Identification of Two Domains in Amelogenin for Enamel Crystal Adhesion

Objectives: Amelogenin (AMG) is the primary protein in developing enamel matrix, which is considered to modulate enamel crystal growth. Previous studies suggest that AMG N- and C-termini were critical for its interactions with other AMG and with apatite crystal. This study aims to test the hypothesis that the N-terminal P2 region and C terminal of AMG cooperate to adhere enamel crystals. Understanding the enamel crystal adhesion could provide insights in tooth regeneration and restoration.
Methods: The recombinant full-length AMG, AMG with P2 sequence deletion (AMG-D-P2), AMG with C-terminal deletion (AMG20), and AMG with both C-terminal and P2 deletions (AMG20-D-P2) were expressed, purified and confirmed by SDS-PAGE. Turbidity assay, which qualitatively determines the sedimentation rates resulted from the inter-particle adhesions between nano-HAP particles, was performed by measuring the UV absorbance value of mixed solutions containing protein and HAP. Same assays were performed using P2 peptide, AMG C-terminal peptide (P24) and a fusion peptide of P2 and P24 (P2P24).
Results: AMG and mutants were successfully constructed, expressed and purified. The HAP crystals displayed the fastest sedimentation rate in the presence of full-length AMG. The deletion of either C-terminus or P2 region of AMG significantly decelerated HAP crystals sedimentations. The inhibitory effect on HAP was more significant for the mutant with double deletions. P2P24 fusion peptide significantly accelerated the crystal-crystal interaction, in contrast, P2 or P24 peptide alone decelerated the interactions between HAP crystals.
Conclusions: Our data provide the evidences to identify two functional domains, P2 and P24, in amelogenin for the adhesion of HAP crystals.