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Origin, Splicing, and Expression of Rodent Amelogenin Exon 8
1 Department of Cytokine Biology, Correspondence: * corresponding author, jbartlett{at}forsyth.org
Amelogenin RNA transcripts undergo extensive alternative splicing, and MMP-20 processes the isoforms following their secretion. Since amelogenins have been ascribed cell-signaling activities, we asked if a lack of proteolytic processing by MMP-20 affects amelogenin signaling and consequently alters amelogenin splice site selection. RT-PCR analyses of amelogenin mRNA between control and Mmp20–/–mice revealed no differences in the splicing pattern. We characterized 3 previously unidentified amelogenin alternatively spliced transcripts and demonstrated that exon-8-encoded amelogenin isoforms are processed by MMP-20. Transcripts with exon 8 were expressed approximately five-fold less than those with exon 7. Analyses of the mouse and rat amelogenin gene structures confirmed that exon 8 arose in a duplication of exons 4 through 5, with translocation of the copy downstream of exon 7. No downstream genomic sequences homologous to exons 4–5 were present in the bovine or human amelogenin genes, suggesting that this translocation occurred only in rodents.
Key Words: enamelysin • enamel • amelogenin
Secretory-stage ameloblasts are responsible for many important processes necessary for enamel formation, including: alternative mRNA splicing, translation and secretion of enamel proteins (i.e., amelogenin, ameloblastin, enamelin, MMP-20), selective reabsorption and degradation of cleavage products, establishment and regulation of the influx of calcium and phosphate ions, and maintenance of extracellular pH and ionic strength (Simmer and Fincham, 1995; Fincham et al., 1999). Disruptions of any one of these processes may cause enamel defects. For example, the Mmp20 null mouse and human kindreds with critical MMP20 mutations have brittle enamel that tends to delaminate from the dentin surface (Caterina et al., 2002; Kim et al., 2005; Ozdemir et al., 2005). It is believed that ameloblasts respond to changes in the extracellular matrix by adjusting their activities to maintain conditions appropriate for biomineralization (Xu et al., 2006). The most abundant enamel protein is amelogenin, which is essential for proper enamel formation (Hart et al., 2002; Wright et al., 2003; Kim et al., 2004). However, the roles of specific alternatively spliced amelogenins (Gibson et al., 1991; Lau et al., 1992; Salido et al., 1992) during enamel development remain unclear. In rodents, amelogenin undergoes extensive alternative splicing, and at least 13 different isoforms have been previously characterized in rats (R) and mice (M). The major amelogenin mRNA encodes an amelogenin protein of 180 amino acids (M180) (Snead et al., 1983, 1985). Other alternatively spliced mouse amelogenin transcripts include: M156, M141, M74, M59 (Lau et al., 1992), M194, M44 (Simmer et al., 1994), M170, and M73 (Hu et al., 1997). These splice products are all derived from amelogenin mRNA transcripts ending in exon 7. Additional alternatively spliced amelogenin mRNAs ending in exons 8 and 9 were identified in the rat, including R203, R179, R82, and R57 (Li et al., 1995, 1998). Similar transcripts have recently been identified in mice (Papagerakis et al., 2005). However, the roles of exons 8 and 9 remain an enigma, because extensive characterizations of pig enamel extracts (Yamakoshi et al., 1994, 2004) have not identified amelogenin C-termini homologous to the rodent exon 8–9 encoded segment. To assess the conservation of exons 8 and 9 among mammalian species, we analyzed mouse, rat, bovine, and human amelogenin genomic sequences downstream of exon 7. We also asked if a relationship exists between amelogenin alternative splicing and proteolysis. If amelogenin cleavage products serve as signaling molecules, as has been proposed to explain the therapeutic effects of Emdogain (Hammarström et al., 1997), and/or if ameloblasts adjust their activities based on the presence of the protein component within the matrix (Xu et al., 2006), then proteolysis within the matrix might affect ameloblast protein expression. To determine if the regulation of alternative splicing by ameloblasts is altered by a failure of MMP-20 to cleave enamel matrix proteins, we compared spliced amelogenin mRNA transcripts and SDS-PAGE enamel protein profiles in mouse molars from control and Mmp20 null mice. We also raised anti-peptide antibodies to the exon-8-encoded sequence and analyzed differences in cleavage patterns of exon-8-containing amelogenins in normal and enamelysin null mouse molars.
The Animal Care Committee of The Forsyth Institute approved the protocol for the handling, care, and usage of animals.
SDS-PAGE
RT-PCR & qPCR
Western Blot Analysis Exon-8-encoded peptide (CAFSPMKWYQGMTRHP) was synthesized and conjugated to the carrier protein KLH. Antibodies were generated in rabbits by 4 immunizations. Serum was purified by protein G affinity chromatography (Pierce Biotechnology, Rockford, IL, USA). After electrophoresis, proteins were transferred to nitrocellulose membranes. The membranes were incubated with exon-8-specific serum overnight at room temperature. Amelogenins encoded by exon 8 were identified by incubation with secondary antibodies labeled with horseradish peroxidase, followed by processing for chemiluminescence (Amersham, Piscataway, NJ, USA).
Identification of Novel Mouse Amelogenin Alternatively Spliced mRNA Transcripts We identified and sequenced 3 novel mouse amelogenin RT-PCR products by use of primer 5'Exon 4w/6bc paired with 3'Exon 9. If we assume, as is the case for every other known mouse amelogenin transcript, that these new transcripts also contain exons 1–3, then the new amelogenin transcripts encode proteins containing 217 (M217), 193 (M193), and 96 (M96) amino acids. This brings to 16 the number of amelogenin alternatively spliced mRNA transcripts in rodents. A summary of these transcripts—including their exon structure, encoded amino acids, protein molecular mass, and protein isoelectric point—is provided in Table 1  . All calculations omit the signal peptide and include the Ser16 phosphate.
A Comparison of Amelogenin Alternative Splicing between Controls and Mmp20–/– Mice
Next, to characterize the relative expression levels of amelogenin transcripts that contain exon 7 vs. those transcripts that contain exons 8 and 9, we performed qPCR on total RNA from first molars extracted from three-, five-, seven-, nine-, and 11-day-old mice. These teeth were in the secretory stage (days 3 & 5), secretory-early maturation stage (day 7), and maturation stages (days 9 & 11) of development. As expected, the overall expression of amelogenin transcripts decreased as development progressed to the maturation stage. The exon-7-containing transcripts were expressed at approximately five-fold-higher levels than the exons-8- and -9-containing transcripts (Fig. 1A  ).
In silico Analysis of Exon 8 and Surrounding Genomic Sequence We performed a mouse genome BLASTN search with 3907 bp from the mouse amelogenin gene (GenBank accession NT_039718.4), immediately downstream of exon 7 and extending to the precise end of exon 9. Two Blast hits were observed. The first was identical to the query sequence, and the second showed 91% (191/209) identity, with no gaps, to another region of mouse Amelx that included exons 4 and 5 (Fig. 1B ). Sequence analysis demonstrated that exon 4 aligned with an identical sequence downstream of exon 7. We designated this second sequence exon 4b. The deduced amino acid sequences for mouse exon 4 and exon 4b were identical: KSHSQAINTDRTAL. Following 96 nucleotides of homologous intron sequence, exon 5 exactly aligned with exon 8. Over half (8/15) of the 15 amino acids encoded by exons 5 and 8 were identical (Fig. 1C). Interestingly, the 7 amino acid changes in mouse exon 8 vs. exon 5 arose from just 8 nucleotide differences (compare Figs. 1B and 1C). A similar result was obtained when the homologous region of rat Amelx was analyzed (GenBank accession NW_048039.1). These results essentially confirm what has recently been reported elsewhere (Li et al., 2006). To expand upon these results, we used BLASTN searches to identify amelogenin exon 8 in other non-rodent species. No second region homologous to the exon-4 exon-5 region was identified in the bovine or human amelogenin genes. No exon 8 sequences were identified in the numerous amelogenin cDNA sequences in the non-redundant NCBI database. These findings suggest that exon 8 was generated from exon 5 in a rodent ancestor by the duplication and downstream insertion of a 200- to 300-bp Amelx segment containing exons 4 and 5. We speculate that this duplication activated a downstream sequence that became exon 9.
Developmental Assessment of Amelogenins in Control and Mmp20–/–First Molars
Western Blot of Proteins Extracted from Mmp20+/– and Mmp20–/–Day 3 Molars To identify amelogenin protein expressed from transcripts containing exons 8 and 9, we used an exon-8-specific antiserum (Fig. 2B ) to visualize extracted mouse molar proteins. Western blot analysis of amelogenin from Mmp20+/– and Mmp20–/–molars revealed the presence of at least 3 different amelogenins between 20 and 30 kDa (Fig. 2C). The Mmp20+/– lane had an additional band of approximately 18 kDa that likely resulted from hydrolysis by MMP-20. The apparent mobilities and relative quantities of the 3 upper bands in the Mmp20–/– lane are consistent with the presence of uncleaved M217, M203, and M179. These bands might be equivalent to the 3 bands previously observed in exon 9 Western blots of rat enamel matrix (Baba et al., 2002). Overall, the amelogenin bands from the Mmp20+/– lane appeared to shift downward by 1–2 kDa, relative to the bands present in the Mmp20–/– lane. This is consistent with cleavage of these amelogenin isoforms by MMP-20 in a way that released only a short peptide, but did not destroy the epitope for the exon 8 antibody. Since enamelysin typically processes amelogenin from its C-terminus, we suspect that the exon-9-encoded sequence was excised.
Sixteen different alternatively spliced amelogenin mRNA transcripts have now been characterized in rodents. All amelogenin mRNAs contain exons 1 through 3, and at least a part of exon 6. Exon 4, when present, is always upstream of exon 6 sequences, so exon 4b, which is downstream of exon 7, is always deleted during splicing. Seven of the amelogenin transcripts contain a novel C-terminus encoded by exons 8 and 9. Exons 8 and 9 are always paired; no transcripts have only exon 8 or only exon 9. Furthermore, exon 7, which encodes only 1 amino acid prior to a stop codon, is never found in the same transcript as exons 8 and 9. Previously, Li et al.(1998) predicted that all amelogenin mRNA transcripts ending with exon 7 might alternatively end with exons 8 and 9. Our results lend support to this claim. The amelogenin alternatively spliced transcripts are expressed by secretory-stage ameloblasts, translated into their corresponding amelogenin isoforms, and secreted together into the enamel matrix. However, the various amelogenin isoforms are not secreted in equal quantities. In fact, most of the amelogenin variants are expressed in minute amounts and have never been detected in enamel protein extracts. Since all of the splice junctions in the amelogenin gene are phase 0 (occur between codons), skipped or included exons always maintain the appropriate reading frame for the downstream exons. While the primary sequences of the predominant amelogenins are highly conserved, the patterns of alternative splicing that generate the less abundant amelogenin isoforms vary widely among mammalian species (Simmer and Snead, 1995). In humans, there are also 2 non-allelic amelogenin genes on the sex chromosomes (Lau et al., 1989), a pattern that is found in many mammalian species. Proteolytic processing increases the heterogeneity further (Bartlett and Simmer, 1999; Simmer and Hu, 2002). The mouse exon-8-deduced amino acid sequence was compared with the exon-5-deduced amino acid sequences from various species (Sire et al., 2005). Although the gene duplication that generated exon 8 was relatively recent (since the divergence of rodents and human, but before the splitting of mouse and rat), the exon-8-deduced protein sequence has even more amino acid substitutions than does the marsupial (opossum) exon-5-encoded sequence, which diverged much earlier and therefore had more time to mutate. Perhaps the few exon 8 mutations that significantly changed the amino acid sequence were positively selected to reduce interference with the function of the exon-5-encoded region. Here, we provide evidence showing that amelogenin exon 8 arose from a relatively recent duplication of exon 5, and that exon 8 is present only in rodent amelogenin genes. We identified 3 previously undetected amelogenin isoforms containing the exon-8- and -9-encoded C-terminus, and we determined that no qualitative differences in amelogenin alternative splicing are evident in the Mmp20–/– null mouse relative to controls. The relative simplicity of the enamel matrix in the Mmp20–/– null mouse was demonstrated, which suggests that analysis of the amelogenin proteins in the null mouse may provide insights into the relative abundance of the various amelogenin isoforms. Finally, we demonstrated that enamelysin processes exon-8-containing amelogenins.
We thank Dr. Carolyn Gibson for suggesting that we quantify the relative abundance of amelogenin isoforms containing exon 7 vs. those containing exons 8 & 9, and Shana Dwyer for technical assistance. This work was supported by National Institute of Dental and Craniofacial Research grant DE14084 (to J.D.B.). Received for publication November 14, 2005. Revision received June 21, 2006. Accepted for publication June 22, 2006.
Journal of Dental Research, Vol. 85, No. 10,
894-899 (2006) This article has been cited by other articles:
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