POSTTRANSLATIONAL MODIFICATION IN RAT BONE OSTEOPONTIN

Osteopontin is a multiply-phosphorylated glycoprotein which contains a n RGD cell adhesion sequence and regions containing a high level of as partic acid, It is abundant in bone, milk, and urine and is also found in malignant tissues, Peptides generated from rat bone osteopontin by digestion with endoprotease Lys-C were analyzed for posttranslational modification by a combination of Edman degradation and matrix-assiste d laser desorption mass spectrometry, In the peptides analyzed in this way (accounting for approximately half the protein), eleven sites wer e found that were variably phosphorylated, Three modified lysines were also detected by virtue of their inability to be cleaved by endoprote ase Lys-C (Lys(19) and Lys(286)), by observation of an anomalous pth-a mino acid during Edman degradation (Lys(19) Lys(29) and Lys(286)) and by a mass difference of 115 +/- 5. Bone osteopontin is extremely heter ogeneous, as none of these modifications were found in 100% of peptide s examined, In this respect, bone osteopontin differs from bovine milk osteopontin in which 28 residues are completely phosphorylated (Soren sen, et at, (1995) Protein Science 4:2040-2049), While the sequences o f bovine and rat osteopontin differ significantly, many conserved seri nes in the N- and C-terminal regions that are phosphorylated in bovine milk osteopontin are at least partially phosphorylated in the rat bon e protein, However, there are also conserved residues in both bovine a nd rat proteins which are phosphorylated in one species but not the ot her.