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Induction of Apatite by the Cooperative Effect of Amelogenin and the 32-kDa Enamelin

Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, 2250 Alcazar Street, Los Angeles, CA 90033;

^* corresponding author, joldak{at}usc.edu

	ABSTRACT

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Extracellular matrix proteins are considered to play essential roles in controlling the nucleation, growth, and organization of hydroxyapatite crystals during enamel formation. The effects of amelogenin and the 32-kDa enamelin proteins on apatite nucleation were investigated by a steady-state gel diffusion device containing 10% gelatin gels loaded with 0, 0.75%, and 1.5% (w/w) native porcine amelogenins. It was found that the induction time for hydroxyapatite precipitation was strongly increased by the presence of amelogenins, suggesting an inhibitory effect of apatite nucleation. Addition of 18 µg/mL of 32-kDa enamelin to 10% gelatin also caused inhibition of nucleation. Remarkably, addition of 18 and 80 µg/mL of 32-kDa enamelin in gels containing 1.5% amelogenin accelerated the nucleation process in a dose-dependent manner. Our observations 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.

KEY WORDS: tooth enamel • amelogenin • enamelin • nucleation • hydroxyapatite

	INTRODUCTION

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Fundamental concepts related to the mechanisms of dental enamel mineralization are common among many mineralized tissues (Lowenstam and Weiner, 1989). Dental enamel is the most highly mineralized tissue in the vertebrate body and is comprised of carbonated hydroxyapatite crystals that are unusually large and elongated compared with apatite crystals in other skeletal tissues (Daculsi et al., 1984). Enamel mineralization is an extracellular event that takes place in an amelogenin-rich matrix constituting more than 90% of the total protein components (Termine et al., 1980). The remaining matrix is composed of acidic enamelins, proteinases, and ameloblastins (Fincham et al., 1999). Numerous structural studies have supported the notion that amelogenins self-assemble to form nanosphere structures that further assemble to form a supramolecular framework serving as a scaffold for the initiation and oriented growth of enamel apatite crystals (Robinson et al., 1998; Fincham et al., 1999; Wen et al., 1999; Paine et al., 2000). While several authors have supported the concept of enamel crystal orientation and habit being controlled through an organized assembly of the constituent proteins of the extracellular matrix, the mechanisms for crystal nucleation remain unclear and controversial. Two different scenarios have been proposed: (1) Crystal nucleation occurs at the DEJ and is promoted by either one of the components of dentin extracellular matrix or dentin crystals (Arsenault and Robinson, 1989; MacDougall et al., 1997); and (2) crystal nucleation is independent of dentin mineral formation and is modulated by the components of the enamel extracellular matrix (Diekwisch et al., 1995). Multiple organized nucleation sites (nanoscale precursor subunits) composed of amelogenins, enamelins, ameloblastins, and calcium-phosphate ions have also been proposed to serve as nucleators of enamel apatite crystals (Robinson et al., 2003). Enamelins that were originally defined as non-amelogenin acidic proteins (Termine et al., 1980) are ameloblastin-specific (Hu et al., 2000). The localization of enamelin proteolytic products has suggested their close association to enamel apatite crystal faces and their potential involvement in controlling nucleation and possibly growth modulation of the crystallites (Uchida et al., 1991). The conservation of enamelin structural features—namely, glycosylation and phosphorylation sites on the 32-kDa enamelin in the human, mouse, and pig—suggests those structural domains to be important functional domains of the protein (Yamakoshi et al., 1998; Hu et al., 2000). The putative spatial relationship of enamelin protein with the apatite crystallographic axis (Jodaikin et al., 1986) and its high affinity to interact with apatite crystals support our hypothesis that, among other enamel proteins of the extracellular matrix, the 32-kDa enamelin is the most appropriate candidate to act as crystal nucleator of enamel apatite (Doi et al., 1984; Tanabe et al., 1990).

Amelogenin and enamelin have been shown to be critical for normal enamel formation, as documented by recent transgenic and null mice model studies (Paine et al., 2000; Gibson et al., 2001), as well as by linkage analysis of human pedigrees with defective tooth enamel formation, called amelogenesis imperfecta (Kindelan et al., 2000; Kida et al., 2002). While such studies prove that these extracellular matrix proteins play critical physiological functions in the formation of enamel formation, clear insight into the mechanism of their action is still lacking.

Considering the scenario that nucleation of enamel crystals is independent of apatite crystal nucleation in dentin, we have implemented a steady-state gel-diffusion in vitro experimental system that was originally developed by Hunter et al.(1986) to assess the effect of the 32-kDa enamelin on hydroxyapatite nucleation in 10% gelatin gel. The present study was aimed at examining the hypothesis that the 32-kDa, the most stable proteolytically cleaved enamelin, has the potential to serve as a nucleator of enamel apatite crystallites. The use of gelatin gel allowed amelogenin and enamelin to be applied at different concentrations, and therefore dose-dependency of nucleation by these proteins could be evaluated. It was observed that while incorporation of amelogenin into the gel caused delay in apatite nucleation, addition of enamelin to the amelogenin/gel mixture system enhanced the induction, suggesting a cooperative effect between the two proteins in promoting the nucleation of apatite crystals.

	MATERIALS & METHODS

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Protein Extraction
Enamel scrapings were collected from unerupted fourth and fifth mandibular molars of fresh six-month-old pig jaws. The pig mandibles were obtained fresh from a local slaughterhouse (Farmer John Clougherty Co., Los Angeles, CA, USA) through Sierra For Medical Science (Santa Fe Springs, CA, USA). The United States Department of Agriculture has inspected and approved the process of sample harvesting. Proteins were isolated from porcine developing enamel by a slight modification of the dissociative extraction technique described by Termine et al.(1980).

Amelogenins were extracted in guanidinium hydrochloride (4M)-Tris (50 mM) at pH 7.4, de-salted, and concentrated by Amicon ultrafiltration, with a 10-kDa cut-off Amicon YM-10 membrane against 0.5% formic acid. The protein solution was lyophilized and stored in -20°C. The extract was characterized by SDS-PAGE to be a mixture of secreted amelogenin (25 K, 7.4%) and its processed products (23 K, 10.7%; 20 K, 49.5%; < 18 kDa, 32.4%) (Wen et al., 1999) (Fig. 1A, lane 2).

View larger version (30K): [in this window] [in a new window]   Figure 1. In vitro experimental set-up for the nucleation of apatite by amelogenins and the 32-kDa enamelin. (A) Sodium dodecyl sulfate-polyacrylamide gel (15% acrylamide) electrophoresis (SDS-PAGE) patterns of extracted amelogenins and the 32-kDa enamelin. Lane 1: Gibco BRL protein molecular-weight standard containing proteins with 43-k, 29-k, 18-k, 14-k, and 5-k molecular weights. Lane 2: amelogenins extracted from the extracellular enamel matrix of developing pig mandibular molars by the dissociative technique as described in MATERIALS & METHODS. The extract was characterized to be a mixture of secreted amelogenin (25 K, 7.4%) and its processed products (23 K, 10.7%; 20 K [the major band], 49.5%; 14–18 kDa, 32.4%) (Wen et al., 1999). Lane 3: The 32-KDa enamelin with an apparent molecular weight of 32 kDa. (B) Schematic representation for the experimental set-up used for monitoring nucleation of apatite crystals. Induction time was determined based on the difference in calcium uptake by the gel between the control (without phosphate) and the samples (with phosphate) as described in Fig. 3.

Apatite Nucleation
The experiments were carried out according to the gel-diffusion technique developed by Hunter et al.(1986) with slight modifications (Fig. 1B). Stock solutions of 2 M NaCl, 1 M Tris-HCl, pH = 8, 2 M CaCl₂, and 2 M NaHPO₄ were prepared in de-ionized water with reagent-grade crystalline reagents. Three types of working solutions containing 0.01% sodium azide were prepared with the following compositions: (Solution A) 10 mM Ca, 50 mM NaCl, 100 mM Tris-HCl, pH = 7.4; (Solution B) 6 mM P, 50 mM NaCl, 100 mM Tris-HCl, pH = 7.4; and (Solution C) 50 mM NaCl, 100 mM Tris-HCl, pH = 7.4.

    Gelatin-amelogenin gels
Using the following procedure, we prepared 10% gelatin gels containing 0, 0.75, and 1.5% amelogenins. Samples (containing phosphate ions) and control (without phosphate ions) were prepared in triplicate. An appropriate amount (30 or 15 mg) of the lyophilized amelogenins was mixed with 0.150 mL of solution B, and the resulting mixture was vortexed for 15 sec. The same procedure was repeated with solution C. Gelatin powder (BioRad, Hercules, CA, USA) was mixed with solution B or C to produce a 10.9% w/v mixture. The pH was adjusted to 7.4 by the addition of NaOH via a combination glass electrode. Finally, 1.85 mL of the 10.9% w/v gelatin gel was mixed with 0.15 mL matrix (solution B with amelogenin), and 0.4 mL of the produced mixture was transferred into plastic wells. The gels were stored overnight at 4°C and used the next day for the nucleation experiments.

    The effect of amelogenin
The gel slabs were equilibrated at room temperature for 3 hrs and overlaid with 2.5 mL of solution A containing 10 mM Ca plus 1.25 µCi ⁴⁵CaCl₂ (Perkin Elmer Life Sciences, Boston, MA, USA). Aliquots of 50 µL of the upper part of each well were transferred into scintillation vials containing 4 mL of scintillation liquid (Bio-Safe RPI, Chicago, IL, USA), and the radioactivity was measured in a Beckman liquid scintillation counter (Beckman LS-5801, Beckman Instruments, Fullerton, CA, USA). Samples were collected every 1–2 hrs during the first day and then after 12 and 24 hrs. All experiments were carried out at room temperature for 5 days. The results are expressed as the ratio of ⁴⁵Ca radioactivity (counts per min) measured at time t to that measured at time zero and represent the mean ± standard deviation of 3 measurements. Comparisons between the sample and the control for each measurement were made by Student’s t test, and statistically significant differences were defined at p < 0.05. Induction time was defined as the time when the comparison between the sample (phosphate-containing gel) and the control for each measurement was statistically significant.

    The effect of the 32-kDa enamelin
Enamelin was adsorbed onto the gelatin gel by 35 µL of enamelin solution (18 or 80 µg/mL) being spread on top of the gel. The gel was then covered with calcium solution containing ⁴⁵Ca, and the calcium uptake was measured as described above. In parallel experiments, phosphovitin was used as a positive control for nucleation.

    Transmission electron microscopy
Apatite precipitates were recovered from the gel-solution interface, heated at 40°C, and centrifuged immediately. The supernatant was discarded, and a small piece of the pellet was re-suspended in ethanol, placed on carbon-backed 300-mesh Parlodion-coated copper grids, and air-dried as previously described (Moradian-Oldak et al., 1991). The specimens were examined by a JEOL TEM (JEM1200-EX, Tokyo, Japan) operated at 80 kV. Selected-area electron diffraction was performed with an aperture size of 20 µm.

	RESULTS

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A thin layer of apatite crystals was formed at the interface between the gel and the liquid overlaid on the surface of the gel (Fig. 1B). A representative TEM image of crystals formed in 10% gelatin gel containing 1.5% amelogenin and the corresponding electron diffraction pattern is presented in Fig. 2. Morphological analysis of the images revealed elongated thin crystals about 100 nm long. The arrowhead reflections 1 and 2 correspond to lattice spacing of 3.44 and 2.74 Å, respectively, which are characteristic of hydroxyapatite (Fig. 2).

View larger version (138K): [in this window] [in a new window]   Figure 2. Transmission electron micrograph of crystals grown in 10% gelatin-1.5% amelogenin gel, indicating the formation of apatite crystals. The insert shows the corresponding electron diffraction pattern. The arrowhead reflections 1 and 2 correspond to lattice spacings of 3.44 and 2.74 Å, respectively, which are characteristic of hydroxyapatite.

View larger version (12K): [in this window] [in a new window]   Figure 3. Uptake of calcium ions from the upper solution by gelatin gel containing 1.5% amelogenin with (, sample) and without (, control) phosphate ions. Comparisons between the sample and the control for each measurement were made by Student’s t test, and statistically significant differences were defined at p < 0.05. Induction time was defined as the time when the comparison between the sample (phosphate-containing gel) and the control for each measurement was statistically significant. Values are based on ratio percentages of 45Ca radioactivity (counts per min) at time t to that measured at time zero as the function of time. The data (13 points) shown are the mean ± SD of 3 separate experiments. The variability ranged between SD = 0.4–1.9 for control and SD = 0.3–2.3 for sample.

	DISCUSSION

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Our present study was aimed at exploring the possibility of enamel apatite nucleation being independent of the presence of dentin apatite crystals (Diekwisch et al., 1995). We hypothesized that the process of crystal nucleation during enamel biomineralization is highly controlled and mediated through molecular interactions, both protein-protein (amelogenin self-assembly and amelogenin-enamelin interactions) and protein-mineral interactions. The best-studied, and apparently the most stable, enamelin cleavage product is the 32-kDa (Yamakoshi, 1995).

We systematically examined the effects of amelogenin and the 32-kDa enamelin on the induction of synthetic apatite crystals precipitated in 10% gelatin gel. Analysis of the data showed that incorporation of amelogenins into gelatin matrix at concentrations of 0.75 and 1.5 w/w% (equivalent to 7.5–15 mg/mL) resulted in inhibition of hydroxyapatite nucleation in a dose-dependent manner (Table). Using the same gel-diffusion system without the presence of phosphate ions, we have previously shown that the presence of amelogenin in gelatin gel did not affect the rate of calcium diffusion into the gel (Moradian-Oldak et al., 2003). We therefore propose the inhibition by amelogenin to be a direct effect of the protein on the process of crystal initiation. This inhibitory effect may result from structural re-organization of the amelogenin/gelatin-gel that may block the clusters of ions needed for the formation of nuclei (Blumenthal et al., 1991). It is noteworthy that the same level of delay was achieved by the 32-kDa enamelin, an acidic glycoprotein, at much lower concentration (18 µg/mL or 0.0018%).

Remarkably, addition of the 32-kDa enamelin (18 µg/mL) to the gelatin/amelogenin gel caused promotion of apatite nucleation almost six-fold, reducing the induction time from 20–48 hrs to 5–6 hrs (Table). It has been shown that many acidic glycoproteins act as strong inhibitors of crystal nucleation and growth in solution but can serve as effective nucleators once they have been adsorbed onto surfaces and adopted defined structures (Lussi et al., 1988). Indeed, enamelin showed a strong affinity to apatite crystals in vitro and was the most potent inhibitor of apatite crystal growth in solution (Doi et al., 1984; Tanabe et al., 1990).

We interpret our data to suggest that amelogenin and enamelin cooperate to promote nucleation of apatite crystals during enamel formation. We propose that the 32-kDa enamelin bound to amelogenin serves as a potential nucleator for apatite crystals through its oriented and structured ion-binding motifs, such as phosphoserines (Saito et al., 2000). We speculate that such structured orientation of the 32-kDa enamelin would not be achieved with gelatin gel only. The pig 32-kDa enamelin has 106 amino acids (residues 174–279), which include two phosphoserines and three glycosylated asparagines (Yamakoshi et al., 1998). The exact structural binding relationship between enamelin and amelogenin at the molecular level has not yet been firmly established. However, enamelin has the potential to assemble with the amelogenin matrix through the tri-tyrosine motif on amelogenin N-terminus, interacting with the N-acetylglucosamine on the 32-kDa enamelin (Ravindranath et al., 1999; Yamakoshi et al., in press). Our in vitro model system was designed in such a way that, after the gel was overlaid with enamelin-containing solution, the molecules of the 32-kDa enamelin will bind with the gel and, more specifically, with amelogenin. In this case, nucleation occurred on the surface of the gel. The observation that apatite formation took place in the outer surface of the gel supports the idea that induction of apatite was promoted by the 32-kDa enamelin bound on the surface of amelogenin/gelatin gel. Further detailed study on the amelogenin/enamelin complex is needed to support the proposed cooperative mechanism.

	ACKNOWLEDGMENTS

 
We thank Dr. Lingli Wang for excellent technical assistant in protein purification. This study was supported by NIH-NIDCR grants DE-12350 and DE-13414 (JMO).

	FOOTNOTES

 
¹ present address, Department of Materials Science, University of Patras, Greece;

Received May 7, 2003; Last revision January 15, 2004; Accepted January 21, 2004

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Arsenault AL, Robinson BW (1989). The dentino-enamel junction: a structural and microanalytical study of early mineralization. Calcif Tissue Int 45:111–121.[ISI][Medline]

Blumenthal NC, Cosma V, Gomes E (1991). Regulation of hydroxyapatite formation by gelatin and type I collagen gels. Calcif Tissue Int 48:440–442.[ISI][Medline]

Daculsi G, Menanteau J, Kerebel LM, Mitre D (1984). Enamel crystals: size, shape, length and growing process; high resolution TEM and biochemical study. In: Tooth enamel IV. Fearnhead RW, Suga S, editors. Bristol: John Wright Ltd., pp. 14–18.

Diekwisch TG, Berman BJ, Gentner S, Slavkin HC (1995). Initial enamel crystals are not spatially associated with mineralized dentine. Cell Tissue Res 279:149–167.[ISI][Medline]

Doi Y, Eanes ED, Shimokawa H, Termine JD (1984). Inhibition of seeded growth of enamel apatite crystals by amelogenin and enamelin proteins in vitro. J Dent Res 63:98–105.[Abstract/Free Full Text]

Fincham A, Moradian-Oldak J, Simmer JP (1999). The structural biology of the developing dental enamel matrix. J Struct Biol 126:270–299.[ISI][Medline]

Gibson CW, Yuan ZA, Hall B, Longenecker G, Chen E, Thyagarajan T, et al. (2001). Amelogenin-deficient mice display an amelogenesis imperfecta phenotype. J Biol Chem 276:31871–31875.[Abstract/Free Full Text]

Hu CC, Hart TC, Dupont BR, Chen JJ, Sun X, Qian Q, et al. (2000). Cloning human enamelin cDNA, chromosomal localization, and analysis of expression during tooth development. J Dent Res 79:912–919.[Abstract/Free Full Text]

Hunter GK, Nyburg SC, Pritzker KP (1986). Hydroxyapatite formation in collagen, gelatin, and agarose gels. Coll Relat Res 6:229–238.[ISI][Medline]

Jodaikin A, Traub W, Weiner S (1986). Protein conformation in rat tooth enamel. Arch Oral Biol 31:685–689.[ISI][Medline]

Kida M, Ariga T, Shirakawa T, Oguchi H, Sakiyama Y (2002). Autosomal-dominant hypoplastic form of amelogenesis imperfecta caused by an enamelin gene mutation at the exon-intron boundary. J Dent Res 81:738–742.[Abstract/Free Full Text]

Kindelan SA, Brook AH, Gangemi L, Lench N, Wong FS, Fearne J, et al. (2000). Detection of a novel mutation in X-linked amelogenesis imperfecta. J Dent Res 79:1978–1982.[Abstract/Free Full Text]

Lowenstam H, Weiner S (1989). On biomineralization. New York: Oxford University Press, p. 175.

Lussi A, Crenshaw MA, Linde A (1988). Induction and inhibition of hydroxyapatite formation by rat dentine phosphoprotein in vitro. Arch Oral Biol 33:685–691.[ISI][Medline]

MacDougall M, Simmons D, Luan X, Nydegger J, Feng J, Gu TT (1997). Dentin phosphoprotein and dentin sialoprotein are cleavage products expressed from a single transcript coded by a gene on human chromosome 4. Dentin phosphoprotein DNA sequence determination. J Biol Chem 272:835–842.[Abstract/Free Full Text]

Moradian-Oldak J, Weiner S, Addadi L, Landis WJ, Traub W (1991). Electron imaging and diffraction study of individual crystals of bone, mineralized tendon and synthetic carbonate apatite. Connect Tissue Res 25:219–228.[ISI][Medline]

Moradian-Oldak J, Tan J, Fincham AG (1998). Interaction of amelogenin with hydroxyapatite crystals; an adherence effect through amelogenion molecular self-association. Biopolymers 46:225–238.[ISI][Medline]

Moradian-Oldak J, Iijima M, Bouropoulos N, Wen HB (2003). Assembly of amelogenin proteolytic products and control of octacalcium phosphate crystal morphology. Connect Tissue Res 44:58–64.

Mullin JW (1997). Crystallization. Oxford: Butterworth-Heinemann, p. 198.

Paine ML, Zhu DH, Luo W, Bringas P Jr, Goldberg M, White SN, et al. (2000). Enamel biomineralization defects result from alterations to amelogenin self-assembly. J Struct Biol 132:191–200.[ISI][Medline]

Ravindranath RM, Moradian-Oldak J, Fincham AG (1999). Tyrosyl motif in amelogenins binds N-acetyl-D-glucosamine. J Biol Chem 274:2464–2471.[Abstract/Free Full Text]

Robinson C, Brookes SJ, Shore RC, Kirkham J (1998). The developing enamel matrix: nature and function. Eur J Oral Sci 106(Suppl 1):282–291.

Robinson C, Shore RC, Wood SR, Brookes SJ, Smith DAM, Wright JT, et al. (2003). Subunit structures in hydroxyapatite crystal dvelopment in enamel: implications for amelogenesis imperfecta. Connect Tissue Res 44:65–71.

Saito T, Yamauchi M, Abiko Y, Matsuda K, Crenshaw MA (2000). In vitro apatite induction by phosphophoryn immobilized on modified collagen fibrils. J Bone Miner Res 15:1615–1619.[ISI][Medline]

Tanabe T, Aoba T, Moreno EC, Fukae M, Shimuzu M (1990). Properties of phosphorylated 32 kd nonamelogenin proteins isolated from porcine secretory enamel. Calcif Tissue Int 46:205–215.[ISI][Medline]

Termine JD, Belcourt AB, Christner PJ, Conn KM, Nylen MU (1980). Properties of dissociatively extracted fetal tooth matrix proteins. I. Principal molecular species in developing bovine enamel. J Biol Chem 20:9760–9768.

Uchida T, Tanabe T, Fukae M, Shimizu M (1991). Immunocytochemical and immunochemical detection of a 32 kDa nonamelogenin and related proteins in porcine tooth germs. Arch Histol Cytol 54:527–538.[ISI][Medline]

Wen HB, Moradian-Oldak J, Leung W, Bringas P Jr, Fincham AG (1999). Micro-structure of an amelogenin gel matrix. J Struct Biol 126:42–51.[ISI][Medline]

Yamakoshi Y (1995). Carbohydrate moieties of porcine 32kDa enamelin. Calcif Tissue Int 56:323–330.[ISI][Medline]

Yamakoshi Y, Pinheiro FH, Tanabe T, Fukae M, Shimizu M (1998). Sites of asparagine-linked oligosaccharides in porcine 32kDa enamelin. Connect Tissue Res 39:39–46.[Medline]

Yamakoshi Y, Hu JC-C, Fukae M, Tanabe T, Oida S, Simmer J. Amelogenin and 32 kDa enamelin protein-protein interactions. In: Proceedings of the 8th International Symposium on Biomineralization "Biomineralization: formation, diversity, evolution and application" September 25 - 28, 2001. Sasagawa I, editor. Kurulawa, Niigata, Japan (in press).

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