Altered Amelogenin Self-assembly Based on Mutations Observed in Human X-linked Amelogenesis Imperfecta (AIH1)

Abstract

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin, a 180-amino acid hydrophobic protein that self-assembles to form nanospheres. We have used independent technical methods, consisting of the yeast twohybrid (Y2H) assay and surface plasmon resonance (SPR), to demonstrate the importance of amelogenin self-assembly domains. In addition, we have analyzed mutations in amelogenin observed in patients with amelogenesis imperfecta who demonstrate defects in enamel formation. Assessments of self-assembly of these mutant amelogenins by either SPR or Y2H assay yield concordant data. These data support the conclusion that the amelogenin amino-terminal self-assembly domain is essential to the creation of an enamel extracellular organic matrix capable of directing mineral formation. It also suggests that a pathway through which point mutations in the amelogenin protein can adversely impact on the formation of the enamel organ is by disturbing self-assembly of the organic matrix. These data support the utilization of the Y2H assay to search for protein interactions among extracellular matrix proteins that contribute to biomineralization and provide functional information on protein-protein and protein-mineral interactions.The self-assembly of a protein complex is a hallmark of biological systems. From processive enzymatic reactions to extracellular matrices, cells achieve control over their microenvironment through the assembly of a mixture of proteins. Protein assemblies are created with a specific stoichiometry. Such assembly precision is achieved through the interaction of protein motifs contained within the constituent protein members. The yeast two-hybrid (Y2H) 1 assay has been used successfully to study protein assembly and offers distinct advantages over many biochemical techniques used previously. An example of the value of the Y2H assay can be seen in the assembly properties of type X collagen. Type X collagen is a homotrimer of ␣1(X) chains encoded by the COL10A1 gene, which is expressed in hypertrophic chondrocytes during the process of endochondral ossification. Dwarfism in domestic pigs is a result of a single change (Gly 590 3 Arg) to the ␣1(X) chain of type X collagen (1). In humans, a similar phenotype has been described for a mutation at the equivalent position (Gly 590 3 Glu) of type X collagen. These patients have Schmid metaphyseal chondrodysplasia, which is a mild skeletal disorder associated with dwarfism and growth plate abnormality. The Y2H assay and in vitro assembly experiments demonstrated that the amino acid substitution interfered with the ability of the mutated collagen molecules to engage in trimerization (1). In this example, confirmation of a valid animal model for a human genetic disease has been possible in part by the Y2H assay. At the same time, the Y2H assay has ...

Altering Biomineralization by Protein Design

“…Thr-21 and Pro-41) mutations within the A-domain of amelogenin are affected with defective enamel formation, a disease known as amelogenesis imperfecta (63,64). Previously, we used plasmon resonance spectroscopy, dynamic light scatter, and atomic force microscopy to show that recombinant amelogenin that phenocopies these single amino acid mutations from man also results in defective selfassembly for amelogenin (22,65). Based upon the present gene replacement studies with engineered amelogenin in mice, we predict that altered interactions between the ameloblasts and the mutated amelogenin in the matrix of humans affected with amelogenesis imperfecta contribute to the defective mineralization and materials properties observed in their enamel.…”

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

“…16 In the case of the porcine species, under low pH aqueous conditions the monomeric form of amelogenin exists in an extended, unfolded state. 9 It is suspected that intrinsic disorder contributes to the structure and function of these proteins in self-assembly 5,9,15,[17][18][19][20][21][22] and in cellmatrix, protein-matrix, and protein-mineral interactions. [5][6][7][8][9][23][24][25] The main challenge is to elucidate how intrinsic disorder affects the molecular behavior and configuration of this unusual series of proteins, and how amelogenin molecular behavior, in turn, affects biomineralization within the enamel matrix.…”

“…Recent NMR studies of recombinant porcine amelogenin (rP172) indicated that there are three primary regions that are structurally distinct. 9 These are the highly conserved N-terminus (P2-W45) 26 or the proposed selfassembly ''A domain'' (P2-M42), [20][21][22] the partially condensed, charged C-terminal domain (D155-D173), 9,[20][21][22] and the extended Pro, Met, Gln-rich central domain (T58-P154) 9,16 that contains the polyproline Type II I70-P89 and P102-P145 sequence regions. 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. 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.…”

NF-Y and CCAAT/Enhancer-binding Protein α Synergistically Activate the Mouse Amelogenin Gene

“…These signals culminate to orchestrate expression of the amelogenin gene, the major organic component of the enamel matrix. Amelogenin plays a key role in regulating proper enamel mineralization and is believed to regulate its own replacement by the mineral phase to create a woven hierarchical architecture that accounts for the unique material properties of enamel (2)(3)(4)(5). Mutations to the human amelogenin gene have been linked to patients with the inherited enamel defect X-linked amelogenesis imperfecta (6).…”