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At least 19 recordsLinked to original sources
PubMed2006

The exon 6ABC region of amelogenin mRNA contribute to increased levels of amelogenin mRNA through amelogenin protein-enhanced mRNA stabilization.

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.

Amelogenin↗
PubMed2005

Reuptake of extracellular amelogenin by dental epithelial cells results in increased levels of amelogenin mRNA through enhanced mRNA stabilization.

Amelogenin is an extracellular matrix protein secreted by ameloblasts and is a major component of enamel matrix. Recently, in addition to their role in enamel formation, the biological activity of enamel proteins in the process of cell differentiation has recently become widely appreciated. In this study, we examined the biological activity of amelogenin on ameloblast differentiation. Recombinant mouse amelogenin (rm-amelogenin) enhanced the expression of endogenous amelogenin mRNA in a cultured dental epithelial cell line (HAT-7), despite a lack of increased amelogenin promoter activity. To solve this discrepancy, we analyzed the effects of rm-amelogenin on the stability of amelogenin mRNA. The half-life of amelogenin mRNA is extremely short, but in the presence of rm-amelogenin its half-life was extended three times longer than the control. Furthermore, we showed the entry of exogenous fluorescein isothiocyanate-conjugated rm-amelogenin into the cytoplasm of HAT-7 cells. It follows from our results that exogenous amelogenin increases amelogenin mRNA levels through stabilization of mRNA in the cytoplasm of HAT-7 cells. Here we speculated that during differentiation, dental epithelial cells utilize a unique mechanism for increasing the production of amelogenin, the reuptake of secreted amelogenin.

3' Untranslated Regions↗
PubMed2003

The small bovine amelogenin LRAP fails to rescue the amelogenin null phenotype.

Amelogenins are the most abundant secreted proteins in developing dental enamel. These evolutionarily-conserved proteins have important roles in enamel mineral formation, as mutations within the amelogenin gene coding region lead to defects in enamel thickness or mineral structure. Because of extensive alternative splicing of the primary RNA transcript and proteolytic processing of the secreted proteins, it has been difficult to assign functions to individual amelogenins. To address the function of one of the amelogenins, we have created a transgenic mouse that expresses bovine leucine-rich amelogenin peptide (LRAP) in the enamel-secreting ameloblast cells of the dental organ. Our strategy was to breed this transgenic mouse with the recently generated amelogenin knockout mouse, which makes none of the amelogenin proteins and has a severe hypoplastic and disorganized enamel phenotype. It was found that LRAP does not rescue the enamel defect in amelogenin null mice, and enamel remains hypoplastic and disorganized in the presence of this small amelogenin. In addition, LRAP overexpression in the transgenic mouse (wildtype background) leads to pitting in the enamel surface, which may result from excess protein production or altered protein processing due to minor differences between the amino acid compositions of murine and bovine LRAP. Since introduction of bovine LRAP into the amelogenin null mouse does not restore normal enamel structure, it is concluded that other amelogenin proteins are essential for normal appearance and function.

Amelogenesis↗
PubMed1998

Interaction of amelogenin with hydroxyapatite crystals: an adherence effect through amelogenin molecular self-association.

At the secretory stage of tooth enamel formation the majority of the organic matrix is composed of amelogenin proteins that are believed to provide the scaffolding for the initial carbonated hydroxyapatite crystals to grow. The primary objective of this study was to investigate the interaction between amelogenins and growing apatite crystals. Two in vitro strategies were used: first, we examined the influence of amelogenins as compared to two other macromolecules, on the kinetics of seeded growth of apatite crystals; second, using transmission electron micrographs of the crystal powders, based on a particle size distribution study, we evaluated the effect of the macromolecules on the aggregation of growing apatite crystals. Two recombinant amelogenins (rM179, rM166), the synthetic leucine-rich amelogenin polypeptide (LRAP), poly(L-proline), and phosvitin were used. It was shown that the rM179 amelogenin had some inhibitory effect on the kinetics of calcium hydroxyapatite seeded growth. The inhibitory effect, however, was not as destructive as that of other macromolecules tested. The degree of inhibition of the macromolecules was in the order of phosvitin > LRAP > poly(L-proline) > rM179 > rM166. Analysis of particle size distribution of apatite crystal aggregates indicated that the full-length amelogenin protein (rM179) caused aggregation of the growing apatite crystals more effectively than other macromolecules. We propose that during the formation of hydroxyapatite crystal clusters, the growing apatite crystals adhere to each other through the molecular self-association of interacting amelogenin molecules. The biological implications of this adherence effect with respect to enamel biomineralization are discussed.

Amelogenin↗
PubMed2001

Sexual dimorphism of porcine amelogenins: male-specific amelogenins have strong adsorption properties onto apatite crystals.

The present studies were undertaken to investigate the sexual dimorphism of porcine amelogenins and to gain information as to whether excesses of male amelogenins, if any, possess functional significance in protein-crystal interactions. Enamel proteins, including the intact full-length amelogenins and their degraded polypeptides, were isolated from the secretory enamel of male and female pigs. To identify the amelogenins among the separated pools of male- and female-matrix proteins, rabbit anti-C13 and C25 peptide sera were used, which reacted specifically with the conserved C-terminal domain. Immunoblotting showed that a few extra members of the amelogenins, sharing common epitopes at the C-terminus, were recognized in male products. The apparent yield of the male amelogenins was only marginal, on the basis of their stained intensities on the gel, but the secreted male amelogenins demonstrated selective (probably the strongest among the amelogenins) adsorption properties onto apatite crystals. Reflecting the general symmetric electrophoretic profiles of the male- and female-enamel proteins in toto, there were no sex-linked differences in the protein-crystal interaction and the resulting regulatory function of crystal precipitation.

Journal Article↗
PubMed2001

Immunohistochemical localization of amelogenin in human odontogenic tumors, using a polyclonal antibody against bovine amelogenin.

In the present study, we investigated the localization of amelogenin in odontogenic tumors, using an anti-amelogenin polyclonal antibody. In order to make the antibody, antisera against an amelogenin fraction obtained from the enamel matrix of unerupted bovine tooth was raised in rabbits. By Western blot analysis, a main band of 25 kDa and six minor bands (6.8, 12, 18, 20, 23, and 27 kDa) were detected under nonreducing conditions. Immunoreactivity for the amelogenin was observed in ameloblasts and in the immature enamel matrix of 4-day-old rats. In odontogenic tumors, positive reactions for amelogenin were localized in limited areas in adenomatoid odontogenic tumor, calcifying odontogenic cyst, primary intraosseous carcinoma and odontoma. The strongest immunoreactions were shown in enamel matrices in odontomas. Small mineralized foci in epithelial nests showed positive reactions, and a few reactions were observed in epithelium adjacent to the mineralized foci. In calcifying odontogenic cysts, some ghost cells in the lining epithelium were strongly stained. The results indicate that the present antibody for amelogenin is useful for the determination of odontogenic tumors, especially in those in which small mineralized foci are present in the epithelial nests.

Amelogenin↗
PubMed1992

Alternative splicing of the mouse amelogenin primary RNA transcript contributes to amelogenin heterogeneity.

A heterogeneous population of amelogenin proteins is derived from a single copy of the mouse amelogenin gene. To investigate the one gene--multiple protein enigma, we designed a study to distinguish between alternative splicing and proteolytic cleavage models. A pulse of [35S]methionine labeling demonstrated that multiple amelogenins are synthesized concurrently, a result consistent with an alternative splicing mechanism. Using reverse transcription and polymerase chain reaction we cloned a segment from the 5' end of a mouse amelogenin mRNA and connected it to a previously isolated abbreviated cDNA clone. Four additional cDNAs derived from alternatively spliced amelogenin mRNAs have been cloned and characterized. The five transcripts encode amelogenins 180, 156, 141, 74, and 59 amino acids in length.

Alternative Splicing↗
PubMed1998

Small-angle X-ray scattering and computer-aided molecular modeling studies of 20 kDa fragment of porcine amelogenin: does amelogenin adopt an elongated bundle structure?

Amelogenins, which are major matrix constituents in the developing tooth, play a regulatory role in the process of enamel crystal formation. Porcine amelogenin with 173 amino acid residues is rich in proline, glutamine, leucine, and histidine. We utilized the small-angle X-ray scattering (SAXS) technique to examine the solution structure of porcine amelogenin. Samples used were two porcine amelogenins with apparent molecular weights of 20 kDa (amino acids 1 to 148) and 13 kDa (amino acids 46 to 148) on SDS-PAGE. Prior to SAXS measurements, the protein samples were dissolved in 2% (v/v) acetic acid to give a concentration range up to 10 mg/ml. Comparison between Rg (the overall radius of gyration) and Rc (the cross-sectional radius of gyration) revealed that the 20 kDa amelogenin exists in this solution as asymmetric particles with a length of about 15 nm, presumably corresponding of dimers. Based on these experimental data and computer-aided molecular modeling studies, we propose that the 20 kDa amelogenin adopts an elongated bundle structure which mainly consists of extended structures similar to polyproline II and/or beta-strand, interspersed with beta-turn or loop.

Amelogenin↗
PubMed2003

[Extraction and purification of porcine amelogenin and preparation for the polyclonal amelogenin antibody].

OBJECTIVE: To prepare the polyclonal antibody to amelogenin. METHODS: The fetal porcine dental enamel was collected. Enamel matrix protein was extracted in 4M guanidine HCl (pH 7.4) with protease inhibitors present. Polyacrylamide gel filtration was included to isolate amelogenin from the initial dissociated extraction. The purified amelogenin conjugated with or without complete/incomplete Freund's adjuvant was then used to immunize the rabbits subcutaneously or intravenously. The specific IgG antibody was further purified by DE-52 cellulose. The working concentration of IgG antibody was determined through ELISA test. RESULTS: The Gel filtration showed that amelogenin components is at molecular weights of 15 kD and 13 kD apparently, which was consistent with those described before. The ELISA results showed that the working concentration for IgG was 1:1000. CONCLUSION: The antibody prepared in this study can be used for the detection of amelogenin.

Amelogenin↗
PubMed1995

Amelogenin signal peptide mutation: correlation between mutations in the amelogenin gene (AMGX) and manifestations of X-linked amelogenesis imperfecta.

Formation of tooth enamel is a poorly understood biological process. In this study we describe a 9-bp deletion in exon 2 of the amelogenin gene (AMGX) causing X-linked hypoplastic amelogenesis imperfecta, a disease characterized by defective enamel. The mutation results in the loss of 3 amino acids and exchange of 1 in the signal peptide of the amelogenin protein. This deletion in the signal peptide probably interferes with translocation of the amelogenin protein during synthesis, resulting in the thin enamel observed in affected members of the family. We compare this mutation to a previously reported mutation in the amelogenin gene that causes a different disease phenotype. The study illustrates that molecular analysis can help explain the various manifestations of a tooth disorder and thereby provide insights into the mechanisms of tooth enamel formation.

Amelogenesis Imperfecta↗
PubMed1993

Amelogenin post-translational modifications: carboxy-terminal processing and the phosphorylation of bovine and porcine "TRAP" and "LRAP" amelogenins.

TRAP and LRAP amelogenin components were isolated by size-exclusion and reversed-phase HPLC from developing dental enamel. Porcine developing enamel contains TRAP and LRAP components analogous to those of bovine. Amino acid composition and mass spectrographic analyses established that, in both species, the carboxy-terminal sequences of the LRAP components are two residues longer than previously reported for bovine LRAP, and that both the TRAPs and LRAPs contained a single phosphorylated residue. These amelogenin polypeptides were the principal components of the enamel protein lower molecular weight fraction. The LRAP sequence data for both species suggests that the mechanism of amelogenin carboxy-terminal processing may differ significantly from that previously suggested.

Amelogenin↗
PubMed1994

Molecular cloning and DNA sequence of rat amelogenin and a comparative analysis of mammalian amelogenin protein sequence divergence.

The developing rat incisor is a common model used in the study of enamel development. It has been impossible to study correlation between rat enamel structure and the sequence of the major developing enamel protein in this species as to date a DNA sequence for rat amelogenin has not been reported. This study presents the first cloning of a full-length cDNA copy of rat amelogenin and its deduced primary sequence. Detailed analysis of this sequence provides evidence that the gene has evolved by internal sequence duplication. Comparison of the rat amelogenin primary sequence with those published for other species provides evidence that this protein, while exhibiting extreme levels of sequence conservation, has been subject to significant structural changes that may be related to alterations in enamel structure in different mammalian groups.

Amelogenin↗
PubMed1990

Amelogenin gene similarity in vertebrates: DNA sequences encoding amelogenin seem to be conserved during evolution.

Mouse amelogenin cDNA was used in hybridization assays with genomic DNA, cut with the restriction enzyme Eco RI, from the edentulous chicken (Gallus domesticus), the monophyodont mouse (as control), diphyodont man, and the polyphyodont fishes Atlantic salmon (Salmo salar) and seawolf (Anarrhichas lupus). The hybridization assay was performed under stringent conditions with non-radioactive probes. Hybridization was obtained with mouse (6.4-kb band), man (9-kb and 13-kb bands), and seawolf (18-kb band) genomic DNA. This demonstrates DNA sequence similarities between these species, and supports the theory that DNA sequences encoding enamel proteins appear to be highly conserved during the evolution of vertebrates. Lack of hybridization in salmon and chicken may be due to sequence divergences or structural differences in an amelogenin gene analog, or it may be that no amelogenin gene is present in these animals.

Amelogenin↗
PubMed1994

Isolation and characterisation of an alternatively-spliced rat amelogenin cDNA: LRAP--a highly conserved, functional alternatively-spliced amelogenin?

A cDNA coding for a 59 amino acid polypeptide containing both the carboxy- and amino-termini, but lacking the central domain, of the rat tooth enamel matrix protein, amelogenin, was cloned and sequenced. The deduced polypeptide sequence indicates that this cDNA was derived from an amelogenin RNA molecule by using an alternative intra-exonic 3' splice acceptor site. This alternatively spliced product is almost identical to products previously identified in both cow and mouse enamel organs: the leucine-rich amelogenin peptide (LRAP). The conservation of this truncated polypeptide across the species suggests that it may have an important role in the formation of tooth enamel.

Alternative Splicing↗
PubMed2006

Comparative calcium binding of leucine-rich amelogenin peptide and full-length amelogenin.

Leucine-rich amelogenin peptide (LRAP) is an alternately spliced amelogenin. LRAP is known to bind to hydroxyapatite, and has been shown to signal mesenchymal cells to proliferate, but its function in enamel formation is unclear. The purpose of this study was to determine the calcium-binding properties and structure of recombinant human LRAP (rLRAP) compared with full-length amelogenin (rH174). rLRAP and rH174 were synthesized in Escherichia coli and purified by affinity chromatography and reverse-phase high-performance liquid chromatography. Calcium binding was measured by isothermal titration calorimetry (ITC) at pH 7.5 and 25 degrees C, and raw data were analyzed by origin 7.0 software. The structure of rLRAP was analyzed by nuclear magnetic resonance (NMR) and circular dichroism (CD) in the absence or presence of Ca2+, pH 7.5 and 4.0, at 25 degrees C. Thermodynamic values showed that rLRAP had a Ca2+-binding affinity approximately 6.4-times greater than rH174. NMR and CD data revealed that rLRAP was randomly coiled, and that this structure was not altered by Ca2+, which bound to rLRAP and rH174 via ionic interactions. Unlike r174 (beta-spiral), rLRAP had a random-coiled structure. The calcium binding and structural differences between rLRAP and rH174 suggest that these proteins have different functions in enamel biomineralization.

Amelogenesis↗
PubMed1995

Recent advances in amelogenin biochemistry.

This paper reviews advances in amelogenin biochemistry in three areas; (i) amelogenin expression; (ii) amelogenin post-translational and post-secretory processing, and (iii) amelogenin structure and function. Recent studies of amelogenin expression have demonstrated that alternative-splicing of mouse amelogenin RNA generates seven distinct mRNAs, coding for amelogenin proteins from 194 to 44 amino acid residues in length. A polyclonal antibody to a sequence of the 194-residue murine amelogenin identified this protein in vivo. While several studies have reported that amelogenins are post-translationally phosphorylated, it has proved difficult to confirm this view. Mass spectrometry studies of bovine and porcine TRAP and LRAP amelogenins have established a phosphoserine residue at position-16 as originally reported by Takagi et al. for a 180-residue bovine amelogenin. Also, we discovered that the detailed mechanism(s) of carboxy-terminal amelogenin proteolytic processing appear different than previously reported. In terms of amelogenin structure, it is well known that amelogenins form aggregated structures. Studies employing a recombinant amelogenin and dynamic light-scattering instrumentation demonstrated aggregate structures of 15-20 nm in radius, corresponding to a mass of 2-3 million daltons. Imaging these aggregates by transmission electron and atomic force microscopy suggested that these structures are equivalent to the "stippled" or "granular" material seen in electron photomicrographs of developing enamel. Collectively, these advances in amelogenin biochemistry lead to a new view of amelogenin structure, processing and functions in enamel biomineralization.

Alternative Splicing↗
PubMed2001

Controlled proteolysis of amelogenins reveals exposure of both carboxy- and amino-terminal regions.

The matrix-mediated enamel biomineralization involves secretion of the enamel specific amelogenin proteins that through self-assembly into nanosphere structures provide the framework within which the initial enamel crystallites are formed. During enamel mineralization, amelogenin proteins are processed by tooth-specific proteinases. The aim of this study was to explore the factors that affect the activity of enamel proteases to process amelogenins. Two factors including amelogenin self-assembly and enzyme specificity are considered. We applied a limited proteolysis approach, combined with mass spectrometry, in order to determine the surface accessibility of conserved domains of amelogenin assemblies. A series of commercially available proteinases as well as a recombinant enamelysin were used, and their proteolytic actions on recombinant amelogenin were examined under controlled and limited conditions. The N-terminal region of the recombinant mouse amelogenin rM179 was found to be more accessible to tryptic digest than the C-terminal region. The endoproteinase Glu-C cleaved amelogenin at both the N-terminal (E18/V) and C-terminal (E178/V) sites. Chymotrypsin cleaved amelogenin at both the carboxy- (F151/S) and amino-terminal (W25/Y) regions. Interestingly, the peptide bond F/S152 was also recognized by the action of enamelysin on recombinant mouse amelogenin whereas thermolysin cleaved the S152/M153 peptide bond in addition to T63/L64 and I159/L160 and M29/I30 bonds. It was then concluded that regions at both the carboxy- and amino-terminal were exposed on the surface of amelogenin nanospheres when the N-terminal 17 amino acid residues were proposed to be protected from proteolysis, presumably as the result of their involvement in direct protein-protein interaction. Cleavage around the FSM locus occurred by recombinant enamelysin under limited conditions, in both mouse (F151/S152) and pig amelogenins (S148/M). Our in vitro observations on the limited proteolysis of amelogenin by enamelysin suggest that enamelysin cleaved amelogenin at the C-terminal region showing a preference of the enzyme to cleave the S/M and F/S bonds. The present limited proteolysis studies provided insight into the mechanisms of amelogenin degradation during amelogenesis.

Amelogenin↗