Developing Enamel Matrix Research Articles

Role of 20-kDa Amelogenin (P148) Phosphorylation in Calcium Phosphate Formation in Vitro

The potential role of amelogenin phosphorylation in enamel formation is elucidated through in vitro mineralization studies. Studies focused on the native 20-kDa porcine amelogenin proteolytic cleavage product P148 that is prominent in developing enamel. Experimental conditions supported spontaneous calcium phosphate precipitation with the initial formation of amorphous calcium phosphate (ACP). In the absence of protein, ACP was found to undergo relatively rapid transformation to randomly oriented plate-like apatitic crystals. In the presence of non-phosphorylated recombinant full-length amelogenin, rP172, a longer induction period was observed during which relatively small ACP nanoparticles were transiently stabilized. In the presence of rP172, these nanoparticles were found to align to form linear needle-like particles that subsequently transformed and organized into parallel arrays of apatitic needle-like crystals. In sharp contrast to these findings, P148, with a single phosphate group on serine 16, was found to inhibit calcium phosphate precipitation and stabilize ACP formation for more than 1 day. Additional studies using non-phosphorylated recombinant (rP147) and partially dephosphorylated forms of P148 (dephoso-P148) showed that the single phosphate group in P148 was responsible for the profound effect on mineral formation in vitro. The present study has provided, for the first time, evidence suggesting that the native proteolytic cleavage product P148 may have an important functional role in regulating mineralization during enamel formation by preventing unwanted mineral formation within the enamel matrix during the secretory stage of amelogenesis. Results obtained have also provided new insights into the functional role of the highly conserved hydrophilic C terminus found in full-length amelogenin.

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.

Evidence for direct amelogenin–target cell interactions using dynamic force spectroscopy

Increasing evidence suggests that amelogenin, long held to be a structural protein of developing enamel matrix, may also have cell signaling functions. However, a mechanism for amelogenin cell signaling has yet to be described. The aim of the present study was to use dynamic chemical force spectroscopy to measure amelogenin interactions with possible target cells. Full-length amelogenin (rM179) was covalently attached to silicon nitride AFM tips. Synthetic RGD peptides and unmodified AFM tips were used as controls. Amelogenin-RGD cell binding force measurements were carried out using human periodontal ligament fibroblasts (HPDF) from primary explants and a commercially available osteoblast-like human sarcoma cell line as the targets. Results indicated a linear logarithmic dependence between loading rate and unbinding force for amelogenin-RGD target cells across the range of loading rates used. For RGD controls, binding events measured at 5.5 nN s-1 force loading rate resulted in a mean force of 60 pN. Values for amelogenin-fibroblast and amelogenin-osteoblast-like cell unbinding forces, measured at similar loading rates, were 50 and 55 pN, respectively. These data suggest that amelogenin interacts with potential target cells with forces characteristic of specific ligand-receptor binding, suggesting a direct effect for amelogenin at target cell membranes.

Tooth developmental biology: disruptions to enamel‐matrix assembly and its impact on biomineralization

Dental enamel is a composite bioceramic material that is the hardest tissue in the vertebrate body, containing long, thin crystallites of substituted hydroxyapatite (HAP). Over a lifetime of an organism, enamel functions under repeated and immense loads, generally without catastrophic failure. Enamel is a product of ectoderm-derived cells called ameloblasts. Recent investigations on the formation of enamel using cell and molecular approaches are now being coupled to biomechanical investigations at the nanoscale and mesoscale levels. For amelogenin, the principal structural protein for forming enamel, we have identified two domains that are required for its proper self-assembly into supramolecular structures referred to as nanospheres. Nanospheres are believed to control HAP crystal habit. Other structural proteins of the enamel matrix include ameloblastin and enamelin, but little is known about their biological importance. Transgenic animals have been prepared to investigate the effect of overexpression of wild-type or mutated enamel proteins on the developing enamel matrix. Amelogenin transgenes were engineered to contain deletions to either of the two self-assembly domains and these alterations produced significant defects in the enamel. Additional transgenic animal lines have been prepared and studied and each gives additional insights into the mechanisms for enamel biofabrication. This study summarizes the observed enamel phenotypes of recently derived transgenic animals. These data are being used to help define the role of each of the enamel structural proteins in enamel and study how each of these proteins impact on enamel biomineralization.

Calbindin Independence of Calcium Transport in Developing Teeth Contradicts the Calcium Ferry Dogma

Cytosolic calcium-binding proteins termed calbindins are widely regarded as a key component of the machinery used to transport calcium safely across cells. Acting as mobile buffers, calbindins are thought to ferry calcium in bulk and simultaneously protect against its potentially cytotoxic effects. Here, we contradict this dogma by showing that teeth and bones were produced normally in null mutant mice lacking calbindin(28kDa). Structural analysis of dental enamel, the development of which depends critically on active calcium transport, showed that mineralization was unaffected in calbindin(28kDa)-null mutants. An unchanged rate of calcium transport was verified by measurements of (45)Ca incorporation into developing teeth in vivo. In enamel-forming cells, the absence of calbindin(28kDa) was not compensated by other cytosolic calcium-binding proteins as detectable by (45)Ca overlay, two-dimensional gel, and equilibrium binding analyses. Despite a 33% decrease in cytosolic buffer capacity, cytotoxicity was not evident in either the null mutant enamel or its formative cells. This is the first definitive evidence that calbindins are not required for active calcium transport, either as ferries or as facilitative buffers. Moreover, in challenging the broader notion of a cytosolic route for calcium, the findings support an alternative paradigm involving passage via calcium-tolerant organelles.

Expression of collagen XVIII and MMP-20 in developing teeth and odontogenic tumors.

Collagen XVIII is a basement membrane (BM) component, whereas MMP-20 (enamelysin) is a matrix metalloproteinase predominantly expressed in teeth. Since MMP-20 was found to degrade collagen XVIII, we studied the co-expression of these proteins in dental tissues. Collagen XVIII surrounded the developing tooth during early and late bell stages and was also present in developing enamel. Western blotting indicated that developing enamel contains collagen XVIII N-terminal fragments of the frizzled variant. Enamelysin was co-localized with collagen XVIII in the developing enamel matrix and stratum intermedium. Electron microscope analysis showed that total mineral, calcium and phosphorus contents of enamel were slightly increased in collagen XVIII null mice but the analysis revealed no visible defects in the enamel or dentin structures. In odontogenic tumors MMP-20 and collagen XVIII were co-localized in the enamel-like tumor matrix. Our results show that collagen XVIII is present in developing teeth, but its absence seems not to be critical for the development of the teeth.

Can premorbid episodes of diminished vagal tone be detected via histological markers in patients with PTSD?

While laboratory methods for estimating genetic susceptibility for adult psychopathology have received much recent attention, laboratory methods for objectively estimating a person's early autonomic nervous system perturbations have been under-researched. Research on heart rate variability suggests that early life episodes of diminished vagal tone may predict poor stress resilience in adults. This article will detail a research method for retrospectively estimating in adults the chronology of diminished vagal tone episodes experienced prior to age 10. This method makes use of the developing enamel matrix, one of very few tissues that cannot recover after being stressed.

Characterization of a Procine Amelogenin Preparation, EMADOGAIN, a Biological Treatment for Periodontal Disease

EMDOGAIN is derived from porcine developing enamel matrix and has been shown to facilitate regeneration of the periodontium, although its mechanism of action is unknown. The aim of the present study was to identify enamel matrix proteins and proteolytic enzymes present in EMDOGAIN and compare them with those extracted from developing porcine enamel itself. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and zymography were used to identify the proteins present and to determine their enzyme activity. The results showed that developing enamel contained amelogenins, albumin, amelin, and enamelin. EMDOGAIN, however, contained only amelogenins. Both metalloendoproteases and serine protease activity were revealed in both EMDOGAIN and developing enamel. The roles of the amelogenin and enzyme components, if any, in periodontal regeneration are unknown.

Elongated growth of octacalcium phosphate crystals in recombinant amelogenin gels under controlled ionic flow.

Amelogenin proteins constitute the primary structural entity of the extracellular protein framework of the developing enamel matrix. Recent data on the interactions of amelogenin with calcium phosphate crystals support the hypothesis that amelogenins control the oriented and elongated growth of enamel carbonate apatite crystals. To exploit further the molecular mechanisms involved in amelogenin-calcium phosphate mineral interactions, we conducted in vitro experiments to examine the effect of amelogenin on synthetic octacalcium phosphate (OCP) crystals. A 10% (wt/vol) recombinant murine amelogenin (rM179, rM166) gel was constructed with nanospheres of about 10- to 20-nm diameter, as observed by atomic force microscopy. The growth of OCP was modulated uniquely in 10% rM179 and rM166 amelogenin gels, regardless of the presence of the hydrophilic C-terminal residues. Fibrous crystals grew with large length-to-width ratio and small width-to-thickness ratio. Both rM179 and rM166 enhanced the growth of elongated OCP crystals, suggesting a relationship to the initial elongated growth of enamel crystals.

Spatially related amelogenin interactions in developing rat enamel as revealed by molecular cross-linking studies

A cleavable cross-linker (dithiobis[succinimidyl propionate], DTSP) was used to investigate the subunit structure of the developing enamel matrix. Intact matrix was cross-linked under conditions chosen to simulate those found in vivo. The cross-linked complexes were isolated by preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and their subunit composition determined by analytical SDS-PAGE following reductive cleavage of the cross-links. Western blotting using antiamelogenin antibodies was used to confirm the identity of the proteins involved. The results showed that nascent amelogenins tended to be cross-linked to other nascent amelogenins while amelogenin-processing products tended to be cross-linked to other processed molecules at the same stage of processing. The results suggest that nascent amelogenins are in close association after secretion and during extracellular processing, and that processed products are not free to associate with nascent molecules, presumably due to diffusion constraints in the tissue. This conclusion implies that individual amelogenin molecules within supramolecular aggregates (nanospheres) are processed in situ and remain in the same nanosphere while all the individual component amelogenins undergo processing. The biological function of amelogenin processing remains unclear but the fact that amelogenin–amelogenin associations are maintained during processing indicates that matrix stability is an important factor while the enamel layer is being deposited.

Microstructures of an Amelogenin Gel Matrix

The thermo-reversible transition (clear↔opaque) of the amelogenin gel matrix, which has been known for some three decades, has now been clarified by microstructural investigations. A mixed amelogenin preparation extracted from porcine developing enamel matrix (containing “25K,” 7.4%; “23K,” 10.7%; “20K,” 49.5%; and smaller peptides, 32.4%) was dissolved in dilute formic acid and reprecipitated by adjusting the pH to 6.8 with NaOH solution. Amelogenin gels were formedin vitroby sedimenting the precipitate in microcentrifuge tubes. The gels were fixed with Karnovsky fixative at 4 and 24°C, which was found to preserve their corresponding clear (4°C) and opaque (24°C) states. Scanning electron microscopy, atomic force microscopy, and transmission electron microscopy were employed for the microstructural characterization of the fixed clear and opaque gels. The amelogenin gel matrix was observed to possess a hierarchical structure of quasi-spherical amelogenin nanospheres and their assemblies. The nanospheres of diameters 8–20 nm assemble to form small spherical assemblies of diameters 40–70 nm that further aggregated to form large spherical assemblies of 70–300 nm in diameter. In the clear gel, most of the large assemblies are smaller than 150 nm, and the nanospheres and assemblies are uniformly dispersed, allowing an even fluid distribution among them. In the opaque gel, however, numerous spherical fluid-filled spaces ranging from 0.3 to 7 μm in diameter were observed with the majority of the large assemblies sized 150–200 nm in diameter. These spaces presumably result from enhanced hydrophobic interactions among nanospheres and/or assemblies as the temperature increased. The high opacity of the opaque (24°C) gel apparently arises from the presence of the numerous fluid-filled spaces observed compared to the low-temperature (4°C) preparation. These observations suggest that the hydrophobic interactions among nanospheres and different orders of amelogenin assemblies are important in determining the structural integrity of the dental enamel matrix.

Characterization of recombinant pig enamelysin activity and cleavage of recombinant pig and mouse amelogenins.

Enamelysin (MMP-20) is a tooth-specific matrix metalloproteinase that is initially expressed by ameloblasts and odontoblasts immediately prior to the onset of dentin mineralization, and continues to be expressed throughout the secretory stage of amelogenesis. During the secretory stage, enamel proteins are secreted and rapidly cleaved into a large number of relatively stable cleavage products. Multiple proteinases are present in the developing enamel matrix, and the precise role of enamelysin in the processing of enamel proteins is unknown. We have expressed, activated, and purified the catalytic domain of recombinant pig enamelysin, and expressed a recombinant form of the major secreted pig amelogenin rP172. These proteins were incubated together, and the digestion products were analyzed by SDS-PAGE and mass spectrometric analyses. We assigned amelogenin cleavage products by selecting among the possible polypeptides having a mass within 2 Daltons of the measured values. The polypeptides identified included the intact protein (amino acids 2-173), as well as 2-148, 2-136, 2-107, 2-105, 2-63, 2-45, 46-148, 46-147, 46-107, 46-105, 64-148, 64-147, and 64-136. These fragments of rP172 include virtually all of the major amelogenin cleavage products observed in vivo. We propose that enamelysin is the predominant proteinase that processes enamel proteins during the secretory phase of amelogenesis.

Proteolytic activity of opossum tooth extracts.

Amelogenins are the main component of the developing enamel matrix. In placental mammals, amelogenins are rapidly cleaved following their secretion. HPLC fractionation of tooth extracts produces a complex chromatographic profile. The fractions are rich in amelogenin cleavage products that generally retain the amino-terminus of the parent protein but have varying lengths of peptide removed from the original carboxyl-terminus. In contrast, HPLC fractionation of opossum tooth extracts produces a simple profile with a single major chromatographic peak. SDS-and Western blot analyses demonstrated that most of the amelogenin consisted of a prominent protein band that migrated at 28 kDa. Mass spectroscopy confirmed the presence of two uncleaved, alternatively spliced forms of opossum amelogenin, Op202 and Op57, but did not detect major amelogenin cleavage products evident in tooth extracts from placental mammals. Amino acid composition analysis supported the conclusion that uncleaved amelogenin is the major component in the developing enamel matrix. Enzymogram analyses using gelatin, casein and recombinant amelogenin as substrates, comparing porcine, rat and opossum tooth extracts, suggested that fewer proteinases are present in opossum. These results identify potentially significant differences in the proteolytic processing of amelogenins between metatherian and eutherian mammals.

Purification and sequencing of a 21 kDa and 25 kDa bovine enamel metalloproteinase.

The developing enamel matrix contains metalloproteinases that are presumed to have a role in hydrolysis of enamel matrix proteins. Determination of the identity and function of these proteinases requires further information such as their amino acid composition and sequence. In this study, we purified the 21 kDa and 25 kDa matrix metalloproteinase from secretory stage bovine enamel matrix. After extraction, these proteinases were further purified by sequential separation by ion exchange, Con A affinity chromatography, and reversed phase HPLC. The proteinases were separated by SDS PAGE, transferred to a PVDF membrane and the N-terminus was sequenced. The N-terminal sequences of both proteinases were the same, and showed homology to the porcine enamelysin (MMP 20) cDNA sequence. A cDNA for bovine MMP 20 was isolated from a bovine enamel organ cDNA library using a probe generated by PCR amplification. The coding regions of bovine and porcine MMP 20 cDNAs were highly homologous and contained the same regions of predicted amino acid sequence homology with the proteinase N-terminal sequences. These results suggest that the 21 kDa and 25 kDa enamel matrix metalloproteinases are cleavage products of the initially secreted MMP 20, and that the sequence for MMP 20 is conserved across species.

Cloning and characterization of porcine enamelin mRNAs.

Dental enamel forms by matrix-mediated biomineralization. The components of the developing enamel matrix are generally specific for that matrix. The primary structures of three enamel proteins-amelogenin, tuftelin, and sheathlin (ameloblastin/amelin)-have been derived from cDNA sequences. Here we report the cloning and characterization of mRNA encoding a fourth enamel protein: enamelin. The longest porcine enamelin cDNA clone has 3907 nucleotides, exclusive of the poly(A) tail. The primary structure of the secreted protein is 1104 amino acids in length. Without post-translational modifications, the secreted protein has an isotope-averaged molecular mass of 124.3 kDa and an isoelectric point of 6.5. Polymerase chain-reaction phenotyping of enamelin cDNA suggests that porcine enamelin transcripts are not alternatively spliced and use a single polyadenylation/cleavage site. Immunohistochemical and Western blot analyses with an affinity-purified antipeptide antibody specific for the enamelin carboxyl terminus demonstrate that enamelin is synthesized and secreted by secretory-phase ameloblasts. The parent protein is a 186-kDa glycoprotein that concentrates along the secretory face of the ameloblast Tomes' process. Intact enamelin and proteolytic cleavage products containing its carboxyl terminus are limited to the most superficial layer of the developing enamel matrix, while other enamelin cleavage products are observed in deeper enamel.

Cloning, characterization, and heterologous expression of exon-4-containing amelogenin mRNAs.

The formation of dental enamel is dependent upon amelogenins, a family of proteins constituting most of the developing enamel matrix. Depending upon the species, these enamel proteins are expressed from either one or two copies of the amelogenin gene. Each gene directs the synthesis of a variety of amelogenin isoforms through alternative splicing of their pre-mRNA transcript(s). Before the role of amelogenins in dental enamel formation can be better understood, one must know the isoforms that are secreted and their biochemical properties. Previously, we cloned and characterized 7 mouse amelogenin RNA messages generated by alternative splicing. The largest amelogenin cDNA encoded a 194-residue amelogenin isoform which was the only clone to contain the 42-nucleotide exon 4 segment. Anti-peptide antibodies raised against the derived translation of this exon revealed an unexpectedly diverse assortment of murine amelogenins, suggesting that additional splicing variants could contain the exon 4 coding region. Using exon-4-specific oligonucleotide primers, we have amplified, cloned, and characterized three different amelogenin RNA messages. These messages encode amelogenin polypeptides (exclusive of signal peptides) 194, 170, and 73 amino acids in length. The isotope-averaged molecular weights for the deduced, single-phosphorylated, proteins are 21,897.1, 19,113.9, and 8176.5 Daltons, respectively. Splice-site selection for the generation of these mRNAs was identical to that of the previously characterized messages for the M180, M156, and M59 except for the inclusion of exon 4. The exon-4-containing amelogenin isoforms were heterologously expressed in E. coli by means of the pET11 expression system (Novagen, Madison, WI).

Alternative splicing of amelogenin mRNA from rat incisor ameloblasts.

Amelogenin proteins are a major component of the developing enamel matrix, and are likely to have a key role in the control of enamel biomineralization. The heterogeneity of amelogenin is in part due to alternative splicing of the amelogenin RNA transcripts. Several patterns of alternative splicing have been described in mouse, bovine, porcine, and human enamel, with all alternatively spliced products having homologous 5' and 3' sequences within the coding regions. In these studies, we have used anchored PCR to identify alternatively spliced amelogenin cDNA sequences in the rat. We found amelogenin cDNAs that could be divided into two groups based on their 3' sequence. Group 1 cDNAs had a novel terminal sequence that has not been previously identified, while group 2 cDNAs were similar to those previously identified in other animal species. We identified a sequence, identical to the novel 3' amelogenin cDNA sequence, in rat genomic DNA downstream from the previously identified exon 7. The putative amelogenin proteins predicted for the two groups differed in their predicted isoelectric points, with the novel group 1 proteins having a more basic isoelectric point.

Purification of nonamelogenin proteins from bovine secretory enamel.

The developing enamel matrix is composed of two groups of proteins that can be generally classified as amelogenins and nonamelogenins. The hydrophobic amelogenins represent the majority of the developing enamel matrix proteins, whereas nonamelogenins include the more hydrophilic enamelins, proteinases, and other minor protein components, which represent a small proportion of the matrix. This report describes the purification and partial amino acid sequences of two previously unknown proteins isolated from developing bovine enamel. These proteins were prepared by extracting bovine secretory stage enamel with low ionic strength buffer, followed by ammonium sulfate fractionation. The proteins were purified by ion-exchange, affinity, and reversed-phase chromatography. We propose to designate the proteins BEgp (a glycoprotein) and BEpa. A partial sequence was also obtained from a third protein (BEpb) which was nearly identical to BEpa. Antibodies were prepared to a synthetic peptide based on the N-terminal sequence of BEpa and subsequent immunoblots of various bovine tissues showed a major component of approximately 25 kDa specifically in enamel and ameloblasts. Little or no cross-reactivity of the antibody was found to bovine proteins extracted from heart, lung, kidney, liver, dental pulp, or bone. Similar analyses of both rat secretory stage and maturation stage enamel showed two bands of 28 kDa and 29 kDa. Immunohistochemical localization in rat incisors, showed specific staining of the enamel, secretory granules, and Golgi apparatus in ameloblasts. No sequence homology with known proteins could be demonstrated for BEgp or BEpa, suggesting that these components of developing enamel are novel tooth-specific proteins.

The amelogenin gene.

Amelogenin is a major protein constituent of the developing enamel matrix. This protein is now well characterized from the data of amino acid sequences which have been shown to be at a high degree of homology between all species investigated to date. The gene structure of this protein has been demonstrated and it is confirmed that there are two amelogenin genes, one on the X-chromosome and the other on the Y-chromosome in humans. The mapping of human amelogenin gene in the p22 region on the X-chromosome and the gene on the Y-chromosome was established. It has been confirmed that several types of X-linked amelogenesis imperfecta are caused by structural defects in the amelogenin gene on the X-chromosome. The physiological importance of amelogenin in the enamel formation is suggested by the symptoms of this inherited disease in addition to inhibition experiments of amelogenin transcription and translation (Couwenhoven et al., J. Craniofac. Genet. Dev. Biol. 13:259-269, 1993; Diekwisch et al., Development 117:471-482, 1993). Recently, an attempt to synthesize recombinant mouse amelogenin by E. coli was also undertaken (Simmer et al., Calcif. Tissue Int. 54:312-319, 1994). The regulation of amelogenin expression is now under investigation (Chen et al., Dev. Dynamics 199:189-198, 1994) and the elucidation of this mechanism will contribute a great deal to the study of tooth development.

Analysis of Amelogenin Proteins Using Monospecific Antibodies to Defined Sequences

Amelogenins are the predominant proteins found in the developing enamel matrix and are believed to play a crucial role in normal mineralization. Although the amelogenin gene is found as a single copy in all species in which it has been examined, multiple amelogenin polypeptides ranging in size from 5 to 25 kDa are obtained upon extraction of developing enamel matrix, making identification and characterization of individual components difficult. This heterogeneity may be ascribed to transcription of divergent genes located on the X and Y chromosomes, alternative splicing of the primary transcripts, physiologic degradative processing, and artefactual degradation. In order to characterize individual components, antibodies were produced to the following peptides: (1) QPLQPMQPMQPLQPLQPL (corresponding to the repeat sequence encoded only in the bovine X chromosome gene), (2) IRHPPLPP (corresponding to a unique sequence generated by alternative splicing found in leucine-rich amelogenin peptide (LRAP), (3) LPDLPLEAWPATDKTKREEVD corresponding to the amelogenin carboxy-terminus. Amelogenin proteins obtained from fetal bovine molars were subjected to SDS PAGE and Western electrotransfer, and immuno-ultrastructural analysis. These analyses demonstrated that: (1) the distribution of amelogenin polypeptides isolated from male fetuses differed appreciably from that of females, (2) the LRAP junctional peptide sequence can be specifically identified, and (3) the LRAP peptide can be immunolocalized in the enamel matrix of both males and females.