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Characterization of Native and Recombinant Bone Sialoprotein: Delineation of the Mineral‐Binding and Cell Adhesion Domains and Structural Analysis of the RGD Domain
Author(s) -
Stubbs John T.,
Mintz Keith P.,
Eanes Edward D.,
Torchia Dennis A.,
Fisher Larry W.
Publication year - 1997
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1359/jbmr.1997.12.8.1210
Subject(s) - bone sialoprotein , recombinant dna , tripeptide , cell adhesion , chemistry , integrin , microbiology and biotechnology , biochemistry , biology , peptide , cell , osteocalcin , gene , alkaline phosphatase , enzyme
Bone sialoprotein is a small, sulfated, and phosphorylated integrin‐binding glycoprotein apparently found only in tissues that eventually mineralize. Nondenatured bone sialoprotein (BSP) purified from rat osteosarcoma cell line (UMR 106–01 BSP) culture media is shown to have a hydroxyapatite K d ≈ 2.6 × 10 −9 M, perhaps the strongest affinity for this mineral of any of the matrix proteins. Both native BSP and a 47 kD fragment of UMR‐BSP (Fragment 1 ∼133A–∼265Y) are more potent inhibitors of seeded hydroxyapatite crystal growth than recombinant human BSP fragments lacking post‐translational modifications. The recombinant proteins, however, do show reproducible inhibitory activity, suggesting that at least some of the strong mineral‐binding properties are encoded directly within the protein sequence itself. BSP facilitates the adhesion of several cell types through its integrin binding (RGD) tripeptide sequence. Nuclear magnetic resonance (NMR) analysis of a 15 N‐enriched 59 amino acid recombinant domain containing the RGD tripeptide shows that the structure of this isolated domain is highly flexible with or without 5 mM calcium. Previous work has also shown that an endogenous fragment of UMR‐BSP (Fragment 1) supports cell adhesion in the absence of the RGD sequence. In this report, non‐RGD cell adhesion sites are localized within conserved amino‐ and carboxy‐terminal tyrosine‐rich domains of recombinant human BSP. Given the proximity of the latter non‐RGD cell adhesion site to the RGD tripeptide, a model of BSP‐receptor interactions is presented.