Effects of Amyloid Domain on the KLK4 Hydrolysis of Amelogenin

Objectives: Amelogenin (AMG) is the dominant protein in developing enamel matrix and its removal by proteinase KLK4 is a key event in proper enamel mineralization. Recently, our lab discovered that a 14-residue region (P2) near the N-terminus of the protein is essential for amelogenin self-assembly into nanoribbons. This domain can form nano-strings possibly through amyloid-like β-sheet structures. The objective of this study is to determine the effects of this assembly-related amyloid domain on the hydrolysis of AMG by KLK4.

Methods: Recombinant wild-type AMG and its mutant protein with P2 deletion were expressed and purified with 3% acetic acid followed by C4 beads purification. Equal amount of wild-type AMG and P2 deleted mutant were incubated with KLK4 in triplicate for zero, three, six, and twenty hours. SDS-PAGE and Coomassie blue staining were used to compare the kinetic digestions of these two proteins. The densities of the bands were analyzed by NIH ImageJ. At each time point, the percentages of non-digested protein remaining after incubation were compared by Student t-test.

Results: The purified AMG protein and its mutants show different digestion rates in the presence of KLK4. Only 9.6% of AMG was digested after three-hour incubation with KLK4 and 59.6% still remained intact after twenty-hour incubation. However, 41.9% of P2-deleted mutants were digested after three-hour incubation and only 13.3% remained after twenty-hour incubation.
Conclusions: The deletions of amyloid assembly sequence P2 significantly accelerated the hydrolysis of AMG by KLK4 (p value < 0.01), which indicates that the existence of amyloid-mediated assembly may play an important role in regulating AMG degradation in tooth enamel development.