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

Abstract

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.

Amelogenin Interacts with Cytokeratin-5 in Ameloblasts during Enamel Growth

“…It is possible that these granules reacting to the anti-amelogenin polyclonal antibodies could be due to other enamel proteins, such as enamelin, for it is known that the antibodies produced against amelogenin cross-react with other enamel proteins such as enamelin (29,(31)(32)(33). In support, the polyclonal antibodies produced against recombinant amelogenin also stain more than 15 fractions, ranging from 14 to 42 kDa (30). Our results suggest avoidance of ovalbumin treatment of ultrathin sections of ameloblasts for transmission electron microscopy and immunolocalization of cytoplasmic or membranebound amelogenins.…”

“…In a number of ultrastructural immunostaining studies involving amelogenin in ameloblasts (29,30), the thin sections were pretreated with 1-4% ovalbumin prior to immunostaining with anti-amelogenin polyclonal antibodies. Because ovalbumin is a GlcNAc carrier with a molecular size twice that of amelogenin, binding of ovalbumin to amelogenin in sections may create a stearic hindrance to antibody binding, even if the epitope recognized by the antibodies is found at the C-terminal end of amelogenin.…”

Immunocytochemical Study of Amelogenin Deposition during the Early Odontogenesis of Molars in Alendronate-treated Newborn Rats

“…Although it is well established that amelogenin is the major enamel protein and most of the knowledge about its functions has been accumulated in recent years [reviewed in Nanci et al (1998); Moradian-Oldak (2001)], many questions remain unsolved. The administration of sodium alendronate, which clearly increased the amelogenin secretion by ameloblasts, may help us to elucidate some aspects of the amelogenin function during amelogenesis.…”

Ameloblastin is not implicated in bone remodelling and repair

“…The MC3T3-E1 cell line used in this study expresses CD63 but the SaOS-2 cell line does not (unpublished), yet no effect on mineral nodule formation was detected with both cell lines. Since positive expression of CD63 has been reported in both cell lines (Pereira et al, 2009;Shapiro et al, 2007), discrepancies in results could be explained by the source of cell lines used and/or cell transformation following multiple passages. We used Bril as a marker of bone formation.…”