Posttranslational modification in rat bone osteopontin.
Osteopontin is a multiply-phosphorylated glycoprotein which contains an RGD cell adhesion sequence and regions containing a high level of aspartic 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 post-translational modification by a combination of Edman degradation and matrix-assisted laser desorption mass spectrometry. In the peptides analyzed in this way (accounting for approximately half the protein), eleven sites were found that were variably phosphorylated. Three modified lysines were also detected by virtue of their inability to be cleaved by endoprotease Lys-C (Lys19 and Lys286), by observation of an anomalous pth-amino acid during Edman degradation (Lys19 Lys29 and Lys286) and by a mass difference of 115 +/- 5. Bone osteopontin is extremely heterogeneous, as none of these modifications were found in 100% of peptides examined. In this respect, bone osteopontin differs from bovine milk osteopontin in which 28 residues are completely phosphorylated (Sørensen, et al., (1995) Protein Science 4:2040-2049). While the sequences of bovine and rat osteopontin differ significantly, many conserved serines in the N- and C-terminal regions that are phosphorylated in bovine milk osteopontin are at least partially phosphorylated in the rat bone protein. However, there are also conserved residues in both bovine and rat proteins which are phosphorylated in one species but not the other.
Neame
P J
PJ
Shriners Hospital, Department of Biochemistry and Molecular Biology, University of South Florida, Tampa 33612, USA. pneame@com1.med.usf.edu
Butler
W T
WT
D000595:Amino Acid Sequence; D000818:Animals; D001842:Bone and Bones / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds; D053495:Osteopontin; D010446:Peptide Fragments / Q000032:analysis*; D011499:Protein Processing, Post-Translational; D051381:Rats; D012795:Sialoglycoproteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C043517:2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinene 2-oxide; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D053495:Osteopontin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase