Janet Moradian-Oldak

Interaction of amelogenin with hydroxyapatite crystals: An adherence effect through amelogenin molecular self‐association

At the secretory stage of tooth enamel formation the majority of the organic matrix is composed of amelogenin proteins that are believed to provide the scaffolding for the initial carbonated hydroxyapatite crystals to grow. The primary objective of this study was to investigate the interaction between amelogenins and growing apatite crystals. Two in vitro strategies were used: first, we examined the influence of amelogenins as compared to two other macromolecules, on the kinetics of seeded growth of apatite crystals; second, using transmission electron micrographs of the crystal powders, based on a particle size distribution study, we evaluated the effect of the macromolecules on the aggregation of growing apatite crystals. Two recombinant amelogenins (rM179, rM166), the synthetic leucine-rich amelogenin polypeptide (LRAP), poly(L-proline), and phosvitin were used. It was shown that the rM179 amelogenin had some inhibitory effect on the kinetics of calcium hydroxyapatite seeded growth. The inhibitory effect, however, was not as destructive as that of other macromolecules tested. The degree of inhibition of the macromolecules was in the order of phosvitin > LRAP > poly(L-proline) > rM179 > rM166. Analysis of particle size distribution of apatite crystal aggregates indicated that the full-length amelogenin protein (rM179) caused aggregation of the growing apatite crystals more effectively than other macromolecules. We propose that during the formation of hydroxyapatite crystal clusters, the growing apatite crystals adhere to each other through the molecular self-association of interacting amelogenin molecules. The biological implications of this adherence effect with respect to enamel biomineralization are discussed.

The Tooth Enamel Protein, Porcine Amelogenin, Is an Intrinsically Disordered Protein with an Extended Molecular Configuration in the Monomeric Form

Amelogenins make up a class of proteins associated with the formation of mineralized enamel in vertebrates, possess highly conserved N- and C-terminal sequence regions, and represent an interesting model protein system for understanding biomineralization and protein assembly. Using bioinformatics, we report here the identification of molecular traits that classify 12 amelogenin proteins as members of the intrinsically disordered or unstructured protein family (IDPs), a group of proteins that normally exist as unfolded species but are capable of transformation to a folded state as part of their overall function. Using biophysical techniques (CD and NMR), we follow up on our bioinformatics studies and confirm that one of the amelogenins, recombinant porcine rP172, exists in an extended, unfolded state in the monomeric form. This protein exhibits evidence of conformational exchange between two states, and this exchange may be mediated by Pro residues in the sequence. Although the protein is globally unfolded, we detect the presence of local residual secondary structure [alpha-helix, extended beta-strand, turn/loop, and polyproline type II (PPII)] that may serve several functional roles within the enamel matrix. The extended, labile conformation of rP172 amelogenin is compatible with the known functions of amelogenin in enamel biomineralization, i.e., self-assembly, associations with other enamel matrix proteins and with calcium phosphate biominerals, and interaction with cell receptors. It is likely that the labile structure of this protein facilitates interactions of amelogenin with other macromolecules or with minerals for achievement of internal protein stabilization.

Protein-mediated enamel mineralization.

Enamel is a hard nanocomposite bioceramic with significant resilience that protects the mammalian tooth from external physical and chemical damages. The remarkable mechanical properties of enamel are associated with its hierarchical structural organization and its thorough connection with underlying dentin. This dynamic mineralizing system offers scientists a wealth of information that allows the study of basic principels of organic matrix-mediated biomineralization and can potentially be utilized in the fields of material science and engineering for development and design of biomimetic materials. This chapter will provide a brief overview of enamel hierarchical structure and properties and the process and stages of amelogenesis. Particular emphasis is given to current knowledge of extracellular matrix protein and proteinases, and the structural chemistry of the matrix components and their putative functions. The chapter will conclude by discussing the potential of enamel for regrowth.

Analysis of self-assembly and apatite binding properties of amelogenin proteins lacking the hydrophilic C-terminal

Amelogenins, the major protein component of the mineralizing enamel extracellular matrix, are critical for normal enamel formation as documented in the linkage studies of a group of inherited disorders, with defective enamel formation, called Amelogenesis imperfecta. Recent cases of Amelogenesis imperfecta include mutations that resulted in truncated amelogenin protein lacking the hydrophilic C-terminal amino acids. Current advances in knowledge on amelogenin structure, nanospheres assembly and their effects on crystal growth have supported the hypothesis that amelogenin nanospheres provide the organized microstructure for the initiation and modulated growth of enamel apatite crystals. In order to evaluate the function of the conserved hydrophilic C-terminal telopeptide during enamel biomineralization, the present study was designed to analyze the self-assembly and apatite binding behavior of amelogenin proteins and their isoforms lacking the hydrophilic C-terminal. We applied dynamic light scattering to investigate the size distribution of amelogenin nanospheres formed by a series of native and recombinant proteins. In addition, the apatite binding properties of these amelogenins were examined using commercially available hydroxyapatite crystals. Amelogenins lacking the carboxy-terminal (native P161 and recombinant rM166) formed larger nanospheres than those formed by their full-length precursors: native P173 and recombinant rM179. These data suggest that after removal of the hydrophilic carboxy-terminal segment further association of the nanospheres takes place through hydrophobic interactions. The affinity of amelogenins lacking the carboxy-terminal regions to apatite crystals was significantly lower than their parent amelogenins. These structure-functional analyses suggest that the hydrophilic carboxy-terminal plays critical functional roles in mineralization of enamel and that the lack of this segment causes abnormal mineralization.

Interactions of Matrix Proteins from Mineralized Tissues with Octacalcium Phosphate

Acidic matrix macromolecules, present in many mineralized tissues, including those of vertebrates, are thought to be involved in controlling crystal formation. Little, however, is known about their in vivo functions, particularly in relation to calcium-phosphate-containing crystals. The manner in which a variety of synthetic and natural acidic macromolecules interact in vitro with crystals of octacalcium phosphate (OCP) has been studied. Interactions were assessed by examining changes in morphology of the crystals resulting from preferential interaction of the additive with some crystal faces and not others. Macromolecules rich in acidic amino acids, with or without polysaccharides, such as polyaspartate and mollusk shell proteins respectively, were shown to interact preferentially with rows of Ca ions exposed on the hydrated plate surface of OCP crystals. In contrast, the phosphorylated proteins, phosphophoryn and phosvitin, interacted specifically with the apatite-like motifs on the OCP side faces. BSP did not interact specifically with OCP, under the experimental conditions used. The observation that these classes of acidic macromolecules recognize different crystal faces should be taken into account when evaluating functions of acidic matrix macromolecules in mineralized tissues.

Elongated Growth of Octacalcium Phosphate Crystals in Recombinant Amelogenin Gels under Controlled Ionic Flow

Amelogenin proteins constitute the primary structural entity of the extracellular protein framework of the developing enamel matrix. Recent data on the interactions of amelogenin with calcium phosphate crystals support the hypothesis that amelogenins control the oriented and elongated growth of enamel carbonate apatite crystals. To exploit further the molecular mechanisms involved in amelogenin-calcium phosphate mineral interactions, we conducted in vitro experiments to examine the effect of amelogenin on synthetic octacalcium phosphate (OCP) crystals. A 10% (wt/vol) recombinant murine amelogenin (rM179, rM166) gel was constructed with nanospheres of about 10- to 20-nm diameter, as observed by atomic force microscopy. The growth of OCP was modulated uniquely in 10% rM179 and rM166 amelogenin gels, regardless of the presence of the hydrophilic C-terminal residues. Fibrous crystals grew with large length-to-width ratio and small width-to-thickness ratio. Both rM179 and rM166 enhanced the growth of elongated OCP crystals, suggesting a relationship to the initial elongated growth of enamel crystals.

How amelogenin orchestrates the organization of hierarchical elongated microstructures of apatite.

Amelogenin (Amel) accelerates the nucleation of hydroxyapatite (HAP) in supersaturated solutions of calcium phosphate (Ca-P), shortening the induction time (delay period), under near-physiological conditions of pH, temperature, and ionic strength. Hierarchically organized Amel and amorphous calcium phosphate (ACP) nanorod microstructures are formed involving a coassembly of Amel-ACP particles at low supersaturations and low protein concentrations in a slow, well-controlled, constant composition (CC) crystallization system. At the earliest nucleation stages, the CC method allows the capture of prenucleation clusters and intermediate nanoclusers, spherical nanoparticles, and nanochains prior to enamel-like nanorod microstructure formations at later maturation stages. Amel-ACP nanoscaled building blocks are formed spontaneously by synergistic interactions between flexible Amel protein molecules and Ca-P prenucleation clusters, and these spherical nanoparticles evolve by orientated aggregation to form nanochains. Our results suggest that, in vivo, Amel may determine the structure of enamel by controlling prenucleation cluster aggregation at the earliest stages by forming stable Amel-ACP microstructures prior to subsequent crystal growth and mineral maturation.

Electron imaging and diffraction study of individual crystals of bone, mineralized tendon and synthetic carbonate apatite

A transmission electron microscope study of carbonate apatite crystals isolated from bone and mineralizing tendon, as well as those produced synthetically under approximated-physiological conditions, shows that they are thin irregular shaped plates. Electron diffraction patterns of individual crystals confirm that the large developed crystal face is (100), and that the longest dimensions of the biogenic crystals are aligned with the crystallographic c axes. As the latter are also aligned with collagen fibril axes, the observations provide additional information on the tissue organization itself. The marked similarity between the biologic and synthetic crystals suggests that the biological environment in which the crystals form may not be primarily responsible for controlling their shape.

Topographic imaging of mineral and collagen in the calcifying Turkey tendon

Topographic imaging, a method of providing a direct view of ultrastructure in three dimensions, has been newly applied to a study of mineral crystals and collagen from calcifying turkey leg tendon. Individual crystals obtained from intact tendon were observed as thin platelets of irregular shape having a relatively smooth surface. Mineralized collagen fibrils isolated singly or examined in thin tissue sections were found to exhibit the characteristic 64-70 nm period and were associated with platelets and needle-like mineral. The crystals were disposed in numerous fashions, notably as small groups of platelets within individual collagen hole zones, as a number of needle-like densities arranged parallel to one another, or as a combination of platelets and needles over entire stretches of single collagen fibrils. The topographic observations of crystals and crystal-collagen interaction clearly demonstrate the plate-like habit of the mineral in calcifying turkey tendon and suggest that these crystals are located both within and on the surface of collagen fibrils. In certain sites such as the collagen hole zones, the crystals appear organized in a specific manner, possibly with a preferred c-axial orientation. Crystals of hydroxyapatite prepared in vitro and examined topographically are similar in habit and texture to the crystals from tendon. When interpretation of this method is corroborated by other independent microscopic techniques, topographic imaging has widespread potential application in many fields of study in which structural surface features of biological tissues or non-biological materials are of interest at the electron microscope level.

Control of octacalcium phosphate and apatite crystal growth by amelogenin matrices

Tooth enamel, the hardest bioceramic composite in the vertebrate body, is the result of a cascade of intra- and extracellular events. Amelogenins, the principal extracellular matrix protein component of mineralizing enamel, have been considered to play substantial roles in controlling the growth and organization of enamel crystals. Considering octacalcium phosphate (OCP) as a precursor phase of enamel apatite crystallites, we have developed in vitro systems to grow OCP and apatite crystals in amelogenin matrices and therefore to investigate amelogenin–OCP and amelogenin–apatite interactions. This paper reviews our current findings on the effect of amelogenin on the morphology, size, phase and orientation of such crystals.