In vivo Functional Studies on the Mineral-binding Activity of Bone Sialoprotein

Bone sialoprotein (BSP) is an anionic phosphoprotein expressed in mineralizing connective tissues, shown in vitro to nucleate hydroxyapatite (HA) crystals. The two-polyglutamic acid regions [poly (E)] are believed to be responsible, at least in part, for this activity, in addition to mediating binding to HA. Objectives: The aim of this study was to test the latter in vivo by comparing the mineral binding activity of recombinant BSP (prBSP) with that of BSP in which the poly (E) domains are replaced by polyalanine [poly (A)] by site-directed mutagenesis (pE1,2A BSP). Methods: Male Wistar rats (~ 100g) were anesthetized and an opening was drilled in the bone on the buccal aspect of the hemimandible near the apical end of the incisor. A 1-day osmotic minipump was connected to this 'bony window' and used to deliver native or mutant BSP tagged with dinitrophenol (DNP) at an estimated concentration of 0.06 mg/ml, for a total of 14 µg over the 24-hour period. Rats were then perfused and hemimandibles embedded in LR White resin. Tissue sections from the drill site were processed for colloidal gold immunolabeling with an anti-DNP antibody to reveal the presence of dinitrophenylated compounds. Results: prBSP was immunodetected along the mineralized bone surfaces of the 'bony window' and exposed osteocyte canaliculi. Few gold particles were observed when pE1,2A BSP was infused. Initial analysis indicated a labeling density of 7 particles/µm² along the exposed bone for prBSP and of 2 particles/µm² for pE1,2A BSP. Conclusions: The results indicate that substitution of poly (E) domains with poly (A) reduces the binding of BSP to mineralized bone matrix by about 70%, a finding consistent with its proposed role derived from in vitro assays. The experimental approach used offers the possibility to uniquely examine functional aspects of matrix proteins within the whole-animal biology. Supported by CIHR.