Vol.
167,
March
No.
30,
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1990
Purification
Pages
of a Calcium
Binding Protein (Rat SPARC) from Primary Enriched Culture Medium
1393-1399
Sertoli cell-
C. Yan ChenglJ 1The Population
Council, 1230 York Avenue, New York, New York 10021
ZThe Rockefeller Received
February
University,
20,
1230 York Avenue, New York, New York 10021
1990
A protein with an apparent Mr of 43,000 was purified from Sertoli cell-enriched culture medium by sequential anion-exchange, gel permeation, C4 reversed-phase, and diphenyl reversed-phase HPLC. N-Terminal sequence analysis of this protein revealed a sequence of NH,-XPQTEAAEEMVAEETVV for the first 17 amino acids. Comparison of this sequence with existing protein data base maintained at the Protein Identification Resource revealed that it shares extensive identity with a previously described protein secreted by mouse embryo parietal endoderm, SPARC, which is equivalent to a protein secreted by a basement-membrane-producing tumor, BM-40; and a bone protein, osteonectin. This protein also possesses similar in vitro biological activity of SPARC in which it binds Ca2+. The possible physiological significance of this protein was discussed. 0 1990Academic Press, Inc.
In the course of studying proteins contained
in Sertoli cell-enriched
culture
medium, it was noted that a protein with an apparent Mr of 43,000 was co-purified with rat clusterin (also referred to as CMB-2l/SGP-2/TRPP/ARP) of the purification
steps. Since its Mr was identical to the
we sought to isolate this protein to determine clusterin through its N-terminal acids from its N-terminus
(1,2) throughout most o subunit of rat clusterin,
if it was a molecular
sequence analysis.
with existing protein
Comparison
variant of rat
of the first 17 amino
data base revealed that it shares
extensive identity with a mouse embryo parietal endodermic protein designated SPARC (3) which is equivalent to a culture shock protein secreted by bovine aortic endothelial cells (4-5); osteonectin (6-7), a Mr 43,000 protein isolated from bone; and BM-40, a calcium
binding
protein
isolated
from basement-membrane-producing
Recent investigations
revealed that SPARC/BM-40/osteonectin
of calcium-dependent
processes in extracellular
tumors
appears to be a regulator
matrix (3, 5, 19), the study of testicular 0006-291x/90
1393
(8).
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
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SPARC might therefore provide new insights in the regulatory function of this protein in the testis. Materials
and Methods
of Sertoli cell-enriched culture medium. Primary Sertoli cell-enriched culture media were prepared from Sprague-Dawley rats at 20 days of age using procedures as previously described (1, 9). A batch of 3 liters of spent media was concentrated and equilibrated against 20 mM Tris, pH 7.4, at 22C using a Millipore Minitan ultrafiltration system equipped with four Minitan plates with an Mr cutoff at 10,000. Media were concentrated to about 50 ml and filtered through a Nalgene 0.2 pm filtering unit. Preparation
Purification of rat testicular SPARC. A. Anion-exchange HPLC. The spent media were pumped onto a Mono Q (HR 10/16, 16 x 100 mm i.d.) preparative column at a flow rate of 4 ml/mm using procedures as previously described (10, 11). Fractions containing testicular SPARC was visualized by SDS-PAGE, pooled, equilibrated against PBS buffer (10 mM sodium phosphate, 0.15 M NaCl, pH 6.8 at 22C), and concentrated to about 200 ~1 using an Amicon Centriconmicroconcentrator. B. Gel permeation HPLC. Sample containing testicular SPARC was loaded onto a Bio-Sil TSK250 preparative size exclusion HPLC column (21.5 x 600 mm) and proteins were eluted using PBS buffer at a flow rate of 3 ml/min (11). C. Vydac C4 reversed-phase HPLC. Fractions containing rat testicular SPARC obtained from the above step were pooled, dialyzed against double distilled water, lyophilized, resuspended in 200~1 of solvent A [5% acetonitrile/95% H,O containing 0.1% TFA (trifluoroacetic acid, v/v)] and loaded onto a Vydac C4 reversed-phase HPLC column (4.6 x 250 mm i.d.) at a flow rate of 1 ml/mm. Proteins were eluted using a linear gradient of lo-80% solvent B (95% acetonitrile/5% HZ 0 containing 0.1% TFA) in 90 min at a flow rate of 1 ml/mm. Fractions containing SPARC were monitored by SDS-PAGE, lyophilized, and resuspended in 200 ~1 of solvent A. D. Vydac diphenyl reversed-phase HPLC, Partially purified testicular SPARC obtained in the above step was injected onto a Vydac diphenyl reversed-phase HPLC column (4.6 x 250 mm i.d.) and proteins were eluted To using a linear gradient of 20-70% solvent B in 90 min at a flow rate of 1 ml/mm. monitor the purity of the protein throughout the entire purification scheme, eluents from the columns were monitored by diode array technology using a LKB Model 2140 Rapid Spectral Detector with a spectrum of wavelengths ranged between 190 and 370 nm and an integration time of 1 second. Analytical polyacrylamide gel electrophoresis (PAGE). Analytical PAGE in the presence of SDS was performed by the method of Laemmli (12) using a 10% T SDSpolyacrylamide gel with 2.6% cross-linker using methylene-bisacrylamide. Testicular SPARC was not readily visible using silver nitrate, gel was therefore stained with Coomassie blue R-250 routinely as previously described (13). N-Terminal sequence analysis of purified rat testicular SPARC. Automated Edman degradation of the native purified protein was performed on an Applied Biosystem Model 473A pulse liquid phase sequencer using procedures as previously described except that BioBrene Plus was used as a sample carrier (1, 14, 20). PTH (phenylthiohydantoin)-amino acids were automatically identified and quantified by reversed-phase HPLC using a Brownlee PTH C-18 (2.1 x 220 mm i.d., 5 pm) column at 55C which was built into the Model 473A sequencer. The repetitive yield was about 96%.
1394
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
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RESEARCH
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Results Purification
of rat testicular SPARC.
43,000 was identified
A protein with a single polypeptide
in Sertoli cell-enriched
of this protein was identical to the
culture medium.
Since the apparent Mr
Q subunit of rat clusterin, we thus sought to purify
this protein to determine if it was a molecular variant of rat clusterin. subsequently shown to be related to a mouse embryo endodermic
20
chain of Mr
40
80
RETENTION
TIME
IWJ
However, it was
protein called SPARC
IZ”
hinl
BO
0
Figs. LA, B. Purification of rat testicular SPARC. (A) Samples containing rat testicular SPARC fractionated by anion-exchange and gel permeation HPLC as described in Materials and Methods were pooled, equilibrated against double distilled water, lyophilized, and resuspended in 200 ~1 of Solvent A (5% acetonitrile/95% H20 containing 0.1% TFA, v/v) and loaded onto a Vydac C4 reversed-phase HPLC column (4.6 x 250 mm i.d.). Proteins were eluted using a linear gradient of N-SO% Solvent B (95% acetonitrile/5% H20, containing 0.1% TFA, v/v) over 90 min. A total of six protein peaks were observed when the eluents were monitored by UV absorbance at 280 rnn. Rat testicular SPARC was located under peak 3 by SDS-PAGE and Coomassie blue staining. Rat testicular SPARC obtained in Fig. 1A was lyophilized and (B) resuspended in 200 ~1 of Solvent A and loaded onto a Vydac diphenyl reversed-phase HPLC column (4.6 x 250 mm i.d.). Proteins were eluted using a linear gradient of 2070% B over 90 min and a total of six proteins peaks were noted. Purified rat testicular SPARC was detected under peak 3 by SDS-PAGE. inj. indicates where sample was injected onto the column; grad. indicates where gradient was started. 1395
Vol.
167,
No.
through N-terminal SPARC.
BIOCHEMICAL
3, 1990
AND
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COMMUNICATIONS
sequence analysis, thus this protein was designated rat testicular
Even though this protein co-eluted with rat clusterin during the steps of
anion-exchange and gel permeation reversed-phase HPLC
HPLC, it was readily separated from rat clusterin by
(Figs. lA, B).
In two 3-liters batches of Sertoli cell-enriched
culture media, we have isolated about 200 pg of purified SPARC protein.
Using a
Vydac C4 reversed-phase HPLC column, rat testicular SPARC was eluted under peak 3 shown in Fig. 1A and clusterin was eluted under peak 5 (Fig. 1A). This protein was
A
B
1
02
2
3
03 Structural analysis of purified rat testicular SPA!X by SDSFigs. 2A, B. polyacrylamidegelsunder reducing (A) and non-reducingconditions(B). In (A), about 3pg of purified rat testicularSPARC obtainedby diphenyl reversed-phase HPLC wasdenaturedin SDScontainingsamplebuffer (0.125M Tris, pH 6.8 at 22C containing 1% SDS, 1.6% 2mercaptoethano1,10% glycerol, and 1 mM EDTA) at 1OOCfor 5 min (lane 1) and resolvedon a 7.5% T SDS-polyacrylamide gel. Lane 2 represents2.5pg eachof BRL prestainedmolecularweightstandardsof myosin,200,000;phosphorylase b, 97,000;bovine serumalbumin, 68,000;ovalbumin,45,000;carbonic anhydrase,29,000. In (B), fractions under peak 3 shownin Fig. 1B which correspondedto lanes 1-3 were resolvedby SDS-PAGE on a lO%T SDS-polyacrylamidegel where the sampleshad been denaturedin the SDS-sample buffer without 2-mercaptoethanol.D, dye-front. Fig. 3. Binding of r6 W+] to rat testicularSPARC. Highly purified ovalbumin( 2.5 pg protein, lane l), rat testicular SPARC (2.5 rg protein lane 2), and BSA (2.5 pg protein, lane3) were loadedonto a SDS-polyacrylamidegel and resolvedunder reducing conditions. Following electrophoresis,proteins were transferred onto nitrocellulose paper,the membranewaswashedthree times(10 mitt each) with 100ml of washbuffer (10 mM imidazole, 50 mM KCl, and 10 mM MgC12,pH 7.4 at 22C). The membrane was then incubatedwith 100 ml of the washbuffer supplementedwith 5 pCi of [45CaClJ(New England Nuclear, SA 37 mCi/mg) for 30 mitt at room temperature, rinsedwith the washbuffer, washedin 100ml of 50% methanolfor 10min, dried, and proteins were then visualizedby autoradiographyusingEastmanKodak X-Omat AR film at -70C.
1396
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further purified on a Vydac diphenyl reversed-phase column 3 (Fig. 1B).
When purified
polyacrylamide
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and was eluted under peak
rat testicular SPARC was resolved on a 10% T SDS-
gel under reducing conditions, it displayed a single band of Mr 43,000
(Fig. 2A). When samples under peak 3 (Fig. 1B) containing purified testicular SPARC were resolved on a 10% T SDS-polyacrylamide
gel under non-reducing
conditions, a
single band of Mr 43,000 was also observed indicating that this protein contained only a single polypeptide vitro biological N-Terminal
chain (Fig. 2B). Purified testicular SPARC also possessed the in
properties of the mouse protein to bind Ca?+ (Fig. 3). sequence analysis of rat testicular
SPARC.
sequence of purified rat testicular SPARC was determined
The N-terminal
amino acid
using a pulse liquid phase
sequencer and the first 17 amino acids were shown in Fig. 4. When this sequence was compared Identification
with the release 19.0 of the protein
sequence data base at the Protein
Resource, it was noted that it displayed extensive identity with a mouse
embryo parietal endodermic protein designated SPARC (3) shown in Fig. 4. The first 17 amino
acids of the mouse SPARC
peptide
sequence derived from the nucleotide
sequence analysis was reported to be the signal peptide (3), thus it appeared that the secreted protein in the mouse has an extra N-terminal
amino acid, Ala, compared with
the rat testicular SPARC. Discussion
N-Terminal
sequence analysis of a Mr 43,000 protein isolated from Sertoli cell-
enriched culture medium revealed that it is structurally unrelated to the a of rat clusterin previously isolated in this laboratory
(1).
Interestingly,
shows extensive identity with a mouse embryo parietal endodermic
NH+?AWIFFLLCl
AGRAdP NH2-
QQ; EVA E EI f P~A~~~~
EEETV V
subunit this protein
protein designated
Mouse SPARC Rat Tes+lcular
SPARC
Fig. 4. Comparison of N-terminal amino acid sequence of rat testicular SPARC with mouse SPARC. The predicted N-terminal amino acid sequence of mouse SPARC was derived from nucleotide sequence of the mouse SPARC cDNAs isolated from parietal endoderm libraries (3). The first 17 amino acid residues of the mouse SPARC sequence wasthe signal sequence (3). 1397
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AND
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SPARC (3). It was of interest to note that these parietal endoderm cells are responsible for the synthesis of a thick basement membrane
(Reichert’s membrane)
surrounding the
developing mouse embryo, however, SPARC, which represents more than 20% of the secreted proteins by these cells, does not appear to be a structural component extracellular
of the
matrix (3). It has been shown that SPARC is synthesized and secreted
in vitro by several murine cell lines, fibroblasts, aortic endothelial
cells, and vascular
smooth muscle cells (3, 4, 15, 16). It is a high affinity albumin-binding whose secretion was increased by as much as 3-fold following
glycoprotein
cellular
injury (5).
SPARC was subsequently shown to be identical to BM-40, an acidic glycoprotein 40,000 first isolated from the basement membrane-producing Swarm) mouse tumor (8). various teratocarcinoma
Immunoassayable
EHS (Engelbreth-Holm-
BM-4O/SPARC
cell lines (such as PYS-2), epithelial
of Mr
was also detectable
in
cell line (such as PAM 212),
and Schwann cells of the peripheral nerve (8). SPARC is also identical to osteonectin, a phosphorylated non-collagenous
glycoprotein with an apparent Mr of 38,000, initially
cloned as a major
protein of the bone in the bull (7), which localized predominantly
osteogenic compartments
of the bone (6).
Both bovine osteonectin and mouse SPARC
have been shown to be coded for by a single gene with similar exon/intron (3, 7).
The functional
significance of SPARC/osteonectin/BM-40
however, studies with circular
dichroism
in the
organization
is not yet clear,
using spectropolarimeter
indicated
that
osteonectin has two putative calcium binding domains, one is located close to the Nterminus and the other one is located near the C-terminus (17).
Northern blot analysis using a cDNA probe prepared against SPARC has revealed that the SPARC/osteonectin the mRNA
mRNA is expressed in mouse embryos and gonads and that
is abundant in the Sertoli cell (TM4)
and Leydig cell (TM3)
lines (18).
Recent studies have indicated that SPARC is indeed synthesized and secreted by Leydig and Sertoli cells in vitro as demonstrated study demonstrates
by immunoprecipitation
that rat primary Sertoli cell-enriched
(19).
The present
cultures also contain a Mr
43,000 protein which shares identity with the mouse SPARC protein demonstrated terminal sequence analysis. This protein might be important testis during germ cell development. 1398
by N-
for calcium transport in the
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Acknowledgments This work was supported by a grant from NIH (HD 13541). The author was also supported by a grant from Istituto Di Ricerca F. Angelini (Rome, Italy). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Cheng CY, Chen CLC, Feng ZM, Marshall A and Bardin CW (1988) Biochem. Biophys. Res. Commun. 155:398-404. Cheng CY, Marshall A, Grima J and Mathur PP (1990) Mol. Androl. 2 : 73- 84. Mason IJ, Taylor A, Williams JG, Sage H and Hogan BLM (1986) EMBO J. 5:1465-1472. Sage H, Johnson C and Bornstein P (1984) J. Biol. Chem. 259:3993-4007. Sage H, Tupper J and Bramson R (1986) J. Cell. Physiol. 127:373-387. Termine JD, Kleinman HK, Whitson SW, Corm KM, McGarvey ML and Martin GR (1981) Cell 26:99-105. Young MF, Bolander ME, Day AA, Ramis CI, Robey PG, Yamada Y and Termine JD (1986) Nucleic Acids Res. 14:4483-4497. Dziadek M, Paulsson M, Aumailley M and Timpl R (1986) Eur. J. Biochem. 161:455-464. Cheng CY, Mather JP, Byer AL and Bardin CW (1986) Endocrinology 118:480-488. Cheng CY and Bardin CW (1986) Biochemistry 25:5276-5288. Cheng CY and Bardin CW (1987) J. Biol. Chem. 262:12768-12779. Laemmli UK (1970) Nature 227:680-685. Cheng CY, Musto NA, Gunsalus GL and Bardin CW (1983) J. Steroid Biochem. 19: 1379-1389. Cheng CY, Mathur PP and Grima J (1988) Biochemistry 27:4079-4088. Sage H, Vernon RB, Decker J, Funk S and Iruela-Arispe ML (1989) J. Histochem. Cytochem. 37:819-829. Holland PWH, Harper SJ, McVey JH and Hogan BLM (1987) J. Cell Biol. 105:473-482. Engel J, Taylor W, Paulsson M, Sage H and Hogan B (1987) Biochemistry 26:6958-6965. Howe CC, Overton GC, Sawicki J, Solter D, Stein P and Strickland S (1988) Differentiation 37:20-25. Vernon RB and Sage H (1989) Biol. Reprod. 40:1329-1340. Cheng CY, Grima J, Stahler, MS, Lockshin RA and Bardin CW (1989) J. Biol. Chem. 264:21386-21393.
1399
167,
March
No.
30,
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1990
Purification
Pages
of a Calcium
Binding Protein (Rat SPARC) from Primary Enriched Culture Medium
1393-1399
Sertoli cell-
C. Yan ChenglJ 1The Population
Council, 1230 York Avenue, New York, New York 10021
ZThe Rockefeller Received
February
University,
20,
1230 York Avenue, New York, New York 10021
1990
A protein with an apparent Mr of 43,000 was purified from Sertoli cell-enriched culture medium by sequential anion-exchange, gel permeation, C4 reversed-phase, and diphenyl reversed-phase HPLC. N-Terminal sequence analysis of this protein revealed a sequence of NH,-XPQTEAAEEMVAEETVV for the first 17 amino acids. Comparison of this sequence with existing protein data base maintained at the Protein Identification Resource revealed that it shares extensive identity with a previously described protein secreted by mouse embryo parietal endoderm, SPARC, which is equivalent to a protein secreted by a basement-membrane-producing tumor, BM-40; and a bone protein, osteonectin. This protein also possesses similar in vitro biological activity of SPARC in which it binds Ca2+. The possible physiological significance of this protein was discussed. 0 1990Academic Press, Inc.
In the course of studying proteins contained
in Sertoli cell-enriched
culture
medium, it was noted that a protein with an apparent Mr of 43,000 was co-purified with rat clusterin (also referred to as CMB-2l/SGP-2/TRPP/ARP) of the purification
steps. Since its Mr was identical to the
we sought to isolate this protein to determine clusterin through its N-terminal acids from its N-terminus
(1,2) throughout most o subunit of rat clusterin,
if it was a molecular
sequence analysis.
with existing protein
Comparison
variant of rat
of the first 17 amino
data base revealed that it shares
extensive identity with a mouse embryo parietal endodermic protein designated SPARC (3) which is equivalent to a culture shock protein secreted by bovine aortic endothelial cells (4-5); osteonectin (6-7), a Mr 43,000 protein isolated from bone; and BM-40, a calcium
binding
protein
isolated
from basement-membrane-producing
Recent investigations
revealed that SPARC/BM-40/osteonectin
of calcium-dependent
processes in extracellular
tumors
appears to be a regulator
matrix (3, 5, 19), the study of testicular 0006-291x/90
1393
(8).
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
SPARC might therefore provide new insights in the regulatory function of this protein in the testis. Materials
and Methods
of Sertoli cell-enriched culture medium. Primary Sertoli cell-enriched culture media were prepared from Sprague-Dawley rats at 20 days of age using procedures as previously described (1, 9). A batch of 3 liters of spent media was concentrated and equilibrated against 20 mM Tris, pH 7.4, at 22C using a Millipore Minitan ultrafiltration system equipped with four Minitan plates with an Mr cutoff at 10,000. Media were concentrated to about 50 ml and filtered through a Nalgene 0.2 pm filtering unit. Preparation
Purification of rat testicular SPARC. A. Anion-exchange HPLC. The spent media were pumped onto a Mono Q (HR 10/16, 16 x 100 mm i.d.) preparative column at a flow rate of 4 ml/mm using procedures as previously described (10, 11). Fractions containing testicular SPARC was visualized by SDS-PAGE, pooled, equilibrated against PBS buffer (10 mM sodium phosphate, 0.15 M NaCl, pH 6.8 at 22C), and concentrated to about 200 ~1 using an Amicon Centriconmicroconcentrator. B. Gel permeation HPLC. Sample containing testicular SPARC was loaded onto a Bio-Sil TSK250 preparative size exclusion HPLC column (21.5 x 600 mm) and proteins were eluted using PBS buffer at a flow rate of 3 ml/min (11). C. Vydac C4 reversed-phase HPLC. Fractions containing rat testicular SPARC obtained from the above step were pooled, dialyzed against double distilled water, lyophilized, resuspended in 200~1 of solvent A [5% acetonitrile/95% H,O containing 0.1% TFA (trifluoroacetic acid, v/v)] and loaded onto a Vydac C4 reversed-phase HPLC column (4.6 x 250 mm i.d.) at a flow rate of 1 ml/mm. Proteins were eluted using a linear gradient of lo-80% solvent B (95% acetonitrile/5% HZ 0 containing 0.1% TFA) in 90 min at a flow rate of 1 ml/mm. Fractions containing SPARC were monitored by SDS-PAGE, lyophilized, and resuspended in 200 ~1 of solvent A. D. Vydac diphenyl reversed-phase HPLC, Partially purified testicular SPARC obtained in the above step was injected onto a Vydac diphenyl reversed-phase HPLC column (4.6 x 250 mm i.d.) and proteins were eluted To using a linear gradient of 20-70% solvent B in 90 min at a flow rate of 1 ml/mm. monitor the purity of the protein throughout the entire purification scheme, eluents from the columns were monitored by diode array technology using a LKB Model 2140 Rapid Spectral Detector with a spectrum of wavelengths ranged between 190 and 370 nm and an integration time of 1 second. Analytical polyacrylamide gel electrophoresis (PAGE). Analytical PAGE in the presence of SDS was performed by the method of Laemmli (12) using a 10% T SDSpolyacrylamide gel with 2.6% cross-linker using methylene-bisacrylamide. Testicular SPARC was not readily visible using silver nitrate, gel was therefore stained with Coomassie blue R-250 routinely as previously described (13). N-Terminal sequence analysis of purified rat testicular SPARC. Automated Edman degradation of the native purified protein was performed on an Applied Biosystem Model 473A pulse liquid phase sequencer using procedures as previously described except that BioBrene Plus was used as a sample carrier (1, 14, 20). PTH (phenylthiohydantoin)-amino acids were automatically identified and quantified by reversed-phase HPLC using a Brownlee PTH C-18 (2.1 x 220 mm i.d., 5 pm) column at 55C which was built into the Model 473A sequencer. The repetitive yield was about 96%.
1394
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Results Purification
of rat testicular SPARC.
43,000 was identified
A protein with a single polypeptide
in Sertoli cell-enriched
of this protein was identical to the
culture medium.
Since the apparent Mr
Q subunit of rat clusterin, we thus sought to purify
this protein to determine if it was a molecular variant of rat clusterin. subsequently shown to be related to a mouse embryo endodermic
20
chain of Mr
40
80
RETENTION
TIME
IWJ
However, it was
protein called SPARC
IZ”
hinl
BO
0
Figs. LA, B. Purification of rat testicular SPARC. (A) Samples containing rat testicular SPARC fractionated by anion-exchange and gel permeation HPLC as described in Materials and Methods were pooled, equilibrated against double distilled water, lyophilized, and resuspended in 200 ~1 of Solvent A (5% acetonitrile/95% H20 containing 0.1% TFA, v/v) and loaded onto a Vydac C4 reversed-phase HPLC column (4.6 x 250 mm i.d.). Proteins were eluted using a linear gradient of N-SO% Solvent B (95% acetonitrile/5% H20, containing 0.1% TFA, v/v) over 90 min. A total of six protein peaks were observed when the eluents were monitored by UV absorbance at 280 rnn. Rat testicular SPARC was located under peak 3 by SDS-PAGE and Coomassie blue staining. Rat testicular SPARC obtained in Fig. 1A was lyophilized and (B) resuspended in 200 ~1 of Solvent A and loaded onto a Vydac diphenyl reversed-phase HPLC column (4.6 x 250 mm i.d.). Proteins were eluted using a linear gradient of 2070% B over 90 min and a total of six proteins peaks were noted. Purified rat testicular SPARC was detected under peak 3 by SDS-PAGE. inj. indicates where sample was injected onto the column; grad. indicates where gradient was started. 1395
Vol.
167,
No.
through N-terminal SPARC.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
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COMMUNICATIONS
sequence analysis, thus this protein was designated rat testicular
Even though this protein co-eluted with rat clusterin during the steps of
anion-exchange and gel permeation reversed-phase HPLC
HPLC, it was readily separated from rat clusterin by
(Figs. lA, B).
In two 3-liters batches of Sertoli cell-enriched
culture media, we have isolated about 200 pg of purified SPARC protein.
Using a
Vydac C4 reversed-phase HPLC column, rat testicular SPARC was eluted under peak 3 shown in Fig. 1A and clusterin was eluted under peak 5 (Fig. 1A). This protein was
A
B
1
02
2
3
03 Structural analysis of purified rat testicular SPA!X by SDSFigs. 2A, B. polyacrylamidegelsunder reducing (A) and non-reducingconditions(B). In (A), about 3pg of purified rat testicularSPARC obtainedby diphenyl reversed-phase HPLC wasdenaturedin SDScontainingsamplebuffer (0.125M Tris, pH 6.8 at 22C containing 1% SDS, 1.6% 2mercaptoethano1,10% glycerol, and 1 mM EDTA) at 1OOCfor 5 min (lane 1) and resolvedon a 7.5% T SDS-polyacrylamide gel. Lane 2 represents2.5pg eachof BRL prestainedmolecularweightstandardsof myosin,200,000;phosphorylase b, 97,000;bovine serumalbumin, 68,000;ovalbumin,45,000;carbonic anhydrase,29,000. In (B), fractions under peak 3 shownin Fig. 1B which correspondedto lanes 1-3 were resolvedby SDS-PAGE on a lO%T SDS-polyacrylamidegel where the sampleshad been denaturedin the SDS-sample buffer without 2-mercaptoethanol.D, dye-front. Fig. 3. Binding of r6 W+] to rat testicularSPARC. Highly purified ovalbumin( 2.5 pg protein, lane l), rat testicular SPARC (2.5 rg protein lane 2), and BSA (2.5 pg protein, lane3) were loadedonto a SDS-polyacrylamidegel and resolvedunder reducing conditions. Following electrophoresis,proteins were transferred onto nitrocellulose paper,the membranewaswashedthree times(10 mitt each) with 100ml of washbuffer (10 mM imidazole, 50 mM KCl, and 10 mM MgC12,pH 7.4 at 22C). The membrane was then incubatedwith 100 ml of the washbuffer supplementedwith 5 pCi of [45CaClJ(New England Nuclear, SA 37 mCi/mg) for 30 mitt at room temperature, rinsedwith the washbuffer, washedin 100ml of 50% methanolfor 10min, dried, and proteins were then visualizedby autoradiographyusingEastmanKodak X-Omat AR film at -70C.
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further purified on a Vydac diphenyl reversed-phase column 3 (Fig. 1B).
When purified
polyacrylamide
RESEARCH
COMMUNICATIONS
and was eluted under peak
rat testicular SPARC was resolved on a 10% T SDS-
gel under reducing conditions, it displayed a single band of Mr 43,000
(Fig. 2A). When samples under peak 3 (Fig. 1B) containing purified testicular SPARC were resolved on a 10% T SDS-polyacrylamide
gel under non-reducing
conditions, a
single band of Mr 43,000 was also observed indicating that this protein contained only a single polypeptide vitro biological N-Terminal
chain (Fig. 2B). Purified testicular SPARC also possessed the in
properties of the mouse protein to bind Ca?+ (Fig. 3). sequence analysis of rat testicular
SPARC.
sequence of purified rat testicular SPARC was determined
The N-terminal
amino acid
using a pulse liquid phase
sequencer and the first 17 amino acids were shown in Fig. 4. When this sequence was compared Identification
with the release 19.0 of the protein
sequence data base at the Protein
Resource, it was noted that it displayed extensive identity with a mouse
embryo parietal endodermic protein designated SPARC (3) shown in Fig. 4. The first 17 amino
acids of the mouse SPARC
peptide
sequence derived from the nucleotide
sequence analysis was reported to be the signal peptide (3), thus it appeared that the secreted protein in the mouse has an extra N-terminal
amino acid, Ala, compared with
the rat testicular SPARC. Discussion
N-Terminal
sequence analysis of a Mr 43,000 protein isolated from Sertoli cell-
enriched culture medium revealed that it is structurally unrelated to the a of rat clusterin previously isolated in this laboratory
(1).
Interestingly,
shows extensive identity with a mouse embryo parietal endodermic
NH+?AWIFFLLCl
AGRAdP NH2-
QQ; EVA E EI f P~A~~~~
EEETV V
subunit this protein
protein designated
Mouse SPARC Rat Tes+lcular
SPARC
Fig. 4. Comparison of N-terminal amino acid sequence of rat testicular SPARC with mouse SPARC. The predicted N-terminal amino acid sequence of mouse SPARC was derived from nucleotide sequence of the mouse SPARC cDNAs isolated from parietal endoderm libraries (3). The first 17 amino acid residues of the mouse SPARC sequence wasthe signal sequence (3). 1397
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SPARC (3). It was of interest to note that these parietal endoderm cells are responsible for the synthesis of a thick basement membrane
(Reichert’s membrane)
surrounding the
developing mouse embryo, however, SPARC, which represents more than 20% of the secreted proteins by these cells, does not appear to be a structural component extracellular
of the
matrix (3). It has been shown that SPARC is synthesized and secreted
in vitro by several murine cell lines, fibroblasts, aortic endothelial
cells, and vascular
smooth muscle cells (3, 4, 15, 16). It is a high affinity albumin-binding whose secretion was increased by as much as 3-fold following
glycoprotein
cellular
injury (5).
SPARC was subsequently shown to be identical to BM-40, an acidic glycoprotein 40,000 first isolated from the basement membrane-producing Swarm) mouse tumor (8). various teratocarcinoma
Immunoassayable
EHS (Engelbreth-Holm-
BM-4O/SPARC
cell lines (such as PYS-2), epithelial
of Mr
was also detectable
in
cell line (such as PAM 212),
and Schwann cells of the peripheral nerve (8). SPARC is also identical to osteonectin, a phosphorylated non-collagenous
glycoprotein with an apparent Mr of 38,000, initially
cloned as a major
protein of the bone in the bull (7), which localized predominantly
osteogenic compartments
of the bone (6).
Both bovine osteonectin and mouse SPARC
have been shown to be coded for by a single gene with similar exon/intron (3, 7).
The functional
significance of SPARC/osteonectin/BM-40
however, studies with circular
dichroism
in the
organization
is not yet clear,
using spectropolarimeter
indicated
that
osteonectin has two putative calcium binding domains, one is located close to the Nterminus and the other one is located near the C-terminus (17).
Northern blot analysis using a cDNA probe prepared against SPARC has revealed that the SPARC/osteonectin the mRNA
mRNA is expressed in mouse embryos and gonads and that
is abundant in the Sertoli cell (TM4)
and Leydig cell (TM3)
lines (18).
Recent studies have indicated that SPARC is indeed synthesized and secreted by Leydig and Sertoli cells in vitro as demonstrated study demonstrates
by immunoprecipitation
that rat primary Sertoli cell-enriched
(19).
The present
cultures also contain a Mr
43,000 protein which shares identity with the mouse SPARC protein demonstrated terminal sequence analysis. This protein might be important testis during germ cell development. 1398
by N-
for calcium transport in the
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Acknowledgments This work was supported by a grant from NIH (HD 13541). The author was also supported by a grant from Istituto Di Ricerca F. Angelini (Rome, Italy). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Cheng CY, Chen CLC, Feng ZM, Marshall A and Bardin CW (1988) Biochem. Biophys. Res. Commun. 155:398-404. Cheng CY, Marshall A, Grima J and Mathur PP (1990) Mol. Androl. 2 : 73- 84. Mason IJ, Taylor A, Williams JG, Sage H and Hogan BLM (1986) EMBO J. 5:1465-1472. Sage H, Johnson C and Bornstein P (1984) J. Biol. Chem. 259:3993-4007. Sage H, Tupper J and Bramson R (1986) J. Cell. Physiol. 127:373-387. Termine JD, Kleinman HK, Whitson SW, Corm KM, McGarvey ML and Martin GR (1981) Cell 26:99-105. Young MF, Bolander ME, Day AA, Ramis CI, Robey PG, Yamada Y and Termine JD (1986) Nucleic Acids Res. 14:4483-4497. Dziadek M, Paulsson M, Aumailley M and Timpl R (1986) Eur. J. Biochem. 161:455-464. Cheng CY, Mather JP, Byer AL and Bardin CW (1986) Endocrinology 118:480-488. Cheng CY and Bardin CW (1986) Biochemistry 25:5276-5288. Cheng CY and Bardin CW (1987) J. Biol. Chem. 262:12768-12779. Laemmli UK (1970) Nature 227:680-685. Cheng CY, Musto NA, Gunsalus GL and Bardin CW (1983) J. Steroid Biochem. 19: 1379-1389. Cheng CY, Mathur PP and Grima J (1988) Biochemistry 27:4079-4088. Sage H, Vernon RB, Decker J, Funk S and Iruela-Arispe ML (1989) J. Histochem. Cytochem. 37:819-829. Holland PWH, Harper SJ, McVey JH and Hogan BLM (1987) J. Cell Biol. 105:473-482. Engel J, Taylor W, Paulsson M, Sage H and Hogan B (1987) Biochemistry 26:6958-6965. Howe CC, Overton GC, Sawicki J, Solter D, Stein P and Strickland S (1988) Differentiation 37:20-25. Vernon RB and Sage H (1989) Biol. Reprod. 40:1329-1340. Cheng CY, Grima J, Stahler, MS, Lockshin RA and Bardin CW (1989) J. Biol. Chem. 264:21386-21393.
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