Altering Biomineralization by Protein Design

Abstract

To create a bioceramic with unique materials properties, biomineralization exploits cells to create a tissue-specific protein matrix to control the crystal habit, timing, and position of the mineral phase. The biomineralized covering of vertebrate teeth is enamel, a distinctive tissue of ectodermal origin that is collagen-free. In forming enamel, amelogenin is the abundant protein that undergoes self-assembly to contribute to a matrix that guides its own replacement by mineral. Conserved domains in amelogenin suggest their importance to biomineralization. We used gene targeting in mice to replace native amelogenin with one of two engineered amelogenins. Replacement changed enamel organization by altering protein-to-crystallite interactions and crystallite stacking while diminishing the ability of the ameloblast to interact with the matrix. These data demonstrate that ameloblasts must continuously interact with the developing matrix to provide amelogenin-specific protein to protein, protein to mineral, and protein to membrane interactions critical to biomineralization and enamel architecture while suggesting that mutations within conserved amelogenin domains could account for enamel variations preserved in the fossil record.In mammals mesenchyme-derived bone is the dominant biomineralized tissue and is composed of substituted hydroxyapatite crystals with specific proteins distributed in an abundant collagen network (1-3). For bone, the mineral composite is dynamic with both the protein and mineral phases being constantly remodeled by cells. A notable exception to such a biomineralization theme is found in enamel, the vertebrate covering of teeth. In teeth, ectoderm-derived ameloblasts synthesize a short-lived, diverse repertoire of proteins that self-assemble to produce a matrix that directs its own replacement by the mineral phase (4, 5). Mature enamel is a bioceramic composed of less than 0.5% protein that is dispersed among the longest hydroxyapatite crystals in the vertebrate body (6 -8). Unlike mesenchyme-derived bone, enamel does not contain collagen, does not have the capacity of self-repair, and does not undergo remodeling. Nonetheless, enamel lasts the lifetime of the organism due its unique material properties and remarkable resistance to failure (9, 10). These properties are due in part to the cell-directed organization of crystallites into bundles called rods. Rods are further woven together during their formation by cell migration to form a unique three-dimensional lattice architecture that resists wear and deformation (9 -14).Amelogenin is the most abundant of the enamel matrix proteins and undergoes self-assembly to form nanospheres (15-17). Amelogenin nanospheres appear to control the habit of hydroxyapatite crystallites by directing mineral growth to the ends (cЈ axis) of the crystallites by providing the space for crystallites to grow within and by buffering the local environment against protons liberated during mineral deposition (5, 7, 18 -20). The amino acid sequence of amelogenin is highly ...

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.…”

Leucine-rich amelogenin peptide induces osteogenesis in mouse embryonic stem cells

“…Cell passage was achieved by treatment of the cells with 0.05% Trypsin and reseeded onto a fresh MEF feeder layer. Amelogenin-null (KO) ES cells were generated as previously described [15] (See supplement and Fig S1) and were maintained in the same condition as the wild-type ES cells.…”

Protein Interaction between Ameloblastin and Proteasome Subunit α Type 3 Can Facilitate Redistribution of Ameloblastin Domains within Forming Enamel

“…Enamel forms in the extracellular space with the replacement of enamel matrix proteins by carbonated HAP crystallites. To provide optimal material properties required for fracture toughness (3,19,61), the crystallites within the rods are woven together with the interrod crystallites to form a continuum. The enamel rod is the smallest divisible repeating structural element of enamel, and each rod is fabricated by a single ameloblast cell that synthesizes and secretes enamel matrix proteins that guide their replacement by the mineral phase.…”