Differential Effects of Posttranslational Modification on Osteopontin-Mediated in vitro Mineralization

Objectives: Extracellular matrix proteins play multifunctional roles, among which is the regulation of the calcification process. To test the hypothesis that posttranslational modification (PTM) of extracellular matrix proteins determines their ability to act as hydroxyapatite (HA) nucleators, or inhibitors of HA formation and growth, we investigated the effects of osteopontin (OPN) fragments on HA formation in a gelatin gel system. Since OPN is enzymatically cleaved in mineralizing tissues this study was designed to provide more detail about the importance of these fragments on OPN-HA interactions.

Methods: OPN from bovine milk (mOPN) was enzymatically cleaved with thrombin and three purified fragments (N-terminal (147 aa), C-terminal (57aa), and central (56 aa) were tested at concentrations of 0-50 ug/ml in the previously described gelatin gel diffusion system (Gericke et al, Calcif Tissues Int, 2005; 77: 45-54). The OPN fractions were then dephosphorylated and the study repeated using 25 ug/ml protein. Comparisons were made to highly phosphorylated mOPN (28 phosphorylated residues), bone OPN (7-9 phosphorylated residues), and fully dephosphorylated OPN based on one way ANOVA and the Tukey-Kramer significance test.

Results: The phosphorylated N- and C-terminal fragments (having 16/28 and 7/28 known phosphorylated residues, respectively), similar to mOPN, promoted HA formation. Dephosphorylation of these fragments eliminated any effect on HA formation and growth in a dose dependent manner. The central fragment (5/28 phosphorylated residues), in contrast, inhibited HA formation and growth, similar to the effects seen for the bone protein.

Conclusion: These studies support our hypothesis both in terms of phosphorylation and cleavage. They also indicate that the central portion of mOPN may bind more tightly to HA than the N- and C- terminal fragments. Studies to validate that suggestion are in progress.

Supported by: NIH-DE04141