Relationship between length variations in Ser/Asp-rich repeats in phosphophoryn and in vitro precipitation of calcium phosphate.

ABSTRACT

OBJECTIVE: Phosphophoryn (PP) is generated from the proteolytic cleavage of dentin sialophosphoprotein (DSPP). PP which contains tandem serine/asparatic acid rich repeats (SDrr) is known to enhance dentin mineralization. The nucleotide sequences coding SDrr are identified in the DSPP genes of toothed animals and the length variations of SDrr between intra- and inter-species have been reported. However, it remains unknown about the relationship between the length variations in SDrr and the functions of PP in matrix mineralization. DESIGN: By utilizing a mammalian expression system, we generated several recombinant PP proteins (rPP) containing SDrr of different lengths and analyzed their effects on the precipitation of calcium phosphate with an in vitro gel diffusion system. RESULTS: rPP-Δ37.6 SDrr and rPP-Δ63.5 SDrr, which possessed shortened SDrr that accounted for 62.4 and 36.5% the length of SDrr in full-length rPP (rPP-full), respectively, induced the precipitation of calcium phosphate similar to that of rPP-full at the same molar concentration, whereas rPP-ΔSDrr, in which SDrr were flipped, did not. Furthermore, rPP-Δ63.5 SDrr significantly increased the accumulation of calcium compared with rPP-full at adjusted concentrations so that the same amounts of SDrr were embedded. The results of an ELISA analysis indicated that the amounts of rPP-Δ37.6 SDrr and rPP-Δ63.5 SDrr secreted from transfected cells were 5.2- and 7.1-fold greater than that of rPP-full, respectively. CONCLUSIONS: The generated rPP-Δ63.5 SDrr which can be substituted for rPP-full may be a candidate for a therapeutic molecule to facilitate hard tissue generation such as reparative dentin formation.