Posttranslational modifications of bovine osteopontin: Identification of twenty‐eight phosphorylation and three O‐glycosylation sites

Abstract

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-XGlu/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 11, 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 0-glycosylated threonines (Thr 115, Thr 124, and Thr 129) have been identified in a threonineand 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 0-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.Keywords: mineralization; 0-glycosylation; osteopontin; phosphorylation; phosphoserine; phosphothreonine; S-ethylcysteine The phosphorylation of serine, threonine, and tyrosine residues in intracellular proteins is an essential and well-documented mechanism in the regulation of cell physiology. Less attention has been directed to the localization and effects of phosphorylation in extracellular proteins. As the number of examples of phosphorylated proteins has intensified, it has become apparent that a majority of these contain multiple phosphorylations (Roach, 1991). In extracellular proteins, these multiple phosphorylations are often of structural importance in the forma-.~

Identification of the Phosphorylated Sites of Metabolically 32P-Labeled Osteopontin from Cultured Chicken Osteoblasts

“…Recently, 27 phosphorylated serines and one phosphorylated threonine were identified in bovine milk OPN (58). The predominant sites for phosphorylation contained recognition sequences for casein kinase II.…”

The Role of Osteopontin in Tumor Progression and Metastasis in Breast Cancer

“…Close to the RGD sequence, a site for thrombin cleavage is conserved in all known osteopontin species (67,89,90). The susceptibility of osteopontin to thrombin cleavage opens the possibility that osteopontin may be cleaved during the course of blood coagulation, and in tissues and fluids exhibiting thrombin-like proteolytic activity.…”

“…It seems to be an important characteristic of the protein because cleavage products have been observed in rat plasma and rat tumors (28), human milk (89), and pig bone (54). Fragments of osteopontin originating from either unknown or other proteolytic activities have also been identified in human milk (67) and in human uterus (91). Functionally, fragments of osteopontin produced by thrombin cleavage amplify the effects of the full-length protein (92).…”

L’ostéopontine, une molécule aux multiples facettes

“…C'est une petite molécule de structure très complexe, d'environ 60 kDa, avec des isoformes multiples correspondant à des épissages alternatifs différents, et surtout à des modifications post-traductionnelles par phosphorylation et glycosylation variables, expliquant les différents poids moléculaires apparents et les différents noms rapportés dans la littérature (sialoprotéine I, gène eta-1, 44 kDa bone pp, 2 ar, pp 69, pp 62, spp 1). L'OPN est aussi un substrat de la transglutaminase, qui permet sa polymérisation en complexes de plus de 4 000 kDa [3,4]. Il existe des formes intracellulaires et extracellulaires, comme dans la matrice extracellulaire de l'os et dans certains fluides (lait, urine, bile) [5].…”