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...
The phosphorylated acidic glycoproteins bone sialoprotein (BSP) and osteopontin (OPN) bind to hydroxyapatite (HA) crystals and may be involved in the regulation of bone mineralization. The HA-binding properties of these proteins have been attributed to glutamic acid-rich sequences in BSP and aspartic acid-rich sequences in OPN. The present study examines the roles of these polycarboxylate sequences in the binding of BSP and OPN to HA. Porcine BSP, OPN and the synthetic polypeptides poly-L-glutamic acid [Poly(Glu)] and poly-L-aspartic acid [Poly(Asp)] were labeled with fluorescein isothiocyanate and their binding to HA determined by fluorimetry. From the binding isotherms, dissociation constants (KDs) for all the reagents tested were determined to be in the micromolar range. The saturation binding capacities of HA for Poly(Glu), Poly(Asp), BSP and OPN were similar (500-600 micrograms/m2). To investigate the role of glutamic acid-rich and aspartic acid-rich sequences in the binding to HA of BSP and OPN, respectively, competitive binding studies with Poly(Glu) and Poly(Asp) were performed. Poly(Glu) was able to displace a maximum of 100% of Poly(Glu), 81% of OPN, 68% of BSP and 65% of Poly(Asp). Poly(Asp) was able to displace a maximum of 100% of Poly(Glu), 99% of Poly(Asp), 95% of OPN and 89% of BSP. These results are consistent with the view that BSP and OPN bind to HA via their polycarboxylate sequences, but suggest a complex mode of interaction between polyelectrolytes and ionic crystals.
Bone sialoprotein (BSP) was shown to be a potent nucleator of hydroxyapatite (HA) in a steady-state agarose gel system (Hunter and Goldberg, 1993, PNAS 90: 8562). Nucleation of HA was also demonstrated with the homopolymer poly-glutamic acid but not with poly-aspartic acid or osteopontin. Since BSP contains contiguous sequences of glutamic acid, it is reasonable to suggest that the HA-nucleating activity of BSP resides within these regions. Purified porcine BSP was treated with trypsin and digests fractionated by gel filtration. In addition to small peptides (P3-5), two peptides of 38 kDa (P1) and 25 kDA (P2) were recovered, and after characterization assigned to the regions within BSP encompassing residues 133-272 (P1) and 42-125 (P2). Each of these peptides contained one of the two glutamic acid-rich regions of porcine BSP. In the steady-state agarose gel system, BSP, P1 and P2 induced HA formation, whereas the pooled small BSP-derived peptides (P3-5) did not. Analysis by circular dichroism spectroscopy revealed that the homopolymer poly-L-glutamic acid assumes a helical structure, while poly-L-aspartic acid does not. These findings suggest that the nucleating activity does not require intact molecules, that the nucleation of HA and BSP appears to require glutamic acid-rich sequences in a helical conformation and that there are two domains in porcine BSP that are each capable of nucleating HA.
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