-
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 -
Journal of Bone and Mineral Research :... Jun 2005The recognized structural proteins of the enamel matrix are amelogenin, ameloblastin, and enamelin. While a large volume of data exists showing that amelogenin...
UNLABELLED
The recognized structural proteins of the enamel matrix are amelogenin, ameloblastin, and enamelin. While a large volume of data exists showing that amelogenin self-assembles into multimeric units referred to as nanospheres, other reports of enamel matrix protein-protein interactions are scant. We believe that each of these enamel matrix proteins must interact with other organic components of ameloblasts and the enamel matrix. Likely protein partners would include integral membrane proteins and additional secreted proteins.
INTRODUCTION
The purpose of this study was to identify and catalog additional proteins that play a significant role in enamel formation.
MATERIALS AND METHODS
We used the yeast two-hybrid assay to identify protein partners for amelogenin, ameloblastin, and enamelin. Once identified, RT-PCR was used to assess gene transcription of these newly identified and potential "enamel" proteins in ameloblast-like LS8 cells.
RESULTS
In the context of this yeast assay, we identified a number of secreted proteins and integral membrane proteins that interact with amelogenin, ameloblastin, and enamelin. Additionally, proteins whose functions range from the inhibition of soft tissue mineralization, calcium ion transport, and phosphorylation events have been identified as protein partners to these enamel matrix proteins. For each protein identified using this screening strategy, future studies are planned to confirm this physiological relationship to biomineralization in vivo.
CONCLUSION
Identifying integral membrane proteins of the secretory surface of ameloblast cells (Tomes' processes) and additional enamel matrix proteins, based on their abilities to interact with the most abundant enamel matrix proteins, will better define the molecular mechanisms of enamel formation at its most rudimentary level.
Topics: Ameloblasts; Amelogenin; Animals; Antigens, CD; Biglycan; Blood Proteins; Calnexin; Cell Membrane; DNA, Complementary; Dental Enamel; Dental Enamel Proteins; Dentin; Extracellular Matrix Proteins; Mice; Models, Biological; Open Reading Frames; Phosphorylation; Platelet Membrane Glycoproteins; Protein Binding; Proteoglycans; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Tetraspanin 30; Time Factors; Transcription, Genetic; Two-Hybrid System Techniques; alpha-2-HS-Glycoprotein; alpha-Fetoproteins
PubMed: 15883644
DOI: 10.1359/JBMR.050111 -
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 -
Cell Proliferation Oct 2012Amelogenins are proposed to be responsible for enamel matrix derivative (EMD)-induced periodontal regeneration; however, heterogeneity of amelogenins makes it...
OBJECTIVES
Amelogenins are proposed to be responsible for enamel matrix derivative (EMD)-induced periodontal regeneration; however, heterogeneity of amelogenins makes it challenging to purify the full-length proteins. This study has been carried out to express and purify a recombinant full-length human amelogenin protein (rHhAm175) in the eukaryotic yeast Pichia pastoris, and further compare biological responses of human periodontal ligament fibroblasts (PDLFs) to rHhAm175 and porcine EMD (pEMD).
MATERIALS AND METHODS
Human cDNA encoding a 175-amino acid amelogenin was subcloned into the pPIC3.5K vector. The rHhAm175 expressed in P. pastoris GS115 (Mut+) was purified and characterized. We examined cell attachment, migration and proliferation responses of human PDLFs to rHhAm175 and pEMD respectively, and characterized associated changes of proliferation-related intracellular signalling molecules, including extracellular signal response kinase (ERK) and Akt kinases/protein kinase B (Akt/PKB) kinases.
RESULTS
The purified rHhAm175 was confirmed to be molecular mass 22 021.13 Da, phosphorylated human amelogenin, and alone significantly promoted proliferation and migration of human PDLFs to an extent comparable to that of pEMD. Cell attachment was increased over the first 60 min incubation with rHhAm175 or pEMD. Both rHhAm175 and pEMD induced PDLF mitogenesis via extracellular signal response kinase (ERK1/2), but not by Akt kinases/protein kinase B (Akt/PKB).
CONCLUSIONS
rHhAm175 modulated cell activities of human PDLFs, to a comparable extent as porcine EMD. These data suggest that rHhAm175 might be used to induce periodontal tissue regeneration.
Topics: Amelogenin; Animals; Cell Movement; Cell Proliferation; Cells, Cultured; Dental Enamel; Dental Enamel Proteins; Humans; MAP Kinase Signaling System; Pichia; Recombinant Proteins; Swine
PubMed: 22834823
DOI: 10.1111/j.1365-2184.2012.00834.x -
Gaceta Medica de Mexico 2019Amelogenesis imperfecta is a group of developmental disorders of the dental enamel that is mainly associated with mutations in the AMELX gene. Clinically, it presents... (Review)
Review
Amelogenesis imperfecta is a group of developmental disorders of the dental enamel that is mainly associated with mutations in the AMELX gene. Clinically, it presents different phenotypes that affect the structure and function of dental enamel both in primary and secondary dentition. The purpose of this study was to conduct a literature review on the AMELX functions and mutations that are related to amelogenesis imperfecta. A literature search was carried out in two databases: PubMed and Web of Science, using the keywords "AMELX", "amelogenin", "amelogenesis imperfecta" and "AMELX mutation". Forty articles were reviewed, with AMELX being found to be the predominant gene in the development of dental enamel and amelogenesis imperfecta by altering the structure of amelogenin. In the past few years, the characteristics of the amelogenesis imperfecta process have been described with different phenotypes of hypoplastic or hypo-mineralized enamel, and different mutations have been reported, by means of which the gene sequencing and the position of mutations have been determined.
Topics: Amelogenesis Imperfecta; Amelogenin; Dental Enamel; Humans; Mutation; Phenotype
PubMed: 30799455
DOI: 10.24875/GMM.18003604 -
Matrix Biology : Journal of the... Jan 2022Studies on animal models with mutations in ameloblastin gene have suggested that the extracellular matrix protein ameloblastin (AMBN) plays important roles in...
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.
Topics: Ameloblasts; Amelogenesis; Amelogenin; Animals; Cell Culture Techniques, Three Dimensional; Cell Line; Dental Enamel Proteins
PubMed: 34813898
DOI: 10.1016/j.matbio.2021.11.002 -
Health Science Reports Jan 2023Odontogenic cysts and tumors often form hard and soft structures that resemble odontogenesis. It is well known that amyloid is produced in Pindborg tumors; however, it...
BACKGROUND AND AIMS
Odontogenic cysts and tumors often form hard and soft structures that resemble odontogenesis. It is well known that amyloid is produced in Pindborg tumors; however, it is still debatable whether it is also formed in other odontogenic tumors and cysts. This study aimed to detect the presence of amyloid in different odontogenic cysts and tumors in correlation to matrix proteins secreted during enamel formation; namely amelogenin and odontogenic ameloblast-associated protein.
METHODS
This study included formalin fixed paraffin embedded tissue blocks of 106 different types of odontogenic cysts and tumors. Congo red and thioflavin T were performed to confirm the presence of amyloid; immunohistochemistry was used to detect amelogenin and odontogenic ameloblast-associated protein.
RESULTS
Amyloid was detected in pindborg tumors (conventional), adenomatoid odontogenic tumors, odontogenic fibroma (Amyloid variant), follicular solid and unicystic ameloblastomas, radicular cysts, dentigerous cysts, dentinogenic ghost cell odontogenic tumor, ameloblastic fibroma, calcifying odontogenic cyst, and primordial Odontogenic tumor. Amelogenin was detected in 95.3% of the cases, while odontogenic ameloblast-associated protein was detected in 93.4% of the cases. The association between odontogenic ameloblast-associated protein and amyloid was highly significant at < 0.01. However, there was no significant relationship between amelogenin and amyloid > 0.05.
CONCLUSION
Although pindborg tumor is the bonafide example of amyloid deposition in odontogenic tumors, this study concluded that amyloid may be deposited in traces to massive amounts in various odontogenic cysts and tumors, and it is significantly linked to odontogenic ameloblast-associated protein but not amelogenin matrix protein, since all amyloid cases were odontogenic ameloblast associated protein positive.
PubMed: 36655142
DOI: 10.1002/hsr2.1061 -
The Anatomical Record Dec 1998Hagfishes lack mineralized tissues and teeth. Part of a cDNA strand, allegedly from amelogenin, the major gene involved in enamel formation in mammals, has recently been... (Comparative Study)
Comparative Study Review
Hagfishes lack mineralized tissues and teeth. Part of a cDNA strand, allegedly from amelogenin, the major gene involved in enamel formation in mammals, has recently been cloned in a hagfish (Slavkin and Diekwish, Anat. Rec., 1996;245:131-150). This cloning is of great interest because it could change the current view about the evolution of mineralized tissues, but no phylogenetic analysis of this piece of DNA has been made by the authors. Phylogenetic analysis of this part of cDNA has been conducted using both phenetic and cladistic methods. The cDNA amplified in hagfish does not fit with a nonmammalian origin but fits well with a degraded rodent sequence. The gene cloned in hagfish is probably of mammalian origin due to contamination during PCR.
Topics: Algorithms; Amelogenin; Animals; DNA, Complementary; Dental Enamel Proteins; Evolution, Molecular; Hagfishes; Humans; Phylogeny; Polymerase Chain Reaction; Species Specificity; Tooth Germ
PubMed: 9845211
DOI: 10.1002/(SICI)1097-0185(199812)252:4<608::AID-AR11>3.0.CO;2-O -
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 -
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