Cloning of a cDNA coding for otolin-1 revealed that the deduced amino-acid sequence contained a collagenous domain in the central part of the protein.. The otolithic membrane, an accesso
Trang 1Fish otolith contains a unique structural protein, otolin-1
Emi Murayama1, Yasuaki Takagi2, Tsuyoshi Ohira1, James G Davis3, Mark I Greene3
and Hiromichi Nagasawa1
1 Laboratory of Bioorganic Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan;
2 Otsuchi Marine Research Center, Ocean Research Institute, The University of Tokyo, Japan; 3 Department of Pathology
and Laboratory Medicine, University of Pennsylvania School of Medicine, PA, USA
A collagen-like protein was identi®ed from the otoliths of the
chum salmon, Oncorhynchus keta The otolith, composed
mainly of calcium carbonate with small amount of organic
matrices, is formed in the inner ear and serves as a part of the
hearing and balance systems Although the organic matrices
may play important roles in the growth of otolith, little is
known about their chemical nature and physiological
func-tion In this study, a major organic component of the otolith,
designated otolin-1, which may serve as a template for
calci®cation, was puri®ed The sequences of two tryptic
peptides from otolin-1 revealed high homology with parts of
a saccular collagen which had been described previously
[Davis, J.G., Oberholtzer, J.C., Burns, F.R & Greene, M.I
(1995) Science 267, 1031±1034] Cloning of a cDNA coding
for otolin-1 revealed that the deduced amino-acid sequence contained a collagenous domain in the central part of the protein Although collagen is the most abundant structural protein in the animal body, otolin-1 mRNA was expressed speci®cally in the sacculus Immunohistochemical studies showed that otolin-1 is synthesized in the transitional epithelium and transferred to the otolith and otolithic membrane This is the ®rst report concerning characteriza-tion of a structural protein containing many tandem repeats
of the sequence, Gly-Xaa-Yaa, typical for collagen from the biomineral composed of calcium carbonate
Keywords: otolith; collagen; calcium carbonate; biomineral-ization; chum salmon
The inner ear of teleost ®shes includes three semicircular
canals and three otolithic organs consisting of the sacculus,
utricle and lagena [1], each of which contains an otolith
called sagitta, asteriscus and lapillus, respectively Being the
largest of the three, sagitta has been the most widely studied
and is often referred to the term ÔotolithÕ The ®sh otolith
(sagitta) is a calci®ed mass that resides in the portion of the
endolymphatic sac called sacculus and participates in ®sh
auditory and vestibular function [2,3] The otolith is
composed principally of calcium carbonate but also contain
small amount of organic matrices Although the organic
matrices are considered to play important roles in otolith
formation [4], little is known about their chemical nature
and function
The otolith has some unique characteristics in
com-parison with other calci®ed tissues First, there are no
interconnected or attached cells in or on the otolith, and it
is attached to the otolithic membrane which, in turn, is
connected to the sensory epithelium of the sacculus
Unlike bones which are continuously re-absorbed and
re-precipitated, otoliths are metabolically inert except
under severe stress [5] Second, otoliths have ®ne incre-ments that are added daily throughout postembryonic life [6,7] probably caused by an alternate deposition of calcium carbonate-rich and organic matrices-rich layers [8,9] Based on these characteristics, otoliths are widely used for age and growth rate determination in ®shes [10] Third, otolith is the only tissue composed of calcium carbonate in the ®sh whereas bones, teeth and scales are composed of calcium phosphate Furthermore, otoliths are the ®rst calci®ed tissue that arise during embryonic development in ®shes [11] In the rainbow trout, Oncorhynchus mykiss, we observed the appearance of plural primordia (otolith nuclei) on approximately the 15th day in postfertilization ®sh reared at 10 °C The composition of endolymph surrounding the otolith is
an important factor for otolith growth This ¯uid is supersaturated with calcium and bicarbonate ions, and its precise composition is critical for calci®cation [12] Local alkaline microenvironments in the endolymph are required
to promote the precipitation of the calcium carbonate [13,14], while endolymph alone does not allow the sponta-neous precipitation of calcium carbonate A pH gradient exists in the sacculus and its regulation is also important for the rate of calcium deposition [15] The otolithic membrane,
an accessory structure that couples the otolith to the sensory epithelium in the sacculus, may be one site for otolith formation and it is composed of a gelatinous layer and a subcupular meshwork Davis et al revealed that the con-stituent of the gelatinous layer of the otolithic membrane contained meshwork-forming collagens referred to as saccular collagen in the bluegill sun®sh Lepomis macrochirus [16,17] However, it is still unknown about the chemical nature of organic matrices of subcupular meshwork and otolith
Correspondence to H Nagasawa, Department of Applied Biological
Chemistry, Graduate School of Agricultural and Life Sciences, The
University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Fax: + 81 3 5841 8022, Tel.: + 81 3 5841 5132,
E-mail: anagahi@mail.ecc.u-tokyo.ac.jp
Abbreviations: sSC, sun®sh saccular collagen; N-NC domain,
N-terminal noncollagenous domain; C-NC domain, C-terminal
noncollagenous domain; OMP-1, otolith matrix protein-1; ABC,
avidin-biotin-peroxidase complex.
(Received 28 August 2001, revised 22 October 2001, accepted 26
November 2001)
Trang 2We previously identi®ed otolith matrix protein-1
(OMP-1), a major component of EDTA-soluble matrix
proteins in otoliths of teleost ®shes [18] OMP-1 has 40%
homology to the C-terminal half of the human
melano-transferrin, a monomeric glycoprotein produced by
human melanoma cells [19] and belonging to the
trans-ferrin family which plays a role in iron metabolism [20]
In the rainbow trout, EDTA-soluble matrix proteins
including OMP-1 are synthesized and secreted from the
transitional and squamous epithelial cells in the sacculus
[21,22] When otoliths are decalci®ed with EDTA,
gelati-nous insoluble materials remain It has been reported that
the primordia (otolith nuclei) before calci®cation also had
gel-like material [11,23] Thus, these gelatinous materials
are thought to be important in the formation of the
otolith
Here, we describe the characterization of a collagen-like
protein identi®ed as a major component of EDTA-insoluble
gelatinous material obtained from the chum salmon
otoliths
E X P E R I M E N T A L P R O C E D U R E S
Fish and otolith
Chum salmon, Oncorhynchus keta, with an average weight
of 3000 g, were captured at the Otsuchi Marine Research
Center, Ocean Research Institute, The University of
Tokyo in Iwate Prefecture Experimental animals were
randomly selected and anesthetized with
2-phenoxyetha-nol Otoliths (sagittae) which weighed 10 mg per single
otolith were collected from the sacculus after decapitation
and stored at room temperature until use For
immuno-histochemical studies, homing adult chum salmon were
caught by a trap net set at the mouth of Otsuchi Bay,
Iwate prefecture, in November, 2000, and transferred to
the outdoor tank of Otsuchi Marine Research Center
They were reared in running seawater at 15 °C under a
natural photoperiod
Extraction of EDTA-insoluble matrix protein from otoliths
Otoliths of chum salmon were rinsed with distilled water
and decalci®ed with 0.5M EDTA (pH 8.0) with
occa-sional shaking The resulting suspension was centrifuged
and the residual precipitate was obtained The
EDTA-insoluble materials were washed with distilled water
extensively, and extracted with 10 mM Chaps at 50 °C
for 2 h
De-N-glycosylation
The Chaps-extracted matrix proteins derived from ®ve
otoliths of the chum salmon were desalted with an ultrafree
cartridge (10 000 cut off, Millipore Co.) and concentrated to
a ®nal volume of 2.5 lL, which was added to a mixture of
2.5 lL of denaturing buffer [1% SDS, 1M Tris/HCl
(pH 8.6), 0.1M2-mercaptoethanol] The resulting solution
was heated at 100 °C for 3 min Then, 13 lL of distilled
water and 1 mU of glycopeptidase F (TaKaRa) were added
to this solution, which was incubated at 37 °C for 15 h
Then, the reaction mixture was concentrated and applied to
SDS/PAGE analysis
N-terminal and internal amino-acid sequence analyses Chaps-extracted matrix proteins from the EDTA-insoluble materials of 10 otoliths were concentrated by ultra®ltration
as described above and applied to SDS/PAGE with 10% polyacrylamide gel according to the method of Laemmli [24] After electrophoresis, the gel was stained with 0.1% Coomasie Brilliant Blue G-250 (Wako, Osaka) or subjected
to electroblotting Chaps-extracted matrix proteins separa-ted on SDS/PAGE were electrically transferred to a poly(vinylidene di¯uoride) (PVDF) membrane (ATTO, Tokyo) and stained with 0.2% Coomasie Brilliant Blue R-350 (Pharmacia) A portion of the membrane carrying a blotted matrix protein with an apparent molecular mass of
100 kDa was cut out and applied to a protein sequencer (Applied Biosystems model 491cLC) in the pulsed-liquid mode On the other hand, the matrix protein was electro-eluted from the gel in an elution buffer (20 mMTris/HCl,
pH 8.0, 0.1% SDS) and the eluate was desalted and applied
to a protein sequencer as described above To analyze internal amino-acid sequences, the gel carrying the matrix protein with an apparent molecular mass of 100 kDa was cut out, crushed into small pieces, and rinsed well with distilled water To a tube containing the crushed gel, 300 lL
of 0.1Mammonium bicarbonate containing 10% acetonit-rile and 1% Triton X-100 was added and 1.5 lL of TPCK-treated trypsin (Promega) solution (1 mgámL)1 in 0.1M ammonium bicarbonate) was added This enzyme solution was incubated at 37 °C for 24 h The mixture was ®ltered to remove small gel pieces, and applied to reverse-phase HPLC using a Capcell Pak C18column (2.0 ´ 150 mm, Shiseido) Separation was performed with a 50-min linear gradient of 10±60% acetonitrile in 0.05% tri¯uoroacetic acid at a ¯ow rate of 0.2 mLámin)1 Fragment peptides were collected manually by monitoring the absorbances at 225 nm Mass spectra of the fragment peptides were measured by MALDI TOF-MS (Voyager Biospectrometry, Applied Biosystems)
in the positive ion mode using a-cyano-4-hydroxycinnamic acid as the matrix Fractions containing more than two peptides were further puri®ed by reverse-phase HPLC under the same conditions as above except for the use of 10 mM ammonium bicarbonate instead of 0.05% tri¯uoroacetic acid Each peak material was collected manually and applied to a protein sequencer as described above PCR ampli®cation
Degenerate oligonucleotide primers sOT5¢-R1 (GCYTGRT CDATRTCYTGICC), designed based on a partial sequence of the fragment peptide obtained in this experi-ment, and bgSC-F (TAYAAYGGCARGGICAYTGG GA), designed on a partial sequence of saccular collagen previously reported by Davis et al [16], were prepared First-strand cDNA was synthesized with a Ready-To-Go T-primed First-Strand kit (Pharmacia) using 1 lg of total RNA which was isolated from sacculi of chum salmon using Isogen (Nippongene) The resulting cDNA was then diluted 100-fold, and 1 lL of the diluted solution was used for the PCR reaction with 1 lM of each primer (sOT5¢-R1 and bgSC-F), 1 ´ LA PCRTM buffer II (TaKaRa), 2.5 mM MgCl2, 400 lMdNTP and 1 U of TaKaRa LA TaqTMin
a total volume of 20 lL reaction The ampli®cation was performed at 95 °C for 2 min at the initial step followed by
Trang 335 cycles at 95 °C for 30 s, 54 °C for 30 s, and 72 °C for
30 s A ®nal extension step was performed at 72 °C for
3 min PCR reactions with only one degenerate primer
(sOT5¢-R1 or bgSC-F) were performed in parallel as
negative control
5¢ and 3¢ RACE
First-strand cDNA was synthesized with a SMARTTM
RACE cDNA Ampli®cation kit (Clontech) using 1 lg of
total RNA which was isolated from sacculi of chum salmon
A speci®c primer, TGCGGCGCGCGGGGCCGGTTGC
GCACG (sOT5¢-R2) corresponding to nucleotides 1370±
1397 in Fig 1A was prepared 5¢ RACE was performed
with this primer and a universal primer mix (UPM,
Clontech) matching the adapter sequence at 5¢ end of
cDNA under the same conditions as described above with
the following changes; dimethyl sulfoxide was added at a
®nal concentration of 5%, and the reactions included ®ve
cycles at 94 °C for 5 s and at 72 °C for 3 min, followed by
®ve cycles at 94 °C for 5 s, at 70 °C for 10 s, and at 72 °C
for 3 min, and 25 cycles at 94 °C for 5 s, at 68 °C for 10 s,
and at 72 °C for 3 min 3¢ RACE was performed with a
cDNA template synthesized with a Ready-To-Go T-primed
First-Strand kit (Pharmacia) as described above A speci®c
primer, TACGGCCAAGACATCGACCA (sOT3¢-F)
corresponding to nucleotides 1443±1462 in Fig 1A was
prepared PCR ampli®cation was performed with this
primer and an RTG primer (Pharmacia) matching the
adapter sequence at 3¢ end of the cDNA under the same
conditions as described above with the following changes;
the reaction cycles were reduced to 25, the annealing step
was performed at 55 °C, and the extension was performed
for 1 min
Nucleotide sequence analysis
Nucleotide sequence analysis was performed for both
strands using the dideoxynucleotide chain termination
method [25] on a Long-Read TowerTM DNA sequencer
(Amersham Pharmacia Biotech) Plasmid DNA was
puri-®ed by the alkaline lysis method [26] A sample of 10 lg of
plasmid DNA was used for sequencing with a Thermo
Sequenase Cy 5 dye terminator cycle sequencing kit
(Amersham Pharmacia Biotech)
Northern blot analysis
Samples of 10 lg each of total RNA prepared from the
chum salmon tissues including the sacculi, semicircular
canals, brain, heart tissue, liver, muscle, skin and scales were
subjected to electrophoresis on a 1% agarose gel in 40 mM
3-(N-morphorino)-propanesulfonic acid (pH 7.0),
contain-ing 18% formamide, then transferred to Hybond N+nylon
membrane (Amarsham Pharmacia Biotech), and baked at
80 °C for 2 h These RNA samples were probed with
otolin-1 cDNA fragment corresponding to nucleotide
1284±1465 in Fig 1A, randomly labeled with [a-32P]dCTP
using a Random Primer DNA Labeling Kit Ver 2
(TaKaRa) Hybridization was performed at 42 °C in 50%
formamide, 6 ´ NaCl/Cit (0.1M NaCl, 0.1M sodium
citrate), 1 ´ Denhardt's solution, 0.5% SDS and
20 lgámL)1 calf thymus DNA for 12 h The membrane
Fig 1 Nucleotide sequence of a cDNA encoding otolin-1 and its deduced acid sequence (A) The nucleotide (upper) and amino-acid (lower) sequences are indicated The putative signal peptide (1±25)
is indicated by white text on black background, and the residues that have been directly microsequenced are indicated by a dotted-underline Putative O-glycosylation sites are marked by circles, and possible N-glycosylation sites are boxed The collagenous domain is indicated
by large boxed-in area Glycine residues in the collagenous domain are shown in bold Brackets enclose the region homologous with the C-NC domains of collagen types VIII and X The182 bp RT-PCR fragment sequence is indicated by dotted rectangles The partial amino-acid sequences obtained from protein analysis are double-underlined An asterisk represents the termination codon, and a consensus ƠAATAAÃ polyadenylation signal is underlined (B) Schematic representation of the predicted domain organization of otolin-1 and the putative gly-cosylation sites The possible N- and O-glygly-cosylation sites are indicated
by squares and circles, respectively The nucleotide sequence was submitted to DDBJ/EMBL/GenBank and has been assigned the accession number AB067770.
Trang 4was washed at 65 °C with 0.1 ´ NaCl/Cit for 10 min and
autoradiographed on an X-ray ®lm with intensifying screen
at )80 °C for 24 h
Western blot analysis
A PVDF membrane carrying the matrix protein with an
apparent molecular mass of 100 kDa was prepared as
described above The membrane was incubated in a
blocking solution (5% skim milk in NaCl/Pi) for 2 h at
room temperature, and then immersed in the blocking
solution containing diluted af®nity-puri®ed sun®sh saccular
collagen reactive immunoglobulins that had been raised
against a synthetic oligopeptide corresponding to the part of
the C-terminal noncollagenous domain [anti-(C-NC) Ig,
where C-NC is a 138-residue C-terminal noncollagenous
domain] [17] at a concentration of 250 ngámL)1for more
than 2 h The speci®city of the immunoglobulins had
already been examined by immunoprecipitation and
West-ern blotting with various kinds of ®sh tissue lysates
including brain, eighth cranial nerve, gill, and semicircular
canals [17], before our experiments were performed The
membrane was washed three times each with NaCl/Pi
containing 0.1% Tween-20 for 15 min and then, incubated
with 1 : 3000 alkaline-phosphatase conjugated goat
anti-(rabbit IgG) Ig (Bio-Rad) diluted in the blocking solution
for 2 h at room temperature The membrane was washed
again as described above and equilibrated with developing
solution (100 mM Tris/HCl/100 mM NaCl/50 mM MgCl2,
pH 9.5) for 5 min The membrane was then incubated with
25 nM each of 5-bromo-4-chloro-3-indolyl phosphate and
4-nitrotetrazolium blue diluted in the developing solution
for 15 min The reaction was stopped by immersing in
distilled water
Immunohistochemistry
Fish were deeply anesthetized in a 0.02% aqueous solution
of 2-phenoxyethanol and decapitated The head was opened
dorsally and the brain was removed using forceps Right
and left sacculi, each containing an otolith, were removed
and ®xed in a mixture of 4% paraformaldehyde and 0.2%
glutaraldehyde in 0.1Mcacodylate buffer (pH 7.5) for 4 h
at room temperature The ®xed sacculi were decalci®ed in
10% EDTA in 10 mM Tris/HCl (pH 7.5) for 2 days at
room temperature The decalci®ed sacculi were post ®xed
with the same ®xative as described above for 3 h at room
temperature The sacculi were then dehydrated in ethanol
and embedded in paraf®n In order to examine the
localization of 100-kDa EDTA-insoluble otolith matrix
protein (otolin-1)-producing cells, undecalci®ed sections of
sacculi were prepared Sacculi were removed as described
above and ®xed in a mixture of 4% paraformaldehyde and
0.2% glutaraldehyde in 0.1Mcacodylate buffer (pH 7.5) for
4 h at room temperature The ®xed sacculi were stored
overnight in 70% ethanol at 4 °C After opening the
posterior end of the sacculus using a scalpel, the otolith was
removed from the sacculus using ®ne forceps The sacculi
were then dehydrated in ethanol and embedded in paraf®n
Transverse sections were cut at 6 lm and mounted on
gelatin-coated slides Deparaf®nized sections were incubated
for 30 min with a 0.6% H2O2 solution to inhibit
endo-genous peroxidase activity and subsequently with NaCl/Pi
containing 2% normal goat serum for 30 min to prevent nonspeci®c binding of immunoglobulins The sections were then incubated overnight at 4 °C with 500 ngámL)1of anti-(C-NC) Ig [17] Localization of immunoglobulins was visualized by the avidin-biotin-peroxidase complex (ABC) method [27] using commercial reagents (Vectastain
ABC-PO Kit, Vector Laboratory, Burlingame, CA, USA) and 3,3¢-diaminobenzidine tetrahydrochloride as a substrate Sections were mounted and observed under a differential interface microscope (Carl Zeiss, Oberkochen, Germany)
R E S U L T S
Extraction and separation of EDTA-insoluble matrix proteins from the salmon otolith
Otoliths from the chum salmon were decalci®ed with an EDTA solution The EDTA-insoluble materials exhibited a gel-like texture and retained the shape of the whole otolith These EDTA-insoluble materials were solubilized in a buffered Chaps solution, then were desalted, concentrated and subjected to SDS/PAGE analysis At least two proteins, one with an apparent molecular mass of 100 kDa and another with 55 kDa were detected (Fig 2A) The former was designated otolin-1 according to the de®nition which had been described by Degens et al [4], and the latter was found to be OMP-1 which we had previously identi®ed and biochemically characterized as a major component of EDTA-soluble matrix proteins [18]
Fig 2 Characterization of otolin-1 extracted from EDTA-insoluble matrix protein of O keta otolith Each sample was subjected to SDS/ PAGE analysis on 10% gel under reduced conditions and stained with Coomasie Brilliant Blue (A) Pro®le of the EDTA-insoluble otolith matrix proteins extracted with Chaps (lane 2) The upper arrow indi-cates otolin-1 and the lower one OMP-1, a major component of EDTA-soluble matrix proteins Lane 1, molecular mass standards (B) Possible N-glycosylation of otolin-1 Lane 1, molecular mass stan-dards Chaps-extracted matrix proteins before (lane 2) and after (lane 3) glycopeptidase-F (GPF) digestion The upper arrow indicates the untreated otolin-1, and the lower one indicates otolin-1 treated with GPF (C) Western blot analysis of Chaps-extracted EDTA-insoluble matrix proteins with anti-(C-NC1) Ig Otolin-1 is indicated by the lower arrow and the upper one indicates high molecular mass proteins (200 kDa).
Trang 5N-terminal and internal amino-acid sequences
The EDTA-insoluble matrix proteins recovered from the
salmon otolith were resolved by SDS/PAGE analysis and
transferred to a PVDF membrane The part of the
membrane that contained otolin-1 was cut out and subjected
to N-terminal protein sequence analysis The N-terminal
seven amino-acid residues were identi®ed except for positions 3 and 4 (Table 1) Otolin-1 obtained from the acrylamide gel by electroelution was desalted and also applied to a protein sequencer The results showed that the N-terminal 15 amino-acid residues were identi®ed except for positions 1±4 (Table 1)
To analyze the internal amino-acid sequences, otolin-1 was digested with trypsin in the acrylamide gel after electrophoresis and then the digested peptides recovered from the gel were carboxymethylated The resulting peptides were then separated by reverse-phase HPLC (Fig 3) As the hatched area (Fig 3A) was found to contain two tryptic fragments by MALDI TOF-MS analysis, they were sepa-rated by reverse-phase HPLC under different conditions (Fig 3B) and sequenced (Table 2).BLAST search analysis [28] revealed that the sequences of both peptides had a high homology to the sun®sh saccular collagen (sSC) [16] Cloning of a cDNA encoding otolin-1
Two degenerate oligonucleotide primers, sOT5¢-R1 and bsSC-F, were designed based on the amino-acid sequence of the internal peptide no 1 and a part of C-terminal sequence
of noncollagenous domain of the sSC, respectively Using these primers, PCR was carried out using ®rst strand cDNA synthesized from chum salmon saccular poly(A)+ RNA as
a template This yielded a single product of 182 bp in length (Fig 1A) Then, a speci®c primer (sOT5¢-R2) was designed based on this PCR product and 5¢ RACE was performed, giving a 1.5 kbp product The deduced amino-acid sequence encoded on this RACE-product contained the N-terminal sequence, con®rming that it corresponded to the otolin-1 protein To identify the 3¢ end of the otolin-1 transcript, an otolin-1-speci®c primer (sOT3¢-F), was designed and 3¢ RACE was performed, generating a 444-bp product that contained a deduced amino-acid sequence of the internal sequence no 1 and 2
Thereby the full length otolin-1 cDNA was completed (Fig 1A) and it was found to contain a 1524 nucleotide ORF followed by a 282 nucleotide 3¢ noncoding region that contained the consensus ƠAATAAÃ polyadenylation signal located 16 nucleotides upstream from the start of the poly(A) tail This ORF was found to encode a 508-residue precursor protein that consisted of a 25-residue signal peptide and a 227-residue collagenous domain ¯anked by a 118-residue N-terminal noncollagenous (N-NC) domain and a 138-residue C-NC domain (Fig 1B) Otolin-1 con-tained the collagenous domain at the position from 119 to
345 and two potential N-glycosylation sites at positions 96 and 391, one in each noncollagenous domain (Fig 1A,B)
As the N-NC domain contained many serine and threonine residues, theNETDGLYC2.0 program [29] was used to predict
Fig 3 Elution pro®le of puri®cation of the tryptic peptides of otolin-1 on
reverse-phase HPLC (A) The ®rst step RP-HPLC Column, Capcell
Pak C 18 column (2.0 ´ 150 mm); solvent, 10±60% acetonitrile in
0.05% tri¯uoroacetic acid; ¯ow rate: 0.2 mLámin )1 ; detection,
absorbance at 225 nm; temperature, 40 °C The concentration of
acetonitrile is indicated by the dotted line The hatched area showed
the fraction containing two tryptic peptides (B) The second step
RP-HPLC Solvent, 10±60% acetonitrile in 10 m M NH 4 HCO 3 ; other
conditions are the same as those described above Peaks 1 and 2
represent internal fragments 1 and 2, respectively.
Table 1 N-terminal amino-acid sequences of otolin-1 Preparation 1:
otolin-1 was prepared on a PVDF membrane Preparation 2: otolin-1
was prepared by electroelution from the gel ?, Unidenti®able value.
Preparation Sequence Position
2 ?-?-?-?-R-R-P-K-P-Q-N-T-K-K-P 1±15
Table 2 Amino-acid sequences of two puri®ed internal peptides of otolin-1 by the second RP-HPLC Degenerate primer (sOT5¢-R1) was designed at the position indicated by the underlined sequence, running right to left Position refers to the position in the predicted amino-acid sequence.
Internal 1 D-S-L-Y-G-Q-D-I-D-Q-A-S-N-L-A-L-L-R 427±444 Internal 2 L-A-S-G-D-Q-V-W-L-E-T-L-R 445±457
Trang 6potential O-glycosylation sites (Fig 1A,B) In vivo
N-glycosylation was evident as glycopeptidase-F digestion
of otolin-1 decreased its apparent molecular mass by
10 kDa as observed on SDS/PAGE (Fig 2B) When a
homology search using the full length of otolin-1 sequence
was conducted using the Swiss-Prot database, it revealed
that otolin-1 has 68% identity to the sun®sh saccular
collagen [16] Furthermore, the C-terminal noncollagenous
domain of otolin-1 also had homology to the C-terminal
noncollagenous domains of the collagen types VIII and X
(Fig 4)
Tissue-speci®c expression of the otolin-1 mRNA
in the sacculi
To examine the expression levels of the otolin-1 mRNA in
various tissues including the sacculi, semicircular canals,
brain, heart tissue, liver, muscle, skin and scales, Northern
blot analysis was performed using a cDNA fragment
encoding a part of otolin-1 (nucleotides 1284±1465, Fig 1A)
as a probe The otolin-1 transcript was only detected in the
mRNA from the sacculus and was approximately 1.9 kb in
length (Fig 5) This transcript size agreed well with that of
the otolin-1 cDNA (Fig 1A)
Localization of otolin-1
To examine the localization of otolin-1, Western blot
analysis and immunohistochemical experiments were
per-formed using anti-(C-NC) Ig [17] that were predicted to
recognize the otolin-1 molecule In the Chaps-soluble matrix
proteins, a band corresponding to otolin-1 and a broader
band of higher molecular mass were detected (Fig 2C) The
presence of the higher molecular mass form suggested the
possible existence of an aggregation product that includes
otolin-1 The upper panel in Fig 6 is schematic
represen-tation of a transverse section of the chum salmon sacculus
The saccular wall is a single-layer epithelium surrounded by
a thin connective tissue layer The thickest part of the
epithelium is the sensory epithelium comprised of sensory
hair cells and supporting cells Next to the sensory
epithelium, the transitional epithelium extends outward
until it to transitions into a squamous epithelium comprised
of small, cuboidal- or ¯at-shaped cells Columnar-shaped
cells are typically found in the transitional epithelium and
are interspersed with mitochondria-rich cells The otolith
membrane is composed of a gelatinous layer and a
subcupular meshwork, and together they af®x the otolith
to the underlying sensory epithelium In decalci®ed sections, the anti-(C-NC) Ig detected the otolin-1 protein in both the otolith and the gelatinous layer of the otolithic membrane, but not in the subcupular meshwork (Fig 6A,B) In addition, the otolin-1 protein did not appear to be uniformly distributed within the otolith: some parts stained intensely, while other parts showed only weak staining The staining
of the gelatinous layer of the otolithic membrane was modest In undecalci®ed sections, the otolin-1 immuno-reactivity was observed in some of the transitional epithelial cells, which were located adjacent to the sensory epithelium (Fig 6C) The immunoreactivity appeared to be more concentrated in the basal aspect of those cells The number
of immunoreactive cells varied depending on the part of the sacculus The sensory epithelium, squamous epithelium and mitochondria-rich cells were negative for otolin-1 staining
D I S C U S S I O N
In different ®sh species, the otolith has a species-speci®c shape that is likely to be due to differential accretion of the
Fig 4 Comparison of the amino-acid sequence of otolin-1 with those of other collagens Alignment of most homologous regions from the C-NC domains of otolin-1 (Oto1), sun®sh saccular collagen (sSC), human type X collagen (hType X) and mouse type VIII collagen (mType VIII) The positions of identical and similar residues are indicated by shaded box and bold type, respectively.
Fig 5 Tissue speci®c expression of otolin-1 mRNA Total RNA (10 lg each) prepared from semicircular canals (SeC), sacculi (Sa), brain (B), heart tissue (H), liver (L), muscle (M), skin (SK) and scales (SC) were subjected to Northern blot analysis The blot was probed with a [a- 32 P]-labeled otolin-1 cDNA as described in Experimental proce-dures Size was determined by comparison of migration with RNA size markers (Gibco) Lower panel shows 18S and 28S rRNA bands stained with ethidium bromide before blotting.
Trang 7inorganic constituents and differential utilization of the matrix proteins that serve within as a framework In this paper, a collagen-like structural protein termed otolin-1 in the salmon otolith has been identi®ed and characterized When salmon otoliths were decalci®ed, residual, gelatinous materials in the shape of the otolith remained and the otolin-1 protein was determined to be a major component of this gelatinous material Thus, it is possible that otolin-1 is part of the internal framework of the otolith where it may,
in part, provide the nucleation site for precipitation of calcium carbonate crystals
Otolin-1 has 68% identity (to sSC) that was originally identi®ed by differential screening of a sun®sh saccular cDNA library [16] Though there is high conservation between both of their C-NC domains, the N-NC domain of otolin-1 was much longer than that of the sSC and appears
to be distinct from that of the sSC The otolin-1 C-NC domain, like that of the sSC, had high homology to the C-NC domain of collagen types VIII [30] and X [31] (Fig 4) Collagen types VIII and X are non®brillar short chain collagens that form three-dimensional meshwork The collagenous domain of otolin-1 is smaller than that of the collagen types VIII and X, and contains 74 perfect Gly-X-Y repeats with three interruptions (imperfect Gly-X-Y repeats) It is also known that C-NC and N-NC domains
of collagen types VIII and X form the nodes, while the collagenous domains form the interconnecting spacers and together collectively they oligomerize supramolecularly into
a three-dimensional, hexagonally arranged lattice [32,33] If otolin-1 aggregates like these collagens, it might possibly provide nucleation sites to facilitate calci®cation
Furthermore, one or both of two potential N-linked glycosylation sites (located at positions of 96 and 391) identi®ed in the otolin-1 protein appear to be utilized in vivo
as glycopeptidase-F digestion was able to reduce the apparent molecular mass from 100 to 90 kDa in our biochemical analyses In addition, many putative O-linked glycosylation sites are observed at the N-NC domain These sugars may facilitate the aggregation of the collagen with each other and/or with other resident extracellular matrix moieties such as proteoglycans Consistent with this possi-bility, our studies have also indicated that the EDTA-insoluble, otolith-derived material contained various kinds
of sugars (data not shown)
Collagens are structural proteins present in many animal species Collagen type VIII is found loosely dispersed in the basement membranes of various tissues [34] while collagen type X is found only in the matrix of the hypertrophic zone
of the epiphyseal growth plate cartilage [35±37], yet is not the primary organic constituent deposited during endo-chondral ossi®cation Thus, collagen type VIII may serve as
a molecular bridge between different types of matrix molecules, whereas collagen type X may serve, in some manner not yet understood, in the process of mineralization [38] In this study, Northern blot analysis revealed that otolin-1 mRNA was expressed only in the sacculus and could not be detected even in the semicircular canals, another sensory structure found in the teleost inner ear Therefore, otolin-1 is a special collagen-like protein whose mRNA distribution is strictly limited in the sacculus
We also examined the localization of otolin-1 among the various structures and cells contained within the salmon sacculus Immunohistochemical analysis using anti-(C-NC)
Fig 6 Schematic representation and light micrographs of chum salmon
sacculus stained with anti-(C-NC) Ig (A) Decalci®ed section Otolith
(OT) and gelatinous layer (GL) Boundary of OT and GL is shown by
the dotted line In OT, some regions are stained intensely while other
regions show only modest staining GL is stained weakly Bar
50 lm (B) Decalci®ed section Otolithic membrane and sensory
epi-thelium (SE) In the otolithic membrane, immunoreactivity is weakly
observed in GL, but not in the subcupular meshwork (SM) SE does
notreactwithanti-(C-NC)Ig.Bar 50 lm.(C)Undecalci®edsection.
SE and transitional epithelium (TE) A part of transitional epithelial
cells, which are located at the periphery of SE, is positively stained with
anti-(C-NC) Ig Mitochondria-rich cell (*) is negative Bar 50 lm.
Trang 8Ig revealed that otolin-1 was distributed in the otolith,
gelatinous layer of the otolithic membrane and in a part of
the transitional epithelial cells Davis et al reported that
anti-(C-NC) Ig reacted with the columnar supporting cells
and the gelatinous layer of the otolithic membrane, but not
with lower ®lamentous subcupular meshwork in the bluegill
sun®sh [17] Our observations are consistent with those
previously reported, but we have extended those
observa-tions by demonstrating that this particular form of collagen
is also incorporated into and/or onto the otolith
Further-more, we have detected otolin-1 as a minor component in
the endolymph (data not shown) Based on these results,
otolin-1 was synthesized and secreted apically from the
transitional epithelium into the endolymph, then
immedi-ately incorporated into the otolith and the gelatinous layer
of the otolithic membrane However, it is still unclear
whether otolin-1 is deposited to the otolith via the otolithic
membrane or not In the otolith, otolin-1 did not appear to
be uniformly distributed as some regions were strongly
immunoreactive while other regions were much less so We
do not yet know how this might be signi®cant concerning
this difference We expected that immunoreactivity was
observed in the daily rings, but it was not con®rmed in this
study Immunoelectron microscopic analyses may be
required to fully de®ne the precise distribution and function
of the otolin-1 protein associated with the salmon otolith
On the basis of these results and the fact that the
primordia (otolith nuclei) are organic materials secreted
from the sensory epithelium [23], it may be that, early on, a
certain part of the primordial otolithic membrane and later
of the gelatinous layer of the otolithic membrane proper,
may serve as a ÔfoundationÕ for the growth of each
primordium Then, additional precipitation may occur on
the outer surface of each primordium staying on the suitable
positions of the gelatinous layer What kind of interaction
does it occur between the primordia and the gelatinous
layer? Do they contain same matrices? Khan et al revealed
that at least nine bands were observed from a sample of the
gelatinous layer of the otolithic membrane from the
rainbow trout, O mykiss using SDS/PAGE [39]
Dunkel-berger et al noted that the ®brous materials of subcuplar
meshwork could penetrate through the gelatinous layer and
incorporated in the overlying otolith in the juvenile
mum-michog, Fundulus heteroclitus [40] They also mentioned that
the gelatinous layer is closely associated with the otolith
surface, but incorporation of the ®bers into the otolith was
not observed In the present study, it is not clear whether the
otolin-1 protein detected in the otolith is continuous with
that detected in the gelatinous layer of the otolithic
membrane or not To understand the mechanism of onset
of otolith formation, a more detailed molecular study of the
interaction between these components at the zone where
new otolith primordia accrues to become ÔnewÕ calci®ed
otolith will be required
These studies suggest the possibility that ®sh might also
make use of collagens to promote the mineralization of
calcium carbonate, i.e in the formation of the otolith just
as they do for bones and dentin Major component of
bones and dentin is ®brillar collagen, while that of otolith
is otolin-1 which is a unique molecule belonging to the
family of a short-chain, meshwork-forming collagen
Thus, otolin-1 may contribute to form biominerals
composed of calcium carbonate in contrast to ®brillar
collagen in bones and dentin made of calcium phosphate
It has been said that the proteinaceous materials contained in the otolith are noncollagenous proteins [41]
In this experiment, we identi®ed a collagen-like protein containing many tandem repeats of the sequence, Gly-Xaa-Yaa, from the otolith, one of the biomineralized tissues composed of calcium carbonate, for the ®rst time
In contrast, it was reported that a part of the matrix protein, Lustrin A, from shell and pearl nacre of Haliotis rufescens, which were also composed of calcium carbon-ate, had some similarity to the type I collagen [42] The region having a homology to the type I collagen is, however, limited in only 30 amino-acid residues, in which two glycine residues were replaced by other amino-acid residues In addition, it is known that the interaction of collagen with other matrix proteins is important for bone formation In the case of otolith matrix proteins, the EDTA-insoluble fraction also contained OMP-1 as shown
in Fig 2A, a major component of EDTA-soluble matrix protein [18] Therefore, OMP-1 may be trapped by the meshwork of otolin-1 Further analyses of these complex intermolecular interactions will be required to understand how otolin-1, in certain saccular microenvironment, contributes to otolith formation
A C K N O W L E D G E M E N T S
We are grateful to Dr Akihisa Urano of Division of Biological Sciences, Hokkaido University for his generous gift of chum salmon We also thank Dr Goro Yoshizaki and Mr Yutaka Takeuchi of Department of Aquatic Biosciences, Tokyo University
of Fisheries for generous discussion about embryonic development This work was supported by Grants-in-Aid for Creative Basic Research (no 12NP0201) and for Scienti®c Research (no 12876025 and 13660176) from the Ministry of Education, Culture, Sports, Science and Technology of Japan E M was supported by Research Fellowships of Japan Society for the Promotion of Science for Young Scientists.
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