Localization of sulfated sialic acids in the dentinal tubules during tooth formation in mice

Summary -like properties of the major enamel protein amelogenin suggest that it binds to glycoconjugates in dentinal tubules released at the dentin–enamel junction (DEJ) during enamel formation. Therefore, a detailed mapping of glycosylation in dentinal tubules during tooth formation was undertaken using histochemistry and lectin-binding assays. The tubular content exhibited sialidase-susceptible γ-metachromasia with Toluidine Blue (pH 2.5) and staining with Alcian Blue (pH 1.0). The presence of sulfate groups was confirmed by benzidine reactions (Bracco-Curtiʹs and tetrazonium assays). α2,3-, α2,6- and α2,8-sialidases entirely abolished staining with the benzidine reactions. The presence of sialic acids in dentinal tubules was confirmed with the Bialʹs reaction and sialidase-susceptible binding of Limax flavus lectin suggesting that sialic acids are the major sulfated sugars in the glycoconjguates. Immunostaining with the monoclonal antibody 5-D-4 before and after treatment with chondroitin-4- and chondroitin-6-sulfatase confirmed the presence of keratan sulfate (KS), a sialylated proteoglycan, in dentinal tubules. We suggest that sulfated sialic acids are part of the KSs. The sulfated glycoconjugates are also found in dentin and the DEJ but not in predentin suggesting that amelogenin binds to the sialoconjugate during enamel formation.