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

Moradian‐Oldak, Janet; Bouropoulos, Nikolaos; Wang, Lingli; Gharakhanian, Narbeh

doi:10.1016/s0945-053x(01)00190-1

Cited by 97 publications

(123 citation statements)

References 31 publications

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“…This organization within the nanosphere would optimize charged interactions between the protein and hydroxyapatite and has been suggested based on the results of several studies (9,10). Consistent with the placement of the COOH terminus on the surface of the nanospheres, experimental studies have also shown that removal of the COOH terminus reduces the ability of the protein to interact with HAP (14,15). Although very promising, these studies do not rule out other contributions such as protein-protein interactions or structural changes playing a key role in the change in interaction mechanism.…”

Section: Resultsmentioning

confidence: 75%

“…These hypotheses are supported by in vitro experiments, reviewed recently by Moradian-Oldak (13), demonstrating the effect of amelogenins on growing calcium phosphate crystals. Experiments by both Aoba et al (14) and Moradian-Oldak et al (15) demonstrate that the loss of the charged carboxyl terminus during proteolytic processing of amelogenin results in reduced affinity to hydroxyapatite and a reduction in ability to inhibit crystal growth. These observations provide some evidence that the COOH terminus is important in the interaction of amelogenin with the calcium phosphate crystal but do not rule out other contributions such as structural change or altered protein-protein interactions.…”

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confidence: 99%

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The COOH Terminus of the Amelogenin, LRAP, Is Oriented Next to the Hydroxyapatite Surface

Shaw

Campbell

Paine

et al. 2004

Journal of Biological Chemistry

134

167

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The organic matrix in forming enamel consists largely of the amelogenin protein self-assembled into nanospheres that are necessary to guide the formation of the unusually long and highly ordered hydroxyapatite (HAP) crystallites that constitute enamel. Despite its ability to direct crystal growth, the interaction of the amelogenin protein with HAP is unknown. However, the demonstration of growth restricted to the c-axis suggests a specific protein-crystal interaction, and the charged COOH terminus is often implicated in this function. To elucidate whether the COOH terminus is important in the binding and orientation of amelogenin onto HAP, we have used solid state NMR to determine the orientation of the COOH terminus of an amelogenin splice variant, LRAP (leucine-rich amelogenin protein), which contains the charged COOH terminus of the full protein, on the HAP surface. These experiments demonstrate that the methyl 13 C-labeled side chain of Ala 46 is 8.0 Å from the HAP surface under hydrated conditions, for the protein with and without phosphorylation. The experimental results provide direct evidence orienting the charged COOH-terminal region of the amelogenin protein on the HAP surface, optimized to exert control on developing enamel crystals.Enamel is composed of unusually long and highly oriented hydroxyapatite (HAP) 1 crystals (1), Ͼ1000 times longer than the HAP crystals found in bone (2). The molecular interactions leading to this highly controlled structure are not currently well understood, but the organic matrix is observed to be essential to the proper formation of enamel (3). Amelogenin proteins constitute Ͼ90% of the protein present in developing enamel (3). Amelogenin knock-out mice experience improper enamel formation, and genetic mutations of amelogenin also result in enamel defects, establishing the importance of amelogenin in enamel formation (3, 4).Despite its importance, mechanisms at the molecular level directing amelogenins control of enamel crystallites are not understood. It is known that under physiological conditions, amelogenin self-assembles into nanospheres of ϳ20 nm in diameter, consisting of multiple monomers, and this is believed to be its functional form (5-7). Transmission electron microscope studies have shown that the amelogenin nanospheres align in beaded rows along the c-axis of the developing enamel crystal (5), exhibiting exquisite control over the resulting morphology. This suggests a specific matrix-crystal interaction, yet the region of the protein interacting with HAP to result in the observed crystal regulation has not been identified. It has been postulated, based on experimental evidence, that both the charged NH 2 and COOH terminus of amelogenin are exposed on the surface of the nanospheres (6, 8 -11), aiding in proteinprotein interactions, increasing the solubility, and enhancing calcium phosphate interactions for the hydrophobic protein (12). These hypotheses are supported by in vitro experiments, reviewed recently by Moradian-Oldak (13), demonstrating the effect ...

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Section: Resultsmentioning

confidence: 75%

mentioning

confidence: 99%

The COOH Terminus of the Amelogenin, LRAP, Is Oriented Next to the Hydroxyapatite Surface

Shaw

Campbell

Paine

et al. 2004

Journal of Biological Chemistry

134

167

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“…The C-terminal amino acids of amelogenin are hydrophilic [21]. The loss of the charged COO -terminal of amelogenin results in a reduction of the affinity to hydroxyapatite [22]. Amelogenin molecules spontaneously self-assemble into spherical structures called nanospheres [23].…”

Section: Discussionmentioning

confidence: 99%

Unbinding Process of Amelogenin and Fibrinogen Adsorbed on Different Solid Surfaces Using AFM

Richert¹,

Boukari²,

Berner³

et al. 2011

JBNB

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The interaction of proteins with solid surfaces is a fundamental phenomenon in the biomaterials field. We investigated, using atomic force microscopy (AFM), the interactions of a recombinant amelogenin with titanium, a biphasic calcium phosphate (BCP) and mica. The unbinding processes were compared to those of an earlier studied protein, namely fibrinogen. Force spectroscopy (AFM) experiments were carried out at 0 ms, 10 2 ms, 10 3 ms and 10 4 ms of contact time. In general, the rupture forces increased as a function of interaction time. The unbinding forces of amelogenin interacting with the BCP surface were always stronger than those of the amelogenin-titanium system. The unbinding forces of fibrinogen interacting with the BCP surface were always much stronger than those of the fibrinogen-titanium system.For the most part, this study provides direct evidence that recombinant amelogenin binds more strongly than fibrinogen on the studied substrates.

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“…It has been found that amelogenin can affect the growth and crystal habit of calcium phosphate by adsorbing onto specific faces of growing seed crystals [5,14,15]. Studies have suggested that the charged amino acids in the C-terminal domain are exposed at the nanosphere surface, promoting amelogenin-calcium phosphate interactions [16]. Recent studies using solid-state NMR [17] and neutron reflectivity and molecular modeling [18] determined that the hydrophilic C-terminus was oriented next to the hydroxyapatite surface, providing direct evidence that the C-terminus of amelogenin is positioned to exert control over crystal growth.…”

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

Cited by 97 publications

References 31 publications

The COOH Terminus of the Amelogenin, LRAP, Is Oriented Next to the Hydroxyapatite Surface

The COOH Terminus of the Amelogenin, LRAP, Is Oriented Next to the Hydroxyapatite Surface

Unbinding Process of Amelogenin and Fibrinogen Adsorbed on Different Solid Surfaces Using AFM

The nucleation and growth of calcium phosphate by amelogenin

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