Effect of phosphorylation on the interaction of calcium with leucine-rich amelogenin peptide

Enamel is the most highly mineralized vertebrate tissue composed of ~96% mineral and 4% organic material and water. During amelogenesis, the ameloblast secretes matrix proteins and is responsible for creating and maintaining an extracellular environment favorable to mineral deposition (1). Amelogenin, the predominant enamel matrix protein, has been shown to undergo self-assembly to form spherical or oblate-shaped nanoparticles (2–4), as well as elongated structures (5–7), and is believed to play an essential role in guiding the formation of ordered arrays of apatitic crystals during enamel development (5, 8–12). In particular, the N-terminal (containing the only phosphate group on serine-16) and the hydrophilic C-terminal domains of the full-length amelogenin (Fig. 1) have been shown to be critical for proper enamel formation (13–16). It is also believed that during enamel mineral growth, the free calcium ion concentration is regulated, in part, by the binding of calcium to enamel proteins and their proteolytic cleavage products (17–18). The Leucine Rich Amelogenin Peptide (LRAP), a 56 amino-acid alternative splice product of the amelogenin gene found throughout amelogenesis and comprised of the first 33 N-terminal and the last 23 C-terminal amino acids of full-length amelogenin (Fig. 1), has recently been shown by us to share similar behavioral properties with amelogenin with respect to self-assembly and its ability to regulate crystal growth in vitro (19–20). As in solution (19), LRAP has been also shown to assemble into nanospheres on fluoroapatite (21) and surfactant-coated gold surfaces (22). Furthermore, it has been shown (23) that non-phosphorylated LRAP and recombinant full-length human amelogenin (rH174) have the same capacity to bind calcium (i.e., 4 – 6 calcium ions per molecule), although the calcium affinity constant for LRAP was greater than that for the full-length amelogenin. Based on similarities of structure and behavior, LRAP has allowed us to investigate the potential role of specific amino-acid domains of amelogenin and phosphorylation in protein self-assembly using Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM). Such studies have illustrated potentially important differences in the self-assembly behavior of phosphorylated and non-phosphorylated LRAP (19). The aim of the present study was to extend these recent findings using Small Angle X-ray Scattering (SAXS), to further investigate the role of phosphorylation in LRAP self-assembly, in the presence and absence of calcium, through comparative studies of phosphorylated (LRAP(+P)) and non-phosphorylated (LRAP(−P)) forms of LRAP (Fig. 1). Figure 1 Amino-acid sequences of the full-length porcine amelogenin (P173) and the phosphorylated (+P) and non-phosphorylated (−P) LRAP peptides