Biosynthesis of osteonectin by fetal porcine calvarial cells in vitro

The biosynthesis of osteonectin, a major glycoprotein of bone, has been studied in vitro using bone cells from fetal porcine calvariae. The calvarial cells, which were shown to produce osteonectin by immunotransfer and immunocytochemical analysis, were pulse labeled with [35S]methionine and the radiolabeled osteonectin in the cell layers and in the chase-medium was isolated by specific immunoprecipitation. The osteonectin, representing 0.1% of the radiolabeled cell layer and 1% of the medium proteins, co-migrated with authentic bovine osteonectin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced and nonreduced conditions (Mr approximately 40,000), and was bound selectively to hydroxyapatite in the presence of 4 M guanidine HCl. The co-migration of the osteonectins indicates that the tissue-extracted osteonectin represents an intact protein and that a pro- form of osteonectin is unlikely. However, a preosteonectin form (Mr approximately 46,000) could be immunoprecipitated from cell-free translation of calvarial mRNA. The minimal secretion time for osteonectin synthesized in serum-containing medium was found to be approximately 40 min but maximal accumulation did not occur until 2 h postlabeling. In serum-free media, extensive degradation of osteonectin, characterized by the presence of two immunoprecipitable fragments (Mr approximately 36,000 and 31,000), was evident.