POSTTRANSLATIONAL MODIFICATIONS OF BOVINE OSTEOPONTIN - IDENTIFICATION OF 28 PHOSPHORYLATION AND 3 O-GLYCOSYLATION SITES

Osteopontin (OPN) is a multiphosphorylated glycoprotein found in bone and other normal and malignant tissues, as well as in the physiologica l fluids urine and milk. The present study demonstrates that bovine mi lk osteopontin is phosphorylated at 27 serine residues and 1 threonine residue. Phosphoamino acids were identified by a combination of amino acid analysis, sequence analysis of S-ethylcysteine-derivatized phosp hopeptides, and mass spectrometric analysis. Twenty-five phosphoserine s and one phosphothreonine were located in Ser/Thr-X-Glu/Ser(P)/Asp mo tifs, and two phosphoserines were found in the sequence Ser-X-X-Glu/Se r(P). These sequence motifs are identical with the recognition sequenc es of mammary gland casein kinase and casein kinase II, respectively. Examination of the phosphorylation pattern revealed that the phosphory lations were clustered in groups of approximately three spanned by unp hosphorylated regions of 11-32 amino acids. This pattern is probably o f importance in the multiple functions of OPN involving interaction wi th Ca2+ and inorganic calcium salts. Furthermore, three O-glycosylated threonines (Thr 115, Thr 124, and Thr 129) have been identified in a threonine- and proline-rich region of the protein. Three putative N-gl ycosylation sites (Asn 63, Asn 85, and Asn 193) are present in bovine osteopontin, but sequence and mass spectrometric analysis showed that none of these asparagines were glycosylated in bovine mammary gland os teopontin. Alignment analysis showed that the majority of the phosphor ylation sites in bovine osteopontin as well as all three O-glycosylati on sites were conserved in other mammalian sequences. This conservatio n of serines, even in otherwise less well-conserved regions of the pro tein, indicates that the phosphorylation of osteopontin at specific si tes is essential for the function of the protein.