RESUMEN
Tooth enamel, the hardest tissue in the body, is formed by the evolutionarily highly conserved biomineralization process that is controlled by extracellular matrix proteins. The intrinsically disordered matrix protein ameloblastin (AMBN) is the most abundant nonamelogenin protein of the developing enamel and a key element for correct enamel formation. AMBN was suggested to be a cell adhesion molecule that regulates proliferation and differentiation of ameloblasts. Nevertheless, detailed structural and functional studies on AMBN have been substantially limited by the paucity of the purified nondegraded protein. With this study, we have developed a procedure for production of a highly purified form of recombinant human AMBN in quantities that allowed its structural characterization. Using size exclusion chromatography, analytical ultracentrifugation, transmission electron, and atomic force microscopy techniques, we show that AMBN self-associates into ribbon-like supramolecular structures with average widths and thicknesses of 18 and 0.34 nm, respectively. The AMBN ribbons exhibited lengths ranging from tens to hundreds of nm. Deletion analysis and NMR spectroscopy revealed that an N-terminal segment encoded by exon 5 comprises two short independently structured regions and plays a key role in self-assembly of AMBN.
Asunto(s)
Proteínas del Esmalte Dental/metabolismo , Exones , Cromatografía en Gel , Dicroismo Circular , Proteínas del Esmalte Dental/química , Proteínas del Esmalte Dental/genética , Electroforesis en Gel de Poliacrilamida , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Ameloblastin is mainly known as a dental enamel protein, synthesized and secreted into developing enamel matrix by the enamel-forming ameloblasts. The function of ameloblastin in tooth development remains unclear, but it has been suggested to be involved in processes varying from regulating crystal growth to activity as a growth factor or partaking in cell signaling. Recent studies suggest that some enamel matrix proteins also might have important functions outside enamel formation. In this context ameloblastin has recently been reported to induce dentin and bone repair, as well as being present in the early bone and cartilage extracellular matrices during embryogenesis. However, what cells express ameloblastin in these tissues still remains unclear. Thus, the expression of ameloblastin was examined in cultured primary mesenchymal cells and in vivo during healing of bone defects in a "proof of concept" animal study. Real time RT-PCR analysis revealed human ameloblastin (AMBN) mRNA expression in human mesenchymal stem cells and primary osteoblasts and chondrocytes. Expression of AMBN mRNA was also confirmed in human CD34 positive cells and osteoclasts. Western and dot blot analysis of cell lysates and medium confirmed the expression and secretion of ameloblastin from mesenchymal stem cells, primary human osteoblasts and chondrocytes. Expression of ameloblastin was also detected in newly formed bone in experimental bone defects in adult rats. Together these findings suggest a role for this protein in early bone formation and repair.
Asunto(s)
Proteínas del Esmalte Dental/metabolismo , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/fisiología , Animales , Western Blotting , Línea Celular , Condrocitos/citología , Condrocitos/metabolismo , Femenino , Humanos , Inmunohistoquímica , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Osteoblastos/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Ameloblastin (AMBN) is a protein expressed mainly during dental hard tissue development. Biochemically, it is classified as an intrinsically disordered protein (IDP). Its biological role remains largely unknown; however, the question of AMBN function will undoubtedly be connected to its structural properties and its potential for protein-protein and protein-cell interactions. A basic biophysical characterization of human recombinant ameloblastin (hrAMBN) and its N- and C-terminal domains by means of circular dichroism spectroscopy and dynamic light scattering showed that under physiological conditions ameloblastin is an IDP with a prevalent polyproline-II (PPII) conformation. Both the N- and C-terminal polypeptides, when expressed independently, showed different structural preferences upon heating as well as different behaviour in the presence of trifluoroethanol and CaCl(2) salt. The N-terminal peptide showed a more ordered structure with a strong tendency to adopt a helical conformation upon the addition of trifluorethanol, whereas the C-terminal domain seemed to be primarily responsible for the structural disorder of the entire AMBN molecule.
Asunto(s)
Proteínas del Esmalte Dental/química , Fenómenos Biofísicos , Cloruro de Calcio/farmacología , Dicroismo Circular , Reactivos de Enlaces Cruzados , Humanos , Concentración Osmolar , Conformación Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Dispersión de Radiación , Análisis de Secuencia de Proteína , Cloruro de Sodio/farmacología , Análisis Espectral , Temperatura , Trifluoroetanol/farmacologíaRESUMEN
In this study, we examined the role of the enamel matrix protein, ameloblastin, in bone growth and remodelling, and attempted to identify some of the molecular mechanisms involved in these processes. The effects of recombinant ameloblastin (rAmbn) were tested in vivo in rats, and in vitro in primary human mesenchymal stem cells, osteoblasts, chondrocytes, and osteoclasts. We used a microarray technique to identify genes that were regulated in human osteoblasts and verified our findings using multiplex protein analysis and real-time RT-PCR. Recombinant ameloblastin was found to stimulate bone healing in vivo, and to enhance the proliferation of mesenchymal stem cells and osteoblasts, as well as the differentiation of osteoclast precursor cells in vitro. The most profound effect was on the regulation of genes related to immune responses as well as on the expression of cytokines and markers of bone cell differentiation, indicating that ameloblastin has an effect on mesenchymal cell differentiation. A receptor has not yet been identified, but we found rAmbn to induce, directly and indirectly, signal transducer and activator of transcription (STAT) 1 and 2 and downstream factors in the interferon pathway.
Asunto(s)
Regeneración Ósea/efectos de los fármacos , Proteínas del Esmalte Dental/fisiología , Factores Inmunológicos/metabolismo , Interferones/biosíntesis , Células Madre Mesenquimatosas/metabolismo , Factor de Transcripción STAT1/biosíntesis , Factor de Transcripción STAT2/biosíntesis , Análisis de Varianza , Animales , Regeneración Ósea/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Proteínas del Esmalte Dental/farmacología , Regulación de la Expresión Génica , Humanos , Interferones/genética , Mandíbula/citología , Mandíbula/cirugía , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Ratas , Proteínas Recombinantes/farmacología , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT2/genética , Estadísticas no ParamétricasRESUMEN
Ameloblastin (AMBN) was originally believed to be an enamel-specific extracellular matrix glycoprotein secreted by ameloblasts. Recently, AMBN expression was also detected in developing mesenchymal dental hard tissues, in trauma-induced reparative dentin, and during early craniofacial bone formation. The function and structure of AMBN still remain ambiguous, and there are no known proteins with similar primary sequences. We therefore performed a bio-informatic analysis of AMBN to model ab initio the three-dimensional structure of the molecule. The results suggest that AMBN is a two-domain, intrinsically unstructured protein (IUP). The analysis did not reveal any regions with structural similarity to known receptor-ligand systems, and did not identify any higher-order structures similar to functional regions in other known sequences. The AMBN model predicts 11 defined regions exposed on the surface, internalizing the rest of the molecule including a human-specific insert. Molecular dynamics analysis identified one specific and several non-specific calcium-binding regions, mostly at the C-terminal part of the molecule. The model is supported by previous observations that AMBN is a bipolar calcium-binding molecule and hints at a possible role in protein-protein interactions. The model provides information useful for further studies on the function of AMBN.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas del Esmalte Dental/química , Modelos Moleculares , Redes Neurales de la Computación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Humanos , Modelos Químicos , Estructura Terciaria de ProteínaRESUMEN
Based on previous results showing the expression of ameloblastin (Ambn; amelin) in the formation of mesenchymal dental hard tissues, we investigated its presence during bone development. Immunohistochemistry (IHC), in situ hybridization (ISH), and reverse transcription-polymerase chain reaction (RT-PCR) were used to investigate the expression of ameloblastin protein and mRNA during craniofacial development in rats. Tissue samples were collected on embryonic day 18 and from days 2-28 postnatally. IHC revealed the expression of ameloblastin during bone formation at embryonic and early postnatal stages with different patterns of expression in intramembranous and endochondral ossification. In intramembranous ossification, ameloblastin expression was detected in the superficial layer of the condensed vascularized primitive connective tissue and in the cellular layer covering the surface of the newly formed woven bone. In endochondral ossification, ameloblastin was expressed within the extracellular matrix of the cartilage templates and in the perichondrium. Between days 2 and 28 the expression decreased markedly, concordant with the maturation of the bone, and disappeared after completion of bone remodeling. The results obtained by IHC were confirmed by ISH and RT-PCR, showing the expression of ameloblastin mRNA during craniofacial bone formation. This study indicates the expression of the putative dental protein ameloblastin during craniofacial bone development in rats.
Asunto(s)
Proteínas del Esmalte Dental/biosíntesis , Maxilares/embriología , Desarrollo Maxilofacial/fisiología , Animales , Ensayo de Inmunoadsorción Enzimática , Regulación del Desarrollo de la Expresión Génica , Técnicas para Inmunoenzimas , Hibridación in Situ , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Observations that amelogenins, in the form of enamel matrix derivative (EMD), have a stimulatory effect on mesenchymal cells and tissues, and on the regeneration of alveolar bone, justified investigations into the effect of EMD on bone-forming cells. The binding and uptake of EMD in primary osteoblastic cells was characterized, and the effect of EMD on osteoblast gene expression, protein secretion, and mineralization was compared with the effect of parathyroid hormone (PTH). Although no specific receptor(s) has yet been identified, EMD appeared to be taken up by osteoblasts through clathrin-coated pits via the interaction with clathrin adaptor protein complex AP-2, the major mechanism of cargo sorting into coated pits in mammalian cells. EMD had a positive effect on factors involved in mineralization in vitro, causing an increased alkaline phosphatase (ALP) activity in the medium as well an as increased expression of osteocalcin and collagen type 1. Several hundred genes are regulated by EMD in primary human osteoblasts. There appear to be similarities between the effects of EMD and PTH on human osteoblasts. The expression pattern of several mRNAs and proteins upon EMD stimulation also indicates a secondary osteoclast stimulatory effect, suggesting that the osteogenic effect of EMD in vivo, at least partly, involves stimulation of bone remodelling.