Tyrosine-rich Amelogenin Peptide Research Articles

Morphological study of remineralization of the eroded enamel lesions by tyrosine-rich amelogenin peptide

BackgroundTyrosine-rich amelogenin peptide (TRAP) is the main amelogenin digestion product in the developmental enamel matrix. It has been shown to promote remineralization of demineralized enamel in our previous study. However, direct evidence of the effect of TRAP on the morphology and nanostructure of crystal growth on an enamel surface has not been reported. This study aimed to examine the effect of TRAP on the morphology of calcium phosphate crystals grown on early enamel erosion using a pH-cycling model.MethodsEroded lesions were produced in human premolars by 30-second immersion in 37% phosphoric acid. Forty-five samples of eroded human premolar enamel blocks were selected and randomly divided into 3 groups: deionized water (DDW, negative control); 100 µg/mL TRAP, and 2 ppm sodium fluoride (NaF, positive control group). For 14 days, the specimens were exposed to a pH-cycling model. Using scanning electron microscopy (SEM) and atomic force microscopy (AFM) methods, the surface morphology, calcium-phosphorus ratio, and enamel surface roughness were examined. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) were used to assess crystal characteristics.ResultsAfter pH-cycling, compared to the two control groups, the surface of the eroded enamel of the peptide TRAP group shows a large number of new, densely arranged rod-like crystals, parallel to each other, regularly arranged, forming an ordered structure, with crystal morphology similar to that of natural enamel. The crystals are mostly hydroxyapatite (HA).ConclusionThis study demonstrates that the peptide TRAP modulates the formation of hydroxyapatite in eroded enamel and that the newly formed crystals resemble natural enamel crystals and promote the remineralization of enamel, providing a promising biomaterial for remineralization treatment of enamel lesions.

Self-assembly and mineralization of full-length human amelogenin and its functional fragments in vitro.

To investigate the dynamic process of the self-assembly behaviors of a full-length human amelogenin (AM) and its functional fragments tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide(LRAP) in vitro and its role in hydroxyapatite (HA) crystal formation. The full-length human AM and its functional fragments, TRAP and LRAP, were reassembled and purified in vitro. The protein solution of 100 µg‧mL-1, pH=8, was prepared in Tris-HCl and incubated at room temperature for 1-15 min. Their self-assembly behaviors were observed and compared under a transmission electron microscope (TEM). The full-length AM was added to artificial saliva and incubated for 3 days. A scanning electron microscope (SEM) was used in observing the morphology of the induced new crystals. Then, TARP and LRAP were added. The resulting solution was incubated for 3 days and then observed again. When pH=8, the full-length human AM and TRAP assembly started spontaneously and formed "nanospheres" after 15 min.The nanospheres formed by TRAP existed independently, with a uniform size but without obvious internal structures. The full-length AM was assembled hierarchically, which formed "nanospheres" and further extended in all directions, formed a chain structure, and then aggregated into a net. The self-assembly behavior of LRAP was not obvious. Proteins mostly existed in the form of monomers without "nanosphere" formation. Only few oligomers were observed. The full-length AM was induced independently for 3 days to form rod-shaped HA crystals. TRAP and LRAP proteins were added, after 3 days the crystal elongation was obvious in the c axis, but the growth in plane A and plane B was poor. The self-assembly and mineralization behaviors of full-length human AM, TRAP, and LRAP were consistent with the directional growth mechanism of HA crystals in vivo, providing a theoretical basis for the role of the fragments in the growth and maturation of HA crystals.

Epitope mapping of anti‐amelogenin IgA in coeliac disease

Previously we detected increased levels of IgA to the enamel matrix protein amelogenin in children with untreated coeliac disease (CeD). The biological impact of autoantibodies to amelogenin may depend on which part of the amelogenin (epitopes) they bind. Sera or blood samples from 146 untreated children with CeD from two different cohorts and 25 non-CeD control children were tested for IgA anti-amelogenin (Emdogain) reactivity. The 32 CeD children and six controls with the highest reactivity were selected for detailed IgA anti-amelogenin (AMELX) epitope mapping using 31 overlapping, 10-22mer peptides in ELISA. The dominating reactivity were to six peptides in a 75-amino-acid (aa)-long central segment (aa 75-150) containing enzymatic cleavage sites for matrix metalloproteinase 20 (MMP-20) and kallikrein-related peptidase 4 (KLK-4) and to two N-terminal peptides (aa 13-41) included in the tyrosine-rich amelogenin peptide fragment, which is important for self-assembly. Only two of the six control children reacted to single, but different peptides. Solid-phase extraction with gliadin- and Emdogain-coated Sepharose revealed a gliadin cross-reactive central region and a putative conformation-dependent, apparently non-cross-reactive N-terminal region. High IgA anti-amelogenin reactivity may interfere with normal amelogenesis by inhibiting amelogenin self-assembly, amelogenin-hydroxyapatite interaction, and/or enzymatic degradation.

Mechanics of amelogenin TRAP protein in the proximity of hydroxyapatite mineral is altered by interfacial water

Enamel is a composite bioceramic that covers teeth. Here, based on molecular dynamic (MD) simulation, we seek a better understanding of the interactions between the enamel matrix proteins with hydroxyapatite (HAP) crystal surfaces. These interactions occur during enamel formation and are responsible for controlling the formation of high aspect ratio carbonated hydroxyapatite nanocrystallites, whereas, in mature enamel, they are implicated to alter the mechanical properties of mature enamel to yield a tough composite that resists fracture during chewing. We choose an archetypal matrix protein known as tyrosine-rich amelogenin peptide (TRAP), which is retained among the HAP crystallites of mature enamel. Our findings provide molecular details to the emerging blueprint needed to engineer a biologically inspired enamel restorative material.

Identification of major matrix metalloproteinase-20 proteolytic processing products of murine amelogenin and tyrosine-rich amelogenin peptide using a nuclear magnetic resonance spectroscopy based method

ObjectiveThe aim of this study was to identify major matrix metalloproteinase-20 (MMP20) proteolytic processing products of amelogenin over time and determine if the tyrosine-rich amelogenin peptide (TRAP) was a substrate of MMP20. DesignRecombinant15N-labeled murine amelogenin and 13C,15N-labeled TRAP were incubated with MMP20 under conditions where amelogenin self-assembles into nanospheres. Digestion products were fractionated by reverse-phase high-performance liquid chromatography at various time points. Product identification took advantage of the intrinsic disorder property of amelogenin that results in little change to its fingerprint 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance spectrum in 2% acetic acid upon removing parts of the protein, allowing cleavage site identification by observing which amide cross peaks disappear. ResultsThe primary product in five out of the six major reverse-phase high-performance liquid chromatography bands generated after a 24 h incubation of murine amelogenin with MMP20 were: S55-L163, P2-L147, P2-E162, P2-A167, and P2-R176. After 72 h these products were replaced with five major reverse-phase high-performance liquid chromatography bands containing: L46-A170, P2-S152, P2-F151, P2-W45, and short N-terminal peptides. TRAP was completely digested by MMP20 into multiple small peptides with the initial primary site of cleavage between S16 and Y17. ConclusionsIdentification of the major MMP20 proteolytic products of amelogenin confirm a dynamic process, with sites towards the C-terminus more rapidly attacked than sites near the N-terminus. This observation is consistent with nanosphere models where the C-terminus is exposed and the N-terminus more protected. One previously reported end-product of the MMP20 proteolytic processing of amelogenin, TRAP, is shown to be an in vitro substrate for MMP20.

Human gingival fibroblast response to enamel matrix derivative, porcine recombinant 21.3-kDa amelogenin and 5.3-kDa tyrosine-rich amelogenin peptide

Enamel matrix derivative (EMD) containing a variety of protein fractions has been used for periodontal tissue regeneration. It is suggested that the proteins contained in EMD positively influence gingival fibroblasts migration and proliferation. Effects of EMD as well as of porcine recombinated 21.3-kDa amelogenin (prAMEL) and 5.3-kDa tyrosine-rich amelogenin peptide (prTRAP) on human gingival fibroblast (HGF-1, ATCC; USA) cell line were investigated. Real-time cell analysis (xCELLigence system; Roche Applied Science) was performed to determine the effects of EMD, prAMEL and prTRAP (12.5–50 μg/mL) on HGF-1 cell proliferation and migration. The effect of treatment on cell cycle was determined using flow cytometry. EMD significantly increased HGF-1 cell proliferation after 24- and 48-h incubation. Individually, prAMEL and prTRAP also increased HGF-1 cell proliferation; however, the difference was significant only for prAMEL 50 µg/mL. prAMEL and TRAP significantly increased HGF-1 cell migration after 60- and 72-h incubation. Cell cycle analysis showed significant decrease of the percentage of cells in the G0/G1 phase and a buildup of cells in the S and M phase observed after EMD and prAMEL stimulation. This process was ligand and concentration-dependent. The various molecular components in the enamel matrix derivative might contribute to the reported effects on gingival tissue regeneration; however, biologic effects of prAMEL and prTRAP individually were different from that of EMD.

Effects of enamel matrix proteins on adherence, proliferation and migration of epithelial cells: A real-time in vitro study.

Enamel matrix derivative (EMD) can mimic odontogenic effects by inducing the proliferation and differentiation of connective tissue progenitor cells, stimulating bone growth and arresting epithelial cells migration. To the best of our knowledge, there is no data indicating that any active component of EMD reduces epithelial cell viability. The present study examines the impact of commercial lyophilized EMD, porcine recombinant amelogenin (prAMEL; 21.3 kDa) and tyrosine-rich amelogenin peptide (TRAP) on the adherence, proliferation and migration of human epithelial cells in real-time. The tongue carcinoma cell line SCC-25 was stimulated with EMD, porcine recombinant AMEL and TRAP, at concentrations of 12.5, 25 and 50 µg/ml. Cell adherence, migration and proliferation were monitored in real-time using the xCELLigence system. No significant effects of EMD on the morphology, adhesion, proliferation and migration of SCC-25 cells were observed. However, porcine recombinant AMEL had a dose-dependent inhibitory effect on SCC-25 cell proliferation and migration. Predominantly, no notable differences were found between control and TRAP-treated cells in terms of cell adhesion and migration, a decrease in proliferation was observed, but this was not statistically significant. EMD and its active components do not increase the tongue cancer cell viability.

Amelogenin-Ameloblastin Spatial Interaction around Maturing Enamel Rods

Amelogenin and ameloblastin are 2 extracellular matrix proteins that are essential for the proper development of enamel. We recently reported that amelogenin and ameloblastin colocalized during the secretory stage of enamel formation when nucleation of enamel crystallites occurs. Direct interactions between the 2 proteins have been also demonstrated in our in vitro studies. Here, we explore interactions between their fragments during enamel maturation. We applied in vivo immunofluorescence imaging, quantitative co-localization analysis, and a new FRET (fluorescence resonance energy transfer) technique to demonstrate ameloblastin and amelogenin interaction in the maturing mouse enamel. Using immunochemical analysis of protein samples extracted from 8-d-old (P8) first molars from mice as a model for maturation-stage enamel, we identified the ~17-kDa ameloblastin (Ambn-N) and the TRAP (tyrosine-rich amelogenin peptide) fragments. We used Ambn-N18 and Ambn-M300 antibodies raised against the N-terminal and C-terminal segments of ameloblastin, as well as Amel-FL and Amel-C19 antibodies against full-length recombinant mouse amelogenin (rM179) and C-terminal amelogenin, respectively. In transverse sections, co-localization images of N-terminal fragments of amelogenin and ameloblastin around the prism boundary revealed the “fish net” pattern of the enamel matrix. Using in vivo FRET microscopy, we further demonstrated spatial interactions between amelogenin and ameloblastin N-terminal fragments. In the maturing mouse enamel, the association of these residual protein fragments created a discontinuity between enamel rods, which we suggest is important for support and maintenance of enamel rods and eventual contribution to unique enamel mechanical properties. We present data that support cooperative functions of enamel matrix proteins in mediating the structural hierarchy of enamel and that contribute to our efforts to design and develop enamel biomimetic material.

Effect of tyrosine-rich amelogenin peptide on behavior and differentiation of endothelial cells.

BackgroundEnamel matrix derivative (EMD) is an effective biomaterial for periodontal tissue regeneration and might stimulate angiogenesis. Tyrosine-rich amelogenin peptide (TRAP) is present in EMD and is thought to contribute in its biological activity. In the present study, we investigated the effect of chemically synthesized TRAP on proliferation, migration, angiogenic structure formation, and differentiation of human umbilical vein endothelial cells (HUVECs) in vitro.Material and methodsThe effects of TRAP isolated from EMD and chemically synthesized TRAP on proliferation/viability, migration, and angiogenic structure formation were investigated. Expression of angiopoietin-2 (ang-2), von Willebrand factor (vWF), E-selectin, intracellular adhesion molecules 1 (ICAM-1), vascular endothelial growth factor (VEGF) receptors FMS-like tyrosine kinase 1 (FLT-1), and kinase insert domain receptor (KDR) was measured on both messenger RNA (mRNA) and protein levels.ResultsThe proliferation/viability of HUVECs was inhibited by TRAP at concentration of 100 μg/ml and slightly stimulated by EMD at similar concentration. Both EMD and TRAP stimulated endothelial cell migration in microchemotaxis chamber. The effect of both TRAP preparations on the migration was significantly higher than that of EMD. All substances stimulated formation of angiogenic structure in vitro. The expression of ICAM-1, E-selectin, FLT-1, KDR, and vWF was significantly increased by both TRAP and EMD at a concentration 50 μg/ml. The expression of ang-2 was not affected by TRAP but was significantly increased by EMD.ConclusionOur in vitro study shows that TRAP confer the most effects of EMD on the endothelial cells.Clinical relevanceTRAP might be used as a basis for development of new approaches for periodontal regeneration.

Proline-Rich Peptide Mimics Effects of Enamel Matrix Derivative on Rat Oral Mucosa Incisional Wound Healing.

Proline-rich peptides have been shown to promote periodontal regeneration. However, their effect on soft tissue wound healing has not yet been investigated. The aim of this study is to evaluate the effect of enamel matrix derivative (EMD), tyrosine-rich amelogenin peptide (TRAP), and a synthetic proline-rich peptide (P2) on acute wound healing after a full-thickness flap procedure in an incisional rat model. This experimental study has a split-mouth, randomized, placebo-controlled design. Test and control wounds were created on the palatal mucosa of 54 Sprague-Dawley rats. Wounds were histologically processed, and reepithelialization, leukocyte infiltration, and angiogenesis were assessed at days 1, 3, and 7 post-surgery. EMD and P2 significantly promoted early wound closure at day 1 (P <0.001 and P = 0.004, respectively). EMD maintained a significant acceleration of reepithelialization at day 3 (P = 0.004). Wounds treated by EMD and P2 showed increased angiogenesis during the first 3 days of healing (P = 0.03 and 0.001, respectively). Leukocyte infiltration was decreased in EMD-treated wounds at day 1 (P = 0.03), and P2 and TRAP induced a similar effect at days 3 (P = 0.002 and P <0.0001, respectively) and 7 (P = 0.005 and P <0.001). EMD and P2 promoted reepithelialization and neovascularization in full-thickness surgical wounds on rat oral mucosa.

Identification of proteins from human permanent erupted enamel

Proteins from the extracellular matrix of enamel are highly specific and necessary for proper enamel formation. Most proteins are removed from the matrix by enamel proteases before complete mineralization is achieved; however, some residual protein fragments persist in the mineralized matrix of erupted enamel. So far, only amelogenin peptides obtained by traditional bottom-up proteomics have been recovered and identified in human permanent erupted enamel. In this study, we hypothesize that other enamel-specific proteins are also found in human permanent enamel, by analysing human erupted third molars. Pulverized enamel was used to extract proteins, and the protein extract was subjected directly to liquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS) without a previous trypsin-digestion step. Amelogenin and non-amelogenin proteins (ameloblastin and enamelin) were succesfully identified. The sequences of the naturally occurring peptides of these proteins are reported, finding in particular that most of the peptides from the amelogenin X-isoform come from the tyrosine-rich amelogenin peptide (TRAP) and that some were identified in all specimens. In conclusion, our LC-MS/MS method without trypsin digestion increased the coverage of identification of the enamel proteome from a few amelogenin peptides to a higher number of peptides from three enamel-specific proteins.

Differential effects of tyrosine-rich amelogenin peptide on chondrogenic and osteogenic differentiation of adult chondrocytes.

Current approaches to treat osteoarthritis (OA) are insufficient. Autologous chondrocyte implantation (ACI) has been used for the past decade to treat patients with OA or focal cartilage defects. However, a number of complications have been reported post-ACI, including athrofibrosis and symptomatic hypertrophy. Thus, a long-term ACI strategy should ideally incorporate methods to ‘prime’ autologous chondrocytes to form a cartilage-specific matrix and suppress hypertrophic mineralization. The objective of this study is to examine the effects of tyrosine-rich amelogenin peptide (TRAP; an isoform of the developmental protein amelogenin) on human articular cartilage cell (HAC) chondrogenic differentiation and hypertrophic mineralization in vitro. Effects of chemically synthesized TRAP on HAC chondrogenic differentiation were determined by assessing: (1) sGAG production; (2) Alcian blue staining for proteoglycans; (3) collagen type II immunostaining; and (4) expression of the chondrogenic genes SOX9, ACAN and COL2A1. Hypertrophic mineralization was assayed by: (1) ALP expression; (2) Alizarin red staining for Ca+2-rich bone nodules; (3) OC immunostaining; and (4) expression of the osteogenic/hypertrophic genes Ihh and BSP. Chemically synthesized TRAP was found to suppress terminal osteogenic differentiation of HACs cultured in hypertrophic mineralization-like conditions, an effect mediated via down-regulation of the Ihh gene. Moreover, TRAP was found to augment chondrogenic differentiation of HACs via induction of SOX9 gene expression when cells were cultured in pro-chondrogenic media. The results obtained from this proof-of-concept study motivate further studies on the use of TRAP as part of a preconditioning regimen in autologous chondrocyte implantation procedures for OA patients and patients suffering from focal cartilage defects.Electronic supplementary materialThe online version of this article (doi:10.1007/s00441-015-2292-7) contains supplementary material, which is available to authorized users.

Interaction of enamel matrix proteins with human periodontal ligament cells.

ObjectivesIt has recently been shown that enamel matrix derivative (EMD) components (Fraction C, containing <6 kDa peptides (mainly a 5.3 kDa tyrosine-rich amelogenin peptide (TRAP)), and Fraction A, containing a mixture of >6 kDa peptides (including a leucine-rich amelogenin peptide (LRAP))) differentially regulate osteogenic differentiation of periodontal ligament (PDL) cells. The present study examined whether EMD and the EMD Fractions (i) bind and internalize into PDL cells and (ii) precipitate and form insoluble complexes on PDL cells.Materials and methodsBiotin-labelled EMD/EMD Fractions were incubated with PDL cells under various different culture conditions and confocal and electron microscopies were carried out to examine the binding and intracellular trafficking of these proteins.ResultsThe results reported here show, for the first time, that at least some components in Fraction A and the TRAP peptide in Fraction C can bind and be internalized by human PDL cells via receptor-mediated endocytosis. In addition, Fraction A was found to form insoluble aggregate-like structures on PDL cells, whereas Fraction C was soluble in culture media.ConclusionSoluble amelogenin isoform TRAP appears to be internalizing into a subset of PDL cells. Moreover, TRAP uptake is most likely controlled by receptor-mediated endocytosis.Clinical relevanceInformation on interaction between PDL cells and EMD/TRAP might prove useful in designing targeted interventions (i.e. use of chemically prepared soluble amelogenin peptides) to repair/regenerate periodontal tissues. Such interventions can also (i) avoid the use of rather crude animal-derived enamel matrix protein (EMP)/EMD preparation and (ii) preparation of cost-effective and more controlled chemically synthesized amelogenin peptides for the clinical use.Electronic supplementary materialThe online version of this article (doi:10.1007/s00784-015-1510-8) contains supplementary material, which is available to authorized users.

A tyrosine-rich amelogenin peptide promotes neovasculogenesis in vitro and ex vivo

The formation of new blood vessels has been shown to be fundamental in the repair of many damaged tissues, and we have recently shown that the adult human periodontal ligament contains multipotent stem/progenitor cells that are capable of undergoing vasculogenic and angiogenic differentiation in vitro and ex vivo. Enamel matrix protein (EMP) is a heterogeneous mixture of mainly amelogenin-derived proteins produced during tooth development and has been reported to be sometimes effective in stimulating these processes, including in clinical regeneration of the periodontal ligament. However, the identity of the specific bioactive component of EMP remains unclear. In the present study we show that, while the high-molecular-weight Fraction A of enamel matrix derivative (a heat-treated form of EMP) is unable to stimulate the vasculogenic differentiation of human periodontal ligament cells (HPC) in vitro, the low-molecular-weight Fraction C significantly up-regulates the expression of the endothelial markers VEGFR2, Tie-1, Tie-2, VE-cadherin and vWF and markedly increases the internalization of low-density lipoprotein. Furthermore, we also demonstrate, for the first time, that the synthetic homolog of the 45-amino acid tyrosine-rich amelogenin peptide (TRAP) present in Fraction C is likely to be responsible for its vasculogenesis-inducing activity. Moreover, the chemically synthesized TRAP peptide is also shown here to be capable of up-regulating the angiogenic differentiation of the HPC, based on its marked stimulation of in vitro cell migration and tubule formation and of blood vessel formation assay in a chick embryo chorioallantoic membrane model ex vivo. This novel peptide, and modified derivatives, might thereby represent a new class of regenerative drug that has the ability to elicit new blood vessel formation and promote wound healing in vivo.

Amelogenin Peptide Extract Increases Differentiation and Angiogenic and Local Factor Production and Inhibits Apoptosis in Human Osteoblasts

Enamel matrix derivative (EMD), a decellularized porcine extracellular matrix (ECM), is used clinically in periodontal tissue regeneration. Amelogenin, EMD’s principal component, spontaneously assembles into nanospheresin vivo, forming an ECM complex that releases proteolytically cleaved peptides. However, the role of amelogenin or amelogenin peptides in mediating osteoblast response to EMD is not clear. Human MG63 osteoblast-like cells or normal human osteoblasts were treated with recombinant human amelogenin or a 5 kDa tyrosine-rich amelogenin peptide (TRAP) isolated from EMD and the effect on osteogenesis, local factor production, and apoptosis assessed. Treated MG63 cells increased alkaline phosphatase specific activity and levels of osteocalcin, osteoprotegerin, prostaglandin E2, and active/latent TGF-β1, an effect sensitive to the effector and concentration. Primary osteoblasts exhibited similar, but less robust, effects. TRAP-rich 5 kDa peptides yielded more mineralization than rhAmelogenin in osteoblastsin vitro. Both amelogenin and 5 kDa peptides protected MG63s from chelerythrine-induced apoptosis. The data suggest that the 5 kDa TRAP-rich sequence is an active amelogenin peptide that regulates osteoblast differentiation and local factor production and prevents osteoblast apoptosis.

Differential Effect of Amelogenin Peptides on Osteogenic DifferentiationIn Vitro: Identification of Possible New Drugs for Bone Repair and Regeneration

Enamel matrix proteins (EMP) have been shown to promote regeneration of periodontal ligament and root cementum, and sometimes to enhance the differentiation of bone-forming cells in vitro and new bone growth in vivo. However, the inconsistent and unpredictable effects of EMP that have been reported for bone regeneration may be due to the highly variable composition of this heterogeneous material, which is comprised mainly of amelogenin and amelogenin-derived peptides. The present study has therefore examined the effects of naturally occurring low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions of Emdogain(®) (EMD; Institut Straumann, Basel, Switzerland), a commercially available form of EMP, on osteogenic differentiation of bone precursor cells in vitro. In addition, the effects of chemically synthesized specific components of LMW and HMW-namely, the tyrosine-rich amelogenin peptide (TRAP), a specific amelogenin isoform derived by proteolytic clipping, and a leucine-rich amelogenin peptide (LRAP), an isoform derived by alternative splicing-on bone-forming cell activity were also investigated. Our findings demonstrate that while TRAP suppressed the formation of bone-like mineralized nodules, LRAP upregulated osteogenic differentiation. Furthermore, synthetically produced TRAP and its unique C-terminal 12 amino acid sequence (TCT) also suppressed bone-forming cells, whereas LRAP and its unique C-terminal 23 amino acid sequence (LCT) markedly enhanced terminal differentiation of bone-forming cells. These findings suggest that the differential effects of amelogenin-derived peptide sequences present in EMP could be of potential clinical value, with the novel bioactive TCT peptide as a useful tool for limiting pathological bone cell growth and the unique LCT sequence having therapeutic benefits in the treatment of periodontal and orthopedic diseases.

Amelogenins promote an alternatively activated macrophage phenotype in vitro

Amelogenins are extracellular matrix proteins used for the topical treatment of chronically inflamed tissues. The influence of amelogenins on human monocyte-derived macrophages was studied by measuring the concentrations of cytokines in culture supernatants. The interactions of cells and protein aggregates were visualised by transmission electron microscopy. The amelogenin treatment of macrophages increased several pro- and anti-inflammatory cytokines, including alternative macrophage activation marker AMAC-1 (p < 0.001) and vascular endothelial growth factor (VEGF; p < 0.001). The levels were independent of cytochalasin B, although amelogenin aggregates were ingested by macrophages. Amelogenin effect was compared with that of tyrosine-rich amelogenin peptide, which apart from augmented VEGF levels (p < 0.05), had no significant influence on the other cytokines analysed. In conclusion, amelogenins increased the macrophage release of key cell mediators involved in tissue repair. The effect was independent of phagocytosis, implying a receptor-mediated signal. The markedly increased levels of AMAC-1 suggest that amelogenins promote a reparative macrophage phenotype.

Extraction and sequencing of human and Neanderthal mature enamel proteins using MALDI-TOF/TOF MS

We report here the first results of a method to extract and sequence mature enamel proteins from modern human and Neanderthal tooth enamel. Using MALDI-TOF/TOF mass spectrometry and a combination of direct sequencing and peptide mass mapping we have sequenced a peptide from the tyrosine-rich amelogenin peptide (TRAP) of the X isoform of the amelogenin protein for modern and recent human samples. We also report our results from two Neanderthal enamel samples where we were also able to recover fragments of the TRAP protein, which had a similar sequence to the modern human samples.

Amelogenins regulate expression of genes associated with cementoblasts in vitro

Amelogenins are major proteins expressed by ameloblasts during development of the crown (enamel and dentin). These matrix proteins guide crystal habits of the mineral phase of developing enamel and are possible regulators of other genes/proteins during development and maturation of crown and root (dentin and cementum). This study focused on defining the effect that a specific proteolytic cleavage product of amelogenin, tyrosine-rich amelogenin peptide (TRAP), has on cementoblast behavior. Immortalized cementoblasts (OCCM-30) were exposed to TRAP in vitro. Cells treated with TRAP were evaluated for cell proliferation, gene expression for osteocalcin (OCN), osteopontin (OPN), and bone sialoprotein (BSP), and induction of mineral nodule formation. No significant difference in cell proliferation was found between vehicle-treated cells and those treated with TRAP for up to 9 d after treatment. Gene expression of OCN, OPN, and BSP in TRAP-treated cementoblasts showed down-regulation, up-regulation, and no significant change, respectively, relative to vehicle control. A marked decrease in mineral nodule formation was found in cells treated with TRAP compared with the vehicle control, in a dose-dependent manner. These data, along with our previous results demonstrating similar activity with full-length amelogenin and leucine-rich amelogenin peptide (LRAP), suggest that amelogenin-like molecules regulate mesenchymal cell behavior.

Overexpression of TRAP in the Enamel Matrix Does Not Alter the Enamel Structural Hierarchy

The secreted, full-length amelogenin is the dominant protein of the forming enamel organ. As enamel mineralization progresses, amelogenin is quickly subjected to proteolytic activity, and eliminated from the enamel environment. Mature enamel contains only traces of structural proteins, including enamelin and the sheath protein ameloblastin. In addition, a proteolytic fragment of amelogenin, known as the tyrosine-rich amelogenin peptide or TRAP, is present in low but isolatable quantities. By overexpressing TRAP during enamel development we sought to determine if such overexpression would result in structural alterations to the mature enamel. We reasoned that overexpressing a protein associated with enamel maturation, at an inappropriate developmental stage, would result in alterations to the enamel protein assembly and hence, alterations in enamel structure and morphology. As judged by transmission and scanning electron microscopy, the enamel formed by overexpressing TRAP showed little morphological differences when compared to the enamel of normal nontransgenic animals. Based on scanning electron-microscopic images, there was modest hypomineralization evident in the interrod enamel of the TRAP-overexpressing animals. However, this finding was inconsistent and inconsequential from a structural and functional perspective. From these results it appears that additional amounts of TRAP protein in the immature enamel matrix are not sufficient to alter the properties of the enamel extracellular matrix to an extent that the hierarchical structure of mature enamel is altered.