Tyrosyl motif in amelogenins binds N-acetyl-D-glucosamine.
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 (GlcNAc), chitobiose, and chitotetraose and by ovalbumin with terminal GlcNAc. The GlcNAc affinity was confirmed by dosimetric binding of rM179 with [14C]GlcNAc, specific binding in relation to varying concentrations of GlcNAc, Scatchard plot analysis and competitive inhibition with cold GlcNAc. The hemagglutination activity and [14C]GlcNAc affinity were retained by the NH2-terminal tyrosine-rich amelogenin peptide (TRAP) but not by the leucine-rich 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 [14C]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. [14C]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.
Ravindranath
R M
RM
Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles, California 90033, USA.
Moradian-Oldak
J
J
Fincham
A G
AG
D000117:Acetylglucosamine / Q000378:metabolism*; D053523:Amelogenin; D000595:Amino Acid Sequence; D000818:Animals; D002241:Carbohydrates / Q000494:pharmacology; D003746:Dental Enamel Proteins / Q000737:chemistry / Q000378:metabolism*; D006023:Glycoproteins / Q000494:pharmacology; D006384:Hemagglutination; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000494:pharmacology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D014909:Wheat Germ Agglutinins / Q000737:chemistry / Q000378:metabolism
D053523:Amelogenin; C507200:Amelx protein, mouse; D002241:Carbohydrates; D003746:Dental Enamel Proteins; D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014909:Wheat Germ Agglutinins; D014443:Tyrosine; D000117:Acetylglucosamine