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1 Department of Orthodontics and Pediatric Dentistry and 2 Department of Biologic and Materials Science, University of Michigan, School of Dentistry, 1011 North University, Ann Arbor, MI 48109-1078, USA;
* corresponding author, University of Michigan Dental Research Laboratory, 1210 Eisenhower Place, Ann Arbor, MI 48108, USA; janhu{at}umich.edu
Abstract Introduction Enamelin Protein Sequences and Immunohistochemistry Enamelin mRNA Expression Cloning Enamelin cDNAs and Genes Enamelin Genes and cDNA Enamelin Post-translational Modifications Enamelin Protein-Protein Interactions Enamelin and Amelogenesis Imperfecta (AI) Current Research Acknowledgments References
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Key words. Enamel, enamelin, amelogenin, ameloblastin, amelogenesis imperfecta, biomineralization, tooth formation
| Introduction |
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— Ellen Glasgow (Urist, 1966)
There has been considerable confusion about what is meant by the term enamelin. This is because the definition of enamelin has changed over time. The classic perspective of amelogenesis held that the enamel matrix of developing mammalian teeth was comprised of two classes of proteins: amelogenins and enamelins (Termine et al., 1980; Deutsch, 1989). The amelogenins did not bind to enamel crystals, and therefore could be extracted from secretory-stage enamel scrapings with guanidine HCl. The enamelins, which consisted of proteins tightly bound to the crystallites, were extracted after the amelogenins by dissolving the mineral, typically with guanidine HCl/EDTA. The classic theory was conceived over 20 years ago, prior to the availability of any significant amino acid, cDNA, or genomic sequence data for the proteins in the enamel matrix. This framework proved useful for over a decade, but became antiquated as the nature of enamel proteins and the genetic mechanisms that produced them were more fully understood (Fincham et al., 1999).
In the early 1980s, partial amino acid sequences for amelogenin were reported from pigs (Fukae et al., 1979, 1980; Fukae and Shimizu, 1983), cattle (Zalut et al., 1980; Fincham et al., 1981; Takagi et al., 1984), and humans (Fincham et al., 1983). The protein data were followed by cDNA sequences, with mouse amelogenin being the first to be cloned (Snead et al., 1983) and characterized (Snead et al., 1985). These advances gave amelogenin a specific identity, and the term amelogenin became restricted to proteins expressed from amelogenin genes, which were found to localize on the sex chromosomes (Lau et al., 1989; Sasaki and Shimokawa, 1995). Alternative RNA splicing (Sasaki, 1984; Gibson et al., 1991; Simmer, 1995) and proteolytic processing (Suga, 1970; Bartlett and Simmer, 1999; Simmer and Hu, 2002) were shown to expand greatly the number of amelogenins present in the enamel matrix. Eventually, the amelogenin gene on the X-chromosome (AMELX) was shown to cause X-linked amelogenesis imperfecta (Lagerström et al., 1991; Hart et al., 2002). Characterizing the non-amelogenin enamel proteins, or "enamelins", lagged ten to 15 years behind amelogenin, but followed the same course of starting as a class of proteins and ending as a specific gene product.
Non-amelogenin enamel proteins combined to account for only 10% of the protein in immature enamel. For a while, it appeared as though there might not be any true enamelins, but that this class of proteins consisted largely of serum albumin (Limeback and Simic, 1989; Limeback et al., 1989; Strawich and Glimcher, 1989, 1990; Strawich et al., 1993). Albumin is not synthesized by ameloblasts (Couwenhoven et al., 1989; Yuan et al., 1996) and is not considered to be a true enamel protein, although it is commonly found in enamel protein preparations. It is still not certain how it gets there. Radiolabeled serum albumin injected into rabbits did not incorporate into the enamel layer, suggesting a physiological barrier between the extravascular fluid and the enamel matrix (Kinoshita, 1979; Kinoshita and Ogura, 1979). Albumin detected in normal enamel is likely to be an artifact of tissue preparation (Chen et al., 1995; Fincham et al., 1999).
A cDNA clone encoding a protein called tuftelin was characterized in the late 1980s and was thought to encode the major enamelin component in developing enamel (Deutsch et al., 1989, 1991). Although the tuftelin clone was isolated from a cDNA expression library using antibodies raised against the major 66-kDa protein in the guanidine/EDTA fraction of developing bovine enamel (Deutsch et al., 1987), the protein encoded by the cDNA was not present in the enamel matrix in appreciable quantities, and has never been isolated or characterized from immature enamel. Immunohistochemistry with tuftelin-specific antibodies showed positive signal at the dentino-enamel junction (DEJ), suggesting a role in enamel crystal nucleation (Deutsch et al., 1997). The term "enamelin" should no longer be used to refer to tuftelin (Brookes et al., 2002).
Today, we know that the two predominant non-amelogenin proteins in immature enamel are ameloblastin (also called amelin and sheathlin) and enamelin, which are the expression products of the AMBN and ENAM genes, respectively. These proteins were discovered by biochemists using a novel way of separating amelogenins from non-amelogenin proteins, which differed from the sequential dissociative extraction scheme used widely elsewhere. They separated porcine enamel fractions on SDS-polyacrylamide gels (SDS-PAGE), and soaked the gels in 25% isopropanol (Fukae and Tanabe, 1985, 1987b). The non-amelogenins remained in the gel, while the amelogenins went into solution. They were able to characterize two distinct parts of the protein we now call ameloblastin. Polypeptides from the N-terminal region of the protein had apparent molecular weights in the range of 13–17 kDa; they were concentrated in the sheath space that partially separates rod and interrod enamel and were designated as "sheath proteins" (Uchida et al., 1991b, 1995). The C-terminus of ameloblastin was characterized, shown to bind calcium, and was described as the 27- and 29-kDa calcium-binding proteins (Fukae and Tanabe, 1987a; Murakami et al., 1997; Yamakoshi et al., 2001). A second group of non-amelogenin enamel proteins was also characterized. A 32-kDa non-amelogenin was isolated from developing enamel that comprised about 1% of total enamel protein and showed many enamelin-like biochemical properties. It was acidic, and separated on 2-D gels into seven constituents having isoelectric points ranging from 3 to 4.5. It also bound strongly to hydroxyapatite (HA) crystals and inhibited HA crystal growth in vitro (Tanabe et al., 1990). Immunohistochemistry with antibodies against the 32-kDa (Uchida et al., 1991a) and the related 89-kDa (Uchida et al., 1991b) non-amelogenin proteins showed a concentration of signal in the crystallite-containing rod and interrod areas of the enamel matrix, suggesting that the proteins were bound to mineral in vivo. It was therefore concluded that this protein represented the enamel matrix component that constituted the enamelin class of proteins, and was specifically designated as enamelin.
The term enamelin is now restricted to the enamelin gene (ENAM) and its protein products. The classic theory, based upon the division of enamel proteins into two groups according to their mineral-binding properties, poorly accommodated the growing molecular data. It turned out that intact amelogenin binds to hydroxyapatite (Aoba et al., 1987; Ryu et al., 1998), as do some enamelin (Tanabe et al., 1990; Brookes et al., 2002) and ameloblastin cleavage products (Brookes et al., 2001). Furthermore, mineral binding is only one biochemical property. Solubility, ion binding, protein-protein interactions, susceptibility to proteolysis, and effects on crystal growth are other important properties potentially related to the function of enamel proteins. As a consequence, enamel matrix components are currently grouped according to the gene that expressed them (AMELX, EMBN, ENAM), so that all of the protein products of the enamelin gene are designated as enamelin, with the different proteolytic cleavage products being distinguished by their apparent molecular weight on SDS-PAGE.
This is the story of enamelin, how it was discovered, where it is expressed, its structure and post-translational modifications, its processing by proteases, its genetics, and how investigations into the composition of the enamel matrix of developing teeth improved our understanding of enamel formation and led to the discovery that defects in the enamelin gene are a major factor in the etiology of autosomal-dominant amelogenesis imperfecta.
| Enamelin Protein Sequences and Immunohistochemistry |
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Antibodies were raised against the 32-kDa enamelin N-terminal sequence and affinity-purified (Uchida et al., 1991a). These antibodies proved to be highly specific. Western blots clearly demonstrated that the 32-kDa enamelin was a proteolytic cleavage product of a much larger protein. The newly formed surface enamel contained enamelin proteins of 140- and 89-kDa apparent molecular weight. The enamelin cleavage products decreased in size with increasing depth toward the DEJ, including molecules with apparent molecular weights of 56, 45, and 32 kDa. Light and electron micrographs of immunostained sections of pig incisors were startling in their clarity and detail. The earliest signal for enamelin protein was observed in differentiating ameloblasts as the basal lamina disappeared, and in predentin at the future DEJ. During the secretory stage, the signal extended from the DEJ to the Tomes’ process of the secretory ameloblast, being especially strong beneath the secretory face of the Tomes’ process, and scarce along the non-secretory face and the sheath space separating rod from interrod enamel (Fig. 1
). The enamelin signal disappeared abruptly during the early maturation stage.
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). These discoveries strengthened the interpretation that the proteolytic cleavage products of enamel proteins are not simply degradation products, but are functional polypeptides, and that their functions are potentially different from those of the intact proteins and other cleavage products (Bartlett and Simmer, 1999; Simmer and Hu, 2002).
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). The antibody against the enamelin N-terminus stained the entire enamel matrix, with no particular concentration at the secretory face of the Tomes’ process (Fig. 3a). The signal decreased from the surface to the DEJ, and electron micrographs detected a greater concentration of the enamelin N-terminus in the sheath space relative to the rod and interrod enamel (Dohi et al., 1998). The 32-kDa antibody showed a slight concentration at the secretory face and a scarcity of signal along the non-secretory face of the Tomes’ process (Fig. 3b). The signal decreased going from the surface to the DEJ, although at the DEJ itself, it was particularly intense (Uchida et al., 1991a). Throughout the entire enamel layer, the 32-kDa enamelin showed a "reverse honeycomb" distribution pattern, being particularly scarce in the sheath space (Uchida et al., 1991a). The anti-peptide antibody specific for the N-terminus of the 34-kDa enamelin cleavage product, which is in the center of the protein, shows a concentration of signal beneath the secretory face of the Tomes’ process (Fig. 3c). The signal diminishes rapidly with depth, indicating that this portion of the enamelin protein is rapidly degraded or re-absorbed into the ameloblast and does not accumulate in the matrix (Hu et al., 1997b). The most restricted localization pattern was observed with the antibody specific for the enamelin C-terminus (Fig. 3d). Signal was observed only beneath the secretory face of the Tomes’ process, at the mineralization front. This highly restricted localization supports the interpretation that intact (uncleaved) enamelin participates in the elongation of enamel crystals, which occurs at the mineralization front (Hu et al., 1997b).
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| Enamelin mRNA Expression |
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The technique that generated the most restricted pattern of enamelin expression was in situ hybridization. In situ hybridization in a day 1 mouse developing incisor detected enamelin mRNA expressed by ameloblasts, but not by odontoblasts or other cells in the dental pulp. Extensive in situ hybridization analyses of enamelin expression in mouse molars from post-natal days 1, 2, 3, 7, 9, 14, and 21 have been reported (Hu et al., 2001a). Enamelin mRNA in maxillary first molars was first observed in pre-ameloblasts on the cusp slopes at day 2. The onset of enamelin expression was approximately synchronous with the initial accumulation of predentin matrix. Enamelin was expressed by ameloblasts throughout the secretory, transition, and early-maturation stages and was terminated in maturation-stage ameloblasts on day 9. No enamelin expression was observed in pulp or bone, or along the developing root.
Northern blot analysis of mRNA obtained from enamel organ epithelia (EOE), dental pulp organ, liver, heart, kidney, brain, spleen, skeletal muscle, and lung detected enamelin mRNA only in the EOE and dental pulp, with expression levels being at least an order of magnitude higher in the EOE (Hu et al., 1998).
Reverse-transcription/polymerase chain-reaction (RT-PCR), as well as Western blots, detected low levels of enamelin expression during root formation. Tissue samples were prepared from the apical portion of the forming root in porcine permanent incisor tooth germs. The specific source of enamelin expression was presumed to be cells derived from Hertwig’s epithelial root sheath (Fukae et al., 2001).
As of November, 2003, six human enamelin cDNAs have been detected in non-dental tissues as expressed sequence tags (ESTs). Two were from eye (BU741192 and BM726916), one from kidney (AI627857), one from a kidney tumor (AW466983), one from lymph (BU428532), and one from prostate (AI675060) tissues.
In summary, enamelin is expressed primarily by secretory ameloblasts and is presumed to function only during dental enamel formation. Enamelin expression by ameloblasts is low compared with that by amelogenin and ameloblastin, with enamelin protein accounting for only a few percent of total protein in the forming enamel layer. Much lower levels of enamelin expression have been observed in dental pulp, presumably secreted by odontoblasts, and along the forming root. Enamelin mRNAs have been detected as ESTs from several tissues, with no suggestion that such expression is physiologically significant.
| Cloning Enamelin cDNAs and Genes |
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| Enamelin Genes and cDNA |
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. The first ATG codon starting from the 5' end of the human, mouse, and pig cDNA sequences are all in the appropriate context for translation, but are followed in four codons by a translation stop signal (TGA), with the true translation initiation codons being located a short distance downstream. This feature has been observed in other genes (Kozak, 1984) and may play a role in the regulation of translation. In every enamelin homologue, translation begins with an amino acid sequence that satisfies the structural criteria for a functional signal peptide (Gierasch, 1989). The appropriate signal peptidase cleavage sites are predicted by computer analyses and, in the case of the pig protein, have been confirmed by Edman sequencing of the enamelin amino-terminus (Fukae, 1989; Fukae et al., 1996).
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) (Hu et al., 2001b). In contrast, the human ameloblastin gene has 13 coding exons (Toyosawa et al., 2000), while the human amelogenin gene has six (Salido et al., 1992). All of the coding exons in these genes have type O splice junctions, meaning that none of the introns splits a codon. Type O splice junctions allow exons to function as modules, so skipping exons by alternative splicing does not shift the reading frame. Despite this feature, no alternatively spliced enamelin cDNAs have ever been identified.
The primary structures of mammalian enamelin from the four known eutherian species show a high degree of sequence homology (Fig. 5), except in a particular segment between the 32-kDa and 25-kDa enamelin cleavage products. In the coding region for this section, there is a 33-nucleotide segment that is tandemly repeated in the mouse and rat sequences, but not in the pig or human enamelin genes. There are 14 copies of the repeat in the mouse and 16 in the rat. The human and pig enamelin cDNAs encode a pre-protein of 1142 amino acids, while the mouse has 1274 amino acids, and rat enamelin is larger still. Enamel proteins seem to be able to accommodate repeated segments into their structures. Exon 6 (the largest exon) in the amelogenin gene contains short repeat sequences that differ in length and sequence in virtually every amelogenin gene characterized (Hu et al., 1996). In the human ameloblastin gene, exons, 7, 8, and 9 each encode homologous 13 amino acid segments (Toyosawa et al., 2000). The repeat sequences in the rodent enamelin genes occur within exon 10, the largest and last exon. The inserted polypeptides significantly affect the amino acid compositions, calculated isoelectric points, and isotope-averaged molecular masses of the rodent enamelin homologues (Table). In practice, though, these differences may not be significant with respect to the proteins’ functional properties. This is due to the high impact of post-translational modifications on the size, structure, charge, stability, and function of enamelin.
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| Enamelin Post-translational Modifications |
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). The 32-kDa enamelin is the most abundant enamelin cleavage product in the enamel matrix, and provides a good example of the influence of post-translational modifications on the character of the protein.
The 32-kDa enamelin has 106 amino acids (extending from Leu174 to Arg279), but has two phosphorylated serines (Ser191 and Ser216) and 3 glycosylated asparagines (Asn245, Asn252, and Asn264) (Fig. 6). The isotope-averaged molecular weight of the 32-kDa phosphoprotein (without glycosylations) is 11,657.6 Daltons; its predicted isoelectric point is 5.27. Based upon the primary structure of the phosphorylated, but unglycosylated, 32-kDa enamelin, the ProtParam tool (http://us.expasy.org/cgi-bin/protparam) computes its instability index (II) as 48.01, classifying it as unstable. The size, isoelectric point, and stability of the 32-kDa enamelin appear to be affected greatly by its high degree of glycosylation. The structures of the N-linked oligosaccharides on the 32-kDa enamelin were determined by a combination of sequential exoglycosidase digestion and two-dimensional sugar mapping (Yamakoshi, 1995; Yamakoshi et al., 1998). Eight different oligosaccharide structures were observed, five biantennary and three triantennary types. The 32-kDa enamelin was cleaved, and glycopeptides containing each of the three glycosylation sites were isolated (Yamakoshi, 1995; Yamakoshi et al., 1998). It was determined that Asn245 can have any of the five biantennary complexes. Asn252 uses two of the biantennary complexes, while Asn264 uses any of the three triantennary complexes. The variability of the glycosylation pattern for the 32-kDa enamelin resulted from differences in the number, site, and mode of linkage of N-acetylneuraminic acid to the core-sugar chains, and in their degree of sialylation. Extensive and variable glycosylation presumably accounts for the large number of distinct 32-kDa enamelin bands that can be distinguished by two-dimensional gel electrophoresis, its lower-than-predicted isoelectric point, its large apparent molecular weight, and its great stability relative to the other enamelin cleavage products. Polyclonal antibodies raised against recombinant 32-kDa enamelin expressed in bacteria could detect the recombinant protein, but not the 32-kDa protein isolated from in vivo (Ryu et al., in press). Post-translational modifications were the only difference between the recombinant and native proteins. Clearly, post-translational modifications can have a profound effect on the structure and function of enamelin.
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| Enamelin Protein-Protein Interactions |
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| Enamelin and Amelogenesis Imperfecta (AI) |
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Alterations in the human amelogenin gene are responsible for X-linked forms of AI (Lagerström et al., 1991; Aldred et al., 1992; Hart et al., 2003), but only 5% of families with AI show an X-linked pattern of inheritance (Bäckman and Holmgren, 1988). The first linkage of an autosomal-dominant form of AI (ADAI) was to chromosome 4q11-q21 (Kärrman et al., 1997). The human ameloblastin gene (AMBN) was shown to localize in this region, and it was concluded that AMBN is linked to ADAI (MacDougall et al., 1997). Subsequent studies, however, localized the enamelin gene (ENAM) within the linked interval (Hu et al., 2000), and mutational studies of families with AI linked to chromosome 4q were determined to have critical defects in the enamelin gene (Rajpar et al., 2001; Mårdh et al., 2002), while no defects in the ameloblastin gene were detected (Mårdh et al., 2001). The ameloblastin gene is still considered to be a candidate gene for AI, but its linkage to the disease is unproven.
At present, there are four published reports of defects in the human enamelin gene that cause autosomal-dominant amelogenesis imperfecta (ADAI) (Rajpar et al., 2001; Kida et al., 2002; Mårdh et al., 2002; Hart et al., 2003). The mutation sites are shown in Fig. 8. The first reported human enamelin mutation was a splice donor site mutation after enamelin codon 178, which resulted in a severe form of thin and smooth hypoplastic AI that is believed to affect only 1.5% of all AI cases (Rajpar et al., 2001). This mutation occurs at the beginning of the intron following the sixth coding exon. It is difficult to predict the effect of this mutation on the enamelin protein structure. It was proposed that skipping coding exon 6 would be the most likely scenario and would delete amino acids 158–178; but failure to delete the intron following this exon seems equally probable and would cause the following extraneous amino acids to be added after Gln178: EKFFFLYTVSEK*, along with the deletion of amino acids 179–1142. It was also proposed that the mutation could interfere with the proteolytic activity of enamelin, but this can be dismissed, since the original data showing that enamelin had proteolytic activity (Moradian-Oldak et al., 1996) were erroneous, the mistaken conclusion being caused by the co-purification of enamel matrix serine proteinase 1 (now known as kallikrein-4, KLK4) with the 32-kDa enamelin cleavage product (Simmer et al., 1998).
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Most recently, a splice donor site mutation after enamelin codon 196 was shown to cause autosomal-dominant hypoplastic AI (Kida et al., 2002). There are normally 6 Gs at the end of coding exon 7, which are followed by a 7th G at the beginning of the adjacent intron. One of these Gs was deleted in a Japanese family with AI. If the splice donor site function is preserved in the mutant condition, the deletion would shift the reading frame at the start of the last coding exon, resulting in the inclusion of the following 80 amino acids after Gly196: ILTLDILDIMALGVALLIIQKKCLNKILKNPKKKILLKQKVQAQNPQLIQQSLRRILPNQILKGVREEMTPAPQETVPQD*. The affected members of the family showed hypoplastic enamel in both their deciduous and permanent teeth that resulted in a yellowish appearance and hypersensitivity to cold stimuli. This same mutation has recently been characterized in a family from Australia (Hart et al., 2003).
| Current Research |
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Perhaps the most exciting new developments involve the characterization of mutations in the human enamelin gene that cause amelogenesis imperfecta. We anticipate that such studies will allow the diagnosis of AI to become gene-based. Whenever the gene and mutation that cause AI in a given family are learned, careful descriptions of the associated clinical phenotypes will allow genotype-phenotype correlations to be identified. By studying the outcomes of different restoration procedures (i.e., bonding and veneers vs. crowns) for each genotype/phenotype condition, practicing dentists will use gene-based diagnoses to choose among various treatment options, and thereby restore the dentition in a way that achieves the best results.
| Acknowledgments |
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