Complete Topographical Distribution of Both thein Vivoandin VitroPhosphorylation Sites of Bone Sialoprotein and Their Biological Implications
Bone sialoprotein (BSP) is a multifunctional, highly phosphorylated, and glycosylated protein with key roles in biomineralization and tissue remodeling. This work identifies the complete topographical distribution and precise location of both thein vitroandin vivophosphorylation sites of bovine BSP by a combination of state-of-the-art techniques and approaches. In vitrophosphorylation of native and deglycosylated BSPs by casein kinase II identified seven phosphorylation sites by solid-phase N-terminal peptide sequencing that were within peptides 12–22 (LEDS(P)EENGVFK), 42–62 (FAVQSSSDSS(P)EENGNGDS(P)S(P)EE), 80–91 (EDS(P)DENEDEES(P)E), and 135–145 (EDES(P)DEEEEEE). Thein vivophosphorylation regions and sites were identified by use of a novel thiol reagent, 1-S-mono[14C]carboxymethyldithiothreitol. This approach identified all of the phosphopeptides defined byin vitrophosphorylation, but two additional phosphopeptides were defined at residues, 250–264 (DNGYEIYES(P)ENGDPR), and 282–289 (GYDS(P)YDGQ). Furthermore, use of native BSP and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified several of the above peptides, including an additional phosphopeptide at residues 125–130 (AGAT(P)GK) that was not defined in either of thein vitroandin vivostudies described above. Overall, 7in vitroand 11in vivophosphorylation sites were identified unequivocally, with natural variation in the quantitative extent of phosphorylation at eachin vivophosphorylation site.