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..
Trang 1protein aggregate associated with collagen in fish otoliths Hidekazu Tohse1,2, Yasuaki Takagi2and 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
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,
ara-gonite 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
tempera-ture 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; how-ever, analyses of the single proteins has not led
to insights into how these morphologies and polymorphisms are formed in the biominerals, as the
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 zebrafish otoliths [So¨llner C, Burgham-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
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.
Trang 2organic 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
forma-tion when they are applied to crystal inducforma-tion 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
polysaccha-rides 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
mineral-associated 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
biomin-eral 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
deposi-tion onto the otolith matrix [11,12] A gel-like
struc-ture containing the framework is observed upon
decalcification, suggesting that the otolith matrix is
constructed from large aggregates of framework
mole-cules and mineral-associated molemole-cules
In the majority of biomineral matrices, not just
mollusk shells and otoliths, high-molecular-weight
(HMW, > 100 kDa) proteins are observed by gel
elec-trophoresis 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
electro-phoresis 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
con-taining 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
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 conta-ining 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 sup-plementary Fig S1) The open reading frame is followed by a 3¢ UTR containing a putative polyaden-ylation 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
omm-64 mRNA is AB213022) The DNA encoding OMM-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 exam-ined as an endogenous control S.C., semicircular canal; W muscle, white muscle; R muscle, red muscle; B kidney, body kidney; H kidney, head kidney.
Trang 3SDS(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
threo-nines 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
oto-lith crystal polymorphism [14] Although the identity
between these proteins was only 25%, some distinctive
domains of starmaker are conserved in OMM-64
(sup-plementary 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
iden-tified 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
N-terminal portion of the protein (including the signal
peptide and small tandem repeats), a large exon
com-prises 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
tran-scribed 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
Fig S3) In the inner ear sacculus, strong hybridization signals were detected, mainly in the cells at the periph-ery of the macula and in transitional epithelial cells except mitochondria-rich cells (ion-transporting iono-cytes, 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 sac-culus The regions magnified in (B)–(D) are indicated by boxes (B) Macula (M) and transitional epithelial (TE) regions Intense hybridiza-tion signals were observed in the cells at the periphery of the mac-ula (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 saccu-lus (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.
Trang 4other 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
hybrid-ization 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 a45Ca overlay assay (Fig 3) Of these recombinant
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 con-clude, 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 anti-serum (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 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
recombi-nant OMM-64 variants separated by SDS–PAGE to detect
nega-tively charged proteins as blue bands (left) and 45 Ca 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.
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 ‘Stains-all staining’ and western blotting (arrowheads) All proteins in the EDTA-soluble (O S ) and -insoluble (O I ) otolith matrix were stained blue using ‘Stains-all’ In these matrices, strong immunoreactions were detected in the high-molecular-weight region (arrows).
Trang 5of 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,
indicat-ing 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
aggre-gate in a concentration-dependent manner (Fig 5C)
These results suggest that OMM-64 is contained in the
otolith matrix aggregate consisting of heparan sulfate
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 anti-rOMM-64-C serum Similar to omm-64 mRNA expres-sion, 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 im-munonegative We found that OMM-64 accumulates
at the apical membrane in macula (Fig 6A) and in the ring-like structures in otoliths (Fig 6C)
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 oto-lith 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
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’ stain-ing showed that the HMW aggregate was not digested completely even after 30 min of treatment (arrowhead) Silver staining indi-cated that the other proteins may be damaged by the 30 min TFMS treatment (C) Heparitinase II digests the HMW aggregate (arrow-head) and separates free OMM-64 (arrow) in a concentration-dependent manner Bovine serum albumin, which was contained in the enzyme solution, was observed at 66 kDa by both silver and
‘Stains-all’ staining.
Trang 6matrix We also observed this interaction when anti-rOtolin-1 affinity beads were used for the same experi-ments However, because non-specific immunoreactive bands were observed in western blotting using anti-rOtolin-1 serum, the 95 and 140 kDa bands could not
be confirmed to be otolin-1 Therefore, MALDI-TOF-TOF tandem mass spectrometry was performed to identify the proteins The tryptic peptide mass finger-printing spectra of these proteins were highly similar (supplementary Fig S4), and both proteins were iden-tified 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
Fig 6 Localization of OMM-64 in the inner ear sacculus by
immu-nohistochemistry (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
transi-tional 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
OMM-64 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
pri-mary 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).
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 ⁄ P i (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.
Trang 7associated 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
biomi-nerals [1,2], numerous studies have investigated
pro-teins, 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
exam-ined proteins contaexam-ined 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
phosphory-lated, 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
calcu-lated 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
OMM-64 by blast search (blastp and tblastn) The relationship between starmaker and dentin sialophos-phoprotein 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 ortho-logs 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 func-tion 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 So¨llner et al [14] also reported that they could not find an ortholog of star-maker 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 glycosaminogly-can chains were not characterized Although it has been suggested that glycosaminoglycan chains are involved in the nanoscale processes of calcium carbon-ate 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 distri-bution 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
Trang 8are concentrated in the proximal endolymph [20].
Therefore, the proximal region of the sacculus
pro-duces 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
pro-teins, 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
incorpo-ration 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
fer-tilization (dpf)] [22] Therefore, it is possible that the
developmental process that underlies aragonite
oto-liths and vaterite otooto-liths 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
for-mation 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
con-tains ear-specific collagen, otolin-1, and forms
frame-work mineral constructs The two proteins, OMM-64
and otolin-1, are expressed in the same cells in the inner ear sacculus and are secreted into the extracellu-lar matrices of the inner ear In the otoliths, they are both localized in the ring-like structures These find-ings identify for the first time proteins with these func-tions that construct matrix aggregates in calcium carbonate biominerals
Experimental procedures
Animals
Rainbow trout (Oncorhynchus mykiss) weighing approxi-mately 1000 g were used They were reared in outdoor ponds at 10–15C 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-sol-uble otolith matrix To identify proteins contained in these aggregates, immunoscreening of a cDNA library was per-formed 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
each clone were induced and transferred to poly(vinylidene difluoride) (PVDF) membranes (Millipore, Billerica, MA, USA), which had been soaked with 20 mm
blocking with 5% fat-free dried milk in NaCl⁄ Tris (50 mm
were incubated with the antiserum at a 1 : 1000 dilution overnight Each immunoreacted membrane was then incu-bated with secondary antibody (horseradish peroxidase con-jugated 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
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 SMART 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
Trang 9(Clontech) was used to clone the region 5¢ to the gene.
PCR products were ligated into pGEM-T Easy vector
(Pro-mega, Madison, WI, USA), and the ligated plasmid DNAs
were transformed into XL1-blue-competent cells
(Strata-gene) After growth and harvest of the Escherichia coli cells,
the amplified DNAs were recovered using a QIAprep
mini-prep kit (Qiagen, Hilden, Germany) and sequenced using a
DNA sequencer (3130xl Genetic Analyzer; Applied
Biosys-tems, 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
con-tamination 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¢
reverse transcription After purification using phenol⁄
chlo-roform, 1 lg of the total RNA was reverse-transcribed
using a first-strand cDNA synthesis kit (Amersham
Bio-sciences, Little Chalfont, UK) Using 1⁄ 100 aliquots of the
first-strand cDNAs as templates, PCR was performed using
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
anti-sense and anti-sense probes labeled with digoxigenin were
pro-duced 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
ISO-GEN (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
hybridized with digoxigenin-labeled sense- and
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
phospha-tase-conjugated anti-digoxigenin Fab fragments (Roche)
coupled with CDP-star alkaline phosphatase substrate
(Roche) according to the manufacturer’s protocol
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
Ala21–Ser628; rOMM-64-V, Arg410–Ser543; rOMM-64-C,
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¢-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-64-C, 5¢-CGCGGATCCGACTCAGTGGATGACCAATCC-3¢
AG-3¢) that had 5¢ adapters corresponding to BamHI (GGATCC) and XhoI (CTCGAG) restriction sites, respec-tively 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 trans-formed again into BL21 E coli cells (Amersham Biosciences)
200 lL of the cells was transferred to 20 mL of new LB
The GST–recombinant protein fusions were then induced
by addition of IPTG at a final concentration of 1 mm After incubation at 37C 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,
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
1.8 mm KH2PO4, pH 7.4) to the extract, and allowed the GST–recombinant protein fusions to bind to the beads at
10 mL of extraction buffer and NaCl⁄ Pi
directly applied to SDS–PAGE under reducing conditions
Trang 10Separated proteins were stained with ‘Stains-all’ (Sigma,
St Louis, MO, USA) [28] or were blotted onto a PVDF
membrane to detect45Ca2+-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
Bio-sciences) at 4C 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
rOMM-64-C had been confirmed by MALDI-TOF mass
spectrometry (4700 Proteomics Analyzer, Applied
Biosys-tems), 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
endo-lymph 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
vigor-ously five times each for 1 min each with 1% SDS and
dis-tilled water, and were immediately decalcified in 0.5 m
was changed every day by centrifugation at 25 000 g for
10 min, and the supernatant was collected The
superna-tants were stored at)30 C After complete decalcification
(approximately 5 days), the stored solutions were
concen-trated 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
centrifu-gation 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,
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
‘Stains-all’ [28] or silver to detect negatively charged pro-teins and all propro-teins, respectively
To detect OMM-64 and otolin-1 by western blotting, anti-rOMM-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 solu-tion containing the antibodies (1 : 1000 dilusolu-tion) 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 second-ary 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 trifluorome-thanesulfonic acid (TFMS) at 0C 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
(5000 Da cut-off) For enzymatic digestion, 10 lg of water-soluble otolith matrix protein, completely dried in a
10 lL of the following deglycosylation enzymes dissolved in buffers described in the manufacturer’s protocol (Seika-gaku, Tokyo, Japan): glycopeptidase A (0.5 munits), chon-droitinase ABC (0.5 units), heparitinase II (0, 1, 2, 3, 5 and
10 munits), hyaluronidase SD (25 munits) or endo-a-N-ac-ethylgalactosaminidase (70 munits) The samples were sub-jected 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-par-affination and rehydration, the sections were incubated at
(1 : 1000) for 1 h and then with anti-rOMM-64 serum
immunoreaction with secondary antibody (1 : 2000, peroxi-dase-conjugated anti-rabbit IgG, Bio-Rad) for 2 h, the sec-tions 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