The cooperative self-assembly of 25 kd and 23 kd amelogenins

Self-assembly of the extracellular matrix protein amelogenin is believed to play an essential role in regulating the growth and organization of enamel crystals during enamel formation. The full-length amelogenin uniquely regulates the growth, shape, and arrangement of enamel crystals. Protein hydrolysis will ultimately facilitate a tissue with high mineral content. The protein processing is however highly specific and suggest a functional role of the cleaved amelogenins in enamel mineral formation. Specific interactions between full-length amelogenin and its cleavage products in the extracellular space need to be understood. Objective: The purpose of this study was to explore if full-length amelogenin and its first MMP-20 cleavage product have cooperative self-assembling properties. Method: Recombinant full-length amelogenin (Amg25) and proteolytic cleavage product (Amg23) were purified using C4-beads and freeze-dried. We studied the effect on protein self-assembly as a function of pH. Protein-solutions at different concentrations and ratios were placed on glass slides and protein was immobilized for 1 h. Microstructure of the gel-matrix was analyzed using atomic force microscopy. Results: Amelogenin self-assembled into nanospheres at all pHs, but at pH around 7.0 and 8.0, respectively, Amg25 and Amg23 formed nanostrings of about 100 nm length. Networks of self-assembled nanostrings were obtained when both proteins Amg25 and Amg23 were present at a 50:50 ratio. The netlike linkings tended to complete without individual nanostrings at incubation times of 3 days and more. Conclusions: Full-length amelogenin and its cleavage product have cooperative self-assembly effect and can generate a fibrous network over time which is different from the initial self-assembly into nanospheres or nanostrings.

Support: by NIH/NIDCR R01-DE017529 and R01-DE015821.