COLLAGEN BINDING and HYDROXYAPATITE NUCLEATION PROPERTIES OF BONE SIALOPROTEIN

Introduction: Bone sialoprotein (BSP) is an acidic phosphoprotein with collagen binding and hydroxyapatite (HA) nucleating properties. Objectives: To elucidate the nature and significance of the BSP-collagen interaction and to examine the role of phosphorylation on BSP-mediated HA-nucleation. Methods: Bone extracted and prokaryotic-expressed recombinant BSP (rBSP) were prepared. Collagen-binding was investigated by ELISA-type solid-phase binding assay and affinity chromatography. HA-nucleation was tested using a steady-state assay system in agarose or collagen gels. Reagents were phosphorylated by treatment with protein kinase CK2 (CK2) and verified by MALDI-TOF MS and ESI-MS. Results: Triple helical (KD = 13.4 ± 0.8 nM), pepsin-treated (KD = 13.1 ± 1.6 nM) and fibrillar (KD = 12.0 ± 0.8 nM) type I collagen showed similar affinity for rBSP while heat-denatured type I collagen (KD = 44.5 ± 4.9 nM) had decreased affinity. Acetonitrile, but not NaCl effectively eluted rBSP from collagen-columns implying a hydrophobic interaction. The BSP-collagen interaction increased the HA-nucleating potency of BSP 10-fold compared to its activity when unbound. Bone-extracted BSP (0.001 nmol) displayed higher potency than unmodified rBSP (0.100 nmol) in agarose gels. However, this potency was decreased upon dephosphorylation. CK2 treatment of rBSP (4.6 phosphates) increased nucleation potency by 10-fold. rBSP(1-100) and rBSP(133-206) each containing one of the 2 Glu-rich regions were treated with CK2. While phosphorylation did not have an effect on the nucleation activity of rBSP(1-100), a 10-fold increase in potency was observed for rBSP(133-206). Conclusions: Optimal binding to BSP requires collagen to be in a native, triple-helical conformation and is not mediated by collagen telopeptides. This interaction is stabilized by hydrophobic forces and increases the HA-nucleation potency of BSP, implying a co-operative effect. Phosphorylation of rBSP increases its HA-nucleation potency, an activity that we have located to the second Glu-rich region. Supported by CIHR.