Recombinant Full-length Amelogenin Research Articles

Glucose-Regulated Protein 78: A Novel Therapeutic Target for Amelogenin- Induced Periodontal Tissue Regeneration

Amelogenin, the major component of a commercial enamel matrix derivative (Straumann® Emdogain), is commonly employed in periodontology. It is mainly used in periodontal surgery to stimulate the regeneration of periodontal tissues, including the cementum, periodontal ligament, and alveolar bone. However, the precise molecular mechanisms underlying amelogenin-induced regeneration have not yet been elucidated. Thus, to gain further insight into how amelogenin induces periodontal tissue regeneration, we performed a protein interaction screen using recombinant full-length amelogenin (rM180) as bait. Coupling affinity chromatography with proteomic analysis identified glucose-regulated protein 78 (Grp78) as a new amelogenin-binding protein. We further demonstrated that the interaction between amelogenin and Grp78 not only contributes to cell proliferation in osteoblastic cells but also enhances cell migration in periodontal ligament stem/progenitor cells. Although the potentiation effects of Grp78 should be further examined in vivo, our findings indicate the significant therapeutic potential for amelogenin-induced periodontal tissue regeneration.

Novel amelogenin-releasing hydrogel for remineralization of enamel artificial caries.

Recently, the use of recombinant full-length amelogenin protein in combination with fluoride has shown promising results in the formation of densely packed enamel-like structures. In this study, amelogenin (rP172)-releasing hydrogels containing calcium, phosphate, and fluoride were investigated for remineralization efficacy using in vitro early enamel caries models. The hydrogels were applied to artificial caries lesions on extracted human third molars, and the remineralization efficacy was tested in different models: static gel remineralization in the presence of artificial saliva, pH cyclic treatment at pH 5.4 acetic buffer and pH 7.3 gel remineralization, and treatment with multispecies oral biofilms grown in a continuous flowing constant-depth film fermenter. The surface microhardness of remineralized enamel increased significantly when amelogenin was released from hydrogel. No cytotoxicity was observed when periodontal ligament cells were cultured with the mineralized hydrogels.

Self-aligning amelogenin nanoribbons in oil–water system

The highly organized microstructure of dental enamel is a result of protein-guided anisotropic growth of apatite nanofibers. It is established that amelogenin proteins, the main constituent of the developing enamel matrix, form nanospheres in vitro, but the amphiphilic nature of the full-length protein conveys the possibility of generating more complex structures as observed with other surfactant-like molecules. This study tested if the use of metastable oil-water emulsions can induce supramolecular assemblies of amelogenin. Recombinant full-length amelogenin, rH174, was mixed into octanol/ethyl acetate preparations of different ratios to form emulsions at pH 4.5 and 7.4. Atomic force and electron microscopy showed the formation of 16.7±1.0nm wide nanoribbons which grew to several micrometer length over a period of days. Nanoribbons formed from reverse micelles by enabling hydrophobic tails of the molecules to interact while preventing the formation of amelogenin nanospheres. Ribbon formation required the presence of calcium and phosphate ions and may be localized at a dark central line along the amelogenin ribbons. The ribbons have a strong tendency to align in parallel maintaining 5-20nm space between each other. The growth rates and number of ribbons were significantly higher at pH 4.5 and related to the metastability of the emulsion. A model for ribbon extension proposes the addition of short segments or amelogenin dimers to the ends of the ribbon. The formation of self-aligning and uniaxially elongating amelogenin structures triggered by the presence of calcium and phosphate may represent a suitable new model for protein controlled mineralization in enamel.

Role of 20-kDa Amelogenin (P148) Phosphorylation in Calcium Phosphate Formation in Vitro

The potential role of amelogenin phosphorylation in enamel formation is elucidated through in vitro mineralization studies. Studies focused on the native 20-kDa porcine amelogenin proteolytic cleavage product P148 that is prominent in developing enamel. Experimental conditions supported spontaneous calcium phosphate precipitation with the initial formation of amorphous calcium phosphate (ACP). In the absence of protein, ACP was found to undergo relatively rapid transformation to randomly oriented plate-like apatitic crystals. In the presence of non-phosphorylated recombinant full-length amelogenin, rP172, a longer induction period was observed during which relatively small ACP nanoparticles were transiently stabilized. In the presence of rP172, these nanoparticles were found to align to form linear needle-like particles that subsequently transformed and organized into parallel arrays of apatitic needle-like crystals. In sharp contrast to these findings, P148, with a single phosphate group on serine 16, was found to inhibit calcium phosphate precipitation and stabilize ACP formation for more than 1 day. Additional studies using non-phosphorylated recombinant (rP147) and partially dephosphorylated forms of P148 (dephoso-P148) showed that the single phosphate group in P148 was responsible for the profound effect on mineral formation in vitro. The present study has provided, for the first time, evidence suggesting that the native proteolytic cleavage product P148 may have an important functional role in regulating mineralization during enamel formation by preventing unwanted mineral formation within the enamel matrix during the secretory stage of amelogenesis. Results obtained have also provided new insights into the functional role of the highly conserved hydrophilic C terminus found in full-length amelogenin.

The cooperative self-assembly of 25 and 23 kDa amelogenins

Self-assembly of the extracellular matrix protein amelogenin is believed to play an essential role in regulating the growth and organization of enamel crystals during enamel formation. The full-length amelogenin uniquely regulates the growth, shape, and arrangement of enamel crystals. Protein hydrolysis will ultimately facilitate a tissue with high mineral content. Protein processing is however highly specific suggesting a functional role of the cleaved amelogenins in enamel maturation. Here we hypothesize that the cooperative self-assembly of the recombinant full-length amelogenin 25kDa and the 23kDa proteolytic cleavage product is a function of pH, mixing ratio and incubation time and is associated with the isoelectric point of the protein. Self-assembly of amelogenin into nanospheres which increased in size with increasing pH was observed by atomic force microscopy. Elongated structures of about 100nm length and 25nm width formed over several days for amelogenin 25 and 23kDa predominantly at pH-values of 6.5 and 7.5, respectively. When both proteins 25 and 23kDa were mixed, self-assembled nanostrings of 200-300nm length consisting of fused nanospheres were obtained at pH around 7.0 within 24h. The protein nanostrings formed links over time and a continuous mesh was obtained after 7 days. Electrical conductivity data also showed gradual changes when both amelogenins were mixed in solutions supporting the idea that elongated structures form over extended periods of time. We propose that due to the difference in the isoelectric point, self-assembled nanospheres composed of 23 or 25kDa amelogenin have opposite ionic charges at pH-values around 7.0 and thus experience ionic attraction that enables cooperative self-assembly.

Enamel inspired nanocomposite fabrication through amelogenin supramolecular assembly

Fabricating the structures similar to dental enamel through the in vitro preparation method is of great interest in the fields of dentistry and material sciences. Developing enamel is composed of calcium phosphate mineral, water, and enamel matrix proteins, mainly amelogenins. To prepare a material mimicking such composition a novel approach of simultaneously assembling amelogenin and calcium phosphate precipitates by electrolytic deposition (ELD) was established. It was found that recombinant full-length amelogenin (rP172) self-assembled into nanochain structures during ELD (following increase in solution pH), and had significant effect on the induction of the parallel bundles of calcium phosphate nanocrystals, grown on semiconductive silicon wafer surface. When a truncated amelogenin (rP148) was used; no nanochain assembly was observed, neither parallel bundles were formed. The coating obtained in the presence of rP172 had improved elastic modulus and hardness when compared to the coating incorporated with rP148. Our data suggest that the formation of organized bundles in amelogenin–apatite composites is mainly driven by amelogenin nanochain assembly and highlights the potential of such composite for future application as dental restorative materials.

Fabrication of enamel-mimicking mineralization composite coating induced by electrolytic deposition (ELD) system

AbstractMature tooth enamel does not remodel nor self-repairs after damage. Therefore, in vitro formation of a uniquely ordered composite similar to enamel is of particular interest. In this study we applied a newly developed electrolytic deposition (ELD) system to promote the amelogenin nano-chain self-assembly and simultaneous calcium phosphate crystallization. Composite coatings of amelogenin-calcium phosphate were prepared on a cathode substrate (Si wafer), from an initial pH of 4-5, and calcium and phosphorus containing solution at room temperature. The effects of a recombinant full-length amelogenin (rP172) and truncated protein without the C-terminal (rP148) on the growth and morphology of the calcium phosphate nano-composite were investigated. A potentiostat was used to control the electrochemical parameters.Following the application of electric current, the local pH around the cathode was increased and resulted in the self-assembly of amelogenin that occurred simultaneously with calcium phosphate mineralization. Transmission and scanning electron micrographs of assembled rp172 amelogenin collected from the 25mM sodium phosphate solution during electrodeposition showed uniform nanospheres and nano-chains structures, while the rP148 formed irregular aggregates. SEM observation of the surface of the nano-composite after electrodeposition, revealed organized nano-rod structures in the presence of rP172, while only nano-sized spherical aggregates were seen in the presence of rP148. ATR-IR confirmed that the mineral phases were mainly octacalcium phosphate, but apatite and amorphous calcium phosphate were also present. Nanoindentation was tested on rP172 and rP148 composite coating. The rP172 composite coating exhibited higher elastic modulus and hardness than rP148 composite. The fracture toughness of rP172 composite was comparable to mature enamel. We therefore conclude that the 24 amino acid hydrophilic C-terminal of amelogenin is essential for the self-assembly, and therefore, for calcium phosphate crystal organization. The new electro-deposition system is effective for fabrication of amelogenin/apatite composites with defined organized structures.

Self‐assembly and effect on crystal growth of the leucine‐rich amelogenin peptide

Amelogenins are a unique group of alternatively spliced proteins. While the full-length amelogenin is known to assemble into nanospheres and alter apatite crystal growth and alignment, the function of the leucine-rich amelogenin peptide (LRAP) in biomineralization is not understood. This study tested the hypothesis that LRAP self-assembles into a supramolecular structure and guides crystal growth similarly to the full-length protein. Synthetic LRAP and recombinant full-length amelogenin (rH175) were used at different concentrations and either immobilized onto fluoroapatite substrates (FAP) or immersed into saturated calcium-phosphate solutions. The structure of the assembled protein and the height of apatite crystals formed on the FAP template were determined using atomic force microscopy. Both LRAP and rH175 assembled into nanospheres. LRAP self-assembly, however, was only observed at concentrations of >0.5 mg ml-1 and limited to sizes between 5 and 30 nm. Apatite crystal growth was not significantly affected by LRAP, while rH175 accelerated crystal growth by up to 50-fold. The increased growth rate was only observed when rH175 precipitated at concentrations of >0.8 mg ml-1. It was concluded that the ability of amelogenins to self-assemble into nanospheres and to bind to apatite in vitro is not inevitably an indication for the ability to control apatite crystal growth.

Amelogenin-guided crystal growth on fluoroapatite glass-ceramics.

The formation of aligned fibrous apatite crystals in enamel is predominantly attributed to the involvement of amelogenin proteins. We developed a model to study interactions of matrix proteins with apatite mineral in vitro and tested the hypothesis that amelogenin solubility affects the ability to induce protein-guided mineralization. Crystal growth experiments were performed on fluoroapatite (FAP) glass-ceramics in mineralizing solutions containing recombinant full-length amelogenin (rH174) at different concentrations. Using atomic force microscopy, we observed that mineral precipitated randomly on the substrate, but also formed thin layers (height, 10 nm) on FAP within 24 hrs. This growth pattern was unaffected when 0.4 mg/mL of rH174 was added. In contrast, crystals grew on FAP at a rate up to 20 times higher, at 1.6 mg/mL protein. Furthermore, nanospheres and mineral bound specifically to FAP and aligned in strings approximately parallel to the c-axis of FAP, leading us to the conclusion that amelogenin proteins indeed control direction and rate of growth of apatite in enamel.