Posttranslational modifications of bovine osteopontin: identification of twenty-eight phosphorylation and three O-glycosylation sites.
Osteopontin (OPN) is a multiphosphorylated glycoprotein found in bone and other normal and malignant tissues, as well as in the physiological fluids urine and milk. The present study demonstrates that bovine milk 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 phosphopeptides, and mass spectrometric analysis. Twenty-five phosphoserines and one phosphothreonine were located in Ser/Thr-X-Glu/Ser(P)/Asp motifs, and two phosphoserines were found in the sequence Ser-X-X-Glu/Ser(P). These sequence motifs are identical with the recognition sequences of mammary gland casein kinase and casein kinase II, respectively. Examination of the phosphorylation pattern revealed that the phosphorylations were clustered in groups of approximately three spanned by unphosphorylated regions of 11-32 amino acids. This pattern is probably of importance in the multiple functions of OPN involving interaction with 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-glycosylation 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 osteopontin. Alignment analysis showed that the majority of the phosphorylation sites in bovine osteopontin as well as all three O-glycosylation sites were conserved in other mammalian sequences. This conservation of serines, even in otherwise less well-conserved regions of the protein, indicates that the phosphorylation of osteopontin at specific sites is essential for the function of the protein.
Sørensen
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ES
Protein Chemistry Laboratory, University of Aarhus, Denmark.
Højrup
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Petersen
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TE
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence; D005260:Female; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008892:Milk; D008969:Molecular Sequence Data; D053495:Osteopontin; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D010769:Phosphothreonine / Q000032:analysis; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D012694:Serine; D012795:Sialoglycoproteins / Q000737:chemistry* / Q000378:metabolism*
D010446:Peptide Fragments; D010750:Phosphoproteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D053495:Osteopontin; D010769:Phosphothreonine; D010768:Phosphoserine; D012694:Serine