Osteopontin (OPN) is an acidic phosphoglycoprotein that is believed to function in the prevention of soft tissue calcification. In vitro studies have shown that OPN can inhibit the formation of hydroxyapatite (HA) and other biologically relevant crystal phases, and that this inhibitory activity requires phosphorylation of the protein; however, it is not known which phosphorylated residues are involved. We have synthesized peptides corresponding to four phosphoserine-containing sequences in rat OPN: OPN7–17, containing phosphoserines 10 and 11; OPN41–52, containing phosphoserines 46 and 47; OPN248–264, containing phosphoserines 250, 257 and 262; and OPN290–301, containing phosphoserines 295–297. The abilities of these peptides to inhibit de novo HA formation were determined using a constant-composition autotitration assay. All four OPN phosphopeptides caused a dose-dependent increase in nucleation lag time, but did not significantly affect subsequent formation of the crystals. However, OPN41–52 (inhibitory constant 73.5 min/µM) and OPN290–301 (72.2 min/µM) were approx. 4 times more potent inhibitors than OPN7–17 (19.7 min/µM) and OPN247–264 (16.3 min/µM). ‘Scrambling’ the amino acid sequence of OPN290–301 resulted in decreased potency (45.6 min/µM), whereas omission of the phosphate groups from this peptide caused a greater decrease (5.20 min/µM). These findings have identified phosphorylated sequences that are important for the ability of rat bone OPN to inhibit HA crystal formation, and suggest that negative-charge density is an important factor in this activity.
Abbreviations used: Fmoc, fluoren-9-ylmethoxycarbonyl; HA, hydroxyapatite; OPN, osteopontin; RP-HPLC, reversed-phase HPLC; TFA, trifluoroacetic acid.