Analysis of Hydroxyapatite Surface Coverage by Amelogenin Nanospheres Following the Langmuir Model for Protein Adsorption

Abstract

The assembly of amelogenin protein into nanospheres is postulated to be a key factor in the stability of enamel extracellular matrix framework, which provides the scaffolding for the initial enamel apatite crystals to nucleate and grow. Adsorption isotherms were evaluated in order to investigate the nature of interactions of amelogenin nanospheres with hydroxyapaite crystals in solution, where their assembly status and particle size distribution are defined. We report that the adsorption isotherm of a recombinant mouse amelogenin (rM179) on synthetic hydroxyapatite crystals can be described using a Langmuir model indicating that amelogenin nanospheres adsorb onto the surface of apatite crystals as binding units with defined adsorption sites. The adsorption affinity and the maximum adsorption sites were 19.7 x 10(5) L/mol and 6.09 x 10(-7) mol/m2, respectively, with an r2 value of 0.99. Knowing the composition and particle size distribution of amelogenin nanospheres under the condition of adsorption experiments, we have calculated the number of nanospheres and the crystal surface area covered by each population of nanospheres at their maximum adsorption. It was found that total maximum binding covers 64% of the area unit. This observation supports the speculation that amelogenin binding onto apatite surface is selective and occurs only at certain sites.

Dissecting Amelogenin Protein Nanospheres

“…The "unstructured" nature of the oligomers makes them ideal for interaction with various targets, such as apatite (52,53), enamelin (53,54), ameloblastin (55), and the cell surface (56). Fluorescence experiments with single tryptophan amelogenins revealed that whereas the C terminus of amelogenin (specifically residue Trp 161 ) resides in an aqueous environment even when assembled into oligomers or nanospheres the N-terminal region around Trp 25 and Trp 45 moves into a more hydrophobic environment as soon as monomers begin to assemble into oligomers.…”

“…At pH 8, the histidine residues would be deprotonated, allowing weak hydrophobic forces to bind the oligomers together in the form of a nanosphere. b, oligomers become stabilized on the positively charged surfaces and in the mildly acidic environments that are achieved during the maturation stage of enamel development (8,51,52). Blue rectangles and red ovals represent N and C termini, respectively.…”

Amelogenin Supra-Molecular Assembly in vitro Compared with the Architecture of the Forming Enamel Matrix

“…Following the description of the 'nanospheres', we have considered the interactions of apatite with amelogenin 'nanospheres' as a binding unit as opposed to amelogenin 'monomers'. Bouropoulos and Moradian-Oldak [2003] reported that the adsorption isotherm of amelogenin nanospheres onto hydroxyapatite could be described with a good correlation coeffi cient (R 2 = 0.99) indicating that amelogenin nanospheres can bind to the surface of apatite crystals with defi ned adsorption sites. Recently, solid-state NMR studies have provided direct evidence for the orientation of the charged C-terminal region of amelogenin protein on the hydroxyapatite surface [Shaw et al, 2004].…”

Reuptake of Extracellular Amelogenin by Dental Epithelial Cells Results in Increased Levels of Amelogenin mRNA through Enhanced mRNA Stabilization

“…It has been suggested that amelogenins are essential for the organization of the crystal pattern and the regulation of enamel thickness (10). Some researchers demonstrated that amelogenin plays an important role in the control of hydroxylapatite nucleation, orientation, and growth habit (11)(12)(13)(14)(15)(16). Amelogenin-null mice exhibit a phenotype similar to human X-linked amelogenesis imperfecta, in which ameloblast differentiation is normal but an abnormally thin layer of enamel is formed.…”