Role of 20-kDa Amelogenin (P148) Phosphorylation in Calcium Phosphate Formation in Vitro

Abstract

The potential role of amelogenin phosphorylation in enamel formation is elucidated through in vitro mineralization studies. Studies focused on the native 20-kDa porcine amelogenin proteolytic cleavage product P148 that is prominent in developing enamel. Experimental conditions supported spontaneous calcium phosphate precipitation with the initial formation of amorphous calcium phosphate (ACP). In the absence of protein, ACP was found to undergo relatively rapid transformation to randomly oriented plate-like apatitic crystals. In the presence of non-phosphorylated recombinant full-length amelogenin, rP172, a longer induction period was observed during which relatively small ACP nanoparticles were transiently stabilized. In the presence of rP172, these nanoparticles were found to align to form linear needle-like particles that subsequently transformed and organized into parallel arrays of apatitic needle-like crystals. In sharp contrast to these findings, P148, with a single phosphate group on serine 16, was found to inhibit calcium phosphate precipitation and stabilize ACP formation for more than 1 day. Additional studies using non-phosphorylated recombinant (rP147) and partially dephosphorylated forms of P148 (dephoso-P148) showed that the single phosphate group in P148 was responsible for the profound effect on mineral formation in vitro. The present study has provided, for the first time, evidence suggesting that the native proteolytic cleavage product P148 may have an important functional role in regulating mineralization during enamel formation by preventing unwanted mineral formation within the enamel matrix during the secretory stage of amelogenesis. Results obtained have also provided new insights into the functional role of the highly conserved hydrophilic C terminus found in full-length amelogenin.Extracellular matrix molecules play a crucial role in the regulation of biological mineralization by controlling crystal size, shape, and organization. An example of this exquisite regulation is in the formation of the highly organized dental enamel tissue that is regulated in part by amelogenin, the major extracellular matrix protein secreted by ameloblasts (1). Although amelogenin is processed by proteinases soon after secretion, the intact full-length parent molecule has been found to be exclusively associated with newly formed enamel mineral (2). Prior studies in our laboratory (3) have also shown that fulllength recombinant mouse amelogenin (rM179) can regulate the formation of parallel arrays of apatitic crystals (a salient feature of developing and mature dental enamel) under conditions of spontaneous precipitation in vitro. This functional capability appears to be related to the specific primary structure of the full-length amelogenin and its unique assembly properties under certain physicochemical conditions of pH and temperature (4). In particular, the conserved hydrophilic C terminus of amelogenin has been shown to play a key role in these processes (3).To date, however, most studies have utilized r...

Hierarchical self-assembly of amelogenin and the regulation of biomineralization at the nanoscale

“…The electron diffraction analysis of these mineralization products suggests that at this stage mineral remains amorphous (Fig. S10D) in agreement with our earlier in vitro mineralization studies, which show that the mineral crystallization occurs later (8,28). In contrast to rM179, rM166 was not able to stabilize prenucleation clusters or guide their organization.…”

“…S9B). The ability of rM179 to stabilize the prenucleation clusters and guide their assembly into linear chains that subsequently fuse into the needle-shaped mineral particles is consistent with the view that the enamel crystals form by controlled assembly of prenuclation clusters (8,28) via a nonclassical crystallization mechanism (29). Finally, after 2 h in the control experiments, large aggregates of plate-like mineral particles were observed (Fig.…”

“…S10). Similar mineral bundles formed in the presence of full-length amelogenin have been previously reported in dehydrated samples (1,8,28). A close examination of the needle-shaped mineral particles in the bundles (Figs.…”

“…The major protein of forming enamel, amelogenin, plays essential roles in regulation of the mineralization and structural organization of this tissue (6,7). Amelogenin transiently stabilizes amorphous calcium phosphate (ACP) and regulates the formation of parallel arrays of mineral crystallites (8,9). It is comprised of three domains: an N-terminal tyrosine-rich amelogenin peptide (TRAP), a charged C-terminal hydrophilic telopeptide (C telopeptide), and a central domain rich in X-Y-Pro repeats (2).…”

“…When secreted, amelogenin undergoes a series of proteolytic cleavages starting at the C telopeptide (15). Although amelogenin lacking the C telopeptide forms nanospheres similar to those of a full-length protein (16), they loose the ability to assemble into chains (12,17) and to organize mineral particles (1,8), suggesting a unique role for the full-length molecule in regulation of mineralization. Recent experiments with transgenic animals also indicate that the full-length molecule is absolutely essential for enamel formation (18).…”