Methods: The interactions between the 32-kDa enamelin and amelogenin were investigated using immunochemical (immunoprecipitation and immunogold labeling) and biophysical methods (circular dichroism (CD) and dynamic light scattering (DLS)). OCP crystals were grown in 10% recombinant rP148 with increasing concentration (0.04%-0.4%) of the 32-kDa enamelin. The morphology of OCP crystals was observed by scanning electron microscopy (SEM) and the dimensional changes of OCP were measured by the mean values of length (L), width (W), and thickness (T), and analyzed by the ratios (L/W and W/T).
Results: Immunoprecipitation studies revealed that the 32-kDa enamelin and amelogenin eluted together from the Protein A column. In situ immunogold dual labeling of developing porcine molars presented co-localization of enamelin and amelogenin within 100 nm radius regions. DLS results showed that the 32-kDa enamelin had a profound effect on amelogenin assembly at pH 8.0, giving rise to partial dissociation of the nanospheres in a dose dependent manner. The appearance of an isodichroic point and the shifting and intensity decrease of the minima in the CD spectra of amelogenin following the addition of enamelin were indicative of conformational changes in amelogenin. These results demonstrated that there is a direct interaction between the two macromolecules. Analysis of the crystals' dimensions indicated that their ratios (L/W) had a tendency to increase with increasing concentration of the 32-kDa enamelin added to 10% amelogenin.
Conclusions: Our results collectively demonstrate that the 32-kDa enamelin has a direct interaction with amelogenin. These two enamel proteins also cooperate to promote elongated growth of OCP crystals in vitro.
Supported By: NIH-NIDCR DE-13414 and DE-15644.