Effects of phosphorylation on the self-assembly of native full-length porcine amelogenin and its regulation of calcium phosphate formation in vitro

Abstract

The self-assembly of the predominant extracellular enamel matrix protein amelogenin plays an essential role in regulating the growth and organization of enamel mineral during early stages of dental enamel formation. The present study describes the effect of the phosphorylation of a single site on the full-length native porcine amelogenin P173 on self-assembly and on the regulation of spontaneous calcium phosphate formation in vitro. Studies were also conducted using recombinant non-phosphorylated (rP172) porcine amelogenin, along with the most abundant amelogenin cleavage product (P148) and its recombinant form (rP147). Amelogenin self-assembly was assessed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Using these approaches, we have shown that self-assembly of each amelogenin is very sensitive to pH and appears to be affected by both hydrophilic and hydrophobic interactions. Furthermore, our results suggest that the phosphorylation of the full-length porcine amelogenin P173 has a small but potentially important effect on its higher-order self-assembly into chain-like structures under physiological conditions of pH, temperature, and ionic strength. Although phosphorylation has a subtle effect on the higher-order assembly of full-length amelogenin, native phosphorylated P173 was found to stabilize amorphous calcium phosphate for extended periods of time, in sharp contrast to previous findings using non-phosphorylated rP172. The biological relevance of these findings is discussed.

Figure 1

Aligned amino acids sequences of recombinant and native porcine amelogenins used in the present study. Native proteins are phosphorylated at S-16, unlike the recombinant counterparts. Recombinant proteins also lack the N-terminal methionine. P148 and rP147 represent native and recombinant protein cleavage products, respectively, which lack 25 C-terminal amino acids, including an 11 amino acidic hydrophilic domain, as indicated.

Figure 2

DLS results for recombinant (rP147, rP172) and native (P148, P173) porcine amelogenins at a concentration of 2mg/mL in Tris-HCl buffer, illustrating the effect of pH on the hydrodynamic radius (R_H) of studied proteins at ionic strengths of IS = 15 mM, 64 mM, and 163 mM. The pH of the sample changes with temperature, as indicated in the x axes, due to the high temperature coefficient of −0.031 ΔpH/°C of Tris-HCl, as described in Materials and methods. Note that higher-order self-assembly, that is the pH/temperature where off scale scattering occurs, appears to be more affected by changes in ionic strength in recombinant amelogenins compared to the native proteins. Differences and similarities in protein behavior are discussed in the text.

Figure 3

TEM results for recombinant and native porcine amelogenins at 2mg/mL in Tris-HCl buffer, pH 7.2 and ionic strength of IS = 163 mM. A) rP147, large aggregate and threadlike structures. B) P148 forms large agglomerates. C) rP172, elongated structures of quite variable length, some isolated spherical particles. D) P173, mostly isolated spherical particles and some small elongated structures. Scale bar = 100 nm.

Figure 4

TEM micrographs of A) P173 at pH = 7.0 in MES buffer, ionic strength IS = 163mM and B) rP172 at pH = 7.2 in Tris-HCl buffer, ionic strength IS = 163mM. Scale bar = 100nm. Note that both full-length native and recombinant amelogenin form elongated structures made up of smaller apparently spherical subunits.

Figure 5

Comparison between native and recombinant pig amelogenins at 2mg/mL in Tris-HCl buffer, pH = 7.2 and ionic strengths of 15 mM (A – D) and 64 mM (E – H). A) and E) rP147; B) and F) P148; C) and G) rP172; D) and H) P173. Scale bar = 100 nm. Note that rP172 prevails as isolated spherical particles at low ionic strength of 15 mM but forms elongated structures at higher ionic strength at the same pH. The change in ionic strength appears to have the opposite effect in P173, with more and longer elongated features at low ionic strength and more isolated spherical particles at higher ionic strength (see also Fig. 2). Additional details are provided in the text and in Table 3.

Figure 6

Changes in pH as a function of time observed during mineralization experiments, in the absence (a, control) and presence of P173 (b), monitored over a 24-h period (B). Changes in pH are presented in an expanded scale (A) for better clarity. Similar results were obtained for multiple repeats (at least n=3) using 5 different preparations of P173. The significance of observed differences is discussed in the text.

Figure 7

TEM micrographs of calcium phosphate mineral products formed in the absence (A, control) and presence (B) of P173 examined at selected times (10 min, 40 min, 1 hour, and 1 day), as described in Materials and methods. As shown (A and B) at 10 min, amorphous calcium phosphate (ACP) was initially formed in the control and in the presence of P173, based on the observed (insets) selected area electron diffraction (SAED) patterns. Subsequent changes in mineral particle shape and organization with time are described in the text. As described, in the control ACP particles form networks that appear to undergo transformation within 1 hour. After 1 day, as shown, randomly arranged plate-like apatitic crystals were found in the control (A, inset – showing circular distribution of apatitic reflections). In contrast, ACP was observed in the presence of P173 even after 1 day (B, inset).

Figure 8

FT-IR spectra of calcium phosphate minerals (after 1 day) produced in the absence (a, control) and presence (b) of P173. FT-IR data for the control are consistent with a poorly crystalline apatitic phase (a), while the spectrum obtained for samples produced in the presence of P173 is characteristic of ACP (b). As discussed in the text, Amide I and Amide II bands can also be seen in the latter spectrum. FT-IR results are consistent with noted SAED findings.