Identification and characterization of the major chicken bone phosphoprotein. Analysis of its synthesis by cultured embryonic chick osteoblasts.

The major phosphoprotein synthesized by cultured chicken embryo osteoblasts had a molecular mass of approximately 66 kDa. The 32P label on the protein was cleaved by acid phosphatase treatment and O-[32P]phosphoserine and O-[32P]phosphothreonine could be identified after partial acid hydrolysis. The phosphoprotein contributed approximately 2.0% of the total protein synthesized by osteoblasts and was shown to be secreted, as shown by its presence in the culture media. Glycosylation was demonstrated by the fact that it could be labelled with [3H]galactosamine. The major approximately 66-kDa phosphoprotein was resolved by isoelectric focusing into three major variants with pI values ranging over 3.7 - 3.9; all three forms appear to be the result of variation in the extent of protein phosphorylation. An identical approximately 66-kDa phosphoprotein could be extracted from chicken bones which had both the same range of pI values and an identical elution position following DEAE-Sephacel chromatography. Analysis of the protein isolated from bone demonstrated the presence of sialic acid and, while amino-terminal sequence analysis and internal tryptic fragment sequence analysis of about 25% of the protein revealed little similarity to the rat phosphoprotein osteopontin, a conserved nine-residue sequence spanning the Arg-Gly-Asp cell-binding site of the rat protein osteopontin, was identified in the approximately 66-kDa chicken protein. Peptide mapping with Staphylococcus aureus V8 protease of the in vivo protein compared to the in vitro synthesized protein demonstrated identical peptide fingerprints. The two proteins also had comparable amino acid compositions. Several smaller-molecular-mass phosphoproteins ranging in size over about 55 - 29 kDa were also observed in the HCl extracts of bone. Peptide mapping of these species demonstrated that the approximately 66-kDa, approximately 55-kDa, and approximately 45-kDa species had a common core of peptide fragments. Pulse/chase experiments in culture revealed no evidence for a defined pathway of intracellular proteolysis associated with the approximately 66-kDa species since this phosphoprotein remained the prevalent species after a 24-h chase. Because of the predominant association of all the smaller-molecular-mass forms with the cell layer and an absence of a quantitative conversion to any of the smaller forms over a 24-h chase, these results suggested that the lower-molecular-mass species were not the result of proteolytic processing during synthesis or secretion, but rather represent proteolysis of the approximately 66-kDa component in the extracellular matrix.(ABSTRACT TRUNCATED AT 400 WORDS)