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Amelotin—a Novel Secreted, Ameloblast-specific Protein

¹ Canadian Institutes for Health Research (CIHR) Group in Matrix Dynamics, University of Toronto, Faculty of Dentistry, 150 College Street, Toronto, ON M5S 3E2, Canada;
² Center for Oral Biology (COB), Karolinska Institute, Huddinge, Sweden;

View larger version (72K): [in a new window]   Figure 1. Amelotin isolation and sequences. (A) Identification of an ameloblasts-specific gene fragment by DD-PCR. Two independent preparations of cDNA from ameloblasts (1), odontoblasts (2), dental pulp cells (3), and alveolar osteoblasts (4) were resolved on a 6% polyacrylamide gel and visualized by radioautography. Arrowheads indicate the bands representing the amelotin fragment. (B) cDNA and conceptually translated protein sequence of amelotin. The fragment identified by DD-PCR is underlined. (C) Protein sequence comparison of mouse and human amelotin. Asterisks indicate identical, semicolons highly conserved, and periods similar amino acids. (D) Exon-intron structure and predicted localization of mouse and human amelotin genes. Open squares indicate exons containing non-coding sequences. The relative location of the ameloblastin (Ambn) and enamelin (Enam), as well as the SIBLING gene cluster (Dspp, Dmp1, BSP. MEPE, OPN), is also indicated.

View larger version (49K): [in a new window]   Figure 2. Northern blot analysis of amelotin expression in whole mouse embryos at various stages of embryonic development (A) and several post-natal and adult tissues (B), relative to ß-actin. Amelotin expression appears to be tooth-specific.

View larger version (82K): [in a new window]   Figure 3. Analysis of amelotin expression by in situ hybridization. (A) Arrowheads indicate the amelotin signal in ameloblasts of the first (M1), second (M2), and third (M3) molar on post-natal days d5 to d15. Bar indicates 30 µm. (B) Amelotin expression during various stages of amelogenesis in the 10-day-old mouse incisor from the incisal end to the cervical loop. Magnified views (a-i) show details of the restricted expression in maturation-stage ameloblasts. Bar indicates 10 µm.

View larger version (85K): [in a new window]   Figure 4. Analysis of amelotin secretion from C2C12 cells by immunocytochemistry and Western blot. Expression of the FLAG peptide alone (cFLAG-pCMV5) shows a strong signal in the cytoplasm; Western analyses of cell lysates (a) and conditioned media (b) show no signal, since the FLAG peptide is too small to be resolved by PAGE. Expression of the amelotin-FLAG fusion protein (cFLAG-Amelotin) shows a weak signal in the cytoplasm and a strong signal in the conditioned media, while the addition of Brefeldin A (cFLAG-Amelotin + Brefeldin A) causes retention of the amelotin fusion protein in the cytosol. Bar indicates 50µm.