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Scientifica 2021Amelogenin is a common sex typing marker encountered in forensic case work. Phenotypically normal males have been reported in the literature who exhibit anomalous...
Amelogenin is a common sex typing marker encountered in forensic case work. Phenotypically normal males have been reported in the literature who exhibit anomalous amelogenin allele. These males express only a single amelogenin peak representing AMEL-X and are called as AMEL-Y-null males. Gender misclassification of such individuals is an obvious consequence of this mutation, as a male sample would falsely appear to be a female sample. This study was aimed to attribute the AMEL-Y-null male DNA profiles encountered in forensic casework in the Pakistani population to appropriate phylogenetic clade based on shared ancestry. A total of 18 null AMEL-Y males were screened out of the sample pool of 5000 male individuals, reflecting mutational frequency of 0.36%. A common phylogenetic ancestor is suggested for 17 individuals, based on computational analysis of the Y-STR haplotypes, shown to be belonging to the J haplogroup while only one sample belonged to the R group. The samples in J groups showed homology with subclades J2b2a M241 and J2b2a PH1648, while R group individual showed 100% homology with R1a. Data are reported after haplotype network development of AMEL-Y-null Pakistani males using Network 10.0 for the study of evolutionary distances and emergence of nodes.
PubMed: 34035976
DOI: 10.1155/2021/5521411 -
Molecules (Basel, Switzerland) Sep 2020Dental caries or tooth decay is a preventable and multifactorial disease that affects billions of people globally and is a particular concern in younger populations.... (Review)
Review
Dental caries or tooth decay is a preventable and multifactorial disease that affects billions of people globally and is a particular concern in younger populations. This decay arises from acid demineralisation of tooth enamel resulting in mineral loss from the subsurface. The remineralisation of early enamel carious lesions could prevent the cavitation of teeth. The enamel protein amelogenin constitutes 90% of the total enamel matrix protein in teeth and plays a key role in the biomineralisation of tooth enamel. The physiological importance of amelogenin has led to the investigation of the possible development of amelogenin-derived biomimetics against dental caries. We herein review the literature on amelogenin, its primary and secondary structure, comparison to related species, and its' in vivo processing to bioactive peptide fragments. The key structural motifs of amelogenin that enable enamel remineralisation are discussed. The presence of several motifs in the amelogenin structure (such as polyproline, N- and C-terminal domains and C-terminal orientation) were shown to play a critical role in the formation of particle shape during remineralization. Understanding the function/structure relationships of amelogenin can aid in the rational design of synthetic polypeptides for biomineralisation, halting enamel loss and leading to improved therapies for tooth decay.
Topics: Amelogenin; Amino Acid Motifs; Animals; Biomimetics; Cattle; Dental Caries; Dental Enamel; Durapatite; Humans; Leucine; Mice; Peptides; Protein Domains; Swine; Tyrosine
PubMed: 32937944
DOI: 10.3390/molecules25184214 -
Frontiers in Physiology 2017The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel...
The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel crystals. Here we have conducted a series of and studies to characterize the changes in matrix shape and organization that take place during the transition from ameloblast intravesicular matrices to extracellular subunit compartments and pericrystalline sheath proteins, and correlated these changes with stages of amelogenin matrix protein posttranslational processing. Our transmission electron microscopic studies revealed a 2.5-fold difference in matrix subunit compartment dimensions between secretory vesicle and extracellular enamel protein matrix as well as conformational changes in matrix structure between vesicles, stippled materials, and pericrystalline matrix. Enamel crystal growth in organ culture demonstrated granular mineral deposits associated with the enamel matrix framework, dot-like mineral deposits along elongating initial enamel crystallites, and dramatic changes in enamel matrix configuration following the onset of enamel crystal formation. Atomic force micrographs provided evidence for the presence of both linear and hexagonal/ring-shaped full-length recombinant amelogenin protein assemblies on mica surfaces, while nickel-staining of the N-terminal amelogenin N92 His-tag revealed 20 nm diameter oval and globular amelogenin assemblies in N92 amelogenin matrices. Western blot analysis comparing loosely bound and mineral-associated protein fractions of developing porcine enamel organs, superficial and deep enamel layers demonstrated (i) a single, full-length amelogenin band in the enamel organ followed by 3 kDa cleavage upon entry into the enamel layer, (ii) a close association of 8-16 kDa C-terminal amelogenin cleavage products with the growing enamel apatite crystal surface, and (iii) a remaining pool of N-terminal amelogenin fragments loosely retained between the crystalline phases of the deep enamel layer. Together, our data establish a temporo-spatial correlation between amelogenin protein processing and the changes in enamel matrix configuration that take place during the transition from intracellular vesicle compartments to extracellular matrix assemblies and the formation of protein coats along elongating apatite crystal surfaces. In conclusion, our study suggests that enzymatic cleavage of the amelogenin enamel matrix protein plays a key role in the patterning of the organic matrix framework as it affects enamel apatite crystal growth and habit.
PubMed: 29089900
DOI: 10.3389/fphys.2017.00790 -
Journal of Dental Research Aug 2016Amelogenin and ameloblastin are 2 extracellular matrix proteins that are essential for the proper development of enamel. We recently reported that amelogenin and...
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.
Topics: Amelogenin; Animals; Dental Enamel; Dental Enamel Proteins; Fluorescence Resonance Energy Transfer; Mice; Molar; Peptide Fragments; Protein Interaction Domains and Motifs
PubMed: 27146703
DOI: 10.1177/0022034516645389 -
Journal of Structural Biology Aug 2013Enamelin and amelogenin are vital proteins in enamel formation. The cooperative function of these two proteins controls crystal nucleation and morphology in vitro. We...
Enamelin and amelogenin are vital proteins in enamel formation. The cooperative function of these two proteins controls crystal nucleation and morphology in vitro. We quantitatively analyzed the co-localization between enamelin and amelogenin by confocal microscopy and using two antibodies, one raised against a sequence in the porcine 32 kDa enamelin region and the other raised against full-length recombinant mouse amelogenin. We further investigated the interaction of the porcine 32 kDa enamelin and recombinant amelogenin using immuno-gold labeling. This study reports the quantitative co-localization results for postnatal days 1-8 mandibular mouse molars. We show that amelogenin and enamelin are secreted into the extracellular matrix on the cuspal slopes of the molars at day 1 and that secretion continues to at least day 8. Quantitative co-localization analysis (QCA) was performed in several different configurations using large (45 μm height, 33 μm width) and small (7 μm diameter) regions of interest to elucidate any patterns. Co-localization patterns in day 8 samples revealed that enamelin and amelogenin co-localize near the secretory face of the ameloblasts and appear to be secreted approximately in a 1:1 ratio. The degree of co-localization decreases as the enamel matures, both along the secretory face of ameloblasts and throughout the entire thickness of the enamel. Immuno-reactivity against enamelin is concentrated along the secretory face of ameloblasts, supporting the theory that this protein together with amelogenin is intimately involved in mineral induction at the beginning of enamel formation.
Topics: Ameloblasts; Amelogenin; Animals; Antibodies; Dental Enamel Proteins; Extracellular Matrix; Mice; Microscopy, Confocal; Molar; Swine
PubMed: 23563189
DOI: 10.1016/j.jsb.2013.03.014 -
Journal of Molecular Histology Oct 2012Amelogenin is the most abundant matrix protein in enamel. Proper amelogenin processing by proteinases is necessary for its biological functions during amelogenesis....
Amelogenin is the most abundant matrix protein in enamel. Proper amelogenin processing by proteinases is necessary for its biological functions during amelogenesis. Matrix metalloproteinase 9 (MMP-9) is responsible for the turnover of matrix components. The relationship between MMP-9 and amelogenin during tooth development remains unknown. We tested the hypothesis that MMP-9 binds to amelogenin and they are co-expressed in ameloblasts during amelogenesis. We evaluated the distribution of both proteins in the mouse teeth using immunohistochemistry and confocal microscopy. At postnatal day 2, the spatial distribution of amelogenin and MMP-9 was co-localized in preameloblasts, secretory ameloblasts, enamel matrix and odontoblasts. At the late stages of mouse tooth development, expression patterns of amelogenin and MMP-9 were similar to that seen in postnatal day 2. Their co-expression was further confirmed by RT-PCR, Western blot and enzymatic zymography analyses in enamel organ epithelial and odontoblast-like cells. Immunoprecipitation assay revealed that MMP-9 binds to amelogenin. The MMP-9 cleavage sites in amelogenin proteins across species were found using bio-informative software program. Analyses of these data suggest that MMP-9 may be involved in controlling amelogenin processing and enamel formation.
Topics: Ameloblasts; Amelogenesis; Amelogenin; Animals; Animals, Newborn; Binding Sites; Cell Line; Gene Expression Regulation, Developmental; Matrix Metalloproteinase 9; Mice; Protein Binding; Tooth
PubMed: 22648084
DOI: 10.1007/s10735-012-9423-1 -
Monographs in Oral Science 2011Dental fluorosis occurs as a result of excess fluoride ingestion during tooth formation. Enamel fluorosis and primary dentin fluorosis can only occur when teeth are... (Review)
Review
Dental fluorosis occurs as a result of excess fluoride ingestion during tooth formation. Enamel fluorosis and primary dentin fluorosis can only occur when teeth are forming, and therefore fluoride exposure (as it relates to dental fluorosis) occurs during childhood. In the permanent dentition, this would begin with the lower incisors, which complete mineralization at approximately 2-3 years of age, and end after mineralization of the third molars. The white opaque appearance of fluorosed enamel is caused by a hypomineralized enamel subsurface. With more severe dental fluorosis, pitting and a loss of the enamel surface occurs, leading to secondary staining (appearing as a brown color). Many of the changes caused by fluoride are related to cell/matrix interactions as the teeth are forming. At the early maturation stage, the relative quantity of amelogenin protein is increased in fluorosed enamel in a dose-related manner. This appears to result from a delay in the removal of amelogenins as the enamel matures. In vitro, when fluoride is incorporated into the mineral, more protein binds to the forming mineral, and protein removal by proteinases is delayed. This suggests that altered protein/mineral interactions are in part responsible for retention of amelogenins and the resultant hypomineralization that occurs in fluorosed enamel. Fluoride also appears to enhance mineral precipitation in forming teeth, resulting in hypermineralized bands of enamel, which are then followed by hypomineralized bands. Enhanced mineral precipitation with local increases in matrix acidity may affect maturation stage ameloblast modulation, potentially explaining the dose-related decrease in cycles of ameloblast modulation from ruffle-ended to smooth-ended cells that occur with fluoride exposure in rodents. Specific cellular effects of fluoride have been implicated, but more research is needed to determine which of these changes are relevant to the formation of fluorosed teeth. As further studies are done, we will better understand the mechanisms responsible for dental fluorosis.
Topics: Ameloblasts; Amelogenesis; Amelogenin; Cariostatic Agents; Chronic Disease; Dental Enamel; Fluorides; Fluorosis, Dental; Humans; Odontogenesis; Tooth Calcification; Tooth Demineralization
PubMed: 21701193
DOI: 10.1159/000327028 -
Stem Cells and Development Aug 2021The growth of long and polarized ameloblast-like cells has long been heralded as a major prerequisite for enamel tissue engineering. In this study, we have designed...
The growth of long and polarized ameloblast-like cells has long been heralded as a major prerequisite for enamel tissue engineering. In this study, we have designed three-dimensional bioreactor/scaffold microenvironments to propagate and assess the ability of cervical loop derivatives to become long and polarized ameloblast-like cells. Our studies demonstrated that cervical loop/periodontal progenitor coculture in a growth-factor-enriched medium resulted in the formation of ameloblast-like cells expressing high levels of amelogenin and ameloblastin. Coculture of cervical loop cells with dental pulp cells on tailored collagen scaffolds enriched with leucine-rich amelogenin peptide (LRAP) and early enamel matrix resulted in singular, elongated, and polarized ameloblast-like cells that expressed and secreted ameloblastin and amelogenin enamel proteins. Bioreactor microenvironments enriched with enamel matrix and LRAP also proved advantageous for the propagation of HAT-7 cells, resulting in a ∼20-fold higher expression of amelogenin and ameloblastin enamel proteins compared with controls growing on plain scaffolds. Together, studies presented here highlight the benefits of microgravity culture systems combined with ameloblast-specific microenvironments and tailored scaffolds for the growth of ameloblast-like cells.
Topics: Ameloblasts; Amelogenin; Bioreactors; Cell Differentiation; Coculture Techniques; Dental Pulp
PubMed: 34060920
DOI: 10.1089/scd.2021.0115 -
Frontiers in Physiology 2014The hardest tooth enamel tissue develops from a soft layer of protein-rich matrix, predominated by amelogenin that is secreted by epithelial ameloblasts in the secretory...
The hardest tooth enamel tissue develops from a soft layer of protein-rich matrix, predominated by amelogenin that is secreted by epithelial ameloblasts in the secretory stage of tooth enamel development. During enamel formation, a well-controlled progressive removal of matrix proteins by resident proteases, Matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), will provide space for the apatite crystals to grow. To better understand the role of amelogenin degradation in enamel biomineralization, the present study was conducted to investigate how the adsorption of amelogenin to hydroxyapatite (HAP) crystals affects its degradation by enamel proteinases, MMP20 and KLK4. Equal quantities of amelogenins confirmed by protein assays before digestions, either adsorbed to HAP or in solution, were incubated with MMP20 or KLK4. The digested samples collected at different time points were analyzed by spectrophotometry, SDS-PAGE, high performance liquid chromatography (HPLC), and LC-MALDI MS/MS. We found that majority of amelogenin adsorbed on HAP was released into the surrounding solution by enzymatic processing (88% for MMP20 and 98% for KLK4). The results show that as compared with amelogenin in solution, the HAP-bound amelogenin was hydrolyzed by both MMP20 and KLK4 at significantly higher rates. Using LC-MALDI MS/MS, more accessible cleavage sites and hydrolytic fragments from MMP20/KLK4 digestion were identified for the amelogenin adsorbed on HAP crystals as compared to the amelogenin in solution. These results suggest that the adsorption of amelogenin to HAP results in their preferential and selective degradation and removal from HAP by MMP20 and KLK4 in vitro. Based on these findings, a new degradation model related to enamel crystal growth is proposed.
PubMed: 25104939
DOI: 10.3389/fphys.2014.00268 -
Proceedings of the National Academy of... Aug 2020As the hardest tissue formed by vertebrates, enamel represents nature's engineering masterpiece with complex organizations of fibrous apatite crystals at the nanometer...
As the hardest tissue formed by vertebrates, enamel represents nature's engineering masterpiece with complex organizations of fibrous apatite crystals at the nanometer scale. Supramolecular assemblies of enamel matrix proteins (EMPs) play a key role as the structural scaffolds for regulating mineral morphology during enamel development. However, to achieve maximum tissue hardness, most organic content in enamel is digested and removed at the maturation stage, and thus knowledge of a structural protein template that could guide enamel mineralization is limited at this date. Herein, by examining a gene-modified mouse that lacked enzymatic degradation of EMPs, we demonstrate the presence of protein nanoribbons as the structural scaffolds in developing enamel matrix. Using in vitro mineralization assays we showed that both recombinant and enamel-tissue-based amelogenin nanoribbons are capable of guiding fibrous apatite nanocrystal formation. In accordance with our understanding of the natural process of enamel formation, templated crystal growth was achieved by interaction of amelogenin scaffolds with acidic macromolecules that facilitate the formation of an amorphous calcium phosphate precursor which gradually transforms into oriented apatite fibers along the protein nanoribbons. Furthermore, this study elucidated that matrix metalloproteinase-20 is a critical regulator of the enamel mineralization as only a recombinant analog of a MMP20-cleavage product of amelogenin was capable of guiding apatite mineralization. This study highlights that supramolecular assembly of the scaffold protein, its enzymatic processing, and its ability to interact with acidic carrier proteins are critical steps for proper enamel development.
Topics: Amelogenesis; Amelogenin; Animals; Apatites; Dental Enamel; Dental Enamel Proteins; Mice; Nanofibers
PubMed: 32737162
DOI: 10.1073/pnas.2007838117