Tyrosine rich amelogenin peptide (TRAP) is one of the most abundant short amelogenin peptides existing in enamel protein matrix. This peptide is considered to be a hydrolytic product of amelogenin, cleaved between W44/L45 by matrix metalloproteinase 20 (MMP-20). A single amino acid mutation of P41 to T41 in a type of X-linked amelogenesis imperfecta (AI) reduces the hydrolysis rate and formation of TRAP. Objective: This study was to further investigate the role of P41 in the formation of TRAP and the pathogenesis of this type of AI. Methods: A series of fluorescent labeled peptides were synthesized in ProteaseSpot plates, including a fluorescent labeled backbone peptide (SYGYEPMGGW/LHHQ) derived from residue 35-48 of the human amelogenin and 20 alternate fluorescent labeled peptides with different P mutations (insertion, replacement and position switch) upstream to cleavage site W/L. To measure the interference of P41 to T41 mutation on the binding affinity between MMP-20 and substrate, another two peptides, P1 (same as backbone peptide without labeling) and T1 (mutant), were synthesized and mixed with a quenched peptide for competition assay. The dynamic digestion of these peptides by MMP-20 were monitored by a fluorometer and the products were analyzed by mass spectrometry (MS). Results: The proline at its original location 41 showed highest cleavage rate by MMP-20, compared to the other mutations. The competition assay indicated that the T1 peptide had significantly lower affinity to MMP-20 than P1. Conclusions: The P41 upstream to the W44/L45 cleavage site in amelogenin is critical for MMP-20 to access and cleave this protein. The P41 to T41 mutation results in the decrease of MMP-20 binding to amelogenin N terminus, and the formation of TRAP, which may lead to this type of AI. Supported by grants R01DE015821 and R01DE13508 from NIDCR.

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