Post-translationally modified residues of native human osteopontin are located in clusters: identification of 36 phosphorylation and five O-glycosylation sites and their biological implications.

OPN (osteopontin) is an integrin-binding highly phosphorylated glycoprotein, recognized as a key molecule in a multitude of biological processes such as bone mineralization, cancer metastasis, cell-mediated immune response, inflammation and cell survival. A significant regulation of OPN function is mediated through PTM (post-translational modification). Using a combination of Edman degradation and MS analyses, we have characterized the complete phosphorylation and glycosylation pattern of native human OPN. A total of 36 phosphoresidues have been localized in the sequence of OPN. There are 29 phosphorylations (Ser8, Ser10, Ser11, Ser46, Ser47, Thr50, Ser60, Ser62, Ser65, Ser83, Ser86, Ser89, Ser92, Ser104, Ser110, Ser113, Thr169, Ser179, Ser208, Ser218, Ser238, Ser247, Ser254, Ser259, Ser264, Ser275, Ser287, Ser292 and Ser294) located in the target sequence of MGCK (mammary gland casein kinase) also known as the Golgi kinase (S/T-X-E/S(P)/D). Six phosphorylations (Ser101, Ser107, Ser175, Ser199, Ser212 and Ser251) are located in the target sequence of CKII (casein kinase II) [S-X-X-E/S(P)/D] and a single phosphorylation, Ser203, is not positioned in the motif of either MGCK or CKII. The 36 phosphoresidues represent the maximal degree of modification since variability at many sites was seen. Five threonine residues are O-glycosylated (Thr118, Thr122, Thr127, Thr131 and Thr136) and two potential sites for N-glycosylation (Asn63 and Asn90) are not occupied in human milk OPN. The phosphorylations are arranged in clusters of three to five phosphoresidues and the regions containing the glycosylations and the RGD (Arg-Gly-Asp) integrin-binding sequence are devoid of phosphorylations. Knowledge about the positions and nature of PTMs in OPN will allow a rational experimental design of functional studies aimed at understanding the structural and functional interdependences in diverse biological processes in which OPN is a key molecule.

[1]  M. Farach-Carson,et al.  Phosphorylated osteopontin promotes migration of human choriocarcinoma cells via a p70 S6 kinase‐dependent pathway , 2005, Journal of cellular biochemistry.

[2]  E. Salih,et al.  Complete Topographical Distribution of Both the in Vivo and in Vitro Phosphorylation Sites of Bone Sialoprotein and Their Biological Implications* , 2004, Journal of Biological Chemistry.

[3]  O. Baba,et al.  Post-translational modifications of sibling proteins and their roles in osteogenesis and dentinogenesis. , 2004, Critical reviews in oral biology and medicine : an official publication of the American Association of Oral Biologists.

[4]  A. Chambers,et al.  Role of osteopontin in tumour progression , 2004, British Journal of Cancer.

[5]  Daphne Vassiliou,et al.  Differential expression of osteopontin and bone sialoprotein in bone metastasis of breast and prostate carcinoma , 2004, Clinical & Experimental Metastasis.

[6]  K. Hollberg,et al.  TRACP as an Osteopontin Phosphatase , 2003, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[7]  A. Johnsen,et al.  Purification and characterization of osteopontin from human milk. , 2003, Protein expression and purification.

[8]  L. Pinna,et al.  One‐thousand‐and‐one substrates of protein kinase CK2? , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[9]  H. Cantor,et al.  Phosphorylation‐dependent interaction of osteopontin with its receptors regulates macrophage migration and activation , 2002, Journal of leukocyte biology.

[10]  Jinxi Wang,et al.  Natural variation in the extent of phosphorylation of bone phosphoproteins as a function of in vivo new bone formation induced by demineralized bone matrix in soft tissue and bony environments. , 2002, The Biochemical journal.

[11]  C. Tye,et al.  Osteopontin posttranslational modifications, possibly phosphorylation, are required for in vitro bone resorption but not osteoclast adhesion. , 2002, Bone.

[12]  A. Boskey,et al.  Osteopontin Deficiency Increases Mineral Content and Mineral Crystallinity in Mouse Bone , 2002, Calcified Tissue International.

[13]  L. Otvos,et al.  Phosphorylated osteopontin peptides suppress crystallization by inhibiting the growth of calcium oxalate crystals. , 2001, Kidney international.

[14]  M. Noda,et al.  Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival. , 2001, The Journal of clinical investigation.

[15]  D. Torchia,et al.  Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin. , 2001, Biochemical and biophysical research communications.

[16]  R. Cook,et al.  Identification and Characterization of the Carboxyl-terminal Region of Rat Dentin Sialoprotein* , 2001, The Journal of Biological Chemistry.

[17]  C. Giachelli,et al.  Osteopontin: a versatile regulator of inflammation and biomineralization. , 2000, Matrix biology : journal of the International Society for Matrix Biology.

[18]  A. Brunati,et al.  Novel consensus sequence for the Golgi apparatus casein kinase, revealed using proline-rich protein-1 (PRP1)-derived peptide substrates. , 2000, The Biochemical journal.

[19]  C. Giachelli,et al.  Phosphorylation of Osteopontin Is Required for Inhibition of Vascular Smooth Muscle Cell Calcification* , 2000, The Journal of Biological Chemistry.

[20]  K. Masuda,et al.  N-Glycan structures of an osteopontin from human bone. , 2000, Biochemical and biophysical research communications.

[21]  M. Glimcher,et al.  Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity. , 2000, Science.

[22]  M D McKee,et al.  Calcification of vascular smooth muscle cell cultures: inhibition by osteopontin. , 1999, Circulation research.

[23]  M. Farach-Carson,et al.  Modulation of Osteopontin Post-translational State by 1,25-(OH)2-Vitamin D3 , 1998, The Journal of Biological Chemistry.

[24]  K. Schey,et al.  Identification of tryptophan oxidation products in bovine α‐crystallin , 1998, Protein science : a publication of the Protein Society.

[25]  M. Farach-Carson,et al.  Modulation of Osteopontin Post-translational State by 1 , 25-( OH ) 2-Vitamin D 3 DEPENDENCE ON Ca , 1998 .

[26]  L. Pinna,et al.  Phosphorylation of osteopontin by Golgi apparatus casein kinase. , 1997, Biochemical and biophysical research communications.

[27]  M. Glimcher,et al.  Identification of the Phosphorylated Sites of Metabolically 32P-Labeled Osteopontin from Cultured Chicken Osteoblasts* , 1997, The Journal of Biological Chemistry.

[28]  G. Kundu,et al.  Altered sialylation of osteopontin prevents its receptor-mediated binding on the surface of oncogenically transformed tsB77 cells. , 1997, Biochemistry.

[29]  M. Glimcher,et al.  Protein kinases of cultured osteoblasts: Selectivity for the extracellular matrix proteins of bone and their catalytic competence for osteopontin , 1996, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[30]  S. Carr,et al.  Phosphopeptide analysis by matrix-assisted laser desorption time-of-flight mass spectrometry. , 1996, Analytical chemistry.

[31]  M. Glimcher,et al.  Phosphorylation of Purified Bovine Bone Sialoprotein and Osteopontin by Protein Kinases* , 1996, The Journal of Biological Chemistry.

[32]  L. Pinna,et al.  Golgi apparatus mammary gland casein kinase: monitoring by a specific peptide substrate and definition of specificity determinants , 1996, FEBS letters.

[33]  W. Butler,et al.  Posttranslational modification in rat bone osteopontin. , 1996, Connective tissue research.

[34]  P. Højrup,et al.  Posttranslational modifications of bovine osteopontin: Identification of twenty‐eight phosphorylation and three O‐glycosylation sites , 1995, Protein science : a publication of the Protein Society.

[35]  D. Heinegård,et al.  Dephosphorylation of osteopontin and bone sialoprotein by osteoclastic tartrate-resistant acid phosphatase. Modulation of osteoclast adhesion in vitro. , 1994, The Journal of biological chemistry.

[36]  T. E. Petersen,et al.  Identification of two phosphorylation motifs in bovine osteopontin. , 1994, Biochemical and biophysical research communications.

[37]  T. E. Petersen,et al.  Phosphorylation, glycosylation and amino acid sequence of component PP3 from the proteose peptone fraction of bovine milk , 1993, Journal of Dairy Research.

[38]  A. Boskey,et al.  Osteopontin-hydroxyapatite interactions in vitro: inhibition of hydroxyapatite formation and growth in a gelatin-gel. , 1993, Bone and mineral.

[39]  E. Neilson,et al.  Inhibition of calcium oxalate crystal growth in vitro by uropontin: another member of the aspartic acid-rich protein superfamily. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[40]  B. B. Mukherjee,et al.  Physiological properties and differential glycosylation of phosphorylated and nonphosphorylated forms of osteopontin secreted by normal rat kidney cells. , 1990, The Journal of biological chemistry.

[41]  B. Kemp,et al.  Protein kinase recognition sequence motifs. , 1990, Trends in biochemical sciences.

[42]  D. Tenen,et al.  Purification of a human milk protein closely similar to tumor-secreted phosphoproteins and osteopontin. , 1989, Biochimica et biophysica acta.

[43]  V. Barkholt,et al.  Amino acid analysis: determination of cysteine plus half-cystine in proteins after hydrochloric acid hydrolysis with a disulfide compound as additive. , 1989, Analytical biochemistry.

[44]  R. Schrohenloher,et al.  Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone. , 1987, The Journal of biological chemistry.

[45]  D. Heinegård,et al.  Isolation and characterization of two sialoproteins present only in bone calcified matrix. , 1985, The Biochemical journal.

[46]  J. Mercier.  Phosphorylation of caseins, present evidence for an amino acid triplet code posttranslationally recognized by specific kinases. , 1981, Biochimie.

[47]  K. Abromeit.  Music Received , 2023, Notes.