N-terminal and C-terminal (CT) domains of amelogenin have been shown to be essential for proper enamel formation. Recent studies have also suggested that although the C-terminus plays an apparent role in protein-mineral interactions, other amelogenin structural domains are involved. The objective was to explore the role of the amelogenin N-terminus in the regulation of calcium phosphate formation in vitro. Spontaneous mineralization studies were carried out using the phosphorylated (+P) and nonphosphorylated (–P) N-terminus of the leucine-rich amelogenin peptide (LRAP) that lacks the hydrophilic CT domain. Mineralization progress was monitored via changes in solution pH. Mineral phases formed were characterized using TEM, selected area electron diffraction, and FT-IR. In controls, amorphous calcium phosphate was initially formed and subsequently transformed to randomly oriented hydroxyapatite (HA) plate-like crystals. In contrast to the control, LRAP(+P)-CT stabilized ACP formation for >1 day, while LRAP(–P)-CT accelerated the transformation of ACP to HA but had little effect on crystal shape or orientation. In conclusion, the N-terminal domain found in LRAP, as in amelogenins, appears to have the capacity to interact with forming calcium phosphate mineral phases. Results suggest that the N-terminal domain of amelogenin may play a direct role in early stages of enamel formation.

Keywords:
Amelogenin, Calcium phosphates, Enamel, LRAP, Mineralization
1.
Aizenberg, J., D.A. Muller, J.L. Grazul, D.R. Hamann (2003) Direct fabrication of large micropatterned single crystals. Science 299: 1205–1208.
2.
Aoba, T., M. Fukae, T. Tanabe, M. Shimizu, E.C. Moreno (1987) Selective adsorption of porcine-amelogenins onto hydroxyapatite and their inhibitory activity on hydroxyapatite growth in supersaturated solutions. Calcif Tissue Int 41: 281–289.
3.
Bartlett, J.D., J.P. Simmer (1999) Proteinases in developing dental enamel. Crit Rev Oral Biol Med 10: 425–441.
4.
Beniash, E., R.A. Metzler, R.S. Lam, P.U. Gilbert (2009) Transient amorphous calcium phosphate in forming enamel. J Struct Biol 166: 133–143.
5.
Beniash, E., J.P. Simmer, H.C. Margolis (2005) The effect of recombinant mouse amelogenins on the formation and organization of hydroxyapatite crystals in vitro. J Struct Biol 149: 182–190.
6.
Eanes, E.D. (2001) Amorphous calcium phosphate; in Chow, L.C., E.D. Eanes (eds): Octacalcium Phosphate. Basel, Karger, p 167.
7.
Fincham, A.G., J. Moradian-Oldak, J.P. Simmer (1999) The structural biology of the developing dental enamel matrix. J Struct Biol 126: 270–299.
8.
Fukumoto, S., T. Kiba, B. Hall, N. Iehara, T. Nakamura, G. Longenecker, P.H. Krebsbach, A. Nanci, A.B. Kulkarni, Y. Yamada (2004) Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts. J Cell Biol 167: 973–983.
9.
Gajjeraman, S., K. Narayanan, J. Hao, C. Qin, A. George (2007) Matrix macromolecules in hard tissues control the nucleation and hierarchical assembly of hydroxyapatite. J Biol Chem 282: 1193–1204.
10.
Gericke, A., C. Qin, L. Spevak, Y. Fujimoto, W.T. Butler, E.S. Sorensen, A.L. Boskey (2005) Importance of phosphorylation for osteopontin regulation of biomineralization. Calcif Tissue Int 77: 45–54.
11.
Gibson, C.W., Z.A. Yuan, B. Hall, G. Longenecker, E. Chen, T. Thyagarajan, T. Sreenath, J.T. Wright, S. Decker, R. Piddington, G. Harrison, A.B. Kulkarni (2001) Amelogenin-deficient mice display an amelogenesis imperfecta phenotype. J Biol Chem 276: 31871–31875.
12.
Hu, J.C., Y. Hu, C.E. Smith, M.D. McKee, J.T. Wright, Y. Yamakoshi, P. Papagerakis, G.K. Hunter, J.Q. Feng, F. Yamakoshi, J.P. Simmer (2008) Enamel defects and ameloblast-specific expression in Enam knock-out/lacz knock-in mice. J Biol Chem 283: 10858–10871.
13.
Kwak, S.Y., F.B. Wiedemann-Bidlack, E. Beniash, Y. Yamakoshi, J.P. Simmer, A. Litman, H.C. Margolis (2009) Role of 20-kDa amelogenin (P148) phosphorylation in calcium phosphate formation in vitro. J Biol Chem 284: 18972–18979.
14.
Loste, E., F. Meldrum (2001) Control of calcium carbonate morphology by transformation of an amorphous precursor in a constrained volume. Chem Commun 10: 901–902.
15.
Mahamid, J., A. Sharir, L. Addadi, S. Weiner (2008) Amorphous calcium phosphate is a major component of the forming fin bones of zebrafish: indications for an amorphous precursor phase. Proc Natl Acad Sci USA 105: 12748–12753.
16.
Margolis, H.C., E. Beniash, C.E. Fowler (2006) Role of macromolecular assembly of enamel matrix proteins in enamel formation. J Dent Res 85: 775–793.
17.
Moradian-Oldak, J., N. Bouropoulos, L. Wang, N. Gharakhanian (2002) Analysis of self- assembly and apatite binding properties of amelogenin proteins lacking the hydrophilic C-terminal. Matrix Biol 21: 197–205.
18.
Moradian-Oldak, J., J. Tan, A.G. Fincham (1998) Interaction of amelogenin with hydroxyapatite crystals: an adherence effect through amelogenin molecular self-association. Biopolymers 46: 225–238.
19.
Nagano, T., A. Kakegawa, Y. Yamakoshi, S. Tsuchiya, J.C. Hu, K. Gomi, T. Arai, J.D. Bartlett, J.P. Simmer (2009) Mmp-20 and Klk4 cleavage site preferences for amelogenin sequences. J Dent Res 88: 823–828.
20.
Paine, M.L., W. Luo, D.H. Zhu, P. Bringas, Jr., M.L. Snead (2003a) Functional domains for amelogenin revealed by compound genetic defects. J Bone Miner Res 18: 466–472.
21.
Paine, M.L., M.L. Snead (1997) Protein interactions during assembly of the enamel organic extracellular matrix. J Bone Miner Res 12: 221–227.
22.
Paine, M.L., H.J. Wang, M.L. Snead (2003b) Amelogenin self-assembly and the role of the proline located within the carboxyl-teleopeptide. Connect Tissue Res 44(suppl 1): 52–57.
23.
Pugach, M.K., Y. Li, C. Suggs, J.T. Wright, M.A. Aragon, Z.A. Yuan, D. Simmons, A.B. Kulkarni, C.W. Gibson (2010) The amelogenin C-terminus is required for enamel development. J Dent Res 89: 165–169.
24.
Shaw, W.J., A.A. Campbell, M.L. Paine, M.L. Snead (2004) The COOH terminus of the amelogenin, LRAP, is oriented next to the hydroxyapatite surface. J Biol Chem 279: 40263–40266.
25.
Shaw, W.J., K. Ferris (2008) Structure, orientation, and dynamics of the C-terminal hexapeptide of LRAP determined using solid-state NMR. J Phys Chem B 112: 16975–16981.
26.
Shaw, W.J., K. Ferris, B. Tarasevich, J.L. Larson (2008) The structure and orientation of the C-terminus of LRAP. Biophys J 94: 3247–3257.
27.
Smith, C.E., R. Wazen, Y. Hu, S.F. Zalzal, A. Nanci, J.P. Simmer, J.C. Hu (2009) Consequences for enamel development and mineralization resulting from loss of function of ameloblastin or enamelin. Eur J Oral Sci 117: 485–497.
28.
Termine, J.D., A.B. Belcourt, P.J. Christner, K.M. Conn, M.U. Nylen (1980) Properties of dissociatively extracted fetal tooth matrix proteins. 1. Principal molecular species in developing bovine enamel. J Biol Chem 255: 9760–9768.
29.
Uchida, T., T. Tanabe, M. Fukae, M. Shimizu, M. Yamada, K. Miake, S. Kobayashi (1991) Immunochemical and immunohistochemical studies, using antisera against porcine 25 kDa amelogenin, 89 kDa enamelin and the 13–17 kDa nonamelogenins, on immature enamel of the pig and rat. Histochemistry 96: 129–138.
30.
Wazen, R.M., P. Moffatt, S.F. Zalzal, Y. Yamada, A. Nanci (2009) A mouse model expressing a truncated form of ameloblastin exhibits dental and junctional epithelium defects. Matrix Biol 28: 292–303.
© 2011 S. Karger AG, Basel
2011
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.