CLONING AND CHARACTERIZATION OF PORCINE ENAMELIN MESSENGER-RNAS

Dental enamel forms by matrix-mediated biomineralization. The componen ts of the developing enamel matrix are generally specific for that mat rix. The primary structures of three enamel proteins-amelogenin, tufte lin, and sheathlin (ameloblastin/amelin)-have been derived from cDNA s equences. Here we report the cloning and characterization of mRNA enco ding a fourth enamel protein: enamelin. The longest porcine enamelin c DNA clone has 3907 nucleotides, exclusive of the poly(A) tail. The pri mary structure of the secreted protein is 1104 amino adds in length. W ithout post-translational modifications, the secreted protein has an i sotope-averaged molecular mass of 124.3 kDa and an isoelectric point o f 6.5. Polymerase chain-reaction phenotyping of enamelin cDNA suggests that porcine enamelin transcripts are not alternatively spliced and u se a single polyadenylation/cleavage site. Immunohistochemical and Wes tern blot analyses with an affinity-purified antipeptide antibody spec ific for the enamelin carboxyl terminus demonstrate that enamelin is s ynthesized and secreted by secretory-phase ameloblasts. The parent pro tein is a 186-kDa glycoprotein that concentrates along the secretory f ace of the ameloblast Tomes' process. Intact enamelin and proteolytic cleavage products containing its carboxyl terminus are limited to the most superficial layer of the developing enamel matrix, while other en amelin cleavage products are observed in deeper enamel.