Identification of osteopontin phosphorylation sites involved in bone remodeling and inhibition of pathological calcification

Abstract

Osteopontin is a noncollagenous, phosphorylated extracellular glycoprotein, expressed in mineralized and nonmineralized tissues, organs and body fluids. The protein contains an RGD tripeptide cell-binding motif, and is subjected to a variety of posttranslational modifications that play important roles in its multiple biological functions, such as bone remodeling and inhibition of pathological calcification. In this study, we have expressed bovine osteopontin in a prokaryotic system and identified the seven amino acid residues phosphorylated in vitro by CKII.

Fig. 1

Expression, purification and thrombin cleavage of GST fused bovine osteopontin. Lane A: Prestained protein marker. Lane B: GST fused OPN, no thrombin. Lane C: The released recombinant OPN at 4 U thrombin per mg of GST fused protein. Lane D: The released recombinant OPN at 2 U thrombin per mg of GST fused protein. A portion of the recombinant protein was mixed with protein loading dye, fractionated onto 10% SDS-PAGE, and stained with Stains-all.

Fig. 2

The phosphorylated tryptic peptides of recombinant bovine osteopontin. The peptides were separated by RP-HPLC on a Vydac C18 column (25 cm × 0.46 cm) using a linear gradient from H₂O and 0.1% (v/v) trifluoroacetic acid to 60% CH₃CN and 0.055% (v/v) trifluoroacetic acid over 80 min at a flow rate of 1 ml/min. Absorbance at 219 nm was recorded continuously. Fractions of 1 ml were collected, and aliquots were counted for ³²P radioactivity. The phosphorylated tryptic peptides are numbered.