Detection of monodisperse aggregates of a recombinant amelogenin by dynamic light scattering

Moradian‐Oldak, Janet; Simmer, James P.; Lau, Eduardo C.; Sarte, Patrick; Slavkin, Harold C.; Fincham, Alan G.

doi:10.1002/bip.360341006

Cited by 133 publications

(153 citation statements)

References 44 publications

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“…Increasing nucleation density with increasing protein concentration up to 6.5 μg/mL may occur because of an increase in the number of available nucleation sites with protein concentration. It has been found that the amelogenin nanosphere size is constant over a wide range of concentrations (0.05-2 mg/ mL, [6]), indicating that only the nanosphere density is changing with protein concentration.…”

Section: Nucleation Behaviormentioning

confidence: 99%

“…Roles in nucleation, growth, regulation of crystal size and shape, and control of crystal-crystal aggregation have been proposed [2][3][4][5]. Amelogenin is unique in that it self-assembles into nanospheres, supra-molecular structures observed in vitro [6][7][8] and in vivo [7,9] which are believed to be critical to the function of the protein. Several in vivo studies using antisense mice [10], knock out mice [11], transgenic mice [12], and hammerhead ribozymes [13] have investigated the role of amelogenin in regulating enamel nucleation and growth.…”

Section: Introductionmentioning

confidence: 99%

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The nucleation and growth of calcium phosphate by amelogenin

Tarasevich

Howard

Larson

et al. 2007

Journal of Crystal Growth

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The nucleation processes involved in calcium phosphate formation in tooth enamel are not well understood but are believed to involve proteins in the extracellular matrix. The ability of one enamel protein, amelogenin, to promote the nucleation and growth of calcium phosphate was studied in an in vitro system involving metastable supersaturated solutions. It was found that recombinant amelogenin (rM179 and rp(H)M180) promoted the nucleation of calcium phosphate compared to solutions without protein. The amount of calcium phosphate increased with increasing supersaturation of the solutions and increasing protein concentrations up to 6.5 μg/mL. At higher protein concentrations, the amount of calcium phosphate decreased. The kinetics of nucleation was studied in situ and in real time using a quartz crystal microbalance (QCM) and showed that the protein reduced the induction time for nucleation compared to solutions without protein. This work shows a nucleation role for amelogenin in vitro which may be promoted by the association of amelogenin into nanosphere templates, exposing charged functionality at the surface.

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Section: Nucleation Behaviormentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The nucleation and growth of calcium phosphate by amelogenin

Tarasevich

Howard

Larson

et al. 2007

Journal of Crystal Growth

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“…Hoang et al (2002) have shown that recombinant amelogenin is responsible for cell-adhesive activity. Native amelogenin self-assembles to form spherical aggregates, the so-called nanospheres, that are believed to function as structural components directly involved in the matrix-mediated enamel biomineralization (Moradian-Oldak et al 1994a, 2003Wen et al 2001;Snead 2003). In a series of articles, Ravindranath et al (1999Ravindranath et al ( , 2000Ravindranath et al ( , 2001Ravindranath et al ( , 2003 have investigated the possible interactions of amelogenin with the ameloblasts, in particular, with some keratins, through the ligand-binding properties of the C-terminal domain, which binds specifically to N-acetyl glucosamine and to N-acetyl glucosamine-mimicking peptide.…”

Section: Introductionmentioning

confidence: 99%

Molecular Evolution of Amelogenin in Mammals

2005

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Abstract. An evolutionary analysis of mammalian amelogenin, the major protein of forming enamel, was conducted by comparison of 26 sequences (including 14 new ones) representative of the main mammalian lineages. Amelogenin shows highly conserved residues in the hydrophilic N-and C-terminal regions. The central hydrophobic region (most of exon 6) is more variable, but it has conserved a high amount of proline and glutamine located in triplets, PXQ, indicating that these residues play an important role. This region evolves more rapidly, and is less constrained, than the other well-conserved regions, which are subjected to strong constraints. The comparison of the substitution rates in relation to the CpG richness confirmed that the highly conserved regions are subjected to strong selective pressures. The amino acids located at important sites and the residues known to lead to amelogenesis imperfecta when substituted were present in all sequences examined. Evolutionary analysis of the variable region of exon 6 points to a particular zone, rich in either amino acid insertion or deletion. We consider this region a hot spot of mutation for the mammalian amelogenin. In this region, numerous triplet repeats (PXQ) have been inserted recently and independently in five lineages, while most of the hydrophobic exon 6 region probably had its origin in several rounds of triplet insertions, early in vertebrate evolution. The putative ancestral DNA sequence of the mammalian amelogenin was calculated using a maximum likelihood approach. The putative ancestral protein was composed of 177 residues. It already contained all important amino acid positions known to date, its hydrophobic variable region was rich in proline and glutamine, and it contained triplet repeats PXQ as in the modern sequences.

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“…A regular buildup regime of the (PSS-PAH) n multilayer is reached after at least three bilayers 37 , therefore a PEM film constituted by four bilayers of PSS-PAH was always used. rM179 (0.25mg/mL in 25 mM Tris, pH8) was then adsorbed onto the positively ending PEI-(PSS-PAH) 4 multilayer film. At these experimental conditions rM179 self assembles to nanospheres.…”

Section: Optical Waveguide Light-mode Spectroscopy (Owls)mentioning

confidence: 99%

“…Once the optical signals leveled off, the protein solution was replaced by buffer solution and the desorption of the proteins (if any) was monitored. To build multilayers from the negatively charged amelogenin nanospheres, PLL as a positively charged polyelectrolyte was adsorbed between the nanosphere adsorbtion steps, until a PEI-(PSS-PAH) 4 -(rM179-PLL) 4 mixed polyelectrolyte/nanosphere architecture was obtained. In other experiments the effect of GlcNac was studied: GlcNac was first adsorbed onto the top of the PEI-(PSS-PAH) 4 film, followed by nanosphere adsorption, until, in theory, a PEI-(PSS-PAH) 4 -(GlcNAc-rM179) 2 architecture was obtained.…”

Section: Optical Waveguide Light-mode Spectroscopy (Owls)mentioning

confidence: 99%

Polyelectrolyte-Mediated Adsorption of Amelogenin Monomers and Nanospheres Forming Mono- or Multilayers

et al. 2007

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We have applied optical waveguide lightmode spectroscopy combined with streaming potential measurements and Fourier transformed infrared spectroscopy to investigate adsorption of amelogenin nanospheres onto polyelectrolytes. The long term objective was to better understand the chemical nature of these assemblies and to gain further insight into the molecular mechanisms involved during self-assembly. It was found that monolayers of monomers and negatively charged nanospheres of a recombinant amelogenin (rM179) irreversibly adsorbed onto a positively charged polyelectrolyte multilayer films. Based on measurements performed at different temperatures it was demonstrated that intermolecular interactions for the formation of nanospheres were not affected by their adsorption onto polyelectrolytes. Consecutive adsorption of nanospheres resulting in the formation of multilayer structures was possible by using cationic poly(L-lysine) as mediators. NAcetyl-D-Glucosamine (GlcNac) did not disturb the nanosphere-assembled protein's structure and it only affected the adsorption of monomeric amelogenin. Infrared spectroscopy of adsorbed amelogenin revealed conformational differences between the monomeric and assembled forms of rM179. While there was a considerable amount of α-helices in the monomers, β-turn and β-sheet structures dominated the assembled proteins. Our work constitutes the first report on a structurally controlled in-vitro buildup of an rM179 nanosphere monolayer-based matrix. Our data support the notion that amelogenin self-assembly is mostly driven by hydrophobic interactions and that amelogenin/PEM interactions are dominated by electrostatic forces. We suggest that similar forces can govern amelogenin interactions with non-amelogenins or the mineral phase during enamel biomineralization.

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Detection of monodisperse aggregates of a recombinant amelogenin by dynamic light scattering

Cited by 133 publications

References 44 publications

The nucleation and growth of calcium phosphate by amelogenin

The nucleation and growth of calcium phosphate by amelogenin

Molecular Evolution of Amelogenin in Mammals

Polyelectrolyte-Mediated Adsorption of Amelogenin Monomers and Nanospheres Forming Mono- or Multilayers

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