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 (poly[E]) regions, predicted to be in an ␣-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. domains, were expressed and tested for nucleating activity. Whereas the peptide encompassing the second poly[E] 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 ␣-helical in conformation.Mineralization of the extracellular matrix in bone, dentin, and cementum is a complex, poorly understood process that is believed to involve both hydroxyapatite-nucleating and -modulating noncollagenous proteins. In bone, it has been postulated that type I collagen acts as a structural matrix, whereas HA nucleation is mediated by an anionic phosphoprotein (1-3). Of the noncollagenous proteins, bone sialoprotein (BSP) 1 is the most likely candidate. The highly anionic nature of BSP and its spatio-temporal pattern of expression have led investigators to propose a role of this protein in the mineralization of bone (1-4).Mammalian BSPs contain an average of 327 amino acids, which includes a 16-residue signal sequence. The protein has a molecular mass of ϳ33-34 kDa. However, post-translational modifications, including both N-and O-linked glycosylation, tyrosine sulfation, and serine and threonine phosphorylation, constitute 50% of the total mature protein weight of ϳ75 kDa. Analysis of the mammalian BSP cDNAs reveals a 45% level of sequence identity, plus an additional 10 -23% in conservative replacements. However, identity of up to 90% is observed in and around two polyglutamic acid sequences (poly[E]); an ArgGly-Asp (RGD) cell-binding motif; sites of phosphorylation, sulfation, and glycosylation; and sequences near the amino and carboxyl termini, which are rich in tyrosine residues (5).Normally BSP expression is limited almost exclusively to mineralized connective tissues, and its expression is localized to areas of bone formation. By in situ hybridization, it has been shown that BSP expression occurs in osteoblasts actively engaged in bone formation and is found at low or undetectable levels in other regions of mineralized tissue (6 -11). Transfection of BSP into nonmineralizin...

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