In-situ and In-vitro Studies of Amelogenin-Ameloblastin Binding

Objectives: Enamel matrix proteins amelogenin (Amel) and ameloblastin (Ambn) colocalize in the secretory stage and their fragments continue to colocalize in the maturation stage of enamel formation. Addition of Ambn changes Amel secondary structure in-vitro, suggesting molecular interactions. However, evidence of direct interactions between Amel and Ambn in enamel formation remains scarce. The objectives of this project are a) to observe Amel-Ambn interactions in-vivo in developing mouse incisor, b) to show direct interaction between recombinant Amel and Ambn in-vitro, c) to identify the domain of Ambn interacting with Amel, and d) to confirm the interactions within native enamel protein extracts.
Methods: In-vivo colocalization between Amel and Ambn was observed through immunohistochemical labeling in 5- and 8-day-old mouse incisors and quantified with a Leica SP8 confocal microscope. In-situ Fluorescence Resonance Energy Transfer (FRET) microscopy was used to determine the distance between Amel and Ambn fragments in day 6 and 8 mouse incisors. To show direct interaction and to identify the domain of Ambn binding to Amel, Pierce co-immunoprecipitation (co-IP) kit was used. Co-IP between recombinant amelogenin and WT Ambn, 2 Ambn mutants lacking exon 5 and exon 6 sequences, and various Ambn peptides was analyzed using Western Blots. Results of the recombinant protein co-IPs were confirmed with native enamel matrix proteins from porcine second molars.
Results: Amel and Ambn colocalized within ameloblasts, at the Tomes’ processes, and within enamel matrix from secretory to maturation stages in 5- and 8-days old mouse incisors. FRET analysis of 6-days mouse incisor sections showed that Amel and Ambn fragments were ~7 nm apart. Amel-Ambn interactions occurred through the N-terminal fragment of the sequence encoded by exon 5 of Ambn. These interactions were also confirmed in native Amel and Ambn extracted from the enamel matrix of porcine second molar.
Conclusions: We have demonstrated direct Amel-Ambn binding in-vitro between recombinant proteins and in porcine native protein extract suggesting that they co-assemble and may play a cooperative role during enamel formation. Together the proteins may control mineralization and maintain enamel prism architecture.