The enamel protein amelogenin binds to GlcNAc (Ravindranath, R. M. H., Moradian-Oldak, R., and Fincham, A.G. (1999) J. Biol. Chem. 274, 2464-2471) and to the GlcNAc-mimicking peptide (GMp) (Ravindranath, R. M. H., Tam, W., Nguyen, P., and Fincham, A. G. (2000) J. Biol. Chem. 275, 39654-39661). The GMp motif in the N-terminal region of the cytokeratin 14 of ameloblasts binds to trityrosyl motif peptide (ATMP) of amelogenin (Ravindranath, R. M. H., Tam, W., Bringas, P., Santos, V., and Fincham, A. G. (2001) J. Biol. Chem. 276, 36586 - 36597). K14 (Type I) pairs with K5 (Type II) in basal epithelial cells; GlcNAc-acylated K5 is identified in ameloblasts. Dosimetric analysis showed the binding affinity of amelogenin to K5 and to GlcNAc-acylated-positive control, ovalbumin. The specific binding of [3H]ATMP with K5 or ovalbumin was confirmed by Scatchard analysis. [3H]ATMP failed to bind to K5 after removal of GlcNAc. Blocking K5 with ATMP abrogates the K5-amelogenin interaction. K5 failed to bind to ATMP when the third proline was substituted with threonine, as in some cases of human X-linked amelogenesis imperfecta or when tyrosyl residues were substituted with phenylalanine. Confocal laser scan microscopic observations on ameloblasts during postnatal (PN) growth of the teeth showed that the K5-amelogenin complex migrated from the cytoplasm to the periphery (on PN day 1) and accumulated at the apical region on day 3. Secretion of amelogenin commences from day 1. K5, similar to K14, may play a role of chaperone during secretion of amelogenin. Upon secretion of amelogenin, K5 pairs with K14. Pairing of K5 and K14 commences on day 3 and ends on day 9. The pairing of K5 and K14 marks the end of secretion of amelogenin.
"The tri-tyrosyl motif has been shown to bind to a conserved N-acetyl-d-glucosamine mimicking sequence present in cytokeratin 14 (33) and N-acetyl-d-glucosamine residues on cytokeratin 5 (19). Amelogenin and cytokeratin 14 co-localize at the apical regions of ameloblasts and dissociate at the Tomes’ processes prior to secretion leading to suggestions of a possible role for cytokeratin 14 in chaperoning amelogenin during amelogenesis (19,33). Failure to bind cytokeratin 14 was demonstrated when the human P → T mutation was introduced into the amelogenin tri-tyrosyl motif or when all three tyrosine residues were replaced by phenylalanine (33). "
[Show abstract][Hide abstract] ABSTRACT: Amelogenesis imperfecta (AI) describes a broad group of clinically and genetically heterogeneous inherited defects of dental enamel bio-mineralization. Despite identification of a number of genetic mutations underlying AI, the precise causal mechanisms have yet to be determined. Using a multi-disciplinary approach, we describe here a mis-sense mutation in the mouse Amelx gene resulting in a Y --> H substitution in the tri-tyrosyl domain of the enamel extracellular matrix protein amelogenin. The enamel in affected animals phenocopies human X-linked AI where similar mutations have been reported. Animals affected by the mutation have severe defects of enamel bio-mineralization associated with absence of full-length amelogenin protein in the developing enamel matrix, loss of ameloblast phenotype, increased ameloblast apoptosis and formation of multi-cellular masses. We present evidence to demonstrate that affected ameloblasts express but fail to secrete full-length amelogenin leading to engorgement of the endoplasmic reticulum/Golgi apparatus. Immunohistochemical analysis revealed accumulations of both amelogenin and ameloblastin in affected cells. Co-transfection of Ambn and mutant Amelx in a eukaryotic cell line also revealed intracellular abnormalities and increased cytotoxicity compared with cells singly transfected with wild-type Amelx, mutant Amelx or Ambn or co-transfected with both wild-type Amelx and Ambn. We hypothesize that intracellular protein-protein interactions mediated via the amelogenin tri-tyrosyl motif are a key mechanistic factor underpinning the molecular pathogenesis in this example of AI. This study therefore successfully links phenotype with underlying genetic lesion in a relevant murine model for human AI.
Human Molecular Genetics 04/2010; 19(7):1230-47. DOI:10.1093/hmg/ddq001 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Frequency of imperfect amelogenesis (IA) varies in different world populations. There is no information on the frequency of this entity in Colombia. This report informs the consanguinity in 3 cases of IA, the mother 36 years old and her two children of 8 and 15 years old. The same condition was found in 4 mother's relatives. This corroborates a hereditary pattern of this condition. The initial treatment for these patients is preventive, controlling them periodically and keeping in mind the appropriate handling of oral hygiene habits. A balanced diet poor in sugar or cariogenic agents and frequent teeth fluorination are the techniques to strengthen enamel traces. Later on these patients must be rehabilitated to recover both aesthetic and teeth functions.
[Show abstract][Hide abstract] ABSTRACT: The primary sequences of human and mouse tuftelin are 89% identical. Both proteins comprise 390 amino acids and produce an acidic protein with an isoelectric point of 5.7, and an unmodified molecular weight of 44 kD. Using fluorescent-tagged tuftelin and amelogenin plasmid constructs we saw little evidence that these two enamel proteins colocalize in ameloblast-like LS-8 cells. Tuftelin is primarily localized to distinct 'speckled' domains within the cell cytoplasm. In an attempt to better define a physiological function for tuftelin during amelogenesis, we have produced transgenic mice that overexpress tuftelin in ameloblasts and subsequently the enamel matrix. Tuftelin overexpression impacts dramatically upon the enamel crystallite habit and the enamel prismatic structure. Overexpressing tuftelin results in gross imperfections in enamel that is evident both at the nanoscale and the mesoscale. The most notable difference observed in the transgenic animals, when compared to wild-type animals, is an apparent loss of restricted growth of enamel crystallites along their a-axis and b-axis. This equates to a change in the crystallite aspect ratio. In the transgenic animals the crystallite structures appear more 'plate'-like in contrast to the symmetric, 'ribbon'-like crystallite morphology that is a characteristic feature of mammalian enamel.
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