mass fingerprinting of two
proteins bound to anti-rOtolin-1 affinity beads.
This material is available as part of the online article
from
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corresponding author for the article.

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2523