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J. Biol. Chem., Vol. 282, Issue 50, 36370-36376, December 14, 2007

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Spatiotemporal Expression of Ameloblastin Isoforms during Murine Tooth Development^*

Rajeswari M. H. Ravindranath¹, Asokan Devarajan, and Takashi Uchida

From the Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California Los Angeles, California 90033 and the Department of Oral Biology, Division of Molecular Medicinal Science, Hiroshima University Graduate, School of Biomedical Sciences, Kasumi 1-2-3, Hiroshima 734, Japan

Ameloblasts synthesize and secrete the enamel matrix proteins (amelogenin, ameloblastin, and enamelin). This investigation examined the profiles of ameloblastin in the ameloblasts and in the enamel matrix during different postnatal (PN) days (days 0-9) of development of mouse molar, using an antibody specific for C-terminal sequence of ameloblastin (Ct; GNKVHQPQVHNAWRF). Ameloblastin is found in three different molecular sizes (37, 55, and 66 kDa) in both ameloblasts and enamel matrix during PN development. In the ameloblasts, the sequence of expression of these fractions varied. The 37-kDa fraction was observed (even before the appearances of mRNA of the proteases, enamelysin and kallikrein-4) on days 0 and 1, persisted until day 3, and was not found thereafter. Other isoforms (55 and 66 kDa) distinctly appeared in ameloblasts after day 1, reached a peak on day 5, and remained thereafter. The Ct-positive granules appeared beaded in the ameloblasts on day 3. In the extracellular matrix, a 37-kDa (but not 66- or 55-kDa) fraction was detected on days 0 and 1 and remained in the matrix throughout the PN days. The larger isoforms (55 and 66 kDa) appeared in the enamel matrix from day 3 onward. On days 0-3, but not later, the 37-kDa isoform co-localizes with amelogenin in Tomes' process and formative enamel, as revealed by laser scan confocal microscopy. Autoradiography confirmed accumulation of ³H-labeled amelogenin trityrosyl motif peptide in the region of Tomes' process and formative enamel from day 0 to 3. These observations suggest that the 37-kDa isoform interacts with amelogenin during early tooth development.

Received for publication, June 8, 2007 , and in revised form, October 4, 2007.

^* This work was supported by NIDCR, National Institutes of Health Grant RO1-DE-13204-05. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, 2250 Alcazar St., Los Angeles, CA 90033. Tel.: 323-442-3171; Fax: 323-442-2981; E-mail: rravindr{at}usc.edu.

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