Protein dynamics of amelogenesis

Abstract

Background: The synthesis, secretion, and fate of matrix proteins released by ameloblasts during enamel formation was studied in continuously erupting rat incisors.

Methods: Computerized image processing was used to quantify silver grain distribution in radioautographs of sections prepared from rats injected with 3H-methionine, and this was correlated with fluorographs defining radiolabeling patterns of proteins in enamel organ cell and enamel homogenates prepared from freeze-dried teeth of rats injected with 35S-methionine and other radioactive amino acids and precursors such as sugar, sulfate, and phosphate. Some rats were also treated with brefeldin A to characterize newly formed proteins blocked from being secreted from ameloblasts.

Results: The results indicate that ameloblasts rapidly synthesize and secrete (minutes) at least five primary enamel matrix proteins, including a 65 kDa sugar-containing sulfated enamel protein and four nonsulfated proteins with molecular weights near 31, 29, 27, and 23 kDa as estimated by SDS-PAGE. The 27 kDa protein appears to correspond to the primary amelogenin described in many species. The cells also appear to release at least one phosphoprotein with molecular weight near 27 kDa, which may be an amelogenin, and up to five cysteine-containing proteins with molecular weights near 94, 90, 72, 55, and 27 kDa. The proteins collectively are released at interrod and rod growth sites where they appear to remain close to their point of release from ameloblasts. The 65 kDa sulfated protein and 31 kDa nonsulfated protein are rapidly converted into lower molecular weight forms (hours), whereas nonsulfated proteins near 29, 27, and 23 kDa are more slowly transformed into fragments near 20, 18, and 10 kDa in molecular weight (days). These fragments do not accumulate but appear to be removed from the enamel layer as they are created.

Conclusions: Enamel proteins seen by Coomassie blue (or silver) staining of one-dimensional polyacrylamide gels, therefore, represent a composite image of newly secreted and derived forms of sulfated and nonsulfated proteins that sometimes have similar molecular weights.