Partial rescue of the amelogenin null dental enamel phenotype

The amelogenins are the most abundant secreted proteins in developing dental enamel. Enamel from amelogenin (Amelx) null mice is hypoplastic and disorganized, similar to that observed in X-linked forms of the human enamel defect amelogenesis imperfecta resulting from amelogenin gene mutations. Both transgenic strains that express the most abundant amelogenin (TgM180) have relatively normal enamel, but strains of mice that express a mutated amelogenin (TgP70T), which leads to amelogenesis imperfecta in humans, have heterogeneous enamel structures. When Amelx null (KO) mice were mated with transgenic mice that produce M180 (TgM180), the resultant TgM180KO offspring showed evidence of rescue in enamel thickness, mineral density, and volume in molar teeth. Rescue was not observed in the molars from the TgP70TKO mice. It was concluded that a single amelogenin protein was able to significantly rescue the KO phenotype and that one amino acid change abrogated this function during development.     

Enamelysin (matrix metalloproteinase 20)-deficient mice display an amelogenesis imperfecta phenotype

Enamelysin is a tooth-specific matrix metalloproteinase that is expressed during the early through middle stages of enamel development. The enamel matrix proteins amelogenin, ameloblastin, and enamelin are also expressed during this same approximate developmental time period, suggesting that enamelysin may play a role in their hydrolysis. In support of this interpretation, recombinant enamelysin was previously demonstrated to cleave recombinant amelogenin at virtually all of the precise sites known to occur in vivo. Thus, enamelysin is likely an important amelogenin-processing enzyme. To characterize the in vivo biological role of enamelysin during tooth development, we generated an enamelysin-deficient mouse by gene targeting. Although mice heterozygous for the mutation have no apparent phenotype, the enamelysin null mouse has a severe and profound tooth phenotype. Specifically, the null mouse does not process amelogenin properly, possesses an altered enamel matrix and rod pattern, has hypoplastic enamel that delaminates from the dentin, and has a deteriorating enamel organ morphology as development progresses. Our findings demonstrate that enamelysin activity is essential for proper enamel development.     

A transgenic animal model resembling amelogenesis imperfecta related to ameloblastin overexpression

Genetic diseases that affect tooth enamel are grouped under the classification amelogenesis imperfecta. Human pedigrees and experiments on transgenic and null mice have all demonstrated that mutations to the secreted proteins amelogenin, enamelin, and enamelysin result in visibly, structurally, or mechanically defective enamel. In an attempt to better define a physiologic function for ameloblastin during enamel formation, we have produced transgenic mice that misexpress the ameloblastin gene. These transgenic animals exhibit imperfections in their enamel that is evident at the nanoscale level. Specifically, ameloblastin overexpression influences enamel crystallite habit and enamel rod morphology. These findings suggest enamel crystallite habit and rod morphology are influenced by the temporal and spatial expression of ameloblastin and may implicate the role of the ameloblastin gene locus in the etiology of a number of undiagnosed autosomally dominant cases of amelogenesis imperfecta.     

The Evolution of Enamel Microstructure: How Important Is Amelogenin?

The shape, size, and orientation of enamel prisms have heretofore been thought to be controlled solely by the shape of the Tomes' process. It is known, however, that amelogenin proteins play an important role in enamel deposition and maturation and it is possible that they contribute independently to enamel structure. Using a phylogenetic framework, we clarify the role of amelogenin proteins in the formation of enamel microstructure. We found a negative association between evolutionary changes in amelogenin protein sequences and enamel complexity: amelogenin evolution slows as enamel complexity increases. This is probably because selective constraints on amelogenin increase as enamel complexity increases. Monotremes, which have lost their adult dentition, have particularly high rates of amelogenin evolution while rodents, which have very complex enamel, have very low rates. There is a positive correlation between the number of different amelogenin proteins in a given species and the complexity of its enamel microstructure. An increased number of amelogenins may be necessary for the formation of multiple enamel types in the same tooth. Alternative splicing of amelogenin exons, which allows multiple protein products to be produced from the same gene, may be a key innovation in the diversification of enamel microstructure.     

Binding of amelogenin to MMP-9 and their co-expression in developing mouse teeth

Amelogenin is the most abundant matrix protein in enamel. Proper amelogenin processing by proteinases is necessary for its biological functions during amelogenesis. Matrix metalloproteinase 9 (MMP-9) is responsible for the turnover of matrix components. The relationship between MMP-9 and amelogenin during tooth development remains unknown. We tested the hypothesis that MMP-9 binds to amelogenin and they are co-expressed in ameloblasts during amelogenesis. We evaluated the distribution of both proteins in the mouse teeth using immunohistochemistry and confocal microscopy. At postnatal day 2, the spatial distribution of amelogenin and MMP-9 was co-localized in preameloblasts, secretory ameloblasts, enamel matrix and odontoblasts. At the late stages of mouse tooth development, expression patterns of amelogenin and MMP-9 were similar to that seen in postnatal day 2. Their co-expression was further confirmed by RT-PCR, Western blot and enzymatic zymography analyses in enamel organ epithelial and odontoblast-like cells. Immunoprecipitation assay revealed that MMP-9 binds to amelogenin. The MMP-9 cleavage sites in amelogenin proteins across species were found using bio-informative software program. Analyses of these data suggest that MMP-9 may be involved in controlling amelogenin processing and enamel formation.     

Conservation and variation in enamel protein distribution during vertebrate tooth development

Vertebrate enamel formation is a unique synthesis of the function of highly specialized enamel proteins and their effect on the growth and organization of apatite crystals. Among tetrapods, the physical structure of enamel is highly conserved, while there is a greater variety of enameloid tooth coverings in fish. In the present study, we postulated that in enamel microstructures of similar organization, the principle components of the enamel protein matrix would have to be highly conserved. In order to identify the enamel proteins that might be most highly conserved and thus potentially most essential to the process of mammalian enamel formation, we used immunoscreening with enamel protein antibodies as a means to assay for degrees of homology to mammalian enamel proteins. Enamel preparations from mouse, gecko, frog, lungfish, and shark were screened with mammalian enamel protein antibodies, including amelogenin, enamelin, tuftelin, MMP20, and EMSP1. Our results demonstrated that amelogenin was the most highly conserved enamel protein associated with the enamel organ, enamelin featured a distinct presence in shark enameloid but was also present in the enamel organ of other species, while the other enamel proteins, tuftelin, MMP20, and EMSP1, were detected in both in the enamel organ and in other tissues of all species investigated. We thus conclude that the investigated enamel proteins, amelogenin, enamelin, tuftelin, MMP20, and EMSP1, were highly conserved in a variety of vertebrate species. We speculate that there might be a unique correlation between amelogenin-rich tetrapod and lungfish enamel with long and parallel crystals and enamelin-rich basal vertebrate enameloid with diverse patterns of crystal organization.     

Amelogenesis: Transformation of a protein-mineral matrix into tooth enamel

During enamel formation, the organic enamel protein matrix interacts with calcium phosphate minerals to form elongated, parallel, and bundled enamel apatite crystals of extraordinary hardness and biomechanical resilience. The enamel protein matrix consists of unique enamel proteins such as amelogenin, ameloblastin, and enamelin, which are secreted by highly specialized cells called ameloblasts. The ameloblasts also facilitate calcium and phosphate ion transport toward the enamel layer. Within ameloblasts, enamel proteins are transported as a polygonal matrix with 5 nm subunits in secretory vesicles. Upon expulsion from the ameloblasts, the enamel protein matrix is re-organized into 20 nm subunit compartments. Enamel matrix subunit compartment assembly and expansion coincide with C-terminal cleavage by the MMP20 enamel protease and N-terminal amelogenin self-assembly. Upon enamel crystal precipitation, the enamel protein phase is reconfigured to surround the elongating enamel crystals and facilitate their elongation in C-axis direction. At this stage of development, and upon further amelogenin cleavage, central and polyproline-rich fragments of the amelogenin molecule associate with the growing mineral crystals through a process termed “shedding”, while hexagonal apatite crystals fuse in longitudinal direction. Enamel protein sheath-coated enamel “dahlite” crystals continue to elongate until a dense bundle of parallel apatite crystals is formed, while the enamel matrix is continuously degraded by proteolytic enzymes. Together, these insights portrait enamel mineral nucleation and growth as a complex and dynamic set of interactions between enamel proteins and mineral ions that facilitate regularly seeded apatite growth and parallel enamel crystal elongation.     

Targeted overexpression of amelotin disrupts the microstructure of dental enamel

We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.     

Amelogenins: assembly, processing and control of crystal morphology.

The remarkable properties of enamel crystals and their arrangements in an extraordinary micro-architecture are clear indications that the processes of crystal nucleation and growth in the extracellular matrix are highly controlled. The major extracellular events involved in enamel formation are: (a) delineation of space by the secretory ameloblasts and the dentino-enamel junction; (b) self-assembly of amelogenin proteins to form the supramolecular structural framework; (c) transportation of calcium and phosphate ions by the ameloblasts resulting in a supersaturated solution; (d) nucleation of apatite crystallites; and (e) elongated growth of the crystallites. Finally, during the 'maturation' step, rapid growth and thickening of the crystallites take place, which is concomitant with progressive degradation and eventual removal of the enamel extracellular matrix components (mainly amelogenins). This latter stage during which physical hardening of enamel occurs is perhaps unique to dental enamel. We have focused our in vitro studies on three major extracellular events: matrix assembly, matrix processing and control of crystal growth. This paper summarizes current knowledge on the assembly, processing and effect on crystal morphology by amelogenin proteins. The correlation between these three events and putative functional roles for amelogenin protein are discussed.     

Physiological implications of DLX homeoproteins in enamel formation

Tooth development is a complex process including successive stages of initiation, morphogenesis, and histogenesis. The role of the Dlx family of homeobox genes during the early stages of tooth development has been widely analyzed, while little data has been reported on their role in dental histogenesis. The expression pattern of Dlx2 has been described in the mouse incisor; an inverse linear relationship exists between the level of Dlx2 expression and enamel thickness, suggesting a role for Dlx2 in regulation of ameloblast differentiation and activity. In vitro data have revealed that DLX homeoproteins are able to regulate the expression of matrix proteins such as osteocalcin. The aim of the present study was to analyze the expression and function of Dlx genes during amelogenesis. Analysis of Dlx2/LacZ transgenic reporter mice, Dlx2 and Dlx1/Dlx2 null mutant mice, identified spatial variations in Dlx2 expression within molar tooth germs and suggests a role for Dlx2 in the organization of preameloblastic cells as a palisade in the labial region of molars. Later, during the secretory and maturation stages of amelogenesis, the expression pattern in molars was found to be similar to that described in incisors. The expression patterns of the other Dlx genes were examined in incisors and compared to Dlx2. Within the ameloblasts Dlx3 and Dlx6 are expressed constantly throughout presecretory, secretory, and maturation stages; during the secretory phase when Dlx2 is transitorily switched off, Dlx1 expression is upregulated. These data suggest a role for DLX homeoproteins in the morphological control of enamel. Sequence analysis of the amelogenin gene promoter revealed five potential responsive elements for DLX proteins that are shown to be functional for DLX2. Regulation of amelogenin in ameloblasts may be one method by which DLX homeoproteins may control enamel formation. To conclude, this study establishes supplementary functions of Dlx family members during tooth development: the participation in establishment of dental epithelial functional organization and the control of enamel morphogenesis via regulation of amelogenin expression.     

The effect of recombinant mouse amelogenins on the formation and organization of hydroxyapatite crystals in vitro

Amelogenin is the most abundant protein in developing dental enamel. It is believed to play an important role in the regulation of the growth and organization of enamel crystals. Amelogenin, unlike many other proteins found in biominerals, is mostly hydrophobic except for a 13 amino acid hydrophilic C-terminal domain. To clarify the role of amelogenin in enamel mineralization, we designed calcium phosphate crystal growth experiments in the presence of recombinant amelogenins with or without the charged C-terminal domain. The shape and organization of the crystals were examined by TEM in bright field and diffraction modes. It was found that both full-length and truncated amelogenin inhibit crystal growth in directions normal to the c-axis. At the same time, crystallites organized into parallel arrays only in the presence of the full-length amelogenin in monomeric form. Pre-assembled amelogenins had no effect on crystals organization. These results imply that the hydrophobic portion of amelogenin plays a role in an inhibition of crystal growth, whereas the C-terminal domain is essential for the alignment of crystals into parallel arrays. Our data also suggest that nascent enamel structure emerges as a result of cooperative interactions between forming crystals and assembling proteins.     

Altered amelogenin self-assembly based on mutations observed in human X-linked amelogenesis imperfecta (AIH1)

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin, a 180-amino acid hydrophobic protein that self-assembles to form nanospheres. We have used independent technical methods, consisting of the yeast two-hybrid (Y2H) assay and surface plasmon resonance (SPR), to demonstrate the importance of amelogenin self-assembly domains. In addition, we have analyzed mutations in amelogenin observed in patients with amelogenesis imperfecta who demonstrate defects in enamel formation. Assessments of self-assembly of these mutant amelogenins by either SPR or Y2H assay yield concordant data. These data support the conclusion that the amelogenin amino-terminal self-assembly domain is essential to the creation of an enamel extracellular organic matrix capable of directing mineral formation. It also suggests that a pathway through which point mutations in the amelogenin protein can adversely impact on the formation of the enamel organ is by disturbing self-assembly of the organic matrix. These data support the utilization of the Y2H assay to search for protein interactions among extracellular matrix proteins that contribute to biomineralization and provide functional information on protein-protein and protein-mineral interactions.     

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

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.     

Analysis of self-assembly and apatite binding properties of amelogenin proteins lacking the hydrophilic C-terminal.

Amelogenins, the major protein component of the mineralizing enamel extracellular matrix, are critical for normal enamel formation as documented in the linkage studies of a group of inherited disorders, with defective enamel formation, called Amelogenesis imperfecta. Recent cases of Amelogenesis imperfecta include mutations that resulted in truncated amelogenin protein lacking the hydrophilic C-terminal amino acids. Current advances in knowledge on amelogenin structure, nanospheres assembly and their effects on crystal growth have supported the hypothesis that amelogenin nanospheres provide the organized microstructure for the initiation and modulated growth of enamel apatite crystals. In order to evaluate the function of the conserved hydrophilic C-terminal telopeptide during enamel biomineralization, the present study was designed to analyze the self-assembly and apatite binding behavior of amelogenin proteins and their isoforms lacking the hydrophilic C-terminal. We applied dynamic light scattering to investigate the size distribution of amelogenin nanospheres formed by a series of native and recombinant proteins. In addition, the apatite binding properties of these amelogenins were examined using commercially available hydroxyapatite crystals. Amelogenins lacking the carboxy-terminal (native P161 and recombinant rM166) formed larger nanospheres than those formed by their full-length precursors: native P173 and recombinant rM179. These data suggest that after removal of the hydrophilic carboxy-terminal segment further association of the nanospheres takes place through hydrophobic interactions. The affinity of amelogenins lacking the carboxy-terminal regions to apatite crystals was significantly lower than their parent amelogenins. These structure-functional analyses suggest that the hydrophilic carboxy-terminal plays critical functional roles in mineralization of enamel and that the lack of this segment causes abnormal mineralization.     

Identification and characterization of a squamate reptilian amelogenin gene: Iguana iguana

As the principal components of the developing tooth enamel matrix, amelogenins play a significant role in tooth enamel formation and organization. In order to elucidate the structure and function of amelogenins in the evolution of enamel, we have selected the Iguana iguana as a squamate model organism. Here we report the first complete squamate amelogenin sequence available as of yet and document unique features of Iguana amelogenins and enamel. Transmission electron microscopy documented randomly oriented Iguana enamel crystals during the elongation phase compared with organized enamel crystal patterns at comparable stages in mammals. Sequencing of PCR amplified products revealed a full-length I. iguana amelogenin cDNA containing 877 nucleotides with a 564 nucleotide coding sequence encoding 187 amino acids. The homologies of the newly discovered I. iguana amelogenin amino acid sequence with the published mouse, caiman (Palaeosuchus), and snake (Elaphe) amelogenin were 41.3%, 53.5%, and 55.5%, respectively. On Western blots one major protein with a molecular weight of 24 kDa, and two minor proteins with molecular weights of 28 and 13.5 kDa, respectively, were detected based on the cross-reactivity of antisera against recombinant Rana pipiens amelogenin proteins. Sequence analysis revealed a moderate sequence homology between mammalian and reptilian amelogenin genes. A significant alteration was the deletion of the hydrophilic GSP sequence from exon 3 in the mouse sequence resulting in a conversion to a hydrophobic region in Iguana. Together, these findings identified a novel amelogenin cDNA sequence in the squamate reptilian I. iguana and functional implications for the evolution of amelogenins and enamel in squamates.     

Initial enamel crystals are not spatially associated with mineralized dentine

During epithelial-mesenchymal interactions associated with mammalian tooth development, epithelially-derived and mesenchymally-derived extracellular matrix molecules form a discrete dentine-enamel junction. The developmental and molecular processes required to form this junction are not known. To address this problem we designed studies to test the hypothesis that ectodermally-derived epithelial cells synthesize and secrete enamel proteins which function to nucleate and regulate the growth of enamel calcium phosphate crystals. Initial enamel crystals were detected separate from the adiacent dentine. Electron-microprobe analyses revealed that early enamel crystals were octacalciumphosphate or tricalciumphosphate rather than hydroxyapatite. Thereafter, enamel crystals became confluent with the adjacent, albeit significantly smaller hydroxyapatite crystals associated with mineralized dentine. Therefore, we interpret our data to indicate that de novo enamel crystal nucleation and growth are independent from the mineralization processes characterized for dentine. We further argue that gene expression of enamel protein appears to have a constitutive function during early enamel formation and that supramolecular aggregates of amelogenin and enamelin provide the microenvironment for the nucleation and crystal growth of the initial enamel matrix.     

Interaction of amelogenin with hydroxyapatite crystals: An adherence effect through amelogenin molecular self-association

At the secretory stage of tooth enamel formation the majority of the organic matrix is composed of amelogenin proteins that are believed to provide the scaffolding for the initial carbonated hydroxyapatite crystals to grow. The primary objective of this study was to investigate the interaction between amelogenins and growing apatite crystals. Two in vitro strategies were used: first, we examined the influence of amelogenins as compared to two other macromolecules, on the kinetics of seeded growth of apatite crystals; second, using transmission electron micrographs of the crystal powders, based on a particle size distribution study, we evaluated the effect of the macromolecules on the aggregation of growing apatite crystals. Two recombinant amelogenins (rM179, rM166), the synthetic leucine-rich amelogenin polypeptide (LRAP), poly(L -proline), and phosvitin were used. It was shown that the rM179 amelogenin had some inhibitory effect on the kinetics of calcium hydroxyapatite seeded growth. The inhibitory effect, however, was not as destructive as that of other macromolecules tested. The degree of inhibition of the macromolecules was in the order of phosvitin < LRAP < poly(L -proline) < rM179 < rM166. Analysis of particle size distribution of apatite crystal aggregates indicated that the full-length amelogenin protein (rM179) caused aggregation of the growing apatite crystals more effectively than other macromolecules. We propose that during the formation of hydroxyapatite crystal clusters, the growing apatite crystals adhere to each other through the molecular self-association of interacting amelogenin molecules. The biological implications of this adherence effect with respect to enamel biomineralization are discussed. © 1998 John Wiley & Sons, Inc. Biopoly 46: 225–238, 1998     

A synthetic, chemically modified ribozyme eliminates amelogenin, the major translation product in developing mouse enamel in vivo.

Ribozymes are small RNA structures capable of cleaving RNA target molecules in a catalytic fashion. Designed ribozymes can be targeted to specific mRNAs, blocking their expression without affecting normal functions of other genes. Because of their specific and catalytic mode of action ribozymes are ideal agents for therapeutic interventions against malfunctioning or foreign gene products. Here we report successful experiments to 'knock out' a major translation product in vivo using synthesized, chemically modified ribozymes. The ribozymes, designed to cleave amelogenin mRNA, were injected close to developing mandibular molar teeth in newborn mice, resulting in a prolonged and specific arrest of amelogenin synthesis not caused by general toxicity. No carriers were required to assist cellular uptake. Amelogenins are highly conserved tissue-specific proteins that play a central role in mammalian enamel biomineralization. Ultrastructural analyses of in vivo ribozyme-treated teeth demonstrated their failure to develop normally mineralized enamel. These results demonstrate that synthesized ribozymes can be highly effective in achieving both timed and localized 'knock-out' of important gene products in vivo, and suggest new possibilities for suppression of gene expression for research and therapeutic purposes.