Post-translational modification and proteolytic processing of urinary osteopontin.
OPN (osteopontin) is a highly phosphorylated glycoprotein present in many tissues and body fluids. In urine, OPN is a potent inhibitor of nucleation, growth and aggregation of calcium oxalate crystals, suggesting that it has a role in the prevention of renal stone formation. The role of OPN in nephrolithiasis is, however, somewhat unclear, as it may also be involved in urinary stone formation, and it has been identified among the major protein components of renal calculi. Most likely, the function of OPN in urine is dependent on the highly anionic character of the protein. Besides a very high content of aspartic and glutamic residues, OPN is subjected to significant PTM (post-translational modification), such as phosphorylation, sulfation and glycosylation, which may function as regulatory switches in promotion or inhibition of mineralization. In the present study, we have characterized the PTMs of intact human urinary OPN and N-terminal fragments thereof. MS analysis showed a mass of 37.7 kDa for the intact protein. Enzymatic dephosphorylation and peptide mass analyses demonstrated that the protein contains approximately eight phosphate groups distributed over 30 potential phosphorylation sites. In addition, one sulfated tyrosine and five O-linked glycosylations were identified in OPN, whereas no N-linked glycans were detected. Peptide mapping and immunoblotting using different monoclonal antibodies showed that the N-terminal fragments present in urine are generated by proteolytic cleavage at Arg(228)-Leu(229) and Tyr(230)-Lys(231).
Christensen
Brian
B
Protein Chemistry Laboratory, Department of Molecular Biology and Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
Petersen
Torben E
TE
Sørensen
Esben S
ES
D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D053495:Osteopontin / Q000652:urine*; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational
D010446:Peptide Fragments; D053495:Osteopontin; D010447:Peptide Hydrolases