Tell Us The Case of AMEL

AMEL Evolution

AMEL is the main component of forming enamel and it plays crucial roles in enamel structure and mineralization [Diekwisch et al., 1993; reviews in Bartlett et al.,

2006b; Margolis et al., 2006]. Mutations of the encoding gene lead to AIH1 [Hart et al., 2002; Kim et al., 2004]. Given this importance it is not surprising that AMEL is the best-known EMP. Over the past years, AMEL studies on model animals have provided information on the gene structure and supposed functions of the various regions of the protein [Fincham et al., 1991; Fincham and Mora-dian-Oldak, 1995; Greene et al., 2002]. AMEL is subject to posttranslational modifications [Fincham and Mora-dian-Oldak, 1993] and it self-assembles to form nano-spheres that are involved in enamel mineralization [Wen et al., 2001; Snead, 2003; Du et al., 2005; Veis, 2005]. The N- and C-terminal regions interact with mineral [Aoba et al., 1989; Aoba, 1996; Hoang et al., 2002; Paine et al., 2003; Snead, 2003] and are involved in adhesion with the ameloblast surface through membrane proteins (e.g. Cd63, annexin A2, and Lamp1 [Wang et al., 2005b; Tompkins et al., 2006]). AMEL interacts also with some keratins in ameloblasts through ligand-binding properties located in the N-terminal region [Ravindranath et al., 1999, 2000, 2001, 2003]. Some splice products have been proposed to be signaling molecules [Veis et al., 2000; Veis, 2003].

From these studies, increasing evidence accumulates to support the idea that the N-terminal, and to a lesser degree the C-terminal, regions are the most important regions for proper AMEL function. This importance is also revealed by several AIH1, caused by mutations modifying the functioning of these regions. The question of a possible role for the central variable region (encoded by most of exon 6) is completely ignored. Is it useless? Certainly not. Evolutionary analyses indicate that this core region of the protein, although intrinsically disordered, could be responsible for the well-ordered microstructure of enamel [Delgado et al., 2005; Sire et al., 2005; 2006]. More data are still needed to understand the relationships between structure and function of this region and, more generally, to reveal the amino acid positions and regions that could play an essential role.

As an alternative to biochemical and in vitro approaches, an evolutionary analysis of mammalian AMEL was performed using 56 sequences constituting a dataset representative of mammalian diversity [Delgado et al., 2005]. Here, we summarize and complete these results in proposing two alignments (fig. 11): one, illustrated with 20 sequences of the N- and C-terminal regions only, reveals the numerous well-conserved residues that are important for the proper function of the protein (interactions with the cell membrane and/or with mineral crystals). The other alignment, comprising 51 sequences, is centered in the variable central region of exon 6, which houses, in mammals, a hot spot of mutation. The putative ancestral sequence has been calculated for both alignments. Briefly, this evolutionary analysis reveals the following points.

(i) A total of 56 residues (out of 74 in the full-length sequence) have remained unchanged in the N- and C-ter-minal regions of AMEL during mammalian evolution, i.e. during 225 million years [van Rheede et al., 2006] (fig. 11a). This indicates that strong functional constraints act on these amino acids, meaning that they certainly play, either alone or with other conserved residues, an important role. Most variants are found in the C-ter-minal region of exon 5.

(ii) The hot spot of mutation (large insertions/deletions of residues) has appeared recently in mammals, and independently in several lineages (fig. 11b). Insertions are found in basal primates (lemurs), in tree shrews, in basal rodents (squirrel and guinea pig), in bovids (cow and goat) and cervids (deer), in only one family of carnivores (ur-sids), in bats (Macrochiroptera), in insectivores (hedgehog), in afrotherians (elephant shrew), and in marsupials (opossums). The perissodactyls (e.g. horse) and proto-therians (platypus and echidna) are the only important lineages in which such large insertions are absent. These insertions contain a variable number of three amino acid (triplet) repeats (e.g. PIQ-PMQ-PLQ). These triplet repeats range from two (in the tree shrew) to 12 (in a fruit bat), in which a total of 36 residues (108 bp) are inserted. Within some lineages, e.g. bovids, the number of repeats can vary in closely related species (8 repeats in the African buffalo, 7 in cattle, and 5 in the other members of the family). It is noteworthy that AMELY, that is expressed at a low level in forming enamel (less than 10% [Salido et al., 1992]), does not show insertions in this region. This illustrates the separate evolution of the two AMEL copies on sex chromosomes [Girondot and Sire, 1998], AMELY being subjected to the particular mode of evolution of the Y chromosome [Iwase et al., 2001; Lahn et al., 2001; Iwase et al., 2003]. The lack of triplet insertions in AMELY versus AMELX exon 6 allows to easily discriminate males from females in lineages possessing the hot spot of mutation, e.g. bovids [Weikard et al., 2006] and ursids [Yamamoto et al., 2002]. Large deletions (>9 residues) are found in dolphin, Weddell seal, panda and roundleaf bat (Microchiroptera). However, we do not know whether these indels have a consequence on enamel microstructure in these species [Delgado et al., 2005]. It is clear, however, that the conservation of such large indels during evolution has no negative results on enamel function as protective tissue.

AMEL_Ancestral Human

Squirrel_monkey

Lemur

Galago

Mouse

Guinea_pig

Squirrel

Goat

Horse

Flying_fox

Hedgehog

Elephant

Tenrec

Hyrax

Opossum

Wallaby

Platypus exon2 | exon3

MGTWILLACL LGAAFAMPLP PHPGHPGYIN

FSYEVLE P:

exon5 LK WYQNMIRQQ'

exon6

PSYGYEPMGG WLHHQIIPVL

DLPLEAWPAT DKTKREE S.

exon6

PSYGYEPMGG WLHHQIIPVL

AMEL_Ancestral

Human

Orangutan

Squirrel_monkey

Lemur

Galago

Marmoset

Tree_shrew

Flying_lemur

Mouse

Hamster

Guinea_pig

Squirrel

Goat

Sheep

African_buffalo

Japanese_serow

Deer

Hippopotamus

Dolphin

Porpoise

Horse

Tapir

Rhinoceros

Wolverine

River_otter

Arctic_fox

Gray_seal

Weddell_seal

Canada_lynx

Tiger

Brown_bear

Panda

Flying_fox

Fruit_bat

Roundleaf_bat

Hedgehog

Shrew

Armadillo

Elephant

Manatee

Tenrec

Golden_mole

Elephant_shrew

Hyrax

Opossum

Aquatic_opossum

Platypus

Echidna

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M.PMQPMQPM QPIQPIQPIQ

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M.PMQPMQPM QPMQPVQ

PLQPMQPL QPLQPLQ

PLQPLQPL QPLQPLQ

PHQPLQPM QPMQPLQPLQ PLQ

PHQPLQPM QPMQPLQPLQ PLQPLQ.

PLQPMQPL QPLQPLQ

PLQPLQPL QPLQPLQ

-PQ APVHPMQPLP PQ-PPLPPMF

M.PMAPMQPM Q.

•PIQPIQPI QPIQPIQPIQ PIQPIQPMQP MQPMQPIQ.. • PQQPVHPI QPQSPVHSMQ L

M.PMQPMHPM H

..PIQPIQPI QPIQPMQPMQ PMQPMQPMQ. ... ..PIQPIQPI QPMQPMQPMQ PLQPMQPMQP MQ.

.L..I.. -L-SLH-.I.. .I.. .I.. .I.. .I.. .I.. .. S.M..I.. .K P.M..I..

QSM

Fig. 11. Alignment of AMEL amino acid sequences in representative mammals. a Well-conserved N- and C-terminal regions in 20 species. Exon 4 is not represented because it is lacking in several species. Partial sequences were removed from this alignment. Vertical bars indicate the limits between exons. Unchanged residues are shown on a gray background. b Central region of exon 6

in 51 species emphasizing the region considered a hot spot of mutation. This region is characterized by amino acid triplet insertions or deletions. Identical sequences were not included in these alignments: e.g. human = chimpanzee and rhesus monkey;

mouse = rat; cow = European bison = Identical residue;

Fig. 12. Amino acid sequence of human amelogenin highlighting the residues which remained unchanged during the 225 million years of mammalian diversification. The importance of amino acids is inferred from the alignment of 60 mammalian sequences representative of the main lineages, as partially shown in figure 11. Exon 4 (14 residues) was not included because it is missing in several species studied. Signal peptide is on gray background. The protein sequence (191 amino acids) is numbered from methionine (1). Bold characters (n = 75) indicate residues unchanged in mammals, italics (n = 35) residues that can be substituted by an amino a{tb}cid from the same group only, small roman characters residues that can be substituted, characters on gray background (n = 5) residues that are known so far to lead to amelogenesis imperfecta when substituted, and underlined characters indicate (n = 31) residues that are unchanged in amniotes (mammals and reptiles) [Delgado et al., in press].

Ex 2 Ex 3 Ex 5 Ex 6 Ex 6

ïx 6 Ex 6 Ex 6 Ex 6 Ex 6 Ex 6 Ex 6

a

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(iii) Although this central region of AMEL exon 6 is variable, it maintains its richness in proline (30%) and glutamine (20%) in all sequences studied. This means that this region is also subject to a functional constraint but that this selective pressure probably acts on the general conservation of the P and Q richness rather than on specific amino acid positions. This strongly suggests that this region could be subject to polymorphism in humans.

(iv) The origin of the largest of AMEL exon 6 has to be found in the repeats of nine nucleotides coding for three residues (triplets) PXQ or PXX [Delgado et al., 2005]. These repeats have not been blurred by substitutions during at least 310 million years of amniote evolution, because such triplet repeats have been identified in crocodile AMEL [Sire et al., 2006]. The triplet insertions found in the hot spot mutation in mammals are probably reminiscent of this mechanism. These repeats are to be found, probably, in the origin of AMEL after AMBN duplication, and also constitute the originality of AMEL compared to the other EMPs and to ameloblast-secreted SCPPs in general. This leads to the hypothesis that AMEL divergence consisted of the loss of most of the C-terminal region of the AMBN ancestor and of the development of exon 6 (probably from AMBN exon 5) through several runs of PXQ triplet repeats. This new protein was posi tively selected during enamel evolution in vertebrates because this hydrophobic region, rich in P and Q, improved the resistance of enamel to wear and microbreaks. This could explain why today AMEL represents 90% of the forming enamel matrix in mammals.

Validation of Mutations and Important Residues

The evolutionary analysis of AMEL in mammals reveals >70 residues (out of 191) that are certainly important for a correct function of AMEL because they have remained unchanged during 225 million years of evolution (fig. 12). The number of conserved residues is reduced to 34 when reptilian AMELs are added to this analysis [Delgado et al., in press]. These 34 positions conserved during 310 million years of amniote evolution are considered crucial residues for enamel formation. All of them are located in the N- and C-terminal regions of AMEL, known to play an important role in relation with the environment (interactions with the ameloblast surface and/or with the mineral crystals). The residues conserved only in mammals could indicate that they play new, important roles for enamel formation in this lineage.

As a consequence of their long-lasting conservation, substitution of the important amino acids revealed in this study could result in enamel defects (AIH1) when substi tuted in humans (fig. 12). The five substitutions leading to AIH1 are validated when using the mammalian, and four of them when using the amniote dataset. Therefore, this list of conserved residues in the human AMEL sequence (fig. 12) can be useful for the clinical diagnosis of AIH1 since it helps to validate any human AMEL mutation, which could be suspected for AIH1.

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