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European Journal of Oral Sciences Dec 2011Amelogenin is critical for enamel formation, and human amelogenin gene (AMELX) mutations cause hypoplastic and/or hypomaturation enamel phenotypes. The Amelx null (AKO)...
Amelogenin is critical for enamel formation, and human amelogenin gene (AMELX) mutations cause hypoplastic and/or hypomaturation enamel phenotypes. The Amelx null (AKO) mouse has a severe hypoplastic phenotype. This study evaluated the effect of amelogenin loss on enamel formation and crystallite morphology. Enamel from AKO and wild-type (WT) mice was used. The AKO mice were mated with transgenic mice expressing the most abundant known amelogenin isoform, TgM180-87, to rescue (KOM180-87) the enamel crystallite phenotype. Molar enamel was embedded, sectioned with a diamond microtome, and images were obtained by transmission electron microscopy. The crystallite sizes from multiple sections were measured using Image J. The mean thicknesses (WT = 26 nm, AKO = 16 nm, and KOM180-87 = 25 nm) and the mean widths (WT = 96 nm, AKO = 59 nm, KOM180-87 = 85 nm) of crystallites were measured. Despite a complete loss of amelogenin in AKO mice, a mineralized enamel layer with well-defined and organized crystallites is formed. In the absence of amelogenin, enamel crystallites were reduced in thickness and width. For the first time we show that introduction of the m180 amelogenin isoform into the AKO mouse through cross-breeding rescues the crystallite phenotype. We conclude that amelogenin is essential for the development of normal crystallite size.
Topics: Amelogenesis; Amelogenin; Animals; Crystallization; Dental Enamel; Dental Enamel Hypoplasia; Female; Male; Mice; Mice, Knockout; Mutation; Protein Isoforms; X Chromosome
PubMed: 22243229
DOI: 10.1111/j.1600-0722.2011.00883.x -
International Journal of Molecular... Nov 2021Biomineralization is a crucial process whereby organisms produce mineralized tissues such as teeth for mastication, bones for support, and shells for protection....
Biomineralization is a crucial process whereby organisms produce mineralized tissues such as teeth for mastication, bones for support, and shells for protection. Mineralized tissues are composed of hierarchically organized hydroxyapatite crystals, with a limited capacity to regenerate when demineralized or damaged past a critical size. Thus, the development of protein-based materials that act as artificial scaffolds to guide hydroxyapatite growth is an attractive goal both for the design of ordered nanomaterials and for tissue regeneration. In particular, amelogenin, which is the main protein that scaffolds the hierarchical organization of hydroxyapatite crystals in enamel, amelogenin recombinamers, and amelogenin-derived peptide scaffolds have all been investigated for in vitro mineral growth. Here, we describe uniaxial hydroxyapatite growth on a nanoengineered amelogenin scaffold in combination with amelotin, a mineral promoting protein present during enamel formation. This bio-inspired approach for hydroxyapatite growth may inform the molecular mechanism of hydroxyapatite formation in vitro as well as possible mechanisms at play during mineralized tissue formation.
Topics: Amelogenin; Biomimetic Materials; Biomimetics; Biomineralization; Crystallization; Dental Enamel; Dental Enamel Proteins; Durapatite; Humans; Nanostructures; Nanotechnology; Protein Engineering; Protein Folding; Recombinant Proteins; Tooth
PubMed: 34830225
DOI: 10.3390/ijms222212343 -
The Journal of Biological Chemistry Oct 2006We recently demonstrated that the reuptake of full-length amelogenin protein results in increased levels of amelogenin mRNA through enhanced mRNA stabilization (Xu, L.,...
We recently demonstrated that the reuptake of full-length amelogenin protein results in increased levels of amelogenin mRNA through enhanced mRNA stabilization (Xu, L., Harada, H., Tamaki, T. Y., Matsumoto, S., Tanaka, J., and Taniguchi, A. (2006) J. Biol. Chem. 281, 2257-2262). Here, we examined the molecular mechanism of enhanced amelogenin mRNA stabilization. To identify the cis-regulatory region within amelogenin mRNA, we tested various reporter systems using a deletion series of reporter plasmids. A deletion at exon 6ABC of amelogenin mRNA resulted in a 2.5-fold increase in the amelogenin mRNA expression level when compared with that of full-length mRNA, indicating that a cis-element exists in exon 6ABC of amelogenin mRNA. Furthermore, Northwestern analysis demonstrated that amelogenin protein binds directly to its mRNA in vitro, suggesting that amelogenin protein acts as a trans-acting protein that specifically binds to this cis-element. Moreover, recombinant mouse amelogenin protein extended the half-life of full-length amelogenin mRNA but did not significantly alter the half-life of exon 6ABC-deletion mutant mRNA. The splice products produced by deletion of exon 6ABC are known as leucine-rich amelogenin peptides and have signaling effects on cells. Our findings also suggest that the regulation of full-length amelogenin protein expression differs from the regulation of leucine-rich amelogenin peptide expression.
Topics: Amelogenin; Animals; Cell Culture Techniques; Cell Line; Dactinomycin; Epithelial Cells; Exons; Genes, Reporter; Half-Life; Luciferases; Plasmids; RNA, Messenger; Rats; Recombinant Proteins; Transfection
PubMed: 16954216
DOI: 10.1074/jbc.M605406200 -
The International Journal of... Feb 1995The central problems of enamel biochemistry are the mechanisms concerned with initiation and development of the mineral crystals, together with their architectural... (Review)
Review
The central problems of enamel biochemistry are the mechanisms concerned with initiation and development of the mineral crystals, together with their architectural arrangement within the tissue. These processes are mediated by the extracellular matrix as well as the composition of the mineral itself. Initial mineral deposition occurs at the dentine surface, nucleated either by dentinal components or early enamel matrix, possibly non-amelogenin molecules. The early crystals are small in size and rich in magnesium and carbonate resulting in relatively poor crystallinity. This is in spite of the fact that fluoride is high at this stage. Crystal development includes a reduction in magnesium, carbonate and fluoride as crystals increase in length following the retreating ameloblasts from the dentine. The matrix acquires increasing concentrations of amelogenin and albumin. Prismatic structure begins to develop together with some growth of crystals in width and thickness. Degradation of amelogenin and non-amelogenin molecules generates a series of specific molecular fragments possibly concerned with modulating crystal growth and morphology and the creation of prismatic and interprismatic structures. Towards the end of secretion, matrix, now almost completely degraded, is replaced by fluid followed by massive crystal growth during maturation. Degradation of albumin also occurs at this stage, probably as a result of comprehensive destruction of molecules which might impair crystal growth. Selective acquisition of magnesium and fluoride at this stage may reflect the hydrated state of the tissue as well as cell changes. Fluid is displaced as crystals grow and the enamel acquires concentrations of mineral characteristic of mature tissue.
Topics: Ameloblasts; Animals; Dental Enamel; Extracellular Matrix; Humans; Minerals
PubMed: 7626401
DOI: No ID Found -
European Journal of Oral Sciences Dec 2011The structures and interactions among macromolecules in the enamel extracellular matrix play vital roles in regulating hydroxyapatite crystal nucleation, growth, and...
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.
Topics: Amelogenesis; Amelogenin; Animals; Calcium Phosphates; Circular Dichroism; Crystallization; Dental Enamel Proteins; Hydrogen-Ion Concentration; Macromolecular Substances; Microscopy, Electron, Scanning; Molecular Weight; Protein Binding; Protein Interaction Mapping; Protein Multimerization; Recombinant Proteins; Scattering, Radiation; Sodium Chloride; Spectrometry, Fluorescence; Sus scrofa; Tooth Calcification
PubMed: 22243267
DOI: 10.1111/j.1600-0722.2011.00916.x -
PloS One 2014Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While...
Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In situ hybridization and immunohistodetection were performed in mandibular tissues using AMELX KO mice as controls. We identified AMELX-producing (RNA-positive) cells lining the adjacent alveolar bone and AMBN and AMELX proteins in the microenvironment surrounding EMPs-producing cells. Western blotting of proteins extracted by non-dissociative means revealed that AMELX and AMBN are not exclusive to mineralized matrix; they are present to some degree in a solubilized state in mandibular bone and presumably have some capacity to diffuse. Our data support the notion that AMELX and AMBN may function as growth factor-like molecules solubilized in the aqueous microenvironment. In jaws, they might play some role in bone physiology through autocrine/paracrine pathways, particularly during development and stress-induced remodeling.
Topics: Amelogenin; Animals; Dental Enamel Proteins; Diffusion; Epithelial Cells; Eye Proteins; Female; Gene Expression Regulation, Developmental; Male; Mandible; Mesoderm; Mice; Mice, Knockout; Muscle Proteins; Organ Specificity; Ovary; RNA, Messenger; Solubility; Testis; Tongue; Viscera
PubMed: 24933156
DOI: 10.1371/journal.pone.0099626 -
Biochemistry Dec 2022Amelogenin, the dominant organic component (>90%) in the early stages of amelogenesis, orchestrates the mineralization of apatite crystals into enamel. The...
Amelogenin, the dominant organic component (>90%) in the early stages of amelogenesis, orchestrates the mineralization of apatite crystals into enamel. The self-association properties of amelogenin as a function of pH and protein concentration have been suggested to play a central role in this process; however, the large molecular weight of the self-assembled quaternary structures has largely prevented structural studies of the protein in solution under physiological conditions using conventional approaches. Here, using perdeuterated murine amelogenin (0.25 mM, 5 mg/mL) and TROSY-based NMR experiments to improve spectral resolution, we assigned the H-N spectra of murine amelogenin over a pH range (5.5 to 8.0) where amelogenin is reported to exist as oligomers (pH ≤∼6.8) and nanospheres (pH ≥∼7.2). The disappearance or intensity reduction of amide resonances in the H-N HSQC spectra was interpreted to reflect changes in dynamics (intermediate millisecond-to-microsecond motion) and/or heterogenous interfaces of amide nuclei at protein-protein interfaces. The intermolecular interfaces were concentrated toward the N-terminus of amelogenin (L3-G8, V19-G38, L46-Q49, and Q57-L70) at pH 6.6 (oligomers) and at pH 7.2 (nanospheres) including the entire N-terminus up to Q76 and regions distributed through the central hydrophobic region (Q82-Q101, S125-Q139, and F151-Q154). At all pH levels, the C-terminus appeared disordered, highly mobile, and not involved in self-assembly, suggesting nanosphere structures with solvent-exposed C-termini. These findings present unique, residue-specific insights into the intermolecular protein-protein interfaces driving amelogenin quaternary structure formation and suggest that nanospheres in solution predominantly contain disordered, solvent-exposed C-termini.
Topics: Animals; Mice; Amelogenin; Magnetic Resonance Spectroscopy; Amides; Solvents; Dental Enamel Proteins
PubMed: 36456190
DOI: 10.1021/acs.biochem.2c00522 -
Orthodontics & Craniofacial Research Aug 2009Emdogain (enamel matrix derivative, EMD) is well recognized in periodontology, where it is used as a local adjunct to periodontal surgery to stimulate regeneration of... (Review)
Review
Emdogain (enamel matrix derivative, EMD) is well recognized in periodontology, where it is used as a local adjunct to periodontal surgery to stimulate regeneration of periodontal tissues lost to periodontal disease. The biological effect of EMD is through stimulation of local growth factor secretion and cytokine expression in the treated tissues, inducing a regenerative process that mimics odontogenesis. The major (>95%) component of EMD is Amelogenins (Amel). No other active components have so far been isolated from EMD, and several studies have shown that purified amelogenins can induce the same effect as the complete EMD. Amelogenins comprise a family of highly conserved extracellular matrix proteins derived from one gene. Amelogenin structure and function is evolutionary well conserved, suggesting a profound role in biomineralization and hard tissue formation. A special feature of amelogenins is that under physiological conditions the proteins self-assembles into nanospheres that constitute an extracellular matrix. In the body, this matrix is slowly digested by specific extracellular proteolytic enzymes (matrix metalloproteinase) in a controlled process, releasing bioactive peptides to the surrounding tissues for weeks after application. Based on clinical and experimental observations in periodontology indicating that amelogenins can have a significant positive influence on wound healing, bone formation and root resorption, several new applications for amelogenins have been suggested. New experiments now confirm that amelogenins have potential for being used also in the fields of endodontics, bone regeneration, implantology, traumatology, and wound care.
Topics: Amelogenin; Calcification, Physiologic; Conserved Sequence; Dental Enamel Proteins; Extracellular Matrix Proteins; Humans; Matrix Metalloproteinases; Osteogenesis; Periodontal Diseases; Regeneration; Root Resorption; Wound Healing
PubMed: 19627527
DOI: 10.1111/j.1601-6343.2009.01459.x -
Frontiers of Oral Biology 2009Amelogenins are the major proteins involved in tooth enamel formation. In the present study, we have cloned and sequenced four novel amelogenins from three amphibian... (Comparative Study)
Comparative Study
Amelogenins are the major proteins involved in tooth enamel formation. In the present study, we have cloned and sequenced four novel amelogenins from three amphibian species in order to analyze similarities and differences between mammalian and non-mammalian amelogenins. The newly sequenced amphibian amelogenin sequences were from a red-eyed tree frog (Litoria chloris) and a Mexican axolotl (Ambystoma mexicanum). We identified two amelogenin isoforms in the Eastern red-backed salamander (Plethodon cinereus). Sequence comparisons confirmed that non-mammalian amelogenins are overall shorter than their mammalian counterparts, contain less proline and less glutamine, and feature shorter polyproline tripeptide repeat stretches than mammalian amelogenins. We propose that unique sequence parameters of mammalian amelogenins might be a pre-requisite for complex mammalian enamel prism architecture.
Topics: Ambystoma mexicanum; Amelogenin; Amino Acid Sequence; Amphibians; Animals; Anura; Base Sequence; DNA, Complementary; Dental Enamel; Evolution, Molecular; Mammals; Molecular Sequence Data; RNA; Sequence Homology
PubMed: 19828974
DOI: 10.1159/000242395 -
National Journal of Maxillofacial... 2014Amelogenins are the major enamel proteins that play a major role in the biomineralization and structural organization of enamel. Aberrations of enamel-related proteins...
BACKGROUND
Amelogenins are the major enamel proteins that play a major role in the biomineralization and structural organization of enamel. Aberrations of enamel-related proteins are thought to be involved in oncogenesis of odontogenic epithelium. The expression of amelogenin is possibly an indicator of differentiation of epithelial cells in the odontogenic lesions.
AIMS AND OBJECTIVES
The present study aimed to observe the expression of amelogenin immunohistochemically in various odontogenic lesions.
MATERIALS AND METHODS
Paraffin sections of 40 odontogenic lesions were stained immunohistochemically with amelogenin antibodies. The positivity, pattern and intensity of expression of the amelogenin antibody were assessed, graded and statistically compared between groups of odontogenic cysts and tumors.
RESULTS
Almost all the odontogenic lesions expressed amelogenin in the epithelial component with the exception of an ameloblastic carcinoma. Differing grades of intensity and pattern were seen between the cysts and tumors. Intensity of expression was uniformly prominent in all odontogenic lesions with hard tissue formation. Statistical analysis however did not indicate significant differences between the two groups.
CONCLUSION
The expression of amelogenin antibody is ubiquitous in odontogenic tissues and can be used as a definitive marker for identification of odontogenic epithelium.
PubMed: 25937729
DOI: 10.4103/0975-5950.154822