ABSTRACT
Lectin-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 gamma-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-Curti's and tetrazonium assays). Alpha2,3-, alpha2,6- and alpha2,8-sialidases entirely abolished staining with the benzidine reactions. The presence of sialic acids in dentinal tubules was confirmed with the Bial's 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.
Subject(s)
Dentin/chemistry , Sialic Acids/analysis , Sulfates/analysis , Tooth/growth & development , Animals , Dental Enamel/chemistry , Dental Enamel/metabolism , Dental Enamel/ultrastructure , Dentin/metabolism , Dentin/ultrastructure , Hydrogen-Ion Concentration , Immunohistochemistry , Keratan Sulfate/biosynthesis , Keratan Sulfate/chemistry , Lectins/chemistry , Mice , Sialic Acids/biosynthesis , Species Specificity , Staining and Labeling , Tooth/metabolism , Tooth/ultrastructureABSTRACT
The enamel protein amelogenin binds to GlcNAc (Ravindranath, R. M. H., Moradian-Oldak, R., and Fincham, A.G. (1999) J. Biol. Chem. 274, 2464-2471) and to the GlcNAc-mimicking peptide (GMp) (Ravindranath, R. M. H., Tam, W., Nguyen, P., and Fincham, A. G. (2000) J. Biol. Chem. 275, 39654-39661). The GMp motif in the N-terminal region of the cytokeratin 14 of ameloblasts binds to trityrosyl motif peptide (ATMP) of amelogenin (Ravindranath, R. M. H., Tam, W., Bringas, P., Santos, V., and Fincham, A. G. (2001) J. Biol. Chem. 276, 36586 - 36597). K14 (Type I) pairs with K5 (Type II) in basal epithelial cells; GlcNAc-acylated K5 is identified in ameloblasts. Dosimetric analysis showed the binding affinity of amelogenin to K5 and to GlcNAc-acylated-positive control, ovalbumin. The specific binding of [3H]ATMP with K5 or ovalbumin was confirmed by Scatchard analysis. [3H]ATMP failed to bind to K5 after removal of GlcNAc. Blocking K5 with ATMP abrogates the K5-amelogenin interaction. K5 failed to bind to ATMP when the third proline was substituted with threonine, as in some cases of human X-linked amelogenesis imperfecta or when tyrosyl residues were substituted with phenylalanine. Confocal laser scan microscopic observations on ameloblasts during postnatal (PN) growth of the teeth showed that the K5-amelogenin complex migrated from the cytoplasm to the periphery (on PN day 1) and accumulated at the apical region on day 3. Secretion of amelogenin commences from day 1. K5, similar to K14, may play a role of chaperone during secretion of amelogenin. Upon secretion of amelogenin, K5 pairs with K14. Pairing of K5 and K14 commences on day 3 and ends on day 9. The pairing of K5 and K14 marks the end of secretion of amelogenin.
