Loss of biological control of enamel mineralization in amelogenin-phosphorylation-deficient mice

Abstract

Amelogenin, the most abundant enamel matrix protein, plays several critical roles in enamel formation. Importantly, we previously found that the singular phosphorylation site at Ser16 in amelogenin plays an essential role in amelogenesis. Studies of genetically knock-in (KI) modified mice in which Ser16 in amelogenin is substituted with Ala that prevents amelogenin phosphorylation, and in vitro mineralization experiments, have shown that phosphorylated amelogenin transiently stabilizes amorphous calcium phosphate (ACP), the initial mineral phase in forming enamel. Furthermore, KI mice exhibit dramatic differences in the enamel structure compared with wild type (WT) mice, including thinner enamel lacking enamel rods and ectopic surface calcifications. Here, we now demonstrate that amelogenin phosphorylation also affects the organization and composition of mature enamel mineral. We compared WT, KI, and heterozygous (HET) enamel and found that in the WT elongated crystals are co-oriented within each rod, however, their c-axes are not aligned with the rods' axes. In contrast, in rod-less KI enamel, crystalline c-axes are less co-oriented, with misorientation progressively increasing toward the enamel surface, which contains spherulites, with a morphology consistent with abiotic formation. Furthermore, we found significant differences in enamel hardness and carbonate content between the genotypes. ACP was also observed in the interrod of WT and HET enamel, and throughout aprismatic KI enamel. In conclusion, amelogenin phosphorylation plays crucial roles in controlling structural, crystallographic, mechanical, and compositional characteristics of dental enamel. Thus, loss of amelogenin phosphorylation leads to a reduction in the biological control over the enamel mineralization process.

Keywords: Amelogenin; Biomineralization; Dental enamel; Phosphorylation.

Figure 1.

SE SEM of etched erupted portions of enamel in WT (A-C), KI (D-F) and HET (G). A – low, B – intermediate and C – high magnification micrographs of the same region of WT enamel. The bulk of WT enamel (EN) comprises highly organized quasiorthogonal pattern of alternating rows of enamel rods with the interrod matter interwoven between the rods. The planes of enamel rods form right angle with the DEJ plane in the direction of the occlusal tip of the incisor (left). In the outer layer of enamel the rods change their direction and become more aligned with the plane of enamel surface. Note: the bright thin coating of the enamel surface is the iron-rich layer in A and B. DE- dentin. D. Low magnification micrograph of KI enamel. The inner portion of the etched enamel has a homogeneous appearance while the outer layer is highly disorganized with deep etched areas and patches of a highly porous matter. String of spherulites is attached to the enamel surface. E. Inner enamel layer adjacent to the dentino-enamel junction (DEJ) (arrows). F. A large magnification image of a spherulite. Note the radial etching pattern. G. HET enamel with KI-like area on the very left of the micrograph and WT-like area occupying the majority of the image. Note that some areas of the WT-like area are more soluble than others, based on the deeper etching profile.

Figure 2.

A-G and I PEEM PIC maps of WT, KI, and HET mature enamel. The key for the color-coding of c-axes orientations is in the lower left corner of panel E. A is low and B, C are high magnification maps of mature WT enamel as indicated by the white boxes in A. D. a map of KI enamel. E. Close up of the area in the white square in D containing a spherulite. F and G are low and high magnification maps of mature HET enamel. The double arrow in E indicates transition from WT-like to KI-like phenotype. H. Polarized light micrograph of a spherulite. The inset is the closeup of the boxed spherulite. I. Surface layer of Het enamel, with a spherulite attached to the enamel surface. The precise locations relative to the tooth cross-section are shown in Fig. S4. DE-Dentin, IEN – inner enamel; OEN– Outer enamel; IR – interrod, R – rod.

Figure 3.

A WT, B KI C minimally defective HET, and D extensively defective HET mouse enamel PIC maps (A1-D1), calcium distribution maps (A2-D2), component maps (A3-D3), and pixels from component maps containing a least 90% Amorphous Calcium Phosphate (ACP) overlaid on the calcium distribution maps (A4-D4). PIC maps show the morphology and crystal orientations of rods and interrod in WT and HET enamel and the aprismatic KI enamel according to the color bar in B1. Calcium distribution maps of the corresponding areas indicate where calcium concentration is higher or lower in the enamel. Component maps indicate the mineral phases detected in each 20-nm pixel and displayed in colors according to the color legend in b3: RGB are ACP, Low Crystallinity-HydroxyAPatite (LC-HAP), and HydroxyAPatite (HAP), respectively. A1-A4 WT mouse enamel is mainly comprised of HAP with distinct areas of ACP concentrated in the interrod. B1-B4 KI mouse enamel has no rods (aka prisms), thus it can all be considered “aprismatic” enamel, and shows a number of areas of ACP throughout. C1-C4 HET mouse enamel has more ACP in the interrod compared to WT enamel. D1-D4 HET in a different region of the same tooth in C1-C4, but with much more ACP, all localized in the interrod. In all panels, pixels that contain ACP in component maps appear black in PIC maps and calcium distribution maps, consistent with no crystallinity. The precise locations of these 4 areas relative to the tooth cross-section are shown in Fig. S4.

Figure 4.

Spectra from reference standards of ACP, LCHAP, HAP (from Beniash et. al. 2009), the component spectra for ACP, LCHAP, HAP, extracted from mouse enamel and used for the component maps in Fig. 2 and single-pixel spectra extracted from WT, HET, and KI enamel for each of the three phases, A-C respectively. D Averaged spectra extracted from only the rod or interrod regions in WT and HET and aprismatic enamel in KI. The WT rod spectrum is mostly HAP, but peak 2 is not as sharp in the WT rod spectrum as it is in the HAP reference spectrum. KI aprismatic enamel is also mostly HAP, but peak 2 is even less sharp than in WT rod or HAP reference spectra, suggesting that KI aprismatic enamel is slightly more disordered than WT rod enamel. WT interrod has a low peak 1 intensity reminiscent of the ACP reference spectrum, but peak 2 is sharper than the ACP reference suggesting that WT interrod is a mixture of ACP and HAP.

Figure 5.

A, B Representative Raman spectra taken from WT (black) and KI (red) mature incisal enamel. A. A spectral region containing major differences between WT and KI enamel from 1200 to 400 cm-1. B. A close up view of the three regions with most prominent differences. The most prominent and important difference is in 1071 cm-1 band, indicating an increase in carbonate level. There are also change in phosphate vibration regions at 620 – 570 cm-1 and 500 – 400 cm-1. C. Box plot of Vickers Hardness values for WT and KI erupted incisal enamel.