Biochemical study on the tissue compartmentalization of phosphophoryn, osteopontin and bone sialoprotein (BSP) in rat incisor dentin

Abstract

Abstract:The tissue compartmentalization of phosphophoryn, osteopontin (OPN) and bone sialoprotein (BSP) in dentin was biochemically analyzed. The radicular portion of rat incisor was excised and sequentially extracted; i.e., proteins non-covalently bound to matrix such as soft tissues were solubilized with 4 M guanidinium chloride (G1), mineral-binding proteins were then extracted with 0.5 M EDTA (E), proteins noncovalently bound to the collagen matrix were extracted with 4 M guanidinium chloride (G2) after E, and finally the proteins covalently bound to the collagen matrix were extracted by bacterial collagenase digestion (C). Stains-All and Rhodamine B staining, which selectively stain calciun~-binding proteins, revealed a 50~80 kDa broad band in the E extract on 10 % polyacrylamide gels. This intense band disappeared after precipitation with CaC12, indicating that the protein is phosphophoryn. After removing phosphophoryn from the E extract, 67 kDa intense and 50 kDa weak bands appeared on the gels stained with Stains-All. The 67 kDa protein was observed similarly in the G2 and C extracts. Western blots using monoclonal antibodies revealed the presence of 67 kDa BSP and 50 kDa OPN. When the tissue compartmentalization was compared, these three proteins were not detected in the G1 extract, phosphophoryn and osteopontin were localized only in the E extract, and BSP was observed not only in the E extract but also in the G2 and C extracts. These findings indicate that phosphophoryn, OPN and BSP are rapidly incorporated into the mineralized fraction and that BSP also binds to collagen-related fraction, suggesting the association with intial mineralization in the dentin.

Contribution of biomimetic collagen-ligand interaction to intrafibrillar mineralization

“…Osteocalcin, one of the most abundant noncollagenous proteins in bone, has been detected in both the gap zone and the overlap zone of collagen fibrils and appear as single spheres or pearl necklace–like strings ( 14 , 15 ). Guanidine and collagenase extraction studies demonstrated that matrix phosphoproteins bind to collagen via ion association or covalent bonding ( 16 ). Collagen secreted by fibroblasts does not mineralize in the presence of mineralization precursors except for areas with ectopic calcification ( 17 ); matrix phosphoproteins are also involved in ectopic mineralization of vascular tissues ( 18 ).…”