Rôle du Leucine Rich Amelogenin Peptide dans la formation et la minéralisation de l’émail

Abstract : Tooth enamel is the outer calcified layer covering the crown of the tooth; it is the most mineralized tissue of the human body. Enamel is synthesized by ameloblast during tooth development, but this cell population degenerates and regresses when the tooth becomes fully functional. The mature enamel is completely devoid of cells and protein content. The amelogenin, the main protein of the enamel has the ability to assemble into highly organized nanochains, which will guide and regulate the deposition of hydroxyapatite crystals during enamel formation. The Leucine Rich Amelogenin Peptide (LRAP) is a product of alternative splicing of the amelogenin gene; it is a short peptide (56kDa), composed of the active N- and C-terminal sequences of the complete amelogenin protein. The aim of this work is to understand is to understand the role of LRAP in enamel formation. We have shown 1) that LRAP presents the same self-assembling properties as the full-length form of amelogenin and its cleavage products; 2) that LRAP exhibits the same calcium phosphate regulating properties as the full-length protein; and 3) that the conformation of the phosphorylated form of LRAP was modified by increasing concentration of calcium in the surrounding medium unlike the dephosphorylated form of LRAP. In the second part of this work, we have studied the peptide signaling properties and confirmed our mineralization results on ameloblast lineage cell culture models (LS8 and ALC) in vitro and on mice first molar germ culture model ex vivo. We have shown that the presence of both forms of LRAP activate the kinetic of differentiation of LS8 and ALC cells in vitro, and results in the formation of organized elongated HAP crystals. In the germ culture experiments, addition of LRAP(+P) in a mineralizing medium induces an increase in the density and volume of the mineral formed whereas in the standard medium, it promotes differentiation of the germs. LRAP(-P) leads to the synthesis by secretory ameloblasts of well organized long and fine HAP crystals. This work may lead the way toward new strategies for enamel tissue regeneration in order to treat alterations in the enamel layer resulting from carious lesions with the LRAP(-P) or genetic disorders such as amelogenesis imperfecta using LRAP(+P).
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Elvire Le Norcy. Rôle du Leucine Rich Amelogenin Peptide dans la formation et la minéralisation de l’émail. Médecine humaine et pathologie. Université Sorbonne Paris Cité, 2015. Français. ⟨NNT : 2015USPCB090⟩. ⟨tel-01535859⟩

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