Is the 32KDa Enamelin Essential for Normal Amelogenesis?

Enamelin is essential for normal amelogenesis. Different mutations in the ENAM gene have been reported in several families with autosomal amelogenesis imperfecta. Developing enamel contains a number of enamelin derived proteins generated by extracellular processing of nascent enamelin. Of these, the 32kDa component is the most abundant in developing porcine enamel and has been shown to have a high affinity for the mineral phase. It has therefore been suggested to be an important molecular component in enamel development though no specific role has yet been proven. However, the proteolytic cleavage site responsible for generating the C-terminal of the 32kDa enamelin is absent in rodents due to amino acid substitution.

Objectives: To investigate whether a 32kDa enamelin homologue is present in rodent amelogenesis.

Methods: Enamelins were extracted from secretory stage murine, rat and porcine enamel using 0.1M phosphate buffer (pH 6.8). Extracts were then subjected to SDS PAGE and western blot probing with antibodies raised against a synthetic peptide corresponding to an internal sequence of the porcine 32kDa enamelin which is also present in rodent enamelin.

Results: SDS PAGE showed that the extracts were dominated by soluble amelogenins. Blotting revealed the presence of 2 heavily stained cross reactive enamelin bands in porcine enamel at ~32 and ~38KDa. In contrast, rodent enamel appeared to only contain enamelins above 50kDa with the most dominant band migrating at ~75kDa.

Conclusions: These data suggest that generation of the specific 32KDa enamelin does not occur in rodents, presumably due to the absence of the appropriate cleavage sequence in these species. The 32kDa enamelin may therefore not be essential for amelogenesis and the functional role of enamelin may be associated with the nascent molecule and/or one of the larger enamelin processing products.

Supported by Wellcome programme grant no. 075945/Z/04/Z