Identification of amelotin- and ODAM-interacting enamel matrix proteins using the yeast two-hybrid system

The formation of dental enamel is a prototype of functional tissue development through biomineralization. Amelotin (AMTN) is a recently discovered secreted enamel protein predominantly expressed during the maturation stage of enamel formation. It accumulates in a basal lamina-like structure at the interface between ameloblasts and enamel mineral and it co-localizes with another recently described enamel protein, odontogenic ameloblast-associated protein (ODAM). The purpose of this study was to determine whether AMTN and ODAM bind to each other and/or to other well-established enamel matrix proteins. The coding sequences of all enamel proteins were cloned into appropriate vectors of the GAL4-based Matchmaker Gold Yeast Two-Hybrid System. The growth of yeast cells on selective media and color induction were used as indicators for reporter gene expression through protein-protein interactions in combinations of prey and bait constructs. We found that AMTN interacts with itself and with ODAM, but not with amelogenin (AMEL), ameloblastin (AMBN), or enamelin (ENAM). Using ODAM as bait, the interaction with AMTN was confirmed. Furthermore, ODAM was found to bind to itself and to AMBN, as well as weakly to AMEL but not to ENAM. We propose a model where the distinct expression of AMTN and ODAM and their interaction are involved in defining the enamel microstructure at the enamel surface.