Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine for an adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N-terminal to the cleavage site for enamel matrix metalloproteinase-20 (MMP-20) in amelogenin. MMP-20 releases the tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate at which MMP-20 hydrolyses mutated amelogenin relative to unmutated amelogenin. A full-length recombinant human amelogenin and a mutated amelogenin with a substitution of proline by threonine were expressed and purified by ammonium sulphate precipitation and reverse phase HPLC. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the recombinant proteins, which resulted in fragments with a mass predicted for TRAP. The proteolytic site was also modelled as substrates by two synthetic peptides, SYGYEPMGGWLHHQ and SYGYETMGGWLHHQ, selected from residues 36 to 49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. These two peptides were labelled at their N- and C-termini respectively by using rhodamine and biotin. After digestion with MMP-20, the truncated peptides were separated by avidin-labelled magnetic Dynal beads and were identified by mass spectrometry. These results demonstrated that both oligopeptides were cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. Enzyme kinetics showed that the k(cat)/K(m) of rMMP-20 against the unmutated amelogenin peptide was 21 times greater than that against the mutated peptide. This study suggests that the reduced rate of TRAP formation by a single amino acid substitution alters enamel matrix hydrolysis by MMP-20, which may result in amelogenesis imperfecta.
