Ameloblastin binding to biomimetic models of cell membranes - A continuum of intrinsic disorder.

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 C- and 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.

Deletion within ameloblastin multitargeting domain reduces its interaction with artificial cell membrane.

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.

Ameloblastin and its multifunctionality in amelogenesis: A review.

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.

Triple Function of Amelogenin Peptide-Chitosan Hydrogel for Dentin Repair.

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 Dynamic Interactions of a Multitargeting Domain in Ameloblastin Protein with Amelogenin and Membrane.

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.

Modeling ameloblast-matrix interactions using 3D cell culture.

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 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.

Coemergence of the Amphipathic Helix on Ameloblastin With Mammalian Prismatic Enamel.

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.

Data from ameloblast cell lines cultured in 3D using various gel substrates in the presence of ameloblastin.

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.

Regulation of Hydroxyapatite Nucleation In Vitro through Ameloblastin-Amelogenin Interactions.

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.

Ameloblastin promotes polarization of ameloblast cell lines in a 3-D cell culture system.

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.

Biomineralization of Enamel and Dentin Mediated by Matrix Proteins.

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.

Co-Immunoprecipitation Reveals Interactions Between Amelogenin and Ameloblastin Their Self-Assembly Domains.

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.

Amelogenin Peptide-Chitosan Hydrogel for Biomimetic Enamel Regrowth.

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 as compared to peptides in solution at room temperature and at 37°C.

Enhancing Collagen Mineralization with Amelogenin Peptide: Towards the Restoration of Dentin.

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.

An Evolutionarily Conserved Helix Mediates Ameloblastin-Cell Interaction.

Ameloblastin (Ambn) has the potential to regulate cell-matrix adhesion through familiar cell-binding domains, but the proposed sequence motifs are not highly conserved across species. Here, we report that Ambn binds to ameloblast-like cell membranes through a highly evolutionary conserved amphipathic helix-forming (AH) motif encoded by exon 5. We applied high-resolution confocal microscopy to show colocalization of Ambn with ameloblast membrane surfaces in developing mouse incisors.

Ameloblastin Binds to Phospholipid Bilayers via a Helix-Forming Motif within the Sequence Encoded by Exon 5.

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.

The Expression and Purification of Recombinant Mouse Ameloblastin in E. coli.

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.

Immunohistochemical Co-Localization of Amelogenin and Ameloblastin in Developing Enamel Matrix.

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.

Peptide-Mediated Biomimetic Regrowth of Human Enamel In Situ.

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.

Control of Calcium Phosphate Nucleation and Transformation through Interactions of Enamelin and Amelogenin Exhibits the "Goldilocks Effect".

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 model to investigate the impact of enamelin interaction with amelogenin on calcium phosphate nucleation for a range of enamelin-to-amelogenin ratios.

Peptide-Based Bioinspired Approach to Regrowing Multilayered Aprismatic Enamel.

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.

Meeting report: a hard look at the state of enamel research.

The Encouraging Novel Amelogenesis Models and Ex vivo cell Lines (ENAMEL) Development workshop was held on 23 June 2017 at the Bethesda headquarters of the National Institute of Dental and Craniofacial Research (NIDCR). Discussion topics included model organisms, stem cells/cell lines, and tissues/3D cell culture/organoids. Scientists from a number of disciplines, representing institutions from across the United States, gathered to discuss advances in our understanding of enamel, as well as future directions for the field.

The Presence of MMP-20 Reinforces Biomimetic Enamel Regrowth.

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.

Amelogenin Affects Brushite Crystal Morphology and Promotes Its Phase Transformation to Monetite.

Amelogenin protein is involved in organized apatite crystallization during enamel formation. Brushite (CaHPO·2HO), one of the precursors of hydroxyapatite mineralization , has been used for fabrication of biomaterials for hard tissue repair. In order to explore its potential application in biomimetic material synthesis, we studied the influence of the enamel protein amelogenin on brushite morphology and phase transformation to monetite.

Ameloblastin peptide encoded by exon 5 interacts with amelogenin N-terminus.

Interactions between enamel matrix proteins are important for enamel biomineralization. In recent studies, we showed that the N-terminal proteolytic product of ameloblastin co-localized with amelogenin around the prism boundaries. However, the molecular mechanisms of such interactions are still unclear.