Vol.
174,
No.
January
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
31, 1991
A 60-kDa
PHOSPHORYLATED 1*
MasanOri
Suzuki
Kentarou
Horiuchi
Department
of
Hamamatsu
PROTEIN
, Akiyoshi 2 , Masaharu
Orthopedic
Uchiyamal
School
of
December
7,
HUMAN
, Kazuhiro Tetsuo
Department
Medicine,
BONE
Kushidal,
and
and
431-31,
Received
FETAL
Takahashil
Surgery1
University
FROM
439-445
1
Inoue
of
2
Chemistry
Hamamatsu,
,
Shizuoka,
Japan
1990
A phosphorylated protein was isolated and purified from fetal human bone. Fetal and adult human bones were decalcified with EDTA, and the extract from the fetal bone was fractionated using Q-Sepharose anion exchange chromatography. The fraction containing Ser(P) was purified by Sephacryl S-200 molecular sieving and C4 reverse-phase HPLC. The purified protein had a molecular weight of 60000 on SDS-PAGE, where the protein was stained with Rhodamine-B. The amino acid composition of this protein was different from any other reported phosphorylated proteins in human bone. However, this phosphorylated protein was difficult to detect in the adult bone extract on SDS-PAGE. cu 1991 AcadPmlc Press, 1°C.
The and of
organic
tissues,
include not
The
the
calcified
exist
only
in
calcified
to
play
writing,
important many
proteins
(2r6.111,
roles
been
protein
(4),
glycoproteins
isolated
phosphothreonine.
from These
(61,
osteonectin
ins.
Most
of
(16), these
but
tissue.
These
are
proteins
have
been
phosphoprotein (17)
and
bone
phosphorylated
have At
been the
been
time
(5-lo),
proteoglycan
to
BSP other
Several
contain
of
acidic
Ser(P)
(2),
and
osteopontin
unnamed various
pos-
identified.
y-carboxyqlutamic
(14).
to
Collagen
non-collaqenous
have
others found
organic
types:
(15), several
composed the
the
(l-3).
glycoprotein and
of
inorganic
(1).
proteins
several
(12,13)
are
some
in
into
bone
mainly while
calcification
acidic
BAG-75
are
of
proteins
tissues,
proteins
protein
consist
hydroxyapatite,
classified
serum-derived
dentin,
non-collaqenous
in
non-collaqenous
have
acid-containing
10%
and substances
called
and
in
tulated
These
collagen
bone
inorganic
crystal
90%
specific
proteins
this
including
substances.
a calcium-phosphate
ones is
calcified
glycoproteextents
and
*
To whom correspondence should be addressed. ethylenediamine tetraacetic acid; Ser(P), Abbreviations: EDTA, serine; HPLC, high-performance liquid chromatography; SDS-PAGE, BSP, bone sialoprotein; sulfate polyacrylamide gel electrophoresis; BAG-75, 75-kDa bone acid glycoprotein; PTH, phenylthiohydantoin.
phosphosodium
Vol.
174,
No.
have
an
have
been
2, 1991
BIOCHEMICAL
affinity
to
AND
hydroxyapatite.
speculated
to
BIOPHYSICAL
Therefore,
be
important
for
of
phosphorylated
RESEARCH
some
the
of
COMMUNICATIONS
these
initiation
of
proteins calcification
(1). Recently, BSP-II, have
four osteonectin
been
and
isolated
from
a phosphorylated is
types
a
human
protein
different
from
any
24-kDa
that other
glycoproteins
including
hydroxyproline-containing bone we
(18,19). isolated
reported
In from
phosphorylated
BSP-I,
phosphoprotein this human
report, fetal proteins
we bone in
describe which human
bone
MATERIALS
AND
METHODS
Tissue and Tissue Extractions: Fetal and adult human long bones were obtained from autopsy and the epiphyseal ends were cut away. The diaphyses were mechanically scraped, cleaned, defatted and ground in a liquid nitrogen-cooled mill. Proteins were extracted from the bones in 0.5 M EDTA at pH 7.8 and 4'C for 1 week (20). The pooled EDTA extracts were dialyzed against distilled water at 4OC and lyophilized. Protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 50 mu e-aminocaproic acid, 5 mM benzamidine hydrochloride, and 1 mM p-hydroxymercuribenzoic acid) were utilized during the preparation of the extracted samples (20). Isolation and Purification: The extracts were dissolved in 0.05 M Tris-HCl buffer, pH 8.2, containing 5 M urea and fractionated by anionexchange in a 2 x 30 cm column of Q-Sepharose (Pharmacia). The column was eluted with the same Tris-HCl buffer using a salt gradient (0 - 1.5 M NaCl) at room temperature at a flow rate of 10 ml/h. Each 2 ml fraction of eluate was monitored automatically at 230 nm absorbance in an Isco flow cell spectrophotometer. The fractions of each peak were separated and dialyzed against distilled water with the aforementioned protease inhibitors at 4OC using a Spectra-pore 3 dialysis tube and were then lyophilized separately. A small amount of each sample was taken and screened for the presence of Ser(P) using an amino acid analyzer as described below. The fractions containing Ser(P) were dissolved in 0.05 M Tris-HCl buffer (pH 8.2) containing 5 M guanidine HCl and fractionated by molecular sieving through a 1.9 x 95 cm column of Sephacryl S-200 (Pharmacia) The column was eluted with 0.05 M Tris HCl buffer (pH 8.2) containing 5 M guanidine HCl at room temperature at a flow rate of 10 ml /h. Fractions (2 ml per fraction) were collected, dialyzed and lyophilized. The major Ser(P)-containing peak was dissolved in 10% acetic acid and separated by HPLC using a C4 reverse phase column (0.46 x 25 cm, Senshu) and a linear gradient of aceto-nitrile (0 - 50%) in 0.1% trifluoroacetic acid. The eluate was monitored by absorbance at 210 nm. SDS-PAGE: The procedure for SDS-PAGE was slightly modified from that described by Laerranli 1211. Two identical 10% gels were run. One was stained with silver according to previously described methods (22). The other ye1 was stained with Rhodamine-B with slight modifications of the procedure described by Debryne (23). The proteins containing phosphoamino acids were specifically stained using this procedure. Amino Acid Analysis: Partial hydrolysis was carried out at llO°C for 6 hours in 4 M HCl in evacuated sealed tubes to determine Ser(P) levels (24). Total amino acid analysis was performed after hydrolysis at llO°C for 24 hours in 6 M HC~. These analyses were made using a Hitachi 835-50 amino acid analyzer. Amino Acid Sequence Analysis: Automated Edman degradation was carried out according to the manufacturer's instructions on a model 477A gas phase sequencer (ABI). PTH derivatives were obtained and identified by a model 120A analyzer (ABI).
440
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS
The
elution
profile
1 (A) .
By
use
of
eluted
at
NaCl
peak
and
the
collected, was S-200
in
a peak
of
by
5 M guanidine
at
fraction
was
against
fractionated
the was
void
eluted this
volume
from at
(shown dialyzed
0.3
peak by
0.8
by and
a bar
in
0.6
Fig.
lyophilized.
in
b'lgure
Fig.
Because
I IA
major 0.8
l(A))
of
M
were
The
Ser(P) l(B)).
were
to
a column
contained in
One
lyophilized.
through
that
M.
from
water
a bar
and
to
(shown
sieving The
shown peaks
concentrations
peak
molecular
is
Ser(P)-containing
distilled
HCl.
collected,
chromatography
gradient, ranging
fractions
dialyzed
then
Q-Sepharose NaCl
concentrations
Ser(P)-containing NaCl,
for
a linear
sample
Sephacryl
emerged This
peak
of
several
after
3
J 0
20
40
FRACTION
60
80
i0
100
NUMBER
4b
FRACTION
sb
sb
NUMBER
MINUTES
Elution profile of the EDTA extract of fetal human bone on F-1(A). Q-Sepharose. The column (2 x 30 cm) was eluted by a linear gradient (----) of O-l.5 M NaCl in 5 M urea at pH 8.2. The eluate was monitored The bar shows the fraction containing 60-kDa phosphorylated at 230 run. protein. Gel filtration of 60-kDa phosphorylated protein-enriched (B) fraction from Q-Sepharose on S-200 Sephacryl. The Column (1 .9 x 95 cm) was eluted with 5 M guanldlne HCl at pH 8.7. The proteln elution was monitored at 230 nm, and 2 ml fractions were collected. The bar shows the fractions contalnlng 60-kDa protein. HPLC of the sample containing 60-kDa phosphorylated protein CC) that was obtained from S-ID0 Sephacryl on a C4 reverse phase column using a TFA/acetonitrlle solvent system. The purified protein 1s shown by a bar.
441
100
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
6OK-
03
02 Line SDS-PAGE. was stained faintly The EDTA-extract from bone.
fia. 7+. Silver-stained phorylated protein present. Line B: extract from fetal Fiq.
A: A single without other adult bone.
band of 60-kDa contaminants Line C: The
phosbeing EDTA--
Rhodamine-B-stained SDS-PAGE. Line A: Phosvitin as a phosphoprotein marker. Line B: A broad single band of 60-kDa protein stained. Line C: The EDTA-extract from adult bone showed no clear 3,
rylated was
bands. additional
bands
further
The
nitrile
because additional
(Fig.
2).
when
stained
l(C)
Ser(P)-containing
a highly
phosphorylated
of
bands In
component
The
amino
acid
expressed composition
data
Ser(P)/lOOO
total
not
acid
sequence release The
of
corresponding SDS-PAGE
in of
EDTA
amino extract this 3);
be
this are
60-kDa
60-kDa
acids
by human
PTH
in
and
SDS-PAGE it
of
The 16
to this
analysis.
other
The
1.
reported
values amino
are acid
residues
of
Hydroxyproline
collaqenous
components,
A determination but
silver
SDS-PAGE
polypeptide.
attempted,
of Edman
the
amino
degradation
showed
derivatives.
adult protein
many
on
had
but
on
weight
Table
protein
aceto-
a faint
believed
residues.
acidic
an
with band
we
protein in
puri-
stained, stained
for
this at
showed
single
based
protein. was
from
however,
an
peak
60000
out
from
faintly
molecular
shown
carried
sharply
when
characteristic
protein
60-kDa
This
The
60-kDa was
out
is
total
this and
are
this this
to (Fig.
of bone
that
which
found
to
was
profile
Therefore,
purified.
residues
came
a broad
3).
residues/1000
showed
HPLC
SDS-PAGE
showed
estimated
acid
hydroxylysine,
were
on
compositions
amino
the
40%.
(Fig.
in
HPLC
protein
peak
highly
proteins as
shows
observed the
was
phase
peak
Rhodamine-B and
reverse
approximately
were
addition, with
non-collagenous
no
Figure major
phosphorylated
purified
and
C4
concentration
band
be
SDS-PAGE,
purification.
fication.
no
on
bands
442
bone
did
when
stained
were
not
observed
clearly with
show
a band
Rhodamine-B when
the
on gel
was
Vol.
174,
No.
BIOCHEMICAL
2, 1991
Table
1
Amino
protem
acid
and
60K P-P
stained
with
three
ASX THR SER GLX PRO GLY ALA VAL CYS MET ILE LEU TYR PHE LYS HIS ARG P-SER
90 52 78 148 40 105 67 52 22 70 21 32 62 77 46 I6
silver
(Fig.
residues
extract
of
from
BAG-75l
ESP-
The
in bone
BSP-II?
60K GP3
117 80 59 274 75 87 29 37 ND ND 21 26 53 30 55 19 40 ND
from
residues,
residues
62K GP’
II4 56 69 118 115 64 105 93 3 I 30 76 22 34 40 23 30 ND
extract
92 49 94 122 75 105 93 62 2 6 33 75 26 29 50 29 36 ND
adult
bone
while of
COMMUNICATIONS
phosphorylated
proteins
I :
total six
RESEARCH
60.kDa
223 40 122 176 76 25 55 42 ND ND 23 60 9 a 64 46 26 ND
2).
had
of
non-collagenous
117 47 III 176 49 102 70 33 9 13 40 29 24 33 29 42 ND
bone
BIOPHYSICAL
composrtlons
other
Ser(P)/lOOO
fetal
AND
the
Ser(P)/lOOO
contained
crude
EDTA
total
residues.
DISCUSSION Bone Some
contains
of
them
many have
undetermined. tions. :
BSP-I
bovine in
human bone
is
that
composition
of
BSP-II,
and
BSP-I
has have
rat
60-kDa
no
similar
(17)
bone
proteins protein
is
amounts
amino
II-I
mammalian
bone
(6,18),
osteonectin
(17),
are
still func-
are
as
folin
phosphoprotein and
60-kDa
protein
described
above.
different
from
(9)
and
62-kDa
described The
at
weight
those
nine
residues 62-kDa
compositions
443
and
protein
time
different
of
from
methionine,
(16) and
and
of
60-kDa
molecular
on
RSP-I, 1).
This
no
hydroxy-
than
an
anion
this while
this acid
glutamate
contains
a different is
in amino
(Table
aspartate
60-kDa
eluted
The acid
physiological
bone
of
our
molecular
methionine.
rest
their
phosphoprotein
addition,
has
the
about
phosphorylated
the
is the
but
(14).
bone.
lower In
and
BAG-75
protein
human
proteins
hydroxyproline-containing
bovine
contains
column
protein.
and
in
Osteonectin(l8)
change
rat
60-kDa
BSP-II.
proline.
(9)
this
known
hydroxyproline-containing
protein and
in
from
characterized,
is
(16,18),
the
different
non-collagenous
proteins
BAG-75 in
of
and
little
bone
(19),
We believe
60-kDa
isolated
BSP-II
glycoproteins
paper
types
non-collagenous and
and
human
(16)
been
Moreover, Reported
lows
different
ex-
bO-kDa our
ho-kDa
glycoproteins weights
on
SDS--
Vol.
174,
No.
2, 1991
PAGE
when
compared
extractions
to
and
Serum-derived have
and
EDTA
in
the
extract to
Rhodamine-B
(Fig.
phosphoprotein our
bound
to
the
the
protein
could
be
difficult
At
least
five
fetal
bone:
phosphoprotein study.
Recent
tions
of
Therefore, in
the
phosphorylated
BSP-I the
mechanism
as
types
BSP-I,
the
osteonectin
proteins of
described calcification
were
of
different. or
amino
did
acid
clearly when
serum
compositions
extract
bone
adult
phosphorylated
may
studies and above in
We believe be
degraded As
bone
a result,
with
proteins
phosphorylated
intra-
dentin
EDTA.
exist
in
hydroxyproline-containing
osteonectin, 60-kDa
bovine
maturation.
from
a band with
(25).
human during
show
stained
that
maturation in
of
not
suggested
bone
immune-histochemical and
the
SDS-PAGE
dentin
BSP-II, and
methods
a2HS-glycoprotein
bone
study
to
COMMUNICATIONS
protein.
on
of
their
times
but
protein matrix
different
(18,19),
such
human
during
tightly
human
60-kDa
degraded
that
elution
protein
A previous
and
the
weights,
adult
RESEARCH
however,
60-kDa
60-kDa
3). is
bone,
present
from our
BIOPHYSICAL
protein;
molecular
from
corresponding
AND
60-kDa
purifications
similar
different The
our
components
albumin, are
BIOCHEMICAL
showed
protein different
extracellularly may
have
from
this
localiza(26,27).
different
functions
bone.
REFERENCES 1. 2. 3. 4. 5.
6.
7. 8.
9. 10. 11. 12. 13. 14.
Glimcher, M.J. (1987) Instr. Course Lect. 36, 49-69. Fisher, L.W. and Termine, J.D. (1985) Clin. Orthop. Relat. Res. 200, 362-385. Termine, J.D. (1988) Ciba Found. Symp. 136, 178-202. Hauschka, P.V., Lian, J.B. and Gallop, P.M. (1975) Proc. Natl. Acad. Sci. USA 72, 3925-3929. Butler, W.T., Sato, S., Rahemtulla, F., Prince, C.W., Tomana, M., Bhown, M., DiMuzio, M.T. and Bronckers, A.L.J.J. (1985) In The Chemistry and Biology of Mineralized Tissues. (Butler, W.T.) pp. 107-112, EBSCO Media, Birmingham, Alabama. Franzen, A. and Heinegard, D. (1985) In The Chemistry and Biology of Mineralized Tissues. (Butler, W.T.) pp. 132-141, EBSCO Media, Birmingham, Alabama. Linde, A., Jontell, M., Lundgren, T., Nilson, B. and Svanberg, u. (1983) J. Biol. Chem. 258, 1698-1705. Prince, C.W., Oosawa, T., Butler, W.T., Tomana, M., Bhown, A.S., Bhown, M. and Schrohenloher, R.E. (1987) J. Biol. Chem. 262, 29002907. Sato, S., Rahemtulla, F., Prince, C.W., Tomana, M. and Butler, W.T. (1985) Connect. Tiss. Res. 14, 51-64. Uchiyama, A., Suzuki, M., Lefteriou, B. and Glimcher, M.J. (1986) Biochemistry. 25, 7572-7583. Sato, S., Rahemtulla, F., Prince, C-W., Tomana, M. and Butler, W.T. (1985) Connect. Tiss. Res. 14, 65-75. Mbuyi, J.M., Dequeker, J., Bloemmen, F., Stevens, E. (1982) Calcif. Tissue Int. 34, 229-231. Triffitt, J.T. and Owen, M. (1977) Calcif. Tissue Res. 23, 303-305. Delmas, P.D., Tracy, R.P., Riggs, B.L. and Mann, K.G. (1984) Calcif. Tissue Int. 36, 308-316. 444
Vol.
174,
15. 16. 17. 18. 19.
20. 21. 22. 23. 24. 25. 26 27.
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Spector, A.R. and Glimcher, M.J. (1972) Biochim. Biophys. Acta 263, 593-603. Termine, J.D., Belcourt, A.B., Ccnn. K.C. and Kleinman, H.K. (1981) J. Biol. Chem. 256, 10403-10408. Gorski, J.P. and Shimizu, K. (1988) Biol. Chem. 263, 15938-15945. Fisher, L.W., Hawkins, G.R., Tuross, N. and Termine, J.D. (1987) J. Biol. Chem. 262, 9702-9708. Fisher, L.W., Robey, P.G., Tuross, N., Otsuka, A.S., Tepen, D.A., Esch, F.S., Shimasaki, S. and Termine, J.D. (1987) J. Biol. Chem. 13457-13463. 262, Lee, S.L. and Glimcher, M.J. (1981) Calcif. Tissue Int. 33, 385-394. Laeamli, U.K. and Frave, M. (1973) J. Mol. Biol. 80, 575-599. Oakley, B.R., Kirsch, D.R. and Morris, N.R. (1980) Anal. Biochem. 105, 361-370. Debruyne, I. (1983) Anal. Biochem. 133, 110-115. Cohen-Solal, L., Lian, J.B., Kossive, D. and Glimcher, M.J. (1978) FEBS Lett. 89, 107-110. Kossiva, D., Glimcher, M.J. (1983) Lee, S.L., Biochemistry. 22, 2596-2601. Mark, M.P., Prince, C.W., Gay, S., Austin, R.L., Bhown, M., Finkelman, R.D. and Butler, W.T. (1987) J. Bone Mineral Res. 2, 337-346. Nomura, S., Wills, A.J., Edwards, D.R., Heath, J.K. and Hogan, B.L.M. (1988) J. Cell Biol. 106, 441-450.
445
174,
No.
January
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
31, 1991
A 60-kDa
PHOSPHORYLATED 1*
MasanOri
Suzuki
Kentarou
Horiuchi
Department
of
Hamamatsu
PROTEIN
, Akiyoshi 2 , Masaharu
Orthopedic
Uchiyamal
School
of
December
7,
HUMAN
, Kazuhiro Tetsuo
Department
Medicine,
BONE
Kushidal,
and
and
431-31,
Received
FETAL
Takahashil
Surgery1
University
FROM
439-445
1
Inoue
of
2
Chemistry
Hamamatsu,
,
Shizuoka,
Japan
1990
A phosphorylated protein was isolated and purified from fetal human bone. Fetal and adult human bones were decalcified with EDTA, and the extract from the fetal bone was fractionated using Q-Sepharose anion exchange chromatography. The fraction containing Ser(P) was purified by Sephacryl S-200 molecular sieving and C4 reverse-phase HPLC. The purified protein had a molecular weight of 60000 on SDS-PAGE, where the protein was stained with Rhodamine-B. The amino acid composition of this protein was different from any other reported phosphorylated proteins in human bone. However, this phosphorylated protein was difficult to detect in the adult bone extract on SDS-PAGE. cu 1991 AcadPmlc Press, 1°C.
The and of
organic
tissues,
include not
The
the
calcified
exist
only
in
calcified
to
play
writing,
important many
proteins
(2r6.111,
roles
been
protein
(4),
glycoproteins
isolated
phosphothreonine.
from These
(61,
osteonectin
ins.
Most
of
(16), these
but
tissue.
These
are
proteins
have
been
phosphoprotein (17)
and
bone
phosphorylated
have At
been the
been
time
(5-lo),
proteoglycan
to
BSP other
Several
contain
of
acidic
Ser(P)
(2),
and
osteopontin
unnamed various
pos-
identified.
y-carboxyqlutamic
(14).
to
Collagen
non-collaqenous
have
others found
organic
types:
(15), several
composed the
the
(l-3).
glycoprotein and
of
inorganic
(1).
proteins
several
(12,13)
are
some
in
into
bone
mainly while
calcification
acidic
BAG-75
are
of
proteins
tissues,
proteins
protein
consist
hydroxyapatite,
classified
serum-derived
dentin,
non-collaqenous
in
non-collaqenous
have
acid-containing
10%
and substances
called
and
in
tulated
These
collagen
bone
inorganic
crystal
90%
specific
proteins
this
including
substances.
a calcium-phosphate
ones is
calcified
glycoproteextents
and
*
To whom correspondence should be addressed. ethylenediamine tetraacetic acid; Ser(P), Abbreviations: EDTA, serine; HPLC, high-performance liquid chromatography; SDS-PAGE, BSP, bone sialoprotein; sulfate polyacrylamide gel electrophoresis; BAG-75, 75-kDa bone acid glycoprotein; PTH, phenylthiohydantoin.
phosphosodium
Vol.
174,
No.
have
an
have
been
2, 1991
BIOCHEMICAL
affinity
to
AND
hydroxyapatite.
speculated
to
BIOPHYSICAL
Therefore,
be
important
for
of
phosphorylated
RESEARCH
some
the
of
COMMUNICATIONS
these
initiation
of
proteins calcification
(1). Recently, BSP-II, have
four osteonectin
been
and
isolated
from
a phosphorylated is
types
a
human
protein
different
from
any
24-kDa
that other
glycoproteins
including
hydroxyproline-containing bone we
(18,19). isolated
reported
In from
phosphorylated
BSP-I,
phosphoprotein this human
report, fetal proteins
we bone in
describe which human
bone
MATERIALS
AND
METHODS
Tissue and Tissue Extractions: Fetal and adult human long bones were obtained from autopsy and the epiphyseal ends were cut away. The diaphyses were mechanically scraped, cleaned, defatted and ground in a liquid nitrogen-cooled mill. Proteins were extracted from the bones in 0.5 M EDTA at pH 7.8 and 4'C for 1 week (20). The pooled EDTA extracts were dialyzed against distilled water at 4OC and lyophilized. Protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 50 mu e-aminocaproic acid, 5 mM benzamidine hydrochloride, and 1 mM p-hydroxymercuribenzoic acid) were utilized during the preparation of the extracted samples (20). Isolation and Purification: The extracts were dissolved in 0.05 M Tris-HCl buffer, pH 8.2, containing 5 M urea and fractionated by anionexchange in a 2 x 30 cm column of Q-Sepharose (Pharmacia). The column was eluted with the same Tris-HCl buffer using a salt gradient (0 - 1.5 M NaCl) at room temperature at a flow rate of 10 ml/h. Each 2 ml fraction of eluate was monitored automatically at 230 nm absorbance in an Isco flow cell spectrophotometer. The fractions of each peak were separated and dialyzed against distilled water with the aforementioned protease inhibitors at 4OC using a Spectra-pore 3 dialysis tube and were then lyophilized separately. A small amount of each sample was taken and screened for the presence of Ser(P) using an amino acid analyzer as described below. The fractions containing Ser(P) were dissolved in 0.05 M Tris-HCl buffer (pH 8.2) containing 5 M guanidine HCl and fractionated by molecular sieving through a 1.9 x 95 cm column of Sephacryl S-200 (Pharmacia) The column was eluted with 0.05 M Tris HCl buffer (pH 8.2) containing 5 M guanidine HCl at room temperature at a flow rate of 10 ml /h. Fractions (2 ml per fraction) were collected, dialyzed and lyophilized. The major Ser(P)-containing peak was dissolved in 10% acetic acid and separated by HPLC using a C4 reverse phase column (0.46 x 25 cm, Senshu) and a linear gradient of aceto-nitrile (0 - 50%) in 0.1% trifluoroacetic acid. The eluate was monitored by absorbance at 210 nm. SDS-PAGE: The procedure for SDS-PAGE was slightly modified from that described by Laerranli 1211. Two identical 10% gels were run. One was stained with silver according to previously described methods (22). The other ye1 was stained with Rhodamine-B with slight modifications of the procedure described by Debryne (23). The proteins containing phosphoamino acids were specifically stained using this procedure. Amino Acid Analysis: Partial hydrolysis was carried out at llO°C for 6 hours in 4 M HCl in evacuated sealed tubes to determine Ser(P) levels (24). Total amino acid analysis was performed after hydrolysis at llO°C for 24 hours in 6 M HC~. These analyses were made using a Hitachi 835-50 amino acid analyzer. Amino Acid Sequence Analysis: Automated Edman degradation was carried out according to the manufacturer's instructions on a model 477A gas phase sequencer (ABI). PTH derivatives were obtained and identified by a model 120A analyzer (ABI).
440
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS
The
elution
profile
1 (A) .
By
use
of
eluted
at
NaCl
peak
and
the
collected, was S-200
in
a peak
of
by
5 M guanidine
at
fraction
was
against
fractionated
the was
void
eluted this
volume
from at
(shown dialyzed
0.3
peak by
0.8
by and
a bar
in
0.6
Fig.
lyophilized.
in
b'lgure
Fig.
Because
I IA
major 0.8
l(A))
of
M
were
The
Ser(P) l(B)).
were
to
a column
contained in
One
lyophilized.
through
that
M.
from
water
a bar
and
to
(shown
sieving The
shown peaks
concentrations
peak
molecular
is
Ser(P)-containing
distilled
HCl.
collected,
chromatography
gradient, ranging
fractions
dialyzed
then
Q-Sepharose NaCl
concentrations
Ser(P)-containing NaCl,
for
a linear
sample
Sephacryl
emerged This
peak
of
several
after
3
J 0
20
40
FRACTION
60
80
i0
100
NUMBER
4b
FRACTION
sb
sb
NUMBER
MINUTES
Elution profile of the EDTA extract of fetal human bone on F-1(A). Q-Sepharose. The column (2 x 30 cm) was eluted by a linear gradient (----) of O-l.5 M NaCl in 5 M urea at pH 8.2. The eluate was monitored The bar shows the fraction containing 60-kDa phosphorylated at 230 run. protein. Gel filtration of 60-kDa phosphorylated protein-enriched (B) fraction from Q-Sepharose on S-200 Sephacryl. The Column (1 .9 x 95 cm) was eluted with 5 M guanldlne HCl at pH 8.7. The proteln elution was monitored at 230 nm, and 2 ml fractions were collected. The bar shows the fractions contalnlng 60-kDa protein. HPLC of the sample containing 60-kDa phosphorylated protein CC) that was obtained from S-ID0 Sephacryl on a C4 reverse phase column using a TFA/acetonitrlle solvent system. The purified protein 1s shown by a bar.
441
100
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
6OK-
03
02 Line SDS-PAGE. was stained faintly The EDTA-extract from bone.
fia. 7+. Silver-stained phorylated protein present. Line B: extract from fetal Fiq.
A: A single without other adult bone.
band of 60-kDa contaminants Line C: The
phosbeing EDTA--
Rhodamine-B-stained SDS-PAGE. Line A: Phosvitin as a phosphoprotein marker. Line B: A broad single band of 60-kDa protein stained. Line C: The EDTA-extract from adult bone showed no clear 3,
rylated was
bands. additional
bands
further
The
nitrile
because additional
(Fig.
2).
when
stained
l(C)
Ser(P)-containing
a highly
phosphorylated
of
bands In
component
The
amino
acid
expressed composition
data
Ser(P)/lOOO
total
not
acid
sequence release The
of
corresponding SDS-PAGE
in of
EDTA
amino extract this 3);
be
this are
60-kDa
60-kDa
acids
by human
PTH
in
and
SDS-PAGE it
of
The 16
to this
analysis.
other
The
1.
reported
values amino
are acid
residues
of
Hydroxyproline
collaqenous
components,
A determination but
silver
SDS-PAGE
polypeptide.
attempted,
of Edman
the
amino
degradation
showed
derivatives.
adult protein
many
on
had
but
on
weight
Table
protein
aceto-
a faint
believed
residues.
acidic
an
with band
we
protein in
puri-
stained, stained
for
this at
showed
single
based
protein. was
from
however,
an
peak
60000
out
from
faintly
molecular
shown
carried
sharply
when
characteristic
protein
60-kDa
This
The
60-kDa was
out
is
total
this and
are
this this
to (Fig.
of bone
that
which
found
to
was
profile
Therefore,
purified.
residues
came
a broad
3).
residues/1000
showed
HPLC
SDS-PAGE
showed
estimated
acid
hydroxylysine,
were
on
compositions
amino
the
40%.
(Fig.
in
HPLC
protein
peak
highly
proteins as
shows
observed the
was
phase
peak
Rhodamine-B and
reverse
approximately
were
addition, with
non-collagenous
no
Figure major
phosphorylated
purified
and
C4
concentration
band
be
SDS-PAGE,
purification.
fication.
no
on
bands
442
bone
did
when
stained
were
not
observed
clearly with
show
a band
Rhodamine-B when
the
on gel
was
Vol.
174,
No.
BIOCHEMICAL
2, 1991
Table
1
Amino
protem
acid
and
60K P-P
stained
with
three
ASX THR SER GLX PRO GLY ALA VAL CYS MET ILE LEU TYR PHE LYS HIS ARG P-SER
90 52 78 148 40 105 67 52 22 70 21 32 62 77 46 I6
silver
(Fig.
residues
extract
of
from
BAG-75l
ESP-
The
in bone
BSP-II?
60K GP3
117 80 59 274 75 87 29 37 ND ND 21 26 53 30 55 19 40 ND
from
residues,
residues
62K GP’
II4 56 69 118 115 64 105 93 3 I 30 76 22 34 40 23 30 ND
extract
92 49 94 122 75 105 93 62 2 6 33 75 26 29 50 29 36 ND
adult
bone
while of
COMMUNICATIONS
phosphorylated
proteins
I :
total six
RESEARCH
60.kDa
223 40 122 176 76 25 55 42 ND ND 23 60 9 a 64 46 26 ND
2).
had
of
non-collagenous
117 47 III 176 49 102 70 33 9 13 40 29 24 33 29 42 ND
bone
BIOPHYSICAL
composrtlons
other
Ser(P)/lOOO
fetal
AND
the
Ser(P)/lOOO
contained
crude
EDTA
total
residues.
DISCUSSION Bone Some
contains
of
them
many have
undetermined. tions. :
BSP-I
bovine in
human bone
is
that
composition
of
BSP-II,
and
BSP-I
has have
rat
60-kDa
no
similar
(17)
bone
proteins protein
is
amounts
amino
II-I
mammalian
bone
(6,18),
osteonectin
(17),
are
still func-
are
as
folin
phosphoprotein and
60-kDa
protein
described
above.
different
from
(9)
and
62-kDa
described The
at
weight
those
nine
residues 62-kDa
compositions
443
and
protein
time
different
of
from
methionine,
(16) and
and
of
60-kDa
molecular
on
RSP-I, 1).
This
no
hydroxy-
than
an
anion
this while
this acid
glutamate
contains
a different is
in amino
(Table
aspartate
60-kDa
eluted
The acid
physiological
bone
of
our
molecular
methionine.
rest
their
phosphoprotein
addition,
has
the
about
phosphorylated
the
is the
but
(14).
bone.
lower In
and
BAG-75
protein
human
proteins
hydroxyproline-containing
bovine
contains
column
protein.
and
in
Osteonectin(l8)
change
rat
60-kDa
BSP-II.
proline.
(9)
this
known
hydroxyproline-containing
protein and
in
from
characterized,
is
(16,18),
the
different
non-collagenous
proteins
BAG-75 in
of
and
little
bone
(19),
We believe
60-kDa
isolated
BSP-II
glycoproteins
paper
types
non-collagenous and
and
human
(16)
been
Moreover, Reported
lows
different
ex-
bO-kDa our
ho-kDa
glycoproteins weights
on
SDS--
Vol.
174,
No.
2, 1991
PAGE
when
compared
extractions
to
and
Serum-derived have
and
EDTA
in
the
extract to
Rhodamine-B
(Fig.
phosphoprotein our
bound
to
the
the
protein
could
be
difficult
At
least
five
fetal
bone:
phosphoprotein study.
Recent
tions
of
Therefore, in
the
phosphorylated
BSP-I the
mechanism
as
types
BSP-I,
the
osteonectin
proteins of
described calcification
were
of
different. or
amino
did
acid
clearly when
serum
compositions
extract
bone
adult
phosphorylated
may
studies and above in
We believe be
degraded As
bone
a result,
with
proteins
phosphorylated
intra-
dentin
EDTA.
exist
in
hydroxyproline-containing
osteonectin, 60-kDa
bovine
maturation.
from
a band with
(25).
human during
show
stained
that
maturation in
of
not
suggested
bone
immune-histochemical and
the
SDS-PAGE
dentin
BSP-II, and
methods
a2HS-glycoprotein
bone
study
to
COMMUNICATIONS
protein.
on
of
their
times
but
protein matrix
different
(18,19),
such
human
during
tightly
human
60-kDa
degraded
that
elution
protein
A previous
and
the
weights,
adult
RESEARCH
however,
60-kDa
60-kDa
3). is
bone,
present
from our
BIOPHYSICAL
protein;
molecular
from
corresponding
AND
60-kDa
purifications
similar
different The
our
components
albumin, are
BIOCHEMICAL
showed
protein different
extracellularly may
have
from
this
localiza(26,27).
different
functions
bone.
REFERENCES 1. 2. 3. 4. 5.
6.
7. 8.
9. 10. 11. 12. 13. 14.
Glimcher, M.J. (1987) Instr. Course Lect. 36, 49-69. Fisher, L.W. and Termine, J.D. (1985) Clin. Orthop. Relat. Res. 200, 362-385. Termine, J.D. (1988) Ciba Found. Symp. 136, 178-202. Hauschka, P.V., Lian, J.B. and Gallop, P.M. (1975) Proc. Natl. Acad. Sci. USA 72, 3925-3929. Butler, W.T., Sato, S., Rahemtulla, F., Prince, C.W., Tomana, M., Bhown, M., DiMuzio, M.T. and Bronckers, A.L.J.J. (1985) In The Chemistry and Biology of Mineralized Tissues. (Butler, W.T.) pp. 107-112, EBSCO Media, Birmingham, Alabama. Franzen, A. and Heinegard, D. (1985) In The Chemistry and Biology of Mineralized Tissues. (Butler, W.T.) pp. 132-141, EBSCO Media, Birmingham, Alabama. Linde, A., Jontell, M., Lundgren, T., Nilson, B. and Svanberg, u. (1983) J. Biol. Chem. 258, 1698-1705. Prince, C.W., Oosawa, T., Butler, W.T., Tomana, M., Bhown, A.S., Bhown, M. and Schrohenloher, R.E. (1987) J. Biol. Chem. 262, 29002907. Sato, S., Rahemtulla, F., Prince, C.W., Tomana, M. and Butler, W.T. (1985) Connect. Tiss. Res. 14, 51-64. Uchiyama, A., Suzuki, M., Lefteriou, B. and Glimcher, M.J. (1986) Biochemistry. 25, 7572-7583. Sato, S., Rahemtulla, F., Prince, C-W., Tomana, M. and Butler, W.T. (1985) Connect. Tiss. Res. 14, 65-75. Mbuyi, J.M., Dequeker, J., Bloemmen, F., Stevens, E. (1982) Calcif. Tissue Int. 34, 229-231. Triffitt, J.T. and Owen, M. (1977) Calcif. Tissue Res. 23, 303-305. Delmas, P.D., Tracy, R.P., Riggs, B.L. and Mann, K.G. (1984) Calcif. Tissue Int. 36, 308-316. 444
Vol.
174,
15. 16. 17. 18. 19.
20. 21. 22. 23. 24. 25. 26 27.
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Spector, A.R. and Glimcher, M.J. (1972) Biochim. Biophys. Acta 263, 593-603. Termine, J.D., Belcourt, A.B., Ccnn. K.C. and Kleinman, H.K. (1981) J. Biol. Chem. 256, 10403-10408. Gorski, J.P. and Shimizu, K. (1988) Biol. Chem. 263, 15938-15945. Fisher, L.W., Hawkins, G.R., Tuross, N. and Termine, J.D. (1987) J. Biol. Chem. 262, 9702-9708. Fisher, L.W., Robey, P.G., Tuross, N., Otsuka, A.S., Tepen, D.A., Esch, F.S., Shimasaki, S. and Termine, J.D. (1987) J. Biol. Chem. 13457-13463. 262, Lee, S.L. and Glimcher, M.J. (1981) Calcif. Tissue Int. 33, 385-394. Laeamli, U.K. and Frave, M. (1973) J. Mol. Biol. 80, 575-599. Oakley, B.R., Kirsch, D.R. and Morris, N.R. (1980) Anal. Biochem. 105, 361-370. Debruyne, I. (1983) Anal. Biochem. 133, 110-115. Cohen-Solal, L., Lian, J.B., Kossive, D. and Glimcher, M.J. (1978) FEBS Lett. 89, 107-110. Kossiva, D., Glimcher, M.J. (1983) Lee, S.L., Biochemistry. 22, 2596-2601. Mark, M.P., Prince, C.W., Gay, S., Austin, R.L., Bhown, M., Finkelman, R.D. and Butler, W.T. (1987) J. Bone Mineral Res. 2, 337-346. Nomura, S., Wills, A.J., Edwards, D.R., Heath, J.K. and Hogan, B.L.M. (1988) J. Cell Biol. 106, 441-450.
445
