Reduced amelogenin-MMP20 interactions in amelogenesis imperfecta

Abstract

Amelogenin with a proline 41 to threonine mutation (P41T) is hydrolyzed at a lower rate by matrix metalloproteinase 20 (MMP20), resulting in an inherited tooth enamel defect, amelogenesis imperfecta (AI). The aim of this study was to elucidate the effect of P41T on the interactions between amelogenin and MMP20, which may contribute to the formation of this type of AI. The interactions of a recombinant wild-type human amelogenin and its P41T mutant with recombinant human MMP20 were compared by substrate competition assay, pull-down assay, and surface plasmon resonance (SPR). The results showed that the binding of the P41T mutant amelogenin for MMP20 was significantly lower than that of wild-type amelogenin. Our study supports a model in which the P41T mutation reduces the interactions between amelogenin and MMP20, leading to decreased degradation of amelogenin by MMP20, and resulting in AI.

Figure 1

Characterizations of inactive E227A mutated rhMMP20. (A) SDS-PAGE analysis of rhMMP20s after purification showed that the 55-kDa intact protein of wild-type active rhMMP20 (left lane) was absent; however, the autolytic products of rhMMP20 were predominantly located at 25 and 21 kDa. A predominant 55-kDa band with minor smaller fragments of inactive E227A rhMMP20 can be seen in the right lane of the SDS-PAGE gel. M, protein standard; Wt MMP, active wild-type rhMMP20; Mt MMP, inactive E227A rhMMP20. (B) Quenched-peptide digestion assay showed that wild-type rhMMP20 (Wt MMP20) was active. However, the enzymatic activity of inactive E227A rhMMP20 (Mt MMP20) was undetectable. The RFU readings depended on the amount of fluorophores released from the quencher in the quenched peptide by rhMMP20 hydrolysis. RFU, relative fluorescence units. (C) SDS-PAGE analysis showed that the wild-type human recombinant amelogenin is digested by wild-type active rhMMP20 (Wt MMP20, upper row), but is not degraded by mutated inactive E227A rhMMP20 (Mt MMP, lower row).

Figure 2

Comparison of the binding affinities of active rhMMP20 for amelogenin peptides by peptide competition assays. The fluorescence intensity observed in a peptide competition assay was directly related to the amount of digested quenched peptides. Therefore, the fluorescent signal was inversely related to the affinity of active rhMMP20 for the competitor peptides (A1 or M1), which competed with the quenched peptide for binding to MMP20. The reaction without any competitors (QP+MMP20) had the highest RFU readings (curve’s slope = 12.34 ± 0.31, n = 3). The wild-type A1 peptide (A1+QP+MMP20) is a significantly stronger competitor (curve’s slope = 8.29 ± 0.46, n = 3) than M1 peptide (M1+QP+MMP20), containing a P-to-T mutation (curve’s slope = 11.14 ± 0.64, n = 3). QP, quenched peptide; A1, peptide without mutation; M1, peptide with mutation; RFU, relative fluorescence units. *p < 0.05. **p < 0.01.

Figure 3

Sensorgrams of surface plasmon resonance (SPR) analysis of the interactions between inactive E227A rhMMP20 and amelogenins (wild-type rh174 or P41T mutant). The binding affinity of the immobilized E227A rhMMP20 for wild-type rh174 amelogenin (left panel) was higher than that for the P41T mutant (right panel) at each concentration (0.2, 0.5, or 1 μM). Triplicate samples were performed at each amelogenin concentration, and the average values were used for construction of sensorgrams, from which K_on (on-rate constant), K_off (off-rate constant), K_a (affinity constant), and their variability were calculated (Table).