From the Molecular Sciences Department, University of
Tennessee, Health Science Center, Memphis, Tennessee 38163, the
¶ Molecular, Cellular, and Developmental Biology Department,
University of California, Santa Barbara, California 93106, and the
Structural Biology Department, St. Jude Children's Research
Hospital, Memphis, Tennessee 38105
Received for publication, November 10, 2002, and in revised form, January 3, 2003
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ABSTRACT |
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Osteocalcin (bone Gla protein) is an
extracellular matrix protein synthesized by osteoblasts that is a
marker of bone. Osteocalcin probably originated in the ancestors
of Teleostei or bony fish and of the Tetrapoda or amphibians,
reptiles, birds, and mammals. We have characterized the
Cyprinus carpio (carp) osteocalcin for mineral binding to
hydroxyapatite, amino acid sequence, and extent of secondary structure.
Hydroxyapatite binding is enhanced in the presence of calcium. The
Bone contains a vitamin K-dependent protein containing
Bone mineral binding, tissue distribution, and structural studies of
osteocalcin have been performed almost exclusively on proteins from
mammals and birds (Tetrapoda). Bony fish (Teleostei) are the most
successful organisms in aquatic environments. The common ancestor of
tetrapods and teleosts evolved bone over 200 million years ago and a
comparison of Oc in the two orders can provide insights into the
evolution of Oc structure and function. For example, the amino acid
sequences of most tetrapods contain a conserved C-terminal RRFYGPV
sequence that is missing in teleosts, while a truncated N terminus and
extended C terminus are missing in tetrapods (3, 4).
The present studies of Cyprinus carpio osteocalcin and
sequences derived from other fish genome sequences (Fugu
rubripes and Tetraodon nigroviridis) confirm the
primary structure differences observed in osteocalcins of tetrapod and
teleost. A higher proportion of carp Oc is Purification of Osteocalcin--
Carp (C. carpio)
bones were removed and cleaned of adherent tissue after placing in
boiling water for 1-1.5 min as described previously (3). Bones were
broken into 8-125-mm3 pieces, water washed for 20 min,
then lyophilized. The dried bone was ground in a blender. Particles
passing through a 0.425-mm sieve were washed in distilled water for
2 h; acetone-, trichloroethylene-, and acetone-washed for 1 h; and then air-dried. Bone proteins were extracted with 20% formic
acid (6 ml/g of bone) at 4 °C for 4 h, the supernatant filtered
through a 0.8-µm Millipore Millex PF filter, then desalted by gel
filtration on Sephadex G-25 equilibrated with 10% formic acid. The
collected protein fraction was diluted to 5% formic acid and then
lyophilized. The lyophilized proteins were dissolved in 5 M
guanidine, 0.1 M Tris, pH 8, and separated by gel
filtration on Sephacryl S-200 equilibrated with the same buffer. Bones
of different species were analyzed by gel filtration to estimate
osteocalcin content. Final purification for sequence analysis was by
high pressure liquid chromatography on a Vydac C18 reversed
phase column with gradient elution on a Beckman 332 gradient liquid
chromatograph equipped with a Beckman 420 Controller, Amersham
Biosciences UV-1 spectrophotometer, and a single channel chart
recorder. The elution gradient was initially 0.1% trifluoroacetic acid, 21% acetonitrile with a linear gradient to 0.1% trifluoroacetic acid, 56% acetonitrile over 25 min with a flow rate of 1.5 ml/min. Protein peaks were detected by absorbance at 280 nm. The single peak
eluting at ~20 min was manually collected. Purity of osteocalcin was
established with recovery of a single Coomassie Blue-stained band on a
15% polyacrylamide native gel, which coincided with the major
Gla-containing protein found by diazobenzenesulfonic acid (DBS)
staining for Gla-containing proteins, as described (3, 9).
Protein Sequence--
Purified Oc was digested by cyanogen
bromide (CNBr), BNPS skatole, or Staphylococcus
aureus V8 protease; the peptides were separated by reversed phase
HPLC using a modified gradient of 7-56% acetonitrile over 35 min.
Samples were lyophilized in microcentrifuge tubes for sequence, amino
acid composition, and mass spectral analyses. Intact protein and
proteolytic peptides were sequenced in a Porton Instruments sequenator
as described previously (3). Amino acid analysis of Oc and peptides was
performed on a Beckman system 6300, and carboxypeptidase Y digestion
was performed by established methods (3). The C-terminal amino acid
sequence was deduced by tandem mass spectroscopy with CID (collisional inactivation decomposition) with CO2 as the bombarding gas
using the purified C-terminal S. aureus V8 protease fragment
of osteocalcin (10, 11).
Conformational Studies by Circular Dichroism--
Purified Oc
was made calcium-free (apoOc) by incubating Oc (1 mg/ml) in 5 mM in EDTA, pH 7.4, for 15 min at room temperature, then
desalting over a 50-ml Sephadex G-25F column equilibrated with 5 mM ammonium bicarbonate to separate apoOc from
Ca2+ and EDTA. The collected apoOc was lyophilized and
resuspended in 1 mM Tris, pH 7.33, and diluted to between 4 and 5 µM. Bovine and carp Oc protein concentrations were
estimated as 1 unit of 230 nm absorbance as equivalent to 200 µg/ml.
Circular dichroism spectra were collected from 195 to 300 nm with data
collection for 10 s averaging in 1-nm increments with 2 repeats at
25 °C. CaCl2 (1 M) was added to adjust
calcium concentration to 5 mM, and the CD spectrum
collected again. An AVIV model 62S spectropolarimeter equipped with
electronic temperature control and a 1-cm pathlength quartz cuvette was
used for circular dichroism studies. Mean molar ellipticity was
calculated by [( Radioimmunoassay for Carp Osteocalcin--
Two New Zealand White
rabbits were inoculated with a mixture of Freund's Adjuvent and
purified osteocalcin (500 µg) at monthly intervals. All experiments
used a 1:16,667 final dilution of M47 4/12 antiserum. Standard curves
were made from serial dilution of purified carp Oc using 5 units of
absorbance at 230 nm to be 1 mg/ml. Iodination of carp Oc, assay
diluent, buffers, and conditions were identical to those published
(14-16). All samples were assayed in triplicate. After determining
bound radioiodinated Oc tracer (Packard Auto-Gamma Model 5002, Packard
Instrument Co.) the concentration of unknown was determined from a
spline-fit curve using Packard Cobra software (16).
Extraction and Assay of Proteins from Mineralized
Tissues--
The Oc content was determined from the supernatant of
10% formic acid extracts neutralized and diluted as described (16). Cross-reactivity of the assay was determined from serial dilutions of
10% formic acid extracts of bone from Lepomis macrochirus
(bluegill), Tilapia aurea (tilapia), Ctenopharyngodon
idella (grass carp), and Ictiobus bubalus (smallmouth,
buffalo). Phosphorus was determined from the dilutions of the extracts
using Sigma Diagnostics 360-3 phosphorus reagent and standards. Calcium
was determined by atomic absorption spectroscopy as described
previously (3).
Hydroxyapatite Binding--
Oc bound to hydroxyapatite and free
Oc were determined by radioimmunoassay or estimated by counting of
purified osteocalcin mixed with trace amounts of iodinated osteocalcin.
Sigma type 1 hydroxyapatite was used for all experiments. The buffer
used contained 5 mM PIPES, pH 7.4, 0.15 M KCl,
0.1% (w/v) bovine serum albumin (Grand Island Biological fraction V)
and was saturated with hydroxyapatite. All assays were performed in 575 µl with different amounts of hydroxyapatite, osteocalcin, and added
cations or antibodies. Incubations were done for 20 h at 4 °C
in a 1.5-ml screw cap microcentrifuge tubes with constant gentle
rotation. Unbound Oc was assayed from the supernatant after spinning
for 3 min at top speed in a Beckman Microfuge 11. 5 µl of
anti-osteocalcin antiserum (1:115 dilution) or an equal amount of
normal rabbit serum were added to test the effect on Oc-hydroxyapatite
binding. 125I-Osteocalcin was diluted to 10 ng/ml with
unlabeled Oc and the bound and free fractions determined by radioactive
counts. Binding was assessed by nonlinear regression analysis using
Graphpad Prism 3.0 software (17).
Determination of F. rubripes and T. nigroviridis Oc Sequences and
Flanking DNA Sequences from Genomic Sequences--
The Fugu
(tiger pufferfish) exon sequences were identified by TBlastN search
using sequences corresponding to carp protein to search the
Fugu data base at the Joint Genome Institute (JGI) at
bahama.igi-psf.org, finding matching sequences within the 8156 bases of
scaffold 10600 (version 1). The Sparus aurata Oc
gene sequence was used to probe for homologous regions of prepro-Oc of
exons 1 and 2. Determination of the intron boundaries were supported by
the presence of consensus splice sites. The Tetraodon (spotted pufferfish) exon sequences were identified by BlastN 2.2.3 within the T. nigroviridis WGS Trace data base to be in 2 sequences, gnl/ti/100718496 G41P615840RE3.T0 (exon 1 and upstream elements) and gnl/ti/100777902 G41P612422RE6.T0 at
www.ncbi.nlm.nih.gov/blast/Blast.cgi. Consensus sequence searches
and confirming translations of exon sequences were performed with the
program Gene Jockey 2. SignalP V1.1 identified signal peptides and
likely cleavage sites (at www.cbs.dtu.dk/services/SignalP/) (18).
Purification of Osteocalcin--
Carp Oc purification followed
established methods (3). Oc was identified within the largest
absorbance peak after Sephacryl S200 gel filtration chromatography of
the formic acid extract of rib bone. The protein stained positively for
gla by diazobenzenesulfonic acid staining of transblotted proteins from
15% polyacrylamide gels (not shown, Ref. 9). Individual fractions
loaded directly on reversed phase HPLC yielded osteocalcin, which
migrated as a single band on 15% PAGE.
Amino Acid Sequence of C. carpio Osteocalcin--
C.
carpio osteocalcin is a 45-amino acid polypeptide (Fig.
1). Sequences were determined by
automated Edman degradation of reversed phase-purified osteocalcin,
S. aureus V8 protease, CNBr cleavage, or BNPS skatole
cleavage peptides of osteocalcin as described previously (3). The
C-terminal amino acids were deduced by a combination of automated
sequencing, carboxy- peptidase Y digestion with amino acid
analysis, and confirmed by tandem mass spectrometry with CID, which
yielded b' and y' ion fragments of decreasing molecular weight (Fig.
1). The corresponding N-terminal fragments with the expected masses
were also observed (not shown). The use of multiple methods was
necessary due to the low yields on automated sequencing for C-terminal
peptides. The Gla at positions 14, 18, and 21 were inferred by sequence
homology with other ostecalcins, plus the absence of amino acid peaks
during automated sequence analysis at those positions, combined with an
expected Glx from acid hydrolysis. Cystines assigned to positions 20 and 26 were inferred by comparison with osteocalcin species and low
recovery of phenylthiohydantoin-derived amino acid at that position.
Amino acid analysis indicated the presence of 2 molar equivalents of Cys per mol of osteocalcin.
Carp ApoOc and Calcium-bound Oc Has Greater Percentage of
Antibody for C. carpio Oc and Radioimmunoassay--
Both rabbits
injected with carp Oc produced antibody after the second challenge.
Antiserum M47 4/12 was chosen for use in the radioimmunoassay after
analysis of several sera for antibody titer. The RIA had a detection
limit of 0.1 ng. Analysis of partially purified Oc from other fish
species showed that the Oc from I. bubalus (buffalo), and
C. idella (grass carp) cross-reacted completely, as they
exhibited a dose dependence similar to that of carp extracts, and
competed completely with antibody binding to 125I-carp Oc
when added at >300 ng. T. aurea was partially recognized, but bluegill was poorly cross-reactive (Fig.
3A). The antiserum recognized
both the 1-29 N-terminal and 31-48 C-terminal CNBr peptides,
indicating that there are at least 2 epitopes, one in the N-terminal
half of the Oc region and one in the 31-48 C-terminal half. Both
peptides inhibit binding to 1-48 Oc but neither proteolytic peptide
alone inhibited binding completely (Fig. 3B).
Distribution of Oc in Mineralized Tissues--
The amount of Oc in
different mineralized tissues was determined by radioimmunoassay and
expressed per dry weight of tissue or per unit of mineral, estimated by
phosphorus assay (Table II). The level of
Oc was highest in skeletal bones such as the dorsal spines, vertebral
projections, and ribs. Oc levels were lower in the operculum than in
any skeletal bone. Fish dermal scales were especially low in Oc,
containing 30-50 times less than other mineralized tissues tested,
confirming previous findings in bluegill (3).
Osteocalcin Hydroxyapatite Binding Is Enhanced by Calcium--
The
HA binding of carp osteocalcin is enhanced in the presence of calcium
ions. Fig. 4 shows Oc-HA binding in the
presence and absence of 2 mM calcium. The equilibrium
dissociation constant Kd of carp Oc was 22 µM while the Kd with calcium was 11 µM. The Kd values were calculated by
nonlinear regression analysis on Prism software (17). The enhanced
affinity with calcium is better seen in a Scatchard analysis (Fig. 4,
inset). The data fit best to a single binding site model. HA
binding by Oc is enhanced approximate 2-fold in the presence of
calcium. Calcium enhancement of hydroxyapatite affinity is a property
of teleost Oc. The data support a conserved
calcium-dependent affinity for hydroxyapatite shared
between teleost and tetrapods.
Identification of the Oc Gene from the Fugu and Tetraodon Genomic
Data bases--
To compare Oc sequences from as many fish species as
possible, the available genomic sequences from genome sequencing
projects were queried for presence of the osteocalcin gene. F. rubripes and T. nigroviridis data bases yielded matches
for the complete mature protein and the signal and pro-Oc sequences.
A TBlastN search of the genome data base revealed that scaffold 10600 contains the Fugu osteocalcin gene (Fig.
5). The mature Oc protein is completely
encoded by 2 exons, exons 3 and 4, with exon 4 containing a stop codon
after the C-terminal phenylalanine. Nucleotides 2075-2080 mark the
consensus AATAAA for the polyadenylation sequence. Putative exon 1 contains the signal sequence and the beginning of the intracellular
pro-Oc. A short putative exon 2 contains pro-Oc sequence. The
assignment of exons is supported by the presence of the TATA box, CCAAT
box, and OSE1 elements upstream of exon 1, and by homology to
the S. aurata, mouse, and chicken Oc
genes. The exon assignment is also supported by the presence of
consensus sequences at the 5'- and 3'-end of introns.
BlastN searches of the Tetraodon data base revealed 2 overlapping sequenced fragments and contained all the exons and introns and the part of the upstream TATA and CCAAT box elements typical for Oc genes (Fig. 6).
The fragments shared a 360-nucleotide intron 1 overlap 335-695 in the
exon 1-containing fragment with the sequence 17-377 in exons 2-, 3-, and 4-containing fragments. The probability of a random match was
<10 Comparison of Osteocalcin Sequences--
Alignment of Oc sequences
from teleosts and tetrapods reveals the identities between osteocalcins
(Fig. 7A). A solid line separates the teleosts and tetrapod osteocalcins. The selected tetrapods range from amphibians to birds and mammals. The Gla region
including the two Cys are highly conserved. Tetrapods and teleosts have
evolved unique highly conserved sequences near the C-terminal. Teleosts
have a highly conserved AAYXAYYGP(I/P), and most tetrapods
have a conserved QEAYRRFYGPV sequence. Teleosts also have a truncated N
terminus and an extended C terminus compared with tetrapods.
Alignment of the known teleost prepro sequences indicates significant
homology between the N-terminal signal and propeptide sequences (Fig.
7B). The SignalP program predicts a signal sequence from
1-17 in Fugu and Tetraodon and a signal sequence
from 1-20 in Sparus.
Abundance of Osteocalcin in Teleosts Is Variable--
A
survey of several teleost fish rib bones reveals that many species have
very high Oc content. In carp, bluegill, and walleye, Oc represents the
most abundant protein extracted from bone. Electrophoresis and
Coomassie Blue staining of peak pools shows that over 95% of the
protein in these peaks is Oc in bluegill (3) and in carp (data not
shown). Oc content per total extracted protein is ~35% for carp and
56% for bluegill. Other species such as tilapia, buffalo, and grass
carp also showed a similar distribution of bone noncollagenous
proteins. Channel catfish were an exception, with about the same Oc
content as that in bovine bone. One potential correlation is that bones
of scaled fish such as carp, bluegill, and walleye have a high Oc
content, while scaleless fish such as catfish have low Oc content. Low
levels of osteocalcin in bone may relate to the need for catfish to
utilize bone as the primary source of calcium for homeostasis. Other
fish primarily use scale calcium for homeostasis, but the lack of
scales in catfish forces the use of bone calcium, which may result in a
tetrapod-like distribution of osteocalcin in scaleless fishes. The
calcium content of catfish bone was 187 ± 21 mg/g, within the
range of calcium content in bluegill 217 ± 19 mg/g, carp 177 ± 6 mg/g, walleye 206 ± 15 mg/g, tilapia 218 ± 16 mg/g,
and grass carp 197 ± 16 mg/g in the same experiments rat bone
calcium content was 182 ± 19 mg/g. The difference in Oc content
was not correlated to calcium concentrations in the bone.
Carp osteocalcin is an abundant component of carp rib bone
comprising over 35% of the total extractable proteins. Carp
osteocalcin is a polypeptide of 45 amino acids, highly homologous to
the other teleost osteocalcins. It also has highly conserved regions in common with tetrapod osteocalcin, including the gla domain with an
invariant pair of cysteine residues, which form a disulfide bond in the
mature Oc (19). A region near the C terminus has a highly conserved
AYXX(Y/F)YGP motif. A truncated N terminus and extended C
terminus are conserved among teleosts, which are lacking the RFYGPV
C-terminal sequence common in tetrapods.
Gene sequences from 2 new teleost osteocalcins (Fugu and
Tetraodon) show that the signal sequence of fishes is highly
conserved, with the N terminus exhibiting especially high homology.
Pro-Oc sequence conservation included the sequence of basic residues that delineates the pro-Oc and mature Oc in all species. Four exons and
three introns are present in the Oc gene as found in all
other species. The Fugu gene spans 1471 bp from the TATA box to the polyadenylation sequence compared with 2875 bp for the same
region in the S. aurata Oc gene (20). Intron
shortening creates smaller Oc genes for both Fugu
and Tetraodon contributing to the smaller genomes of these
fishes. Searches of the available Danio rerio data base have
been negative as yet, except for portions of exon 4.
Upstream elements found include TATA and CCAAT boxes.
Fugu contains an upstream consensus OSE1 element that binds
an osteoblast-specific transcription factor present in osteoblasts
called OSF (20). Tetraodon regulatory sequences upstream of
exon 1 could not be identified, as an overlapping DNA fragment was not
available at this time. Similarly, the inability to identify an
overlapping fragment containing downstream sequences after exon 4 precluded identification of a polyadenylation site for
Tetraodon, although one was noted in Fugu.
Antibodies raised are cross-reactive to a number of species, which
should facilitate future studies of osteocalcin metabolism in teleosts.
The assay cross-reacts completely with the osteocalcin from I. bubalus (smallmouth buffalo, Fig. 4) with which it shares very
high homology with only 3 amino acid
differences.2
Furthermore, the assay is completely cross-reactive with C. auratus (goldfish), a commonly used teleost
model.3
Immunoreactive osteocalcin content varies in different mineralized
tissues of carp with dorsal spines highest and scales the lowest. This
result confirms assessment of osteocalcin content observed in bluegill
with non-immunochemical methods (3).
Carp apoOc appears to have a higher content of Oc is the most abundant noncollagenous bone protein of most teleosts.
Oc is the dominant protein component of the acid extracts of carp,
bluegill, walleye, grass carp, tilapia, and buffalo (fish) bone.
Similar findings are reported in Sparus (4). One exception is channel catfish bone, which has the same level of Oc as tetrapods. A
possible explanation for low levels of osteocalcin in bone may relate
to the need for catfish to utilize bone as the primary source of
calcium for homeostasis. Other fish primarily use scale calcium for
homeostasis, but the lack of scales in catfish forces the use of bone
calcium resulting in a different distribution of osteocalcin in
scaleless fishes. The hypothesis that high levels of Oc is due to
acellularity of fish bone has been disproven (3). The selection of
fishes tested included acellular and cellular bone, but there was no
correlation for Oc levels with cellularity. These results eliminate
presence of embedded osteocytes in bone as a reason for high levels of Oc.
-helical content of teleost osteocalcin increases and
-sheet
structure decreases upon calcium binding, similar to findings in calf
osteocalcin. The gene structure and primary sequence of
prepro-osteocalcin from 2 pufferfish compared with carp shows that
there are many conserved features in teleost osteocalcin genes. Using
an immunoassay for carp osteocalcin, we determined that the relative
content of osteocalcin is highest in dorsal fin spines and other bones
and lowest in scales. The carp osteocalcin antibodies,
cross-reactive to other species of fish, were used to study the role of
osteocalcin in teleost model systems.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carboxyglutamic acid
(Gla)1 called osteocalcin
(Oc) or bone Gla protein (1, 2). Bony fishes (Teleostei) and land
vertebrates (Tetrapoda) contain the protein Oc (2). Protein sequence
comparisons reveal the highest sequence conservation in the
Gla-containing domain (3, 4). Gla and the conformation conferred by the
Gla domain appear necessary for Oc to bind to hydroxyapatite (HA) (5,
6). Calcium binding causes a conformational change that coincides with
increased affinity for hydroxyapatite (7, 8).
-helical when compared
with the calcium-free bovine Oc. The studies also characterize mineral
binding behavior and tissue distribution of teleost Oc using a C. carpio Oc radioimmunoassay.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) (100)]/[(c)(n)(l)]; units are deg-cm2/decimol (
is ellipticity in millidegrees, c is
concentration mol/liter, n is number of amino acid residues
per protein, and l is pathlength in cm). The estimated secondary
structure was determined from the molar ellipticity from 200 to 240 nm
for each unknown and the K2d program for estimation of secondary
structure from circular dichroism data, which gives estimated structure and the error estimate (kal-el.ugr.es/k2d/k2d.html) (12, 13).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequence of purified carp
osteocalcin. Dashed lines indicate sequences from purified
S. aureus V8 peptides; solid lines indicate
sequences from purified CNBr peptides; arrows indicate the
sequence determined by the N-terminal sequence of the intact protein;
colons indicate sequence from purified BNPS skatole
peptides; arrow outline symbols show y' ion fragments
deduced by tandem mass spectrometry with CID of the C-terminal S. aureus V8 protease peptide of original mass 2332.2 Da. The mass of
C-terminal fragments is shown by numbers in parentheses.
Assignment of Ile at position 46 was possible because amino acid
composition revealed an unassigned Ile as one of the final 3 amino
acids in the C-terminal. ***, amino acid analysis of carboxypeptidase Y
digest.
-Helix--
The circular dichroism spectrum of carp apoOc indicates
that carp apoOc has a higher percentage of
-helical secondary
structure compared with either bovine apoOc or calcium-bound bovine Oc
(Fig. 2, Table
I). The bovine Oc spectrum undergoes a
change in the molar ellipticity of bovine osteocalcin at 222 nm as
reported previously (8). Spectral analysis showed a change from 8 to 9%
-helix content. Carp Oc exhibited a higher percentage of
-helix than bovine Oc in both the calcium-bound form with 5 mM calcium and the apoOc form without added calcium. ApoOc
was prepared by gel filtration desalting from solutions containing 5 mM EDTA as described in "Experimental Procedures."
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Fig. 2.
Circular dichroism spectra of carp and bovine
apoOc and calcium-bound carp and bovine Oc. The solid
lines represent carp and the dashed lines bovine Oc.
A, spectra for apoOc; B, spectra for the
calcium-bound form with 5 mM calcium.
Calculated secondary structure of carp and bovine Oc
-,
- and random
percentage values divided by three.
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Fig. 3.
Radioimmunoassay standard curve and
cross-reactivity. % B/Bo is calculated as
(100)(bound) (background)/(maximum bound)
(background).
A, cross-reactivity of RIA for other fish species.
Filled square, C. carpio (I. bubalus
is also represented by the filled square as its dose-diluted
results were indistinguishable from carp); open square,
C. idella; circle, T. aurea;
triangle, L. macrochirus. B,
cross-reactivity of RIA to proteolytic fragments of carp Oc.
Filled square, intact Oc; triangle, N-terminal
CNBr fragment 1-29; circle, C-terminal CNBr fragment
31-48; diamond, C-terminal CNBr fragment 30-48.
Osteocalcin content in mineralized tissues of carp
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Fig. 4.
Hydroxyapatite binding by carp
osteocalcin. The binding isotherm of purified carp Oc for
hydroxyapatite in the absence of added calcium (open
squares); binding in the presence of 5 mM calcium
(closed circles). Error bars represent S.D.
Experiments were performed as described under "Experimental
Procedures." Inset shows the Scatchard analysis. Results
of the average of duplicate experiments are shown.
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Fig. 5.
F. rubripes osteocalcin gene.
Fugu gene sequence and the available 5'-flanking region. The
figure shows the reversed and complemented sequence of scaffold 10600 consisting of 8156 nucleotides. Nucleotide 1 corresponds to 8156 in
scaffold for the sequence shown. Nucleotides are numbered in the
left margin, and the predicted amino acid sequence is shown
above the coding sequence. Intron boundary consensus
sequences are marked in bold underlining.
Asterisks indicate the in-frame stop codon. The OSE1 element
(TTACATA), TATA box, CCAAT box, and AATAAA polyadenylation signal
consensus sequences are underlined. The correspondence of
the translated sequences of the Fugu, Tetraodon,
and carp to other fish Oc sequences verifies the assignment of the exon
sequences (see below). The intron length from Fugu and
Tetraodon is much shorter than for Sparus, the
only other fish Oc gene sequence available.
64 (<e
146). The assignment of introns
was based on homology with the S. aurata gene sequences,
plus the presence of consensus splice site sequences at the 5'- and
3'-end of introns.
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Fig. 6.
T. nigroviridis osteocalcin gene.
Tetraodon gene sequences contained in 2 short DNA fragments.
The figure shows the reversed and complemented sequence of the 716 nucleotide-long fragment gnl/ti/100718496 G41P615840RE3.T0 (exon 1 and
upstream elements), with nucleotide 1 corresponding to 716. Below
the dashed line, the 740 nucleotide-long sequence of
gnl/ti/100777902 G41P612422RE6.T0 (exons 2, 3, and 4) is shown as
sequenced. Nucleotides are numbered in the left margin, and
the predicted amino acid sequence is shown above the coding sequence.
Intron boundary consensus sequences are marked in bold
underline. Asterisks indicate the in-frame stop codon.
The TATA box and CCAAT box consensus sequences are
underlined. The two fragments share a 360-nucleotide overlap
from intron 1 through part of intron 2.
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Fig. 7.
Comparison of sequences from teleosts and
tetrapods. A, alignment of the mature Oc from six teleost
and four tetrapods. Carp, Fugu, and Tetraodon are
new sequences derived from the current study. Sequences are
aligned N-terminal amino acid of the mature, secreted form of human Oc
chosen as the starting point with spaces every 10 residues.
CAR, carp (C. carpio); FUG, fugu
(F. rubripes); TET, freshwater puffer (T. nigroviridis); BLG, bluegill (L. macrochirus
from Ref. 3); SPA, seabream (S. aurata from Ref.
4); SWF, Swordfish (X. gladius from Ref. 19);
HUM, human (Homo sapiens from Ref. 19);
BOV, bovine (B. taurus from Ref. 19);
EMU, (Dromaius novaehollandiae from Ref. 21);
XEN, xenopus (Xenopus laevis from Ref.
4). A solid line separates the sequences of teleosts from
tetrapods. Asterisks above indicate conserved sequence in
fishes. Asterisks below indicate conserved sequences in all
species. Note that Gla residues are not distinguished from Glu in this
figure. B, alignment of prepro sequences of Oc from the
Sparus (20) with Fugu and Tetraodon.
The initiator methionine is indicated with a number 1 below
the sequences. Asterisks indicate identities,
lines indicate conservative replacements, and
dashes indicate gaps for best alignment. The cleavage
between pro and mature protein is indicated as a space prior
to the AAG in the N terminus of the mature, secreted Oc of the three
fish. Analysis of sequences by SignalP predicts the N terminus is a
signal peptide with cleavage between Ser18 and
Met19 for Fugu and Tetraodon and
between Ala20 and Ser21 for Sparus
(18).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix in comparison
to tetrapod apoOc, 24% compared with 8%. Conversely, bovine apoOc has
46%
-sheet compared with 19% in carp. The addition of calcium has
been reported to induce conformational changes in osteocalcin, which is
correlated with a calcium-dependent increase in
hydroxyapatite affinity (8). The change in structure of osteocalcin may
similarly account for the increased hydroxyapatite affinity of carp Oc
compared with apoOc, although the change in conformation for carp is
very small, as measured by circular dichroism. The high content of
-helical structure may make carp Oc a good candidate for
two-dimensional NMR studies to determine the Oc solution structure.
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ACKNOWLEDGEMENTS |
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We thank F. D. Robinson for technical assistance with the calcium assays. We thank Dr. S. Bhattacharya for his assistance with the atomic absorption spectrophotometer. We thank Sue Denzine at the MAMA Fish Hatchery, WI, for Walleye (S. vitreum), Dr. Ken Davis, University of Memphis, for Tilapia (T. aurea), the Buffalo Fish Co., Memphis TN, for carp (C. carpio) and smallmouth buffalo (I. bubalus), and the Southern Aquatech Hatchery, Memphis TN for grass carp (C. idella).
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The Protein Information Resource accession number for Cyprinus carpio osteocalcin protein is A59458.
§ To whom correspondence should be addressed. Tel.: 901-448-8753; Fax: 901-448-7360; E-mail: snishimoto@utmem.edu.
** Recipient of support from the American Lebanese Syrian Associated Charities (ALSAC) and National Institutes of Health Grant CA21765.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M211449200
2 S. K. Nishimoto and D. Lehane, unpublished data.
3 D. Lehane, personal communication.
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ABBREVIATIONS |
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The abbreviations used are:
Gla, -carboxyglutamic acid;
OC, osteocalcin;
HA, hydroxyapatite;
PIPES, 1,4-piperazinediethanesulfonic acid;
HPLC, high performance liquid
chromatography;
CID, collisional inactivation decomposition;
BNPS, 3-bromo-3-methyl-2-(2-nitrophyl- mercapto)-3H-indole.
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REFERENCES |
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