DEGRADATION OF HAMSTER AMELOGENINS DURING SECRETORY STAGE ENAMEL FORMATION IN ORGAN-CULTURE

Increasing amelogenin heterogeneity during pre-eruptive enamel formation has been explained by proteolytic cleavage of a parent amelogenin, differences in posttranslational modicifactions, translation of multiple alternative spliced mRNA transcripts or combinations of these possibilities. We investigated the possibility of proteolytic degradation of amelogenins during secretory amelogenesis by pulse-labelling amelogenins with [H-3]proline followed by a pulse chase, all under organ culture conditions. The results indicate that during pulse chase, hamster molar tooth explants rapidly released substantial amounts of the radioactivity into the culture medium, as non-trichloroacetic-acid precipitable, noncollagenous H-3-activity at the expense of radioactivity associated with the proteins in the enamel space. Simultaneously, there was a continuous mineralization of the forming enamel in vitro as shown by an increase in total calcium content of the explants. Western blotting, microdissection studies and fluorography of radiolabelled matrix proteins after SDS-PAGE indicated that after an 8-h labelling, three radioactive amelogenin species could be extracted from forming enamel, one prominent species of molecular mass 26 kDa and two less prominent ones of 28 and 22 kDa. During pulse chase more amelogenin bands with lower molecular mass became apparent, a pattern similar to that observed in vivo. Examination of amelogenin blots with the glycan assay showed that none of the hamster amelogenins stained for carbohydrate. We conclude that changes in the amelogenin profiles during enamel development of cultured hamster explants are similar to those observed in vivo. Although the formation of differently sized amelogenins from alternatively spliced mRNA transcripts cannot be ruled out, the in vitro pulse-chase data obtained in this study suggest that amelogenin degradation per se is probably responsible for most of the changes seen in the amelogenin profiles.