Degradation and loss of matrix proteins from developing enamel
Authors: Smith C. E. , Pompura J. R. , Borenstein S. , Fazel A. , Nanci A. 
Published: February 2005

The pattern and timing of the breakdown and loss of matrix proteins were studied in developing rat incisor enamel using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), fluorography, radioautography, and in vitro incubations of proteins isolated from freshly dissected, crushed pieces of enamel. For biochemical studies, the technique of Robinson et al. (1974, 1977, 1983) was used to transect the enamel organ and enamel into a series of strips at 1 mm intervals along the length of the tooth. The proteins in each strip were extracted and either quantified by Lowry analysis or applied to 12% slab (enamel) or 5‐15% continuous gradient (enamel organ) SDS‐polyacrylamide gels and separated by electrophoresis. The biochemical studies indicated that the amount of protein contained within an enamel strip increased gradually by volume across the secretory stage, reached a peak early during the maturation stage, and then declined rapidly thereafter. The distribution of enamel proteins on SDS‐polyacrylamide gels changed markedly throughout this period. These changes included increases and decreases in the intensity of staining of proteins at certain molecular weights (e.g., 18 kDa) and the appearance and disappearance of some proteins not seen clearly near the start of the secretory stage of amelogenesis (e.g., 32 and 10 kDa). Labeling studies with 35S‐methionine suggested that the “stacked” arrangement of proteins typical of forming enamel (secretory stage) actually represented a very dynamic association of proteins, with new ones being added at the top of the stack and then breaking down with time to become those seen at lower molecular weights. Across the secretory stage, new proteins were always added to the top of the stack, but during early maturation this activity slowed dramatically, allowing the breakdown of aging proteins to be visualized more clearly. Radioautographic studies with 3H‐methionine indicated that the breakdown of newly secreted proteins also was correlated with a movement of label from the site of secretion into deeper, previously unlabeled, areas of forming enamel. In vitro studies revealed that the rate and degree of breakdown of enamel proteins varied markedly, depending on the stage of amelogenesis from which the proteins were extracted. Secretory stage enamel proteins showed slow in vitro degradation with accumulation of proteins near 18 kDa. Early maturation stage enamel proteins showed more rapid breakdown with little accumulation of proteins near 18 kDa, whereas late maturation stage enamel proteins showed complete degradation by 2 days of incubation in vitro. Degradation of maturation stage enamel proteins could be effectively inhibited with aprotinin. These results support the concept that amelogenins are degraded into small polypeptides through the action of extracellular proteinases, at least one of which appears to be a trypsinlike serine proteinase. These results further suggest that amelogenins begin to break down within hours of their secretion. There also appears to be an increase in the activity and/or amount or type(s) of proteinases involved in the degradation of amelogenins between the secretory and maturation stages of amelogenesis.