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.     

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.     

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.     

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.     

Human and mouse cementum proteins immunologically related to enamel proteins

SDS-polyacrylamide gel electrophoresis, immunoblot and amino acid composition analyses were applied to human and mouse acellular cementum proteins immunologically related to enamelins and amelogenins. In this analysis, anti-mouse amelogenin, anti-human enamelin and synthetic peptide (e.g., -LPPHPGHPGYIC-) antibodies were shown to cross-react with tooth crown-derived enamelin with a molecular mass of 72,000 Da (72 kDa), amelogenins (26 kDa), and also to four human cementum proteins (72, 58, 50 and 26 kDa) and two mouse cementum proteins (72 and 26 kDa). Each of the antibodies recognized tooth root-derived cementum polypeptides which share one or more epitopes with tooth crown-derived enamel proteins. The molecular mass and isoelectric points for crown-derived and root-derived enamel-related proteins were similar. Analysis of human and mouse cementum proteins revealed a characteristic amino acid composition enriched in glutamyl, serine, glycine, alanine, proline, valine and leucine residues; compared to the major enamel protein amelogenin, cementum proteins were low in proline, histidine and methionine. The human and mouse putative intermediate cementum proteins appear to represent a distinct class of enamel-related proteins. Moreover, these results support the hypothesis that epithelial root sheath epithelia express several cementum proteins immunologically related to canonical enamel proteins.     

Immunochemical and immunohistochemical studies, using antisera against porcine 25 kDa amelogenin, 89 kDa enamelin and the 13-17 kDa nonamelogenins, on immature enamel of the pig and rat

Enamel proteins were extracted from the newly formed layer of immature porcine enamel, and the 25 kDa amelogenin, 89 kDa enamelin and 13-17 kDa nonamelogenins were purified. Specific antisera were raised against these proteins. Antibodies specific to the C-terminal region (residues 149-173) of the 25 kDa amelogenin were generated by absorption of the anti-25 kDa amelogenin serum with 20 kDa amelogenin, which contains residues 1-148 of the antigen. Immunoelectro-transfer blotting of the extracted porcine enamel proteins showed that the anti-25 kDa amelogenin serum recognized the 25 kDa and other low and high molecular weight amelogenins. The C-terminal specific anti-25 kDa amelogenin serum reacted only with amelogenins having molecular weights over 23 kDa. The anti-89 kDa enamelin serum recognized the 89 kDa enamelin and lower molecular weight proteins, but neither the amelogenins nor the 13-17 kDa nonamelogenins. The antiserum against the 13-17 kDa nonamelogenins showed no cross reactivity to the 89 kDa enamelin, but recognized higher molecular weight nonamelogenins. In immunohistochemical preparations of the porcine tooth germs, the 25 kDa amelogenin-like immunoreactivity over immature enamel decreased in a gradient from the enamel surface to the middle layer. In the inner layer immunoreactivity was concentrated over the prism sheaths. The C-terminal specific 25 kDa amelogenin-like immunoreactivity was intense at the outer layer of immature enamel and decreased sharply toward the middle layer. Prism sheaths were intensely stained by the antiserum to the 13-17 kDa nonamelogenins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amelogenin-deficient mice display an amelogenesis imperfecta phenotype.

Dental enamel is the hardest tissue in the body and cannot be replaced or repaired, because the enamel secreting cells are lost at tooth eruption. X-linked amelogenesis imperfecta (MIM 301200), a phenotypically diverse hereditary disorder affecting enamel development, is caused by deletions or point mutations in the human X-chromosomal amelogenin gene. Although the precise functions of the amelogenin proteins in enamel formation are not well defined, these proteins constitute 90% of the enamel organic matrix. We have disrupted the amelogenin locus to generate amelogenin null mice, which display distinctly abnormal teeth as early as 2 weeks of age with chalky-white discoloration. Microradiography revealed broken tips of incisors and molars and scanning electron microscopy analysis indicated disorganized hypoplastic enamel. The amelogenin null phenotype reveals that the amelogenins are apparently not required for initiation of mineral crystal formation but rather for the organization of crystal pattern and regulation of enamel thickness. These null mice will be useful for understanding the functions of amelogenin proteins during enamel formation and for developing therapeutic approaches for treating this developmental defect that affects the enamel.     

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     

Immunochemical and immunohistochemical studies,using antisera against porcine 25 kDa amelogenin, 89 kDa enamelin and the 13–17 kDa nonamelogenins,on immature enamel of the pig and rat

Summary Enamel proteins were extracted from the newly formed layer of immature porcine enamel, and the 25 kDa amelogenin, 89 kDa enamelin and 13–17 kDa nonamelogenins were purified. Specific antisera were raised against these proteins. Antibodies specific to the C-terminal region (residues 149–173) of the 25 kDa amelogenin were generated by absorption of the anti-25 kDa amelogenin serum with 20 kDa amelogenin, which contains residues 1–148 of the antigen. Immunoelectrotransfer blotting of the extracted porcine enamel proteins showed that the anti-25 kDa amelogenin serum recognized the 25 kDa and other low and high molecular weight amelogenins. The C-terminal specific anti-25 kDa amelogenin serum reacted only with amelogenins having molecular weights over 23 kDa. The anti-89 kDa enamelin serum recognized the 89 kDa enamelin and lower molecular weight proteins, but neither the amelogenins nor the 13–17 kDa nonamelogenins. The antiserum against the 13–17 kDa nonamelogenins showed no cross reactivity to the 89 kDa enamelin, but recognized higher molecular weight nonamelogenins. In immunohistochemical preparations of the porcine tooth germs, the 25 kDa amelogenin-like immunoreactivity over immature enamel decreased in a gradient from the enamel surface to the middle layer. In the inner layer immunoreactivity was concentrated over the prism sheaths. The C-terminal specific 25 kDa amelogenin-like immunoreactivity was intense at the outer layer of immature enamel and decreased sharply toward the middle layer. Prism sheaths were intensely stained by the antiserum to the 13–17 kDa nonamelogenins. The 89 kDa enamelin-like immunoreactivity over enamel prisms was intense at the outer layer and decreased toward the middle layer. Staining by the anti-89 kDa enamelin serum of prism sheaths was faint. In immature rat incisor enamel, the C-terminal specific 25 kDa amelogenin antiserum demonstrated a staining pattern similar to that in the immature enamel of the pig. Distinct 13–17 kDa nonamelogenin-like and 89 kDa enamelin-like immunoreactivities were found especially in the layer adjacent to the Tomes' process. We conclude that some enamel proteins are degraded soon after their secretion from the secretory ameloblast in the rat and the pig. The specific enamel proteins which reacted with the antiserum to the 13–17 kDa nonamelogenins seem to be involved with the formation of prism sheaths in immature porcine enamel, but not in rat incisor enamel.     

The receptor activator of nuclear factor-kappa B ligand-mediated osteoclastogenic pathway is elevated in amelogenin-null mice

Amelogenins, major components of developing enamel, are predominantly involved in the formation of tooth enamel. Although amelogenins are also implicated in cementogenesis, their precise spatial expression pattern and molecular role are not clearly understood. Here, we report for the first time the expression of two alternate splice forms of amelogenins, M180 and the leucine-rich amelogenin peptide (LRAP), in the periodontal region of mouse tooth roots. Lack of M180 and LRAP mRNA expression correlated with cementum defects observed in the amelogenin-null mice. The cementum defects were characterized by an increased presence of multinucleated cells, osteoclasts, and cementicles. These defects were associated with an increased expression of the receptor activator of the nuclear factor-kappa B ligand (RANKL), a critical regulator of osteoclastogenesis. These findings indicate that the amelogenin splice variants, M180 and LRAP, are critical in preventing abnormal resorption of cementum.     

DNA sequence for cloned cDNA for murine amelogenin reveal the amino acid sequence for enamel-specific protein

Enamel is the unique and highly mineralized extracellular matrix that covers vertebrate teeth. Amelogenin proteins represent the predominate subfamily of gene products found in developing mammalian enamel, and are implicated in the regulation of the formation of the largest hydroxyapatite crystals in the vertebrate body. Previous attempts to isolate, purify and characterize amelogenins extracted from developing matrix have proven difficult. We now have determined the DNA sequence for a cDNA for the 26-kDa class of murine amelogenin and deduced its corresponding amino acid sequence. The murine amino acid sequence is homologous to bovine or porcine amelogenins extracted from developing enamel matrices. However, an additional 10-residues were found at the carboxy terminus of the murine amelogenin. This is the most complete sequence database for amelogenin peptides and the only DNA sequence for enamel specific genes.     

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.     

Fine structural and immunohistochemical detection of collar enamel in the teeth of <Emphasis Type="Italic">Polypterus senegalus</Emphasis>, an actinopterygian fish

This is the first detailed report about the collar enamel of the teeth of Polypterus senegalus. We have examined the fine structure of the collar enamel and enamel organ of Polypterus during amelogenesis by light and transmission electron microscopy. An immunohistochemical analysis with an antibody against bovine amelogenin, an antiserum against porcine amelogenin and region-specific antibodies or antiserum against the C-terminus, middle region and N-terminus of porcine amelogenin has also been performed to examine the collar enamel matrix present in these teeth. Their ameloblasts contain fully developed Golgi apparatus, rough endoplasmic reticulum and secretory granules. During collar enamel formation, an amorphous fine enamel matrix containing no collagen fibrils is found between the dentin and ameloblast layers. In non-demineralized sections, the collar enamel (500 nm to 1 μm thick) is distinguishable from dentin, because of its higher density and differences in the arrangement of its crystals. The fine structural features of collar enamel in Polypterus are similar to those of tooth enamel in Lepisosteus (gars), coelacanths, lungfish and amphibians. The enamel matrix shows intense immunoreactivity to the antibody and antiserum against mammalian amelogenins and to the middle-region- and C-terminal-specific anti-amelogenin antibodies. These findings suggest that the proteins in the enamel of Polypterus contain domains that closely resemble those of bovine and porcine amelogenins. The enamel matrix, which exhibits positive immunoreactivity to mammalian amelogenins, extends to the cap enameloid surface, implying that amelogenin-like proteins are secreted by ameloblasts as a thin matrix layer that covers the cap enameloid after enameloid maturation.     

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.     

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.