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 (poly[E]) 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 poly[E] regions in full-length BSP was performed, replacing the poly[E] with either polyaspartic acid (poly[D]) or polyalanine (poly[A]) to examine role of charge and conformation, respectively, in HA nucleation. Replacement of single domains with either poly[A] or poly[D] did not alter nucleating activity nor did replacement of both domains with poly[D]. Replacement of both domains with poly[A], however, significantly decreased nucleating activity. In addition, two recombinant peptides, each encompassing one of the two poly[E] 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 alpha-helical in conformation.
Tye
Coralee E
CE
Canadian Institutes of Health Research Group in Skeletal Development and Remodeling, Division of Oral Biology and Department of Biochemistry, School of Dentistry, University of Western Ontario, London, Canada.
Rattray
Kevin R
KR
Warner
Kevin J
KJ
Gordon
Jonathan A R
JA
Sodek
Jaro
J
Hunter
Graeme K
GK
Goldberg
Harvey A
HA
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D017886:Durapatite / Q000378:metabolism*; D058574:Integrin-Binding Sialoprotein; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012542:Scattering, Radiation; D012795:Sialoglycoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; D017886:Durapatite