IJPCDR

An10.5005/jp-journals-10052-0018 Overview of Enamel Matrix Review Article

An Overview of Enamel Matrix Proteins 1Chaitradevi Saxena, 2Kartikay Saxena, 3Vikas Bhakhar, 4M Vidya, 5Mohsin Ghanchi, 6Dhaval Jani

ABSTRACT non-collagenous proteins only and contains several Enamel, one of the hardest tissues of the body, consists of enamel proteins and enzymes. The major part of these 96% inorganic and 4% organic content. The inorganic content enamel proteins is composed of a heterogeneous group is made up of hydroxyapatite crystals, while the organic of low-molecular-weight proteins known as amelo- content is composed of non-collagenous proteins, the major genins, which may constitute as much as 90% of the part of which is enamel proteins. The understanding of enamel total. The remaining 10% consists of non-. proteins has undergone a sea change in the last few years. and ameloblastin are the two most important Primarily, enamel proteins are divided into and 2,3 non-amelogenin families. Amelogenins are believed to regulate non-amelogenins. the growth, thickness, and width of hydroxyapatite crystals. The The extracellular matrix of the enamel is now reason- expression of amelogenins has been shown to have a role in ably well defined in terms of its major compo- sex determination as well. Non-amelogenins include other pro- nents. Unlike other mineralized tissues, a forming enamel teins like ameloblastin, enamelin, enamelysin, and APin. They does not exhibit a distinct unmineralized matrix like are shown to be involved in cell matrix interactions, periodontal osteoid or predentin. The hydroxyapatite crystals grow regeneration, and proper enamel development. Recent studies have also shown the role of enamel proteins in understanding directly against the secretory surfaces of the . the nature of odontogenic tumors. Although the background matrix formed by the margin- ally soluble amelogenins may provide some physical Keywords: Ameloblastin, Amelogenin, Enamel, Enamelin, Enamelysin, Protein support, enamel proteins likely do not play any structur- ing function.1 Morphologically, the organic matrix of a How to cite this article: Saxena C, Saxena K, Bhakhar V, Vidya M, Ghanchi M, Jani D. An Overview of Enamel Matrix Proteins. Int forming enamel appears uniform in decalcified histologic J Prev Clin Dent Res 2016;3(1):79-84. preparations. However, immunohistochemical studies reveal that enamel proteins are differentially distributed Source of support: Nil across the enamel layer. It was found that intact or rela- Conflict of interest: None tively intact non-amelogenin molecules are concentrated near the cell surface, whereas mostly degraded fragments INTRODUCTION are found in the deeper enamel.4 Enamel has evolved as an epithelially derived protective The exact nature and function of enamel proteins is covering for teeth. Being one of the hardest tissues of the a subject that is still under extensive research. Multiple body, it along with dentin serves to protect the underlying studies have been conducted to elucidate the exact pulp. Fully formed enamel is the most highly mineralized effects of these proteins on the development of the tooth extracellular matrix known, consisting of approximately structure. In spite of this, many questions about the 96% mineral and 4% organic material and water.1 enamel proteins are still unanswered. The inorganic content of enamel is crystalline calcium phosphate (hydroxyapatite) substituted with carbonate MELOGENIN FAMILY ions. The organic matrix of the enamel is made from This consists exclusively of the protein amelogenin, which forms the bulk of the total enamel proteins.

1,2,6Reader, 3Senior Lecturer, 4,5Professor and Head Amelogenins 1Department of Conservative Dentistry, College of Dental Sciences, Bhavnagar, Gujarat, India Heterogeneous amelogenins are hydrophobic proteins rich 2,3,5,6Department of Oral Pathology, College of Dental Sciences in proline, histidine, and glutamine. Their heterogeneity Bhavnagar, Gujarat, India is brought about in three ways: 4Department of Oral Pathology, Yenapoya Dental College • The responsible for transcribing amelogenin are Mangaluru, Karnataka, India found on X and Y , and because these Corresponding Author: Vikas Bhakhar, Senior Lecturer 2 genes are not 100% homologous, a sexual heteroge- Department of Oral Pathology, College of Dental Sciences neity exists from the outset.5 Amargadh, Bhavnagar, Gujarat, India, Phone: +919638983058 • The amelogenin contains at least 7 exons, which e-mail: [email protected] can be spliced in numerous ways to produce mature International Journal of Preventive and Clinical Dental Research, January-March 2016;3(1):79-84 79 Chaitradevi Saxena et al

messenger ribonucleic acids that may include all (amplicon), and the Y gene, AMELY, a 112 bp 7 exons or a lack of some of them.6 amplicon. Hence, the AMELX contains a 6 bp deletion in • Amelogenins undergo short-term and long-term the intron 1. Therefore, when the amplicons are run on an extracellular processing by proteolytic enzymes into agarose gel, samples from male sources (XY) will show lower-molecular-weight fragments. Of these, the two bands on an agarose gel (one for the 106 bp fragment tyrosine-rich amelogenin polypeptide (TRAP) and and another for the 112 bp fragment), while females (XX) leucine-rich amelogenin polypeptide (LRAP) are sig- will show only one band. Thus, this process allows for nificant because they constitute the bulk of the final sex determination of unknown samples.9,14 organic matrix of a maturing enamel.7,8 However, mutations in the Y-derived fragment of the gene may result in amplification failure of the Y allele, Applications of Amelogenins causing misidentification of the biological sample as of a female. The error rate is not much. Indians, however, seem Although not completely understood, the function of to have an unusually high rate of amelogenin deletion in amelogenins is believed to be in organizing enamel rods Y chromosomes. In one study, Thangaraj et al3 studied a during tooth development. The latest research indicates total of 270 male samples, of which 5 showed a deletion that this protein regulates the initiation and growth of of Y chromosome–specific amelogenin (1.85%). hydroxyapatite crystals during the mineralization of Amelogenin has also been shown to be a cell adhesion enamel.9 They are supposed to regulate the growth in protein. It has been shown that an amelogenin-containing thickness and width of the crystals. Other studies show preparation, emdogain, possesses cell-adhesive activity. that the amelogenin has an inhibitory effect on the crystal Studies showed that amelogenin and emdogain can growth, and this is achieved by the selective adsorp- promote the adhesion of many cell types via a divalent tion of 25 kDa amelogenin on the surface of the apatite cation-dependent mechanism. It is known that amelogenin crystals. The interaction of amelogenins with enamel binds to minerals; studies have shown that amelogenin crystals may affect the progress of enamel mineralization does not bind to collagen or heparin under physiological in several ways: conditions. Amelogenin shows characteristics similar to • The habit of enamel crystals could be determined in the surface adherent material (SAM) class of cell adhesion part by the preferential adsorption of the molecules proteins. It is not membrane intercalated and requires onto either the basal or the prism face, yielding different divalent cations for activity. While the SAMs often have growth kinetics between these crystallographic a binding site for collagen, amelogenin apparently does planes.10 not. In contrast, amelogenin does bind to hydroxyapatite • The apatitic surfaces might cause an accelerated and not only supports cell adhesion but also promotes cell degradation of the adsorbed molecules or indirectly spreading.15 The results of various studies showed that control the enzymatic degradation of amelogenins; since amelogenin binds to hydroxyapatite and is present most likely the conformation of amelogenins on the in the organic matrix of developing teeth, amelogenin crystal surfaces is different from those of the molecules may mediate the adhesion of ameloblasts and other cell 11 in liquid phase. types to the mineral component of a developing teeth.16 • The removal of degraded products of amelogenins Apart from their roles related to enamel in particular, might be retarded through their adsorption onto amelogenins have been shown to have an effect on the forming or formed crystals, especially if the other dental tissues as well.7 As gene expression assays adsorption affinities of amelogenin moieties are became more sensitive, expression was also noted in enhanced by changes in the nature of the crystal tissues not involved with enamel formation leading to 12 surfaces during successive developmental stages. hypotheses concerning additional roles for these proteins. Another use of amelogenin is in sex determination. As In vitro approaches led to the discovery that some of the mentioned earlier, the genes responsible for transcribing amelogenins are able to regulate gene expression and to amelogenins are found on X and Y chromosomes.5 This participate in cellular signaling. An extract containing heterogeneity is used to help in sex determination. For predominately amelogenins has been used clinically in the sex determination using amelogenin, the technique the treatment of certain forms of periodontal disease with used is polymerase chain reaction. The gene present on regenerative results noted originally in animal models, the X chromosome is designated as AMELX, while the one but later in human patients as well.17 present on Y chromosome is designated as AMELY.13 Using Studies have also been conducted focusing on the primers specific for intron 1 of the gene, the gene sequence role of amelogenins in certain odontogenic tumors. for the intron can be amplified. The X chromosome gene, Amelogenins were detected immunohistochemically AMELX, gives rise to a 106 bp amplification product using a monoclonal antibody. They were strongly 80 IJPCDR

An Overview of Enamel Matrix Proteins expressed in amyloid-like materials, ghost cells, and localizes near the cell surface and not in the deep enamel the cells surrounding ghost cells of calcifying epithelial matrix layer. Recently it was reported that transgenic mice odontogenic tumors and cysts, whereas calcified overexpressing ameloblastin in ameloblasts resemble bodies within the tumors and cysts showed negative imperfect (AI), suggesting the importance staining. The expression of amelogenins was also of ameloblastin in enamel formation.23 Ameloblastin is positive in tumor cells of ameloblastoma, adenomatoid secreted along with amelogenin, and both are contained odontogenic tumor, squamous odontogenic tumor, within the same secretory granule. Given that they are and ameloblastic fibroma. Peripheral tumor cells of the co-secreted, their segregation at growth sites is an intrigu- follicular ameloblastoma were positive with relatively ing phenomenon.1 Investigators say that it may result intense staining. Undifferentiated or flattened tumor cells from microenvironmental conditions or physicochemical of adenomatoid odontogenic tumor and non-keratinized properties of the proteins themselves. tumor cells of the squamous odontogenic tumor showed marked staining. Reduced ameloblasts in the odontoma Applications of Ameloblastin 18,19 displayed the strongest staining for amelogenins. The exact nature and function of ameloblastin is still an In another study, the association of amelogenins and area of research. Studies to understand the ameloblastin ameloblastic fibro-odontoma was studied. The investi- role have primarily been conducted in mice. Since it gators examined immunohistochemically a case of this was observed that ameloblasts detach from the matrix tumor in which an enamel having prism structures was in the absence of ameloblastin, it was hypothesized developed in the absence of odontoblast differentiation that ameloblastin may be involved in cell matrix but was in contact with mesenchymal matrices. His- interactions. In a study conducted for this purpose, tological examination showed diverse morphological investigators generated mice with a null mutation at the features of epithelial tumor cells, e.g., cuboidal cells ameloblastin locus to determine the role of ameloblastin comprising tooth bud–like projections, and in amelogenesis. The mutant mice showed several specific stellate reticulum-like cells, and residual cells in the form anomalies of tooth development including the lack of of extended cords or islands of odontogenic epithelium. enamel formation.7 Immunostaining with anti-amelogenin sera proved that In ameloblastin-null mice, the dental epithelium the intracellular production of amelogenins was initiated differentiates from the matrix surface at the secre- at the tooth bud–like stage. The secreted amelogenins tory stage and loses cell polarity. Mutant ameloblasts were detected almost exclusively in the induced enamel resume proliferation and accumulate to form multiple and dentinoid areas, as well as in the core region of cell layers, producing abnormal, unstructured, and cementicle-like spheres deposited in the encapsulating calcified matrix. Ameloblastin binds specifically to stroma. The results obtained indicate that the odonto- ameloblasts and inhibits cell proliferation of mutant genic tumor epithelia and its products, i.e., amelogenins, ameloblasts. In mutant teeth, ameloblasts regain some participate in multifaceted aspects of dental hard tissue early phenotypes of undifferentiated dental epithelial formation that takes place during oncogenesis.20 cells, and the abnormalities occur when the cells detach. The results indicated that ameloblastin was a key adhe- THE NON-AMELOGENIN FAMILY sion molecule for enamel formation and suggested that ameloblastin played an important role by binding to and This includes other proteins like ameloblastin, enamelin, maintaining the differentiated phenotype of secretory enamelysin, amelotin, APin, etc. Of these, ameloblastin ameloblasts.23,24 In another similar study on mutant forms the bulk of all the proteins. The non-amelogenins mice, it was seen that the expression of amelogenin was are broadly believed to promote and guide the formation specifically reduced in mutant ameloblasts. More than of enamel crystals.21 20% of amelobastin-null mice developed odontogenic tumors.25 Ameloblastin Some investigators have also tried to ascertain the role The most important member of the non-amelogenin of ameloblastin in the periodontal regeneration process. family, ameloblastins form about 5 to 10% of the total They used the commercially available emdogain, which enamel proteins.22 They are secreted by the ameloblasts they said was composed not only of amelogenin pep- during the early secretory to late maturation stages of tides but also of several ameloblastin peptides.26 Using amelogenesis. Ameloblastin is also transiently expressed immunohistochemistry, they identified the presence of in dentin matrix and Hertwig’s root sheath epithelial ameloblastin also in rat mature cementum and alveolar cells, but its role in dentin and cementum formation has bone. In their study, two models were employed to check not been established. Unlike amelogenin, ameloblastin the possible regenerating activity of the recombinant International Journal of Preventive and Clinical Dental Research, January-March 2016;3(1):79-84 81 Chaitradevi Saxena et al human ameloblastin.14 The first model was a 4 mm cir- classification of hypoplastic AI into pitted, local, smooth, cular parietal bone defect in rat calvarias that was treated and rough subtypes. Hypomaturation type AI is char- with the recombinant human ameloblastin protein in acterized by opaque white enamel with a ground glass polyglycolic acid (PGA) carrier, with and without the appearance. Although the thickness of the enamel is not use of a resorbable membrane. The second model was a affected, it is slightly softer than normal, often leading to periodontal window wound in rat mandibles, which was abrasions on the incisal and occlusal surfaces. In hypo- to be treated with the recombinant human ameloblastin calcified type AI, the enamel initially develops with a protein in PGA carrier. Both models were analyzed by normal thickness, but the enamel matrix is poorly calci- micro-computed tomography (µCT), histology, immuno- fied. At least 7 mutations in the ENAM gene have been histochemistry, and in situ hybridization. The results of identified in people with autosomal-dominant forms of their studies showed that ameloblastin may be involved AI. Autosomal-dominant inheritance means that one in bone regeneration process, but further analysis by copy of the ENAM gene in each cell is altered.32 using more comprehensive studies is warranted to make Mutations in the ENAM gene have a variety of effects a conclusive assumption about the possible role of amelo- on enamel formation. Some mutations reduce the amount blastins in bone regeneration.27 of enamelin produced by the gene. Other mutations In addition to cell adhesion, ameloblastin may have lead to the production of an abnormally short version of another activity during enamel formation as suggested enamelin that lacks critical regions. Altered or missing in a transgenic mouse model proposed by Paine et al enamelin can lead to severe problems with a developing in 2003.28 The abnormal matrix accumulations and enamel or cause milder defects, such as shallow pits or discontinuous calcified tissue in the mutant enamel horizontal grooves in the teeth. Mutations in the ENAM organ do not contain typical enamel crystal structures. gene also have been found in people with an autosomal- Ameloblastin may provide a scaffolding to organize recessive form of AI. Autosomal-recessive inheritance enamel matrix protein structures to initiate crystal means that two copies of the ENAM gene in each cell are formation and growth. Alternatively, enamel crystal altered. These mutations result in the production of an formation may require close contact of the enamel organ abnormal version of enamelin that prevents enamel from with the newly forming dentin.23 developing properly. People who inherit two mutated copies of the ENAM gene have severe defects in their Enamelin enamel; as a result, this protective covering may be very thin or completely absent.33 Another member of the non-amelogenin family, enamelin is a protein required for enamel formation, and it plays Enamelysin a key role in the formation and growth of crystals in a Enamelysin is a tooth-specific matrix metalloproteinase developing enamel.29 Enamelin is the largest and least that is expressed during the early through middle stages of abundant of the 3 principal enamel matrix proteins, enamel development. The enamel matrix proteins amelo- representing between 1 and 5% of the total matrix genin, ameloblastin, and enamelin are also expressed protein. Proteolytic processing gives rise to multiple during this same approximate developmental time period, enamelin cleavage products, which accumulate in suggesting that enamelysin may play a role in their hydro- different parts of the enamel matrix.30 The gene that lysis. In support of this interpretation, recombinant enam- provides instructions for making enamelin is referred elysin was previously demonstrated to cleave recombinant to as ENAM gene. Enamelin was described in the older amelogenin at virtually all of the precise sites known to literature as an EDTA-soluble enamel protein. It turned occur in vivo. Thus, it was stated that enamelysin is likely out in recent research to be albumin derived from blood an important amelogenin-processing enzyme.34 contamination.31 Studies have been conducted to further understand the role of enamelysin in tooth development. Most studies Applications of Enamelin are done on mice models. To characterize the in vivo The study about this protein assumes importance biological role of enamelysin during tooth development, because it has been reported that defects in the human investigators generated an enamelysin-deficient mouse enamelin gene cause autosomal-dominant AI. Based by gene targeting.35 Although mice heterozygous for the on clinical appearance, cases of AI are roughly classi- mutation have no apparent phenotype, it was seen that fied into hypoplastic type, hypomaturation type, and the enamelysin-null mouse has a severe and profound hypocalcified (hypomineralized) type. Hypoplastic AI tooth phenotype. Specifically, the null mouse did not is characterized by a reduced thickness of the enamel. process amelogenin properly, possessed an altered The appearance of the enamel surface allows further enamel matrix and rod pattern, had hypoplastic enamel 82 IJPCDR

An Overview of Enamel Matrix Proteins that delaminated from the dentin, and had a deteriorating affected by any abnormality of enamel proteins. Apart enamel organ morphology as development progressed.35 from that, certain odontogenic tumors too have shown These findings demonstrated that enamelysin activity correlation in their activity to that of some enamel was essential for proper enamel development. proteins. The enamel proteins are not only associated with Others pathologies but may also have a role in periodontal Some other important enamel proteins include amelotin regeneration, a field where there is immense scope of and APin. Amelotin was recently described as an further research. The sex-linked expression of amelogenin ameloblast-specific gene, highly but transiently expressed makes it a valuable tool in sex determination. in ameloblasts of the maturation-stage enamel organ REFERENCES before tooth eruption. 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