Determination of the hydroxyapatite-nucleating region of bone sialoprotein.
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.
Goldberg
H A
HA
Faculty of Dentistry, University of Western Ontario, London, Canada. hagoldbe@julian.uwo.ca
Warner
K J
KJ
Stillman
M J
MJ
Hunter
G K
GK
D000818:Animals; D001665:Binding Sites; D017886:Durapatite / Q000378:metabolism*; D058574:Integrin-Binding Sialoprotein; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry; D012795:Sialoglycoproteins / Q000737:chemistry* / Q000378:metabolism; D013552:Swine
D058574:Integrin-Binding Sialoprotein; D010455:Peptides; D011506:Proteins; D012795:Sialoglycoproteins; D017886:Durapatite