COMPARATIVE IMMUNOCHEMICAL ANALYSES OF THE DEVELOPMENTAL EXPRESSION AND DISTRIBUTION OF AMELOBLASTIN AND AMELOGENIN IN RAT INCISORS

Mineralized tissues are unique in using proteins to attract and organi ze calcium and phosphate ions into a structured mineral phase. A preci se knowledge of the expression and extracellular distribution of matri x proteins is therefore very important in understanding their function . The purpose of This investigation was to obtain comparative informat ion on the expression, intracellular and extracellular distribution, a nd dynamics of proteins representative of the two main classes of enam el 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, ameli n, 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 a melogenin, but not for ameloblastin, during the early presecretory and mid- to late maturation stages, during which mRNA signals were detect ed but no proteins appeared to be secreted. Extracellular amelogenin i mmunoreactivity was generally weak near secretory surfaces, increasing over a distance of about 1.25 mu 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 patte rn, with relatively more gold particles near secretory surfaces and mu ch fewer deeper into the enamel layer. Administration of brefeldin A a nd cycloheximide to stop protein secretion revealed that the immunoblo tting pattern of amelogenin was relatively stable, whereas ameloblasti n broke down rapidly into lower molecular weight fragments. The distan ce from the cell surface at which immunolabeling for amelogenin stabil ized generally corresponded to the point at which that for ameloblasti n started to show a net reduction. These data suggest a correlation be tween the distribution of amelogenin and ameloblastin and that intact ameloblastin has a transient role in promoting/stabilizing crystal elo ngation.