Amelogenin sequence and enamel biomineralization in Rana pipiens

The amelogenin gene contributes the majority of tooth enamel proteins and plays a significant role in enamel biomineralization. While several mammalian and reptilian amelogenins have been cloned and sequenced, basal vertebrate amelogenin evolution remains to be understood. In order to start elucidating the structure and function of amelogenins in the evolution of enamel, the leopard frog (Rana pipiens) was used as a model. Tissues from Rana pipiens teeth were analyzed for enamel structure and RNA extracts were processed for sequence analysis. Electron microscopy revealed that Rana pipiens enamel contains long and parallel crystals similar to mammalian enamel, while immunoreactions confirmed the site-specific localization of cross-reactive amelogenins in Rana pipiens enamel. Sequencing of amelogenin PCR products revealed a 782bp cDNA with a 546-nucleotide coding sequence encoding 181 amino acids. The homology of the newly discovered Rana pipiens amelogenin nucleotide and amino acid sequence with the published mouse amelogenin was 38.6% and 45%, respectively. These findings report the first complete amelogenin cDNA sequence in amphibians and indicate a close homology between mammalian enamel formation and Rana pipiens enamel biomineralization.     

Identification and characterization of a squamate reptilian amelogenin gene: Iguana iguana

As the principal components of the developing tooth enamel matrix, amelogenins play a significant role in tooth enamel formation and organization. In order to elucidate the structure and function of amelogenins in the evolution of enamel, we have selected the Iguana iguana as a squamate model organism. Here we report the first complete squamate amelogenin sequence available as of yet and document unique features of Iguana amelogenins and enamel. Transmission electron microscopy documented randomly oriented Iguana enamel crystals during the elongation phase compared with organized enamel crystal patterns at comparable stages in mammals. Sequencing of PCR amplified products revealed a full-length I. iguana amelogenin cDNA containing 877 nucleotides with a 564 nucleotide coding sequence encoding 187 amino acids. The homologies of the newly discovered I. iguana amelogenin amino acid sequence with the published mouse, caiman (Palaeosuchus), and snake (Elaphe) amelogenin were 41.3%, 53.5%, and 55.5%, respectively. On Western blots one major protein with a molecular weight of 24 kDa, and two minor proteins with molecular weights of 28 and 13.5 kDa, respectively, were detected based on the cross-reactivity of antisera against recombinant Rana pipiens amelogenin proteins. Sequence analysis revealed a moderate sequence homology between mammalian and reptilian amelogenin genes. A significant alteration was the deletion of the hydrophilic GSP sequence from exon 3 in the mouse sequence resulting in a conversion to a hydrophobic region in Iguana. Together, these findings identified a novel amelogenin cDNA sequence in the squamate reptilian I. iguana and functional implications for the evolution of amelogenins and enamel in squamates.     

Heterogeneity of amelogenin mRNA in the bovine tooth germ

The amelogenins are a complex mixture of hydrophobic proteins that are the major organic component of developing enamel. To study the molecular mechanisms underlying the heterogeneity of the amelogenins we isolated cDNA clones encoding these proteins. The clones were definitively identified by hybrid-selected translation experiments and by comparison of the DNA sequence with the protein-derived amino acid sequence. Southern hybridization of bovine genomic DNA indicated that amelogenin is a single copy gene. However, Northern hybridization experiments distinctly showed two major species of mRNA, each of which were sufficiently large enough to encode the highest known molecular weight species of amelogenin proteins. Furthermore, immunoprecipitation of hybrid-selected translation products using isolated amelogenin cDNA showed multiple, translated protein products. These data are supportive of a differential mRNA processing mechanism involved in generating a heterogeneous family of amelogenin matrix proteins from a single gene.     

Amelogenins. Sequence homologies in enamel-matrix proteins from three mammalian species.

Partial amino acid sequences for selected amelogenin polypeptides isolated from the developing enamel of cow, pig and human foetuses are reported. It was found that there was an identity of sequence for the initial 28 residues of the polypeptides analysed, irrespective of their origin or size. A tyrosine-rich polypeptide was shown to be the N-terminal fragment of the principal higher-molecular-weight amelogenins, although a leucine-rich polypeptide of similar size was not identified in any other amelogenin structure. The findings demonstrate a striking degree of sequence conservation for the amelogenin proteins of the extracellular enamel matrix and support the concept of a discrete fragmentation of an initial 30 000 Da amelogenin molecule during the mineralization of the enamel.     

Selachian tooth development: II. Immunolocalization of amelogenin polypeptides in epithelium during secretory amelogenesis in Squalus acanthias

We have determined the distribution of amelogenin polypeptides in an order of elasmobranchs using indirect immunofluorescence with rabbit polyclonal antibodies prepared to purified murine amelogenins. We find that amelogenins are definitely present within the inner enamel epithelium prior to the production of the extracellular matrix component termed "enameloid" (row II developing tooth organs). During subsequent stages of selachian tooth development (row III tooth organs), immunofluorescence staining data indicated localization of amelogenin antigens within epithelium as well as the enameloid extracellular matrix. The results from these immunohistochemical studies suggest that the 16-20 kdalton amelogenins, which are characteristic of murine inner enamel epithelial cells undergoing terminal biochemical differentiation into secretory ameloblasts, may also be regarded as molecular markers for amelogenesis in developing teeth in the spiny dogfish, Squalus acanthias.     

The presence and possible functions of the matrix metalloproteinase collagenase activator protein in developing enamel matrix.

The developing enamel matrix contains mostly amelogenins, which are hydrophobic proline-rich proteins. During amelogenesis, the amelogenins are presumably hydrolysed and removed from the enamel. Recently a number of metalloproteinases that may be important in amelogenesis have been identified in zymograms of the developing enamel matrix. In the present study an antibody specific for the matrix metalloproteinase collagenase activator protein (CAP) was characterized and used to identify this metalloproteinase in enamel. Immunoblotting showed that the CAP proteinase was present in the enamel matrix. Immunohistochemistry confirmed that the proteinase is localized in the enamel matrix, most specifically along the dentino-enamel junction. Purified CAP was found to hydrolyse amelogenin protein. Possible functions of the proteinase in the enamel matrix are discussed.     

Altering biomineralization by protein design

To create a bioceramic with unique materials properties, biomineralization exploits cells to create a tissue-specific protein matrix to control the crystal habit, timing, and position of the mineral phase. The biomineralized covering of vertebrate teeth is enamel, a distinctive tissue of ectodermal origin that is collagen-free. In forming enamel, amelogenin is the abundant protein that undergoes self-assembly to contribute to a matrix that guides its own replacement by mineral. Conserved domains in amelogenin suggest their importance to biomineralization. We used gene targeting in mice to replace native amelogenin with one of two engineered amelogenins. Replacement changed enamel organization by altering protein-to-crystallite interactions and crystallite stacking while diminishing the ability of the ameloblast to interact with the matrix. These data demonstrate that ameloblasts must continuously interact with the developing matrix to provide amelogenin-specific protein to protein, protein to mineral, and protein to membrane interactions critical to biomineralization and enamel architecture while suggesting that mutations within conserved amelogenin domains could account for enamel variations preserved in the fossil record.     

Self-assembly properties of recombinant engineered amelogenin proteins analyzed by dynamic light scattering and atomic force microscopy

Dynamic light scattering (DLS) analysis together with atomic force microscopy (AFM) imaging was applied to investigate the supramolecular self-assembly properties of a series of recombinant amelogenins. The overall objective was to ascertain the contribution of certain structural motifs in amelogenin to protein-protein interactions during the self-assembly process. Mouse amelogenins lacking either amino- or carboxy-terminal domains believed to be involved in self-assembly and amelogenins having single or double amino acid mutations identical to those found in cases of amelogenesis imperfecta were analyzed. The polyhistidine-containingfull-length recombinant amelogenin protein [rp(H)M180] generated nanospheres with monodisperse size distribution (hydrodynamic radius of 20.7 +/- 2.9 nm estimated from DLS and 16.1 +/- 3.4 nm estimated from AFM images), comparable to nanospheres formed by full-length amelogenin rM179 without the polyhistidine domain, indicating that this histidine modification did not interfere with the self-assembly process. Deletion of the N-terminal self-assembly domain from amelogenin and their substitution by a FLAG epitope ("A"-domain deletion) resulted in the formation of assemblies with a heterogeneous size distribution with the hydrodynamic radii of particles ranging from 3 to 38 nm. A time-dependent dynamic light scattering analysis of amelogenin molecules lacking amino acids 157 through 173 and containing a hemagglutinin epitope ("B"-domain deletion) resulted in the formation of particles (21.5 +/- 6.8 nm) that fused to form larger particles of 49.3 +/- 4.3 nm within an hour. Single and double point mutations in the N-terminal region resulted in the formation of larger and more heterogeneous nanospheres. The above data suggest that while the N-terminal A-domain is involved in the molecular interactions for the formation of nanospheres, the carboxy-terminal B-domain contributes to the stability and homogeneity of the nanospheres, preventing their fusion to larger assemblies. These in vitro findings support the notion that the proteolytic cleavage of amelogenin at amino- and carboxy-terminii occurring during enamel formation influences amelogenin to amelogenin interactions during self-assembly and hence alters the structural organization of the developing enamel extracellular matrix, thus affecting enamel biomineralization.     

The effect of recombinant mouse amelogenins on the formation and organization of hydroxyapatite crystals in vitro

Amelogenin is the most abundant protein in developing dental enamel. It is believed to play an important role in the regulation of the growth and organization of enamel crystals. Amelogenin, unlike many other proteins found in biominerals, is mostly hydrophobic except for a 13 amino acid hydrophilic C-terminal domain. To clarify the role of amelogenin in enamel mineralization, we designed calcium phosphate crystal growth experiments in the presence of recombinant amelogenins with or without the charged C-terminal domain. The shape and organization of the crystals were examined by TEM in bright field and diffraction modes. It was found that both full-length and truncated amelogenin inhibit crystal growth in directions normal to the c-axis. At the same time, crystallites organized into parallel arrays only in the presence of the full-length amelogenin in monomeric form. Pre-assembled amelogenins had no effect on crystals organization. These results imply that the hydrophobic portion of amelogenin plays a role in an inhibition of crystal growth, whereas the C-terminal domain is essential for the alignment of crystals into parallel arrays. Our data also suggest that nascent enamel structure emerges as a result of cooperative interactions between forming crystals and assembling proteins.     

High yield of biologically active recombinant human amelogenin using the baculovirus expression system

The amelogenins are secreted by the ameloblast cells of developing teeth; they constitute about 90% of the enamel matrix proteins and play an important role in enamel biomineralization. Recent evidence suggests that amelogenin may also be involved in the regeneration of the periodontal tissues and that different isoforms may have cell-signalling effects. During enamel development and mineralization, the amelogenins are lost from the tissue due to sequential degradation by specific proteases, making isolation of substantial purified quantities of full-length amelogenin challenging. The aim of the present study was to express and characterize a recombinant human amelogenin protein in the eukaryotic baculovirus system in quantities sufficient for structural and functional studies. Human cDNA coding for a 175 amino acid amelogenin protein was subcloned into the pFastBac HTb vector (Invitrogen), this system adds a hexa-histidine tag and an rTEV protease cleavage site to the amino terminus of the expressed protein, enabling effective one-step purification by Ni2+-NTA affinity chromatography. The recombinant protein was expressed in Spodoptera frugiperda (Sf9) insect cells and the yield of purified his-tagged human amelogenin (rHAM+) was up to 10 mg/L culture. Recombinant human amelogenin (rHAM+) was characterized by SDS-PAGE, Western blot, ESI-TOF spectrometry, peptide mapping, and MS/MS sequencing. Production of significant amounts of pure, full-length amelogenin opened up the possibility to investigate novel functions of amelogenin. Our recent in vivo regeneration studies reveal that the rHAM+ alone could bring about regeneration of the periodontal tissues; cementum, periodontal ligament, and bone.     

Partial rescue of the amelogenin null dental enamel phenotype

The amelogenins are the most abundant secreted proteins in developing dental enamel. Enamel from amelogenin (Amelx) null mice is hypoplastic and disorganized, similar to that observed in X-linked forms of the human enamel defect amelogenesis imperfecta resulting from amelogenin gene mutations. Both transgenic strains that express the most abundant amelogenin (TgM180) have relatively normal enamel, but strains of mice that express a mutated amelogenin (TgP70T), which leads to amelogenesis imperfecta in humans, have heterogeneous enamel structures. When Amelx null (KO) mice were mated with transgenic mice that produce M180 (TgM180), the resultant TgM180KO offspring showed evidence of rescue in enamel thickness, mineral density, and volume in molar teeth. Rescue was not observed in the molars from the TgP70TKO mice. It was concluded that a single amelogenin protein was able to significantly rescue the KO phenotype and that one amino acid change abrogated this function during development.     

Progressive accretion of amelogenin molecules during nanospheres assembly revealed by atomic force microscopy.

Amelogenin proteins, the principal components of the developing dental enamel matrix, self-assemble to form nanosphere structures that are believed to function as structural components directly involved in the matrix mediated enamel biomineralization. The self-assembly behavior of a recombinant murine amelogenin (rM179) was investigated by atomic force microscopy (AFM) for further understanding the roles of amelogenin proteins in dental enamel biomineralization. Recombinant rM179 amelogenin was dissolved in a pH 7.4 Tris-HCl buffer at concentrations ranging from 12.5 to 300 microg/ml. The solutions were adsorbed on mica, fixed with Karnovsky fixative and rinsed thoroughly with water for atomic force microscopy (AFM). At low concentrations (12.5-50 microg/ml), nanospheres with diameters varying from 7 to 53 nm were identified while at concentrations ranging between 100-300 microg/ml the size distribution was significantly narrowed to be steadily between 10 and 25 nm in diameter. These nanospheres were observed to be the basic building blocks of both engineered rM179 gels and of the developing enamel extracellular matrix. The stable 15-20-nm nanosphere structures generated in the presence of high concentrations of amelogenins were postulated to be of great importance in facilitating the highly organized ultrastructural microenvironment required for the formation of initial enamel apatite crystallites.     

Complete amino acid sequence of amelogenin in developing bovine enamel

Pure amelogenin protein in developing bovine incisor enamel was isolated and its primary structure was investigated by sequencing the peptides obtained after clostripain and chymotrypsin digestions and CNBr degradation with an automated Edman sequencer. The enamel protein was found to be composed of 170 amino acid residues with one phosphate having a molecular weight of 19,350 and its complete amino acid sequence was elucidated. This protein has no sequence homology with any other tissue or secretory protein of known structure.     

Identification and characterization of enamel proteinases isolated from developing enamel. Amelogeninolytic serine proteinases are associated with enamel maturation in pig.

During tooth formation nearly all of the protein matrix of enamel is removed before final mineralization. To study this process, enamel proteins and proteinases were extracted from pig enamel at different stages of tooth development. In the enamel maturation zones, the major enamel matrix proteins, the amelogenins, were rapidly processed and removed. Possibly associated with this process in vivo are two groups of proteinases which were identified in the enamel extracts by enzymography using amelogenin-substrate and gelatin-substrate polyacrylamide gels and by the degradation in vitro of guanidinium chloride-extracted amelogenins. One group of proteinases with gelatinolytic activity consisted of several neutral metalloendoproteinases having Mr values from 62,000 to 130,000. These proteinases were inactive against amelogenins, casein and albumin, and were present in approximately equal proportions in enamel at all developmental stages. In the other group, two serine proteinases, with apparent non-reduced Mr of 31,000 and 36,000 exhibited amelogeninolytic activity. The substrate preference of the enamel serine proteinases was indicated by their limited degradation of casein and their inability to degrade gelatin and albumin. Contrasting with the distribution of the metalloendoproteinase enzymes, the serine proteinases were found only in the enamel scrapings taken from late-maturing enamel. The amelogenin degradation patterns in vivo, observed in the enamel scrapings, were similar to those produced in assays in vitro using partially purified fractions of enamel proteinases and amelogenin substrate. Together, these data strongly indicate an important role for the serine proteinases, and possibly the gelatinolytic proteinases, in the organized processing of the enamel protein matrix during enamel formation.     

Maturation ameloblasts of the porcine tooth germ do not express amelogenin

 Amelogenins are the most abundant constituent in the enamel matrix of developing teeth. Recent investigations of rodent incisors and molar tooth germs revealed that amelogenins are expressed not only in secretory ameloblasts but also in maturation ameloblasts, although in relatively low levels. In this study, we investigated expression of amelogenin in the maturation stage of porcine tooth germs by in situ hybridization and immunocytochemistry. Amelogenin mRNA was intensely expressed in ameloblasts from the differentiation to the transition stages, but was not detected in maturation stage ameloblasts. C-terminal specific anti-amelogenin antiserum, which only reacts with nascent amelogenin molecules, stained ameloblasts from the differentiation to the transition stages. This antiserum also stained the surface layer of immature enamel at the same stages. At the maturation stage, no immunoreactivity was found within the ameloblasts or the immature enamel. These results indicate that, in porcine tooth germs, maturation ameloblasts do not express amelogenins, suggesting that newly secreted enamel matrix proteins from the maturation ameloblast are not essential to enamel maturation occurring at the maturation stage. Accepted: 14 January 1999     

Histidine tag fusion increases expression levels of active recombinant amelogenin in Escherichia coli

Amelogenin is a dental enamel matrix protein involved in formation of dental enamel. In this study, we have expressed two different recombinant murine amelogenins in Escherichia coli: the untagged rM179, and the histidine tagged rp(H)M180, identical to rM179 except that it carries the additional N-terminal sequence MRGSHHHHHHGS. The effects of the histidine tag on expression levels, and on growth properties of the amelogenin expressing cells were studied. Purification of a crude protein extract containing rp(H)M180 was also carried out using IMAC and reverse-phase HPLC. The results of this study showed clearly that both growth properties and amelogenin expression levels were improved for E. coli cells expressing the histidine tagged amelogenin rp(H)M180, compared to cells expressing the untagged amelogenin rM179. The positive effect of the histidine tag on amelogenin expression is proposed to be due to the hydrophilic nature of the histidine tag, generating a more hydrophilic amelogenin, which is more compatible with the host cell. Human osteoblasts treated with the purified rp(H)M180 showed increased levels of secreted osteocalcin, compared to untreated cells. This response was similar to cells treated with enamel matrix derivate, mainly composed by amelogenin, suggesting that the recombinant protein is biologically active. Thus, the histidine tag favors expression and purification of biologically active recombinant amelogenin.     

Analysis of self-assembly and apatite binding properties of amelogenin proteins lacking the hydrophilic C-terminal.

Amelogenins, the major protein component of the mineralizing enamel extracellular matrix, are critical for normal enamel formation as documented in the linkage studies of a group of inherited disorders, with defective enamel formation, called Amelogenesis imperfecta. Recent cases of Amelogenesis imperfecta include mutations that resulted in truncated amelogenin protein lacking the hydrophilic C-terminal amino acids. Current advances in knowledge on amelogenin structure, nanospheres assembly and their effects on crystal growth have supported the hypothesis that amelogenin nanospheres provide the organized microstructure for the initiation and modulated growth of enamel apatite crystals. In order to evaluate the function of the conserved hydrophilic C-terminal telopeptide during enamel biomineralization, the present study was designed to analyze the self-assembly and apatite binding behavior of amelogenin proteins and their isoforms lacking the hydrophilic C-terminal. We applied dynamic light scattering to investigate the size distribution of amelogenin nanospheres formed by a series of native and recombinant proteins. In addition, the apatite binding properties of these amelogenins were examined using commercially available hydroxyapatite crystals. Amelogenins lacking the carboxy-terminal (native P161 and recombinant rM166) formed larger nanospheres than those formed by their full-length precursors: native P173 and recombinant rM179. These data suggest that after removal of the hydrophilic carboxy-terminal segment further association of the nanospheres takes place through hydrophobic interactions. The affinity of amelogenins lacking the carboxy-terminal regions to apatite crystals was significantly lower than their parent amelogenins. These structure-functional analyses suggest that the hydrophilic carboxy-terminal plays critical functional roles in mineralization of enamel and that the lack of this segment causes abnormal mineralization.