Mammalian Amelogenins Research Articles

Immunohistochemical and Western Blotting Analyses of Ganoine in the Ganoid Scales of Lepisosteus oculatus: an Actinopterygian Fish.

In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin.

Immunohistochemical and Western blot analyses of collar enamel in the jaw teeth of gars, Lepisosteus oculatus, an actinopterygian fish

Although most fish have no enamel layer in their teeth, those belonging to Lepisosteus (gars), an extant actinopterygian fish genus, do and so can be used to study amelogenesis. In order to examine the collar enamel matrix in gar teeth, we subjected gar teeth to light and electron microscopic immunohistochemical examinations using an antibody against bovine amelogenin (27 kDa) and antiserum against porcine amelogenin (25 kDa), as well as region-specific antibodies and antiserum against the C-terminus and middle region, and N-terminus of porcine amelogenin, respectively. The enamel matrix exhibited intense immunoreactivity to the anti-bovine amelogenin antibody and the anti-porcine amelogenin antiserum in addition to the C-terminal and middle region-specific antibodies, but not to the N-terminal-specific antiserum. These results suggest that the collar enamel matrix of gar teeth contains amelogenin-like proteins and that these proteins possess domains that closely resemble the C-terminal and middle regions of porcine amelogenin. Western blot analyses of the tooth germs of Lepisosteus were also performed. As a result, protein bands with molecular weights of 78 kDa and 65 kDa were clearly stained by the anti-bovine amelogenin antibody as well as the antiserum against porcine amelogenin and the middle-region-specific antibody. It is likely that the amelogenin-like proteins present in Lepisosteus do not correspond to the amelogenins found in mammals, although they do possess domains that are shared with mammalian amelogenins.

Fine structural and immunohistochemical detection of collar enamel in the teeth of Polypterus senegalus, an actinopterygian fish

This is the first detailed report about the collar enamel of the teeth of Polypterus senegalus. We have examined the fine structure of the collar enamel and enamel organ of Polypterus during amelogenesis by light and transmission electron microscopy. An immunohistochemical analysis with an antibody against bovine amelogenin, an antiserum against porcine amelogenin and region-specific antibodies or antiserum against the C-terminus, middle region and N-terminus of porcine amelogenin has also been performed to examine the collar enamel matrix present in these teeth. Their ameloblasts contain fully developed Golgi apparatus, rough endoplasmic reticulum and secretory granules. During collar enamel formation, an amorphous fine enamel matrix containing no collagen fibrils is found between the dentin and ameloblast layers. In non-demineralized sections, the collar enamel (500 nm to 1 μm thick) is distinguishable from dentin, because of its higher density and differences in the arrangement of its crystals. The fine structural features of collar enamel in Polypterus are similar to those of tooth enamel in Lepisosteus (gars), coelacanths, lungfish and amphibians. The enamel matrix shows intense immunoreactivity to the antibody and antiserum against mammalian amelogenins and to the middleregion- and C-terminal-specific anti-amelogenin antibodies. These findings suggest that the proteins in the enamel of Polypterus contain domains that closely resemble those of bovine and porcine amelogenins. The enamel matrix, which exhibits positive immunoreactivity to mammalian amelogenins, extends to the cap enameloid surface, implying that amelogenin-like proteins are secreted by ameloblasts as a thin matrix layer that covers the cap enameloid after enameloid maturation.

Expression and characterization of a Rana pipiens amelogenin protein

Amelogenin, the major protein of developing enamel matrix, controls enamel crystal growth via unique supermolecular features. While much has been contributed to our understanding of mammalian amelogenin function, little is known about how amelogenin and its unique physico-chemical features have evolved among vertebrates. Here we report, for the first time, amphibian amelogenin recombinant protein expression and characterization in Rana pipiens. In order to characterize R. pipiens amelogenin, the newly discovered amelogenin coding sequence was amplified, subcloned, and expressed in Eshcerichia coli. Our newly generated R. pipiens amelogenin-specific antisera resolved a major 19-kDa band on western blots of frog tooth extracts and revealed an enamel organ tissue-specific localization pattern using immunohistochemistry. Using mass spectroscopy, a single major compound with a molecular weight of 21.6 kDa was detected, which corresponded to the amino acid sequence-based molecular weight prediction of the His fusion recombinant protein. Dynamic light scattering studies resolved 41-nm radius subunits compared with 14-nm radius subunits from mouse recombinant amelogenin controls. Transmission electron microscopy revealed defined spherical subunits in R. pipiens matrix self-assembly in contrast with a homogeneous 'stippled' matrix in mouse amelogenin matrix self-assembly. Our data suggest that R. pipiens amelogenin is distinguished from mammalian amelogenins by a number of unique physico-chemical properties which may be related to specific modes of crystal formation in frog enamel.

Molecular Evolution of Amelogenin in Mammals

An evolutionary analysis of mammalian amelogenin, the major protein of forming enamel, was conducted by comparison of 26 sequences (including 14 new ones) representative of the main mammalian lineages. Amelogenin shows highly conserved residues in the hydrophilic N- and C-terminal regions. The central hydrophobic region (most of exon 6) is more variable, but it has conserved a high amount of proline and glutamine located in triplets, PXQ, indicating that these residues play an important role. This region evolves more rapidly, and is less constrained, than the other well-conserved regions, which are subjected to strong constraints. The comparison of the substitution rates in relation to the CpG richness confirmed that the highly conserved regions are subjected to strong selective pressures. The amino acids located at important sites and the residues known to lead to amelogenesis imperfecta when substituted were present in all sequences examined. Evolutionary analysis of the variable region of exon 6 points to a particular zone, rich in either amino acid insertion or deletion. We consider this region a hot spot of mutation for the mammalian amelogenin. In this region, numerous triplet repeats (PXQ) have been inserted recently and independently in five lineages, while most of the hydrophobic exon 6 region probably had its origin in several rounds of triplet insertions, early in vertebrate evolution. The putative ancestral DNA sequence of the mammalian amelogenin was calculated using a maximum likelihood approach. The putative ancestral protein was composed of 177 residues. It already contained all important amino acid positions known to date, its hydrophobic variable region was rich in proline and glutamine, and it contained triplet repeats PXQ as in the modern sequences.

Amelogenin sequence and enamel biomineralization inRana pipiens

The amelogenin gene contributes the majority of tooth enamel proteins and plays a significant role in enamel biomineralization. While several mammalian and reptilian amelogenins have been cloned and sequenced, basal vertebrate amelogenin evolution remains to be understood. In order to start elucidating the structure and function of amelogenins in the evolution of enamel, the leopard frog (Rana pipiens) was used as a model. Tissues from Rana pipiens teeth were analyzed for enamel structure and RNA extracts were processed for sequence analysis. Electron microscopy revealed that Rana pipiens enamel contains long and parallel crystals similar to mammalian enamel, while immunoreactions confirmed the site-specific localization of cross-reactive amelogenins in Rana pipiens enamel. Sequencing of amelogenin PCR products revealed a 782bp cDNA with a 546-nucleotide coding sequence encoding 181 amino acids. The homology of the newly discovered Rana pipiens amelogenin nucleotide and amino acid sequence with the published mouse amelogenin was 38.6% and 45%, respectively. These findings report the first complete amelogenin cDNA sequence in amphibians and indicate a close homology between mammalian enamel formation and Rana pipiens enamel biomineralization.

Amelogenin: lessons from evolution

Amelogenin plays a crucial role in enamel structure and mineralization, but the function of its various domains is far to be understood. Evolutionary analysis seems to be a promising way to approach structure/function relationships. In this paper, we review the knowledge of amelogenin with a particular focus on what we have learnt from evolution, and we bring new data on the origin and evolution of this molecule. The comparison of amniote (reptiles and mammals) amelogenin sequences reveals that, in contrast to the well-conserved C- and N-terminal domains, the central region (most of exon 6) is highly variable. The evolutionary analysis indicates that it was created by repeated insertion of three amino acids (triplets ProXGlu or ProXX). In several mammalian lineages a new run of triplet insertions and deletions has occurred independently in a locus considered a hot spot of mutation for mammalian amelogenin. In lizard and snake amelogenin evolves rapidly. Sequence alignment reveals that several residues in the N- and C-terminal regions were kept unchanged during 250 million years (MY), proving their importance for amelogenin structure and function. This alignment permits a rapid validation of the amelogenin mutations in human. Genome sequencing and gene mapping permitted to refine the amelogenin story, in relation to the common location (chromosome 4 in human) of several genes coding for dental proteins and SPARCL1, a SPARC (osteonectin) relative. Amelogenin shares a similar organisation with these genes and a blast search in databanks indicates a strong relationship between amelogenin, ameloblastin and enamelin. Taken together these data suggest that amelogenin could have originated from either ameloblastin or enamelin, themselves being created from SPARCL1, which itself originated from a SPARC duplication, 600 millions years ago.

The amelogenin loci span an ancient pseudoautosomal boundary in diverse mammalian species.

The mammalian amelogenin (AMEL) genes are found on both the X and Y chromosomes (gametologous). Comparison of the genomic AMEL sequences in five primates and three other mammals reveals that the 5' portion of the gametologous AMEL loci began to differentiate in the common ancestor of extant mammals, whereas the 3' portion differentiated independently within species of different mammals. The boundary is marked by a transposon insertion in intron 2 and is shared by all species examined. In addition, 540-kb DNA sequences from the short arm of the human X chromosome are aligned with their Y gametologous sequences. The pattern and extent of sequence differences in the 5' portion of the AMEL loci extend to a proximal region that contains the ZFX locus, and those in the 3' portion extend all the way down to the pseudoautosomal boundary (PAB)1. We concluded that the AMEL locus spans an ancient PAB, and that both the ancient and present PABs were determined by chance events during the evolution of mammals and primates. Sex chromosome differentiation likely took place in a region that contains the male-determining loci by suppressing homologous recombination.

Immunodetection of amelogenin-like proteins in the ganoine of experimentally regenerating scales of Calamoichthys calabaricus, a primitive actinopterygian fish.

The account of the present study is to test our previous hypothesis that ganoine, a highly mineralized layer found at the scale surface of primitive actinopterygian fish, could be homologous with the enamel covering the crown of vertebrate teeth. Immunocytochemical techniques have been carried out on regenerating scales of a primitive polypterid, Calamoichthys calabaricus, with three antibodies to mammalian amelogenins. The present study provides the first evidence that ganoine contains molecules which cross-react with mammalian amelogenin proteins. This result is consistent with our previous findings that ganoine and enamel can be considered as homologous tissues. Moreover, the presence in ganoine of a primitive actinopterygian of amelogenin-like proteins, which share epitopes with amelogenins of mammalian enamel, indicates that the gene(s) coding for these proteins appeared earlier than previously suggested and supports the hypothesis that amelogenins show a highly conserved structure through vertebrate evolution.

Molecular Cloning and DNA Sequence of Rat Amelogenin and a Comparative Analysis of Mammalian Amelogenin Protein Sequence Divergence

The developing rat incisor is a common model used in the study of enamel development. It has been impossible to study correlation between rat enamel structure and the sequence of the major developing enamel protein in this species as to date a DNA sequence for rat amelogenin has not been reported. This study presents the first cloning of a full-length cDNA copy of rat amelogenin and its deduced primary sequence. Detailed analysis of this sequence provides evidence that the gene has evolved by internal sequence duplication. Comparison of the rat amelogenin primary sequence with those published for other species provides evidence that this protein, while exhibiting extreme levels of sequence conservation, has been subject to significant structural changes that may be related to alterations in enamel structure in different mammalian groups.

Common epitopes of mammalian amelogenins at the C-terminus and possible functional roles of the corresponding domain in enamel mineralization.

The present studies were undertaken to investigate the presence of common epitopes of mammalian amelogenins at the C-terminus and the possible functional importance of the conserved C-terminal domain in enamel mineralization during mammalian amelogenesis. Enamel proteins, including the intact amelogenins and their degraded polypeptides, were isolated from the secretory enamel of pig, cow, rat, and rabbit incisors. Rabbit and rat antipeptide sera, as well as rat anti-25 kD and 20 kD pig amelogenin sera, were used to identify the amelogenins among the isolated matrix proteins of each of the animal species. The antipeptide sera were developed previously (Aoba et al. [19]) using as immunogens the two synthetic peptides, C13 and C25, which correspond to the last 12 (plus Cys for KLH-conjugation) and 25 amino acid residues of pig intact amelogenin, respectively. Reactivity of the enamel proteins with each antiserum was examined by Western blot analysis. The results of immunoblotting showed that a few enamel matrix proteins in each of the mammalian species were recognized by the anti-C13 serum, specifically, pig amelogenin at 25 kD (and trace components at 27, 22, and 18 kD), cow amelogenin at 28 kD (trace components at 26, 22, 19, and 14 kD), rat amelogenins at 28 and 26 kD (and a trace component at 20 kD), and rabbit amelogenins at 24 and 21 kD (and a trace at 13 kD). The anti-C25 serum reacted additionally with pig amelogenin at 23 kD, cow amelogenin at 27 kD (a major matrix constituent), and rabbit protein at 19 kD.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible roles of partial sequences at N- and C-termini of amelogenin in protein-enamel mineral interaction.

The purpose of this study was to assess the functional significance of homologous sequences of mammalian amelogenins at their N- and C-termini. A porcine 5-kDa fragment corresponding to the N-terminal 45 residues of amelogenins was purified from the secretory enamel. The decapeptide TDKTKREEVD corresponding to the C-terminal 10 residues of amelogenins was synthesized according to conventional solid-phase procedures. The inhibitory activity of both moieties on apatite crystal growth was determined in a supersaturated solution having an ionic composition similar to that of the fluid phase separated from porcine secretory enamel. The 5-kDa amelogenin fragment was sparingly soluble in neutral solutions and (in condensed forms because of aggregation) showed no significant inhibition of crystal growth, whereas the fragment molecules pre-adsorbed onto the seed crystals yielded modest inhibition of hydroxyapatite precipitation. However, their inhibitory activity was significantly lower than that of parent porcine amelogenin (25-kDa molecular mass). The high solubility of synthesized decapeptide allowed us to determine the adsorption isotherm onto hydroxyapatite at 37 degrees C, at an ionic strength similar to that of the enamel fluid. The obtained adsorption isotherm was described by a Langmuir model; the adsorption affinity and the maximum adsorption sites were 6.2 mL/mumol and 0.53 mumol/m2, respectively. As expected from the low adsorption affinity, the peptide showed a much weaker inhibition of apatite crystal growth than the parent amelogenin.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis and characterization of rabbit tooth enamel extracellular-matrix proteins.

Tooth enamel biomineralization is mediated by enamel proteins synthesized by ameloblast cells. Two classes of proteins have been described: enamelins and amelogenins. In lower vertebrates the absence of amelogenins is believed to give rise to aprismatic enamel; however, rabbit teeth, which apparently do not synthesize amelogenins, form prismatic enamel. The present study was designed to characterize the enamel proteins present in rabbit tooth organs and to gain an insight into the process of biomineralization. Rabbit enamel extracellular-matrix proteins were isolated and characterized during sequential stages of rabbit tooth organogenesis. The biosynthesis of enamel proteins was analysed by metabolic 'pulse-chase' experiments as well as mRNA-translation studies in cell-free systems. Our results indicated that rabbit enamel extracellular matrix contains 'amelogenin-like' proteins. However, these proteins are not synthesized as typical amelogenins, as in other mammalian species, thus suggesting that they are the processing products of higher-molecular-mass precursors. An N-terminal amino acid sequence of 29 residues, considered characteristic of mammalian amelogenins, was present in the rabbit 'amelogenin-like' proteins. By using anti-peptide antibodies to this region, similar epitopes were detected in all nascent enamel proteins, including enamelins. These studies suggest that the N-terminal sequence might be characteristic of all enamel proteins, not only amelogenins.

ENAMEL-LIKE ANTIGENS IN HAGFISH: POSSIBLE EVOLUTIONARY SIGNIFICANCE.

The enamel proteins are a family of acidic glycoproteins which represent two groups of polypeptides: (i) amelogenins of approximately 10-30 kd (kilodaltons) which represent 80% of the total enamel protein in most mammalian species and (ii) enamelins of approximately 40-72 kd which represent -5-20% of total protein (Termine et al., 1980; Zeichner-David et al., in press). During mammalian tooth development, the inner enamel epithelial cells of the enamel organ differentiate into ameloblasts which form the extracellular matrix (see extensive reviews by Poole, 1967, 1971; Slavkin, 1974; Slavkin et al., 1981). Comparable features of morphogenesis and differentiation have been described for tooth development in Chondrichthyes (Moss, 1968, 1977) as well as in Teleostei (Shellis, 1975, 1978). It is generally agreed that during terminal epithelial differentiation of ameloblasts, enamel proteins are synthesized and secreted into the enamel matrix (Orvig, 1967; Poole, 1967; Peyer, 1968; Slavkin et al., 1981). To determine the function and structural roles of enamel proteins during vertebrate tooth development, various biochemical comparisons of the enamel polypeptides have been made. For the most part, these comparisons have emphasized the similarities amongst enamel acidic glycoproteins. Partial amino acid sequences of several different mammalian amelogenins indicate remarkable homology in primary structure when comparing 4-5 kd (kilodaltons) polypeptides with 25-30 kd amelogenins (Fukae et al., 1980; Fincham et al., in press). Comparative data on the amino acid compositions and sizes of the principal amelogenins of the developing enamel of cow, hamster, human, pig and sheep show homology (Fincham et al., 1982). In contrast to the similarities reported for amelogenins in various mammals, very little is known about enamelins in different mammalian species (Termine etal., 1980). We have begun to investigate the developmental process of enamel formation in a number of different vertebrates through the use of indirect immunofluorescent microscopy and biochemistry. Several recent studies have reported that rabbit antibodies raised against several different sources of mammalian amelogenins are antigenically cross-reactive with the enamel matrices of lower and higher vertebrates including Chondrichthyes, Teleostei, Amphibia, Reptilia and Mammalia (Graver et al., 1978; Graham et al., 1980; Herold et al., 1980; Slavkin et al., 1982). We now show that rabbit antimouse amelogenin antisera are antigenically cross-reactive with Pacific hagfish (Eptatretus stoutii). The cyclostomes comprise lampreys and hagfish (or myxinoids), which are eel-like aquatic vertebrates, considered as the remaining survivors of a nearly extinct group of agnathan or jawless Paleozoic fish known as ostracoderms (Stensio, 1968; Hardisty and Potter, 1971; Hardisty, 1978). Further, cyclostomes possess a cartilaginous skeleton which is not mineralized (Hardisty, 1978). Therefore, the presence of enamel-like antigens in the outer covering of hagfish teeth, and the presence of a relatively high molecular weight class of enamel-like Droteins, suggests remark-