BIOSYNTHESIS OF BONE PROTEINS [SPP-1 (SECRETED PHOSPHOPROTEIN-1, OSTEOPONTIN), BSP (BONE SIALOPROTEIN) AND SPARC (OSTEONECTIN)] IN ASSOCIATION WITH MINERALIZED-TISSUE FORMATION BY FETAL-RAT CALVARIAL CELLS IN CULTURE

To determine the relationship between the expression of bone proteins and the formation of mineralized-tissue matrix, the biosynthesis of non-collagenous bone proteins was studied in cultures of fetal-rat calvarial cells, which form mineralized nodules of bone-like tissue in the presence of beta-glycerophosphate. The temporal pattern of protein synthesis in both mineralizing and non-mineralizing cultures was studied by metabolic labelling with [S-35]methionine, (SO42-)-S-35 or (PO43-)-P-32 over a 5-day period. After a 24 h labelling period, the culture media were harvested and the cell layers extracted sequentially with aq. 0.5 M-NH3, followed by 4 M-guanidinium chloride (GdmCl), 0.5 M-EDTA and a second extraction with 4 M-GdmCl. Protein associated with collagenous bone matrix was analysed after digestion with bacterial collagenase. On the basis of [S-35]methionine labelling, the major proteins extracted from the mineralizing matrix were secreted phosphoprotein-1 (SPP-1; osteopontin), bone sialoprotein (BSP) and a 14 kDa phosphoprotein. The presence of SPP-1 and BSP in the conditioned media of both mineralizing and non-mineralizing cultures and their incorporation into the mineralizing nodules indicated that these proteins associate with preformed mineral crystals. However, some BSP was also present in GdmCl extracts and, together with a 35 kDa sulphated protein, was released from a bacterial-collagenase digestion of the tissue residue in both non-mineralizing and mineralizing cultures. Two forms of sulphated SPP-1 were identified, a highly phosphorylated 44 kDa species being the predominant form in the mineralized matrix. The BSP was more highly sulphated but less phosphorylated than SPP-1. Bone SPARC (secreted protein, acid and rich in cysteine) protein (osteonectin) was present almost entirely in the conditioned media and did not incorporate (PO43-)-P-32 or (SO42-)-S-35. The SPP-1 and the 14 kDa protein were susceptible to thrombin digestion, the 44 kDa SPP-1 being specifically cleaved into 28 and 26 kDa fragments. The fragments were labelled uniformly with [S-35]methionine, but the 28 kDa fragment incorporated more (SO42-)-S-35, but less (PO43-)-P-32, than the 26 kDa fragment. These studies demonstrate that SPP-1 and BSP are the major osteoblast-derived bone proteins to bind to the bone mineral. The BSP also binds to the collagenous bone matrix indicates a potential role for this protein in linking the hydroxyapatite with collagen.