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Objective: A 37-residue amino acid sequence corresponding to the segment encoded by exon-5 of murine ameloblastin (Ambn), AB2 (Y67-Q103), has been implicated with membrane association, ameloblastin self-assembly, and amelogenin-binding. Our aim was to characterize, at the residue level, the structural behavior of AB2 bound to chemical mimics of biological membranes using NMR spectroscopy., Design: To better define the structure of AB2 using NMR-based methods, recombinant 13 C- and 15 N-labelled AB2 (*AB2) was prepared and data collected free in solution and with deuterated dodecylphosphocholine (dPC) micelles, deuterated bicelles, and both small and large unilamellar vesicles., Results: Amide chemical shift and intensity perturbations observed in 1 H- 15 N HSQC spectra of *AB2 in the presence of bicelles and dPC micelles suggest that a region of *AB2, S6-E36 (murine Ambn S68 - E98), associates with the membrane biomimetics. A CSI-3 analysis of the NMR chemical shift assignments for *AB2 free in solution and bound to dPC micelles indicated the peptide remains disordered except for the adoption of a short, 12-residue α-helix, F10-G21 (murine Ambn F72-G83). In dPC micelles, the NOE NMR data was void of patterns characteristic of long-lived helical structure indicating this helix was transient in nature., Conclusions: A continuum of intrinsic disorder in the membrane-bound state may be responsible for ameloblastin's ability to dynamically interact with multiple partners at the same site during amelogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

In human, mutations in the gene encoding the enamel matrix protein ameloblastin (Ambn) have been identified in cases of amelogenesis imperfecta. In mouse models, perturbations in the Ambn gene have caused loss of enamel and dramatic disruptions in enamel-making ameloblast cell function. Critical roles for Ambn in ameloblast cell signaling and polarization as well as adhesion to the nascent enamel matrix have been supported. Recently, we have identified a multitargeting domain (MTD) in Ambn that interacts with cell membrane, with the majority enamel matrix protein amelogenin, and with itself. This domain includes an amphipathic helix (AH) motif that directly interacts with cell membrane. In this study, we analyzed the sequence of the MTD for evolutionary conservation and found high conservation among mammals within the MTD and particularly within the AH motif. We computationally predicted that the AH motif lost its hydrophobic moment upon deleting hydrophobic but not hydrophilic residues from the motif. Furthermore, we rationally designed peptides that encompassed the Ambn MTD and contained deletions of largely hydrophobic or hydrophilic stretches of residues. To assess their AH-forming and membrane-binding abilities, we combined those peptides with synthetic phospholipid membrane vesicles and performed circular dichroism, membrane leakage, and vesicle clearance measurements. Circular dichroism showed retention of α-helix formation in all peptides except the one with the largest deletion of eleven amino acids including seven that were hydrophobic. This same peptide variant failed to cause leakage or clearance of synthetic membranes, while smaller deletions yielded intermediate membrane interaction as measured by leakage and clearance assays. Our data revealed that deletion of key hydrophobic residues from the AH leads to the most dramatic loss of Ambn-membrane interaction. Pinpointing roles of residues within the MTD has important implications for the multifunctionality of Ambn., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Biomimetic strategies like peptide-guided collagen mineralization promise to enhance the effectiveness of dentin remineralization. We recently reported that rationally designed amelogenin-derived peptides P26 and P32 promoted apatite nucleation, mineralized collagen, and showed potential in enamel regrowth and dentin remineralization. To facilitate the clinical application of amelogenin-derived peptides and to uncover their effectiveness in repairing dentin, we have now implemented a chitosan (CS) hydrogel for peptide delivery and have investigated the effects of P26-CS and P32-CS hydrogels on dentin remineralization using 2 in situ experimental models that exhibited different levels of demineralization. The efficacy of the peptide-CS hydrogels in dentin repair was evaluated by characterizing the microstructure, mineral density, mineral phase, and nanomechanical properties of the remineralized samples. The new strategy of atomic force microscopy PeakForce quantitative nanomechanical mapping was used for direct visualization and nanomechanical analysis of repaired dentin lesions across the lesion depth. Results from the 2 models indicated the potential triple functions of peptide-CS hydrogels for dentin repair: building a highly organized protective mineralized layer on dentin, occluding dentinal tubules by peptide-guided in situ mineralization, and promoting biomimetic dentinal collagen remineralization. Importantly, peptides released from the CS hydrogel could diffuse into the dentinal matrix and penetrate the dentinal tubules, leading to both surface and subsurface remineralization and tubule occlusion. Given our previous findings on peptide-CS hydrogels' potential for remineralizing enamel, we see further promise for hydrogels to treat tooth defects involving multiple hard tissues, as in the case of noncarious cervical lesions. [ABSTRACT FROM AUTHOR]

Extracellular matrix proteins play crucial roles in the formation of mineralized tissues like bone and teeth via multifunctional mechanisms. In tooth enamel, ameloblastin (Ambn) is one such multifunctional extracellular matrix protein implicated in cell signaling and polarity, cell adhesion to the developing enamel matrix, and stabilization of prismatic enamel morphology. To provide a perspective for Ambn structure and function, we begin this review by describing dental enamel and enamel formation (amelogenesis) followed by a description of enamel extracellular matrix. We then summarize the established domains and motifs in Ambn protein, human amelogenesis imperfecta cases, and genetically engineered mouse models involving mutated or null Ambn. We subsequently delineate in silico, in vitro, and in vivo evidence for the amphipathic helix in Ambn as a proposed cell-matrix adhesive and then more recent in vitro evidence for the multitargeting domain as the basis for dynamic interactions of Ambn with itself, amelogenin, and membranes. The multitargeting domain facilitates tuning between Ambn-membrane interactions and self/co-assembly and supports a likely overall role for Ambn as a matricellular protein. We anticipate that this review will enhance the understanding of multifunctional matrix proteins by consolidating diverse mechanisms through which Ambn contributes to enamel extracellular matrix mineralization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Biomineralization of enamel, dentin, and bone involves the deposition of apatite mineral crystals within an organic matrix. Bone and teeth are classic examples of biomaterials with unique biomechanical properties that are crucial to their function. The collagen-based apatite mineralization and the important function of noncollagenous proteins are similar in dentin and bone; however, enamel is formed in a unique amelogenin-containing protein matrix. While the structure and organic composition of enamel are different from those of dentin and bone, the principal molecular mechanisms of protein–protein interactions, protein self-assembly, and control of crystallization events by the organic matrix are common among these apatite-containing tissues. This review briefly summarizes enamel and dentin matrix components and their interactions with other extracellular matrix components and calcium ions in mediating the mineralization process. We highlight the crystallization events that are controlled by the protein matrix and their interactions in the extracellular matrix during enamel and dentin biomineralization. Strategies for peptide-inspired biomimetic growth of tooth enamel and bioinspired mineralization of collagen to stimulate repair of demineralized dentin and bone tissue engineering are also addressed. [ABSTRACT FROM AUTHOR]

Amelogenin (Amel) and ameloblastin (Ambn) are two primary extracellular enamel matrix proteins that play crucial roles for proper thickness, prismatic structure, and robust mechanical properties. Previous studies have shown that Amel and Ambn bind to each other, but the effect of their coassembly on the nucleation of hydroxyapatite (HAP) is unclear. Here, we systematically investigated the coassembly of recombinant mouse Amel and Ambn in various ratios using in situ atomic force microscopy, dynamic light scattering, and transmission electron microscopy. The size of protein particles decreased as the Ambn:Amel ratio increased. To define the coassembly domain on Ambn, we used Ambn-derived peptides and Ambn variants to examine their effects on the amelogenin particle size distribution. We found that the peptide sequence encoded by exon 5 of Ambn affected Amel self-assembly but the variant lacking this sequence did not have any effect on Amel self-assembly. Furthermore, through monitoring the pH change in bulk mineralization solution, we tracked the nucleation behavior of HAP in the presence of Ambn and Amel and found that their coassemblies at different ratios showed varying abilities to stabilize amorphous calcium phosphate. These results demonstrated that Ambn and Amel coassemble with each other via a motif within the sequence encoded by exon 5 of Ambn and cooperate in regulating the nucleation of HAP crystals, enhancing our understanding of the important role of enamel matrix proteins in amelogenesis.

Biomimetic synthesis of artificial enamel is a promising strategy for the prevention and restoration of defective enamel. We have recently reported that a hydrogel system composed of chitosan-amelogenin (CS-AMEL) and calcium phosphate is effective in forming an enamel-like layer that has a seamless interface with natural tooth surfaces. Here, to improve the mechanical system function and to facilitate the biomimetic enamel regrowth, matrix metalloproteinase-20 (MMP-20) was introduced into the CS-AMEL hydrogel. Inspired by our recent finding that MMP-20 prevents protein occlusion inside enamel crystals, we hypothesized that addition of MMP-20 to CS-AMEL hydrogel could reinforce the newly grown layer. Recombinant human MMP-20 was added to the CS-AMEL hydrogel to cleave full-length amelogenin during the growth of enamel-like crystals on an etched enamel surface. The MMP-20 proteolysis of amelogenin was studied, and the morphology, composition, and mechanical properties of the newly grown layer were characterized. We found that amelogenin was gradually degraded by MMP-20 in the presence of chitosan. The newly grown crystals in the sample treated with MMP-20-CS-AMEL hydrogel showed more uniform orientation and greater crystallinity than the samples treated with CS-AMEL hydrogel without MMP-20. Stepwise processing of amelogenin by MMP-20 in the CS-AMEL hydrogel prevented undesirable protein occlusion within the newly formed crystals. As a result, both the modulus and hardness of the repaired enamel were significantly increased (1.8- and 2.4-fold, respectively) by the MMP-20-CS-AMEL hydrogel. Although future work is needed to further incorporate other enamel matrix proteins into the system, this study brings us one step closer to biomimetic enamel regrowth. [ABSTRACT FROM AUTHOR]

The enamel matrix protein Ameloblastin (Ambn) has critical physiological functions, including regulation of mineral formation, cell differentiation, and cell-matrix adhesion. We investigated localized structural changes in Ambn during its interactions with its targets. We performed biophysical assays and used liposomes as a cell membrane model. The xAB2N and AB2 peptides were rationally designed to encompass regions of Ambn that contained self-assembly and helix-containing membrane-binding motifs. Electron paramagnetic resonance (EPR) on spin-labeled peptides showed localized structural gains in the presence of liposomes, amelogenin (Amel), and Ambn. Vesicle clearance and leakage assays indicated that peptide-membrane interactions were independent from peptide self-association. Tryptophan fluorescence and EPR showed competition between Ambn-Amel and Ambn-membrane interactions. We demonstrate localized structural changes in Ambn upon interaction with different targets via a multitargeting domain, spanning residues 57 to 90 of mouse Ambn. Structural changes of Ambn following its interaction with different targets have relevant implications for the multifunctionality of Ambn in enamel formation.

The distinct morphology adopted by ameloblasts during amelogenesis is highly stage specific and involved intimately with the development of a hierarchical enamel microstructure. The molecular mechanisms that govern the development of an elongated and polarized secretory ameloblast morphology and the potential roles played by the enamel matrix proteins in this process are not fully understood. Thus far, the in vitro models that have been developed to mimic these early cell-matrix interactions have either been unable to demonstrate direct morphological change or have failed to adapt across ameloblast cell lines. Here, we use a recently established 3D cell culture model to examine the interactions between HAT-7 cells and the major enamel matrix proteins, amelogenin and ameloblastin. We demonstrate that HAT-7 cells selectively respond to functional EMPs in culture by forming clusters of tall cells. Aspect ratio measurements from three-dimensional reconstructions reveal that cell elongation is 5-times greater in the presence of EMPs when compared with controls. Using confocal laser scanning microscopy, we observe that these clusters are polarized with asymmetrical distributions of Par-3 and claudin-1 proteins. The behavior of HAT-7 cells in 3D culture with EMPs is comparable with that of ALC and LS-8 cells. The fact that the 3D model presented here is tunable with respect to gel substrate composition and ameloblast cell type highlights the overall usefulness of this model in studying ameloblast cell morphology in vitro ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Visakan, Bapat, Su and Moradian-Oldak.)

To investigate correlation between the ameloblastin (Ambn) amino acid sequence and the emergence of prismatic enamel, a notable event in the evolution of ectodermal hard tissues, we analyzed Ambn sequences of 53 species for which enamel microstructures have been previously reported. We found that a potential amphipathic helix (AH) within the sequence encoded by Exon 5 of Ambn appeared in species with prismatic enamel, with a few exceptions. We studied this correlation by investigating synthetic peptides from different species. A blue shift in fluorescence spectroscopy suggested that the peptides derived from mammalian Ambn interacted with liposomes. A downward shift at 222 nm in circular dichroism spectroscopy of the peptides in the presence of liposomes suggested that the peptides of mammals with prismatic enamel underwent a transition from disordered to helical structure. The peptides of species without prismatic enamel did not show similar secondary structural changes in the presence of liposomes. Peptides of mammals with prismatic enamel caused liposome leakage and inhibited LS8 and ALC cell spreading regulated by full-length Ambn. RT-PCR showed that AH is involved in Ambn's regulation of cell polarization genes: Vangl2, Vangl1, Prickle1, ROCK1, ROCK2, and Par3. Our comprehensive sequence analysis clearly demonstrates that AH motif is closely related to the emergence of enamel prismatic structure, providing insight into the evolution of complex enamel microstructure. We speculate that the AH motif evolved in mammals to interact with cell membrane, triggering signaling pathways required for specific changes in cell morphology associated with the formation of enamel prismatic structure., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

This article contains data related to the research article in this issue titled ameloblastin promotes polarization of ameloblast cell lines in a 3D cell culture system (Visakan et al., 2022). In the process of amelogenesis, the organic matrix components are pivotal to the establishment of ameloblast-matrix adhesion. Here we employ immortalized ameloblast cell lines and analyse their morphological changes in 3D cell culture when cultured in the presence of recombinant enamel matrix proteins- ameloblastin and amelogenin compared with controls. The recombinant proteins that were purified using high-performance liquid chromatography (HPLC) were characterized using SDS-gel electrophoresis. A 3D-on-top culture technique was employed, and the cells were analysed 24 and 72 h post inoculation using fluorescent and confocal microscopy for qualitative and quantitative changes. Aspect ratio of cells was measured and used as the parameter to compare between test proteins and controls. Repeated measurements of aspect ratio were recorded to analyse for statistical significance. Additionally, three distinct gel substrates were studied to examine the effect of composition and stiffness of the substrate on cell behaviour. The cells in the 3D culture were fixed and labelled using antibodies to junctional complex, polarity and tight junctional proteins following protocols for whole culture fixation. Co-localization between membrane and specific antibody labels were examined under confocal microscopy., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Studies on animal models with mutations in ameloblastin gene have suggested that the extracellular matrix protein ameloblastin (AMBN) plays important roles in controlling cell-matrix adhesion and ameloblast polarization during amelogenesis. In order to examine the function of AMBN in cell polarization and morphology, we developed an in vitro 3D cell culture model to examine the effect of AMBN and amelogenin (AMEL) addition on ameloblast cell lines. We further used high resolution confocal microscopy to detect expression of polarization markers in response to AMBN addition. Addition of AMBN to the 3D culture matrix resulted in the clustering and elongation (higher aspect ratio) of ALC in a dose dependent manner. The molar concentration of AMEL required to exact this response from ALC was 2.75- times greater than that of AMBN. This polarization effect of ameloblastin was attributable directly to an evolutionary conserved domain within its exon 5-encoded region. The lack of exon 6-encoded region also influenced AMBN-cell interactions but to a lesser extent. The clusters formed with AMBN were polarized with expression of E-cadherin, Par3 and Cldn1 assembly at the nascent cell-cell junctions. The elongation effect was specific to epithelial cells of ameloblastic lineage ALC and LS8 cells. Our data suggest that AMBN may play critical signaling roles in the initiation of cell polarity by acting as a communicator between cell-cell and cell-matrix interactions. Our investigation has important implications for understanding the function of ameloblastin in enamel-cell matrix adhesion and the outcomes may contribute to efforts to develop strategies for enamel tissue regeneration., Competing Interests: Declaration of competing interests None, (Copyright © 2021 Elsevier B.V. All rights reserved.)

The formation of enamel apatite crystals involves extracellular molecular events among which matrix assembly, interactions with growing crystals, and protein processing and removal are the subject of numerous investigations. Following the description of amelogenin nanospheres and the evidence for their presence in vivo as the principal structural component of developing dental enamel, we have focused our studies on investigating at the molecular level the process of nanosphere assembly and evaluating the effects of amelogenin on crystal growth and morphology. This paper is a short review of our recent studies with a focus on the assembly of amelogenin proteolytic products and their modulating effect on octacalcium phosphate (OCP) crystal morphology. In addition, we report that incorporation of amelogenins into 10% gelatin gel does not affect diffusion of calcium. This remarkable finding indicates that the observed modulation effect by amelogenin on OCP crystal morphology is not due to alteration of calcium diffusion into the gels but is the result of direct amelogenin-mineral interactions. [ABSTRACT FROM AUTHOR]

We designed synthetic peptides that have demonstrated an effective remineralization potential to restore incipient enamel decay. In order to develop a clinically viable approach we incorporated the amelogenin-derived peptides P26 and P32 into chitosan hydrogel and examined their efficacy in the remineralization of enamel. Peptides in chitosan exhibited increased stability in vitro as compared to peptides in solution at room temperature and at 37°C. Tooth models for enamel erosion (sections) and white spot lesions (blocks) were subject to periods of demineralization. Treatment groups were subjected to remineralization in artificial saliva in the presence of P26 and P32 in solution and in chitosan hydrogel (P26-CS and P32-CS). Quantitative light-induced fluorescence (QLF) was employed to analyze mineral density following demineralization and remineralization across all the treatment groups. Scanning electron microscopy and nanoindentation were used to characterize the surface structure and mechanical strength of regrown enamel. Control enamel sections treated in artificial saliva demonstrated randomly distributed, tiny, needle-shaped crystals with a low packing density and porosities displaying mineralization defects. In samples treated with P26-CS or P32-CS a denser coating of organized hydroxyapatite (HAP) crystals was formed covering the entire surfaces of demineralized enamel window. The hardness and modulus of enamel surfaces were increased after treatment with P26-CS and P32-CS with no significant difference in the mechanical properties between the two peptide hydrogels. Analysis of mineral density by QLF showed that in enamel sections P26 peptide alone or P26-CS significantly enhanced the remineralization. In enamel blocks P26 in solution had a better efficacy than P26-CS., Competing Interests: Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Macromolecular assembly of extracellular enamel matrix proteins (EMPs) is intimately associated with the nucleation, growth, and maturation of highly organized hydroxyapatite crystals giving rise to healthy dental enamel. Although the colocalization of two of the most abundant EMPs amelogenin (Amel) and ameloblastin (Ambn) in molar enamel has been established, the evidence toward their interaction is scarce. We used co-immunoprecipitation (co-IP) to show evidence of direct molecular interactions between recombinant and native Amel and Ambn. Ambn fragments containing Y/F-x-x-Y/L/F-x-Y/F self-assembly motif were isolated from the co-IP column and characterized by mass spectroscopy. We used recombinant Ambn (rAmbn) mutants with deletion of exons 5 and 6 as well as Ambn derived synthetic peptides to demonstrate that Ambn binds to Amel via its previously identified Y/F-x-x-Y/L/F-x-Y/F self-assembly motif at the N-terminus of its exon 5 encoded region. Using an N-terminal specific anti-Ambn antibody, we showed that Ambn N-terminal fragments colocalized with Amel from secretory to maturation stages of enamel formation in a single section of developing mouse incisor, and closely followed mineral patterns in enamel rod interrod architecture. We conclude that Ambn self-assembly motif is involved in its interaction with Amel in solution and that colocalization between the two proteins persists from secretory to maturation stages of amelogenesis. Our in vitro and in situ data support the notion that Amel and Ambn may form heteromolecular assemblies that may perform important physiological roles during enamel formation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Bapat, Su and Moradian-Oldak.)

Mammalian teeth primarily consist of two distinct calcified tissues, enamel and dentin, that are intricately integrated by a complex and critical structure, the dentin-enamel junction (DEJ). Loss of enamel exposes the underlying dentin, increasing the risk of several irreversible dental diseases. This paper highlights the significance of utilizing the functional domains of a major enamel matrix protein, amelogenin, intrinsic to tooth enamel and the DEJ interface, to rationally design smaller bioinspired peptides for regeneration of tooth microstructures. Using this strategy, we designed a synthetic peptide, P26, that demonstrates a remarkable dual mineralization potential to restore incipient enamel decay and mineralization defects localized in peripheral dentin below the DEJ. As a proof of principle, we demonstrate that interaction between P26 and collagen prompts peptide self-assembly, followed by mineralization of collagen fibrils in vitro . P26-mediated nucleation of hydroxyapatite (HAP) crystals on demineralized dentin in situ significantly facilitates the recovery of mineral density and effectively restores the biomechanical properties of dentin to near-native levels, suggesting that P26-based therapy has promising applications for treating diverse mineralized tissue defects in the tooth., Competing Interests: Conflict of Interest The authors declare no conflict of interest.

Functionalization of Titanium implants using adequate organic molecules is a proposed method to accelerate the osteointegration process, which relates to topographical, chemical, mechanical, and physical features. This study aimed to assess the potential of a peptide derived from cementum attachment protein (CAP-p15) adsorbed onto aTiO 2 surfaces to promote the deposition of calcium phosphate (CaP) minerals and its impact on the adhesion and viability of human periodontal ligament cells (hPDLCs). aTiO 2 surfaces were synthesized by magnetron sputtering technique. The CAP-p15 peptide was physically attached to aTiO 2 surfaces and characterized by atomic force microscopy, fluorescence microscopy, and water contact angle measurement. We performed in vitro calcium phosphate nucleation assays using an artificial saliva solution (pH 7.4) to simulate the oral environment. morphological and chemical characterization of the deposits were evaluated by scanning electronic microscopy (SEM) and spectroscopy molecular techniques (Raman Spectroscopy, ATR-FTIR). The aTiO 2 surfaces biofunctionalized with CAP-p15 were also analyzed for hPDLCs attachment, proliferation, and in vitro scratch-healing assay. The results let us see that the homogeneous amorphous titanium oxide coating was 70 nanometers thick. The CAP-p15 (1 μg/mL) displayed the ability to adsorb onto the aTiO 2 surface, increasing the roughness and maintaining the hydrophilicity of the aTiO 2 surfaces. The physical adsorption of CAP-p15 onto the aTiO 2 surfaces promoted the precipitation of a uniform layer of crystals with a flake-like morphology and a Ca/P ratio of 1.79. According to spectroscopy molecular analysis, these crystalline deposits correspond to carbonated hydroxyapatite. Regarding cell behavior, the biofunctionalized aTiO 2 surfaces improved the adhesion of hPDLCs after 24 h of cell culture, achieving 3.4-fold when compared to pristine surfaces. Moreover, there was an increase in cell proliferation and cell migration processes. Physical adsorption of CAP-p15 onto aTiO 2 surfaces enhanced the formation of carbonate hydroxyapatite crystals and promoted the proliferation and migration of human periodontal ligament-derived cells in in vitro studies. This experimental model using the novel bioactive peptide CAP-p15 could be used as an alternative to increasing the osseointegration process of implants., (© 2024 Wiley Periodicals LLC.)

Amelogenin and ameloblastin are 2 extracellular matrix proteins that are essential for the proper development of enamel. We recently reported that amelogenin and ameloblastin colocalized during the secretory stage of enamel formation when nucleation of enamel crystallites occurs. Direct interactions between the 2 proteins have been also demonstrated in our in vitro studies. Here, we explore interactions between their fragments during enamel maturation. We applied in vivo immunofluorescence imaging, quantitative co-localization analysis, and a new FRET (fluorescence resonance energy transfer) technique to demonstrate ameloblastin and amelogenin interaction in the maturing mouse enamel. Using immunochemical analysis of protein samples extracted from 8-d-old (P8) first molars from mice as a model for maturation-stage enamel, we identified the ~17-kDa ameloblastin (Ambn-N) and the TRAP (tyrosine-rich amelogenin peptide) fragments. We used Ambn-N18 and Ambn-M300 antibodies raised against the N-terminal and C-terminal segments of ameloblastin, as well as Amel-FL and Amel-C19 antibodies against full-length recombinant mouse amelogenin (rM179) and C-terminal amelogenin, respectively. In transverse sections, co-localization images of N-terminal fragments of amelogenin and ameloblastin around the prism boundary revealed the "fish net" pattern of the enamel matrix. Using in vivo FRET microscopy, we further demonstrated spatial interactions between amelogenin and ameloblastin N-terminal fragments. In the maturing mouse enamel, the association of these residual protein fragments created a discontinuity between enamel rods, which we suggest is important for support and maintenance of enamel rods and eventual contribution to unique enamel mechanical properties. We present data that support cooperative functions of enamel matrix proteins in mediating the structural hierarchy of enamel and that contribute to our efforts to design and develop enamel biomimetic material. [ABSTRACT FROM AUTHOR]

Two enamel proteases, matrix metalloproteinase-20 (MMP-20) and kallikrein 4 (KLK4), are known to cleave amelogenin and are necessary for proper enamel formation. However, the effect of hydroxyapatite (HAP) on the proteolytic activity of these enzymes remains unclear. To investigate whether apatite affects normal amelogenin proteolysis, we used 2 different isoforms of amelogenin combined with the appropriate enzymes to analyze proteolytic processing rates in the presence or absence of synthetic hydroxyapatite (HAP) crystals (N = 3). We found a distinct dose-dependent relationship between the amount of HAP present in the proteolysis mixture and the rate of rP172 degradation by rpMMP-20, whereas the effect of HAP on proteolysis of either rP172 or rP148 by rhKLK4 was less prominent. [ABSTRACT FROM AUTHOR]

Organic matrix degradation and crystal maturation are extracellular events that occur simultaneously during enamel biomineralization. We hypothesized that enamel proteases control amelogenin-mineral interaction, which, in turn can affect crystal nucleation, organization, and growth. We used a recombinant amelogenin (rP172), a homolog of its major cleavage product (rP148), and a native amelogenin lacking both N- and C-termini (13k). We compared apatite binding affinity between amelogenins and their digest products during proteolysis. We further compared binding affinity among the 3 amelogenins using a Langmuir model for protein adsorption. Amelogenin-apatite binding affinity was progressively reduced with the proteolysis at the C- and N- termini by recombinant pig MMP-20 (rpMMP-20) and recombinant human kallikrein-4 (rhKLK4), respectively. The binding affinity of amelogenin to apatite was found to be in the descending order of rP172, rP148, and 13k. Analysis of our data suggests that, before its complete degradation during enamel maturation, stepwise processing of amelogenin by MMP-20 and then KLK4 reduces amelogenin-apatite interaction. [ABSTRACT FROM AUTHOR]

Recent studies have highlighted the potential role of the metalloproteinase enamelysin (MMP-20) in controlling some of the most critical stages during enamel development. This study was aimed to assess the selectivity of enamelysin to the three most abundant cleavage sites on the amelogenin sequence, and to gain insight into the factors that control the pattern of amelogenin processing during enamel mineralization. Three deca-peptides with sequences based on pig amelogenin and including the proteolytic cleavage sites W/L, S/M, and P/A were synthesized as substrates. Statistical analysis revealed no significant differences in the rates of cleavage among the three peptides, indicating comparable selectivity of enamelysin for these peptide bonds. Considering the selective appearance of amelogenin proteolytic products, we suggest that amelogenin folding and assembly are the primary factors in controlling the pattern of its proteolysis during the secretory stage of enamel development. [ABSTRACT FROM AUTHOR]

This article is a short review of our recent study on controlled proteolysis of amelogenins by a series of commercially available proteinases as well as the tooth-specific metalloproteinase enamelysin. A limited proteolysis approach and mass spectrometry were applied in order to determine the surface accessibility of conserved domains of amelogenin nanospheres. Furthermore, this study was aimed at exploring the factors that affect the activity of enamel proteases to process amelogenins and at providing insight into the mechanisms of amelogenin degradation during amelogenesis. We found that, under limited conditions, certain amino acid residues at both the C- and N-termini of amelogenin are accessible to proteolytic action by a series of proteinases, suggesting that these regions are exposed on the surface of amelogenin nanospheres. Recombinant enamelysin cleaved amelogenin at the C-terminal region, showing a preference of the enzyme to cleave the S/M and F/S bonds. This result of enamelysin activity on amelogenin explains the abundance of the p148 (20k) pig amelogenin during the secretory stage of amelogenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In vitro studies on interactions between amelogenins and calcium phosphate crystals are critical for elucidating biomineralization mechanisms of tooth enamel. This work was aimed at investigating the effects of native porcine amelogenins on octacalcium phosphate (OCP) crystal growth in a gelatin gel. We prepared OCP mineral discs by circulating calcium and phosphate solutions on the opposite ends of the gels loaded with 0-2% amelogenin for one week. A dose-dependent modulation of OCP crystal habit by amelogenins was observed by scanning electron microscopy. While the incorporation of 0.125, 0.25, or 0.5% amelogenins showed no significant effect on the crystal morphology, in the presence of 1 and 2% amelogenins, the crystals were remarkably longer, having an average aspect ratio 3-5 times greater than that of those formed in the control gels. Transmission electron microscopy and atomic force microscopy suggested that amelogenin assemblies selectively blocked b-axial development, resulting in the c-axial elongation of OCP crystals. [ABSTRACT FROM AUTHOR]

Ameloblastin (Ambn), the most abundant non-amelogenin enamel protein, is intrinsically disordered and has the potential to interact with other enamel proteins and with cell membranes. Here, through multiple biophysical methods, we investigated the interactions between Ambn and large unilamellar vesicles (LUVs), whose lipid compositions mimicked cell membranes involved in epithelial cell-extracellular matrix adhesion. Using a series of Ambn Trp/Phe variants and Ambn mutants, we further showed that Ambn binds to LUVs through a highly conserved motif within the sequence encoded by exon 5. Synthetic peptides derived from different regions of Ambn confirmed that the sequence encoded by exon 5 is involved in LUV binding. Sequence analysis of Ambn across different species showed that the N-terminus of this sequence contains a highly conserved motif with a propensity to form an amphipathic helix. Mutations in the helix-forming sequence resulted in a loss of peptide binding to LUVs. Our in vitro data suggest that Ambn binds the lipid membrane directly through a conserved helical motif and have implications for biological events such as Ambn-cell interactions, Ambn signaling, and Ambn secretion via secretory vesicles., Competing Interests: The authors declare no competing financial interest.

Quantitative co-localization analysis, combined with other in vivo and in vitro techniques, can provide valuable information about the interaction and cooperative function of two proteins. Here we describe in detail the technique of quantitative co-localization analysis of two enamel matrix proteins, amelogenin and ameloblastin, in developing mouse enamel.

Ameloblastin is the second most abundant enamel matrix protein, and is thought to be essential for ameloblast cell polarization, cell adhesion, and enamel mineralization. However, studies of ameloblastin's function and its molecular mechanism have been limited due to difficulty in obtaining recombinant ameloblastin in vitro. Here, we present a protocol for successful ameloblastin expression and purification in E. coli.

Mimicking the dynamics of mineral loss and gain involved in dental caries formation can help us evaluate and compare the mineralization efficacy of different treatment agents used in enamel remineralization. Here, we offer an abridged study design outlining the preparation of tooth samples, creation of artificial dental lesions, application of a peptide, and characterization of the regrown enamel-like mineral layer.

Although amelogenin comprises the vast majority of the matrix that templates calcium phosphate nucleation during enamel formation, other proteins, particularly enamelin, are also known to play an important role in the formation of enamel's intricate architecture. However, there is little understanding of the interplay between amelogenin and enamelin in controlling processes of mineral nucleation and growth. Here, we used an in vitro model to investigate the impact of enamelin interaction with amelogenin on calcium phosphate nucleation for a range of enamelin-to-amelogenin ratios. We found that amelogenin alone is a weak promoter of nucleation, but addition of enamelin enhanced nucleation rates in a highly nonlinear, nonmonotonic manner reaching a sharp maximum at a ratio of 1:50 enamelin/amelogenin. We provide a phenomenological model to explain this effect that assumes only isolated enamelin proteins can act as sites of enhanced nucleation, while enamelin oligomers cannot. Even when interaction is random, the model reproduces the observed behavior, suggesting a simple means to tightly control the timing and extent of nucleation and phase transformation by amelogenin and enamelin., Competing Interests: The authors declare no competing financial interest.

Amelogenins are a group of extracellular enamel matrix proteins which are believed to be involved in the regulation of the size and habits of forming enamel crystals. The aim of this study was to compare the solubility properties of several amelogenins at various pH (from 4.0 to 9.0) at constant ionic strength (IS), and to examine the influence of buffer composition, IS, and divalent metal ions (including Ca2+, Mg2+, and Zn2+) on amelogenin solubility. The solubility of the recombinant murine amelogenin ("rM179") was minimum near its isoelectric point and increased rapidly below and above, regardless of buffer composition. A similar trend was observed for the native porcine ("25K") amelogenin. Porcine "23K" amelogenin was only sparingly soluble from pH of 4.0 to 9.0, in contrast to the analogous recombinant "rM166", which was more soluble in acidic solutions. The synthetic amelogenin polypeptide "TRAP" was extremely insoluble, while synthetic LRAP was readily soluble. Porcine "20K" amelogenin solubility increased strikingly as the solution pH was lowered from 7.0 to 6.0. Increasing IS decreased the solubility of rM179. While Zn2+ reduced rM179 solubility, Ca2+ and Mg2+ showed no significant effects. We conclude that the solubility of amelogenin was dependent on the primary structure, solution pH, and IS, and the low solubility of amelogenins under physiological conditions may result from their tendency to form quaternary (aggregate) structures in vivo. [ABSTRACT FROM AUTHOR]

The gradual discovery of functional domains in native enamel matrix proteins has enabled the design of smart bioinspired peptides for tooth enamel mimetics and repair. In this study, we expanded upon the concept of biomineralization to design smaller amelogenin-inspired peptides with conserved functional domains for clinical translation. The synthetic peptides displayed a characteristic nanostructured scaffold reminiscent of 'nanospheres' seen in the enamel matrix and effectively controlled apatite nucleation in vitro resulting in the formation of smaller crystallites. Following application of the peptides to sectioned human molar teeth, a robust, oriented, synthetic aprismatic enamel was observed after 7 days of incubation in situ. There was a two-fold increase in the hardness and modulus of the regrown enamel-like apatite layers and an increase in the attachment of the tooth-regrown layer interface compared to control samples. Repeated peptide applications generated multiple enamel-like hydroxyapatite (HAP) layers of limited thickness produced by epitaxial growth in which c -axis oriented nanorods evolved on the surface of native enamel. We conclude that peptide analogues with active domains can effectively regulate the orientation of regenerated HAP layers to influence functional response. Moreover, this enamel biofabrication approach demonstrates the peptide-mediated growth of multiple microscale HAP arrays of organized microarchitecture with potential for enamel repair., Competing Interests: The authors declare no competing financial interest.

The aim of this study was to examine the effect of fluoride ion concentration on the morphology of calcium phosphate crystals grown on acid-etched enamel as a model for tooth enamel erosion. Samples were immersed in calcification solution for 16 h and changes in crystal morphology were monitored by field emission scanning electron microscopy. Without fluoride, plate-like octacalcium phosphate crystals (20 nm thick, 2–10 μm wide) were formed. With 1–10 mg/l fluoride, arrays of denser needle-like nanocrystals (20–30 nm wide, >500 nm in length) were formed. We conclude that there is a minimal fluoride concentration (1 mg/l) that dramatically affects the morphology of calcium phosphate crystals grown on etched enamel in vitro. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]