Skip to main content

Advertisement

SpringerLink
Log in

Synthesis of a Potentially Bioactive, Hydroxyapatite-Nucleating Molecule

  • Published:
Calcified Tissue International Aims and scope Submit manuscript

Abstract

A human phosphophoryn (PP) cDNA was previously cloned from immature root apex total RNA in our laboratory. This cDNA comprises 2,364 bp, encoding 788 amino acids. More than 80% of the sequences are arranged as (DSS) n (n = 1–16), DS, and NSS motifs. We hypothesize that the capability of PP to bind Ca2+ and nucleate hydroxyapatite may depend on these repeated sequences. Two polypeptides were synthesized based on the human PP cDNA sequence to test the hypothesis. One polypeptide has the amino acid sequence DDPNSSDESNGNDD (synthetic polypeptide 1, SP1), which is from the N-terminal end of PP; the other polypeptide, DSKSDSSKSESDSS (synthetic polypeptide 2, SP2), is the PP repeated sequence motif. Phosphorylation of the polypeptides was accomplished by reacting them with adenosine triphosphate and casein kinases I and II. The ability of these molecules to cause mineralization was tested in a steady-state agarose gel system. The results show that phosphorylated SP2 (P-SP2) precipitated approximately 60% of the total Ca + PO4 precipitated by PP. P-SP1 precipitated about 23% of that precipitated by PP and was similar to the amount precipitated in the control gel, that is, without added peptides. Transmission electron microscopy and X-ray diffraction analysis showed that the precipitate formed in the P-SP2-containing gel was hydroxyapatite. The capability of P-SP2 to nucleate Ca + PO4 and precipitate hydroxyapatite is a result of the repeated sequence motif, which contains a high percentage of phosphorylated serine. This molecule could be used in the repair and regeneration of dental tissue.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Linde A (1989) Dentin matrix proteins: composition and possible functions in calcification. Anat Rec 224:154–166

    Article  CAS  PubMed  Google Scholar 

  2. Veis A (1993) Mineral-matrix interactions in bone and dentin. J Bone Miner Res 8(suppl 2):S493–S497

    PubMed  Google Scholar 

  3. Butler WT, Ritchie H (1995) The nature and functional significance of dentin extracellular matrix proteins. Int J Dev Biol 39:169–179

    CAS  PubMed  Google Scholar 

  4. Butler WT, Bhown M, DiMuzio MT, Cothran WC, Linde A (1983) Multiple forms of rat dentin phosphoproteins. Arch Biochem Biophys 225:178–186

    Article  CAS  PubMed  Google Scholar 

  5. Takagi Y, Veis A (1984) Isolation of phosphophoryn from human dentin organic matrix. Calcif Tissue Int 36:259–265

    Article  CAS  PubMed  Google Scholar 

  6. Chang SR, Chiego D Jr, Clarkson BH (1996) Characterization and identification of a human dentin phosphophoryn. Calcif Tissue Int 59:149–153

    Article  CAS  PubMed  Google Scholar 

  7. Nawrot CF, Campbell DJ, Schroeder JK, Van Valkenburg M (1976) Dental phosphoprotein-induced formation of hydroxylapatite during in vitro synthesis of amorphous calcium phosphate. Biochemistry 15:3445–3449

    Article  CAS  PubMed  Google Scholar 

  8. Zanetti M, de Bernard B, Jontell M, Linde A (1981) Ca2+-binding studies of the phosphoprotein from rat-incisor dentine. Eur J Biochem 113:541–545

    Article  CAS  PubMed  Google Scholar 

  9. Termine JD, Eanes ED, Conn KM (1980) Phosphoprotein modulation of apatite crystallization. Calcif Tissue Int 31:247–251

    Article  CAS  PubMed  Google Scholar 

  10. Lussi A, Crenshaw MA, Linde A (1988) Induction and inhibition of hydroxyapatite formation by rat dentine phosphoprotein in vitro. Arch Oral Biol 33:685–691

    Article  CAS  PubMed  Google Scholar 

  11. Linde A, Lussi A (1989) Mineral induction by polyanionic dentin and bone proteins at physiological ionic conditions. Connect Tissue Res 21:197–203

    CAS  PubMed  Google Scholar 

  12. Boskey AL, Maresca M, Doty S, Sabsay B, Veis A (1990) Concentration-dependent effects of dentin phosphophoryn in the regulation of in vitro hydroxyapatite formation and growth. Bone Miner 11:55–65

    Article  CAS  PubMed  Google Scholar 

  13. Hunter GK, Hauschka PV, Poole AR, Rosenberg LC, Goldberg HA (1996) Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins. Biochem J 317:59–64

    CAS  PubMed  Google Scholar 

  14. D’Souza RN, Cavender A, Sunavala G, Alvarez J, Ohshima T, Kulkarni AB, MacDougall M (1997) Gene expression patterns of murine dentin matrix protein 1 (Dmp1) and dentin sialophosphoprotein (DSPP) suggest distinct developmental functions in vivo. J Bone Miner Res 12:2040–2049

    Article  CAS  PubMed  Google Scholar 

  15. George A, Sabsay B, Simonian PA, Veis A (1993) Characterization of a novel dentin matrix acidic phosphoprotein. Implications for induction of biomineralization. J Biol Chem 268:12624–12630

    CAS  PubMed  Google Scholar 

  16. He G, Dahl T, Veis A, George A (2003) Dentin matrix protein 1 initiates hydroxyapatite formation in vitro. Connect Tissue Res 44(suppl 1):240–245

    CAS  PubMed  Google Scholar 

  17. He G, Dahl T, Veis A, George A (2003) Nucleation of apatite crystals in vitro by self-assembled dentin matrix protein 1. Nat Mater 2:552–558

    Article  CAS  PubMed  Google Scholar 

  18. Tartaix PH, Doulaverakis M, George A, Fisher LW, Butler WT, Qin C, Salih E, Tan M, Fujimoto Y, Spevak L, Boskey AL (2004) In vitro effects of dentin matrix protein-1 on hydroxyapatite formation provide insights into in vivo functions. J Biol Chem 279:18115–18120

    Article  CAS  PubMed  Google Scholar 

  19. van den Bos T, Steinfort J, Beertsen W (1993) Effect of bound phosphoproteins and other organic phosphates on alkaline phosphatase-induced mineralization of collagenous matrices in vitro. Bone Miner 23:81–93

    PubMed  Google Scholar 

  20. Narayanan K, Ramachandran A, Hao J, He G, Park KW, Cho M, George A (2003) Dual functional roles of dentin matrix protein 1 Implications in biomineralization and gene transcription by activation of intracellular Ca2+ store. J Biol Chem 278:17500–17508

    Article  CAS  PubMed  Google Scholar 

  21. Wallwork ML, Kirkham J, Chen H, Chang SX, Robinson C, Smith DA, Clarkson BH (2002) Binding of dentin noncollagenous matrix proteins to biological mineral crystals: an atomic force microscopy study. Calcif Tissue Int 71:249–255

    Article  CAS  PubMed  Google Scholar 

  22. Wu CB, Pelech SL, Veis A (1992) The in vitro phosphorylation of the native rat incisor dentin phosphophoryns. J Biol Chem 267:16588–16594

    CAS  PubMed  Google Scholar 

  23. Zeichner-David M, Hall F, Williams R, Thiemann F, Yen S, MacDougall M, Slavkin HC (1995) Characterization of protein kinases involved in dentinogenesis. Connect Tissue Res 33:87–95

    CAS  PubMed  Google Scholar 

  24. Ritchie HH, Wang LH (1996) Sequence determination of an extremely acidic rat dentin phosphoprotein. J Biol Chem 271:21695–21698

    Article  CAS  PubMed  Google Scholar 

  25. Veis A, Wei K, Sfeir C, George A, Malone J (1998) Properties of the (DSS)n triplet repeat domain of rat dentin phosphophoryn. Eur J Oral Sci 106(suppl 1):234–238

    CAS  PubMed  Google Scholar 

  26. George A, Bannon L, Sabsay B, Dillon JW, Malone J, Veis A, Jenkins NA, Gilbert DJ, Copeland NG (1996) The carboxyl-terminal domain of phosphophoryn contains unique extended triplet amino acid repeat sequences forming ordered carboxyl-phosphate interaction ridges that may be essential in the biomineralization process. J Biol Chem 271:32869–32873

    Article  CAS  PubMed  Google Scholar 

  27. George A, Srinivasan R, Thotakura S, Veis A (1998) The phosphophoryn gene family: identical domain structures at the carboxyl end. Eur J Oral Sci 106(suppl 1):221–226

    CAS  PubMed  Google Scholar 

  28. Thotakura SR, Mah T, Srinivasan R, Takagi Y, Veis A, George A (2000) The non-collagenous dentin matrix proteins are involved in dentinogenesis imperfecta type II (DGI-II). J Dent Res 79:835–839

    CAS  PubMed  Google Scholar 

  29. MacDougall M, Simmons D, Luan X, Nydegger J, Feng J, Gu TT (1997) Dentin phosphoprotein and dentin sialoprotein are cleavage products expressed from a single transcript coded by a gene on human chromosome 4. Dentin phosphoprotein DNA sequence determination. J Biol Chem 272:835–842

    Article  CAS  PubMed  Google Scholar 

  30. Ritchie H, Wang LH (1997) A mammalian bicistronic transcript encoding two dentin-specific proteins. Biochem Biophys Res Commun 231:425–428

    Article  CAS  PubMed  Google Scholar 

  31. Gu K, Chang S, Ritchie HH, Clarkson BH, Rutherford RB (2000) Molecular cloning of a human dentin sialophosphoprotein gene. Eur J Oral Sci 108:35–42

    Article  CAS  PubMed  Google Scholar 

  32. Ritchie HH, Berry JE, Somerman MJ, Hanks CT, Bronckers AL, Hotton D, Papagerakis P, Berdal A, Butler WT (1997) Dentin sialoprotein (DSP) transcripts: developmentally-sustained expression in odontoblasts and transient expression in pre-ameloblasts. Eur J Oral Sci 105:405–413

    CAS  PubMed  Google Scholar 

  33. Butler WT, Bhown M, Brunn JC, D’Souza RN, Farach-Carson MC, Happonen RP, Schrohenloher RE, Seyer JM, Somerman MJ, Foster RA (1992) Isolation, characterization and immunolocalization of a 53-kDa dentin sialoprotein (DSP). Matrix 12:343–351

    CAS  PubMed  Google Scholar 

  34. D’Souza RN, Bronckers AL, Happonen RP, Doga DA, Farach-Carson MC, Butler WT (1992) Developmental expression of a 53 kD dentin sialoprotein in rat tooth organs. J Histochem Cytochem 40:359–366

    CAS  PubMed  Google Scholar 

  35. Bronckers AL, Farach-Carson MC, Van Waveren E, Butler WT (1994) Immunolocalization of osteopontin, osteocalcin, and dentin sialoprotein during dental root formation and early cementogenesis in the rat. J Bone Miner Res 9:833–841

    Article  CAS  PubMed  Google Scholar 

  36. Boskey A, Spevak L, Tan M, Doty SB, Butler WT (2000) Dentin sialoprotein (DSP) has limited effects on in vitro apatite formation and growth. Calcif Tissue Int 67:472–478

    Article  CAS  PubMed  Google Scholar 

  37. Butler WT, Bhown M, DiMuzio MT, Linde A (1981) Non-collagenous proteins of dentin Isolation and partial characterization of rat dentin proteins and proteoglycans using a three-step preparative method. Collagen Relat Res 1:187–199

    CAS  Google Scholar 

  38. Chen PS, Toribara TY,Warner H (1956) Microdetermination of phosphorus. Anal Chem. 28:1756–1758

    Article  CAS  Google Scholar 

  39. Leventouri T, Bunaciu CE, Perdikatsis V (2003) Neutron powder diffraction studies of silicon-substituted hydroxyapatite. Biomaterials 24:4205–4211

    Article  CAS  PubMed  Google Scholar 

  40. Fujisawa R, Wada Y, Nodasaka Y, Kuboki Y (1996) Acidic amino acid-rich sequences as binding sites of osteonectin to hydroxyapatite crystals. Biochim Biophys Acta 1292:53–60

    PubMed  Google Scholar 

  41. Tye CE, Rattray KR, Warner KJ, Gordon JA, Sodek J, Hunter GK, Goldberg HA (2003) Delineation of the hydroxyapatite-nucleating domains of bone sialoprotein. J Biol Chem 278:7949–7955

    Article  CAS  PubMed  Google Scholar 

  42. Hunter GK, Goldberg HA (1993) Nucleation of hydroxyapatite by bone sialoprotein. Proc Natl Acad Sci USA 90:8562–8565

    CAS  PubMed  Google Scholar 

  43. Rezania A, Thomas CH, Branger AB, Waters CM, Healy KE (1997) The detachment strength and morphology of bone cells contacting materials modified with a peptide sequence found within bone sialoprotein. J Biomed Mater Res 37:9–19

    Article  CAS  PubMed  Google Scholar 

  44. Bearinger JP, Castner DG, Healy KE (1998) Biomolecular modification of p(AAm-co-EG/AA) IPNs supports osteoblast adhesion and phenotypic expression. J Biomater Sci Polym Ed 9:629–652

    CAS  PubMed  Google Scholar 

  45. Barber TA, Golledge SL, Castner DG, Healy KE (2003) Peptide-modified p(AAm-co-EG/AAc) IPNs grafted to bulk titanium modulate osteoblast behavior in vitro. J Biomed Mater Res 64A:38–47

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Dr. Kai Sun and Dr. John F. Mansfield for their help with the TEM analysis. We also thank Dr. Qiming Jin for his help in improving the TEM image. This investigation was supported by NIH grant DE 12899.

Author information

Authors and Affiliations

  1. Department of Cariology, Restorative Sciences, and Endodontics, University of Michigan, Ann Arbor, MI, USA

    S. Chang, H. Chen, J. Liu & B. Clarkson

  2. Division of Restorative Dentistry, University of Leeds, Leeds, United Kingdom

    D. Wood & P. Bentley

Authors
  1. S. Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H. Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D. Wood
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. P. Bentley
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. B. Clarkson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to S. Chang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chang, S., Chen, H., Liu, J. et al. Synthesis of a Potentially Bioactive, Hydroxyapatite-Nucleating Molecule. Calcif Tissue Int 78, 55–61 (2006). https://doi.org/10.1007/s00223-005-0118-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00223-005-0118-4

Keywords

Search

Navigation