SUMMARYMineralized tissues are unique in using proteins to attract and organize calcium and phosphate ions into a structured mineral phase. A precise knowledge of the expression and extracellular distribution of matrix proteins is therefore very important in understanding their function. The purpose of this investigation was to obtain comparative information on the expression, intracellular and extracellular distribution, and dynamics of proteins representative of the two main classes of enamel matrix proteins. Amelogenins were visualized using an antibody and an mRNA probe prepared against the major alternatively spliced isoform in rodents, and nonamelogenins by antibodies and mRNA probes specific to one enamel protein referred to by three names: ameloblastin, amelin, and sheathlin. Qualitative and quantitative immunocytochemistry, in combination with immunoblotting and in situ hybridization, indicated a correlation between mRNA signal and sites of protein secretion for amelogenin, but not for ameloblastin, during the early presecretory and midto late maturation stages, during which mRNA signals were detected but no proteins appeared to be secreted. Extracellular amelogenin immunoreactivity was generally weak near secretory surfaces, increasing over a distance of about 1.25 m to reach a level slightly above an amount expected if the protein were being deposited evenly across the enamel layer. Immunolabeling for ameloblastin showed an inverse pattern, with relatively more gold particles near secretory surfaces and much fewer deeper into the enamel layer. Administration of brefeldin A and cycloheximide to stop protein secretion revealed that the immunoblotting pattern of amelogenin was relatively stable, whereas ameloblastin broke down rapidly into lower molecular weight fragments. The distance from the cell surface at which immunolabeling for amelogenin stabilized generally corresponded to the point at which that for ameloblastin started to show a net reduction. These data suggest a correlation between the distribution of amelogenin and ameloblastin and that intact ameloblastin has a transient role in promoting/stabilizing crystal elongation. A meloblasts , like all hard tissue-forming cells, release an intricate set of extracellular matrix proteins optimized for promoting the development of a closely associated mineral phase (reviewed in Deutsch et al.
The organic matrix in forming enamel consists largely of the amelogenin protein self-assembled into nanospheres that are necessary to guide the formation of the unusually long and highly ordered hydroxyapatite (HAP) crystallites that constitute enamel. Despite its ability to direct crystal growth, the interaction of the amelogenin protein with HAP is unknown. However, the demonstration of growth restricted to the c-axis suggests a specific protein-crystal interaction, and the charged COOH terminus is often implicated in this function. To elucidate whether the COOH terminus is important in the binding and orientation of amelogenin onto HAP, we have used solid state NMR to determine the orientation of the COOH terminus of an amelogenin splice variant, LRAP (leucine-rich amelogenin protein), which contains the charged COOH terminus of the full protein, on the HAP surface. These experiments demonstrate that the methyl 13 C-labeled side chain of Ala 46 is 8.0 Å from the HAP surface under hydrated conditions, for the protein with and without phosphorylation. The experimental results provide direct evidence orienting the charged COOH-terminal region of the amelogenin protein on the HAP surface, optimized to exert control on developing enamel crystals.Enamel is composed of unusually long and highly oriented hydroxyapatite (HAP) 1 crystals (1), Ͼ1000 times longer than the HAP crystals found in bone (2). The molecular interactions leading to this highly controlled structure are not currently well understood, but the organic matrix is observed to be essential to the proper formation of enamel (3). Amelogenin proteins constitute Ͼ90% of the protein present in developing enamel (3). Amelogenin knock-out mice experience improper enamel formation, and genetic mutations of amelogenin also result in enamel defects, establishing the importance of amelogenin in enamel formation (3, 4).Despite its importance, mechanisms at the molecular level directing amelogenins control of enamel crystallites are not understood. It is known that under physiological conditions, amelogenin self-assembles into nanospheres of ϳ20 nm in diameter, consisting of multiple monomers, and this is believed to be its functional form (5-7). Transmission electron microscope studies have shown that the amelogenin nanospheres align in beaded rows along the c-axis of the developing enamel crystal (5), exhibiting exquisite control over the resulting morphology. This suggests a specific matrix-crystal interaction, yet the region of the protein interacting with HAP to result in the observed crystal regulation has not been identified. It has been postulated, based on experimental evidence, that both the charged NH 2 and COOH terminus of amelogenin are exposed on the surface of the nanospheres (6, 8 -11), aiding in proteinprotein interactions, increasing the solubility, and enhancing calcium phosphate interactions for the hydrophobic protein (12). These hypotheses are supported by in vitro experiments, reviewed recently by Moradian-Oldak (13), demonstrating the effect ...
Enamel forms the outer surface of teeth, which are of complex shape and are loaded in a multitude of ways during function. Enamel has previously been assumed to be formed from discrete rods and to be markedly aniostropic, but marked anisotropy might be expected to lead to frequent fracture. Since frequent fracture is not observed, we measured enamel organization using histology, imaging, and fracture mechanics modalities, and compared enamel with crystalline hydroxyapatite (Hap), its major component. Enamel was approximately three times tougher than geologic Hap, demonstrating the critical importance of biological manufacturing. Only modest levels of enamel anisotropy were discerned; rather, our measurements suggest that enamel is a composite ceramic with the crystallites oriented in a complex three-dimensional continuum. Geologic apatite crystals are much harder than enamel, suggesting that inclusion of biological contaminants, such as protein, influences the properties of enamel. Based on our findings, we propose a new structural model.
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