LS-2-P-2626 Colocalization of Amelotin, Odontogenic-Ameloblast Associated and Secretory Calcium-Binding Phosphoprotein-Proline-Glutamine-rich 1 in the basal lamina at the interface of ameloblasts and maturing enamel

An atypical basal lamina (BL) is present between the apical plasma membrane of post-secretory ameloblasts and the surface of maturing enamel. Unlike elsewhere in the body, this BL binds to mineral rather than connective tissue, and likely has had to adapt by integrating specialized molecules. It is rich in glycoconjugates and contains laminin-332 but not γ1 chain laminins, type IV and VII collagens. However, its precise composition still remains elusive. Screening of the secretome of ameloblasts has led our lab to identify genes encoding for three novel proteins called odontogenic ameloblast-associated (ODAM), amelotin (AMTN) and secretory calcium-binding phosphoprotein-Proline-Glutamine-rich 1 (SCPPPQ1). These genes reside in the secretory calcium-binding phosphoprotein (SCPP) gene cluster on human chromosome 4 (5 in rat and mouse) that encodes for proteins involved in the regulation of mineral deposition. We have previously demonstrated that AMTN and ODAM localize at the cell-tooth interface but still little is known about the distribution of SCPPPQ1 and the relationship between the three proteins. Objective: To compare the localization of AMTN, ODAM and SCPPPQ1 during amelogenesis. Methods: Mice and rats were perfused, hemimandibles were decalcified and processed for embedding in paraffin or LR White resin. Immunohistochemistry with rabbit antibodies against rat AMTN, ODAM and SCPPPQ1 was performed on paraffin sections. Single or double postembedding colloidal gold immunolabeling was carried out on ultrathin resin sections. Results: Immunolabeling confirmed that ODAM and AMTN were found in the BL at the cell-enamel interface throughout maturation. Labeling for ODAM seems to appear slightly earlier than that for AMTN. SCPPPQ1 was also detected at the cell-tooth interface but only from the later part of mid-maturation, and reactivity intensified into late maturation. Co-localization of the three proteins in the BL was confirmed by dual immunogold labeling. Both ODAM and SCPPPQ1 exhibited intense Golgi reactions in ameloblasts while cellular labeling for AMTN was extremely weak and seen only at the very beginning of maturation. Conclusions: The ultrastructural localization of AMTN, ODAM and SCPPPQ1 indicates that they are novel components of the BL associated with maturation stage ameloblasts. The sustained Golgi labeling for ODAM and SCPPPQ1 and the very restricted AMTN cellular reaction suggest that ODAM and SCPPPQ1 undergo renewal throughout maturation, while AMTN seems to be produced during a narrow time-frame and to remain stable. While the function of these proteins remains to be determined, their temporospatial distribution suggests that they may contribute to the mechanism binding ameloblasts to enamel, and/or to enamel maturation events.