In vivo functional analysis of polyglutamic acid domains in recombinant bone sialoprotein.
Bone sialoprotein (BSP) is an anionic phosphoprotein expressed in mineralizing connective tissues that binds to hydroxyapatite and nucleates its formation in vitro. Two polyglutamic acid regions (poly [E]) are believed to participate in these activities. The aim of this study was to evaluate the contribution of these acidic regions to the binding of prokaryote recombinant BSP (prBSP(E)) within an actual in vivo environment. Full-length prBSP(E) and prBSP(E) in which the poly [E] domains were replaced by polyalanine (prBSP(A)) were tagged with dinitrophenol (DNP). Tagged preparations comprised intact molecules and some fragmented forms. They were infused through a surgically created hole in the bone of rat hemimandibles and detected using immunogold labeling with anti-DNP antibodies. prBSP(E)-DNP was consistently immunodetected along exposed mineralized bone surfaces and osteocyte canaliculi at the surgical site. Few gold particles were observed on these surfaces when prBSP(A)-DNP was infused. Quantitative analyses showed significant differences in labeling between prBSP(E)-DNP (5.04 +/- 0.73 particles/micro m2) and prBSP(A)-DNP (1.37 +/- 0.35 particles/micro m2). These results indicate that poly [E] domains influence binding of prBSP(E) to surfaces presenting a mixture of mineral and proteins bathed by tissue fluids and suggest that they may similarly mediate the interaction of native BSP in the bone environment.
Wazen
Rima M
RM
Laboratory for the Study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dentistry, Université de Montréal, Station Centre-Ville, Montreal, QC, Canada.
Tye
Coralee E
CE
Goldberg
Harvey A
HA
Hunter
Graeme K
GK
Smith
Charles E
CE
Nanci
Antonio
A
D000818:Animals; D001031:Apatites / Q000378:metabolism; D004140:Dinitrophenols; D006801:Humans; D058574:Integrin-Binding Sialoprotein; D008334:Mandible / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011099:Polyglutamic Acid / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000378:metabolism; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism*
D001031:Apatites; D004140:Dinitrophenols; C546871:IBSP protein, human; C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D010455:Peptides; D010750:Phosphoproteins; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; C019529:polyalanine; D011099:Polyglutamic Acid