Posttranslational modification and proteolytic processing of urinary osteopontin
Résumé
Osteopontin (OPN) 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 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 upon the highly anionic character of the protein. Besides a very high content of aspartic- and glutamic acid residues, OPN is subjected to significant posttranslational modification (PTM), such as phosphorylation, sulphation 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. Mass spectrometric (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 sulphated 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}.
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