Elongated growth of octacalcium phosphate crystals in recombinant amelogenin gels under controlled ionic flow

Amelogenin proteins constitute the primary structural entity of the extracellular protein framework of the developing enamel matrix. Recent data on the interactions of amelogenin with calcium phosphate crystals support the hypothesis that amelogenins control the oriented and elongated growth of enamel carbonate apatite crystals. To exploit further the molecular mechanisms involved in amelogenin-calcium phosphate mineral interactions, we conducted in vitro experiments to examine the effect of amelogenin on synthetic octacalcium phosphate (OCP) crystals. A 10% (wt/vol) recombinant murine amelogenin (rM179, rM166) gel was constructed with nanospheres of about 10- to 20-nm diameter, as observed by atomic force microscopy. The growth of OCP was modulated uniquely in 10% rM179 and rM166 amelogenin gels, regardless of the presence of the hydrophilic C-terminal residues. Fibrous crystals grew with large length-to-width ratio and small width-to-thickness ratio. Both rM179 and rM166 enhanced the growth of elongated OCP crystals, suggesting a relationship to the initial elongated growth of enamel crystals.     

Assembly and processing of an engineered amelogenin proteolytic product (rP148)

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 .     

Control of apatite crystal growth by the co-operative effect of a recombinant porcine amelogenin and fluoride

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.     

Regulation of calcium phosphate formation by amelogenins under physiological conditions

Amelogenin is essential for proper enamel formation. The present in vitro study extends our previous work at low (10 mM) ionic strength (IS) by examining the effect of amelogenin on mineralization under higher (162 mM) IS conditions found in developing enamel. Full-length phosphorylated (P173) and non-phosphorylated (rP172) amelogenins were examined, along with P148 and rP147 that lack the hydrophilic C-terminus. Calcium phosphate formation was assessed by pH change, while the minerals formed were characterized using transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy. Amelogenin self-assembly was also studied using dynamic light scattering and TEM. The results indicate that IS does not influence the effects of rP147, rP172, and P173 on mineralization. However, in contrast to the findings for low IS, where both P173 and P148 stabilize initially formed amorphous calcium phosphate (ACP) nanoparticles for >1 d, elongated hydroxyapatite crystals were observed after 24 h using P148 at high IS, unlike that seen with P173. Differences in self-assembly help explain these findings, which suggest that P173 and P148 may play different roles in regulating enamel mineral formation. The present data support the notion that proteolytic processing of P173 is required in vivo to induce the transformation of initial ACP phases to apatitic enamel crystals.     

Amelogenin-enamelin association in phosphate-buffered saline

The structures and interactions among macromolecules in the enamel extracellular matrix play vital roles in regulating hydroxyapatite crystal nucleation, growth, and maturation. We used dynamic light scattering (DLS), circular dichroism (CD), fluorescence spectroscopy, and transmission electron microscopy (TEM) to investigate the association of amelogenin and the 32-kDa enamelin, at physiological pH 7.4, in phosphate-buffered saline (PBS). The self-assembly behavior of amelogenin (rP148) was altered following addition of the 32-kDa enamelin. Dynamic light scattering revealed a trend for a decrease in aggregate size in the solution following the addition of enamelin to amelogenin. A blue-shift and intensity increase of the ellipticity minima of rP148 in the CD spectra upon the addition of the 32-kDa enamelin, suggest a direct interaction between the two proteins. In the fluorescence spectra, the maximum emission of rP148 was red-shifted from 335 to 341 nm with a marked intensity increase in the presence of enamelin as a result of complexation of the two proteins. In agreement with DLS data, TEM imaging showed that the 32-kDa enamelin dispersed the amelogenin aggregates into oligomeric particles and stabilized them. Our study provides novel insights into understanding the possible cooperation between enamelin and amelogenin in macromolecular co-assembly and in controlling enamel mineral formation.     

Self-assembly and effect on crystal growth of the leucine-rich amelogenin peptide

Amelogenins are a unique group of alternatively spliced proteins. While the full-length amelogenin is known to assemble into nanospheres and alter apatite crystal growth and alignment, the function of the leucine-rich amelogenin peptide (LRAP) in biomineralization is not understood. This study tested the hypothesis that LRAP self-assembles into a supramolecular structure and guides crystal growth similarly to the full-length protein. Synthetic LRAP and recombinant full-length amelogenin (rH175) were used at different concentrations and either immobilized onto fluoroapatite substrates (FAP) or immersed into saturated calcium-phosphate solutions. The structure of the assembled protein and the height of apatite crystals formed on the FAP template were determined using atomic force microscopy. Both LRAP and rH175 assembled into nanospheres. LRAP self-assembly, however, was only observed at concentrations of > 0.5 mg ml⁻¹ and limited to sizes between 5 and 30 nm. Apatite crystal growth was not significantly affected by LRAP, while rH175 accelerated crystal growth by up to 50-fold. The increased growth rate was only observed when rH175 precipitated at concentrations of > 0.8 mg ml⁻¹. It was concluded that the ability of amelogenins to self-assemble into nanospheres and to bind to apatite in vitro is not inevitably an indication for the ability to control apatite crystal growth.     

Characterization of recombinant pig enamelysin activity and cleavage of recombinant pig and mouse amelogenins

Enamelysin (MMP-20) is a tooth-specific matrix metalloproteinase that is initially expressed by ameloblasts and odontoblasts immediately prior to the onset of dentin mineralization, and continues to be expressed throughout the secretory stage of amelogenesis. During the secretory stage, enamel proteins are secreted and rapidly cleaved into a large number of relatively stable cleavage products. Multiple proteinases are present in the developing enamel matrix, and the precise role of enamelysin in the processing of enamel proteins is unknown. We have expressed, activated, and purified the catalytic domain of recombinant pig enamelysin, and expressed a recombinant form of the major secreted pig amelogenin rP172. These proteins were incubated together, and the digestion products were analyzed by SDS-PAGE and mass spectrometric analyses. We assigned amelogenin cleavage products by selecting among the possible polypeptides having a mass within 2 Daltons of the measured values. The polypeptides identified included the intact protein (amino acids 2-173), as well as 2-148, 2-136, 2-107, 2-105, 2-63, 2-45, 46-148, 46-147, 46-107, 46-105, 64-148, 64-147, and 64-136. These fragments of rP172 include virtually all of the major amelogenin cleavage products observed in vivo. We propose that enamelysin is the predominant proteinase that processes enamel proteins during the secretory phase of amelogenesis.     

Porcine Amelogenin : Alternative Splicing, Proteolytic Processing, Protein - Protein Interactions, and Possible Functions

Amelogenin is the major secretory product of ameloblasts and is critical for proper tooth enamel formation. Amelogenin isoforms and their cleavage products comprise over 80% of total secretory stage enamel protein. We have isolated and characterized four secreted amelogenin isoforms from developing porcine enamel : P190 (27-kDa), P173 (25-kDa), P132 (18-kDa) and P56 (6.5-kDa ; leucine rich amelogenin polypeptide or LRAP). P190 and P132 are low abundance amelogenins that contain a novel exon 4-encoded segment of lack the exon 3-encoded segment, respectively. P173 is the most abundant (major) amelogenin isoform. Cleavage of P173 by matrix metalloproteinase 20 (Mmp20) occurs at specific sites that generates a set of N-terminal cleavage products : P162 (23-kDa), P148 (20-kDa), P62/P63 (11-kDa), and Trp(45) (6-kDa, tyrosine rich amelogenin polypeptide or TRAP). P148 is the most abundant protein in developing enamel and influences the conversion of amorphous calcium phosphate into hydroxyapatite in vitro. Mmp20 cleaves LRAP, the second abundant amelogenin isoform after Pro(45) and Pro(40). Processing by Mmp20 allows amelogenin cleavage products to serve separate functions. Over time, Mmp20 catalyzes additional cleavages that facilitate the progressive replacement of amelogenin by mineral, so enamel crystals thicken and widen with depth. Besides proteolytic processing, amelogenin protein-protein interactions are critical for function. Far-Western analyses demonstrate that the larger amelogenins (P173, P162, and P148) are only able to interact with larger amelogenins. No amelogenin-amelogenin interactions are observed for the smaller amelogenin cleavage products, TRAP or LRAP Amelogenin doesn't interact with the 32-kDa glycosylated enamelin cleavage product, unless it it partially deglycosylated.     

Evidence for direct amelogenin–target cell interactions using dynamic force spectroscopy

Increasing evidence suggests that amelogenin, long held to be a structural protein of developing enamel matrix, may also have cell signaling functions. However, a mechanism for amelogenin cell signaling has yet to be described. The aim of the present study was to use dynamic chemical force spectroscopy to measure amelogenin interactions with possible target cells. Full-length amelogenin (rM179) was covalently attached to silicon nitride AFM tips. Synthetic RGD peptides and unmodified AFM tips were used as controls. Amelogenin–RGD cell binding force measurements were carried out using human periodontal ligament fibroblasts (HPDF) from primary explants and a commercially available osteoblast-like human sarcoma cell line as the targets. Results indicated a linear logarithmic dependence between loading rate and unbinding force for amelogenin–RGD target cells across the range of loading rates used. For RGD controls, binding events measured at 5.5 nN s⁻¹ force loading rate resulted in a mean force of 60 pN. Values for amelogenin–fibroblast and amelogenin–osteoblast-like cell unbinding forces, measured at similar loading rates, were 50 and 55 pN, respectively. These data suggest that amelogenin interacts with potential target cells with forces characteristic of specific ligand–receptor binding, suggesting a direct effect for amelogenin at target cell membranes.     

Dynamic light scattering study of an amelogenin gel-like matrix 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 ≈ 30°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.     

Altered self-assembly and apatite binding of amelogenin induced by N-terminal proline mutation

Objective

A single Pro-70 to Thr (p.P70T) mutation of amelogenin is known to result in hypomineralised amelogenesis imperfecta (AI). This study aims to test the hypothesis that the given mutation affects the self-assembly of amelogenin molecules and impairs their ability to conduct the growth of apatite crystals.

Design

Recombinant human full-length wild-type (rh174) and p.P70T mutated amelogenins were analysed using dynamic light scattering (DLS), protein quantification assay and atomic force microscopy (AFM) before and after the binding of amelogenins to hydroxyapatite crystals. The crystal growth modulated by both amelogenins in a dynamic titration system was observed using AFM.

Results

As compared to rh174 amelogenin, p.P70T mutant displayed significantly increased sizes of the assemblies, higher binding affinity to apatite, and decreased crystal height.

Conclusion

Pro-70 plays an important structural role in the biologically relevant amelogenin self-assembly. The disturbed regularity of amelogenin nanospheres by this single mutation resulted in an increased binding to apatite and inhibited crystal growth.     

Self-assembly of amelogenin proteins at the water-oil interface

Self-assembly of amelogenin plays a key role in controlling enamel biomineralization. Recently, we generated self-aligning nanoribbons of amelogenin in water-in-oil emulsions stabilized by the full-length protein (rH174). Here, we tested the hypothesis that the hydrophilic C-terminus is critical for self-assembly of amelogenin into nanoribbons. The self-assembled structures of two amelogenin cleavage products, rH163 and rH146, were compared with structures of rH174 at different pH values and degrees of saturation using atomic force microscopy, electron microscopy, and dynamic light scattering. We observed that the number density of rH174 nanoribbons increased significantly when the initial pH was raised from 4.5 to 5.6. Nanoribbons, as well as unique helical nanostructures, were also readily observed when amelogenin rH146 was used, but showed little tendency for parallel alignment and did not bundle into fibrils like rH174. In contrast, rH163 rarely formed nanoribbons but predominantly assembled into nanospheres under the same conditions. We conclude that the presence of a hydrophilic C-terminus may not be a prerequisite for nanoribbon formation but may be critical for ribbon alignment and subsequent fibril formation. These results highlight the contribution of the hydrophobic domain in the self-assembly of elongated structures of amelogenins. Molecular mechanisms governing these processes based on the formation of reverse micelles are discussed.     

Expression and characterization of a Rana pipiens amelogenin protein

Amelogenin, the major protein of developing enamel matrix, controls enamel crystal growth via unique supermolecular features. While much has been contributed to our understanding of mammalian amelogenin function, little is known about how amelogenin and its unique physico-chemical features have evolved among vertebrates. Here we report, for the first time, amphibian amelogenin recombinant protein expression and characterization in Rana pipiens . In order to characterize R. pipiens amelogenin, the newly discovered amelogenin coding sequence was amplified, subcloned, and expressed in Eshcerichia coli . Our newly generated R. pipiens amelogenin-specific antisera resolved a major 19-kDa band on western blots of frog tooth extracts and revealed an enamel organ tissue-specific localization pattern using immunohistochemistry. Using mass spectroscopy, a single major compound with a molecular weight of 21.6 kDa was detected, which corresponded to the amino acid sequence-based molecular weight prediction of the His fusion recombinant protein. Dynamic light scattering studies resolved 41-nm radius subunits compared with 14-nm radius subunits from mouse recombinant amelogenin controls. Transmission electron microscopy revealed defined spherical subunits in R. pipiens matrix self-assembly in contrast with a homogeneous 'stippled' matrix in mouse amelogenin matrix self-assembly. Our data suggest that R. pipiens amelogenin is distinguished from mammalian amelogenins by a number of unique physico-chemical properties which may be related to specific modes of crystal formation in frog enamel.     

Amelogenins: Multi-Functional Enamel Matrix Proteins and Their Binding Partners

Amelogenins are the most abundant extracellular matrix proteins secreted by ameloblasts during tooth development and are important for enamel formation. Recently, amelogenins have been detected not only in ameloblasts, which are differentiated from the epithelial cell lineage, but also in other tissues, including mesenchymal tissues at low levels, suggesting that amelogenins possess other functions in these tissues. The therapeutic application of an enamel matrix derivative rich in amelogenins resulted in the regeneration of cementum, alveolar bone, and periodontal ligament (PDL) in the treatment of experimental or human periodontitis, indicating the attractive potential of amelogenin in hard tissue formation. In addition, a full-length amelogenin (M180) and leucine-rich amelogenin peptide (LRAP) regulate cementoblast/ PDL cell proliferation and migration in vitro. Interestingly, amelogenin null mice show increased osteoclastogenesis and root resorption in periodontal tissues. Recombinant amelogenin proteins suppress osteoclastogenesis in vivo and in vitro, suggesting that amelogenin is involved in preventing idiopathic root resorption. Amelogenins are implicated in tissue-specific epithelial-mesenchymal or mesenchymal-mesenchymal signaling ; however, the precise molecular mechanism has not been characterized.In this review, we first discuss the emerging evidence for the additional roles of M180 and LRAP as signaling molecules in mesenchymal cells. Next, we show the results of a yeast two-hybrid assay aimed at identifying protein-binding partners for LRAP We believe that gaining further insights into the signaling pathway modulated by the multifunctional amelogenin proteins will lead to the development of new therapeutic approaches for treating dental diseases and disorders.     

Amelogenins in human developing and mature dental pulp

Amelogenins are a group of heterogenous proteins first identified in developing tooth enamel and reported to be present in odontoblasts. The objective of this study was to elucidate the expression and function of amelogenins in the human dentin-pulp complex. Developing human tooth buds were immunostained for amelogenin, and mRNA was detected by in situ hybridization. The effects of recombinant amelogenins on pulp and papilla cell proliferation were measured by Brd U immunoassay, and differentiation was monitored by alkaline phosphatase expression. Amelogenin protein was found in the forming dentin matrix, and amelogenin mRNA was localized in the dentin, presumably in the odontoblast processes. Proliferation of papilla cells was enhanced by recombinant human amelogenin rH72 (LRAP+ exon 4), while pulp cells responded to both rH72 and rH58 (LRAP), with no effect by rH174. These studies suggest that odontoblasts actively synthesize and secrete amelogenin protein during human tooth development, and that low-molecular-weight amelogenins can enhance pulp cell proliferation.     

Micelle structure of amelogenin in porcine secretory enamel

Even during the secretory stage of amelogenesis, enamel crystals thicken as amelogenins (the major protein component) decrease. To explain this phenomenon, we propose a model for amelogenin structure and function based upon the hypothesis that amelogenin forms micelles. Solubility and hydrophobicity analyses suggest that all but the hydrophilic amelogenin C-terminal regions aggregate via hydrophobic bonds to form a micelle core. Amelogenin micelles may form super-assemblies via their C-termini (KTKREEVD), which contain complementary positive (KTKR) and negative (EEVD) elements. Disassembly of the micelles through controlled proteolysis provides space for crystal growth. Initial cleavage (by enamelysin) removes the surface-accessible amelogenin C-terminus, exposing the middle portion to cleavage (by EMSP1). As a result, the 13-kDa amelogenin, a rod-shaped domain based upon ultrafiltration and transmission electron microscopy studies, is released. This model explains how amelogenin is able to 'space' and support the ribbon-like crystals and continuously yield space as the crystals thicken, until they are sufficiently mature to support themselves.     

Amelogenin post-secretory processing during biomineralization in the postnatal mouse molar tooth

The primary structures, molecular genetics and biosynthesis of the amelogenin protein of the developing tooth are established, but knowledge of their subsequent post-secretory processing and its relation to enamel biomineralization is fragmentary. Preparations of tooth matrix proteins were isolated from molars (M1) of mice from birth to 15 days and analysed by SDS-PAGE and immunochemical methods. Amelogenin proteins, isolated and partially purified by HPLC, were characterized by amino acid analysis and SDS-PAGE. At birth a 26 kDa amelogenin was present that during subsequent developmental stages generated a series of 20-25 kDa amelogenins differing in apparent size by approximately 1 kDa. Amino acid analyses showed that all these amelogenins have amino-terminal TRAP sequences; analyses for both glycosylation and phosphorylation were negative. It is suggested that these post-secretory amelogenins are generated by a sequence of specific carboxy-terminal cleavages, and that the observed post-secretory processing of amelogenin is functionally linked to the structure of the enamel matrix and the control of crystallite development.     

Hydrolysis of amelogenin by matrix metalloprotease-20 accelerates mineralization in vitro

In the following respects, tooth enamel is a unique tissue in the mammalian body: (a) it is the most mineralized and hardest tissue in it comprising up to 95 wt% of apatite; (b) its microstructure is dominated by parallel rods composed of bundles of 40–60 nm wide apatite crystals with aspect ratios reaching up to 1:10,000 and (c) not only does the protein matrix that gives rise to enamel guides the crystal growth, but it also conducts its own degradation and removal in parallel. Hence, when mimicking the process of amelogenesis in vitro, crystal growth has to be coupled to proteolytic digestion of the amelogenin assemblies that are known to play a pivotal role in conducting the proper crystal growth. Experimental settings based on controlled and programmable titration of amelogenin sols digested by means of MMP-20 with buffered calcium and phosphate solutions were employed to imitate the formation of elongated, plate-shaped crystals. Whilst amelogenin can act as a promoter of nucleation and crystal growth alone, in this study we show that proteolysis exerts an additional nucleation- and growth-promoting effect. Hydrolysis of full-length amelogenin by MMP-20 decreases the critical time needed for the protein and peptides to adhere and to cover the substrate. The formation and immobilization of a protein layer subsequently reduces the time for calcium phosphate crystallization. Coupling the proteolytic reaction to titration in the presence of 0.4 mg/ml rH174 has been shown to have the same effect on the crystal growth promotion as quadrupling the concentration of rH174 to 1.6 mg/ml. Controlling the rate and the extent of the proteolytic cleavage can thus be used to control the nucleation and growth rates in a protein-guided crystallization system.