| The mechanism of biomineralization in bone has yet to be elucidated. Bone sialoprotein (BSP) is expressed in mineralized tissues and is capable of binding to collagen and nucleating hydroxyapatite (HA) in vitro. In this study, the chemical nature of the BSP-collagen interaction and the effect this interaction has on BSP-mediated HA nucleation were investigated. Involvement of phosphorylation in BSP-mediated HA nucleation was studied and a critical site of modification identified. The information gained from these studies was used to generate a collagen-binding, HA-nucleating fusion peptide of BSP.;Native, bone-extracted BSP (nBSP) is a more potent HA nucleator than unmodified, recombinant BSP (rBSP). Dephosphorylation of nBSP decreases its HA-nucleation potency. Conversely, protein kinase CK2 treatment of rBSP enhances HA-nucleation activity at least 10-fold. Phosphorylation of Ser136 was demonstrated to be the critical site of modification in both full-length rBSP and the derived peptides. Molecular-dynamics simulations of HA-nucleating peptide 133-148 binding to the {100} face of HA shows an alternating-residue pattern during interaction with the crystal surface, which is more pronounced when Ser136 is phosphorylated. This alternating residue pattern is postulated to be important for BSP-mediated nucleation capability.;An rBSP(R26A) fusion peptide containing the collagen-binding region (residues 18 to 45) and nucleation-active region (residues 135 to 159) of BSP was generated. Both rBSP(R26A) and the fusion peptide bound type I collagen with similar affinity as wild-type, full-length rBSP. HA-nucleation activity of the fusion peptide was increased 10-fold upon binding to collagen and 4-fold by CK2-treatment. Future work will investigate the efficacy of this peptide in bone repair.;Keywords: biomineralization, bone, bone sialoprotein, collagen binding, hydroxyapatite, nucleation, phosphorylation, SIBLINGs;The BSP-collagen interaction is hydrophobic in nature and was found not to involve electrostatic interactions. BSP binds equally well to triple-helical collagen, with and without telopeptides, and to fibrillar collagen, but has lower affinity to heat-denatured collagen. Therefore, optimal binding requires collagen to be in a native, triple-helical conformation. Thrombin cleavage of rat BSP Arg26-Tyr27 abolishes binding, but mutation of Arg26 to Ala prevents digestion and maintains type I collagen affinity. Upon binding to collagen, BSP has 10-fold higher HA-nucleation potency. |
