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Biochemical study on the tissue compartmentalization of phosphophoryn, osteopontin and bone sialoprotein (BSP) in rat incisor dentin

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Abstract

The tissue compartmentalization of phosphophoryn, osteopontin (OPN) and bone sialoprotein (BSP) in dentin was biochemically analyzed. The radicular portion of rat incisor was excised and sequentially extracted; i.e., proteins non-covalently bound to matrix such as soft tissues were solubilized with 4 M guanidinium chloride (G1), mineral-binding proteins were then extracted with 0.5 M EDTA (E), proteins non-covalently bound to the collagen matrix were extracted with 4 M guanidinium chloride (G2) after E, and finally the proteins covalently bound to the collagen matrix were extracted by bacterial collagenase digestion (C). Stains-All and Rhodamine B staining, which selectively stain calcium-binding proteins, revealed a 50∼80 kDa broad band in the E extract on 10% polyacrylamide gels. This intense band disappeared after precipitation with CaCl2, indicating that the protein is phosphophoryn. After removing phosphophoryn from the E extract, 67 kDa intense and 50 kDa weak bands appeared on the gels stained with Stains-All. The 67 kDa protein was observed similarly in the G2 and C extracts. Western blots using monoclonal antibodies revealed the presence of 67 kDa BSP and 50 kDa OPN. When the tissue compartmentalization was compared, these three proteins were not detected in the G1 extract, phosphophoryn and osteopontin were localized only in the E extract, and BSP was observed not only in the E extract but also in the G2 and C extracts. These findings indicate that phosphophoryn, OPN and BSP are rapidly incorporated into the mineralized fraction and that BSP also binds to collagen-related fraction, suggesting the association with intial mineralization in the dentin.

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References

  1. Linde A: Dentin matrix proteins: Composition and possible functions in calcification. Anatom Rec 224: 154–166, 1989

    CAS  Google Scholar 

  2. Butler WT, Sato S, Rahemtulla F et al.: Glycoprotein of bone and dentin. In: Buter WT (ed.) The Chemistry and Biology of Mineralized Tissues. Birmingham: Ebsco Media: pp. 107–112, 1985

    Google Scholar 

  3. Veis A: Phosphoproteins of dentin and bone. In: Butler WT (ed.) The Chemistry and Biology of Mineralized Tissues. Birmingham: Ebsco Media: pp. 170–176, 1985

    Google Scholar 

  4. Bronckers ALJJ, Lyaruu DM, Woltgens JHM: Immunohistochemistry of extracellular matrix proteins during various stages of dentinogenesis. Connect Tissue Res 22: 65–70, 1989

    CAS  PubMed  Google Scholar 

  5. Veis A: Acidic proteins as regulators of biomineralization in vertebrates. In: Davidovitch Z (ed.) The Biological Mechanisms of Tooth Movement and Craniofacial Adaptation. Birmingham: Ebsco Media: pp. 115–119, 1992

    Google Scholar 

  6. Macdougall M, Zeichener-David M, Slavkin HC: Characterization of extracellular and nascent dentin phosphoproteins. Conn Tissue Res 22: 71–77, 1989

    CAS  Google Scholar 

  7. Butler WT: Sialoproteins of bone and dentin. J Biol Buccale 19: 83–89, 1991

    CAS  PubMed  Google Scholar 

  8. Prince CW, Oosawa T, Butler WT et al.: Isolation, characterization and biosynthesis of a phosphorylated glycoprotein from rat bone. J Biol Chem 262: 29000–29006, 1986

    Google Scholar 

  9. Oldberg A, Franzen A, Heinegard D: Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cDNA reveals an arg-gly-asp cell-binding sequence. Proc Natl Acad Sci USA 83: 8819–8823, 1986

    CAS  PubMed  Google Scholar 

  10. Fisher LW, Whitson SW, Avioli LV et al.: Matrix sialoprotein of developing bone. J Biol Chem 258: 12723–12727, 1983

    CAS  PubMed  Google Scholar 

  11. Termine JD, Belcourt AB, Conn KM et al.: Mineral and collagen-binding proteins of fetal calf bone. J Biol Chem 256: 10403–10408, 1981

    CAS  PubMed  Google Scholar 

  12. Negata T, Todescan R, Goldberg HA et al.: Sulphation of secreted phosphoprotein I (SPPI, osteopontin) is associated with mineralized tissue formation. Biochem Biophys Res Commun 165: 234–240, 1989

    Google Scholar 

  13. Nagata T, Goldberg HA, Zhang Q et al.: Biosynthesis of bone proteins by fetal porcine calvariaein vitro. Rapid association of sulfated sialoproteins (secreted phosphoprotein-1 and bone sialoprotein) and chondroitin sulfate proteoglycan (CS-PGIII) with bone mineral. Matrix 11: 86–100, 1991

    CAS  PubMed  Google Scholar 

  14. Nagata T, Bellows CG, Kasugai S, et al.: Biosynthesis of bone proteins [SPP-1 (secreted phosphoprotein-1, osteopontin), BSP (bone sialoprotein) and SPARC (osteonectin)] in association with mineralized-tissue formation by fetal-rat calvarial cells in culture. Biochem J 274: 513–520, 1991

    CAS  PubMed  Google Scholar 

  15. Kasugai S, Nagata T, Sodek J: Temporal studies on the tissue compartmentalization of bone sialoprotein (BSP), osteopontin (OPN), and SPARC protein during bone formationin vitro. J Cell Physiol 152: 467–477, 1992

    Article  CAS  PubMed  Google Scholar 

  16. Campbell KP, MacLennan DH, Jorgensen AO: Staining of the Ca2+-binding proteins, calsequestin, calmodulin, troponin C, and S-100, with the cationic carbocyanine dye “Stains-all”. J Biol Chem 258: 11267–11273, 1983

    CAS  PubMed  Google Scholar 

  17. Debruyne I: Staining of alkali-labile phosphoproteins and alkaline phosphatases on polyacrylamide gels. Analytical Biochem 133: 110–115, 1983

    CAS  Google Scholar 

  18. Kuwata F, Maeno M, Yao K-L et al.: Characterization of monoclonal antibody recognizing small collagenous proteins in fetal bone. Collagen Rel Res 7: 39–55, 1987

    CAS  Google Scholar 

  19. Butler WT, Bhown M, Dimuzio MT et al.: Non-collagenous proteins of dentin. Isolation and partial characterization of rat dentin proteins and proteoglycans using a three-step preparation method. Collagen Res 1: 187–199, 1981

    CAS  Google Scholar 

  20. Kuboki Y, Fujisawa R, Aoyama K et al.: Calcium-specific precipitation of dentin phosphoprotein: a new method of purification and significance for the mechanism of calcification. J Dent Res 58: 1926–1932, 1979

    CAS  PubMed  Google Scholar 

  21. Butler WT: Dentin-specific proteins. Methods in Enzymol 145: 290–303, 1987

    CAS  Google Scholar 

  22. Rahima MM, Sabsay B, Wu CB et al.: Characterization of the molecular weights of bovine molar, rat incisor, and other phosphophoryns. Conn Tissue Res 22: 79–90, 1989

    CAS  Google Scholar 

  23. Sodek J, Chen J, Kasugai S et al.: Sialoproteins in bone remodeling. In: Davidovitch Z (ed.) The Biological Mechanisms of Tooth Movement and Craniofacial Adaptation. Birmingham: Ebsco Media: pp. 127–136, 1992

    Google Scholar 

  24. Kubota T, Zhang Q, Wrana JL et al.: Multiple forms of SppI (secreted phosphoprotein, osteopontin) synthesized by normal and transformed rat bone cell populations: Regulation by TGF-β. Biochem Biophys Res Commun 162: 1453–1459, 1989

    CAS  PubMed  Google Scholar 

  25. Fujisawa R, Butler Brunn JC, Zhou HY et al.: Differences in composition of cell-attachment sialoproteins between dentin and bone. J Dent Res 72: 1222–1226, 1993

    CAS  PubMed  Google Scholar 

  26. Senger DR, Peruzzi CA, Papadopoulos A et al.: Secreted phosphoprotein associated with neoplastic transformation. Close homology with plasma proteins cleaved during blood coagulation. Cancer Res 48: 5770–5774, 1989

    Google Scholar 

  27. Butler WT, Bhown M, Brunn J et al.: Isolation, characterization and immunolocalization of a 53-kDal dentin sialoprotein (DSP). Matrix 12: 343–351, 1992

    CAS  PubMed  Google Scholar 

  28. Mark MP, Butler WT, Prince CW et al.: Developmental expression of 44-kDa bone phosphoprotein (osteopontin) and bone γ-carboxyglutamic acid (Gla)-containing protein (osteocalcin) in calcifying tissue of rat. Differentiation 37: 123–136, 1988

    CAS  PubMed  Google Scholar 

  29. Yokota M, Nagata T, Ishida H et al.: Clonal dental pulp cells (RDP4-1, RPC-C2A) synthesize and secrete osteopontin (SPP1, 2ar). Biochem Biophys Res Commun 189: 892–898, 1992

    Article  CAS  PubMed  Google Scholar 

  30. Boskey AL, Maresca M, Doty S et al.: Concentration-dependent effects of dentin phosphophoryn in the regulation of in vitro hydroxyapatite formation and growth. Bone Miner 11: 55–65, 1990

    Article  CAS  PubMed  Google Scholar 

  31. Stetler-Stevenson WG, Veis A: Type I collagen shows a specific binding affinity for bovine dentin phosphophoryn. Calcif Tissue Int 38: 135–141, 1986

    CAS  PubMed  Google Scholar 

  32. Traub W, Jodaikin A, Arad T et al.: Dentin phosphophoryn binding to collagen fibrils. Matrix 12: 197–201, 1992

    CAS  PubMed  Google Scholar 

  33. Chen J, Zhang Q, McCulloch CAG et al.: Immunohistochemical localization of bone sialoprotein (BSP) in fetal porcine bone tissues: Comparison with secreted phosphoprotein 1 (SPP-1, osteopontin) and SPARC (osteonectin). Histochem J 23: 281–289, 1991

    Article  CAS  PubMed  Google Scholar 

  34. Chen J, Shapiro HS, Sodek J: Developmental expression of bone sialoprotein mRNA in rat mineralized connective tissue. J Bone Miner Res 7: 987–997, 1992

    CAS  PubMed  Google Scholar 

  35. Chen J, McCulloch CAG, Sodek J: Bone sialoprotein in developing porcine dental tissues: Cellular expression and comparison of tissue localization with osteopontin and osteonectin. Archs Oral Biol 38: 241–249, 1993

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Periodontology and Endodontology, Tokushima University School of Dentistry, 3-18-15 Kuramoto, 770, Tokushima, Japan

    Toshihiko Nagata, Atsuko Kawahara, Chika Kasahara, Mika Yokota, Seiji Nishikawa, Yoichi Wakano, Hiroshi Ishida & Toshihiko Nagata

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  1. Toshihiko Nagata
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  2. Atsuko Kawahara
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  3. Chika Kasahara
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  4. Mika Yokota
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  5. Seiji Nishikawa
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  6. Yoichi Wakano
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  7. Hiroshi Ishida
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  8. Toshihiko Nagata
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Nagata, T., Kawahara, A., Kasahara, C. et al. Biochemical study on the tissue compartmentalization of phosphophoryn, osteopontin and bone sialoprotein (BSP) in rat incisor dentin. J Bone Miner Metab 12 (Suppl 2), 7–13 (1994). https://doi.org/10.1007/BF02383380

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