Tyrosyl Motif in Amelogenins BindsN-Acetyl-d-glucosamine

Abstract

Ameloblasts secrete amelogenins on the pre-existing enamel matrix glycoproteins at the dentine-enamel junction. The hypothesis that amelogenins may interact with enamel matrix glycoproteins is tested by hemagglutination of purified, native (porcine) and recombinant murine amelogenins (rM179 and rM166) and hemagglutination inhibition with sugars. Amelogenin agglutination of murine erythrocytes was specifically inhibited by N-acetylglucosamine ( 14 C]GlcNAc did indeed bind to this "amelogenin tyrosyl motif peptide" but not when the tyrosyl residues were substituted with phenylalanine or when the third proline was replaced by threonine. Significantly, this latter modification mimics a point mutation identified in a case of human X-linked amelogenesis imperfecta. The amelogenin tyrosyl motif peptide sequence showed a similarity to the secondary GlcNAc-binding site of wheat germ agglutinin.

The Enamel Protein Amelogenin Binds to the N-Acetyl-d-glucosamine-mimicking Peptide Motif of Cytokeratins

“…Previously, we have hypothesized that amelogenins may bind to sugar residues of enamel matrix glycoproteins facilitating the biomineralization process (5). The hypothesis was supported by identification of a stoichiometric interaction specifically between amelogenins and the GlcNAc residues of glycoconjugates (5). Further, we have identified the glycobinding locus of the amelogenin structure in a highly conserved motif (-PYPSYGY-) located at the carboxyl-terminal of the tyrosine-rich amelogenin polypeptide (TRAP).…”

“…Although ameloblasts synthesize several other proteins, including cytokeratin 14 prior to synthesis of amelogenins (1), the amelogenins constitute some 90% of the secretory stage enamel matrix proteins (2)(3)(4). Previously, we have hypothesized that amelogenins may bind to sugar residues of enamel matrix glycoproteins facilitating the biomineralization process (5). The hypothesis was supported by identification of a stoichiometric interaction specifically between amelogenins and the GlcNAc residues of glycoconjugates (5).…”

“…A typical reverse phase HPLC profile of rM179 and electrophoretic homogeneity of rM179, rM166, and the synthetic peptides TRAP and LRAP used in this investigation were shown and described earlier (5). The purified preparations (rM166) were devoid of or had negligible contamination with vector proteins.…”

“…Peptides were purified by reversed-phase HPLC (C4 -214TP54 column or C18 -291HS54 column; Vydac/The Separations Group, Hesperia, CA) with a gradient of 35-50% B in 60 min (buffer B contained 60% (v/v) aqueous acetonitrile in 0.1% (v/v) trifluoroacetic acid, and buffer A contained 0.1% trifluoroacetic acid) at a flow rate of 1.0 or 0.5 ml/min for small peptides depending on their size (5).…”

Amelogenin-deficient Mice Display an Amelogenesis Imperfecta Phenotype

“…Transgenic mice that express an amelogenin protein with a mutated N or C terminus further demonstrated the importance of these proteins in enamel biomineralization (37). In addition, the N-terminal region of amelogenin shows lectin-like activity in vitro (38) and, therefore, may be involved in binding to the glycosylated enamelin proteins found at the enamel junction with dentin. Amelogenin could be functionally important in defining and developing the structural stability required at the enamel dentin junction.…”