Tyrosyl motif in amelogenins binds N-acetyl-D-glucosamine

Ameloblasts secrete amelogenins on the pre-existing enamel matrix glycoprot eins at the dentine-enamel junction. The hypothesis that amelogenins may in teract with enamel matrix glycoproteins is tested by hemagglutination of pu rified, native (porcine) and recombinant murine amelogenins (rM179 and rM16 6) and hemaggIutination inhibition with sugars. Amelogenin agglutination of murine erythrocytes was specifically inhibited by N-acetylglucosamine (Glc NAc), chitobiose, and chitotetraose and by ovalbumin with terminal GlcNAc. The GlcNAc affinity was confirmed by dosimetric binding of rM179 with [C-14 ]GlcNAc, specific binding in relation to varying concentrations of GlcNAc, Scatchard plot analysis and competitive inhibition with Gold GlcNAc, The he magglutination activity and [C-14]GlcNAc affinity were retained by the NH2- terminal tyrosine-rich amelogenin peptide (TRAP) but not by the leucine-ric h amelogenin peptide, LRAP (a polypeptide sharing 33 amino acid residues of TRAP), or by the C-terminal 13 residue polypeptide of amelogenin (rM179). Since TRAP but not the 33-residue sequence of the TRAP shared by LRAP bound to [C-14]GlcNAc, we inferred that the GlcNAc binding motif was located in the 13-residue tyrosyl C-terminal domain of TRAP (PYPSYGYEPMGGW), which was absent from LRAP, [C-14]GlcNAc did indeed bind to this '"amelogenin tyrosy l motif peptide" but not when the tyrosyl residues were substituted with ph enylalanine or when the third proline was replaced by threonine. Significan tly, this latter modification mimics a point mutation identified in a case of human X-linked amelogenesis imperfecta. The amelogenin tyrosyl motif pep tide sequence showed a similarity to the secondary GlcNAc-binding site of w heat germ agglutinin.