Regulation of calcium phosphate formation by amelogenins under physiological conditions

Abstract

Amelogenin is essential for proper enamel formation. The present in vitro study extends our previous work at low (10 mM) ionic strength (IS) by examining the effect of amelogenin on mineralization under higher (162 mM) IS conditions found in developing enamel. Full-length phosphorylated (P173) and non-phosphorylated (rP172) amelogenins were examined, along with P148 and rP147 that lack the hydrophilic C-terminus. Calcium phosphate formation was assessed by pH change, while minerals formed were characterized using transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy. Amelogenin self-assembly was also studied using dynamic light scattering and TEM. Results indicate that IS does not influence the effects of rP147, rP172 and P173 have on mineralization. However, in contrast to low IS findings, where both P173 and P148 stabilize initially formed amorphous calcium phosphate (ACP) nanoparticles for >1 d, elongated hydroxyapatite crystals were observed after 24 h using P148 at high IS, unlike that seen with P173. Differences in self-assembly help explain these findings, which suggest that P173 and P148 may play different roles in regulating enamel mineral formation. Present data support the notion that proteolytic processing of P173 is required in vivo to induce the transformation of initial ACP phases to apatitic enamel crystals.

MMP20 Proteolysis of Native Amelogenin Regulates Mineralization In Vitro

“…Although the inhibitory effect of truncated P148 on in vitro mineralization was also found to be concentration dependent, P148 did not similarly guide ordered mineral formation when present at lower concentrations, like full-length P173 . This difference in effects on mineralization may again be related to prior findings showing that truncated P148 and its recombinant analogues from pig (rP147) and mouse (rM166) form random protein assemblies (Fang et al 2011;Kwak et al 2011;Wiedemann-Bidlack et al 2011). In contrast, full-length P173, rP172, and rM179 (from mouse) undergo a stepwise hierarchical self-assembly process from monomers to nanospheres (Fang et al 2011;Fang et al 2013) to more highly ordered chain-like structures (Aichmayer et al 2005;Aichmayer et al 2010;Wiedemann-Bidlack et al 2011).…”

“…Calcium and pH-adjusted phosphate (pH 10.9) solutions were sequentially added to protein solutions, as previously described (Kwak et al 2009;Kwak et al 2011). The final solutions contained 2.5 mM calcium (CaCl 2 ), 1.5 mM phosphate (KH 2 PO 4 ), and 1 mg/mL amelogenin (final volume = 0.03 mL).…”

Regulation of calcium phosphate formation by native amelogenins in vitro

“…The precise pH value was selected by design so that the reaction solution would have an initial pH ∼7.4 at 37°C upon mixing all solution components. A liquots of calcium and pH-adjusted phosphate solution were sequentially added to protein solutions to yield final concentrations of 2.5 mM Ca 2+ , 1.5 mM P i , and 0.2 - 2.0 mg/mL protein, with a final volume of 60 μL, as previously reported (3, 5). Samples were then placed in a thermostatic water bath adjusted to 37°C.…”

“…Although phosphorylation has been found to affect the self-assembly full-length amelogenin in subtle but potentially important ways, it has a marked effect on crystal formation ( e.g ., 3, 4). While oriented, well-organized bundles of crystals are observed in the presence of rP172, P173 was found to stabilize initially formed nano-particles of amorphous calcium phosphate (ACP) and prevent HA formation for up to 3 days in vitro (3, 4). Recently, however, we have found that P173 cleavage by MMP20 (enamelysin) can induce the transformation of stabilized ACP into ordered bundles of HA crystals, suggesting that the formation of the hierarchical enamel structure may be regulated by the proteolysis of full-length native amelogenin, during the early stages of enamel formation [unpublished findings].…”

Amelogenin and enamel biomimetics

“…In light of these amelogenin-MMP-20 interactions, MMP-20 was proposed to: (i) control amelogenin self-assembly, (ii) decrease amelogenin-apatite binding affinity, (iii) control ACP to apatite phase transformation by amelogenin, 110 and/or (iv) prevent unwanted protein occlusion inside apatite crystals. 111 …”