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Amelotin—a Novel Secreted, Ameloblast-specific Protein
K. Iwasaki1,3,
E. Bajenova1,
E. Somogyi-Ganss2,
M. Miller1,
V. Nguyen1,
H. Nourkeyhani1,
Y. Gao1,4,
M. Wendel2 and
B. Ganss1,*
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;
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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.
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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 β-actin. Amelotin expression appears to be tooth-specific.
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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.
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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.
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Journal of Dental Research, Vol. 84, No. 12,
1127-1132 (2005)
DOI: 10.1177/154405910508401207
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