Ganoin and acrodin formation on scales and teeth in spotted gar: A vital role of enamelin in the unique process of enamel mineralization.

Gars and bichirs develop scales and teeth with ancient actinopterygian characteristics. Their scale surface and tooth collar are covered with enamel, also known as ganoin, whereas the tooth cap is equipped with an enamel-like tissue, acrodin. Here, we investigated the formation and mineralization of the ganoin and acrodin matrices in spotted gar, and the evolution of the scpp5, ameloblastin (ambn), and enamelin (enam) genes, which encode matrix proteins of ganoin. Results suggest that, in bichirs and gars, all these genes retain structural characteristics of their orthologs in stem actinopterygians, presumably reflecting the presence of ganoin on scales and teeth. During scale formation, Scpp5 and Enam were initially found in the incipient ganoin matrix and the underlying collagen matrix, whereas Ambn was detected mostly in a surface region of the well-developed ganoin matrix. Although collagen is the principal acrodin matrix protein, Scpp5 was detected within the matrix. Similarities in timings of mineralization and the secretion of Scpp5 suggest that acrodin evolved by the loss of the matrix secretory stage of ganoin formation: dentin formation is immediately followed by the maturation stage. The late onset of Ambn secretion during ganoin formation implies that Ambn is not essential for mineral ribbon formation, the hallmark of the enamel matrix. Furthermore, Scpp5 resembles amelogenin that is not important for the initial formation of mineral ribbons in mammals. It is thus likely that the evolution of ENAM was vital to the origin of the unique mineralization process of the enamel matrix.

Peptide analysis of tooth enamel - A sex estimation tool for archaeological, anthropological, or forensic research.

Proteomics has become an attractive method to study human and animal material, biological profile, and origin as an alternative to DNA analysis. It is limited by DNA amplification in ancient samples and its contamination, high cost, and limited preservation of nuclear DNA. Currently, three approaches are available to estimate sex-osteology, genomics, or proteomics, but little is known about the relative reliability of these methods in applied settings. Proteomics provides a new, seemingly simple, and relatively non-expensive way of sex estimation without the risk of contamination. Proteins can be preserved in hard teeth tissue (enamel) for tens of thousands of years. It uses two sexually distinct forms of the protein amelogenin in tooth enamel detectable by liquid chromatography-mass spectrometry; the protein amelogenin Y isoform is present in enamel dental tissue only in males, while amelogenin isoform X can be found in both sexes. From the point of view of archaeological, anthropological, and forensic research and applications, the reduced destruction of the methods used is essential, as well as the minimum requirements for sample size.

Ift88 regulates enamel formation via involving Shh signaling.

OBJECTIVES: The ciliopathies are a wide spectrum of human diseases, which are caused by perturbations in the function of primary cilia. Tooth enamel anomalies are often seen in ciliopathy patients; however, the role of primary cilia in enamel formation remains unclear. MATERIALS AND METHODS: We examined mice with epithelial conditional deletion of the ciliary protein, Ift88, (Ift88fl / fl ;K14Cre). RESULTS: Ift88fl / fl ;K14Cre mice showed premature abrasion in molars. A pattern of enamel rods which is determined at secretory stage, was disorganized in Ift88 mutant molars. Many amelogenesis-related molecules expressing at the secretory stage, including amelogenin and ameloblastin, enamelin, showed significant downregulation in Ift88 mutant molar tooth germs. Shh signaling is essential for amelogenesis, which was found to be downregulated in Ift88 mutant molar at the secretory stage. Application of Shh signaling agonist at the secretory stage partially rescued enamel anomalies in Ift88 mutant mice. CONCLUSION: Findings in the present study indicate that the function of the primary cilia via Ift88 is critical for the secretory stage of amelogenesis through involving Shh signaling.

Protein nanoribbons template enamel mineralization.

As the hardest tissue formed by vertebrates, enamel represents nature's engineering masterpiece with complex organizations of fibrous apatite crystals at the nanometer scale. Supramolecular assemblies of enamel matrix proteins (EMPs) play a key role as the structural scaffolds for regulating mineral morphology during enamel development. However, to achieve maximum tissue hardness, most organic content in enamel is digested and removed at the maturation stage, and thus knowledge of a structural protein template that could guide enamel mineralization is limited at this date. Herein, by examining a gene-modified mouse that lacked enzymatic degradation of EMPs, we demonstrate the presence of protein nanoribbons as the structural scaffolds in developing enamel matrix. Using in vitro mineralization assays we showed that both recombinant and enamel-tissue-based amelogenin nanoribbons are capable of guiding fibrous apatite nanocrystal formation. In accordance with our understanding of the natural process of enamel formation, templated crystal growth was achieved by interaction of amelogenin scaffolds with acidic macromolecules that facilitate the formation of an amorphous calcium phosphate precursor which gradually transforms into oriented apatite fibers along the protein nanoribbons. Furthermore, this study elucidated that matrix metalloproteinase-20 is a critical regulator of the enamel mineralization as only a recombinant analog of a MMP20-cleavage product of amelogenin was capable of guiding apatite mineralization. This study highlights that supramolecular assembly of the scaffold protein, its enzymatic processing, and its ability to interact with acidic carrier proteins are critical steps for proper enamel development.

The Dynamic Interactions of a Multitargeting Domain in Ameloblastin Protein with Amelogenin and Membrane.

The enamel matrix protein Ameloblastin (Ambn) has critical physiological functions, including regulation of mineral formation, cell differentiation, and cell-matrix adhesion. We investigated localized structural changes in Ambn during its interactions with its targets. We performed biophysical assays and used liposomes as a cell membrane model. The xAB2N and AB2 peptides were rationally designed to encompass regions of Ambn that contained self-assembly and helix-containing membrane-binding motifs. Electron paramagnetic resonance (EPR) on spin-labeled peptides showed localized structural gains in the presence of liposomes, amelogenin (Amel), and Ambn. Vesicle clearance and leakage assays indicated that peptide-membrane interactions were independent from peptide self-association. Tryptophan fluorescence and EPR showed competition between Ambn-Amel and Ambn-membrane interactions. We demonstrate localized structural changes in Ambn upon interaction with different targets via a multitargeting domain, spanning residues 57 to 90 of mouse Ambn. Structural changes of Ambn following its interaction with different targets have relevant implications for the multifunctionality of Ambn in enamel formation.

Residue-Specific Insights into the Intermolecular Protein-Protein Interfaces Driving Amelogenin Self-Assembly in Solution.

Amelogenin, the dominant organic component (>90%) in the early stages of amelogenesis, orchestrates the mineralization of apatite crystals into enamel. The self-association properties of amelogenin as a function of pH and protein concentration have been suggested to play a central role in this process; however, the large molecular weight of the self-assembled quaternary structures has largely prevented structural studies of the protein in solution under physiological conditions using conventional approaches. Here, using perdeuterated murine amelogenin (0.25 mM, 5 mg/mL) and TROSY-based NMR experiments to improve spectral resolution, we assigned the 1H-15N spectra of murine amelogenin over a pH range (5.5 to 8.0) where amelogenin is reported to exist as oligomers (pH ≤∼6.8) and nanospheres (pH ≥∼7.2). The disappearance or intensity reduction of amide resonances in the 1H-15N HSQC spectra was interpreted to reflect changes in dynamics (intermediate millisecond-to-microsecond motion) and/or heterogenous interfaces of amide nuclei at protein-protein interfaces. The intermolecular interfaces were concentrated toward the N-terminus of amelogenin (L3-G8, V19-G38, L46-Q49, and Q57-L70) at pH 6.6 (oligomers) and at pH 7.2 (nanospheres) including the entire N-terminus up to Q76 and regions distributed through the central hydrophobic region (Q82-Q101, S125-Q139, and F151-Q154). At all pH levels, the C-terminus appeared disordered, highly mobile, and not involved in self-assembly, suggesting nanosphere structures with solvent-exposed C-termini. These findings present unique, residue-specific insights into the intermolecular protein-protein interfaces driving amelogenin quaternary structure formation and suggest that nanospheres in solution predominantly contain disordered, solvent-exposed C-termini.

How prenatal environmental factors affect rat molar enamel formation?

Amelogenin (AMELX) and ameloblastin (AMBN) are crucial for enamel formation, and interruptions in the production of these proteins may cause enamel defects. We investigated how prenatal environmental factors (chronic stress, bisphenol A (BPA), amoxicillin, and lipopolysaccharide (LPS)) affect AMELX and AMBN production of ameloblasts. Fifteen pregnant Sprague-Dawley rats were divided into four experimental groups and a control group. Chronic-stress group rats were exposed to a 12:12 light/light cycle (LL) from day E18 until delivery. BPA group rats were orally administered 5 µg/kg BPA daily from day E1 until delivery. Amoxicillin group rats were injected 100 mg/kg amoxicillin daily from day E18 until delivery. LPS-infection group rats were injected 125 µg/kg bacterial LPS once on day E18. Seven pups from the control group and ten pups from the experimental groups were euthanized on P10. Sections were stained with hematoxylin and eosin (H&E) and Gomori's one-step trichrome staining (GT) and incubated with rabbit polyclonal antibodies to AMELX and AMBN, to evaluate staining intensity at ameloblast stages. The surface morphology was evaluated with a stereomicroscope. AMELX (p = 0.008, p = 0.0001, p = 0.009) and AMBN (p = 0.002, p = 0.001, p = 0.0001) staining of all groups were significantly lower than that of the control group in the secretory, transitional, and maturation stages. Abnormal enamel matrix formation was observed in the H&E and GT staining sections of all experimental groups. Yellowish coloration of the amoxicillin group was observed in morphologic evaluation.

The power of weak ion-exchange resins assisted by amelogenin for natural remineralization of dental enamel: an in vitro study.

This study aims to develop an innovative dental product to remineralize dental enamel by a proper combination of ion-exchange resins as controlled release of mineral ions that form dental enamel, in the presence of amelogenin to guide the appropriate crystal growth. The novel product proposed consists of a combination of ion-exchange resins (weak acid and weak base) individually loaded with the remineralizing ions: Ca2+, PO43- and F-, also including Zn2+ in a minor amount as antibacterial, together with the protein amelogenin. Such cocktail provides onsite controlled release of the ions necessary for enamel remineralization due to the weak character of the resins and at the same time, a guiding tool for related crystal growth by the indicated protein. Amelogenin protein is involved in the structural development of natural enamel and takes a key role in controlling the crystal growth morphology and alignment at the enamel surface. Bovine teeth were treated by applying the resins and protein together with artificial saliva. Treated teeth were evaluated with nanoindentation, scanning electron microscopy and energy-dispersive X-ray spectroscopy. The innovative material induces the dental remineralization creating a fluorapatite layer with a hardness equivalent to sound enamel, with the appropriate alignment of corresponding nanocrystals, being the fluorapatite more acid resistant than the original mineral. Our results suggest that the new product shows potential for promoting long-term remineralization leading to the inhibition of caries and protection of dental structures.

Amelogenesis: Transformation of a protein-mineral matrix into tooth enamel.

During enamel formation, the organic enamel protein matrix interacts with calcium phosphate minerals to form elongated, parallel, and bundled enamel apatite crystals of extraordinary hardness and biomechanical resilience. The enamel protein matrix consists of unique enamel proteins such as amelogenin, ameloblastin, and enamelin, which are secreted by highly specialized cells called ameloblasts. The ameloblasts also facilitate calcium and phosphate ion transport toward the enamel layer. Within ameloblasts, enamel proteins are transported as a polygonal matrix with 5 nm subunits in secretory vesicles. Upon expulsion from the ameloblasts, the enamel protein matrix is re-organized into 20 nm subunit compartments. Enamel matrix subunit compartment assembly and expansion coincide with C-terminal cleavage by the MMP20 enamel protease and N-terminal amelogenin self-assembly. Upon enamel crystal precipitation, the enamel protein phase is reconfigured to surround the elongating enamel crystals and facilitate their elongation in C-axis direction. At this stage of development, and upon further amelogenin cleavage, central and polyproline-rich fragments of the amelogenin molecule associate with the growing mineral crystals through a process termed "shedding", while hexagonal apatite crystals fuse in longitudinal direction. Enamel protein sheath-coated enamel "dahlite" crystals continue to elongate until a dense bundle of parallel apatite crystals is formed, while the enamel matrix is continuously degraded by proteolytic enzymes. Together, these insights portrait enamel mineral nucleation and growth as a complex and dynamic set of interactions between enamel proteins and mineral ions that facilitate regularly seeded apatite growth and parallel enamel crystal elongation.

Sp6/Epiprofin is a master regulator in the developing tooth.

Tooth development involves the coordinated transcriptional regulation of extracellular matrix proteins produced by ameloblasts and odontoblasts. In this study, whole-genome ChIP-seq analysis was applied to identify the transcriptional regulatory gene targets of Sp6 in mesenchymal cells of the developing tooth. Bioinformatic analysis of a pool of Sp6 target peaks identified the consensus nine nucleotide binding DNA motif CTg/aTAATTA. Consistent with these findings, a number of enamel and dentin matrix genes including amelogenin (Amelx), ameloblastin (Ambn), enamelin (Enam) and dental sialophosphoprotein (Dspp), were identified to contain Sp6 target sequences. Sp6 peaks were also found in other important tooth genes including transcription factors (Dlx2, Dlx3, Dlx4, Dlx5, Sp6, Sp7, Pitx2, and Msx2) and extracellular matrix-related proteins (Col1a2, Col11a2, Halpn1). Unsupervised UMAP clustering of tooth single cell RNA-seq data confirmed the presence of Sp6 transcripts co-expressed with many of the identified target genes within ameloblasts and odontoblasts. Lastly, transcriptional reporter assays using promoter fragments from the Hapln1 and Sp6 gene itself revealed that Sp6 co-expression enhanced gene transcriptional activity. Taken together these results highlight that Sp6 is a major regulator of multiple extracellular matrix genes in the developing tooth.

Stage-Specific Role of Amelx Activation in Stepwise Ameloblast Induction from Mouse Induced Pluripotent Stem Cells.

Amelogenin comprises ~90% of enamel proteins; however, the involvement of Amelx transcriptional activation in regulating ameloblast differentiation from induced pluripotent stem cells (iPSCs) remains unknown. In this study, we generated doxycycline-inducible Amelx-expressing mouse iPSCs (Amelx-iPSCs). We then established a three-stage ameloblast induction strategy from Amelx-iPSCs, including induction of surface ectoderm (stage 1), dental epithelial cells (DECs; stage 2), and ameloblast lineage (stage 3) in sequence, by manipulating several signaling molecules. We found that adjunctive use of lithium chloride (LiCl) in addition to bone morphogenetic protein 4 and retinoic acid promoted concentration-dependent differentiation of DECs. The resulting cells had a cobblestone appearance and keratin14 positivity. Attenuation of LiCl at stage 3 together with transforming growth factor ß1 and epidermal growth factor resulted in an ameloblast lineage with elongated cell morphology, positivity for ameloblast markers, and calcium deposition. Although stage-specific activation of Amelx did not produce noticeable phenotypic changes in ameloblast differentiation, Amelx activation at stage 3 significantly enhanced cell adhesion as well as decreased proliferation and migration. These results suggest that the combination of inducible Amelx transcription and stage-specific ameloblast induction for iPSCs represents a powerful tool to highlight underlying mechanisms in ameloblast differentiation and function in association with Amelx expression.

Amelogenin-Derived Peptides in Bone Regeneration: A Systematic Review.

Amelogenins are enamel matrix proteins currently used to treat bone defects in periodontal surgery. Recent studies have highlighted the relevance of amelogenin-derived peptides, named LRAP, TRAP, SP, and C11, in bone tissue engineering. Interestingly, these peptides seem to maintain or even improve the biological activity of the full-length protein, which has received attention in the field of bone regeneration. In this article, the authors combined a systematic and a narrative review. The former is focused on the existing scientific evidence on LRAP, TRAP, SP, and C11's ability to induce the production of mineralized extracellular matrix, while the latter is concentrated on the structure and function of amelogenin and amelogenin-derived peptides. Overall, the collected data suggest that LRAP and SP are able to induce stromal stem cell differentiation towards osteoblastic phenotypes; specifically, SP seems to be more reliable in bone regenerative approaches due to its osteoinduction and the absence of immunogenicity. However, even if some evidence is convincing, the limited number of studies and the scarcity of in vivo studies force us to wait for further investigations before drawing a solid final statement on the real potential of amelogenin-derived peptides in bone tissue engineering.

Trafficking and secretion of keratin 75 by ameloblasts in vivo.

A highly specialized cytoskeletal protein, keratin 75 (K75), expressed primarily in hair follicles, nail beds, and lingual papillae, was recently discovered in dental enamel, the most highly mineralized hard tissue in the human body. Among many questions this discovery poses, the fundamental question regarding the trafficking and secretion of this protein, which lacks a signal peptide, is of an utmost importance. Here, we present evidence that K75 is expressed during the secretory stage of enamel formation and is present in the forming enamel matrix. We further show that K75 is secreted together with major enamel matrix proteins amelogenin and ameloblastin, and it was detected in Golgi and the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) but not in rough ER (rER). Inhibition of ER-Golgi transport by brefeldin A did not affect the association of K75 with Golgi, whereas ameloblastin accumulated in rER, and its transport from rER into Golgi was disrupted. Together, these results indicate that K75, a cytosolic protein lacking a signal sequence, is secreted into the forming enamel matrix utilizing portions of the conventional ER-Golgi secretory pathway. To the best of our knowledge, this is the first study providing insights into mechanisms of keratin secretion.

Novel LRAP-binding partner revealing the plasminogen activation system as a regulator of cementoblast differentiation and mineral nodule formation in vitro.

Amelogenin isoforms, including full-length amelogenin (AMEL) and leucine-rich amelogenin peptide (LRAP), are major components of the enamel matrix, and are considered as signaling molecules in epithelial-mesenchymal interactions regulating tooth development and periodontal regeneration. Nevertheless, the molecular mechanisms involved are still poorly understood. The aim of the present study was to identify novel binding partners for amelogenin isoforms in the cementoblast (OCCM-30), using an affinity purification assay (GST pull-down) followed by mass spectrometry and immunoblotting. Protein-protein interaction analysis for AMEL and LRAP evidenced the plasminogen activation system (PAS) as a potential player regulating OCCM-30 response to amelogenin isoforms. For functional assays, PAS was either activated (plasmin) or inhibited (ε-aminocaproic acid [aminocaproic]) in OCCM-30 cells and the cell morphology, mineral nodule formation, and gene expression were assessed. PAS inhibition (EACA 100 mM) dramatically decreased mineral nodule formation and expression of OCCM-30 differentiation markers, including osteocalcin (Bglap), bone sialoprotein (Ibsp), osteopontin (Spp1), tissue-nonspecific alkaline phosphatase (Alpl) and collagen type I (Col1a1), and had no effect on runt-related transcription factor 2 (Runx2) and Osterix (Osx) mRNA levels. PAS activation (plasmin 5 µg/µl) significantly increased Col1a1 and decreased Bglap mRNA levels (p < .05). Together, our findings shed new light on the potential role of plasminogen signaling pathway in the control of the amelogenin isoform-mediated response in cementoblasts and provide new insights into the development of targeted therapies.

Optimization of Method for Human Sex Determination Using Peptidome Analysis of Teeth Enamel from Teeth of Different Biological Generation, Archeological Age, and Degrees of Taphonomic Preservation.

Determination of biological sex to human remains is a fundamental requirement in anthropological, archeological, and forensic anthropological studies. Sex determination based on morphological criteria is significantly limited in the cases of juvenile remains and adult skeletons in a poor state of preservation. Regular attempts have been made to use alternative techniques to resolve this issue, including analysis of tooth enamel peptides by liquid chromatography/mass spectrometry. Optimization of this method involving acid etching of tooth enamel for 10 min followed by desalting of the products of etching on SDB-RPS StageTips microcolumns and analysis of desalted sample (1/3) by liquid chromatography/mass spectrometry allowed reliable sex determination to fossil remains within a wide range of archeological and biological ages without destructing analyzed teeth. Increasing the duration of enamel etching ensured a 2 to 3-fold increase in the total number of identified peptides and, more importantly, in the number of identified fragments of amelogenin Y isoform specific for male teeth, which facilitated reliable sex determination of fossil remains. The suggested technique was tested with 8 permanent and 15 deciduous teeth of different archaeological age and different degree of preservation. Two amelogenin Y-specific peptide sequences were identified. One of these peptides [SM(+15.99)IRPPYS)] was found in all male-derived samples without exception; the other peptide [IRPPYSS(+79.97)], which contained phosphorylated Ser66 residue, was found only in the enamel from deciduous teeth, which suggests that phosphorylation of Ser66 plays a role in the enamel formation in deciduous teeth.

Intrinsically disordered proteins drive enamel formation via an evolutionarily conserved self-assembly motif.

The formation of mineralized tissues is governed by extracellular matrix proteins that assemble into a 3D organic matrix directing the deposition of hydroxyapatite. Although the formation of bones and dentin depends on the self-assembly of type I collagen via the Gly-X-Y motif, the molecular mechanism by which enamel matrix proteins (EMPs) assemble into the organic matrix remains poorly understood. Here we identified a Y/F-x-x-Y/L/F-x-Y/F motif, evolutionarily conserved from the first tetrapods to man, that is crucial for higher order structure self-assembly of the key intrinsically disordered EMPs, ameloblastin and amelogenin. Using targeted mutations in mice and high-resolution imaging, we show that impairment of ameloblastin self-assembly causes disorganization of the enamel organic matrix and yields enamel with disordered hydroxyapatite crystallites. These findings define a paradigm for the molecular mechanism by which the EMPs self-assemble into supramolecular structures and demonstrate that this process is crucial for organization of the organic matrix and formation of properly structured enamel.

Sex determination of human remains from peptides in tooth enamel.

The assignment of biological sex to archaeological human skeletons is a fundamental requirement for the reconstruction of the human past. It is conventionally and routinely performed on adults using metric analysis and morphological traits arising from postpubertal sexual dimorphism. A maximum accuracy of ∼95% is possible if both the cranium and os coxae are present and intact, but this is seldom achievable for all skeletons. Furthermore, for infants and juveniles, there are no reliable morphological methods for sex determination without resorting to DNA analysis, which requires good DNA survival and is time-consuming. Consequently, sex determination of juvenile remains is rarely undertaken, and a dependable and expedient method that can correctly assign biological sex to human remains of any age is highly desirable. Here we present a method for sex determination of human remains by means of a minimally destructive surface acid etching of tooth enamel and subsequent identification of sex chromosome-linked isoforms of amelogenin, an enamel-forming protein, by nanoflow liquid chromatography mass spectrometry. Tooth enamel is the hardest tissue in the human body and survives burial exceptionally well, even when the rest of the skeleton or DNA in the organic fraction has decayed. Our method can reliably determine the biological sex of humans of any age using a body tissue that is difficult to cross-contaminate and is most likely to survive. The application of this method will make sex determination of adults and, for the first time, juveniles a reliable and routine activity in future bioarcheological and medico-legal science contexts.

Sexing of pre-implantation ovine embryos through polymerase chain reaction-based amplification of GAPDH, SRY and AMEL genes.

The ability to identify the sex of embryo and control of sex ratio has a great commercial importance to livestock industry. Prediction of embryonic sex could be useful in the management decisions of sex selection in breeding programs. Several methods have been attempted to determine the sex but the polymerase chain reaction (PCR)-based sexing method is generally favoured, as it is cost effective, simple and reliable. The aim of the present study was to identify sex of sheep embryos produced in vitro through amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sex-determining region Y (SRY) and amelogenin genes present in genomic DNA (gDNA) of embryos through PCR. To avoid false interpretation of the result by no amplification of SRY in female embryos, a duplex PCR was approached to amplify combinedly SRY and GAPDH genes. Sex-specific blood was used in PCR as positive control. In vitro sheep embryos were produced as per standardized protocol of laboratory. Sexing of sex-specific blood and in vitro produced embryos were approached though PCR to amplify the respective genes using gDNA present in the sample without its traditional isolation. The accuracy of sex prediction for embryos was 100% by this procedure.

Hydroxyapatite Formation Coexists with Amyloid-like Self-Assembly of Human Amelogenin.

Tooth enamel is formed in an extracellular environment. Amelogenin, the major component in the protein matrix of tooth enamel during the developing stage, could assemble into high molecular weight structures, regulating enamel formation. However, the molecular structure of amelogenin protein assembly at the functional state is still elusive. In this work, we found that amelogenin is able to induce calcium phosphate minerals into hydroxyapatite (HAP) structure in vitro at pH 6.0. Assessed using X-ray diffraction (XRD) and 31P solid-state NMR (SSNMR) evidence, the formed HAP mimics natural enamel closely. The structure of amelogenin protein assembly coexisting with the HAP was also studied using atomic force microscopy (AFM), transmission electron microscopy (TEM) and XRD, indicating the ß-amyloid structure of the protein. SSNMR was proven to be an important tool in detecting both the rigid and dynamic components of the protein assembly in the sample, and the core sequence 18EVLTPLKWYQSI29 was identified as the major segment contributing to the ß-sheet secondary structure. Our research suggests an amyloid structure may be an important factor in controlling HAP formation at the right pH conditions with the help of other structural components in the protein assembly.

Amyloid structure of high-order assembly of Leucine-rich amelogenin revealed by solid-state NMR.

High-order assemblies of amelogenin, the major protein in enamel protein matrix, are believed to act as the template for enamel mineral formation. The Leucine-rich amelogenin (LRAP) is a natural splice-variant of amelogenin, a functional protein in vivo, containing conserved domains of amelogenin. In this work, we showed LRAP aggregates hierarchically into assemblies with various sizes including scattered beads, beads-on-a-string and gel-like precipitations in the presence of both calcium and phosphate ions. Solid-state NMR combined with X-ray diffraction and microscopic techniques, was applied to give a picture of LRAP self-assemblies at the atomic level. Our results, for the first time, confirmed LRAP assemblies with different sizes all contained a consistent rigid segment with ß-sheet secondary structure (residues 12-27) and the ß-sheet segment would further assemble into amyloid-like structures.