Amelogenin (Amel) and ameloblastin (Ambn) are two primary extracellular enamel matrix proteins that play crucial roles for proper thickness, prismatic structure, and robust mechanical properties. Previous studies have shown that Amel and Ambn bind to each other, but the effect of their coassembly on the nucleation of hydroxyapatite (HAP) is unclear. Here, we systematically investigated the coassembly of recombinant mouse Amel and Ambn in various ratios using in situ atomic force microscopy, dynamic light scattering, and transmission electron microscopy. The size of protein particles decreased as the Ambn:Amel ratio increased. To define the coassembly domain on Ambn, we used Ambn-derived peptides and Ambn variants to examine their effects on the amelogenin particle size distribution. We found that the peptide sequence encoded by exon 5 of Ambn affected Amel self-assembly but the variant lacking this sequence did not have any effect on Amel self-assembly. Furthermore, through monitoring the pH change in bulk mineralization solution, we tracked the nucleation behavior of HAP in the presence of Ambn and Amel and found that their coassemblies at different ratios showed varying abilities to stabilize amorphous calcium phosphate. These results demonstrated that Ambn and Amel coassemble with each other via a motif within the sequence encoded by exon 5 of Ambn and cooperate in regulating the nucleation of HAP crystals, enhancing our understanding of the important role of enamel matrix proteins in amelogenesis.
Keywords: ameloblastin; amelogenin; coassembly; enamel; hydroxyapatite; mineralization.