Delineation of the Hydroxyapatite-nucleating Domains of Bone Sialoprotein

Bone sialoprotein (BSP) is a highly modified, anionic phosphoprotein that is expressed almost exclusively in mineralizing connective tissues and has been shown to be a potent nucleator of hydroxyapatite (HA). Two polyglutamic acid (polyE) regions, predicted to be in an alpha-helical conformation and located in the amino-terminal half of the molecule, are believed to be responsible for this activity. Using a prokaryotic expression system, full-length rat BSP was expressed and tested for HA nucleating activity in a steady-state agarose gel system. The unmodified protein is less potent than native bone BSP, indicating a role for the post-translational modifications in HA nucleation. Site-directed mutagenesis of the polyE regions in full-length BSP was performed, replacing the polyE with either polyaspartic acid (polyD) or polyalanine (polyA) to examine role of charge and conformation, respectively, in HA nucleation. Replacement of single domains with either polyA or polyD did not alter nucleating activity nor did replacement of both domains with polyD. Replacement of both domains with polyA, however, significantly decreased nucleating activity. In addition, two recombinant peptides, each encompassing one of the two polyE domains, were expressed and tested for nucleating activity. Whereas the peptide encompassing the second polyE domain was capable of nucleating HA, the first domain peptide showed no activity. The conformation of the wild-type and mutated proteins and peptides were studied by circular dichroism and small angle x-ray scattering, and no secondary structure was evident. These results demonstrate that a sequence of at least eight contiguous glutamic acid residues is required for the nucleation of HA by BSP and that this nucleating "site" is not alpha-helical in conformation.