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Molecular cloning and sequencing of cDNA encoding urinary stone protein, which is identical to osteopontin
Vol.
184,
No.
April
30,
1992
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
859-864
MOLECULAR CLONING AND SEQUENCING OF cDNA ENCODING URINARY OSTEOPONTIN STONE PROTEIN , WHICH IS IDENTICAL TO
Kenjiro KOHRI, Yasuyuki SUZUKI, Koji YOSHIDA, Kazuhiko YAMAMOTO, Naoya AMASAKI, Takanori YAMATE, Tohru UMEKAWA, Masanori IGUCHI, Hyogo SINOHARA and Takashi KURITA
Department
of Urology and Biochemistry, Kinki University, Osaka
Received
March
23,
School 589,Japan
of
Medicine,
1992
SUMMARY We have sequenced a cDNA of urinary stone protein. cDNA sequences show complete homology between urinary stone protein and human osteopontin(bone and 1183-1424). sialoprotein) (nucleotides 265-886 Osteopontin is a recently discovered bone matrix protein which has been implicated in mediating mineral formation within bone extracellular matrix. This result shows that presumably involved in stone formation as osteopontin is stone matrix. 0 1992 Academicpress, 1nc
The composition each
calculus
and
a
comprised
noncrystalline
component, stone
is
of urinary
a
Organic
concretions
and accounts
total
weight
complex appears urine established
organic
of diverse
(l-4).
Boyce the
is at
It
from
present
in
2-3
per
consists
of
a
uromucoids
of
coworkers of
stone
the
phase latter
a
urinary
all
urinary
cent
of
their
macromolecular (1,5,6) kidney
(l-3) matrix
as
This as
or glycoproteins
and his
importance
known
least
mucoproteins
to be derived (5-8).
matrix
is
twofold,
or mineral phase.
moiety
for
is
a crystalline
or
proteinaceous
matrix.
dry
of
concretions
and and/or
defined and
and
postulated 0006-291X/92
859
$1.50
Copyright 0 1992 hi Academic Press. Inc. All righrs of r-eproductiom m at?\: form reserved.
Vol.
184,
No.
2,
1992
their
BIOCHEMICAL
active
urinary
architectonic
stones.
comprised
AND
of
Matrix
role itself
approximately
glucosamine,
10% bound
The
mucoprotein
has proven
component
from
urine
the
investigators in
the
others
doubt
number
of
matrix
in
structure
that
of
this
role
of
the
now report
protein.
the The
the
of
mechanism
MATERIALS
stone
of
sequence
sequence
of urinary
remain the
growth,
stone
a better
part while
considerable recent
years
of urinary
stone
composition poorly
and
understood. urinary
stone
osteopontin
urinary
allows
a
cDNA for
of
Some
an active
in
role
isolated
(l-3).
chemical
also
encoded this
been
Although
the
(1).
immunologically
stone
appeared
sugars,
ash
plays
and
The
of material
formers
matrix
have
sequencing
sialoprotein)(ll,l2) Knowledge
and has
matrix
formation
12% organic
matrix
(l-3,9,10).
COMMUNICATIONS
9% nonamino
most
concerning
stone
the
to be the
stone
formation.
RESEARCH
heterogenous
and
lithogenesis
continues stone
water
same
publications
controversy
We
of
process
is a
in stone
believe
in
64% protein,
5%
prominent
BIOPHYSICAL
stone
(bonematrix.
understanding
of
formation.
AND METHODS
Construction of polyclonal antibody for urinary stone protein Urinary stones were collected from patients, washed in distilled water and dried. Constitutions of stones were analyzed by infrared spectrometry. Ten g of renal calcium oxalate stones were pulverized to a fine powder,' and extracted with one liter of 0.2 M ethylenediaminetetraacetic acid (EDTA), pH 7.5, at 4°C for 48 hours, with the latter procedure repeated twice. After stirring overnight at 4"C, the suspension was spun at 6000rpm for 30min and exhaustive dialysis was carried out on the After supernatant. dialysis, the retentate was lyophilized. One ml of complete Freund's adjuvant emulsified with 0.5 mg of dry lyophilized stone protein was injected into subcutaneous and intracutaneous tissues of a rabbit weighing 3 Kg twice, and 1 ml of incomplete Freund's adjuvant 5 times weekly. Antiserum was obtained 1 week later, and stored at -2O'C. Sequence analysis The commercial human kidney cDNA library(Clontech) was screened as described by Sambrook et, al (13) with the stone 860
Vol.
184,
No.
2,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
isolated protein polyclonal antibody. Positive clones were were and the cDNA inserts were subcloned into M13mp19 which used for the construction of a series of deletion mutants various lengths of the inserts by the Cyclone containing The cDNA system (IBI) according to Dale et. al(14). chain were analyzed by the dideoxynucleotide sequence Sanger et. al using the T7 termination method of (15) Sequence analysis was performed sequencing Kit (Pharmacia). Search, Genetyx-CD with a computer program, Fast Homology ver. 16 (SDC-Software Development Co, Tokyo).
RESULTS Initial
antibody
recombinant the
plaques
positive
of
identified
clones
approximately
16 positive
was subjected
to
The cDNA was 1. 2 kilobase
analysis.
Computer-assisted
homology
library
revealed
human
osteopontin
(16)
screening
from
search
complete (bone
nucleotide
long(Fig
of
886,
One of
this
sequence 1).
the
sialoprotein)
265 to
clones. nucleotide
with
identity
50,000
sequence sequence
with
cDNA coding and
1183 to
data the
sequence
1424.
DISCUSSION The
present
osteopontin is
a
encodes
and a 41.5kDa
aspartic
anchor tripeptide, integrin
urinary
acids although
is
and/or
bone
attachment osteoclasts which receptor
to is
postulated family
to
(20)
861
It
and is via
to bind
(12).
have
Osteopontin
(17,18,19)
bone
about
of osteopontin
resorption.
is
matrix
phosphorylated
for
postulated
in vitro
bone
acid/glutamine
account
of
Osteopontin
and
glutamic
The function
it
sequence
protein.
sialated,
residues
(11,12).
cDNA
non-collagenous
serine,
acid/asparagine
that
stone
acidic,
in which
mineralization fibroblast
shows
discovered
glycoprotein,
known,
the
recently
protein,
amino
study
its
and 50% of is
not
roles
in
enhances thought
to
Arg-Gly-Asp
to a member
of
not
whether
known
the
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
gaccagactc
gtctcaggcc
agttgcagcc
ttctcagcca
aacgccgacc
aaggaaaact
cactaccatg
agaattgcag
tgatttgctt
ttgcctccta
ggcatcacct
gtgccatacc
120
agttaaacag
gctgattctg
gaagttctga
ggaaaagcag
ctttacaaca
aatacccaga
180
tgctgtggcc
acatggctaa
accctgaccc
atctcagaag
cagaatctcc
tagccccaca
340
gaatgctgtg
tcctctgaag
aaaccaatga
ctttaaacaa
gagacccttc
caagtaagtc
300
caacgaaagc
catgaccaca
tggatgatat
ggatgatgaa
gatgatgatg
accatgtgga
360
cagccaggac
tccattgact
cgaacgactc
tgatgatgta
gatgacactg
atgattctca
420
ccagtctgat
gagtctcacc
attctgatga
atctgatgaa
ctggtcactg
attttcccac
480
ggacctgcca
gcaaccgaag
ttttcactcc
agttgtcccc
acagtagaca
catatgatgg
540
ccgaggtgat
agtgtggttt
atggactgag
gtcaaaatct
aagaagtttc
gcagacctga
600
catccagtac
cctgatgcta
cagacgagga
catcacctca
cacatggaaa
gcgaggagtt
060
gaatggtgca
tacaaggcca
tccccgttgc
ccaggacctg
aacgcgcctt
ctgattggga
720
cagccgtggg
aaggacagtt
atgaaacgag
tcagctggat
gaccagagtg
ctgaaaccca
780
cagccacaag
cagtccagat
tatataagcg
gaaagccaat
gatgagagca
atgagcattc
840
cgatgtgatt
gatagtcagg
aactttccaa
agtcagccgt
gaattccaca
gccatgaatt
300
tcacagccat
gaagatatgc
tggttgtaga
ccccaaaagt
aaggaagaag
ataaacacct
960
gaaatttcgt
atttctcatg
aattagatag
tgcatcttct
gaggtcaatt
aaaaggagaa
1020
aaaatacaat
ttctcacttt
gcatttagtc
aaaagaaaaa
atgctttata
gcaaaatgaa
1080
agagaacatg
aaatgcttct
ttctcagttt
attggttgaa
tgtgtatcta
tttgagtctg
1140
gaaataacta
atgtgtttga
taattagttt
agtttgtggc
ttcatggaaa
ctccctgtaa
I 200
actaaaagct
tcagggttat
gtctatgttc
attctataga
agaaatgcaa
actatcactg
260
tattttaata
tttgttattc
tctcatgaat
agaaatttat
gtagaagcaa
acaaaatact
1320
tttacccact
taaaaagaga
atataacatt
ttatgtcact
ataatctttt
gttttttaag
ttagtgtata
ttttgttgtg
attatctttt
tgtggtgtga
ataa
Fig. 1. Comparison of encoding osteopontin (from protein stone established reveals complete identity.
osteopontin that
however,
are
binds
also that
to
present osteopontin
the
60
380
nucleotide sequences of cDNAs Kiefer et al., 1989) and urinary in this study (solid lines)
matrix
extracellular
in the
bone matrix. binds
862
extremely
components It
is tightly
known, to
Vol.
184,
No.
2,
BIOCHEMICAL
1992
hydroxyapatite
(12).
osteopontin known.
are
specific
hydroxyapatite (21).
osteopontin aspartic
acid
conformation
the
with
region
via
osteopontin
this
binding
is
not
interacts
with acid
binding
nine
region
can
assume
in osteocalcin
numerous affinity
for
a
as the
and bind
Another sialic
in
consecutive
arrangements
mechanism.
(22,23).
to
possibility
acid-containing hydroxyapatite
of
gene
to
dihydroxyvitamin
cent
contains
by
in
D3
is
strongly
related
activated
of
in kidney.
oxalate renal
of acidic
acid)
which
are
the
regulation
of
(28).
to
calcium containing accounts
Urinary
stone
acids
(glutamic
main
3
1,25
phosphate
amino the
D
milk(27).
Calcium
and calcium
stones
by
1,25-dihydroxyvitamin
presence
an abundance
aspartic
and by the
in
between
indicated
its
as calcium
80 per
protein
is
by
and is
such
the
observed
relationship
metabolism
expression
and
metabolism
mRNA is mainly
A possible
calcium
distribution
(24,25,26)
and
the
of
this
osteopontin
and
osteopontin
for
in
(20).
and kidney
stone
regions
carboxyl-group
confer
Interestingly,
tissue
or
mineral
consisting
the
COMMUNICATIONS
7-carboxyglutamic for
residues
that
oligosaccharides
bone
via
a similar
RESEARCH
osteocalcin
Perhaps
acid
hydroxyapatite
in
region
similar
T-carboxyglutamic
osteopontin
involved
likely
residues.
thought
region
A potential
is
BIOPHYSICAL
protein
most
residues
is
What
molecule The bone
AND
amino
matrix acid
acids
in
osteopontin(12,29,32). Based reasonable
on these to
observations
presume
principal
role
a similar
mechanism
that
in urinary to
and the osteopontin
stone that
formation
of bone
863
present
data,
it
in kidney
plays
as stone
matrix
formation.
is a in
Vol.
184,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
and Garvery,F.K. Boyce,W.H., (1956)J.Uro1.,76,213-227. Boyce,W.H., and King,J.S. (1959)J.Uro1.,81,35-365. Boyce,W.H. (1968)Am.J.Med.,45,673-683. Finlayson,B., Vermeulen,C.W., and Stewart,E.J. (1961) J.Uro1.,86,355-363. (1974)Urol.Clin,N.Am.,1,181-212. Finlayson,B. King,J.S. (1971)Clin.Chem.,l7,971-982. Malek,R.S., and Boyce,W.H. (1973)J.Uro1.,109,551-555. Vermeulen,C.W., and Lyon,E.S. (1968)Am.J.Med,45, 684-692. Sutor,D.J., and O'Flynn,D.J. (1973)I.n Urinary Calculi,p.280. S.Karger,Madrid. Howard,J.E. (1981)Urol.Survey.,3l,87-91. Franzen,A., and Heinegard,D. (1985)Biochem.J.,232, 715-724. Oldberg,A., Franzen,A., and Heinegard,D. (1986) Proc.Nath.Acad.Sci.USA.,83,8819-8823. Sambrook,J., Fritsch,Z.F., and Maniatis,T. (1989) Molecular Cloning.: A Laboratory Manual, 2nd ed. Vols.l-3, Cold Spring Harbor Laboratory, Cold Spring Harbor,NY. Dale,R.M.K., McClure,B.A., and Houchins,J.P. (1985) Plasmid 13,31-41. Sanger,F., Nicklen,S., and Coulson,A.R. (1977) Proc. Natl.Acad.Sci.U.S.A.,74,5463-5467. Kiefer,M.C., Bauer,D.M., and Barr,P.J. (1989)Nucleic Acids Res.17,3306. Somerman,M.J., Prince,C.W., Sauk,J.J., Foster,R.A., and Butler,W.T. (1987)J.Bone,Min.Res.,2,259-265. Somerman,M.J., Fisher,L.W., Foster,R.A., and Sauk, J.J. (1988)Calcif.Tissue.Int.,43,50-53. Somerman,M.J., Prince,C.W., Butler,W.T., Foster,R.A., (1989)Matrix,9,49-54. Moehring,J.M., and Sauk,J.J. Reinholt,F.P., Hultenby,K., Oldberg,A., and Heinegard, D. (1990)Proc.Nath.Acad.Sci.USA.,87,4473-4475. Poster,J.W., and Price,P.A. (1979)J.Biol.Chem.,254, 431-436. Yoon,Y., Buenaga,R., and Rodan,G.A. (1987)Biochem. Biophys.Res.Commun.,l57,166-173. Kerr,J.M., Fisher,L.W., Termine,J.D., and Young,M.F. (1991)Gene,108,237-243. PrinceC.W., and Butler,W.T. (1987)Collagen Relat.Res., 7,305-313. (1987)Biochem. Yoon,K., Buenaga,R., and Rodan,G.A. Biophys.Res.Commun.,l48,1129-1136. Wrana,J.L., Kubota,T., Zhang,Q., Overall,C.M., Aubin, J.E., Butler,W.T., and Sodek,J. (1991)Biochem.J.,273, 523-531. Senger,D.R., Peruzzi,C.A., Papadopoulos,A., and Tenen,D.G. (1989)Biochem.Biophys.Acta.,996,43-49. Yoshida,O., and Okada,Y. (1990)Urol.Int.,45,1O4-111. Spector,A.R., Gray,A., and Prien,E.L. (1976)Invest. Urol., 13,387-389. Gotoh,Y., Pierschbacher,M.D., Grzesiak,J.J., Gerstenfeld,L., and Glimcher,M.J. (1990)Biochem. Biophy.Res.Commun.,l73,471-479.
864
184,
No.
April
30,
1992
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
859-864
MOLECULAR CLONING AND SEQUENCING OF cDNA ENCODING URINARY OSTEOPONTIN STONE PROTEIN , WHICH IS IDENTICAL TO
Kenjiro KOHRI, Yasuyuki SUZUKI, Koji YOSHIDA, Kazuhiko YAMAMOTO, Naoya AMASAKI, Takanori YAMATE, Tohru UMEKAWA, Masanori IGUCHI, Hyogo SINOHARA and Takashi KURITA
Department
of Urology and Biochemistry, Kinki University, Osaka
Received
March
23,
School 589,Japan
of
Medicine,
1992
SUMMARY We have sequenced a cDNA of urinary stone protein. cDNA sequences show complete homology between urinary stone protein and human osteopontin(bone and 1183-1424). sialoprotein) (nucleotides 265-886 Osteopontin is a recently discovered bone matrix protein which has been implicated in mediating mineral formation within bone extracellular matrix. This result shows that presumably involved in stone formation as osteopontin is stone matrix. 0 1992 Academicpress, 1nc
The composition each
calculus
and
a
comprised
noncrystalline
component, stone
is
of urinary
a
Organic
concretions
and accounts
total
weight
complex appears urine established
organic
of diverse
(l-4).
Boyce the
is at
It
from
present
in
2-3
per
consists
of
a
uromucoids
of
coworkers of
stone
the
phase latter
a
urinary
all
urinary
cent
of
their
macromolecular (1,5,6) kidney
(l-3) matrix
as
This as
or glycoproteins
and his
importance
known
least
mucoproteins
to be derived (5-8).
matrix
is
twofold,
or mineral phase.
moiety
for
is
a crystalline
or
proteinaceous
matrix.
dry
of
concretions
and and/or
defined and
and
postulated 0006-291X/92
859
$1.50
Copyright 0 1992 hi Academic Press. Inc. All righrs of r-eproductiom m at?\: form reserved.
Vol.
184,
No.
2,
1992
their
BIOCHEMICAL
active
urinary
architectonic
stones.
comprised
AND
of
Matrix
role itself
approximately
glucosamine,
10% bound
The
mucoprotein
has proven
component
from
urine
the
investigators in
the
others
doubt
number
of
matrix
in
structure
that
of
this
role
of
the
now report
protein.
the The
the
of
mechanism
MATERIALS
stone
of
sequence
sequence
of urinary
remain the
growth,
stone
a better
part while
considerable recent
years
of urinary
stone
composition poorly
and
understood. urinary
stone
osteopontin
urinary
allows
a
cDNA for
of
Some
an active
in
role
isolated
(l-3).
chemical
also
encoded this
been
Although
the
(1).
immunologically
stone
appeared
sugars,
ash
plays
and
The
of material
formers
matrix
have
sequencing
sialoprotein)(ll,l2) Knowledge
and has
matrix
formation
12% organic
matrix
(l-3,9,10).
COMMUNICATIONS
9% nonamino
most
concerning
stone
the
to be the
stone
formation.
RESEARCH
heterogenous
and
lithogenesis
continues stone
water
same
publications
controversy
We
of
process
is a
in stone
believe
in
64% protein,
5%
prominent
BIOPHYSICAL
stone
(bonematrix.
understanding
of
formation.
AND METHODS
Construction of polyclonal antibody for urinary stone protein Urinary stones were collected from patients, washed in distilled water and dried. Constitutions of stones were analyzed by infrared spectrometry. Ten g of renal calcium oxalate stones were pulverized to a fine powder,' and extracted with one liter of 0.2 M ethylenediaminetetraacetic acid (EDTA), pH 7.5, at 4°C for 48 hours, with the latter procedure repeated twice. After stirring overnight at 4"C, the suspension was spun at 6000rpm for 30min and exhaustive dialysis was carried out on the After supernatant. dialysis, the retentate was lyophilized. One ml of complete Freund's adjuvant emulsified with 0.5 mg of dry lyophilized stone protein was injected into subcutaneous and intracutaneous tissues of a rabbit weighing 3 Kg twice, and 1 ml of incomplete Freund's adjuvant 5 times weekly. Antiserum was obtained 1 week later, and stored at -2O'C. Sequence analysis The commercial human kidney cDNA library(Clontech) was screened as described by Sambrook et, al (13) with the stone 860
Vol.
184,
No.
2,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
isolated protein polyclonal antibody. Positive clones were were and the cDNA inserts were subcloned into M13mp19 which used for the construction of a series of deletion mutants various lengths of the inserts by the Cyclone containing The cDNA system (IBI) according to Dale et. al(14). chain were analyzed by the dideoxynucleotide sequence Sanger et. al using the T7 termination method of (15) Sequence analysis was performed sequencing Kit (Pharmacia). Search, Genetyx-CD with a computer program, Fast Homology ver. 16 (SDC-Software Development Co, Tokyo).
RESULTS Initial
antibody
recombinant the
plaques
positive
of
identified
clones
approximately
16 positive
was subjected
to
The cDNA was 1. 2 kilobase
analysis.
Computer-assisted
homology
library
revealed
human
osteopontin
(16)
screening
from
search
complete (bone
nucleotide
long(Fig
of
886,
One of
this
sequence 1).
the
sialoprotein)
265 to
clones. nucleotide
with
identity
50,000
sequence sequence
with
cDNA coding and
1183 to
data the
sequence
1424.
DISCUSSION The
present
osteopontin is
a
encodes
and a 41.5kDa
aspartic
anchor tripeptide, integrin
urinary
acids although
is
and/or
bone
attachment osteoclasts which receptor
to is
postulated family
to
(20)
861
It
and is via
to bind
(12).
have
Osteopontin
(17,18,19)
bone
about
of osteopontin
resorption.
is
matrix
phosphorylated
for
postulated
in vitro
bone
acid/glutamine
account
of
Osteopontin
and
glutamic
The function
it
sequence
protein.
sialated,
residues
(11,12).
cDNA
non-collagenous
serine,
acid/asparagine
that
stone
acidic,
in which
mineralization fibroblast
shows
discovered
glycoprotein,
known,
the
recently
protein,
amino
study
its
and 50% of is
not
roles
in
enhances thought
to
Arg-Gly-Asp
to a member
of
not
whether
known
the
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
gaccagactc
gtctcaggcc
agttgcagcc
ttctcagcca
aacgccgacc
aaggaaaact
cactaccatg
agaattgcag
tgatttgctt
ttgcctccta
ggcatcacct
gtgccatacc
120
agttaaacag
gctgattctg
gaagttctga
ggaaaagcag
ctttacaaca
aatacccaga
180
tgctgtggcc
acatggctaa
accctgaccc
atctcagaag
cagaatctcc
tagccccaca
340
gaatgctgtg
tcctctgaag
aaaccaatga
ctttaaacaa
gagacccttc
caagtaagtc
300
caacgaaagc
catgaccaca
tggatgatat
ggatgatgaa
gatgatgatg
accatgtgga
360
cagccaggac
tccattgact
cgaacgactc
tgatgatgta
gatgacactg
atgattctca
420
ccagtctgat
gagtctcacc
attctgatga
atctgatgaa
ctggtcactg
attttcccac
480
ggacctgcca
gcaaccgaag
ttttcactcc
agttgtcccc
acagtagaca
catatgatgg
540
ccgaggtgat
agtgtggttt
atggactgag
gtcaaaatct
aagaagtttc
gcagacctga
600
catccagtac
cctgatgcta
cagacgagga
catcacctca
cacatggaaa
gcgaggagtt
060
gaatggtgca
tacaaggcca
tccccgttgc
ccaggacctg
aacgcgcctt
ctgattggga
720
cagccgtggg
aaggacagtt
atgaaacgag
tcagctggat
gaccagagtg
ctgaaaccca
780
cagccacaag
cagtccagat
tatataagcg
gaaagccaat
gatgagagca
atgagcattc
840
cgatgtgatt
gatagtcagg
aactttccaa
agtcagccgt
gaattccaca
gccatgaatt
300
tcacagccat
gaagatatgc
tggttgtaga
ccccaaaagt
aaggaagaag
ataaacacct
960
gaaatttcgt
atttctcatg
aattagatag
tgcatcttct
gaggtcaatt
aaaaggagaa
1020
aaaatacaat
ttctcacttt
gcatttagtc
aaaagaaaaa
atgctttata
gcaaaatgaa
1080
agagaacatg
aaatgcttct
ttctcagttt
attggttgaa
tgtgtatcta
tttgagtctg
1140
gaaataacta
atgtgtttga
taattagttt
agtttgtggc
ttcatggaaa
ctccctgtaa
I 200
actaaaagct
tcagggttat
gtctatgttc
attctataga
agaaatgcaa
actatcactg
260
tattttaata
tttgttattc
tctcatgaat
agaaatttat
gtagaagcaa
acaaaatact
1320
tttacccact
taaaaagaga
atataacatt
ttatgtcact
ataatctttt
gttttttaag
ttagtgtata
ttttgttgtg
attatctttt
tgtggtgtga
ataa
Fig. 1. Comparison of encoding osteopontin (from protein stone established reveals complete identity.
osteopontin that
however,
are
binds
also that
to
present osteopontin
the
60
380
nucleotide sequences of cDNAs Kiefer et al., 1989) and urinary in this study (solid lines)
matrix
extracellular
in the
bone matrix. binds
862
extremely
components It
is tightly
known, to
Vol.
184,
No.
2,
BIOCHEMICAL
1992
hydroxyapatite
(12).
osteopontin known.
are
specific
hydroxyapatite (21).
osteopontin aspartic
acid
conformation
the
with
region
via
osteopontin
this
binding
is
not
interacts
with acid
binding
nine
region
can
assume
in osteocalcin
numerous affinity
for
a
as the
and bind
Another sialic
in
consecutive
arrangements
mechanism.
(22,23).
to
possibility
acid-containing hydroxyapatite
of
gene
to
dihydroxyvitamin
cent
contains
by
in
D3
is
strongly
related
activated
of
in kidney.
oxalate renal
of acidic
acid)
which
are
the
regulation
of
(28).
to
calcium containing accounts
Urinary
stone
acids
(glutamic
main
3
1,25
phosphate
amino the
D
milk(27).
Calcium
and calcium
stones
by
1,25-dihydroxyvitamin
presence
an abundance
aspartic
and by the
in
between
indicated
its
as calcium
80 per
protein
is
by
and is
such
the
observed
relationship
metabolism
expression
and
metabolism
mRNA is mainly
A possible
calcium
distribution
(24,25,26)
and
the
of
this
osteopontin
and
osteopontin
for
in
(20).
and kidney
stone
regions
carboxyl-group
confer
Interestingly,
tissue
or
mineral
consisting
the
COMMUNICATIONS
7-carboxyglutamic for
residues
that
oligosaccharides
bone
via
a similar
RESEARCH
osteocalcin
Perhaps
acid
hydroxyapatite
in
region
similar
T-carboxyglutamic
osteopontin
involved
likely
residues.
thought
region
A potential
is
BIOPHYSICAL
protein
most
residues
is
What
molecule The bone
AND
amino
matrix acid
acids
in
osteopontin(12,29,32). Based reasonable
on these to
observations
presume
principal
role
a similar
mechanism
that
in urinary to
and the osteopontin
stone that
formation
of bone
863
present
data,
it
in kidney
plays
as stone
matrix
formation.
is a in
Vol.
184,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
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