Brazilian Journal of Oral Sciences Piracicaba Dental School - UNICAMP EISSN: 1677-3225 Vol. 4, Num. 13, 2005, pp. 716-724

Brazilian Journal of Oral Sciences, Vol. 4, No. 13, April./June. 2005, pp. 716-724

The enamel organic matrix: structureand function

Alexandre Ribeiro do Espírito Santo¹ Sérgio Roberto Peres Line²

¹DDS, MS, Department of Morphology, Piracicaba Dental School, State University of Campinas, Piracicaba-São Paulo, Brazil. ²DDS, MS, PhD, Professor, Department of Morphology, Piracicaba Dental School, State University of Campinas, Piracicaba-São Paulo, Brazil.

Correspondence to:Sérgio Roberto Peres Line Faculdade de Odontologia de Piracicaba -UNICAMP Avenida Limeira, 901, Areião CEP: 13.414-903 Caixa Postal: 52 Piracicaba-SP, Brasil Phone:+55-19-3412-5333 E-mail: serglin@fop.unicamp.br

Received for publication: December 17, 2004
Accepted: March 04, 2005

Code Number: os05013

Abstract

Dental enamel is the most mineralized tissue in the vertebrate body and contains the largest known biologically formed hydroxyapatite crystals. Its formation occurs extracellularly through the collaboration of a proteic transient framework (the enamel organic matrix), which controls hydroxyapatite crystal growth, morphology and orientation. This matrix is deposited with a small amount of mineral during the secretory stage of amelogenesis. The organic components begin to be degraded in the transition stage and are extensively corrupted, and almost entirely replaced by the inorganic crystallites during maturation stage. The present paper reviews current knowledge on the structural biology of the enamel organic matrix.

Key Words: dental enamel, extracellular matrix, amelogenesis, dental enamel proteins, dental enamel proteinases.

Dental enamel is originated from ectoderm and is the hardest, most mineralized tissue in the vertebrate body, containing the largest known biologically formed hydroxyapatite crystals^1-2. Enamel is noncollagenous and does not undergo resorption and remodeling³. This striking example of a highly mineralized structure is exquisitely adapted to absorb essential mechanical and abrasive stresses throughout the lifetime of the organism. Enamel formation occurs extracellularly through the collaboration of proteins that assemble in order to form a transient framework, which is important to control hydroxyapatite crystal growth, morphology and orientation^2,4. This transient protein framework is known as the enamel organic matrix. It is synthesized, secreted and organized by specialized cells of the developing tooth organ, called ameloblasts. As these cells migrate outwards they leave behind a ribbon of secreted proteins in their wake and play active role in transport of ions including Ca²⁺, HPO₄^2-, OH^-and H^+5-6. Enamel development is didactically divided into secretory, transition and maturation stages. The enamel organic matrix is deposited with little amount of mineral during the secretory stage, begins degrading in the transition one, and is extensively corrupted and almost entirely replaced by the inorganic crystallites during maturation^6-8. Thus, despite an embryonic origin in protein, mature enamel is a stiff-, brittle-ceramic composed of hydroxyapatite crystallites embedded within a small amount of organic material distributed among the crystallites. These crystallites are roughly organized into bundles called rods or prisms and thus each rod is made of many small individual hydroxyapatite crystals. This paper specifically presents a review of current knowledge on the structural biology of the enamel organic matrix.

Great advances have been made regarding the identification and cloning of structural enamel matrix proteins and enamel proteinases. The enamel matrix proteins are generally grouped into three classes sharing common features, these are: the amelogenin class of circa 20-kDa hydrophobic proteins; the enamelin class of circa 65-kDa acidic proteins including tuftelin; and the non-amelogenin, non-enamelin class, represented by ameloblastin (also called amelin/ sheathlin)^3,9-11. All three protein classes are proline rich¹². Enamel matrix proteins assemble, interacting with each other and with the hydroxyapatite crystallites. These biochemical interactions are important to guide hydroxyapatite crystals growth and modulate their morphology. Two major enamel proteinases have been identified: matrix metalloproteinase20 (MMP-20), also known as enamelysin, and kallikrein 4 (KLK4), which is a serine proteinase^12-17. These proteinases are expressed in the developing enamel at different times and have different functions. Their roles are to modify and/or to eliminate enamel matrix proteins, which affects the way enamel proteins interact with each other and with the developing enamel crystallites¹². Sulphated proteins, serum albumin and lipids have also been reported as residents of the enamel organic matrix^3,10. Detailed information about these matrix components and their roles is presented in the following sections.

Four genes of enamel structural proteins have already been cloned and characterized in several species, these are: amelogenin^18-20, tuftelin^21-24, enamelin^25-27 and ameloblastin^28-30.

Amelogenins are the most studied enamel matrix proteins. They account for more than 90% of the matrix proteins in the secretory stage of enamel formation⁹, cromprising the major components of the supramolecular transient framework, which is absolutely necessary for normal enamel crystals growth and architecture. This condition may be an explanation for significant advances in the knowledge on characteristics and roles of the amelogenins.

Amelogenins may exhibit various forms due to three main reasons: there are two distinct copies of their gene localized in X and Y chromosomes, different amelogenin mRNAs are produced by alternative splicing and they are proteolytic processed after being secreted by ameloblasts. There is an important debate in the literature regarding the way enamel proteins are arranged in order to interact with each other and with the developing hydroxyapatite crystals^4,12,31. Recent knowledge on amelogenin’s primary and quaternary structures has allowed the development of in vitro experimental systems for the study of amelogenin-mineral interactions and interpretation of the function of this structural protein during amelogenesis^4,32-34. Supramolecular assembly of amelogenin has been assumed to be critical for a competent enamel organic matrix formation. Full-length amelogenins contain two clearly definable self-assembly domains: the amino-terminal hydrophobic domain-A comprising amino-acid residues 1-42; and the carboxyterminal hydrophilic domain-B comprising amino-acid residues 157-173². Amelogenin binds apatite crystals through its hydrophilic domain-B^35-36, also known as leucin-rich amelogenin polypeptide (LRAP)³⁶. Moradian-Oldak et al.³⁷ have recently found that the apatite-binding domain can be narrowed down to the C-terminal 12 or 13 amino acids and further digestion from the C-terminal does not affect this binding affinity. It seems that the function of domain-B is crucial only during the early stage of enamel formation since it is cleaved soon after the protein is secreted into the extracellular space⁴. Prevention of premature crystal-crystal fusion at the very early stage of mineral formation is the most likely function for the full-length amelogenin containing the C-terminal^32,38. While the presence of this domain appears to be critical for the apatite binding affinity of amelogenin, it does not seem to be necessary for the specific modulating effects on crystal morphology. Some studies indicate that the lack of hydrophilic C-terminal affects proteins assembly and results in the fusion of the hydrophobic nanospheres, which are suggested to arrange in order to provide the supramolecular structural framework for the controlled growth of enamel crystals^4,39. These findings are in agreement with previous report⁴⁰, which shows that amelogenin is found in all compartments throughout the entire thickness of developing enamel and that intact amelogenins and their C-terminal cleavage products are only detected within 40 µm of the enamel matrix surface. This finding indicates that full-length amelogenins containing the hydrophilic C-terminal and their hydrophobic N-terminal proteolytic products have distinct functions.

The ablation of amelogenin gene in knockout mice results in severe disruptions in enamel crystallite architecture, even though a functional mineralized enamel does form^41-42. This is also true in the enamel of patients with the genetic disease amelogenesis imperfecta in which particular mutations of amelogenin have been identified⁴³. These observations imply that the presence of this protein is not an absolute requirement for enamel mineralization⁴². However, other evidences indicate a critical dependency of amelogenin self-assembly on the microstructural organization of enamel. This process involves amino-terminal hydrophobic domain-A and carboxy-terminal hydrophilic domain-B. Amelogenin’s amino acid sequence is highly conserved across evolution and this conservation is particularly obvious among the amino-terminal residues 1-51 and again in the carboxyl-terminal residues 160-180^6,44-45. Conservation of amino acid sequence often implies physiologic relevance. The physiologic relevance of the highly conserved amelogenin’s amino-terminus was identified by Lench and Winter⁴⁶ and confirmed by Collier et al.⁴⁷. These authors focused their genetic studies on two unrelated human pedigrees for amelogenesis imperfecta and identified single amino acid changes, occuring within the highly conserved amino-terminus of amelogenin, as the causative factor for the phenotypic changes in the resulting enamel. Some experiments with transgenic animals, containing domain-A, domain-B or both domains deleted, indicated that a highly organized enamel organic matrix (essential to normal enamel formation) results from undisturbed amelogenin self-assembly^42,48. Mutations in human amelogenin gene located on the X chromosome induce amelogenesis imperfecta (AIH1) with enamel phenotypes broadly characterized as hypoplastic or hypomineralized. Documented cases of AIH1 with mutations in the C terminus of amelogenin have resulted in a hypoplastic phenotype, whereas mutations located within the N terminus have resulted in hypomineralized enamel^49-50. Single amino acid in vitro mutations, identical to those appearing in AIH1, were able to reduce amelogenin self-assembly⁵¹. According to Paine et al.⁵², a possible explanation for this genotype/ phenotype relationship observed in vivo may be related to a reduction in the rate of amelogenin hydrolysis⁵³ and the subsequent events of mineralization. The proline residue at position 169 of mouse amelogenin (M180) seems to play a significant role in amelogenin hydrophobic self-assembly, since this process does not occur after removal of the C-terminal including the referred amino acid⁴². However, the importance of this amino acid has not been well established and some reasons for this fact are the conflicting results of investigations which show that mutating proline-169 within M180 to either a lysine or threonine does not influence amelogenin’s domain-A assembly⁴².

Both native and recombinant amelogenins are reported to hemagglutinate mouse red blood cells^3,54. This hemagglutination is inhibited by monomers, dimers and tetramers of N-acetylglucosamine (GlcNAc) but not by Nacetylgalactosamine or related sugars. This activity is retained by TRAP (tyrosine-rich amelogenin polypeptide 45 residues), which results from N-terminal cleavage of amelogenin by enamelysin (MMP-20). It was shown that [¹⁴C]GlcNAc binds to the N-terminal sequence of TRAP (PYPSYGYEPMGGW) but not when the three tyrosyl residues are substituted with phenylalanine or if the third proline is substituted with threonine³. This latter modification mimics the point mutation identified in a case of human X-linked amelogenesis imperfecta⁴⁷. This activity of the TRAP motif of amelogenin is known as lectin-like property and may be functionally involved in interactions with enamel matrix glycoproteins (enamelin, tuftelin or ameloblastin), promoting structural stability of the matrix³ or, alternatively, functioning in a signaling role through recognition by cell surface glycoproteins⁵⁵. Recent studies indicate that amelogenin may interact with ameloblastin to form a heteromolecular assembly as a consequence from the presence of GlcNAcmimicking peptides (GMps) at intermittent sites of ameloblastin and from the recognized amelogenin-trityrosylmotif-peptide (ATMP), which is a GlcNAc/GMp-binding domain in amelogenin⁵⁶. In vitro experiments conducted by Bouropoulos and Moradian-Oldak⁵⁷ strongly suggest that the 32-kDa enamelin and amelogenins cooperate to promote nucleation of apatite crystals and propose a possible novel mechanism of mineral nucleation during enamel biomineralization.

The enamelins comprise a class of enamel acidic proteins that include enamelin and tuftelin. They account for about 2% of all enamel matrix proteins¹².

The major secretory product of the human enamelin gene has 1103 amino acids and is post-translationally modified, secreted and processed by proteases shortly after being secreted⁵⁸. Unlike other enamel proteins, no enamelin isoforms that are translated from alternatively spliced RNA transcripts have been observed³. Porcine enamelin proteolytic products were first isolated and characterized by Fukae and Tanabe⁵⁹. Intact enamelin is only found on the enamel surface, within a micrometer of the ameloblast cell membrane, and many enamelin cleavage products appear to be rapidly degraded and are only found in the outer enamel layer¹². However, stable enamelin proteolytic products are found throughout the entire thickness of developing enamel. The best-studied stable enamelin cleavage product is the 32-kDa enamelin^3,60-63. The porcine 32 kDa-enamelin has 106 amino acids (residues 174-279), which include two phosphoserines and three glycosylated asparagines^62-64. Variable glycosilation contributes to the heterogeneity of this enamel protein³. 32-kDa enamelin is concentrated in the rod and interrod enamel and is absent from the sheath space¹². The functional significance of this specific spacial organization in the enamel layer is not yet understood. Recent in vitro evidences indicate that 32-kDa enamelin and amelogenin cooperation promotes nucleation of apatite crystals⁵⁷. The importance of enamelin for normal enamel formation is indicated by an investigation that shows correlation between a mutation in its gene and an autosomal-dominant form of amelogenesis imperfecta (AI)⁶⁵. The mutation is a single-G deletion within a series of 7 G residues at the exon 9-intron 9 boundary of the enamelin gene. Another publication reports that mutations in this gene cause a severe form of autosomal-dominant smooth hypoplastic AI that represents 1.5%, and a mild form of autosomal-dominant local hypoplastic AI that accounts for 27% of AI cases in Sweden⁵⁸.

A long recognized structure in enamel is the tuft^6,66, which may represent residual enamel matrices that include proteins responsible for crystals nucleation^3,67-68. Enamel tufts occur at the dentine enamel junction (DEJ) and appear as ‘tornadolike swirls’ rising from the DEJ and into the aprismatic enamel^6,68. Protein was recovered from these tufts by microdissection and characterized⁶⁹. Sixteen years later, a cDNA clone with amino acid sequences that matched the composition of the tuft protein⁶⁹ was isolated and characterized²², and so the name tuftelin was adopted. Since tuftelin is an acidic enamel protein, it joined the enamelin class of enamel protein⁶. Full-length mouse tuftelin cDNA has been characterized²⁴. The function of this enamel protein remains unknown but a predicted function, as we referred at the beginning of this paragraph, is to nucleate hydroxyapatite crystal formation. This prediction is made because of tuftelin’s anionic character, its localization to the DEJ and its expression prior to amelogenins during development^6,22,70-71. Tuftelin can be divided into two domains with distinct physical-chemical properties: the carboxyl-terminal domain mediating self-assembly and the amino-terminal domain containing a pronounced anionic motif, which is consistent with tuftelin’s proposed role as a crystal nucleator⁶. The bovine tuftelin gene was also cloned and characterized by Bashir et al.²³, showing a cDNA sequence that was different from the one reported by Deutsch et al.²² at its carboxyl-terminus. The former reported a C-terminus with only 42 amino acids²³ and the second with 92²². Tuftelin thus exists as at least two isoforms: the tuftelin A form²² and the tuftelin B form²³. The self-assembly properties of bovine tuftelin A and B isoforms and mouse tuftelin have been confirmed by Paine et al.⁷². Tuftelin interacting proteins (TIPs) have been discovered^73-74 and one TIP protein, encoding a 39-kDa protein (TIP 39), shows enrichment to the secretory surface of Tomes’ processes⁷², being a candidate molecule linking the ameloblast secretory surface to the assembling enamel organic matrix. The abundance of TIP 39 and tuftelin at the DEJ suggests that these two proteins may also participate in forming specialized enamel at the DEJ.

In an attempt to better define a physiological function for tuftelin during amelogenesis, a recent investigation was carried out with transgenic mice that overexpress tuftelin in ameloblasts and subsequently in the enamel matrix⁷⁵. Overexpression of this protein was shown to impact dramatically upon the enamel crystallite and enamel prismatic structure, resulting in gross imperfections in enamel, which apparently reflects from loss of restricted growth of enamel crystallites along their a-axis and b-axis.

This third class of enamel proteins is represented by ameloblastin (also called amelin/sheathlin)^3,9-11, which accounts for about 5% of total enamel proteins¹². Its amino and carboxyl ends are biochemically different and were discovered separately during investigations of pig enamel proteins³. The carboxyl end was represented by two polypeptides with apparent molecular weights of 27 and 29 kDa, isolated during a search for enamel proteins that bound calcium⁵⁹. The amino-terminal end of ameloblastin was discovered in the same research that isolated and characterized enamelin cleavage products⁷⁶. Ameloblastin’s amino-terminus is represented in the enamel matrix by a group of low molecular weight proteolytic products in the range 13-17 kDa with aggregative properties⁷⁶. Two research groups independently cloned and characterized cDNAs encoding the rat homologue of the protein that had been studied by Fukae and Tanabe^59,76. The protein was termed ameloblastin²⁹ and amelin⁷⁷. Later, the cDNA from pig was cloned and designated as sheathlin²⁵. A cDNA encoding the mouse homologue was cloned and also called ameloblastin³⁰, which is the name used in this paper. Human ameloblastin has already been cloned and characterized^28,78. Hu et al.²⁵ observed that ameloblastin transcripts undergo a limited amount of alternative splicing, generating two isoforms that differ by the deletion or inclusion of a 15 amino acid segment, which is absolutely conserved between the pig and the rat. Ameloblastin also is proteolyticly processed after secretion into enamel matrix. Intact ameloblastin and its cleavage products containing the C-terminal half of the protein are only found in the outer developing enamel, concentrated among the crystallites in the rod and interrod enamel^12,79. Proteolytic products containing the amino-terminal side of the protein are found at all depths within the enamel layer, but they are not distributed randomly, being concentrated in the sheath space¹². Ameloblastin self-assembly could not be demonstrated⁷⁴, but interactions between ameloblastin and amelogenin have already been suggested⁵⁶. While amelogenin and tuftelin appear to be restricted in their expression to ameloblasts engaged in forming enamel, ameloblastin is expressed by ameloblasts during amelogenesis, as well as by cells of Hertwig’s epithelial root sheath during cementogenesis^80-81. This spatial pattern of expression suggests that ameloblastin protein participates in the genesis of both tissues^74,80-81.

The significant role for ameloblastin during amelogenesis has been indicated by experiments that place its gene loci in the critical region of autosomal-dominant forms of amelogenesis imperfecta⁸². Ameloblastin overexpression in mice has recently been shown to influence enamel crystallite and enamel rod morphology, implicating the role of ameloblastin gene locus in the etiology of a number of undiagnosed autosomally dominant cases of amelogenesis imperfecta⁸³. Mutant ameloblastin transcripts have also been reported to be expressed in human ameloblastomas²⁸.

Proteinases are present in low abundance in the developing enamel matrix and are not likely to participate directly in the mineralization process¹². They cleave enamel proteins by catalyzing the hydrolysis of peptide bonds. The nature and sequence of proteolytic activities during enamel biomineralization are critical and somehow unique to this mineralizing tissue³. First, these activities cause changes in structural and physicochemical properties of amelogenins, affecting the way they interact with each other and with the developing enamel crystallites^15,84. Second, the almost complete protein degradation appears to be essential for rapid crystal growth, leading to enamel hardening^85-86. Several proteinases have been detected in the enamel extracellular matrix⁸⁷. However, two enzymes, enamelysin (matrix metalloproteinase-20, MMP-20) and enamel matrix serine proteinase 1 (EMSP1), which is now officially designated kallikrein 4 (KLK4), are major enamel matrix proteinases¹².

Enamelysin mRNA has been cloned from pig⁸⁸, human⁸⁹, cow⁹⁰ and mouse⁹¹. It exhibits two forms (41 and 45 kDa) in zymograms^87-88,92. Catalytic domain fragments (21 and 25 kDa) of enamelysin have also been described⁹⁰. This proteinase is expressed during the early through middle stages of enamel development^12,93 and seems to participate in the proteolytic events that allow the crystals to grow in length but not in width or thickness⁸⁷. In vitro, enamelysin catalyzes all of the amelogenin cleavages that are known to occur during the secretory stage in vivo^12,92, and it is probably the enzyme responsible for the processing of all enamel proteins¹². Enamelysin’s main function seems to be the gradual removal of amelogenin C terminus, changing the physicochemical properties of this protein. This cleavage generates the hydrophobic tyrosine-rich amelogenin polypeptide (TRAP)⁹⁴, which is important in the arrangement of amelogenin nanospheres directly involved in the regulation of enamel crystals elongation, controlling their growth in width and thickness⁴. Experiments with recombinant enamelysin also indicate that this enamel proteinase can degrade itself^94-95.

The essential role of enamelysin during enamel formation was evidenced by a recent investigation that showed an amelogenesis imperfecta phenotype caused by enamelysin gene deletion in mice⁹³. The mice homozygous for this mutation did not process amelogenin properly, showed altered enamel matrix and rod pattern, had hypoplastic enamel and deteriorating enamel organ morphology as development progressed. These alterations, however, were not beared by heterozygous mice (enamelysin^+/-), indicating that the phenotype observed for mutations in the enamelysin gene is autosomal-recessive. These findings are in accordance with previous data of Thompson et al.⁹⁶, which indicates that the most autosomal-recessive anomalies are caused by mutations in enzyme genes. The lack of amelogenin processing in the enamelysin null mice observed by Caterina et al.⁹³ likely eliminates necessary changes in the physicochemical properties of amelogenin that are essential for the proper enamel development.

Kallikrein 4 (KLK4) has been cloned from pig⁹⁷, human⁹⁸ and mouse⁹⁹. The first KLK4 cDNA was isolated from a pig-tooth-specific cDNA library and designated as enamel matrix serine proteinase 1 (EMSP1)^12,97. This proteinase is found in the enamel matrix exhibiting these two forms: 30 and 34 kDa^12,100. Different from enamelysin, KLK4 degrades enamel proteins during early maturation stage of amelogenesis, which facilitates their removal from the matrix and makes way for hardening of the enamel layer¹². In vitro incubation experiments using a fraction rich in KLK4 activity have demonstrated complete degradation of the recombinant amelogenin substrate to peptides as small as 147 Da^3,101. Theoretically, if KLK4 failed to function enamel matrix would not be reabsorbed efficiently and the crystallites would not be able to thicken fully, resulting in a hypomaturation form of amelogenesis imperfecta¹². Experiments with engineered KLK4 knockout mice, which can prove this hypothesis, have not been published yet.

Serum albumin has been detected in the enamel organic matrix especially throughout the secretory and transition stages of amelogenesis^102-105. However, this protein is not synthesized or secreted by ameloblasts¹⁰⁶. Radiolabeled serum albumin injected into rabbits did not incorporate into the enamel layer, suggesting a physiological barrier between the extravascular fluid and the enamel matrix¹⁰⁷. Furthermore, ingress of albumin into enamel from dentin is restricted, particularly during the secretory stage¹⁰⁸. As Shapiro and Amdur¹⁰⁹ demonstrated that red pigmentation observed in extracted developing bovine teeth was probably derived from hemoglobin adsorbed post mortem from the dental sac fluid, Fincham et al.³ suggest that albumin would be likely to exhibit the same behavior.

Lipids have been demonstrated to represent some 0.2% of the developing enamel matrix³. Entombed membrane fragments of the Tomes’ processes in the matrix were suggested to contribute to the overall lipid content in this tissue¹¹⁰. A group of short-lived sulfated proteins (49 and 25 kDa), which are rapidly degraded after secretion, has also been identified in the enamel matrix and suggested to interact functionally with nascent amelogenins¹¹¹. The role of lipids and sulfated proteins in the enamel organic matrix remains poorly understood³.

Enamel formation is a highly complex process. The major enamel organic matrix proteins and proteinases have already been cloned and characterized, and their association with several cases of amelogenesis imperfecta has evidenced their importance in the development of enamel. However, there is an important debate in the literature about the enamel proteins processing and the way they arrange in order to interact with each other and with the developing enamel crystallites. Amelogenin supramolecular self-assemblies appear to be essential for normal enamel formation. On the other hand, the mechanisms by which non-amelogenin proteins and enamel proteinases contribute to this formation should be better established.

Alexandre Ribeiro do Espírito Santo was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq - Brazil.

The enamel organic matrix: structure and function

1. Eisenmann DR. Amelogenesis. In: Ten Cate AR, editor. Oral Histology: development, structure and function. 5th ed. Saint Louis: Mosby; 1998. p. 197-217.
2. Paine ML, Snead ML. Protein interactions during assembly of the enamel organic extracellular matrix. J Bone Miner Res. 1997; 12: 221-7.
3. Fincham AG, Moradian-Oldak J, Simmer JP. The structural biology of the developing dental enamel matrix. J Struct Biol. 1999; 126: 270-99.
4. Moradian-Oldak J, Iijima M, Bouropoulos N, Wen HB. Assembly of amelogenin proteolytic products and control of octacalcium phosphate crystal morphology. Connect Tissue Res. 2003; 44: 58-64.
5. Aoba T, Moreno EC. The enamel fluid in the early secretory stage of porcine amelogenesis: chemical composition and saturation with respect to enamel mineral. Calcif Tissue Int. 1987; 41: 86-94.
6. Paine ML, White SN, Luo W, Fong H, Sarikaya M, Snead ML. Regulated gene expression dictates enamel structure and tooth function. Matrix Biol. 2001; 20: 273-92.
7. Robinson C, Briggs HD, Aktinson PJ, Weatherell JA. Matrix and mineral changes in developing enamel. J Dent Res. 1979; 58: 871-80.
8. Bronckers AL, Bervoets TJ, Lyaruu DM, Woltgens JH. Degradation of hamster amelogenins during secretory stage of enamel formation in organ culture. Matrix Biol. 1995; 14: 533-41.
9. Termine JD, Belcourt AB, Christner PJ, Conn KM, Nylen MU. Properties of dissociatively extracted foetal tooth matrix proteins. I. Principle molecular species in developing bovine enamel. J Biol Chem. 1980; 255: 9760-8.
10. Simmer JP, Fincham AG. Molecular mechanisms of dental enamel formation. Crit Rev Oral Biol Med. 1995; 6: 84-108.
11. Fincham AG, Luo W, Moradian-Oldak J, Paine ML, Snead ML, Zeichner-David M. Enamel biomineralization: the assembly and dissassembly of the protein extracellular organic matrix. In: Teaford MF, Meredith-Smith M, Ferguson MWJ. Editors. Development, function and evolution of teeth. Cambridge: Cambridge University Press; 2000. p. 37-61.
12. Simmer JP, Hu JC. Expression, structure, and function of enamel proteinases. Connect Tissue Res. 2002; 43: 441-9.
13. Carter J, Smillie AC, Shepherd MG. Purification and properties of a protease from developing porcine dental enamel. Arch Oral Biol. 1989; 34: 195-202.
14. Denbesten PK, Heffernan LM. Enamel proteases in secretory and maturation enamel of rats ingesting 0 and 100 ppm of fluoride in drinking water. Adv Dent Res. 1989; 3: 199-202.
15. Moradian-Oldak J, Simmer PJ, Sarte PE, Zeichner-David M, Fincham AG. Specific cleavage of a recombinant murine amelogenin at the carboxy-terminal region by a proteinase fraction isolated from developing bovine tooth enamel. Arch Oral Biol. 1994; 39: 647-56.
16. Fukae M, Tanabe T, Uchida T, Lee SK, Ryu OH, Murakami C et al. Enamelysin (matrix metalloproteinase-20): localization in the developing tooth and effects of pH and calcium on amelogenin hydrolysis. J Dent Res. 1998; 77: 1580-8.
17. Cotrim P, de Andrade CR, Line S, de Almeida OP, Coletta RD. Expression and activity of matrix metalloproteinase-2 (MMP2) in the development of the rat first molar tooth germ. Braz Dent J. 2002; 13: 97-102.
18. Cheng L, Lei JQ, Zhu QQ, Wang HH, Shu R. Cloning of human amelogenin gene encoding mature peptide. Shanghai Kou Qiang Yi Xue. 2004; 13: 126-9.
19. Snead ML, Zeichner-David M, Chandra T, Robson KJ, Woo SL, Slavkin HC. Construction and identification of mouse amelogenin cDNA clones. Proc Natl Acad Sci USA. 1983; 80: 7254-8.
20. 20. Gibson CW, Golub E, Abrams W, Shen G, Ding W, Rosenbloom J. Bovine amelogenin message heterogeneity: alternative splicing and Y-chromosomal gene transcription. Biochemistry. 1992; 31: 8384-8.
21. Mao Z, Shay B, Hekmati M, Fermon E, Taylor A, Dafni L et al. The human tuftelin gene: cloning and characterization. Gene. 2001; 279: 181-96.
22. Deutsch D, Palmon A, Fisher LW, Kolodny N, Termine JD, Young MF. Sequencing of bovine enamelin (‘tuftelin’) a novel acidic enamel protein. J Biol Chem. 1991; 266: 16021-8.
23. Bashir MM, Abrams WR, Rosenbloom J. Molecular cloning and characterization of the bovine tuftelin gene. Arch Oral Biol. 1997; 42: 489-96.
24. Dodds A, Simmons D, Gu TT, Zeichner-David M, MacDougall M. Identification of murine tuftelin cDNA. J Dent Res. 1996; 75: 72.
25. Hu C-C, Fukae M, Uchida T, Qian Q, Zhang CH, Ryu OH et al Sheathlin: cloning, cDNA/polypeptide sequences, and immunolocalization of porcine enamel sheath proteins. J Dent Res. 1997; 76: 648-57.
26. Hu C-C, Simmer JP, Bartlett JD, Qian Q, Zhang C, Ryu OH et al. Murine enamelin: cDNA and derived protein sequences. Connect Tissue Res. 1998; 39: 47-62.
27. Hu C-C, Qian Q, Zhang C, Simmer JP. Cloning of human enamelin. In: Robinson C, Goldberg M, editors. Chemistry and biology of mineralized tissues: Proceedings of the Sixth International Conference [abstract 82]. Vittel: American Academy of Orthopaedic Surgeons; 1998.
28. Toyosawa S, Fujiwara T, Ooshima T, Shintani S, Sato A, Ogawa Y et al. Cloning and characterization of human ameloblastin gene. Gene. 2000; 256: 1-11.
29. Krebsbach PH, Lee SK, Matsuki Y, Kozac C, Yamada KM, Yamada Y. Full-length sequence, localization, and chromosome mapping of ameloblastin: a novel tooth-specific gene. J Biol Chem. 1996; 271: 4431-5.
30. Simmons D, Gu TT, Krebsbach PH, Yamada Y, MacDougall M. Identification and characterization of a cDNA for mouse ameloblastin. Connect Tissue Res. 1998; 39: 3-12; discussion 63-7.
31. Moradian-Oldak J. Amelogenins: assembly, processing and control of crystal morphology. Matrix Biol. 2001; 20: 293-305.
32. Moradian-Oldak J, Tan J, Fincham AG. Interaction of amelogenin with hydroxyapatite crystals: an adherence effect through amelogenin self-association. Biopolymers. 1998; 46: 225-38.
33. Wen HB, Moradian-Oldak J, Fincham AG. Modulation of apatite crystal growth on Bioglass by recombinant amelogenin. Biomaterials. 1999; 20: 1717-25.
34. Simmer JP, Lau EC, Hu C-C, Bringas P, Santos V, Aoba T et al. Isolation and characterization of a mouse amelogenin expressed in E-coli. Calcif Tissue Int. 1994; 54: 312-9.
35. . Aoba T, Tanabe T, Moreno EC. Function of amelogenins in porcine enamel mineralization during secretory stage of amelogenesis. Adv Dent Res. 1987; 1: 252-60.
36. Shaw WJ, Campbell AA, Paine ML, Snead ML. The COOH terminus of the amelogenin, LRAP, is oriented next to the hydroxyapatite surface. J Biol Chem. 2004; 279: 40263-6.
37. Moradian-Oldak J, Bouropoulos N, Wang L, Gharakhanian N. Analysis of self-assembly and apatite binding properties of amelogenin proteins lacking the hydrophilic C-terminal. Matrix Biol. 2002; 21: 197-205.
38. Warshawsky H. Organization of crystals in enamel. Anat Rec. 1989; 224: 242-62.
39. Fincham AG, Moradian-Oldak J, Diekwisch TGH, Layaruu DM, Wright JT, Bringas P et al. Evidence for amelogenin “nanospheres” as functional components of secretory stage enamel matrix. J Struct Biol. 1995; 115: 50-9.
40. Uchida T, Tanabe T, Fukae M, Shimizu M, Yamada M, Miake K et al. Immunochemical and immunohistochemical studies using antisera against porcine 25 kDa amelogenin, 89 kDa enamelin and the 13-17 kDa nonamelogenins, on immature enamel of pig and rat. Histochemistry. 1991; 96: 129-38.
41. Gibson CW, Yuan Z-A, Hall B, Longenecker G, Chen E, Thyagarajan T et al. Amelogenin-deficient mice display an amelogenesis imperfecta phenotype. J Biol Chem. 2001; 276: 31871-5.
42. Paine ML, Wang H-J, Snead ML. Amelogenin self-assembly and the role of the proline located within the carboxyl-teleopeptide. Connect Tiss Res. 2003; 44 (Suppl. 1): 52-7.
43. Ravassipour DB, Hart PS, Ritter AV, Yamauchi M, Gibson C, Wright JT. Unique enamel phenotype associated with amelogenin gene (AMELX) codon 41 point mutation. J Dent Res. 2000; 79: 1476-81.
44. Snead ML, Lau EC, Zeichner-David M, Fincham AG, Woo SL, Slavkin HC. DNA sequence for cloned cDNA for murine amelogenin reveals the amino acid sequence for enamel specific protein. Biochem Biophys Res Commun. 1985; 129: 812-8.
45. Simmer JP, Snead ML. Molecular biology of the amelogenin gene. In: Robinson C, Kirkham J, Shore R. Editors. Dental enamel: formation to destruction. Boca Raton: CRC Press; 1995. p. 59- 84.
46. Lench NJ, Winter GB. Characterization of molecular defects in X-linked amelogenesis imperfecta (AIH1). Hum Mut. 1995; 5: 252-9.
47. Collier PM, Sauk JJ, Rosenbloom J, Yuan ZA, Gibson CW. An amelogenin gene defect associated with human X-linked amelogenesis imperfecta. Arch Oral Biol. 1997; 42: 235-42.
48. Paine ML, Zhu D-H, Luo W, Bringas PJ, Goldberg M, White SN et al. Enamel biomineralization defects result from alterations to amelogenin self-assembly. J Struct Biol. 2000; 132: 191-200.
49. Hart PS, Hart TC, Simmer JP, Wright JT. A nomenclature for Xlinked amelogenesis imperfecta. Arch Oral Biol. 2002; 47: 255- 60.
50. . Snead ML. Amelogenin protein exhibits a modular design: implications for form and function. Connect Tissue Res. 2003; 44 (Suppl. 1): 47-51.
51. Paine ML, Lei Y-P, Dickerson K, Snead ML. Altered amelogenin self-assembly based on mutations observed in human X-linked amelogenesis imperfecta (AIH1). J Biol Chem. 2002; 277: 17112-6.
52. Paine ML, Luo W, Zhu D-H, Bringas PJ, Snead ML. Functional domains for amelogenin revealed by compound genetic defects. J Bone Miner Res. 2003; 18: 466-72.
53. Li W, Gibson CW, Abrams WR, Andrews DW, Denbesten PK. Reduced hydrolysis of amelogenin may result in X-linked amelogenesis imperfecta. Matrix Biol. 2001; 19: 755-60.
54. Ravindranath MHR, Moradian-Oldak J, Fincham AG. Tyrosyl motif in amelogenin binds N-acetyl-D-glucosamine. J Biol Chem. 1999; 274: 2464-71.
55. Akita H, Fukae M, Shimoda S, Aoba T. Localization of glycosylated matrix proteins in the secretory porcine enamel and their possible functional roles in enamel mineralization. Arch Oral Biol. 1992; 37: 953-62.
56. Ravindranath HH, Chen LS, Zeichner-David M, Ishima R, Ravindranath RM. Interaction between the enamel matrix proteins amelogenin and ameloblastin. Biochem Biophys Res Commun. 2004; 323: 1075-83.
57. Bouropoulos N, Moradian-Oldak J. Induction of apatite by the cooperative effect of amelogenin and the 32-kDa enamelin. J Dent Res. 2004; 83: 278-82.
58. Hu JC-C, Yamakoshi Y. Enamelin and autosomal-dominant amelogenesis imperfecta. Crit Rev Oral Biol Med. 2003; 14: 387-98.
59. Fukae M, Tanabe T. Nonamelogenin components of porcine enamel in the protein fraction free from the enamel crystals. Calcif Tissue Int. 1987; 40: 286-93.
60. Tanabe T, Aoba T, Moreno EC, Fukae M, Shimuzu M. Properties of phosphorylated 32 kDa nonamelogenin proteins isolated from porcine secretory enamel. Calcif Tissue Int. 1990; 46: 205-15.
61. Uchida T, Tanabe T, Fukae M, Shimizu M. Immunocytochemical and immunochemical detection of a 32 kDa nonamelogenin and related proteins in porcine tooth germs. Arch Histo Cytol. 1991; 54: 527-38.
62. Yamakoshi Y. Carbohydrate moieties of porcine 32kDa enamelin. Calcif Tissue Int. 1995; 56: 323-30.
63. Yamakoshi Y, Pinheiro FH, Tanabe T, Fukae M, Shimizu M. Sites of asparagine-linked oligosaccharides in porcine 32 kDa enamelin. Connect Tissue Res. 1998; 39: 39-46.
64. Fukae M, Tanabe T, Murakami C, Dohi N, Uchida T, Shimizu M. Primary structure of porcine 89 kDa enamelin. Adv Dent Res. 1996; 10: 111-8.
65. Kida M, Ariga T, Shirakawa T, Oguchi H, Sakiyama Y. Autosomaldominant hypoplastic form of amelogenesis imperfecta caused by an enamelin gene mutation at the exon-intro boundary. J Dent Res. 2002; 81: 738-42.
66. Osborn JW. The three-dimentional morphology of tufts in human enamel. Acta Anat. 1969; 73: 481-9.
67. Palamara J, Phakey PP, Rachinger WA, Orams HJ. Ultrastructure of spindles and tufts in human dental enamel. Adv Dent Res. 1989; 3: 249-57.
68. Robinson C, Shore RC, Kirkham J. Tuft protein: its relationship with the keratins and the developing enamel matrix. Calcif Tissue Int. 1989; 44: 393-8.
69. Robinson C, Lowe NR, Weatherell JA. Amino acid composition, distribution and origin of ‘tuft’ protein in human and bovine dental enamel. Arch Oral Biol. 1975; 20: 29-42.
70. Zeichner-David M, Diekwisch T, Fincham A, Lau E, MacDougall M, Moradian-Oldak J et al. Control of ameloblast differentiation. Int J Dev Biol. 1995; 39: 69-92.
71. Zeichner-David M, Vo H, Tan H, Diekwisch T, Berman B, Thiemann F et al. Timing of the expression of enamel gene products during mouse tooth development. Int J Dev Biol. 1997; 41: 27-38.
72. Paine CT, Paine ML, Luo W, Okamoto CT, Lyngstadaas SP, Snead ML. A tuftelin-interacting protein (TIP39) localizes to the apical secretory pole of mouse ameloblasts. J Biol Chem. 2000; 275: 22284-92.
73. . Paine CT, Paine ML, Snead ML. Identification of tuftelin- and amelogenin-interacting proteins using the yeast two-hybrid system. Connect Tissue Res. 1998; 38: 257-67.
74. Paine ML, Krebsbach PH, Chen LS, Paine CT, Yamada Y, Deutsch D et al. Protein-to-protein interactions: criteria defining the assembly of the enamel organic matrix. J Dent Res. 1998; 77: 496-502.
75. Luo W, Wen X, Wang HJ, MacDougall M, Snead ML, Paine ML. In vivo overexpression of tuftelin in the enamel organic matrix. Cells Tissues Organs. 2004; 177: 212-20.
76. Fukae M, Tanabe T. 45Ca labeled proteins found in porcine developing dental enamel at an early stage of development. Adv Dent Res. 1987; 1: 286-93.
77. Cerny R, Slaby I, Hammarström L, Wurtz T. A novel gene expressed in rat ameloblasts codes for proteins with cell biding domains. J Bone Miner Res. 1996; 11: 883-91.
78. McDougall M, Simmons D, Gu TT, Forsman-Semb K, Mardh CK, Mesbah M et al. Cloning, characterization and immunolocalization of human ameloblastin. Eur J Oral Sci. 2000; 108: 303-10.
79. Murakami C, Dohi N, Fukae M, Tanabe T, Yamakoshi Y, Wakida K et al. Immunochemical and immunohistochemical study of 27 and 29 kDa calcium binding proteins and related proteins in the porcine tooth germ. Histochem Cell Biol. 1997; 107: 485- 94.
80. Fong CD, Slaby I, Hammarström L. Amelin: an enamel related protein, transcribed in the cells of epithelial root sheath. J Bone Miner Res. 1996; 11: 892-8.
81. Lee SK, Krebsbach PH, Matsuki Y, Nanci A, Yamada KM, Yamada Y. Ameloblastin expression in rat incisor and human tooth germs. Int J Dev Biol. 1996; 40: 1141-50.
82. McDougall M, DuPont BR, Simmons D, Reus B, Krebsbach P, Karrman C et al. Ameloblastin gene (AMBN) maps within the critical autosomal dominant amelogenesis imperfecta region at chromosome 4q21. Genomics. 1997; 41: 115-8.
83. Paine ML, Wang HJ, Luo W, Krebsbach PH, Snead ML. A transgenic animal model resembling amelogenesis imperfecta related to ameloblastin overexpression. J Biol Chem. 2003; 278: 19447-52.
84. Tanabe T, Fukae M, Uchida T, Shimuzu M. The localization and characterization of proteinases for the inicial cleavage of porcine amelogenin. Calcif Tissue Int. 1992; 51: 213-7.
85. Eastoe JE. Enamel protein chemistry: past, present and future. J Dent Res. 1979; 58B: 753-64.
86. Robinson C, Kirkham J. Dynamics of amelogenesis as revealed by protein compositional studies. In: Butler WT, editor. The chemistry and biology of mineralized tissues. Birmingham: EBSCO Media; 1985. p. 248-63.
87. Bartlett JD, Simmer JP. Proteinases in developing dental enamel. Crit Ver Oral Biol. 1999; 10: 425-41.
88. Bartlett JD, Simmer JP, Xue J, Margolis HC, Moreno EC. Molecular cloning and mRNA tissue distribution of a novel matrix mettaloproteinase isolated from porcine enamel organ. Gene. 1996; 183: 123-8.
89. Llano E, Pendás AM, Knäuper V, Sorsa T, Salo T, Salido E et al. Identification and structural and functional characterization of human enamelysin (MMP-20). Biochemistry. 1997; 36: 15101- 8.
90. Denbesten PK, Punzi JS, Li W. Purification and sequencing of a 21 kDa and 25kDa bovine enamel metalloproteinase. Eur J Oral Sci. 1998; 106: 345-9.
91. Caterina NC, Shi J, Sun X, Qian Q, Yamada S, Liu Y et al. Cloning, characterization, and expression analysis of mouse enamelysin. J Dent Res. 2000; 79: 1697-703.
92. Bartlett JD, Ryu OH, Xue J, Simmer JP, Margolis HC. Enamelysin mRNA displays a developmentally defined pattern of expression and encodes a protein which degrades amelogenin. Connect Tissue Res. 1998; 39: 405-13.
93. Caterina JJ, Skobe Z, Shi J, Ding Y, Simmer JP, Birkedal-Hansen H et al. Enamelysin (matrix metalloproteinase 20)-deficient mice display an amelogenesis imperfecta phenotype. J Biol Chem. 2002; 277: 49598-604.
94. Ryu OH, Fincham AG, Hu C-C, Zhang C, Qian Q, Bartlett JD et al. Characterization of recombinant enamelysin activity and cleavage of recombinant pig and mouse amelogenin. J Dent Res. 1999; 78: 743-50.
95. Li W, Machule D, Gao C, DenBesten PK. Activation of recombinant bovine matrix metalloproteinase-20 and its hydrolysis of two amelogenin oligopeptides. Eur J Oral Sci. 1999; 107: 352-9.
96. Thompsom MW, Mciness RR, Willard HF. Genetics in Medicine. 5th ed. Philadelphia: WB Saunders; 1991. p. 56.
97. Tanabe T. Purification and characterization of proteolytic enzymes in porcine immature enamel. Tsurumi U Dent J. 1984; 10: 443-52.
98. Simmer JP, Ryu OH, Qian Q, Zhang C, Cao X, Sun X. Cloning and characterization of a cDNA encoding human EMSP1. In: Goldberg M, Robinson C, editors. Chemistry and biology ofmineralized tissues: Proceedings of the Sixth International Conference, Vittel, France, 1998. Vittel: American Academy of Orthopaedic Surgeons; 2000. p. 205-8.
99. Hu C-C, Zhang C, Sun X, Yang Y, Cao X, Ryu O et al. Characterization of the mouse and human PRSS17 genes, their relationship to other proteases, and expression in developing incisors. Gene. 2000; 251: 1-8.
100. Simmer JP, Fukae M, Tanabe T, Yamakoshi Y, Uchida T, Xhu J et al. Purification, characterization and cloning of enamel matrix serine proteinase 1. J Dent Res. 1998; 77: 377-86.
101. Moradian-Oldak J, Leung W, Tan J, Fincham AG. Effect of apatite crystals on the activity of amelogenin degrading enzymes in vitro. Calcif Tissue Int. 1998; 39: 131-40.
102. Okamura K. Localization of serum albumin in dentin and enamel. J Dent Res. 1983; 62: 100-4.
103. Limeback H, Sakarya H, Chu W, MacKinnon M. Serum albumin and its acid hydrolysis peptides dominate preparations of mineral-bound enamel proteins. J Bone Miner Res. 1989; 4: 235-41.
104. Strawich E, Seyer J, Glimcher MJ. Immuno-identification of two non-amelogenin proteins of developing bovine enamel isolated by affinity chromatography. Further proof that tooth enamelins are mainly serum proteins. Connect Tissue Res. 1993; 29: 163-9.
105. Robinson C, Brookes SJ, Kirkham J, Bonass WA, Shore RC. Crystal growth in dental enamel: the role of amelogenins and albumin. Adv Dent Res. 1996; 10: 179-80.
106. Yuan ZA, Collier PM, Rosenbloom J, Gibson CW. Analysis of amelogenin mRNA during bovine tooth development. Arch Oral Biol. 1996; 41: 205-13.
107. Kinoshita Y. Incorporation of serum albumin into the developing dentine and enamel matrix in the rabbit incisor. Calcif Tissue Int. 1979; 29: 41-6.
108. Shore RC, Robinson C, Kirkham J, Brookes SJ. Structure of developing enamel. In: Robinson C, Kirkham J, Shore RC, editors. Dental enamel, formation to destruction. Boca Raton: CRC Press; 1995. p. 135-50.
109. Shapiro IM, Amdur BH. Enamel matrix pigmentation in the developing bovine tooth. Arch Oral Biol. 1965; 10: 1015-8.
110. Goldberg M, Vermelin L, Mostermans P, Lécolle S, Septier D, Godeau C et al. Fragmentation of the distal portion of Tome’s processes of secretory ameloblasts in the forming enamel of rat incisors. Connect Tissue Res. 1998; 38: 159-69.
111. Smith CE, Chen WY, Issid M, Fazel A. Enamel matrix protein turnover during amelogenesis: basic biochemical properties of short-lived sulfated enamel proteins. Calcif Tissue Int. 1995; 57: 133-44.