COMMON EPITOPES OF MAMMALIAN AMELOGENINS AT THE C-TERMINUS AND POSSIBLE FUNCTIONAL ROLES OF THE CORRESPONDING DOMAIN IN ENAMEL MINERALIZATION

The present studies were undertaken to investigate the presence of common epitopes of mammalian amelogenins at the C-terminus and the possible functional importance of the conserved C-terminal domain in enamel mineralization during mammalian amelogenesis. Enamel proteins, including the intact amelogenins and their degraded polypeptides, were isolated from the secretory enamel of pig, cow, rat, and rabbit incisors. Rabbit and rat antipeptide sera, as well as rat anti-25 kD and 20 kD pig amelogenin sera, were used to identify the amelogenins among the isolated matrix proteins of each of the animal species. The antipeptide sera were developed previously (Aoba et al. [19]) using as immunogens the two synthetic peptides, C13 and C25, which correspond to the last 12 (plus Cys for KLH-conjugation) and 25 amino acid residues of pig intact amelogenin, respectively. Reactivity of the enamel proteins with each antiserum was examined by Western blot analysis. The results of immunoblotting showed that a few enamel matrix proteins in each of the mammalian species were recognized by the anti-C13 serum, specifically, pig amelogenin at 25 kD (and trace components at 27, 22, and 18 kD), cow amelogenin at 28 kD (trace components at 26, 22, 19, and 14 kD), rat amelogenins at 28 and 26 kD (and a trace component at 20 kD), and rabbit amelogenins at 24 and 21 kD (and a trace at 13 kD). The anti-C25 serum reacted additionally with pig amelogenin at 23 kD, cow amelogenin at 27 kD (a major matrix constituent), and rabbit protein at 19 kD. The anti-pig 20 kD amelogenin (lacking the last 25 amino acid residues at the C-terminus) serum reacted with a large number of pig, cow, and rat amelogenins but, interestingly, with none of the rabbit enamel proteins. Probing of rat enamel proteins with Maclura pomifera lectin showed the heterogeneity of glycosylation of rat amelogenins, particularly between the 28 and 26 kD intact amelogenins. In parallel adsorption studies, part of the enamel protein samples isolated from each of the species was used as adsorbates to investigate the selective adsorption of amelogenins onto hydroxyapatite. Immunoblot analysis of the proteins adsorbed onto the crystals revealed that the mammalian amelogenins having the common epitopes at the C-terminus, in general, adsorb preferentially onto hydroxyapatite. The adsorption affinity of the degraded amelogenins decreased significantly with the loss of reactivity toward the anti-C13 serum. The overall results support the contention that the intact mammalian amelogenins, including rat and rabbit amelogenins, share common epitopes at the C-terminus and that the conserved C-terminal domain plays an important role in setting the molecular structures of the intact amelogenins so as to facilitate the protein-enamel mineral interaction.