Degradation and loss of matrix proteins from developing enamel

Abstract

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.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative Immunochemical Analyses of the Developmental Expression and Distribution of Ameloblastin and Amelogenin in Rat Incisors

“…The situation is more complex for amelogenins which, for a long time, have been assumed to form a thixotropic gel that allows free mixing of components and, hence, should lead to their homogeneous distribution (discussed in Smith et al 1989;Smith 1998). Consistent with this theory, an early immunolocalization study of enamel proteins using a general antiamelogenin antibody suggested a uniform distribution of matrix constituents (Nanci et al 1985).…”

Hypomaturation Enamel Defects in Klk4 Knockout/LacZ Knockin Mice

“…The ␤-Galactosidase Expression Assay-Mouse heads were collected from postnatal (PN) days 5,6,8,12, and 14, fixed in 4% paraformaldehyde (PFA) overnight, and processed for ␤-galactosidase staining (67)(68)(69). To improve tissue fixation in mice 7 days old or later, the thorax and the right ventricle were opened, phosphate-buffered saline (PBS, 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4.)…”

Enamelysin (Matrix Metalloproteinase 20)-deficient Mice Display an Amelogenesis Imperfecta Phenotype

“…Amelogenin is the predominant component and comprises ϳ90% of total enamel matrix protein (10). Interestingly, the full-length enamel proteins are found only at the mineralizing front, suggesting that they participate in crystal elongation (11)(12)(13)(14)(15)(16)(17)(18)(19). In contrast, the protein cleavage products are found throughout the enamel layer, suggesting that they prevent crystallite growth in width and thickness (16).…”