Evolutionary analysis of selective constraints identifies ameloblastin (AMBN) as a potential candidate for amelogenesis imperfecta

Abstract

BackgroundAmeloblastin (AMBN) is a phosphorylated, proline/glutamine-rich protein secreted during enamel formation. Previous studies have revealed that this enamel matrix protein was present early in vertebrate evolution and certainly plays important roles during enamel formation although its precise functions remain unclear. We performed evolutionary analyses of AMBN in order to (i) identify residues and motifs important for the protein function, (ii) predict mutations responsible for genetic diseases, and (iii) understand its molecular evolution in mammals.ResultsIn silico searches retrieved 56 complete sequences in public databases that were aligned and analyzed computationally. We showed that AMBN is globally evolving under moderate purifying selection in mammals and contains a strong phylogenetic signal. In addition, our analyses revealed codons evolving under significant positive selection. Evidence for positive selection acting on AMBN was observed in catarrhine primates and the aye-aye. We also found that (i) an additional translation initiation site was recruited in the ancestral placental AMBN, (ii) a short exon was duplicated several times in various species including catarrhine primates, and (iii) several polyadenylation sites are present.ConclusionsAMBN possesses many positions, which have been subjected to strong selective pressure for 200 million years. These positions correspond to several cleavage sites and hydroxylated, O-glycosylated, and phosphorylated residues. We predict that these conserved positions would be potentially responsible for enamel disorder if substituted. Some motifs that were previously identified as potentially important functionally were confirmed, and we found two, highly conserved, new motifs, the function of which should be tested in the near future. This study illustrates the power of evolutionary analyses for characterizing the functional constraints acting on proteins with yet uncharacterized structure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0431-0) contains supplementary material, which is available to authorized users.

Fast, Accurate and Automatic Ancient Nucleosome and Methylation Maps with epiPALEOMIX

“…These genes were not part of the top 95th quantile in the hair Palaeo-Eskimo individual, which instead returned Keratin 17, 32, 33a , Periplaktin , and Plectin , all representing proteins related to hair and hair follicle, in line with findings by Pedersen et al (2014). Unfortunately, we could not investigate genes highly expressed in tooth enamel ( AMELX , TUFT1 , and AMBN) (Delsuc et al 2015) and tooth dentine ( Oc and Dspp ) (Hoffmann et al 2001), as they did not pass our filtering thresholds. Altogether, these analyses plead in favor of the tissue-specificity of R s scores.…”

AMBN mutations causing hypoplastic amelogenesis imperfecta and Ambn knockout‐NLS‐lacZ knockin mice exhibiting failed amelogenesis and Ambn tissue‐specificity

“…Transcript 1 includes (while transcript 2 omits) a 45 nucleotide segment encoding 15 amino acids at the 5' end of Exon 6 (Cerny et al, ; Hu et al, ). Alignment of 53 functional AMBN sequences, covering over 200 million years (MY) of evolution, identified 80 unchanged and 81 highly conserved amino acid residues (out of 447 residues in the human AMBN protein, inclusive of the signal peptide; Delsuc, Gasse, & Sire, ). All 15 amino acids encoded by the 5'‐end of Exon 6 that can be deleted during RNA splicing are highly or absolutely conserved, and this peptide also includes an O‐glycosylation (human secreted protein Ser 112 ), suggesting that there may be an important functional difference between the two ameloblastin isoforms generated by alternative splicing (Kobayashi et al, ).…”

“…All 15 amino acids encoded by the 5'‐end of Exon 6 that can be deleted during RNA splicing are highly or absolutely conserved, and this peptide also includes an O‐glycosylation (human secreted protein Ser 112 ), suggesting that there may be an important functional difference between the two ameloblastin isoforms generated by alternative splicing (Kobayashi et al, ). Evolutionary analysis suggested that ancestral mammalian AMBN likely possessed 11 exons (corresponding to exons 1 to 7 and 10 to 13 in humans, as Exons 8 and 9 were added in the human line by tandem duplications of Exon 7; Delsuc et al, ). All AMBN introns are phase 0 (located between complete codons), so duplication and deletion of exons during evolution, and alternative mRNA splicing, does not shift the downstream reading frame.…”

Phosphorylation Modulates Ameloblastin Self-assembly and Ca2+ Binding

“…Despite the numerous predicted phosphor acceptor sites in AMBN (Toyosawa et al, 2000; Lee et al, 2003), few residues of AMBN have been found to be phosphorylated (reviewed in Delsuc et al, 2015). It is possible that phosphorylation of AMBN is more efficient in vivo , but is effectively removed by alkaline phosphatases before it can be detected.…”