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J. Biol. Chem., Vol. 276, Issue 39, 36586-36597, September 28, 2001

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Amelogenin-Cytokeratin 14 Interaction in Ameloblasts during Enamel Formation^*

Rajeswari M. H. Ravindranath, Wai-Yin Tam, Pablo Bringas Jr., Valentino Santos, and Alan G. Fincham

From the Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles, California 90033

The enamel protein amelogenin binds 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 is found in the N-terminal region of CK14, a differentiation marker for ameloblasts. The binding affinity of CK14 and amelogenin was confirmed by dosimetric binding of CK14 to recombinant amelogenin (rM179), and to the tyrosine-rich amelogenin polypeptide. The specific binding site for CK14 was identified in the amelogenin trityrosyl motif peptide (ATMP) of tyrosine-rich amelogenin polypeptide and specific interaction between CK14 and [³H]ATMP was confirmed by Scatchard analysis. Blocking rM179 with GlcNAc, GMp, or CK14 with ATMP abrogates the CK14-amelogenin interaction. CK14 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. Morphometry of developing teeth distinguished three phases of enamel formation; growth initiation phase (days 0-1), prolific growth phase (days 1-7), and growth cessation phase (post-day 7). Confocal microscopy revealed co-assembly of CK14/amelogenin in the perinuclear region of ameloblasts on day 0, migration of the co-assembled CK14/amelogenin to the apical region of the ameloblasts from day 1, reaching a peak on days 3-5, and a collapse of the co-assembly. Autoradiography with [³H]ATMP and [³H]GMp corroborated the dissociation of the co-assembly at the ameloblast Tomes' process. It is proposed that CK14 play a chaperon role for nascent amelogenin polypeptide during amelogenesis.

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