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Importance of Phosphorylation for Osteopontin Regulation of Biomineralization

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Abstract

Previous in vitro and in vivo studies demonstrated that osteopontin (OPN) is an inhibitor of the formation and growth of hydroxyapatite (HA) and other biominerals. The present study tests the hypotheses that the interaction of OPN with HA is determined by the extent of protein phosphorylation and that this interaction regulates the mineralization process. Bone OPN as previously reported inhibited HA formation and HA-seeded growth in a gelatin-gel system. A transglutaminase-linked OPN polymer had similar effects. Recombinant, nonphosphorylated OPN and chemically dephosphorylated OPN, had no effect on HA formation or growth in this system. In contrast, highly phosphorylated milk OPN (mOPN) promoted HA formation. The mOPN stabilized the conversion of amorphous calcium phosphate (a noncrystalline constituent of milk) to HA, whereas bone OPN had a lesser effect on this conversion. Mixtures of OPN and osteocalcin known to form a complex in vitro, unexpectedly promoted HA formation. To test the hypothesis that small alterations in protein conformation caused by phosphorylation account for the differences in the observed ability of OPN to interact with HA, the conformation of bone OPN and mOPN in the presence and absence of crystalline HA was determined by attenuated total reflection (ATR) infrared (IR) spectroscopy. Both proteins exhibited a predominantly random coil structure, which was unaffected by the addition of Ca2+. Binding to HA did not alter the secondary structure of bone OPN, but induced a small increase of β-sheet (few percent) in mOPN. These data taken together suggest that the phosphorylation of OPN is an important factor in regulating the OPN-mediated mineralization process.

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Acknowledgements

The authors would like to thank the members of their research laboratories for their assistance with this study. Support was provided by NIH grants DE04141 (ALB); DE05092 (WTB & CQ), and NSF CHE-0215970 (AG). The support of Arla Foods, the Danish Dairy Research Foundation, and the Danish Research and Development Programme for Food Technology is acknowledged (ESS).

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Authors and Affiliations

  1. Department of Chemistry, Kent State University, Kent, OH, USA

    A. Gericke

  2. Department of Endodontics and Peridontics, University of Texas Health Science Center at Houston, Dental Branch, Houston, TX, USA

    C. Qin & W. T. Butler

  3. Mineralized Tissues Laboratory, Research Division, Hospital for Special Surgery, New York, NY, USA

    L. Spevak, Y. Fujimoto & A. L. Boskey

  4. Protein Chemistry Laboratory, Department of Molecular Biology, University of Aarhus, Denmark

    E. S. Sørensen

  5. Department of Biochemistry, Weil Medical College of Cornell University and Program in Physiology, Biophysics, Systems Biology, Graduate School of Medical Sciences, New york, NY, USA

    A. L. Boskey

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  1. A. Gericke
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Gericke, A., Qin, C., Spevak, L. et al. Importance of Phosphorylation for Osteopontin Regulation of Biomineralization. Calcif Tissue Int 77, 45–54 (2005). https://doi.org/10.1007/s00223-004-1288-1

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