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Changes in levels of osteonectin in bovine dentine during tooth development
Archs oral Bid Vol. 34,No. 2. pp. 89-92, 1989 Printed in Great Britain. All rights reserved
Copyright 0
0003s9969/89 $3.00+O.OO 1989 Pergamon Press plc
CHANGES IN LEVELS OF OSTEONECTIN IN BOVINE DENTINE DURING TOOTH DEVELOPMENT R. FUJISAWA and Y. KUBOKI Department of Biochemistry, School of Dentistry, Hokkaido University, Sapporo 060, Japan (Received I5 March 1988; accepted 19 August 1988) Summary-Bovine incisors were classified into three developmental stages and non-collagenous proteins extracted from them. Sodium dodecyl sulphate gel electrophoresis of the extracts showed a reduction in osteonectin with the various stages. The reduction was confirmed by enzyme immunoassay using antiserum against bone osteonectin. This change is in contrast to dentine phosphoprotein, indicating functional diflerences between these two proteins.
used for the comparisons. These incisors were freed of pulp, soft tissues and soft enamel, and then ground into powder. The powders were washed with 4 M guanidine hydrochloride (Gdn/HCl), 50 mM trisHCl, pH 7.4. Demineralization was carried out with 0.6 M HCl (1 litre/g of powdered teeth) at 4°C for 4 days (Tagaki and Veis, 1984). Completeness of demineralization was checked by measuring the calcium content of the demineralized matrix (less than 0.01 pg/mg of matrix). Non-collagenous proteins were extracted from the demineralized matrix residue with 4 M Gdn/HCl, 50 mM tris-HCl, pH 7.4 (40 ml/g of demineralized matrix) at 4°C for 4 days with agitation. Femurs of calves were demineralized and extracted by the same procedures.
Dentine contains several non-collagenous proteins, some of which are also present in bone (reviewed by
Butler, 1984). These proteins were studied intensively by Leaver and his co-workers (Jones and Leaver, 1974a, 1974b; Thomas and Leaver, 1975, 1977; Smith and Leaver, 1979) who classified them as anionic glycoproteins, less acidic glycoproteins and proteoglycans. Later investigators reported a less complex composition as precautions were taken to avoid proteolysis (Termine et al., 1980; Linde, Bhown and Butler, 1980; B’utler et al., 1981a, b). The most abundant non-collagenous protein in dentine is phosphophoryn, which is a highly acidic protein rich in phosphoserine and aspartic acid. Among the other non-collagenous proteins, Gla proteins (Linde ef al., 1982), dentine proteoglycan (Rahemtulla, Prince and Butler, 1984), 9.5kdalton glycoprotein (Butler et al., 198 la, b), osteonectm (Termine et al., 198 1a, b; Tung et al., 1985) and several serum proteins have been identified. The composition ‘ofthese non-collagenous proteins changes with the maturation of dentine; the amino acid composition of total extracts of non-collagenous proteins changes significantly (Smith and Leaver, 1981). There are qualitative (Fujisawa, Kuboki and Sasaki, 1985) and quantitative (Fujisawa and Kuboki, 1988) changes in phosphophoryn with dentine formation. We have now investigated quantitative developmental changes in dentine osteonectin.
Electrophoresis
Sodium dodecyl sulphate (SDS) gel electrophoresis was carried out according to Laemmli (1970), using 11.5% acrylamide gels. The first dimension of two-dimensional electrophoresis was isoelectric focusing (Wrigley, 1971) using 4% acrylamide gels containing 2% Pharmalyte 3-10 (Pharmacia) and 8 M urea. The second dimension was the SDS gel electrophoresis.
MATERIIALSAND METHODS Extraction of non-o~llagenous proteins
The extraction procedures were essentially those of Fujisawa and Kuboki (1988). First permanent incisors of 2-year-old (calves were classified into three development stages, stages I, II and III, before crown completion, just after crown completion, and near root completion, respectively (Fig. 1). As the composition of non-collagenous proteins differs between crown and root dentine (Takagi, Nagai and Sasaki, 1988), the root was removed and only the crown was
Stage II
Stage 111 Fig. 1. Classification of the bovine incisor into three developmental stages. 89
90
R. FUJISAWAand Y. KUBOKI
Immunochemical procedures
Bone osteonectin was purified from Gdn/HCl
ex-
tracts of demineralized calf femur (Sato et al., 1985). Fifty to eighty milligrammes of the extracts were applied to a Sepharose CL-6B (Pharmacia) column (2.6 x 100 cm) and eluted with 4 M Gdn/HCl, 50 mM
tris-HCl, pH 8.2, at a flow rate of 22 ml/h. The peak corresponding to osteonectin was further purified by DEAE-cellulose (Whatman DE-52, 2.5 x 1Ocm) chromatography and rechromatography, equilibrated with 6 M urea, 50 mM tris-HCl, pH 8.2, and eluted with a linear NaCl gradient up to 0.5 M over 600ml of the buffer at a flow rate of 80ml/h. Chromatography was performed at room temperature and monitored with absorption at 230 nm, and fractions of 5 ml were collected. Purity of the preparation was checked by two-dimensional electrophoresis. Antisera were raised against osteonectin by injecting rabbits subcutaneously with 500 pg of the osteonectin preparation mixed with Freund’s complete adjuvant. Injections of the mixture of osteonectin and Freund’s incomplete adjuvant were repeated five times at l-week intervals. The rabbits were bled a week after the final injection. Enzyme-linked immunosorbent assay (ELISA) was carried out (Termine et al., 1981a, b). Microtitre plates (Corning) were coated with 500 ng of osteo-
PH 5.0 5.560 65 ZO 75
4’5 -I ,
I
,.I#
I
A
1-94 & -
-2
-67
4 0
-43 5.56.0 6.5 7.0 Z5 - 30
5.0 B
‘0
-67 ;
50 C
4
2
4
DISTANCE
6 MIGRATED
8
(cm)
Fig. 3. Densitometric tracings of SDS gel electrophoresis of dentine Gdn/HCl extracts of stages I, 11and III, respectively. Samples run on 11.5% gels and stained with Coomassie brilliant blue R-250. K = kdalton.
nectin in 20mM sodium bicarbonate. Samples were incubated with antisera diluted in phosphate-buffered saline containing 0.05% Tween 20 (PBS-Tween), 1:3000, at 4°C overnight. The mixtures were transferred to the wells coated with osteonectin. After 2 h of incubation, wells were washed twice with PBS-Tween. The binding of antibody to immobilized antigen was detected using goat anti-rabbit IgG antibody conjugated with alkaline phosphatase (Bio-Rad). The colour development was measured in a Titertec Uniscan filter photometer (Flow Laboratories) at 405 nm. RESULTS
-940
-
0
-43 3 I 55 6~657.OoZ5 3o =
3
= -30 Fig. 2. Two-dimensional electronhoresis of bone and den-
tine Gdn/HCI extracts. Gels were stained with Coomassie brilliant blue R-250. (Al Bone GdniHCl extracts. (Bl Purified bone osteonecun: (C) Dcntine’ Gdn/HCl extracts: l-Collagen a 1 and a2 chains; 2-serum albumin; 3-a2-HS-glycoprotein; 4-osteonectin.
Non-collagenous proteins insoluble in 0.6 M HCl were recovered in Gdn/HCl extracts. Several proteins were identified by two-dimensional electrophoresis (Fig. 2); identification was based on the report by Delmas et al. (1984). Major spots were common to both bone and dentine extracts. Among these were serum albumin, 012HS-glycoprotein and osteonectin. The Gdn/HCl extracts from the dentines at different stages were analysed by electrophoresis; a densitometric tracing of each gel is shown in Fig. 3. Some major bands were observed at apparent molecular weights of 67, 40 and 28 kdalton. Osteonectin migrated with an apparent molecular weight of 40 kdalton after reduction (Sato et al., 1985; Romberg et al., 1985). In stage III dentine, the relative amount of osteonectin decreased prominently. To determine the amounts of osteonectin, an ELISA was developed using antisera against bone osteonectin. Osteonectin was purified from calf bone (Fig. 2B) and used as antigen. The antisera showed a single precipitin line with bone Gdn/HCl extracts or osteonectin in an Ouchterlony test or on immunoelectrophoresis. The inhibition curve in ELISA
91
Changes in dentine osteonectin
B i= 5 50I z z
ol 0.01
gjc__&~-._:*2d:$ 0.1
1 lNHtBITORS
, 10
loo
(pg)
Fig. 4. Inhibition ELIISA of osteonectin; osteonectin (O), serum albumin (0) or bone Gla-protein (A). The ordinate indicates percent inhibition of colour development comparing with the control.
(Fig. 4) showed no cross-reaction with serum albumin or osteocalcin. Using this technique, osteonectin was quantified in Gdn/HCl extracts from dentine of the three stages (Table 1). Osteonectin was detected only in Gdn/HCl extracts and not in HCl extracts. The level of osteonectin in stage III was almost half of that in stage I or II. The total amount of proteins extractable from dentine, however, did not decrease. DlSCUSSlON
The composition of matrix proteins in dentine is known to change with mineralization and maturation. The most drastic change occurs between predentine and dentine (Linde, 1984). Phosphophoryn (Jontell and Linde, 1983; Takagi, Fujisawa and Sasaki, 1986) and dentine Gla protein (Jontell and Linde, 1983; Bronckers et al., 1985) are absent in predentine but present in dentine. Predentine is, in contrast, rich in proteoglycans (Linde, 1973; Hjerpe and Engfeldt, 1976; Jontell and Linde, 1983). The changes continue even into the later stages of dentine development. Cros&linkings and cleavages are introduced into phosphophoryn (Kuboki et al., 1984; Fujisawa et al., 1985; Masters, 1985) resulting in a shift of the distribution of its molecular weight to lower values (Lee, Kossiva and Glimcher, 1983). In spite of this degradation, the total amounts of phosphophoryn have been shown to increase in stage III (Fujisawa and Kuboki, 1988). We also found changes in the composition of Gdn/HCl extracts in stage III (Fig. 3). Among these changes, reduction in osteonectin was significant (Fig. 3 and Table 1). Osteonectin is a phosphorylated
glycoprotein of bone (Termine et al., 198la, b). Recently, its primary structure was deduced and shown to be homologous with mouse SPARC (Bolander et al., 1988). It is synthesized by osteoblasts (Whitson et al., 1984; Otsuka et al., 1984) and is rich in mineralizing osteoid (Termine et al., 198la, b). Osteonectin is associated with early mineral deposition (Bianco et al., 1985). Its content is higher in lamellar bone than in woven bone (Conn and Termine, 1985). Osteonectin mRNA level is lower in osteocytes than in osteoblasts (Holland et al., 1987) and is expressed in an earlier stage of embryonic bone formation than other non-collagenous proteins (Yoon, Buenaga and Rodan, 1987; Nomura et al., 1988). Osteonectin or its mRNA is also detected in non-mineralizing tissues, including platelets (Stenner et al., 1986), basement membrane and steroid-producing glands (Holland et al., 1987). In dentine, odontoblasts synthesize and secrete osteonectin (Tung et al., 1985; Holland et al., 1987). In contrast to phosphophoryn, osteonectin has been detected in predentine immunohistochemically (Tung et al., 1985). The reduction in osteonectin in later developmental stages indicates that this protein is not synthesized in such large amounts by mature odontoblasts and/or is degraded during maturation. At this stage phosphophoryn is the predominant product. The wide distribution of osteonectin in various tissues, and its early expression in bone and dentine development indicate that this protein has a more general role than specialized proteins such as phosphophoryn. Acknowledgements-We acknowledge the helpful scientific discussion of Dr. W. T. Butler and the assistance of MS Karen Stewart in typing of the text. This work was supported by a grant from the Ministry of Education of Japan.
REFERENCES
Bianco P., Hayashi Y., Silvestrini G., Termine J. D. and Bonucci E. (1985) Osteonectin and Gla-protein in calf bone: ultrastructural immunohistochemical localization using the protein A-gold method. Cult. Tim. ht. 37, 684-686. Bolander M. E., Young M. F., Fisher L. W., Yamada Y. and Termine J. D. (1988) Osteonectin cDNA sequence reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (Ovomucoid). Proc. nafn. Acad. Sci. U.S.A. 85, 29 19-2923.
Table I. Levels of dentine proteins at three stages of development @g/mg of insoluble collagen remaining after demineralization and extraction, means + SD of four different extracts) Dentine
4 M Gdn/HCI extracts Osteonectin in Gdn/HCl extracts* Osteonectin in HCl extracts* Phosphophoryn**
Stage I
Stage II
Stage III
Bone
41*3 4.6 f 0.7 0.0 15+ 1
42 f 3 5.6 + 1.1 0.0 22* 1
53 f 3*** 2.6 f I.O*** 0.0 33 * 5
39 * 5 8.1 f I.0 0.0 -
lOsteonectin was quantified by ELISA using antisera against bone osteonectin. **Fujisawa and Kuboki (1988). ***Significantly different from values at stages I and II (Student’s f-test; p < 0.05).
92
R. FUJISAWAand Y. KUBOKI
Bronckers A. L., Gay S., Dimuzio M. T. and Butler W. T. (1985) Immunolocalization of y-carboxyglutamic acidcontaining proteins in developing molar tooth germs of the rat. Collagen Rel. Res. 5, 17-22. Butler W. T. (1984) Matrix macromolecules of bone and dentine. Collagen Rel. Res. 4, 297-307. Butler W. T., Bhown M., Dimuzio M. T. and Linde A. (1981a) Noncollagenous proteins of dentin. Isolation and partial characterization of rat dentin proteins and proteoglycans using a three-step preparative method. Collagen Rel. Res. 1, 187-199. Butler W. T., Bhown M., Tomana M., Fretweh B. and Schrohenloher R. E. (198 1b) Characterization of a unique dentin glycoprotein. In: The Chemistry and Biology-of Mineralized Connective Tissues (Edited bv Veis A.) D. 399. Elsevier/North-Holland, New York. _ ’. Conn K. M. and Termine J. D. (1985) Matrix protein profiles in calf bone development. Bone 6, 33-36. Delmas P. D., Tracy R. P., Riggs B. L. and Mann K. G. (1984) Identification of the noncohagenous proteins of bovine bone by two-dimensional gel electrophoresis. Calc. Tiss. Int. 36, 308-316.
Fujisawa R. and Kuboki Y. (1988) Increase of dentin phosphophoryn with dentin formation. Connect. Tiss. Res. 17, 231-238. Fujisawa R., Kuboki Y. and Sasaki S. (1985) In uivo cleavage of dentin phosphophoryn following g-elimination of its phosphoserine residues. Archs Biochem. Bioohvs. 243, 619623.
Hjerpe A. and Engfeldt B. (1976) Proteoglycans of dentine and nredentine. Calc. Tiss. Res. 22. 173-182. Holland P. W. H., Harper S. J., McVey J. H. and Hogan B. L. M. (1987) In ;ivo expression of mRNA for the Ca + + -bindine urotein SPARC (osteonectin) revealed bv in situ hybridization. J. Cell Bidl. 105, 473282. _ Jones I. L. and Leaver A. G. (1974a) Studies on the minor components of the organic matrix of human dentine. Archs oral Biol. 19, 371-380. Jones I. L. and Leaver A. G. (1974b) Glycosaminoglycans of human dentine. Calc. Tiss. Res. 16, 3744. Jontell M. and Linde A. (1983) Non-collagenous proteins of predentin from dentinogenically active bovine teeth. Biochem. J. 214, 769~-776.
Kuboki Y., Fujisawa R., Tsuzaki M., Liu C. F. and Sasaki S. (I 984) Presence of lysinoalanine and histidinoalanine in bovine dentin phosphoprotein. Calc. Tiss. Int. 36, 126-128. Laemmh U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 68&685.
Lee S. L., Kossiva D. and Glimcher M. J. (1983) Phosphoproteins of bovine dentin: evidence for polydispersity during tooth maturation. Biochemistry 22, 2596260
Chem. 259, 9805-98 12.
Rahemtulla F., Prince C. W. and Butler W. T. (1984) Isolation and partial characterization of proteoglycans from rat incisors. Biochem. J. 218, 877-885. Romberg R. W., Werness P. G., Lollar P., Riggs B. L. and Mann K. G. (1985) Isolation and characterization of native adult osteonectin. J. biol. Chem. 260, 2728-2736. Sato S., Rahemtulla F., Prince C. W., Tomana M. and Butler W. T. (1985) Acidic glycoproteins from bovine compact bone. Connect. Tiss. Res. 14, 51-64. Smith A. J. and Leaver A. G. (1979) Non-collagenous components of the organic matrix of rabbit incisor dentine. Archs oral Biol. 24, 449454. Smith A. J. and Leaver A. G. (1981) Distribution of the EDTA-soluble non-collagenous organic matrix components of rabbit incisor dentine. Archs oral Biol. 26, 643451.
Stenner D. D., Tracy R. P., Riggs B. L. and Mann K. G. (1986) Human platelets contain and secrete osteonectin, a major protein of mineralized bone. Proc. natn. Acad. Sci. U.S.A. 83, 6892-6896.
Takagi Y. and Veis A. (1984) Isolation of phosphophoryn from human dentin organic matrix. Calc. Tiss. Int. 36, 259-265.
Takagi Y., Fujisawa R. and Sasaki S. (1986) Identification of dentin phosphophoryn localization by histochemical stainings. Connect. Tiss. Res. 14, 279-292. Takagi Y., Nagai H. and Sasaki S. (1988) Difference in noncollagenous matrix composition between crown and root dentin of bovine incisor. Calc. Tiss. Inf. 42, 97-103. Termine J. D., Belcourt A. B., Miyamoto M. S. and Conn K. M. (1980) Properties of dissociatively extracted fetal tooth matrix proteins. II. Separation and purification of fetal bovine dentin phosphoprotein. J. biol. Chem. 255, 9769-9772.
Termine J. D., Belcourt A. B., Conn K. M. and Kleinman H. K. (1981a) Mineral and collagen-binding proteins of fetal calf bone. J. biol. Chem. 256, 10,403-10,408. Termine J. D., Kleinman H. K., Whitson S. W., Conn K. M., McGarvey M. L. and Martin G. R. (1981b) Osteonectin, a bone-specific protein linking mineral to collagen. Cell 26, 99-105. Thomas M. and Leaver A. G. (1975) Identification and estimation of plasma proteins in human dentine. Archs oral Biol. 20, 217-218.
1.
Linde A. (1973) Glycosaminoglycans of the odontoblastspredentine layer in dentinogenically active porcine teeth. Calc. Tiss. Res. 12, 281-294.
Thomas M. and Leaver A. G. (1977) The less-acidic glycoproteins of the organic matrix of human dentine. Archs oral Biol. 22, 545-549.
Linde A. (I 984) Noncollagenous proteins and proteoglycans in dentinogenisis. In: Dentin and Dentinogenesis (Edited by Linde A.) Chap. 9, Vol. II, p. 55, CRC Press, Boca Raton, Fla. Linde A., Bhown M. and Butler W. T. (1980) Noncollagenous proteins of dentin. A re-examination of proteins from rat incisor dentin utilizing techniques to avoid artifacts. J. biol. Chem. 225, 593 l-5942. Linde A., Bhown M., Cothran W. C., Hoglund A. and Butler W. T. (1982) Evidence for several y-carboxyglutamic acid-containing proteins in dentin. Biochim. biophys. Acta 704, 235-239.
Masters P. M. (1985) In uiuo decomposition
phoserine and serine in noncollagenous protein from human dentine. Calc. Tiss. Int. 37, 236241. Nomura S., Wills A. J., Edwards D. R., Heath J. K. and Hogan B. L. M. (1988) Developmental expression of 2ar (osteopontin) and SPARC (osteonectin) RNA as revealed by in situ hybridization. J. Cell Biol. 106, 44450. Otsuka K., Yao K. L., Wasi S., Tung P. S., Aubin J. E.. Sodek J. and Termine J. D. (1984) Biosynthesis of osteonectin by fetal porcine calvarial cells in vitro. J. biol.
of phos-
Tung P. S., Domenicucci C., Wasi S. and Sodek J. (1985) Specific immunohistochemical localization of osteonectin and collagen types I and III in fetal and adult porcine dental tissues. J. Histochem. Cyrochem. 33, 531-540. Whitson S. W., Harrison W., Dunlap M. K., Bowers D. E. Jr, Fisher L. W., Robey P. G. and Termine J. D. (1984) Fetal bovine bone cells synthesize bone-specific matrix proteins. J. Cell Biol. 99, 607614. Wrigley C. W. (1971) Gel electrofocusing. Meth. Enzymol. 22, 559-564.
Yoon K., Buenaga R. and Rodan G. A. (1987) Tissue specificity and developmental expression of rat osteonectin. Biochem. biophys. Res. Commun. 148, 1129-l 136.
Copyright 0
0003s9969/89 $3.00+O.OO 1989 Pergamon Press plc
CHANGES IN LEVELS OF OSTEONECTIN IN BOVINE DENTINE DURING TOOTH DEVELOPMENT R. FUJISAWA and Y. KUBOKI Department of Biochemistry, School of Dentistry, Hokkaido University, Sapporo 060, Japan (Received I5 March 1988; accepted 19 August 1988) Summary-Bovine incisors were classified into three developmental stages and non-collagenous proteins extracted from them. Sodium dodecyl sulphate gel electrophoresis of the extracts showed a reduction in osteonectin with the various stages. The reduction was confirmed by enzyme immunoassay using antiserum against bone osteonectin. This change is in contrast to dentine phosphoprotein, indicating functional diflerences between these two proteins.
used for the comparisons. These incisors were freed of pulp, soft tissues and soft enamel, and then ground into powder. The powders were washed with 4 M guanidine hydrochloride (Gdn/HCl), 50 mM trisHCl, pH 7.4. Demineralization was carried out with 0.6 M HCl (1 litre/g of powdered teeth) at 4°C for 4 days (Tagaki and Veis, 1984). Completeness of demineralization was checked by measuring the calcium content of the demineralized matrix (less than 0.01 pg/mg of matrix). Non-collagenous proteins were extracted from the demineralized matrix residue with 4 M Gdn/HCl, 50 mM tris-HCl, pH 7.4 (40 ml/g of demineralized matrix) at 4°C for 4 days with agitation. Femurs of calves were demineralized and extracted by the same procedures.
Dentine contains several non-collagenous proteins, some of which are also present in bone (reviewed by
Butler, 1984). These proteins were studied intensively by Leaver and his co-workers (Jones and Leaver, 1974a, 1974b; Thomas and Leaver, 1975, 1977; Smith and Leaver, 1979) who classified them as anionic glycoproteins, less acidic glycoproteins and proteoglycans. Later investigators reported a less complex composition as precautions were taken to avoid proteolysis (Termine et al., 1980; Linde, Bhown and Butler, 1980; B’utler et al., 1981a, b). The most abundant non-collagenous protein in dentine is phosphophoryn, which is a highly acidic protein rich in phosphoserine and aspartic acid. Among the other non-collagenous proteins, Gla proteins (Linde ef al., 1982), dentine proteoglycan (Rahemtulla, Prince and Butler, 1984), 9.5kdalton glycoprotein (Butler et al., 198 la, b), osteonectm (Termine et al., 198 1a, b; Tung et al., 1985) and several serum proteins have been identified. The composition ‘ofthese non-collagenous proteins changes with the maturation of dentine; the amino acid composition of total extracts of non-collagenous proteins changes significantly (Smith and Leaver, 1981). There are qualitative (Fujisawa, Kuboki and Sasaki, 1985) and quantitative (Fujisawa and Kuboki, 1988) changes in phosphophoryn with dentine formation. We have now investigated quantitative developmental changes in dentine osteonectin.
Electrophoresis
Sodium dodecyl sulphate (SDS) gel electrophoresis was carried out according to Laemmli (1970), using 11.5% acrylamide gels. The first dimension of two-dimensional electrophoresis was isoelectric focusing (Wrigley, 1971) using 4% acrylamide gels containing 2% Pharmalyte 3-10 (Pharmacia) and 8 M urea. The second dimension was the SDS gel electrophoresis.
MATERIIALSAND METHODS Extraction of non-o~llagenous proteins
The extraction procedures were essentially those of Fujisawa and Kuboki (1988). First permanent incisors of 2-year-old (calves were classified into three development stages, stages I, II and III, before crown completion, just after crown completion, and near root completion, respectively (Fig. 1). As the composition of non-collagenous proteins differs between crown and root dentine (Takagi, Nagai and Sasaki, 1988), the root was removed and only the crown was
Stage II
Stage 111 Fig. 1. Classification of the bovine incisor into three developmental stages. 89
90
R. FUJISAWAand Y. KUBOKI
Immunochemical procedures
Bone osteonectin was purified from Gdn/HCl
ex-
tracts of demineralized calf femur (Sato et al., 1985). Fifty to eighty milligrammes of the extracts were applied to a Sepharose CL-6B (Pharmacia) column (2.6 x 100 cm) and eluted with 4 M Gdn/HCl, 50 mM
tris-HCl, pH 8.2, at a flow rate of 22 ml/h. The peak corresponding to osteonectin was further purified by DEAE-cellulose (Whatman DE-52, 2.5 x 1Ocm) chromatography and rechromatography, equilibrated with 6 M urea, 50 mM tris-HCl, pH 8.2, and eluted with a linear NaCl gradient up to 0.5 M over 600ml of the buffer at a flow rate of 80ml/h. Chromatography was performed at room temperature and monitored with absorption at 230 nm, and fractions of 5 ml were collected. Purity of the preparation was checked by two-dimensional electrophoresis. Antisera were raised against osteonectin by injecting rabbits subcutaneously with 500 pg of the osteonectin preparation mixed with Freund’s complete adjuvant. Injections of the mixture of osteonectin and Freund’s incomplete adjuvant were repeated five times at l-week intervals. The rabbits were bled a week after the final injection. Enzyme-linked immunosorbent assay (ELISA) was carried out (Termine et al., 1981a, b). Microtitre plates (Corning) were coated with 500 ng of osteo-
PH 5.0 5.560 65 ZO 75
4’5 -I ,
I
,.I#
I
A
1-94 & -
-2
-67
4 0
-43 5.56.0 6.5 7.0 Z5 - 30
5.0 B
‘0
-67 ;
50 C
4
2
4
DISTANCE
6 MIGRATED
8
(cm)
Fig. 3. Densitometric tracings of SDS gel electrophoresis of dentine Gdn/HCl extracts of stages I, 11and III, respectively. Samples run on 11.5% gels and stained with Coomassie brilliant blue R-250. K = kdalton.
nectin in 20mM sodium bicarbonate. Samples were incubated with antisera diluted in phosphate-buffered saline containing 0.05% Tween 20 (PBS-Tween), 1:3000, at 4°C overnight. The mixtures were transferred to the wells coated with osteonectin. After 2 h of incubation, wells were washed twice with PBS-Tween. The binding of antibody to immobilized antigen was detected using goat anti-rabbit IgG antibody conjugated with alkaline phosphatase (Bio-Rad). The colour development was measured in a Titertec Uniscan filter photometer (Flow Laboratories) at 405 nm. RESULTS
-940
-
0
-43 3 I 55 6~657.OoZ5 3o =
3
= -30 Fig. 2. Two-dimensional electronhoresis of bone and den-
tine Gdn/HCI extracts. Gels were stained with Coomassie brilliant blue R-250. (Al Bone GdniHCl extracts. (Bl Purified bone osteonecun: (C) Dcntine’ Gdn/HCl extracts: l-Collagen a 1 and a2 chains; 2-serum albumin; 3-a2-HS-glycoprotein; 4-osteonectin.
Non-collagenous proteins insoluble in 0.6 M HCl were recovered in Gdn/HCl extracts. Several proteins were identified by two-dimensional electrophoresis (Fig. 2); identification was based on the report by Delmas et al. (1984). Major spots were common to both bone and dentine extracts. Among these were serum albumin, 012HS-glycoprotein and osteonectin. The Gdn/HCl extracts from the dentines at different stages were analysed by electrophoresis; a densitometric tracing of each gel is shown in Fig. 3. Some major bands were observed at apparent molecular weights of 67, 40 and 28 kdalton. Osteonectin migrated with an apparent molecular weight of 40 kdalton after reduction (Sato et al., 1985; Romberg et al., 1985). In stage III dentine, the relative amount of osteonectin decreased prominently. To determine the amounts of osteonectin, an ELISA was developed using antisera against bone osteonectin. Osteonectin was purified from calf bone (Fig. 2B) and used as antigen. The antisera showed a single precipitin line with bone Gdn/HCl extracts or osteonectin in an Ouchterlony test or on immunoelectrophoresis. The inhibition curve in ELISA
91
Changes in dentine osteonectin
B i= 5 50I z z
ol 0.01
gjc__&~-._:*2d:$ 0.1
1 lNHtBITORS
, 10
loo
(pg)
Fig. 4. Inhibition ELIISA of osteonectin; osteonectin (O), serum albumin (0) or bone Gla-protein (A). The ordinate indicates percent inhibition of colour development comparing with the control.
(Fig. 4) showed no cross-reaction with serum albumin or osteocalcin. Using this technique, osteonectin was quantified in Gdn/HCl extracts from dentine of the three stages (Table 1). Osteonectin was detected only in Gdn/HCl extracts and not in HCl extracts. The level of osteonectin in stage III was almost half of that in stage I or II. The total amount of proteins extractable from dentine, however, did not decrease. DlSCUSSlON
The composition of matrix proteins in dentine is known to change with mineralization and maturation. The most drastic change occurs between predentine and dentine (Linde, 1984). Phosphophoryn (Jontell and Linde, 1983; Takagi, Fujisawa and Sasaki, 1986) and dentine Gla protein (Jontell and Linde, 1983; Bronckers et al., 1985) are absent in predentine but present in dentine. Predentine is, in contrast, rich in proteoglycans (Linde, 1973; Hjerpe and Engfeldt, 1976; Jontell and Linde, 1983). The changes continue even into the later stages of dentine development. Cros&linkings and cleavages are introduced into phosphophoryn (Kuboki et al., 1984; Fujisawa et al., 1985; Masters, 1985) resulting in a shift of the distribution of its molecular weight to lower values (Lee, Kossiva and Glimcher, 1983). In spite of this degradation, the total amounts of phosphophoryn have been shown to increase in stage III (Fujisawa and Kuboki, 1988). We also found changes in the composition of Gdn/HCl extracts in stage III (Fig. 3). Among these changes, reduction in osteonectin was significant (Fig. 3 and Table 1). Osteonectin is a phosphorylated
glycoprotein of bone (Termine et al., 198la, b). Recently, its primary structure was deduced and shown to be homologous with mouse SPARC (Bolander et al., 1988). It is synthesized by osteoblasts (Whitson et al., 1984; Otsuka et al., 1984) and is rich in mineralizing osteoid (Termine et al., 198la, b). Osteonectin is associated with early mineral deposition (Bianco et al., 1985). Its content is higher in lamellar bone than in woven bone (Conn and Termine, 1985). Osteonectin mRNA level is lower in osteocytes than in osteoblasts (Holland et al., 1987) and is expressed in an earlier stage of embryonic bone formation than other non-collagenous proteins (Yoon, Buenaga and Rodan, 1987; Nomura et al., 1988). Osteonectin or its mRNA is also detected in non-mineralizing tissues, including platelets (Stenner et al., 1986), basement membrane and steroid-producing glands (Holland et al., 1987). In dentine, odontoblasts synthesize and secrete osteonectin (Tung et al., 1985; Holland et al., 1987). In contrast to phosphophoryn, osteonectin has been detected in predentine immunohistochemically (Tung et al., 1985). The reduction in osteonectin in later developmental stages indicates that this protein is not synthesized in such large amounts by mature odontoblasts and/or is degraded during maturation. At this stage phosphophoryn is the predominant product. The wide distribution of osteonectin in various tissues, and its early expression in bone and dentine development indicate that this protein has a more general role than specialized proteins such as phosphophoryn. Acknowledgements-We acknowledge the helpful scientific discussion of Dr. W. T. Butler and the assistance of MS Karen Stewart in typing of the text. This work was supported by a grant from the Ministry of Education of Japan.
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Table I. Levels of dentine proteins at three stages of development @g/mg of insoluble collagen remaining after demineralization and extraction, means + SD of four different extracts) Dentine
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92
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