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ENAMELIN AND AUTOSOMAL-DOMINANT AMELOGENESIS IMPERFECTA
J.C.-C. Hu1,* and
Y. Yamakoshi2
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;
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Figure 1. Consecutive sections of a developing porcine incisor showing the stages of enamel formation. The top section is stained with toluidine blue, and the bottom section is immunostained with the 32-kDa enamelin antibody. Enamelin signal is observed in the enamel matrix throughout the matrix formation (secretory) stage and persists at the DEJ (arrowheads) during enamel maturation. Key: enamel, E; dentin, D; matrix formation stage, F; transition stage, T; maturation stage, M; arrowheads mark the DEJ. Adapted from reference (Uchida et al., 1991a).
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Figure 2. Immunohistochemistry of the porcine secretory-stage enamel showing immunolocalization of enamel proteins using light microscopy (Uchida et al., 1991b). The four antibodies used (from left to right) were a polyclonal antibody raised against intact (25 kDa) amelogenin, an anti-peptide antibody specific for the amelogenin C-terminus, polyclonal antibodies raised against 13- to 17-kDa ameloblastin cleavage products, and polyclonal antibodies raised against the 89-kDa enamelin. The amelogenin C-terminal antibody, indicative of the intact protein, was restricted to the surface enamel. The ameloblastin antibody produced a honeycomb pattern over the entire thickness of the immature enamel. The enamelin antibody generated a reverse honeycomb pattern. Key: secretory ameloblasts (Am), immature enamel (E), dentin (D), Golgi (arrowheads). Approximate magnification, 520x.
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Figure 3. Electron micrographs of porcine secretory-stage enamel showing immunolocalization of enamelin at the enamel surface near the ameloblast Tomes’ process. The results from four affinity-purified anti-peptide antibodies are shown: (a) enamelin N-terminus (Dohi et al., 1998); (b) 32-kDa N-terminus (Uchida et al., 1991a); (c) 34-kDa N-terminus; and (d) enamelin C-terminus (Hu et al., 1997b). Below the histology is a diagram showing the different enamelin cleavage products and the positions of the sequences used to make antibodies. Pig enamelin has 1142 amino acids. The first 38 amino acids constitute the signal peptide. The secreted protein has an apparent molecular weight of 186 kDa (amino acids 39–1142); partially characterized enamelin cleavage products are the 155 kDa (39-unknown), 142 kDa (39 to unknown), 89 kDa (39 to 665), 32 kDa (174–279), 25 kDa (515–665), and the 34 kDa (670-unknown) (Fukae et al., 1996). Key: Tomes’ processes (TP), enamel matrix (E), secretory granules (SG), secretory face (SF), non-secretory face (NF) of Tomes’ process endoplasmic reticulum (ER), terminal web (TW), stippled material (SM). Bar = 1.0 µm.
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Figure 4. Structures of the human ameloblastin (AMBN) and enamelin (ENAM) genes and their chromosomal localizations. Exons are indicated by numbered boxes, introns by a line. The numbers below each exon show the range of amino acids encoded by that exon. The AMBN gene has 13 exons, all coding. AMBN exons 7 through 9 are repeat sequences (Toyosawa et al., 2000). The human ENAM gene is shown with 10 exons for consistency with the mouse enamelin gene (Hu et al., 2001b). It is not known if exon 2, which is noncoding, is used in humans. The enamelin gene has 8 coding exons. The AMBN and ENAM genes are located together on the long arm of chromosome 4. The order of the genes is centromere, AMBN, ENAM, DSPP, DMP1, teleomere.
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Figure 5. Alignment of the derived protein sequences of pig, human, mouse, and rat enamelin. The number of the last amino acid in each row is indicated on the right. Amino acid residues at the amino- or carboxyl-termini of known porcine enamelin cleavage products are labeled above the pig sequence. Potentially modified amino acids are in bold. An asterisk indicates an amino acid that is known to be modified in pig enamelin. Known polymorphisms in the human enamelin protein are underlined: (Arg/Gln)286, (Ile/Thr)648, (Arg/Gln)763, and (Gly/Asp)948.
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Figure 6. Structures of the pyridylaminated-oligosaccharides liberated from the 32-kDa enamelin by glycopeptidase F digestion (Yamakoshi, 1995). Asn245 uses all five of the biantennary types shown on the right. Asn252 uses the top two biantennary types. Asn264 uses the three triantennary types shown on the left (Yamakoshi et al., 1998). Key: fucose, F; galactose, G; N-acetylglucosamine, GN; mannose, M; N-acetylneuraminic acid, S. Diagram showing the position of the 32-kDa enamelin cleavage product in the 186-kDa secreted enamelin protein and the positions of the two phosphorylations (P) and glycosylations (Gly).
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Figure 7. Digestion of the 32-kDa enamelin by enamelysin (MMP-20) and kallikrein 4 (KLK4). The 32-kDa enamelin, the 25-kDa amelogenin, MMP-20, and KLK4 were isolated from developing pig teeth. Cleavage of the 32-kDa enamelin by MMP-20 was not detected even after 72 hrs of incubation at a substrate:enzyme ratio of 50:1 (w/w). MMP-20 did digest amelogenin (data not shown). KLK4 fully degraded the 32-kDa enamelin after 24 hrs of incubation at a substrate:enzyme ratio of 100:1 (w/w).
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Figure 8. Sites of human enamelin gene mutations identified in families with AI. Exons are indicated by numbered boxes, introns by a line. The numbers below each exon show the range of amino acids encoded by that exon. Lines indicate the location of defined mutation sites in the enamelin gene. An "X" indicates that the mutation created a stop codon. Mutations affecting splice junctions are indicated by "spl jctn".
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Critical Reviews in Oral Biology & Medicine, Vol. 14, No. 6,
387-398 (2003)
DOI: 10.1177/154411130301400602
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