Purification, Characterization, and Studies on Biosynthesis of a 59 ...

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Vol. 266, No. 22, Issue of August 5, pp. 14636-14645.1991 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 8 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Purification, Characterization, andStudies on Biosynthesis of a 59-kDa Bone Sialic Acid-containing Protein (BSP) from Rat Mandible Using a Monoclonal Antibody EVIDENCE THAT 59-kDa BSP MAY BE THE RATCOUNTERPART OF HUMAN a2-HS GLYCOPROTEIN AND ISSYNTHESIZED BY BOTHHEPATOCYTES AND OSTEOBLASTS* (Received for publication, January 28,

1991)

Tomokazu Ohnishi, Naokatu Arakaki,Osamu Nakamura, Shuichi Hirono, and Yasushi DaikuharaS From the Department of Biochemistry, Kagoshima University Dental School, 35-1 Sakuragaoka 8, Kagoshima 890, Japan

A monoclonal antibody was raised against a miner- and to be synthesized by the liver. alized tissue-specific sialoprotein containing no phosphorus using partially purified noncollagenous bone matrix proteins from ratsas antigen. Then the sialoprotein was purified by high performance liquid chro-Recently, extracellular matrix molecules have been found matography fromrat mandibulae usingthe monoclonal antibody as a marker. The sialoprotein (59-kDabone to be important in determining not only the physical propersialoprotein (BSP)) witha molecular weight of 59,000 ties of tissues but also the adhesion and differentiated funccontained 1.4% sialic acid but no detectable phospho- tions of cells (1, 2). Mineralized connective tissues, such as bone and dentin, clearly differ from most other tissues in rus. Immunohistochemical studies with the antibody showed that the protein was specific to mineralized their content of crystalline calcium phosphate and hydroxytissues such as bone and dentin, and present inosteo- apatite, and attention has recently been focused on noncolblasts, osteocytes, and bone matrix. No other soft tis- lagenous matrix proteins of the tissues because no apparent sues, such as the cartilage, liver, kidney, and perios- tissue specificity of collagen, which is the major organic conteum, were stained. However, Western blot analysis stituent, has been found (3-5). These noncollagenous matrix proteins are usually classified according to their content of showed that plasma contained immunoreactive 59-kDa BSP. The quantitative amino acidcomposition of 59- phosphorus and sialic acid. One of the best characterized bone kDa BSPresembled that of human a2-HS glycoprotein sialoproteins is osteopontin, which is thought to be involved (a2-HSG) (Lee, C.-C., Bowman, B.H., and Yang, F. in mineralization because of its high affinity to hydroxyapatite (1987) Proc. Natl.Acad. Sei. U.S. A. 84,4403-4407; and the cell surface and because of its highly specific distriKellermann, J., Haupt, H., Auerswald, E.-A,, and bution in bone tissue (6-8). Several other bone-specific sialMuller-Esterl, W. (1989)J. Biol. Chem. 264, 14121- oproteins have also been isolated, and some of these noncol14128) andrat 64-kDa protein (Franzen,A., and Hei- lagenous sialoproteins are known to be synthesized by cells negird, D. (1985) in The Chemistry and Biology of in these tissues (6, 8-13). Most of these sialoproteins, howMineralized Tissues(Butler, W. T., ed), p. 132, EBSCO ever, including osteopontin, which is also known as 44-kDa Media, Birmingham, AL). Amino acid sequence anal- acidic glycoprotein (8), 2ar (14), or SppI (15), arereported to yses of the amino-terminal region and four peptide be phosphorylated (6-8,10,11,14,15). Therefore, our purpose fragments of 59-kDa BSP revealed that about 50%of in this study was to purify a noncollagenous bone matrix the amino acids were homologous with thoseof human a2-HSG, whichis known to be synthesized by the liver, sialoprotein that did not contain phosphorus. transported in the bloodstream, and incorporated into Purification of a proteinfrom mineralized tissues is difficult calcified tissues. But when newborn rat calvaria, pri- because these proteins usually do not have any detectable biological activity in vitro (e.g. enzymatic activity) that can mary cultures of osteoblast-rich cells, and adult rat purification. Monoclonal hepatocytes were incubated with radioactive leucine, be used to monitor them during their antibodies are now widely used in many biological fields (16). immunoreactive 59-kDa BSP was detected in their conditioned medium by fluorography. Several charac- An advantage in use of monoclonal antibody directed against a certain protein is that the antigen need not necessarily be teristics, including the amino acid sequence, suggest that 59-kDa BSP may bethe rat counterpart of human homogeneous because the hybridomas can be screened in a2-HSG. However,rat 59-kDa BSPis a single peptide various ways, such as by immunoblotting. In this study, we and synthesized by both osteoblasts and hepatocytes, first immunized mice with partially purified noncollagenous whereas humana2-HSG is known to bea heterodimer sialoprotein(s) from rat mandibulae and raised a monoclonal antibody that was specific for a sialic acid-containing M , = * This work was supported in part by Grant-in-Aid for Scientific 59,000 protein (59-kDa bone sialoprotein (BSP)’) and for the Research 01480435 from the Ministry of Education, Science, and Culture of Japan. Thecosts of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed Dept. of Biochemistry, Kagoshima University Dental School, 35-1 Sakuragaoka 8, Kagoshima 890,Japan. Tel.: 992-64-2211(ext. 5230);Fax: 992-645738.

The abbreviations used are: BSP, bone sialoprotein; az-HSG, (YZ-

HS glycoprotein; CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol-1-propanesulfonate; MEM, minimum Eagle’s medium; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PBS, Dulbecco’s phosphate buffered saline; PBS(-), Ca2+-and Mg‘“free PBS;SDS, sodium dodecyl sulfate; TFMS, trifluoromethanesulfonic acid.

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of a Bone Sialoprotein

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(p-amidinopheny1)methanesulfonylfluoride hydrochloride as protease inhibitors.After removal of the teeth, the bone was broken into small pieces with a pestle and mortar, and soaked for 4 days in 3 liters of 4 M guanidine HCl (pH 7.4) containing protease inhibitors. The insoluble material collected by filtration with nylon mesh was then placed in a dialysis bag and decalcified by dialysis for 4 days against 2 liters of 0.6 N HCl containing protease inhibitors with daily changing of the outer solution. The decalcified material in the dialysis bag was centrifuged at 12,000 X g for 20 min at 2 "C, and the precipitate was washed with 50 ml ofdistilled water by centrifugation. The combined supernatant was recentrifuged at 105,000 X g for 60 min at 2 "C, and the supernatantwas dialyzed thoroughly against 20 mM Tris-HC1 (pH 8.0) containing all the protease inhibitors described above, except benzamidine (buffer A) for 5 days with daily changing of the outer solution. Benzamidine was omitted from buffer A because it has high absorbance at 280 nm. crude noncollagenous tooth proteins were extracted from rat incisors by the same procedure. Purification of 59-kDa Bone Sialoprotein-The dialyzed material was applied to a column of Q-Sepharose (2.5 X 20 cm) equilibrated with buffer A. Materials were eluted with 500 ml of a linear gradient of 0-1.0 M NaCl in buffer A, and the absorbance of the eluate at 280 EXPERIMENTAL PROCEDURES nm was monitored. Fractions of5.4mlwere collected and their Materials-~-[4,5-~H]Leucine (2.22 TBq/mmol) and carrier-free contents of sialic acid and organic phosphate were measured as (:"S)sulfuric acid were obtained from American Radiolabeled Chem- described below. The sialic acid-enriched fractionscontaining no icals Inc., St. Louis, MO and Du Pont-New England Nuclear, respec- organic phosphate were combined, dialyzed thoroughly against distively. N-Ethylmaleimide, 6-amino-n-caproic acid, benzamidine hy- tilled water, and lyophilized. This partially purified bone matrix drochloride, N-acetylneuramic acid (sialic acid), Nonidet P-40, and protein(s) was used as antigen for preparation of a monoclonal guanidine hydrochloride were from Nacalai Tesque Inc., Kyoto, Ja- antibody as described below. pan; (p-amidinopheny1)methanesulfonylfluoride hydrochloride, AchFinal purification of 59-kDa BSP was achieved by reverse phase romobacter proteinase I (lysyl bond specific proteinase (EC high performance liquid chromatography (model CCPM, Tosoh 3.4.21.50)), acetonitrile (HPLC grade), trifluoroacetic acid, trifluoro- Corp., Tokyo, Japan) with a TSK-gel phenyl-5PW R P column (4.6 methanesulfonic acid (TFMS), anisole, and peroxidase-conjugated X 75 mm) connected with a TSK-gel guard column SW XL (7.5 X 75 rabbit anti-mouse IgG (Fc) antibody were from Wako Pure Chemical mm). A sample of 500 pgof the partially purified bone matrix Industrials, Osaka, Japan; mouse IgG, rabbit anti-rat serum albumin protein(s) described above was dissolved in 100 pl of 62.5 mM Trisantibody, and sheep anti-mouse IgG (IgG fraction) were from Cappel HCl (pH 6.8) containing 0.25% (w/v) CHAPS and3% (v/v) 2Organ Teknika Corp., West Chester, C A CHAPS was from Dojindo mercaptoethanol, and heated at 100 "C for 90 s. Then 100 p1 of this Laboratories, Kumamoto, Japan; gelatin (enzyme immunoassay pretreated sample was applied to the column, which had been equiligrade) and Tween-20 were from Nippon Bio-Rad Laboratories, To- brated with 5% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid kyo, Japan; type I collagen from rat tail was from Sigma; crystallized in water (solvent A). Materials were eluted with a linear gradient of bovine serum albumin was from Miles Scientific, Naperville, IL; 80% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid in water rabbit anti-human a,-HS glycoprotein antiserum was from Behring- (solvent B) by the following schedule: 0% solvent B for 10 min, 0werke AG, Marburg, Federal Republic of Germany; peptide-N4-(N- 43% solvent B for 5 min, 43-48% solvent B for 50 min, 48-100% acetyl-0-glucosaminy1)asparagineamidase (N-oligosaccharide glyco- solvent B for 10 min, and finally, 100% solvent B for 5 min at a flow peptidase, EC 3.5.1.52) was from Genzyme Corp., Boston, MA;Qrate of 0.7 ml/min at room temperature. The absorbance at 214 nm Sepharose and protein A-Sepharose CL-4B were from Pharmacia was monitored, and each peak was collected and analyzed by Western LKB Biotechnology, Uppsala, Sweden; TSK-gel columns for high blotting followed by sodium dodecyl sulfate-polyacrylamide gel elecperformance liquid chromatography (HPLC) were from Tosoh Corp., trophoresis (SDS-PAGE). Then fractions containing 59-kDa BSP Tokyo, Japan; semifetal calf serum (semi-FCS) was from Mitsubishi werepooled and dried under reduced pressure with a centrifugal Kasei Corp., Tokyo, Japan; minimum Eagle's medium (MEM) and concentrator (model CC-100, Tomy Seiko Co., Tokyo, Japan). leucine-free MEM were from Nissui Pharmaceutical Co. Ltd., Tokyo, Quantitative Amino Acid Analysis and Deglycosylation of 59-kDa Japan; trypsin was from Difco Labs., Detroit, MI; and x-ray film for BSP-Purified 59-kDa BSP (100 pg) was hydrolyzed with 6 N HC1 fluorography was from Konica Corp., Tokyo, Japan. Zysorbin (fixed at 110"C for 24 h, and amino acid analysis was performed in an and killed Staphylococcus aureus) and a kit for isotyping mouse amino acid analyzer (model 655, Hitachi, Tokyo, Japan) with chlormonoclonal antibody (Mouse Mono Ab-ID/EIA) were from Zymed amine T, o-phthalaldehyde, and 2-mercaptoethanol, as described Labs. Inc., South San Francisco, CA; a kit for Vectastain ABC was previously (21). from Vector Labs. Inc., Burlingame, CA; and a kit for silver-staining Enzymatic deglycosylation of 59-kDa BSP was performed as sugwas from Kanto Chemical Co., Inc., Tokyo, Japan. Other reagents gested by the manufacturer of N-oligosaccharide glycopeptidase. for preparation of monoclonal antibody and of rat hepatocytes in Briefly, a sample of 200 pg (dry weight) of purified 59-kDa BSP was primary culture and for electrophoresis, amino acid analysis, and dissolved in 100 pl of20 mM sodium phosphate buffer (pH 8.6) histochemical studies were obtained as described previously (21-23). containing 1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol. The Tissue Preparation and Extraction-Mandibulae were quickly ex- sample was boiled for 3 min, 900 pl of sodium phosphate buffer (pH cised under light anesthesia from Sprague-Dawley rats (Clea Japan, 8.6) containing 1.4% (v/v) Nonidet P-40and 1unit of N-oligosacchaInc., Tokyo, Japan) weighing about 200 g and were kept a t -20 "C ride glycopeptidase was added, and the mixture was incubated at until use. About 120 g (wet weight) of manidbulae obtained from 200 37 "C for 18 h. rats was used as starting material for a typical preparation. Chemical deglycosylation with TFMS was performed as described Crude noncollagenous bone matrix proteins were extracted from by Edge et al. (24). A sample of 500 pg (dry weight) of purified 59the mandibulae under conditions similar to those describedpreviously kDa BSP was dissolved in 100 r l of TFMS/anisole (2:l by volume) (22). Unless otherwise noted, all procedures were carried out at 4 'C mixture and incubated at 25 "C for 5 h. The deglycosylated protein or on ice. Briefly, the mandibulae were separated from surrounding was freed from the solvent in a stream of dried N, gas and low soft tissue and soaked for 2 days in 1 liter of 4 M guanidine HCl molecular weight sugars wereremoved by precipitation with 10% (adjusted to pH 7.4 with NaOH) containing 50 mM 6-aminocaproic (v/v) diethylether in n-hexane. acid, 2.5 mM benzamidine HCl, 0.5 mM N-ethylmaleimide, and 3 p~ Preparation of Monoclonal Antibodies against 59-kDa ESP-Partially purified 59-kDa BSP obtained by Q-Sepharose column chro'The nomenclature for a2-HS glycoprotein follows the original matography as described above was used as antigen. BALB/c mice proposal by Schultze et al. (57), referring to its electrophoretic mo- (Clea Japan, Inc., Tokyo, Japan) were immunized subcutaneously bility (ao)and the initials of its original codiscoverers (H. for Here- with 200 pg ofthe antigen in 0.25 ml of Dulbecco's phosphate-buffered mans (56) and s. for Schmid (52)). saline (PBS) emulsified with an equal volume of Freund's incomplete

mineralized tissues bone and dentin. Then we purified this protein using the monoclonal antibody as a specific marker of the protein. Amino acid sequence analyses of the aminoterminal and four peptide fragments generated of this sialoprotein revealed that about 50% of the amino acids were homologous with those of human a2-HS glycoprotein' (apHSG). In this paper, we also report that 59-kDa BSP was synthesized and secreted by both rat osteoblast-rich cells and hepatocytes in primary culture. a2-HSGis a major human plasma sialoprotein (17), a member of the cystatin superfamily (18), and is thought to be involved in many biological processes (17), including bone formation and resorption (19, 20). But, to our knowledge, no protein(s) related to human a2-HSG has been isolated from experimental animals. Rat 59-kDa BSP and the monoclonal antibody tothis protein may be useful for study of the biological role of a2-HSG.

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adjuvant. 2 and 4 weeks later, booster injections of 200 pg of antigen in 0.25mlof PBS with Freund's complete adjuvant weregiven intraperitoneally. Cell fusion and cloning of the hybridomas were performed essentially as described previously (21). Briefly, spleen cells were collected 4 days after the final immunization and fused with NS-I mouse myeloma line cells using 42.3% polyethyleneglycol and 5% dimethyl sulfoxide in water. The hybridized cells were cultured above a macrophage feeder layer, and an aliquot of the culture medium wasscreened for anti-59-kDa BSP immunoglobulin by Western blot analysis and immunohistochemically as described below. A hybridoma producing antibody to 59-kDa BSP was then cloned. A dose of lo7 log phase hybridoma cells was injected intraperitoneally into syngeneic mice "primed" as described previously (21), and ascites fluid was collected 3 weeks later and mixed with an equal volume of PBS. Then the antibody was purified on a column (2.5 X 30 cm) of protein A-Sepharose CL-4B, according to the procedure suggested by the manufacturer. Fractions of eluate containing immunoglobulins were combined and stored at -40 "C. In this way, 35 mg of purified immunoglobulin was obtained from 1 ml of ascites fluid, assuming that the extinction coefficient (1%w/v, 1cm) was 14 a t 280 nm (25). Gel Electrophoresis-SDS-PAGE was performed essentially as described by Laemmli (26) in 4-20% linear gradient separating gel of 1-mm thickness with 3% stacking gel. The running conditions were as described previously (21), and gels were stained with silver with a staining kit as originally described by Wray et al. (27). The molecular weight standards used for SDS-PAGE were myosin (Mr = 200,000), @-galactosidase(116,000), phosphorylase b (97,400), bovine serum albumin (66,000), ovalbumin (45,000), carbonic anhydrase (31,000), and lysozyme (14,400). Isoelectrophoresis was performed using a PhastSystem'" (Pharmacia LKB Biotechnology, Uppsala, Sweden) on precast PhastGel'" IEF 3-9. The procedure suggested by the manufacturer was followed, and thegels were routinely stained with silver as described above. Two-dimensional gel electrophoresis was performed as originally described by Martini and Gould (28). Briefly, samples were separated by 8%PAGE without SDS in the first dimension, and the gel strip was equilibrated with SDS-PAGE sample buffer (26) containing 3% (v/v) 2-mercaptoethanol for 10 min at room temperature. The gel was then placed horizontally in contact with 3% stacking and 8% separating gel, and electrophoresed again in the second dimension. The gelswere either stained with silver or subjected to Western blotting as described below. Western Blot Analysis-After separation by SDS-PAGE on 4-20% polyacrylamide gel, proteins were transferred electrophoretically to a nitrocellulose membrane (Nippon Bio-Rad)in 192 mM Tris-HC1 buffer (pH 7.0) overnight at 0.09 A. The membrane was blocked with 3% gelatin in Ca2+-and Mg2+-freePBS (PBS(-)) and then treated with anti-59-kDa BSP monoclonal antibody. It was then washed with PBS(-) containing 0.05% Tween-20, and antibody specifically bound to 59-kDa BSP was located using a Vectastain ABC kit essentially as described previously (21). Enzyme-linked Immunosorbent Assay (ELISA) for 59-kDa BSPThe ELISA for 59-kDa BSP is a competitive binding method with the use of immobilized antigen on solid phase (29). Purified 59-kDa BSP was used as standard. Samples to be tested and standard were suitably diluted with 50 mM sodium carbonate-bicarbonate buffer (pH 9.6) containing protease inhibitors as used for "tissue preparation and extraction," and then 100 pl of the diluted standards or samples were dispensed into 96-well microtiter plates (Corning Lab. Sciences, Corning, NY). The plates were incubated for 1h at room temperature. The sample solution wasremoved, the wellswere washed with PBS(-) containing 0.05% (v/v) Tween-20 (washing solution), and then 0.2mlof 3% (w/v) gelatin in PBS(-) was added for blocking. After standing for 1 h at room temperature, the wells were washed three times with the washing solution and then received 100 p1 (-10 ng) of purified anti-59-kDa BSP monoclonal antibody in PBS(-) containing 1%gelatin and 0.05% Tween-20. The plate was incubated a t 37 "C for 1h. It was washed three times with the washing solution, and then theantibody bound specifically to the antigen was quantitated using a Vectastain ABC kit for mouse IgG. The color development was performed as follows. After addition of 0.1 ml of 0.4 mg/ml o-phenylenediamine dihydrochloride and 0.015% hydrogen peroxide in 0.1 M citric acid/Na,HPO, buffer (pH 5.0), the plate was incubated for 10 minat room temperature. The reaction was stopped by addition of 50 p1 of 1 N H2S04.Absorbance values were measured by a twowavelength microplate reader (model MPR-A4i, Tosoh Corp., Tokyo, Japan) at492 nm, with a reference wavelength of 630 nm. The control

of a Sialoprotein Bone well received 100 pl of the same solvent used for dilution of samples and was then treatedin the same way as described above. The sensitivity of this ELISA for 59-kDa BSP was 20-2,000 ng/ml (2200 ng/well). Limited Digestion and Amino Acid Sequence Analysis of Peptide Fragments of 59-kDa BSP-Approximately 1 nmol of purified 59kDa BSP was dissolved in 200 p1 of 50 mM Tris-HC1 buffer (pH 9.0) containing 4 M urea and digested with Achromobacter proteinase I at a molar ratio to 59-kDa BSP of 1:200 at 37 "C for 6 h. The reaction was terminated by additions of final concentrations of 1% (v/v) formic acid and 0.1% (v/v), 2-mercaptoethanol. Then the mixture was fractionated by reverse-phase HPLC on a Cosmosil5C18 column (4.6 X 250 mm) at a flow rate of1.0 ml/min at room temperature. The eluants used were the same as those described above for purification of 59-kDa BSP,and materials were eluted with a linear gradient by the following schedule: 0% solvent B for 5 min, 0-25% solvent B for 5 min, 25-65% solvent B for 50 min, 65-100% solvent B for 20 min, and finally, 100% solvent B for 5 min. Absorbance at 214 nm was monitored, and fractions in peaks were collected and concentrated with a centrifugal concentrator. Amino acid sequence analyses were performed with 300-700 pmol of purified 59-kDa BSP, and peptide fragments were obtained as described above in a gas-phase sequencer (model 477A connected to a model120A phenylthiohydantoinamino acid analyzer, Applied Biosystems, Inc., Foster City, CA), as described by Hunkapiller et al. (30). Preparation of Organ Cultures and Cells in Primary CultureOsteoblast-rich cells were prepared from newborn rat calvaria by digestions with collagenase and trypsin, as described by Martin et al. (31). Osteoblast-richpopulations were combined and plated into 12well plastic dishes (Costar Corp., Cambridge, MA) of 2.2-cm diameter MEM at an initial density of 1 X lo5 cells/ml/cm* in 0.75mlof containing 10% semi-FCS. Osteoblast-rich cells were then cultured to confluency, which took 4 days. Organ culture of calvaria from newborn rats was performed as follows. The periosteum was removed and thetissue was incubated in MEM containing 10% semi-FCS plus antibiotics, as originally described by Dietrich et al. (32). Primary cultures of adult rat hepatocytes were prepared as described previously (23). The cells were suspended in Williams medium E supplemented with 5% FCS plus dexamethasone and antibiotics (23) and were plated (0.75 ml/well) in the dishes described above at a cell density of 1.0 X lo5cells/0.2 ml/cm2. Four hours after seeding, the medium was changed to Williams medium E as described above except for serum, which was 10% semi-FCS. The cells were incubated for an additional 24 h before use. The cells and calvaria were maintained at 37 "C in a humidified atmosphere of 5% CO, in air. Immunoprecipitation of 59-kDa BSP-Before experiments, the cells and calvaria were preincubated in leucine-free MEM supplemented with 10% semi-FCS plus 10 p~ L-leucine for 1 h. For hepatocytes, the preincubation medium described above was supplemented with 1 p~ dexamethasone (28 h after seeding). Incubations were started by changing to thesame medium containing [3H]leucine at 3.7 MBq/ml or ("S)sulfate a t 1.85 MBq/ml, and aliquots (0.6 ml) of conditioned medium were collected at the indicated times. The remaining cells were washed three times with cold PBS containing 10 p~ L-leucine and solubilized in 1%Triton X-100. Immunoprecipitation was carried out as described by Sambrook et al. (33). Briefly, the medium or solubilized cells were mixed with purified anti-59-kDa BSP monoclonal antibody and stood overnight at 4 "C. After addition of anti-mouse IgG, the mixture was incubated at 37 "C for 2 h. Then the immunocomplex was precipitated by addition of70 r l of 10% (w/v) Zysorbin in PBS. The immunoprecipitate with anti-59-kDa BSP monoclonal antibody was washed twice with 20 mM Tris-HC1 buffer (pH 7.4) containing detergents (33) by centrifugation and solubilized in "SDS-gel-loading buffer" (33) by boiling. Aliquots of the solution were subjected to fluorography followedby enhanced SDS-PAGE using an autoradiography enhancer (Du Pont-New England Nuclear). The radioactivity of immunoprecipitates was determined by scanning the exposed film with a densitometer (model CS910, Shimazu Corp., Kyoto, Japan) and was expressed in arbitrary units per microgram of DNA. Cellular DNA was measured by the diphenylamine method described by Schneider (34). For measurement of the rateof total protein synthesis, conditioned medium obtained as described above wastreated with trichloroacetic acid at a final concentration of 10%. The samples stood for 30 min at 0 "C, and then insoluble material was collected by centrifugation at 8,000 X g for 5 min at 4 "C and washed in succession twice with 0.75 ml of ice-cold 10%trichloroacetic acid containing 10 p M I-leucine,

Purification and Characterization of a Bone Sialoprotein twice with 0.75 ml of HC1-acetone (0.5 ml of 12 N HCI in 100 ml of acetone), and twice with 0.75 ml of acetone. Then it was dried under reduced pressure, and the residue was solubilized in 0.3 mlof 4 N NaOH and neutralized with 4 N HCl. The radioactivity of the solution was then measured in a liquid scintillation counter(model LSC 903S, Aloka Corp., Tokyo, Japan). ImmunohistochemicalAnalysis-Femora were quickly excised from 3 week-old Sprague-Dawley rats under anesthesia, fixed with 4% paraformaldehyde in PBS(-) for 12 h at room temperature, and decalcified by treatment with Plank-Rychlo solution (35) overnight at 4 “C. Then they were neutralized and washed for 12 h a t 4 “C with several changes of PBS, as described previously (21). The diaphyseal portion of the femur was cut away, and the epiphyseal portion was dehydrated in an alcohol series, embedded in paraffin, and sectioned a t 4 - ~ mthickness. Endogenous peroxidase was inactivated as described previously (22). The sections were blocked with 3% gelatin in PBS for 20 min, placed in a solution of anti-59-kDa BSP monoclonal antibody diluted 1:5,000 (v/v) with PBS containing 1.5% gelatin overnight at room temperature in a moist chamber, and then stained immunohistochemically with peroxidase-conjugated anti-mouse IgG antibody as thesecond antibody. After color development as described previously (22), sections were counterstained with methyl green for 3 h. Control sections were treated in the same way, except that thefirst antibodies used were 1) mouse IgG, 2) ascites from mice inoculated with NS-I myeloma cells, 3) 1.5% gelatin in PBS, and 4) purified anti-59-kDa BSP monoclonal antibody that had been treated with excess purified 59-kDa BSP. All sections were examined by light microscopy. Other Assays-For determination of organic phosphate, samples were hydrolyzed with 0.2 N NaOH for 24 h at 37 “C. The resulting inorganic phosphate was assayed by the method of Fiske and Subbarow, as described by Gerlach and Hiby (36). Protein was assayed by the method of Lowry et al. (37), with crystallized bovine serum albumin as standard. Sialic acid was assayed by the method described by Jourdian et al. (38), and concentrations of NaCl were determined by measurement of conductivity. RESULTS

Preparation of Antigen and Characterization of a Monoclonal Antibody to 59-kDa BSP-A typical Q-Sepharose elution profile of proteins extracted from rat mandibulae is shown in Fig. 1. Two major peaks containing sialic acid were eluted with about 0.2 M (peak I) and 0.45 M (peak 11) NaCl at pH 8.0. Peak I contained sialic acid, but its phosphate content was below the limit of detection. The molecular weight of the major component in peak I appeared to be about 59,000 on SDS-PAGE under reducing conditions, but anumber of minor bands were seen by silver staining (Fig. 2). Since we wanted to purify 59-kDa BSP containing no phosphorus, we next prepared a monoclonal antibody to the protein using this FIG. 1. Chromatographic profile of extract from decalcified mandibulae on Q-Sepharose. An extract from about 120 g (wet weight) of rat mandibulae was applied to a column of Q-Sepharose (2.5 X 20 cm) equilibrated with 20 mM Tris-HC1 (pH 8.0) containing protease inhibitors, as described under “Experimental Procedures,” and materials were eluated with 500 ml of a linear gradient of0-1.0 M NaCl in the same buffer. Aliquots of each fraction (5.4 ml) were used for measurement of the absorbance at 280 nm (0)and of the content of sialic acid (0)and organic phosphate (columns). The salt gradient (-- -) was measured with a conductivity meter. The fractions under the bar of peak I were pooled, dialyzed, lyophilized, and used as partially purified 59-kDa BSP. Other experimental conditions were as described under “Experimental Procedures.”

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partially purified preparation (peak I) as anantigen. We obtained four clones (1-A, 4-A,4-C, and6-C) that produced a monoclonal antibody that specifically recognized 59-kDa BSP transblotted from either crude extract of bones or peak I from a Q-Sepharose column onto nitrocellulose membranes (Fig. 2). Immunohistochemical examination showed that these antibodies were also specific to mineralized tissues, bone and dentin, asdescribed below. Characterization of these monoclonal antibodies revealed that all four had the same types of heavy and light chains, IgGl and K , respectively, and showed no cross-reactivity with rat serum albumin or rat type I collagen (data not shown). Thus, these four antibodies seemed to be the same, and we used the monoclonal antibody 6-C in further studies. By an ELISA for 59-kDa BSP, fractions in the peak I1 shown in Fig. 1contained asmall amount of 59-kDa BSP, but more than 95% of this sialoprotein was eluted in peak I (data not shown). Next, we examined the tissue distribution of the protein(s) recognized by the antibody. Fig. 3, A-D, shows decalcified sections of the epiphysial part of rat femur stained immunohistochemically with the purified monoclonal antibody. The region of endochondral ossification was stained immunohistochemically, butthe cartilage and periosteum were not stained (Fig. 3B). Fig. 3,C and D,shows higher magnifications of portions close to, and far from, the cartilage, respectively, of the section in Fig.3B; osteoblasts and osteocytes were stained intensely and the bone matrix was slightly stained, butthe cartilage matrix between the trabeculae was not stained. In acontrol section (Fig. 3A) treated in the same way but with control ascites from mice inoculated with NS-I cells as the first antibody, none of these structures were stained. The three othercontrol procedures described under “Experimental Procedures” also resulted in no staining (figures not shown). No otherrat tissues, such asthe liver, kidney, spleen, muscle, or mucous membranes, including gingival tissue, were stained with the antibody, but dentin was stained immunohistochemically (data not shown). Western blot analysis of extracts from rat teeth and ratserum showed that these two tissues contained proteinrecognized by the antibody (Fig. 4). The molecular weight of the protein from both tissues was estimated as 59,000 in the presence of SDS under reducing conditions. However, when rat serum was subjected to PAGE without SDS under nonreducing conditions, immunoreactive 59-kDa BSP migrated in a position of much higher molecular

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Purification Characterization and

14640 1

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of a Sialoprotein Bone

7

200k.

116k. 97k.

45k 31 k14k-

FIG. 2. SDS-PAGE and Western blot analysis of crude extract from rat mandibulae and partially purified 59-kDa BSP with four monoclonal antibodies from cloned hybridomas. Samples (2.5 pg, dry weight, for lanes 1-3 and 250 ng for lanes 4-7) of crude extract from rat mandibulae (lanes 1 and 3) and 59-kDa BSP partially purified .on Q-Sepharose (lanes 2 and 4-7) were separated by SDS-PAGE (4-20%), and lanes l and 2 were stained with silver. Lanes 3-7 were transferred electrophoretically to a nitrocellulose membrane and then stained immunochemically with purified monoclonal antibody (lanes 3 and 7, clone 6-C; lane 4 , clone I-A; lane 5,clone 4-A; and lane 6,clone 4-C) as the firstantibody by the ABC method (21). Molecular masses of marker proteins are indicated in daltons on the left.

mass (>200 kDa), suggesting that 59-kDa BSP in serum was present ina multiunit form or was bound to some other protein(s) such as albumin. The results described above indicate that our monoclonal antibody was specific for 59-kDa BSP, and that thisprotein is one of the main noncollagenous proteins inbone tissue. Further Purification and Characterizationof 59-kDa BSPWhen the peak I fraction from the Q-Sepharose column was subjected to PAGE without SDS (native PAGE) under nonreducing conditions, 6 major bands with R, values of 0.130.53were detectable by silver staining (Fig. 5A) and, after transblotting to a nitrocellulose membrane, all these bands showed reactivity with both anti-59-kDa BSP and anti-rat serum albumin antibodies (data not shown). However, when these six bands were subjected to SDS-PAGE in the second dimension under reducing conditions, they all converged to two bands with M , = 66,000 and 59,000 (Fig. 5B), of which the former reacted only with anti-rat serum albumin antibody and the latteronly with anti-59-kDa BSP antibody (data not shown). These results indicated that 59-kDa BSP molecules in peak I from the Q-Sepharose column associated with each other, presumably by disulfide bonds, and also boundto serum albumin. Therefore, we pretreated the sample with CHAPS and 2-mercaptoethanol and purified 59-kDa BSP further by HPLC with anti-59-kDa BSP antibody as a marker. As shown in Fig. 6, on HPLC on a TSK-gel phenyl-5PW RP column, the sample gave several peaks. The fraction eluted withabout 40% acetonitrile and 0.1% trifluoroacetic acid in water was found to contain 59-kDa BSP recognized by anti-59-kDa BSP monoclonal antibody but not anti-rat serum albumin antibody. The chromatographic profiles on SDS-PAGE and Western blot analysis of the final preparation areshown in Fig. 7. Crude extract from 120 g (wet weight) of mandibulae contained 751 mg of total protein and 111mg of 59-kDa BSP by an ELISA for this sialoprotein, and we obtained about 20 mg

FIG. 3. Photomicrographs of decalcified sections of t h e epiphyseal portionof rat femur stained by the immunohistochemical method and counterstained. A, control staining with mouse ascites. R,staining with a monoclonal antibody against 59-kDa BSP, showing positive staining of bone tissue. C and D,portions of I? at higher magnification. Note that osteoblasts (arrows),osteocytes (arrowheads) and bone matrix ( R m )are positively stained. B, bone; c, cartilage; P, periosteum. Magnification, X94 in A and R, and X375 in C and D.

Purification and Characterization of a Bone Sialoprotein 1

2

3

14641

4

200k-

116k97k66k45k31 k14k.

FIG. 4. Presence of immunoreactive 59-kDa BSP in rat tooth and serum. Aliquots (1 pg, dry weight) of rat tooth extract (lanes I and 2 ) or 10 pl of diluted (1:500) rat serum (lanes3 and 4 ) were separated by SDS-PAGE (4-20%). Then even-numbered lanes were electroblotted onto a nitrocellulose membrane, and odd-numberedlanes were stained withsilver.Even-numbered lanes were stained immunochemically with monoclonal antibody against 59-kDa BSP, as described in the legend for Fig. 2. Molecular masses of marker proteins are indicated in daltons on the left. 1st dimension

:rigin

A

kip

BP

-

front

Time (min)

FIG. 6. Purification of59-kDa BSPby HPLC. After pretreatment with CHAPS and 2-mercaptoethanol, the sample(500 pg, dry weight) of partially purified 59-kDa BSP was loaded on a TSK-gel SW XL column (7.5 X 75 mm) connected with a TSK-gel phenyl5PW RP column (4.6 X 75 mm) equilibrated with 5% acetonitrile and 0.1% trifluoroacetic acid in water (solvent A). Materials were eluted with a linear gradient of 5-80% acetonitrile in 0.1% trifluoroacetic acid in water (solvent B) using a gradient programmer. Solid and dotted lines represent the absorbancea t 214 nm and the percentage of solvent B in solventA, respectively. Material in fractions under the bar was found t o contain 59-kDa BSP. Details of other experimentalconditions were as described under“ExperimentalProcedures.”

h) 3

P

:

(D

3

E. 0

66k59k-

3

I

FIG.5. Two-dimensional PAGE of partially purified 59kDa BSP. A, a sample (10 pg, dryweight)obtained froma QSepharose column was separated by 8% PAGE without SDS or 2mercaptoethanol and stained withsilver. R, the resulting gel was resubjected to SDS-PAGE (4-20%) under reducing conditions and then stained with silver. Details of other experimental conditions were as described under“Experimental Procedures.”Molecular masses of marker proteins are indicatedin daltons on the left.

200k

-

116k97k66 k45k31 k-

14k(dry weight) of purified 59-kDa BSP from 120 g (wet weight) of rat mandibulae; the yield was about 18%. FIG. 7. SDS-PAGE and Western blot analysis of purified Purified 59-kDa BSP that had been enzymatically deglycosylated with N-oligosaccharide glycopeptidase gave a single 59-kDa BSP. The final preparation of 59-kDa BSP (2.5 pg, dry weight) obtained by HPLC (fractions under the bar in Fig. 6) was band with a M , 45,000 on SDS-PAGE detected by staining separated by SDS-PAGE (4-20%). Lane 1, silver-staining. Lanes 2 with Coomassie Brilliant Blue. This deglycosylated band re- and 3 were electroblotted ontoa nitrocellulose membrane and stained acted with the antibody (data not shown), suggesting that the immunochemically with anti-59-kDa BSPmonoclonal antibody (lane N-glycosylated carbohydrate chain(s) of this protein does not 2) or anti-rat serum albuminpolyclonal antibody ( l a m 3 ) . Molecular contribute to the epitope for the antibody. The isoelectric masses of marker proteins are indicated in daltons on the left. point of purified 59-kDa BSP was calculated to be 4.8 on a precast PhastGel IEF (data not shown). Thus, the 59-kDa fragments obtained by limited digestion of 59-kDa BSP with BSP is an acidic glycoprotein with an N-glycosylated carbo- Achromobacter proteinase I. As shown in Fig. 8, about 50% of hydrate content of about 24%. its amino acids were homologous with those of human 02The amino acid composition and the contentof sialic acid HSG. In addition, anti-rat 59-kDa BSP antibody showed weak and phosphate in purified 59-kDa BSP were compared with cross-reactivity with normal human plasma, which is known those of other known bone sialoproteins of M , = 50,000- to containa2-HSG by immunoblotting followedby SDS70,000 (Table I). The 59-kDa BSP seemed to be very similar PAGE under reducing conditions, and a band appeareda t M , to human an-HSG (18)and rat64-kDa protein (9). The amino= 48,000, which corresponds to theA (heavy)chain of human acid sequence of the rat 64-kDa protein was not known, but a2-HSG (data notshown), suggesting that theepitope of our that of human a,-HSG has been reported (18,39). Therefore, anti-59-kDa BSP monoclonal antibody would bethe sequence we determined the amino-terminal amino acid sequence of corresponding to the A chain of a2-HSG. However, antithe purified 59-kDa BSP and the sequences of four peptide human a2-HSG antiserumfailed to show the cross-reactivity

-

Purification Characterization and

14642

TABLE I Amino acid compositions and contents of phosphate and sialic acid i n 59-kDa BSP and other known sialoproteinsisolated from bone Human

64-kDa proteinb

32

Asxd Thr Ser Glxd Pro G~Y Ala CY5 Val Met Ile Leu TYr Phe LYS His Arg

57

63

89 56 38 117 93 86 106 6 107

-

29

Phosphate

1.4 Sialic acid

29 82 14 49 42 46 40

ND'

**

(m)APHGsLIYRQPNCDDPEI

az-HS glycoprotein'

residues/1000 amino acids

57

(m)AXEGAGLGFXQW(NNP

N

Rat 59-kDa BSP"

of a Bone Sialoprotein

97 45 121 98 79 120 -

86 -

86 16 46 41 28 48 % of dry weight

0.05

86 57

XLLQGFRQILNQIDK

*

*****

-QNLPWGYKEITLNQIDEVI U 2

115 112

TWNAQNXGTYFK

********* * *

"NAQNNGSNFQL-

103 35 98 3 83 20 46 35 35

I A147

ATLIQFLGGQQVSXAX

***

I A213

VGEPGDAGAAGPVAPLXPGRVR

*

'Data from this paper. I, See Ref. 9.

Calculated from the amino acid sequences of the A plus B chains reported by Kellermann et a/. (18) because rat 59-kDa BSP contains both A- and B-like chains of az-HSG in a single peptide, as described in the "Discussion." See Refs. 18 and 52. Asx and Glx represent Asp plus Asn and Glu plus Gln, respectively. '' Not determined. Not detected.

with purified rat 59-kDa BSP by Western blot analysis. A computer-aided homology search employing the data base of Swiss-PROT (European Molecular Biology Laboratory) revealed no significant similarity with any otherknown protein sequences except rat collagen a-1 (I) whichshowed some similarity because 59-kDa BSP contains collagen-like sequences, as discussed below. Biosynthesis of 59-kDa BSP-The results described above suggest that 59-kDa BSP is likely to be the rat counterpart of human a2-HSG. Human a2-HSG is known to be a plasma protein (17),which is synthesized by the liver (40) and incorporated into the matrix of calcified tissues (41, 42). But in rats, not only the bone matrix, butalso osteoblasts, are stained immunohistochemically with the anti-59-kDa BSP monoclonal antibody (Fig. 3), suggesting that osteoblasts also produce this protein. Therefore, we next examined whether the liver and bone tissues of rats actually synthesize 59-kDa BSP. In this experiment, we used cell cultures of hepatocytes from adult rats and organ cultures of newborn rat calvaria and determined the molecular weight and radioactivity of the specific immunoprecipitate with 59-kDa BSP monoclonal antibody in the conditioned media by fluorography followed by SDS-PAGE. When hepatocytes were labeled with [3H]leucine, the immunocomplexesgave a major band of M , = 59,000, with a minor band of about M , = 235,000 under reducing conditions (Fig. 9A). In contrast, when calvaria were labeled with radioactive leucine, the major band of immunocomplexes appeared at M , = 235,000 with a minor band at M , = 176,000, but no band was seen at M, = 59,000 under reducing conditions (Fig. 9A). Conditioned medium of an osteoblast-rich population from newborn rat calvaria in primary culture gave similar results to thatof medium of organ cultures of calvaria. During

***** *

*** *

(h'K-ITWQPSVGAAAGPWPPCPGRIRH-

'

1.4

*** *

-KATLSEKLGGAEVAVTCT-

B1

0

t

t

FIG. 8. Amino acid sequence similarities of rat 59-kDa BSP and human (r2-HSG. The upper linesshow sequences of the aminoterminal and four peptide fragments generated from rat 59-kDa BSP, and the lower lines show the corresponding sequences of human a2HSG deduced from that of its cDNA (39), which has been confirmed by sequencing the protein (18). Amino acids are expressed by the standard one-letter code, and matched amino acids are shown by asterisks (*). Numbers indicate relative positions of the amino acid residues from the amino terminiof the A (heavy) and B (light) chains of maturehuman a,-HSG. X , amino acids not identified in the automated aminoacid sequencer, some of which could behalf-cystine (C) residues or glycosylated amino acids, ( N H , ) , amino-termini of the proteins or subunits; arrows, carbohydrate attachment sites of collagen-like sehuman aZ-HSG (54, 55); underlinedsequences, quences, Gly(G)-Pro(P)-X,in human aZ-HSG.

purification of 59-kDa BSP, however, we treated ratmandible with 0.6 N HC1 for 4 days for extraction of noncollagenous proteins and obtained a sialoprotein of M, = 59,000. Therefore, we dialyzed the conditioned medium from osteoblasts against 0.6 N HCl for 2 days and againstPBS (bothcontained protease inhibitors as for extraction of tissues) for an additional 2 days at 4 "C before preparing the immunoprecipitation by the same procedure as used for Fig. 9A. After pretreatment with HC1, the radioactive band of immunoprecipitate migrated to M, = 59,000 under bothnonreducing and reducing conditions (Fig. 9B, lanes 3 and 4 ) . However, without acid pretreatment, the M , of the immunocomplex from osteoblasts was 282,000, and again, no band was seen at M, = 59,000 under nonreducing conditions (Fig. 9B, lane 1). These findings suggest that three to four 59-kDa BSP molecules bound to each other weakly before or after their secretion from osteoblasts and that themolecules also bound to some other protein(s) through disulfide a bond(s). Solubilizedhepatocytes and osteoblast-rich cell layers, and an extract of decalcified calvaria with 0.6 N HC1 did not contain anydetectable radioactive material immunoprecipitated with anti-59-kDa BSP. Conditioned medium not immunoprecipitated with anti-59kDa BSP and zero time control media also did not give any detectable bands on fluorograms (data not shown). 59-kDa BSP might be a proteoglycan of the bone matrix because a proteoglycan of about M, = 200,000 has been isolated from the bone (43, 44) and 59-kDa BSP contains sialic acid. Therefore, we examined (35S)sulfateincorporation into thematerial immunoprecipitated with anti-59-kDa BSP antibody from the conditioned medium of the osteoblast-rich

Purification and Characterization of a Bone Sialoprotein

14643

activity in uitro. In uitro immunization (48) should also be a useful technique for preparing monoclonal antibody when the amount of antigen available is not sufficient for in uiuo immunization. Because this 59-kDa BSP seemed to readily form multiunit complexes and to bind to other proteins such as serum albu200k200kmin (Fig. 5), we pretreated the sample with CHAPS and 2mercaptoethanol before subjecting it to HPLC. During the 116k116kpurification of this protein, we also found that 59-kDa BSP 97k97 kshowed some affinity to hydrophilic bonded phase silica, the packing material used in the gel filtration column for HPLC. 66k66kTaking advantage of this affinity of the protein for the gel, I we used a guard column of TSK-gel SW XL just before the column for reverse phase HPLC (phenyl-5PW RP) and so 45 k45 kobtained high resolution of 59-kDa BSP (Fig. 6). Withoutuse of the guard column and pretreatment of the sample, the sialoprotein could not be separated from serum albumin. Immunohistochemical examination of the tissue distribution of 59-kDa BSP showed that this protein was specific to mineralized tissuesand plasma (Figs. 3and 4), and that FIG. 9. Biosynthesis of 59-kDa BSP by hepatocytes and osteoblasts, osteocytes, and thebone matrix were stained with osteoblast-rich cells in primary culture and an organ culture the monoclonal antibody but thatchondrocytes in the area of of calvaria from rats. Cells or calvaria were labeled for 24 h with either ["Hlleucine or [:"S]sulfate. Then the conditioned media were cartilage-to-bone transition were not stained(Fig. 3B). These immunoprecipitated with anti-59-kDa BSP antibody as described findings showed that 59-kDa BSP is not the same as the under "Experimental Procedures." The immunoprecipitates were sol- protein (MI = 59,000) containing sialic acid that has been ubilized and subjected to SDS-PAGE, and the radioactive materials isolated from bovine articular cartilage (49). Several other were located by fluorography. Panel A, all lanes were rununder noncollagenous bone proteins containingsialic acid and phosreducing conditions. The samples were conditioned medium from phate have been isolated from rats, namely osteopontin (bone [:'H]leucine-labeledhepatocytes (lane 1 ), ['H]leucine-labeled calvaria (lane 2), [:'H]leucine-labeled osteoblasts (lane 3 ) , and [:"S]sulfate- sialoprotein I, M , = 44,000) (9), bone acidic glycoprotein 75 labeled osteoblasts (lane 4 ) . Panel B, all samples were conditioned (Mr = 75,000) (ll),bone sialoprotein I1 (Mr= 67,000 (9) or medium from [''Hlleucine-labeled osteoblasts. SDS-PAGE was per- 33,600, according to theamino acid composition deduced from formed under nonreducing conditions (lanes 1 and 3 ) or reducing the cDNA sequence (50)), and 64-kDa protein (Mr = 64,000) conditions (lams 2 and 4 ) . For lam 3 and 4, the medium was dialyzed (9). Unlike these proteins, the purified 59-kDa BSP had against 0.6 N HCI for 2 days and then against PBS for 2 days a t 4 "C before immunoprecipitation. Molecular masses of marker proteins a phosphate content below the limit of detection (