Perturbed Amelogenin Secondary Structure Leads to Uncontrolled Aggregation in Amelogenesis Imperfecta Mutant Proteins

Abstract

Mutations in amelogenin sequence result in defective enamel, and the diverse group of genetically altered conditions is collectively known as amelogenesis imperfecta (AI). Despite numerous studies, the detailed molecular mechanism of defective enamel formation is still unknown. In this study, we have examined the biophysical properties of a recombinant murine amelogenin (rM180) and two point mutations identified from human DNA sequences in two cases of AI (T21I and P41T). At pH 5.8 and 25°C, wild type (WT) rM180 and mutant P41T existed as monomers, and mutant T21I formed lower order oligomers. CD, dynamic light scattering, and fluorescence studies indicated that rM180 and P41T can be classified as a premolten globule-like subclass protein at 25°C. Thermal denaturation and refolding monitored by CD ellipticity at 224 nm indicated the presence of a strong hysteresis in mutants compared with WT. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that WT transformed to a partially folded conformation upon heating and remained stable. The partially folded conformation formed by P41T, however, readily converted into a heterogeneous population of aggregates. T21I existed in an oligomeric state at room temperature and, upon heating, rapidly formed large aggregates over a very narrow temperature range. Thermal denaturation and refolding studies indicated that the mutants are less stable and exhibit poor refolding ability compared with WT rM180. Our results suggest that alterations in self-assembly of amelogenin are a consequence of destabilization of the intrinsic disorder. Therefore, we propose that, like a number of other human diseases, AI appears to be due to the destabilization of the secondary structure as a result of amelogenin mutations.Tooth enamel is one of the hardest and most heavily mineralized vertebrate tissues that can withstand wear without catastrophic failure during the entire life span of an organism (1, 2). The formation of enamel takes place in an extracellular environment, including an array of complex proteins and proteases in three main stages, namely the secretory, transition, and maturation stages (3). As the enamel development progresses, proteases such as enamelysin (MMP-20) and later kallikrein 4 (KLK-4) cleave the proteins, which are removed from the mineralization site in the extracellular matrix enabling the growth of enamel crystals and resulting in enamel hardness. Thus, during amelogenesis, the soft mineralized tissue formed during the early stages becomes hard and tough and almost devoid of any proteins at the maturation stage. Amelogenin is the major constituent (ϳ90%) of the protein matrix during the secretory stage and, together with other proteins in enamel, is responsible for the hierarchical structure observed in the enamel prisms (4 -6).In vitro amelogenin self-assembles into spherical structures, and this property depends on pH, ionic strength, and protein concentration (7). Using CD spectropolarimetry and NMR, we have reported that recomb...

Probing the self‐association, intermolecular contacts, and folding propensity of amelogenin

“…Note that it was necessary to perform the experiments at pH 5.5 and at lower TFE concentrations to be able to reliably measure the hydrodynamic radius of amelogenin monomers. 17,18,27 If the pH is too acidic, the positive charge on the surface of the monomers can cause accelerated diffusion due to charge-charge repulsion. 17,18,27 Similarly, we found that, despite thorough mixing, TFE content > 10% v/v increases light scattering and leads to errors in interpretation.…”

“…17,18,27 If the pH is too acidic, the positive charge on the surface of the monomers can cause accelerated diffusion due to charge-charge repulsion. 17,18,27 Similarly, we found that, despite thorough mixing, TFE content > 10% v/v increases light scattering and leads to errors in interpretation. The introduction of 10% v/v TFE lead to an increase in the average hydrodynamic radius of rP172 (i.e., from 3.66 6 1.69 nm in the absence of TFE to 6.04 6 2.25 nm in the presence of 10% TFE).…”

“…9 Both terminal domains exhibited evidence of residual secondary structure, 9 and these terminal regions may represent putative regions for folding during amelogenin-target interactions or self-assembly. 27 It is experimentally challenging to assess the folding propensities of the three major regions of porcine amelogenin in the presence of matrix targets or during the self-assembly process. One of the major reasons for this is that the enamel matrix is a complex environment [2][3][4][5][6][20][21][22][23][24][25] and the true identity of amelogenin-specific targets, solution conditions, and other important features are not known at present.…”

“…One of the major reasons for this is that the enamel matrix is a complex environment [2][3][4][5][6][20][21][22][23][24][25] and the true identity of amelogenin-specific targets, solution conditions, and other important features are not known at present. The other reason is that the amelogenin oligomerization process itself is quite complex 5,15,18,[20][21][22][23]27 and results in the formation of high-order high-molecular weight complexes that are difficult to study with conventional structural techniques such as NMR. To circumvent this problem, we turn to techniques and approaches that can offer insights into the native folding of IDPs and create conditions that mimic the self-assembly process in a more tractable fashion.…”

The Cooperation of Enamelin and Amelogenin in Controlling Octacalcium Phosphate Crystal Morphology

“…The mixtures were incubated for 20-30 min before measurements and data were analyzed as previously described [Moradian-Oldak et al, 2000;Fan et al, 2008, Laksminarayanan et al, 2010.…”