Tuning of Crystal Nucleation and Growth by Proteins: Molecular Interactions at Solid-Liquid Interfaces in Biomineralization

The mineralized tissues of a bivalve mollusk and a sea urchin are both composed of calcium carbonate crystals that are intimately associated with acidic glycoproteins. In vitro studies in which carboxylate-, carbonate- and phosphate-containing crystals are grown in the presence of partially purified acidic glycoproteins from these two tissues show that some of these macro- molecules are able to interact specifically with certain crystal faces. Significantly all the affected crystal faces contain a common stereochemical motif. Interesting differences, however, were observed in the modes of interaction between the mollusk and sea urchin derived acidic glycoproteins. Only the former can induce oriented calcite nucleation in vitro and only the latter can interact from solution with specific calcite crystal faces. These differences are ascribed in part to the fact that the mollusk macromolecules are much more acidic than those from the sea urchin. Some of the acidic glycoproteins are also occluded inside the growing crystals. In the case of the sea urchin, and not of the mollusk, the proteins are preferentially located at specific crystal planes and their presence influences the mechanical properties of the crystal. A detailed study of these composite crystals by X-ray synchrotron radiation shows how the presence of the protein influences the crystal mosaicity. The interactions revealed by these studies follow well defined stereochemical rules, tuned by electrostatic forces. They, in turn, provide new \u27insight into some of the basic underlying processes occurring in biomineralization

Assembly in vitro of rhodococcus jostii RHA1 encapsulin and peroxidase DypB to form a nano-compartment

Rhodococcus jostii RHA1 peroxidase DypB has been recently identified as a bacterial lignin peroxidase. The dypB gene is co-transcribed with a gene encoding an encapsulin protein, shown in Thermotoga maritima to assemble to form a 60-subunit nano-compartment, and DypB contains a C-terminal sequence motif thought to target the protein to the encapsulin nanocompartment. R. jostii RHA1 encapsulin protein has been overexpressed in R. jostii RHA1, and purified as a high molecular weight assembly (Mr >106). The purified nanocompartment can be denatured to form a low molecular weight species by treatment at pH 3.0, and can be re-assembled to form the nanocompartment at pH 7.0. Recombinant DypB can be assembled in vitro with monomeric encapsulin to form an assembly of similar size and shape to the encapsulin-only nanocompartment, assessed by dynamic light scattering. The assembled complex shows enhanced lignin degradation activity per mg DypB present, compared with native DypB, using a nitrated lignin UV-vis assay method. The measured stoichiometry of 8.6 µmoles encapsulin/µmol DypB in the complex is comparable to the value of 10 predicted from the crystal structure

Preparative SDS PAGE as an Alternative to His–Tag Purification of Recombinant Amelogenin

Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens and in the development of therapeutics. Recombinant proteins are typically engineered with “tags” that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralisation researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond

Structural Analysis of a Repetitive Protein Sequence Motif in Strepsirrhine Primate Amelogenin

Strepsirrhines are members of a primate suborder that has a distinctive set of features associated with the development of the dentition. Amelogenin (AMEL), the better known of the enamel matrix proteins, forms 90% of the secreted organic matrix during amelogenesis. Although AMEL has been sequenced in numerous mammalian lineages, the only reported strepsirrhine AMEL sequences are those of the ring-tailed lemur and galago, which contain a set of additional proline-rich tandem repeats absent in all other primates species analyzed to date, but present in some non-primate mammals. Here, we first determined that these repeats are present in AMEL from three additional lemur species and thus are likely to be widespread throughout this group. To evaluate the functional relevance of these repeats in strepsirrhines, we engineered a mutated murine amelogenin sequence containing a similar proline-rich sequence to that of Lemur catta. In the monomeric form, the MQP insertions had no influence on the secondary structure or refolding properties, whereas in the assembled form, the insertions increased the hydrodynamic radii. We speculate that increased AMEL nanosphere size may influence enamel formation in strepsirrhine primates

Amelogenin Nanoparticles in Suspension: Deviations from Spherical Shape and pH-Dependent Aggregation

It is well-known that amelogenin self-assembles to form nanoparticles, usually referred to as amelogenin nanospheres, despite the fact that not much is known about their actual shape in solution. In the current paper, we combine SAXS and DLS to study the three-dimensional shape of the recombinant amelogenins rP172 and rM179. Our results show for the first time that amelogenins build oblate nanoparticles in suspension using experimental approaches that do not require the proteins to be in contact with a support material surface. The SAXS studies give evidence for the existence of isolated amelogenin nano-oblates with aspect ratios in the range of 0.45-0.5 at pH values higher than pH 7.2 and show an aggregation of these nano-oblates at lower pH values. The role of the observed oblate shape in the formation of chain-like structures at physiological conditions is discussed as a key factor in the biomineralization of dental enamel

Amelogenin Supramolecular Assembly in Nanospheres Defined by a Complex Helix-Coil-PPII Helix 3D-Structure

Tooth enamel, the hardest material in the human body, is formed within a self-assembled matrix consisting mostly of amelogenin proteins. Here we have determined the complete mouse amelogenin structure under physiological conditions and defined interactions between individual domains. NMR spectroscopy revealed four major amelogenin structural motifs, including an N-terminal assembly of four α-helical segments (S9-V19, T21-P33, Y39-W45, V53-Q56), an elongated random coil region interrupted by two 310 helices (∼P60-Q117), an extended proline-rich PPII-helical region (P118-L165), and a charged hydrophilic C-terminus (L165-D180). HSQC experiments demonstrated ipsilateral interactions between terminal domains of individual amelogenin molecules, i.e. N-terminal interactions with corresponding N-termini and C-terminal interactions with corresponding C-termini, while the central random coil domain did not engage in interactions. Our HSQC spectra of the full-length amelogenin central domain region completely overlapped with spectra of the monomeric Amel-M fragment, suggesting that the central amelogenin coil region did not involve in assembly, even in assembled nanospheres. This finding was confirmed by analytical ultracentrifugation experiments. We conclude that under conditions resembling those found in the developing enamel protein matrix, amelogenin molecules form complex 3D-structures with N-terminal α-helix-like segments and C-terminal PPII-helices, which self-assemble through ipsilateral interactions at the N-terminus of the molecule

One-Step Purification of Recombinant Human Amelogenin and Use of Amelogenin as a Fusion Partner

Amelogenin is an extracellular protein first identified as a matrix component important for formation of dental enamel during tooth development. Lately, amelogenin has also been found to have positive effects on clinical important areas, such as treatment of periodontal defects, wound healing, and bone regeneration. Here we present a simple method for purification of recombinant human amelogenin expressed in Escherichia coli, based on the solubility properties of amelogenin. The method combines cell lysis with recovery/purification of the protein and generates a >95% pure amelogenin in one step using intact harvested cells as starting material. By using amelogenin as a fusion partner we could further demonstrate that the same method also be can explored to purify other target proteins/peptides in an effective manner. For instance, a fusion between the clinically used protein PTH (parathyroid hormone) and amelogenin was successfully expressed and purified, and the amelogenin part could be removed from PTH by using a site-specific protease

Meeting report: a hard look at the state of enamel research.

The Encouraging Novel Amelogenesis Models and Ex vivo cell Lines (ENAMEL) Development workshop was held on 23 June 2017 at the Bethesda headquarters of the National Institute of Dental and Craniofacial Research (NIDCR). Discussion topics included model organisms, stem cells/cell lines, and tissues/3D cell culture/organoids. Scientists from a number of disciplines, representing institutions from across the United States, gathered to discuss advances in our understanding of enamel, as well as future directions for the field

Regulation of the Stem Cell–Host Immune System Interplay Using Hydrogel Coencapsulation System with an Anti-Inflammatory Drug

The host immune system is known to influence mesenchymal stem cell (MSC)-mediated bone tissue regeneration. However, the therapeutic capacity of hydrogel biomaterial to modulate the interplay between MSCs and T-lymphocytes is unknown. Here it is shown that encapsulating hydrogel affects this interplay when used to encapsulate MSCs for implantation by hindering the penetration of pro-inflammatory cells and/or cytokines, leading to improved viability of the encapsulated MSCs. This combats the effects of the host pro-inflammatory T-lymphocyte-induced nuclear factor kappaB pathway, which can reduce MSC viability through the CASPASE-3 and CAS-PASE-8 associated proapoptotic cascade, resulting in the apoptosis of MSCs. To corroborate rescue of engrafted MSCs from the insult of the host immune system, the incorporation of the anti-inflammatory drug indomethacin into the encapsulating alginate hydrogel further regulates the local microenvironment and prevents pro-inflammatory cytokine-induced apoptosis. These findings suggest that the encapsulating hydrogel can regulate the MSC-host immune cell interplay and direct the fate of the implanted MSCs, leading to enhanced tissue regeneration