Characterization of inter-crystallite peptides in human enamel rods reveals contribution by the Y allele of amelogenin.

Proteins of the inter-rod sheath and peptides within the narrow inter-crystallite space of the rod structure are considered largely responsible for visco-elastic and visco-plastic properties of enamel. The present study was designed to investigate putative peptides of the inter-crystallite space. Entities of 1-6 kDa extracted from enamel rods of erupted permanent teeth were analysed by mass spectrometry (MS) and shown to comprise N-terminal amelogenin (AMEL) peptides either containing or not containing exon 4 product. Other dominant entities consisted of an N-terminal peptide from ameloblastin (AMBN) and a series of the most hydrophobic peptides from serum albumin (ALBN). Amelogenin peptides encoded by the Y-chromosome allele were strongly detected in Enamel from male teeth. Location of N-terminal AMEL peptides as well as AMBN and ALBN, between apatite crystallites, was disclosed by immunogold scanning electron microscopy (SEM). Density plots confirmed the relative abundance of these products including exon 4+ AMEL peptides that have greater capacity for binding to hydroxyapatite. Hydrophilic X and Y peptides encoded in exon 4 differ only in substitution of non-polar isoleucine in Y for polar threonine in X with reduced disruption of the hydrophobic N-terminal structure in the Y form. Despite similarity of X and Y alleles of AMEL the non-coding region upstream from exon 4 shows significant variation with implications for segregation of processing of transcripts from exon 4. Detection of fragments from multiple additional proteins including keratins (KER), fetuin A (FETUA), proteinases and proteinase inhibitors, likely reflect biochemical events during enamel formation.

Correlation of ameloblastin with enamel mineral content.

In enamel formation, the deposition of minerals as crystallites starts when the mineralization front first forms at the start of the secretory stage. During maturation, the enamel layer accumulates significant amounts of new mineral as the crystallites grow in volume. Inversely related to mineral gain is loss of protein and water from the forming enamel. Both ameloblastin (Ambn) and enamelin are essential components for formation of a functional enamel layer. The aim of this study was to quantify the proportion of mineral and non-mineral material present in developing enamel relative to Ambn concentration using Ambn mutant mice mated with others overexpressing full-length Ambn from the mouse amelogenin promoter at lower (+), similar (++) or higher (+++) concentration than normal. Mandibular incisors (age: 7 weeks, n = 8) were imaged by micro-computed tomography and the enamel was analyzed from the apical region to the incisal edge in sequential 1.0 mm volumes of interest. Mineral density was determined using a series of hydroxyapatite (HA) phantoms to calibrate enamel density measurements. At the site where the mandibular incisor emerged into the oral cavity, the enamel volume, mineral weight, and mineral density were reduced when Tg Ambn was expressed at lower or higher levels than normal. While in wild-type the % mineral was >95%, it was negligible in Ambn-/-, 22.3% in Ambn-/-, Tg(+), 75.4% in Ambn-/-, Tg(++), and 45.2% in Ambn-/-, Tg(+++). These results document that the deposition of mineral and removal of non-mineral components are both very sensitive to expressed Ambn concentrations.

Enamelin is critical for ameloblast integrity and enamel ultrastructure formation.

Mutations in the human enamelin gene cause autosomal dominant hypoplastic amelogenesis imperfecta in which the affected enamel is thin or absent. Study of enamelin knockout NLS-lacZ knockin mice revealed that mineralization along the distal membrane of ameloblast is deficient, resulting in no true enamel formation. To determine the function of enamelin during enamel formation, we characterized the developing teeth of the Enam-/- mice, generated amelogenin-driven enamelin transgenic mouse models, and then introduced enamelin transgenes into the Enam-/- mice to rescue enamel defects. Mice at specific stages of development were subjected to morphologic and structural analysis using ß-galactosidase staining, immunohistochemistry, and transmission and scanning electron microscopy. Enamelin expression was ameloblast-specific. In the absence of enamelin, ameloblasts pathology became evident at the onset of the secretory stage. Although the aggregated ameloblasts generated matrix-containing amelogenin, they were not able to create a well-defined enamel space or produce normal enamel crystals. When enamelin is present at half of the normal quantity, enamel was thinner with enamel rods not as tightly arranged as in wild type suggesting that a specific quantity of enamelin is critical for normal enamel formation. Enamelin dosage effect was further demonstrated in transgenic mouse lines over expressing enamelin. Introducing enamelin transgene at various expression levels into the Enam-/- background did not fully recover enamel formation while a medium expresser in the Enam+/- background did. Too much or too little enamelin abolishes the production of enamel crystals and prism structure. Enamelin is essential for ameloblast integrity and enamel formation.

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

Enamel is a highly organized hierarchical nanocomposite, which consists of parallel arrays of elongated apatitic crystallites forming an intricate three-dimensional microstructure. Amelogenin, the major extracellular matrix protein of dental enamel, regulates the formation of these crystalline arrays via cooperative interactions with forming mineral phase. Using cryoelectron microscopy, we demonstrate that amelogenin undergoes stepwise hierarchical self-assembly. Furthermore, our results indicate that interactions between amelogenin hydrophilic C-terminal telopeptides are essential for oligomer formation and for subsequent steps of hierarchical self-assembly. We further show that amelogenin assemblies stabilize mineral prenucleation clusters and guide their arrangement into linear chains that organize as parallel arrays. The prenucleation clusters subsequently fuse together to form needle-shaped mineral particles, leading to the formation of bundles of crystallites, the hallmark structural organization of the forming enamel at the nanoscale. These findings provide unique insight into the regulation of biological mineralization by specialized macromolecules and an inspiration for bottom-up strategies for the materials design.

Cryogenic transmission electron microscopy study of amelogenin self-assembly at different pH.

Cryogenic transmission electron microscopy (cryo-EM) was used to explore the self-assembly of recombinant murine amelogenin (rM179) in vitro. Our cryo-EM data showed that amelogenin self-assembly is a strongly pH-dependent process. At pH 4.4 the main fraction of the protein exists in a monomeric form, although some peculiar structures consisting of chains of monomers were also observed. At pH 5.8 large nanospheres comprising ring-like structures ~50 nm in diameter were the most abundant particle class. Similarly, at pH 8.0 amelogenins self-assembled into ring-like oligomers of different sizes, which subsequently assembled into nanospheres 15-20 nm in diameter. Furthermore, at pH 7.2, which is close to a physiological pH, branched chains of nanospheres were observed. Our results show that amelogenin assembly is a multistep hierarchical process and provides new insight into the control of enamel mineralization.

Potential role of the amelogenin N-terminus in the regulation of calcium phosphate formation in vitro.

N-terminal and C-terminal (CT) domains of amelogenin have been shown to be essential for proper enamel formation. Recent studies have also suggested that although the C-terminus plays an apparent role in protein-mineral interactions, other amelogenin structural domains are involved. The objective was to explore the role of the amelogenin N-terminus in the regulation of calcium phosphate formation in vitro. Spontaneous mineralization studies were carried out using the phosphorylated (+P) and nonphosphorylated (-P) N-terminus of the leucine-rich amelogenin peptide (LRAP) that lacks the hydrophilic CT domain. Mineralization progress was monitored via changes in solution pH. Mineral phases formed were characterized using TEM, selected area electron diffraction, and FT-IR. In controls, amorphous calcium phosphate was initially formed and subsequently transformed to randomly oriented hydroxyapatite (HA) plate-like crystals. In contrast to the control, LRAP(+P)-CT stabilized ACP formation for >1 day, while LRAP(-P)-CT accelerated the transformation of ACP to HA but had little effect on crystal shape or orientation. In conclusion, the N-terminal domain found in LRAP, as in amelogenins, appears to have the capacity to interact with forming calcium phosphate mineral phases. Results suggest that the N-terminal domain of amelogenin may play a direct role in early stages of enamel formation.

Folding, assembly, and aggregation of recombinant murine amelogenins with T21I and P41T point mutations.

Two point mutations (T21I and P40T) within amelogenin have been identified from human DNA sequences in 2 instances of amelogenesis imperfecta. We studied the folding and self-assembly of recombinant amelogenin (rM180) compared to the T21I and P40T mutants analogs. At pH 5.8 and 25°C, rM180 and the P41T mutant existed as monomers, whereas the T21I mutant formed small oligomers. At pH 8 and 25°C, all of the amelogenin samples formed nanospheres with hydrodynamic radii (R(H)) of around 15-16 nm. Upon heating to 37°C, particles of P41T increased in size (R(H) = 18 nm). During thermal denaturation at pH 5.8, both of the mutant proteins refolded more slowly than the wild-type (WT) rM180. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that the WT transformed to a partially folded conformation upon heating and remained stable. Thermal denaturation and refolding studies indicated that the mutants were less stable and exhibit a greater ability to prematurely aggregate compared to the WT. Our data suggest that in the case of P41T, alterations in the self-assembly of amelogenin are a consequence of destabilization of the secondary structure, while in the case of T21I they are a consequence of change in the overall hydrophobicity at the N-terminal region. We propose that alterations in the assembly (i.e. premature aggregation) of mutant amelogenins may have a profound effect on intra- and extracellular processes such as amelogenin secretion, proteolysis, and its interactions with nonamelogenins as well as with the forming mineral.