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Amelogenin self-assembles to form an extracellular protein matrix, which serves as a template for the continuously growing enamel apatite crystals. To gain further insight into the molecular mechanism of amelogenin nanosphere formation, we manipulated the interactions between amelogenin monomers by altering pH, temperature, and protein concentration to create isolated metastable amelogenin oligomers. Recombinant porcine amelogenins (rP172 and rP148) and three different mutants containing only a single tryptophan (Trp(161), Trp(45), and Trp(25)) were used. Dynamic light scattering and fluorescence studies demonstrated that oligomers were metastable and in constant equilibrium with monomers. Stable oligomers with an average hydrodynamic radius (R(H)) of 7.5 nm were observed at pH 5.5 between 4 and 10 mg · ml(-1). We did not find any evidence of a significant increase in folding upon self-association of the monomers into oligomers, indicating that they are disordered. Fluorescence experiments with single tryptophan amelogenins revealed that upon oligomerization the C terminus of amelogenin (around residue Trp(161)) is exposed at the surface of the oligomers, whereas the N-terminal region around Trp(25) and Trp(45) is involved in protein-protein interaction. The truncated rP148 formed similar but smaller oligomers, suggesting that the C terminus is not critical for amelogenin oligomerization. We propose a model for nanosphere formation via oligomers, and we predict that nanospheres will break up to form oligomers in mildly acidic environments via histidine protonation. We further suggest that oligomeric structures might be functional components during maturation of enamel apatite.

Aberrant protein aggregation causes numerous neurological diseases including Creutzfeldt-Jakob disease (CJD), but the aggregation mechanisms remain poorly understood. Here, we report AFM results on the formation pathways of β-oligomers and nonfibrillar aggregates from wild-type full-length recombinant human prion protein (WT) and an insertion mutant (10OR) with five additional octapeptide repeats linked to familial CJD. Upon partial denaturing, seeds consisting of 3-4 monomers quickly appeared. Oligomers of ~11-22 monomers then formed through direct interaction of seeds, rather than by subsequent monomer attachment. All larger aggregates formed through association of these β-oligomers. Although both WT and 10OR exhibited identical aggregation mechanisms, the latter oligomerized faster due to lower solubility and, hence, thermodynamic stability. This novel aggregation pathway has implications for prion diseases as well as others caused by protein aggregation.

Amelogenins are an intrinsically disordered protein family that plays a major role in the development of tooth enamel, one of the most highly mineralized materials in nature. Monomeric porcine amelogenin possesses random coil and residual secondary structures, but it is not known which sequence regions would be conformationally attractive to potential enamel matrix targets such as other amelogenins (self-assembly), other matrix proteins, cell surfaces, or biominerals. To address this further, we investigated recombinant porcine amelogenin (rP172) using "solvent engineering" techniques to simultaneously promote native-like structure and induce amelogenin oligomerization in a manner that allows identification of intermolecular contacts between amelogenin molecules. We discovered that in the presence of 2,2,2-trifluoroethanol (TFE) significant folding transitions and stabilization occurred primarily within the N- and C-termini, while the polyproline Type II central domain was largely resistant to conformational transitions. Seven Pro residues (P2, P127, P130, P139, P154, P157, P162) exhibited conformational response to TFE, and this indicates these Pro residues act as folding enhancers in rP172. The remaining Pro residues resisted TFE perturbations and thus act as conformational stabilizers. We also noted that TFE induced rP172 self-association via the formation of intermolecular contacts involving P4-H6, V19-P33, and E40-T58 regions of the N-terminus. Collectively, these results confirm that the N- and C-termini of amelogenin are conformationally responsive and represent potential interactive sites for amelogenin-target interactions during enamel matrix mineralization. Conversely, the Pro, Gln central domain is resistant to folding and this may have important functional significance for amelogenin., (Copyright © 2011 The Protein Society.)

Strepsirrhines are members of a primate suborder that has a distinctive set of features associated with the development of the dentition. Amelogenin (AMEL), the better known of the enamel matrix proteins, forms 90% of the secreted organic matrix during amelogenesis. Although AMEL has been sequenced in numerous mammalian lineages, the only reported strepsirrhine AMEL sequences are those of the ring-tailed lemur and galago, which contain a set of additional proline-rich tandem repeats absent in all other primates species analyzed to date, but present in some non-primate mammals. Here, we first determined that these repeats are present in AMEL from three additional lemur species and thus are likely to be widespread throughout this group. To evaluate the functional relevance of these repeats in strepsirrhines, we engineered a mutated murine amelogenin sequence containing a similar proline-rich sequence to that of Lemur catta. In the monomeric form, the MQP insertions had no influence on the secondary structure or refolding properties, whereas in the assembled form, the insertions increased the hydrodynamic radii. We speculate that increased AMEL nanosphere size may influence enamel formation in strepsirrhine primates.

Enamel matrix proteins, including the most abundant amelogenin and lesser amounts of enamelin, ameloblastin, and proteinases, play vital roles in controlling crystal nucleation and growth during enamel formation. The cooperative action between amelogenin and the 32-kDa enamelin is critical to regulating the growth morphology of octacalcium phosphate crystals. Using biophysical methods, we investigated the interaction between the 32-kDa enamelin and recombinant pig amelogenin 148 (rP148) at pH 6.5 in phosphate-buffered saline (PBS). Dynamic light scattering results showed a trend of increasing particle size in the mixture with the addition of enamelin to amelogenin. Upon addition of the 32-kDa enamelin, the shift and intensity decrease in the ellipticity minima of rP148 in the circular dichroism spectra of rP148 illustrated a direct interaction between the 2 proteins. In the fluorescence spectra, the maximum emission of rP148 was blue shifted from 335 to 333 nm in the presence of enamelin as a result of complexation of the 2 proteins. Our results demonstrate that the 32-kDa enamelin has a close association with amelogenin at pH 6.5 in PBS buffer. Our present study provides novel insights into the possible cooperation between enamelin and amelogenin in macromolecular coassembly and in controlling enamel mineral formation., (Copyright © 2011 S. Karger AG, Basel.)

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., (Copyright © 2011 S. Karger AG, Basel.)

Mutations in amelogenin sequence result in defective enamel, and the diverse group of genetically altered conditions is collectively known as amelogenesis imperfecta (AI). Despite numerous studies, the detailed molecular mechanism of defective enamel formation is still unknown. In this study, we have examined the biophysical properties of a recombinant murine amelogenin (rM180) and two point mutations identified from human DNA sequences in two cases of AI (T21I and P41T). At pH 5.8 and 25 °C, wild type (WT) rM180 and mutant P41T existed as monomers, and mutant T21I formed lower order oligomers. CD, dynamic light scattering, and fluorescence studies indicated that rM180 and P41T can be classified as a premolten globule-like subclass protein at 25 °C. Thermal denaturation and refolding monitored by CD ellipticity at 224 nm indicated the presence of a strong hysteresis in mutants compared with WT. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that WT transformed to a partially folded conformation upon heating and remained stable. The partially folded conformation formed by P41T, however, readily converted into a heterogeneous population of aggregates. T21I existed in an oligomeric state at room temperature and, upon heating, rapidly formed large aggregates over a very narrow temperature range. Thermal denaturation and refolding studies indicated that the mutants are less stable and exhibit poor refolding ability compared with WT rM180. Our results suggest that alterations in self-assembly of amelogenin are a consequence of destabilization of the intrinsic disorder. Therefore, we propose that, like a number of other human diseases, AI appears to be due to the destabilization of the secondary structure as a result of amelogenin mutations.

To examine the hypothetical cooperative role of enamelin and amelogenin in controlling the growth morphology of enamel crystals in the post-secretory stage, we applied a cation selective membrane system for the growth of octacalcium phosphate (OCP) in the truncated recombinant porcine amelogenin (rP148) with and without the 32kDa enamelin fragment. Enamelin alone inhibited the growth in the c-axis direction more than rP148, yielding OCP crystals with the smallest aspect ratio of all conditions tested. When enamelin was added to the amelogenin "gel-like matrix", the inhibitory action of the protein mixture on the growth of OCP in the c-axis direction was diminished, while that in the b-axis direction was increased. As a result, the length to width ratio (aspect ratio) of OCP crystal was markedly increased. Addition of enamelin to amelogenin enhanced the potential of amelogenin to stabilize the amorphous calcium phosphate (ACP) transient phase. The ratio of enamelin and amelogenin was crucial for stabilization of ACP and the growth of OCP crystals with larger aspect ratio. The cooperative regulatory action of enamelin and amelogenin was attained, presumably, through co-assembling of enamelin and amelogenin. These results have important implications in understanding the growth mechanism of enamel crystals with large aspect ratio.

Amelogenin (Amel) accelerates the nucleation of hydroxyapatite (HAP) in supersaturated solutions of calcium phosphate (Ca-P), shortening the induction time (delay period), under near-physiological conditions of pH, temperature, and ionic strength. Hierarchically organized Amel and amorphous calcium phosphate (ACP) nanorod microstructures are formed involving a coassembly of Amel-ACP particles at low supersaturations and low protein concentrations in a slow, well-controlled, constant composition (CC) crystallization system. At the earliest nucleation stages, the CC method allows the capture of prenucleation clusters and intermediate nanoclusers, spherical nanoparticles, and nanochains prior to enamel-like nanorod microstructure formations at later maturation stages. Amel-ACP nanoscaled building blocks are formed spontaneously by synergistic interactions between flexible Amel protein molecules and Ca-P prenucleation clusters, and these spherical nanoparticles evolve by orientated aggregation to form nanochains. Our results suggest that, in vivo, Amel may determine the structure of enamel by controlling prenucleation cluster aggregation at the earliest stages by forming stable Amel-ACP microstructures prior to subsequent crystal growth and mineral maturation.

Amelogenin is a proline-rich enamel matrix protein known to play an important role in the oriented growth of enamel crystals. Amelogenin self-assembles to form nanospheres and higher order structures mediated by hydrophobic interactions. This study aims to obtain a better insight into the relationship between primary-secondary structure and self-assembly of amelogenin by applying computational and biophysical methods. Variable temperature circular dichroism studies indicated that under physiological pH recombinant full-length porcine amelogenin contains unordered structures in equilibrium with polyproline type II (PPII) structure, the latter being more populated at lower temperatures. Increasing the concentration of rP172 resulted in the promotion of folding to an ordered beta-structured assembly. Isothermal titration calorimetry dilution studies revealed that at all temperatures, self-assembly is entropically driven due to the hydrophobic effect and the molar heat of assembly (DeltaH(A)) decreases with temperature. Using a computational approach, a profile of domains in the amino acid sequence that have a high propensity to assemble and to have PPII structures has been identified. We conclude that the assembly properties of amelogenin are due to complementarity between the hydrophobic and PPII helix prone regions., (2009 Wiley-Liss, Inc.)

Enamel extracelluar matrix components play vital roles in controlling crystal nucleation and growth during enamel formation. We investigated the interaction between the 32 kDa enamelin fragment and amelogenin using immunochemical and biophysical methods. Immunoprecipitation studies revealed that the 32 kDa enamelin and amelogenin eluted together from a Protein A column. Dynamic light scattering results showed that the 32 kDa enamelin had a profound effect on amelogenin assembly at pH 8.0, causing partial dissociation of the nanospheres, in a dose-dependent manner. The appearance of an isodichroic point and the shifting and intensity decrease of the ellipticity minima in the circular dichroism spectra of amelogenin following the addition of the 32 kDa enamelin were indicative of conformational changes in amelogenin and of a direct interaction between the two macromolecules. Our results collectively demonstrate that the 32 kDa enamelin has a direct interaction with amelogenin in vitro. Our current studies provide novel insights into understanding possible cooperation between enamelin and amelogenin in macromolecular self-assembly and in controlling enamel mineral formation.

Amelogenins make up a class of proteins associated with the formation of mineralized enamel in vertebrates, possess highly conserved N- and C-terminal sequence regions, and represent an interesting model protein system for understanding biomineralization and protein assembly. Using bioinformatics, we report here the identification of molecular traits that classify 12 amelogenin proteins as members of the intrinsically disordered or unstructured protein family (IDPs), a group of proteins that normally exist as unfolded species but are capable of transformation to a folded state as part of their overall function. Using biophysical techniques (CD and NMR), we follow up on our bioinformatics studies and confirm that one of the amelogenins, recombinant porcine rP172, exists in an extended, unfolded state in the monomeric form. This protein exhibits evidence of conformational exchange between two states, and this exchange may be mediated by Pro residues in the sequence. Although the protein is globally unfolded, we detect the presence of local residual secondary structure [alpha-helix, extended beta-strand, turn/loop, and polyproline type II (PPII)] that may serve several functional roles within the enamel matrix. The extended, labile conformation of rP172 amelogenin is compatible with the known functions of amelogenin in enamel biomineralization, i.e., self-assembly, associations with other enamel matrix proteins and with calcium phosphate biominerals, and interaction with cell receptors. It is likely that the labile structure of this protein facilitates interactions of amelogenin with other macromolecules or with minerals for achievement of internal protein stabilization.

Reconstructing enamel-like structures on teeth have been an important topic of study in the material sciences and dentistry. The important role of amelogenin in modulating the mineralization of organized calcium phosphate crystals has been previously reported. We used amelogenin and utilized a modified biomimetic deposition method to remineralize the surface of etched enamel to form mineral layers containing organized needle-like fluoridated hydroxyapatite crystals. The effect of a recombinant amelogenins (rP172) on the microstructure of the mineral in the coating was analyzed by SEM, XRD and FT-IR. At rP172 concentrations below 33 microg/mL, no significant effect was observed. In the presence of 1 mg/L F and at a concentration of 33 microg/mL rP172, formation of fused crystals growing from the enamel surface was initiated. Amelogenin promoted the oriented bundle formation of needle-like fluoridated hydroxyapatite in a dose dependent manner. Biomimetic synthesis of the amelogenin fluoridated hydroxyapatite nano-composite is one of the primary steps towards the development and design of novel biomaterial for future application in reparative and restorative dentistry.

The present study describes a method using immunohistochemical labeling in combination with high-resolution imaging (field emission scanning electron microscopy) to investigate the spatial localization of amelogenins on apatite crystallites in developing porcine enamel. Cross-sections of developing enamel tissue from freeze-fractured pig third molar were treated with antiserum against recombinant mouse amelogenin and immunoreactivity confirmed by Western blot analysis. The samples were then treated with the goat anti-rabbit IgG conjugated with 10-nm gold particles. The control samples were treated with the secondary antibody only. The in-lens secondary electrons detector and quadrant back-scattering detector were employed to reveal the high-resolution morphology of enamel structures and gold particle distribution. The immunolabeling showed a preference of the gold particle localization along the side faces of the ribbon-like apatite crystals. The preferential localization of amelogenin in vivo on enamel crystals strongly supports its direct function in controlling crystal morphology., (Copyright 2008 S. Karger AG, Basel.)

The 32 kDa hydrophilic and acidic enamelin, the most stable cleavage fragment of the enamel specific glycoprotein, is believed to play vital roles in controlling crystal nucleation or growth during enamel biomineralization. Circular dichroism and Fourier transform infrared spectra demonstrate that the secondary structure of the 32 kDa enamelin has a high content of alpha-helix (81.5%). Quantitative analysis on the circular dichroism data revealed that the 32 kDa enamelin undergoes conformational changes with a structural preference to beta-sheet with increasing concentration of calcium ions. We suggest that the increase of beta-sheet conformation in the presence of Ca(2+) may allow preferable interaction of the 32 kDa enamelin with apatite crystal surfaces during enamel biomineralization. The calcium association constant (K(a)=1.55 (+/-0.13)x10(3)M(-1)) of the 32 kDa enamelin calculated from the fitting curve of ellipticity at 222 nm indicated a relatively low affinity. Our current biophysical studies on the 32 kDa enamelin structure provide novel insights towards understanding the enamelin-mineral interaction and subsequently the functions of enamelin during enamel formation.

Under near-physiological pH, temperature, and ionic strength, amelogenin (Amel) accelerates hydroxyapatite (HAP) nucleation kinetics, decreasing the induction time in a concentration-dependent manner. Hierarchically organized apatite microstructures are achieved by self-assembly involving nucleated nanocrystallites and Amel oligomers and nanospheres at low supersaturations and protein concentrations in a slow and well-controlled constant composition (CC) system. The CC method allows the capture of an intermediate structure, the nanorod, following the formation of the critical nuclei at the earliest nucleation stages of calcium phosphate crystallization. The nanorod building blocks form spontaneously by synergistic interactions between flexible Amel protein assemblies and rigid calcium phosphate nanocrystallites. These intermediate structures further assemble by a self-epitaxial growth mechanism to form the final hierarchically organized microstructures that are compositionally and morphologically similar to natural enamel. This in vitro observation provides direct evidence that Amel promotes apatite crystallization and organization. We interpret our observations to propose that in vivo Amel may maximally exert an influence on the structural control of developing enamel crystals at the earliest nucleation stages.

Amelogenin, the major extracellular enamel matrix protein, plays critical roles in controlling enamel mineralization. This generally hydrophobic protein self-assembles to form nanosphere structures under certain solution conditions. To gain clearer insight into the mechanisms of amelogenin self-assembly, we first investigated the occurrences of secondary structures within its sequence. By applying isothermal titration calorimetry (ITC), we determined the thermodynamic parameters associated with protein-protein interactions and with conformational changes during self-assembly. The recombinant porcine full length (rP172) and a truncated amelogenin lacking the hydrophilic C-terminal (rP148) were used. Circular dichroism (CD) measurements performed at low concentrations (<5 microM) revealed the presence of the polyproline-type II (PPII) conformation in both amelogenins in addition to alpha-helix and unordered conformations. Structural transition from PPII/unordered to beta-sheet was observed for both proteins at higher concentrations (>62.5 microM) and upon self-assembly. ITC measurements indicated that the self-assembly of rP172 and rP148 is entropically driven (+DeltaS(A)) and energetically favorable (-DeltaG(A)). The magnitude of enthalpy (DeltaH(A)) and entropy changes of assembly (DeltaS(A)) were smaller for rP148 than rP172, whereas the Gibbs free energy change of assembly (DeltaG(A)) was not significantly different. It was found that rP172 had higher PPII content than rP148, and the monomer-multimer equilibrium for rP172 was observed in a narrower protein concentration range when compared to rP148. The large positive enthalpy and entropy changes in both cases are attributed to the release of ordered water molecules and the associated entropy gain (due to the hydrophobic effect). These findings suggest that PPII conformation plays an important role in amelogenin self-assembly and that rP172 assembly is more favorable than rP148. The data are direct evidence for the notion that hydrophobic interactions are the main driving force for amelogenin self-assembly.

Fabricating the structures similar to dental enamel through the in vitro preparation method is of great interest in the fields of dentistry and material sciences. Developing enamel is composed of calcium phosphate mineral, water, and enamel matrix proteins, mainly amelogenins. To prepare a material mimicking such composition a novel approach of simultaneously assembling amelogenin and calcium phosphate precipitates by electrolytic deposition (ELD) was established. It was found that recombinant full-length amelogenin (rP172) self-assembled into nanochain structures during ELD (following increase in solution pH), and had significant effect on the induction of the parallel bundles of calcium phosphate nanocrystals, grown on semiconductive silicon wafer surface. When a truncated amelogenin (rP148) was used; no nanochain assembly was observed, neither parallel bundles were formed. The coating obtained in the presence of rP172 had improved elastic modulus and hardness when compared to the coating incorporated with rP148. Our data suggest that the formation of organized bundles in amelogenin-apatite composites is mainly driven by amelogenin nanochain assembly and highlights the potential of such composite for future application as dental restorative materials.

We have applied optical waveguide lightmode spectroscopy combined with streaming potential measurements and Fourier-transformed infrared spectroscopy to investigate adsorption of amelogenin nanospheres onto polyelectrolytes. The long-term objective was to better understand the chemical nature of these assemblies and to gain further insight into the molecular mechanisms involved during self-assembly. It was found that monolayers of monomers and negatively charged nanospheres of a recombinant amelogenin (rM179) irreversibly adsorbed onto a positively charged polyelectrolyte multilayer films. On the basis of measurements performed at different temperatures, it was demonstrated that intermolecular interactions for the formation of nanospheres were not affected by their adsorption onto polyelectrolytes. Consecutive adsorption of nanospheres resulting in the formation of multilayer structures was possible by using cationic poly(l-lysine) as mediators. N-Acetyl-d-glucosamine (GlcNac) did not disturb the nanosphere-assembled protein's structure, and it only affected the adsorption of monomeric amelogenin. Infrared spectroscopy of adsorbed amelogenin revealed conformational differences between the monomeric and assembled forms of rM179. While there was a considerable amount of alpha-helices in the monomers, beta-turn and beta-sheet structures dominated the assembled proteins. Our work constitutes the first report on a structurally controlled in vitro buildup of an rM179 nanosphere monolayer-based matrix. Our data support the notion that amelogenin self-assembly is mostly driven by hydrophobic interactions and that amelogenin/PEM interactions are dominated by electrostatic forces. We suggest that similar forces can govern amelogenin interactions with non-amelogenins or the mineral phase during enamel biomineralization.

The organic matrix in forming enamel consists largely of the amelogenin protein self-assembled into nanospheres that play a pivotal role in controlling the oriented and elongated growth of highly ordered apatitic crystals during enamel biomineralization. However, the mechanisms of amelogenin-mediated mineralization have not yet been fully elucidated. Here we report that amelogenin dramatically accelerates the nucleation kinetics by decreasing the induction time in a dose-dependent manner in a controlled constant composition (CC) in vitro crystallization system. Remarkably, at very low protein concentrations, elongated microstructures which are similar in appearance to apatitic crystals in enamel were formed at relatively low supersaturations, through interfacial structural match/synergy between structured amelogenin assemblies and apatite nanocrystallites. This heterogeneous crystallization study provides experimental evidence to support the concept that templating by amelogenin very early in the crystallization process facilitates the formation of developing enamel crystals.

We recently reported the remarkable spontaneous self-assembly and hierarchical organization of amelogenin 'microribbons' and their ability to facilitate oriented growth of apatite crystals in vitro. In a letter of correction we communicated the finding that the X-ray diffraction pattern reported in our original report was that of cellulose contaminant and not amelogenin microribbon. We have re-evaluated our data and confirmed the protein nature of the microribbons using Fourier transform infrared and Raman microspectroscopy. Some microribbons were remarkably similar in their morphology to that of cellulose fibers. The size distribution of amelogenin microribbons was wider, particularly in width and length, and generally smaller than those originally reported. Here we present additional detailed information on the formation of a series of intermediate hierarchical structures of amelogenin assemblies prior to the formation of microribbon. The most significant finding was that full-length amelogenin nanospheres had a tendency to assemble into collinear arrays whose function is assumed to be critical at the initial stage of enamel mineral deposition. The present data gives an insight into the step-by-step assembly process of amelogenin from nanometer scale molecules to micrometer scale organized structures that can be used as templates for controlled and oriented growth of apatite mineralization in vitro.

Amelogenin self-assembly is critical for the structural organization of apatite crystals during enamel mineralization. The aim of the present study was to investigate the influence of temperature and protein concentration on the aggregation of amelogenin nanospheres at high protein concentrations (>4.4 mg ml-1) in order to obtain an insight into the mechanism of amelogenin self-assembly to form higher-order structures. Amelogenins were extracted from enamel scrapings of unerupted mandibular pig molars. The dynamics of protein solutions were measured using dynamic light scattering (DLS) as a function of temperature and at acidic pH. At pH 4-5.5, three kinds of particles were observed, ranging in size from 3 to 80 nm. At pH 6, heating the solution above approximately 30 degrees C resulted in a drastic change in the solution transparency, from clear to opaque. Low pH showed no aggregation effect, whilst solutions at a slightly acidic pH exhibited diffusion dynamics associated with the onset of aggregation. In addition, at the same temperature range, the hydrodynamic radii of the aggregates increased drastically, by almost one order of magnitude. These observations support the view that hydrophobic interactions are the primary driving force for the pH- and temperature-sensitive self-assembly of amelogenin particles in a 'gel-like' matrix. The trend of self-assembly in a 'gel-like matrix' is similar to that in solution.

The purpose of this study was to express, characterize, and investigate the self-assembly of a recombinant porcine amelogenin lacking the hydrophilic 24 C-terminal amino acids (rP148). To gain further insight into the function of amelogenin processing during enamel mineralization, this protein was also used as a substrate to examine the action of matrix metalloproteinase-20 (MMP-20). The assembly properties of rP148 were monitored by dynamic light scattering (DLS). In general, rP148 molecules assemble into monomers, dimers, oligomers, and some nanosphere-like particles. Depending on the solution conditions, large aggregates were also observed. Matrix metalloproteinase-20 cleaved the rP148 molecule at a few sites, creating a number of different products, including the tyrosine-rich amelogenin polypeptide (TRAP). Our data suggest that although rP148 self-assembles into small particles, its assembly properties are different from those of the full-length rP172, indicating that the C-terminal 24 amino acids play a critical role in nanosphere assembly. We further demonstrate that MMP-20 digests rP148 in a manner that generates a similar proteolytic pattern, as would be expected to occur in vivo.

Recently, we used native amelogenins extracted from developing pig enamel to examine the combined effect of fluoride and amelogenins on the growth of octacalcium phosphate (OCP) and apatite crystals. The purpose of the present study was to investigate this combined effect using a highly purified recombinant amelogenin. We applied porcine amelogenin (rP172) and fluoride in a dual-membrane system as a model for tooth enamel formation. The combination of rP172 and fluoride in this system resulted in the formation of rod-like apatite crystals. On the other hand, without fluoride, rod-like OCP crystals of a comparable size were formed, and rather large hexagonal prisms of mixed crystals of OCP and apatite grew without amelogenins. Thus, highly purified and homogeneous recombinant amelogenin, in co-operation with F, regulated the mineral phase, habit, and size of crystals in the same manner as the extracted heterogeneous porcine amelogenins. We suggest that in both cases the control over the crystal phase and morphology was a direct effect of amelogenin protein serving as a scaffold for apatite mineralization.

Tooth regeneration presents many challenges to researchers in the fields of biology, medicine and material science. This review considers the opportunities for biomedical material research to contribute to this multidisciplinary endeavor. We present short summaries and an overview on the collective knowledge of tooth developmental biology, advances in stem-cell research, and progress in the understanding of the tooth biomineralization principles as they provide the foundation for developing strategies for reparative and regenerative medicine. We emphasize that various biomaterials developed via biomimetic strategies have great potential for tooth tissue engineering and regeneration applications. The current practices in tooth tissue engineering approaches and applications of biomimetic carriers or scaffolds are also discussed.

Extracellular matrix proteins control the formation of the inorganic component of hard tissues including bone, dentin, and enamel. The structural proteins expressed primarily in the enamel matrix are amelogenin, ameloblastin, enamelin, and amelotin. Other proteins, like biglycan, are also present in the enamel matrix as well as in other mineralizing and nonmineralizing tissues of mammals. In addition, the presence of sulfated enamel proteins, and "tuft" proteins has been examined and discussed in relation to enamel formation. The structural proteins of the enamel matrix must have specific protein-protein interactions to produce a matrix capable of directing the highly ordered structure of the enamel crystallites. Protein-protein interactions are also likely to occur between the secreted enamel proteins and the plasma membrane of the enamel producing cells, the ameloblasts. Such protein-protein interactions are hypothesized to influence the secretion of enamel proteins, establish short-term order of the forming matrix, and to mediate feedback signals to the transcriptional machinery of these cells. Membrane-bound proteins identified in ameloblasts, and which interact with the structural enamel proteins, include Cd63 (cluster of differentiation 63 antigen), annexin A2 (Anxa2), and lysosomal-associated glycoprotein 1 (Lamp1). These and related data help explain the molecular and cellular mechanisms responsible for the removal of the organic enamel matrix during the events of enamel mineralization, and how the enamel matrix influences its own fate through signaling initiated at the cell surface. The knowledge gained from enamel developmental studies may lead to better dental and nondental materials, or materials inspired by Nature. These data will be critical to scientists, engineers, and dentists in their pursuits to regenerate an entire tooth. For tooth regeneration to become a reality, the protein-protein interactions involving the key dental proteins must be identified and understood. The scope of this review is to discuss the current understanding of protein-protein interactions of the developing enamel matrix, and relate this knowledge to enamel biomineralization.

Although both tooth enamel and bone are composed of organized assemblies of carbonated apatite crystals, enamel is unusual in that it does not contain collagen nor does it remodel. Self-assembly of amelogenin protein into nanospheres has been recognized as a key factor in controlling the oriented and elongated growth of carbonated apatite crystals during dental enamel biomineralization. We report the in vitro formation of birefringent microribbon structures that were generated through the supramolecular assembly of amelogenin nanospheres. These microribbons have diffraction patterns that indicate a periodic structure of crystalline units along the long axis. The growth of apatite crystals orientated along the c axis and parallel to the long axes of the microribbons was observed in vitro. The linear arrays (chains) of nanospheres observed as intermediate states before the microribbon formation give an important indication as to the function of amelogenin in controlling the oriented growth of apatite crystals during enamel mineralization.

Tooth enamel is formed in the extracellular space within an organic matrix enriched in amelogenin proteins. Amelogenin nanosphere assembly is a key factor in controlling the oriented and organized growth of enamel apatite crystals. Recently, we have reported the formation of higher ordered structures resulting from organized association and self-orientation of amelogenin nanospheres in vitro. This remarkable hierarchical organization includes self-assembly of amelogenin molecules into subunits of 4-6 nm in diameter followed by their assembly to form nanospheres of 15-25 nm in radii. Chains of >100 nm length are then formed as the result of nanosphere association. These linear arrays of nanospheres assemble to form the microribbons that are hundreds of microns in length, tens of microns in width, and a few microns in thickness. Here, we review the step by step process of amelogenin self-assembly during the formation of microribbon structures in vitro. Assembly properties of selected amelogenins lacking the hydrophilic C terminus will then be reviewed. We will consider amelogenin as a template for the organized growth of crystals in vitro. Finally, we will compare the structures formed in vitro with globular and periodic structures observed earlier, in vivo, by different sample preparation conditions. We propose that the alignment of amelogenin nanospheres into long chains is evident in vivo, and is an important indication for the function of this protein in controlling the oriented and elongated growth of apatite crystals during enamel biomineralization., (2005 S. Karger AG, Basel)

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.

The objective of the present study was to determine the effect of apatite crystals on the activity of amelogenin degrading enzymes in vitro. Current experimental data, together with previous reports support the view that among the different proteinases present in the enamel extracellular matrix, serine proteinase(s) are responsible for the massive degradation of amelogenins during the maturation stage. For our in-vitro experiments we used the recombinant amelogenin M179 as substrate and a "65%-satd. (NH4)2SO4" fraction of enamel proteins as well as chymotrypsin as sources for serine-proteinase activity. We report preliminary experiments of amelogenin proteolysis in the presence of apatite crystals resulting in a different proteolysis pattern when compared to amelogenin proteolysis without apatite crystals. Quantitative analysis of the HPLC peaks corresponding to the proteolysis products indicates that the presence of apatite crystals in the proteolysis solution inhibits the ability of the serine-proteinases to degrade amelogenin. The present observations support the hypothesis that amelogenin degradation correlates with apatite crystal growth during enamel maturation.

We have proposed that these nanosphere structures are functionally involved in the organization and control of initial enamel biomineralization at the ultrastructural level. Based on the observed nanosphere hydrodynamic radii (18-20 nm diameter) computation suggests these structures to be compounded of some 100 amelogenin monomers, raising the question as to the possible molecular mechanism for the assembly of such structures? Based on recent dynamic light scattering experiments using the recombinant murine amelogenin M179, and employing a newer size distribution algorithm we now report that the size distribution data for M179 are better described by a bimodal distribution model, than the monomodal distribution as previously described. We suggest that amelogenin nanosphere assembly proceeds through intermediate structures (perhaps represented in vivo by "stippled material") of some 4-5 nm hydrodynamic radius, and computed to comprise 4-6 amelogenin monomers. We suggest that such intermediary, sub-unit structures, assemble through inter-molecular hydrophobic interactions to generate the 20 nm diameter nanospheres observed by TEM in the secretory stage enamel matrix.

During enamel formation the proteins of the extracellular matrix, particularly amelogenins, are removed prior to maturation. In order to investigate this process and to improve our understanding of the function of proteinases during enamel maturation, proteinase fractions were isolated from developing pig enamel and assayed for proteolytic activity in vitro. A recombinant murine amelogenin, M179, was used as a substrate. Two major groups of enamel proteinases were defined as high-molecular-mass ['high-molecular-weight' in Moradian-Oldak, Simmer, Sarte, Zeichner-David and Fincham (1994) Arch. Oral Biol.39, 647-656] and low-molecular-mass proteinases. Here we report the characterization of one of the proteinases present in the low-molecular-mass group. We demonstrate that this proteinase is a serine proteinase capable of degradation of M179 following cleavage of the tyrosine-rich amelogenin polypeptide from the N-terminal region. A partial N-terminal sequence of the proteinase was obtained (LPHVPHRIPPGYGRPXTXNEEGXNPYFXFFXXHG). An anti-peptide antibody directed against a synthetic peptide corresponding to the first 14 amino acids of the above sequence was produced. The presence of the proteinase in the acetic acid extract was confirmed by Western blotting. Searching using the amino acid sequence determined in this study showed it to be also present in the 32 kDa and 89 kDa enamelin proteins reported by Fukae, Tanabe, Murakami and Tohi [(1996) Adv. Dent. Res., in the press]. We therefore identify the 32 kDa enamelin as an enamel proteinase ('ameloprotease-I') which is responsible for amelogenin degradation in maturing enamel. We propose that the 89 kDa enamelin is a precursor of ameloprotease-I, the first enamel protein for which a function has been defined.

This paper is a short review of our recent studies on amelogenin proteolysis in vitro using a recombinant mouse amelogenin M179 as a substrate. The specific aims of this study were to identify, isolate and characterize the proteinases in the enamel extracellular matrix. We identified two classes of enamel proteinases; 1) the high molecular weight proteinase (60-68 kDa) cleaves the c-terminal segment of M179 and is a calcium dependent metalloproteinase with an optimum pH of 8.2) The low molecular weight proteinase (approximately 30 kDa) removes the TRAP (Tyrosine Rich Amelogenin Polypeptide) sequence and causes further degradation of M179. The latter was identified to be a serine proteinase with an optimal activity at pH 6. These data support the notion that enamel proteinases cleave amelogenin through specific and highly controlled mechanisms and that they may fulfill direct roles during enamel maturation.

This study was undertaken to examine probable initial pathway(s) of amelogenin proteolysis, making comparisons between species and thus searching for a common theme. Specimens of developing dental enamel matrix were obtained from (i) mouse, 6 days post natal, (ii) male pig, (iii) female bovine, (iv) rat, and (v) female human. In collaboration with the Mass Spectrometry Facility of the School of Pharmacy, University of California, San Francisco, samples of the lyophilized proteins were analyzed by liquid chromatography-mass spectrometry. The results were complex, a large number (15-30 components) being identified in each case. Mass values obtained for each sample were compared with computed values derived from segments of the known amino acid sequences for the principal amelogenins of the five species. Putative identity with an experimental figure was accepted when the mass numbers agreed to within +/-2.0 daltons. In each case it was found that some components could be identified with sequences of the parent amelogenin. In the case of the mouse and rat strong evidence was obtained for sequential proteolytic processing from the carboxy-terminus of both the 180 and 156 residue amelogenins. A comparison between the five species showed, a fragment (cow, man, pig and mouse) uniquely identified as being derived by the processing of the parent amelogenin to the first proline residue from the carboxy-terminus, leading to the cleavage of 11 residues of the anionic carboxy-terminal sequence. In addition, it was found in each case, that mass identity of experimental data with the known sequences was only obtained assuming the presence of a single phosphorylated residue.

This review highlights a number of advances towards understanding the sequential developmental cascade of events beginning in the oral ectodermally-derived odontogenic placode and culminating in the formation of the mineralized enamel extracellular matrix. Recent discoveries of growth factors, growth factor receptors and transcription factors associated with instructive epithelial-mesenchymal interactions and subsequent controls for ameloblast cell differentiation are reviewed. The relationship between ameloblast cytology, terminal differentiation and biochemical phenotype are discussed. The tissue-specific gene products characteristic of the ameloblast phenotype as well as their possible functions in formation of the enamel matrix are analyzed as well as the role of maturation-stage ameloblast cells in controlling enamel biomineralization. Finally, pathological conditions in which alterations in the ameloblast or specific gene products result in an abnormal enamel phenotype are reviewed. Clearly, the scientific progress achieved in the last few years concerning the molecular determinants involved in tooth development has been remarkable. However, there remains considerable lack of knowledge regarding the precise mechanisms that control ameloblast differentiation and enamel biomineralization. Anticipated progress continues to require increased international cooperation and collaborations as well as increased utilization of structural biology investigations of enamel extracellular matrix proteins.

This paper reviews advances in amelogenin biochemistry in three areas; (i) amelogenin expression; (ii) amelogenin post-translational and post-secretory processing, and (iii) amelogenin structure and function. Recent studies of amelogenin expression have demonstrated that alternative-splicing of mouse amelogenin RNA generates seven distinct mRNAs, coding for amelogenin proteins from 194 to 44 amino acid residues in length. A polyclonal antibody to a sequence of the 194-residue murine amelogenin identified this protein in vivo. While several studies have reported that amelogenins are post-translationally phosphorylated, it has proved difficult to confirm this view. Mass spectrometry studies of bovine and porcine TRAP and LRAP amelogenins have established a phosphoserine residue at position-16 as originally reported by Takagi et al. for a 180-residue bovine amelogenin. Also, we discovered that the detailed mechanism(s) of carboxy-terminal amelogenin proteolytic processing appear different than previously reported. In terms of amelogenin structure, it is well known that amelogenins form aggregated structures. Studies employing a recombinant amelogenin and dynamic light-scattering instrumentation demonstrated aggregate structures of 15-20 nm in radius, corresponding to a mass of 2-3 million daltons. Imaging these aggregates by transmission electron and atomic force microscopy suggested that these structures are equivalent to the "stippled" or "granular" material seen in electron photomicrographs of developing enamel. Collectively, these advances in amelogenin biochemistry lead to a new view of amelogenin structure, processing and functions in enamel biomineralization.

The amelogenins of the extracellular matrix of developing dental enamel, comprise a family of tissue-specific proteins which are postulated to play a central role in the biomineralization of dental enamel [1]. The primary structures of amelogenins derived from cow, pig, human, mouse and rat have now been elucidated by the interpretation of cDNA sequences or by direct amino acid sequence determinations [2-6] demonstrating a high degree of sequence homology between species [1]. However, the nature of post-translational modification of these proteins is less clear. In particular, early reports of amelogenin phosphorylation [7-8] have proved to be difficult to confirm by direct chemical analyses [1]. Using mass spectrographic analysis, we recently [9], reported that the lower molecular weight (5-7 kDa) bovine and porcine amelogenin polypeptides (TRAP and LRAP) contained a single phospho-serine residue at position 16Ser and, since these polypeptides are derived by proteolytic processing from the higher molecular weight "parent" amelogenins (18-25 kDa), we concluded that these precursor molecules must also be phosphorylated, as has previously been suggested [10]. In contrast to these observations, an extensive amino acid sequencing study of porcine amelogenins has recently reported no evidence for such phosphorylation [1]. We now report that a new analysis of the major porcine ("20K") amelogenin provides positive evidence for porcine amelogenin phosphorylation.

TRAP and LRAP amelogenin components were isolated by size-exclusion and reversed-phase HPLC from developing dental enamel. Porcine developing enamel contains TRAP and LRAP components analogous to those of bovine. Amino acid composition and mass spectrographic analyses established that, in both species, the carboxy-terminal sequences of the LRAP components are two residues longer than previously reported for bovine LRAP, and that both the TRAPs and LRAPs contained a single phosphorylated residue. These amelogenin polypeptides were the principal components of the enamel protein lower molecular weight fraction. The LRAP sequence data for both species suggests that the mechanism of amelogenin carboxy-terminal processing may differ significantly from that previously suggested.

Control over crystal growth by acidic matrix macromolecules is an important process in the formation of many mineralized tissues. Earlier studies on the interactions between acidic macromolecules and carboxylate- and carbonate-containing crystals showed that the proteins recognize a specific stereochemical motif on the interacting plane. Here we show that a similar stereochemical motif is recognized by acidic mollusc shell macromolecules interacting with four different organic calcium phosphate-containing crystals. In addition, an acidic protein from vertebrate tooth dentin was also observed to recognize a similar structural motif in one of the crystals. The characteristic motif recognized is composed of rows of calcium ions and phosphates arranged in a plane defined by two free oxygens and a phosphorus atom emerging perpendicular to the affected face. These observations may have a direct bearing on the manner in which control over crystal growth is exerted on carbonate apatite crystals commonly found in vertebrate tissues.

Topographic imaging, a method of providing a direct view of ultrastructure in three dimensions, has been newly applied to a study of mineral crystals and collagen from calcifying turkey leg tendon. Individual crystals obtained from intact tendon were observed as thin platelets of irregular shape having a relatively smooth surface. Mineralized collagen fibrils isolated singly or examined in thin tissue sections were found to exhibit the characteristic 64-70 nm period and were associated with platelets and needle-like mineral. The crystals were disposed in numerous fashions, notably as small groups of platelets within individual collagen hole zones, as a number of needle-like densities arranged parallel to one another, or as a combination of platelets and needles over entire stretches of single collagen fibrils. The topographic observations of crystals and crystal-collagen interaction clearly demonstrate the plate-like habit of the mineral in calcifying turkey tendon and suggest that these crystals are located both within and on the surface of collagen fibrils. In certain sites such as the collagen hole zones, the crystals appear organized in a specific manner, possibly with a preferred c-axial orientation. Crystals of hydroxyapatite prepared in vitro and examined topographically are similar in habit and texture to the crystals from tendon. When interpretation of this method is corroborated by other independent microscopic techniques, topographic imaging has widespread potential application in many fields of study in which structural surface features of biological tissues or non-biological materials are of interest at the electron microscope level.