RESUMEN
The enamel matrix protein amelogenin is secreted by ameloblasts into the extracellular space to guide the formation of highly ordered hydroxyapatite mineral crystallites, and, subsequently, is almost completely removed during mineral maturation. Amelogenin interacts with the transmembrane proteins CD63 and LAMP (lysosome-associated membrane protein) 1, which are involved in endocytosis. Exogenously added amelogenin has been observed to move rapidly into CD63/LAMP1-positive vesicles in cultured cells. In the present study, we demonstrate the protein region defined by amino acid residues 103-205 for CD63 interacts not only with amelogenin, but also with other enamel matrix proteins (ameloblastin and enamelin). A detailed characterization of binding regions in amelogenin, CD63 and LAMP1 reveals that the amelogenin region defined by residues PLSPILPELPLEAW is responsible for the interaction with CD63 through residues 165-205, with LAMP1 through residues 226-251, and with the related LAMP2 protein through residues 227-259. We predict that the amelogenin binding region is: (i) hydrophobic; (ii) largely disordered; and (iii) accessible to the external environment. In contrast, the binding region of CD63 is likely to be organized in a '7' shape within the mushroom-like structure of CD63 EC2 (extracellular domain 2). In vivo, the protein interactions between the secreted enamel matrix proteins with the membrane-bound proteins are likely to occur at the specialized secretory surfaces of ameloblast cells called Tomes' processes. Such protein-protein interactions may be required to establish short-term order of the forming matrix and/or to mediate feedback signals to the transcriptional machinery of ameloblasts and/or to remove matrix protein debris during enamel biomineralization.
Asunto(s)
Amelogenina/metabolismo , Antígenos CD/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Amelogenina/química , Secuencia de Aminoácidos , Animales , Antígenos CD/química , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Proteínas de Membrana de los Lisosomas/química , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Glicoproteínas de Membrana Plaquetaria/química , Conformación Proteica , Homología de Secuencia de Aminoácido , Tetraspanina 30 , Técnicas del Sistema de Dos HíbridosRESUMEN
The established structural proteins of the enamel matrix are amelogenin, ameloblastin, and enamelin. Historically, tuftelin and tuftelin-interacting protein 11 (TFIP11) have also been discussed as possible enamel proteins. Protein complexes are achieved by protein-protein interactions, and it is protein complexes that control biomineralization. The purpose of our recent studies was to catalog protein partners for these proteins that are, or have been, implicated in tooth formation. We used the sensitive yeast two-hybrid assay to identify proteins that interact directly with amelogenin, ameloblastin, enamelin, the leucine-rich amelogenin peptide (LRAP) and TFIP11. In this manuscript we refer to, or document, potential protein partners for the proteins listed above. The yeast two-hybrid assay may ultimately prove to be a valuable proteomics methodology for using to decipher molecular events that ultimately result in enamel biomineralization.
Asunto(s)
Proteínas del Esmalte Dental/análisis , Proteínas Nucleares/análisis , Proteínas de Transporte Vesicular/análisis , Amelogenina , Animales , Vectores Genéticos/genética , Ratones , Sistemas de Lectura Abierta/genética , Plásmidos/genética , Mapeo de Interacción de Proteínas , Proteoma/análisis , Empalme del ARN , Factores de Empalme de ARN , Proteínas de Unión al ARN , Ratas , Análisis de Secuencia de Proteína , Levaduras/genéticaRESUMEN
Diversity in gene expression is commonly observed as a result of alternative splicing of RNA transcripts. This is true in the case of amelogenin, one of the enamel matrix proteins. Our hypothesis is that additional amelogenin mRNA transcripts are generated in vivo, but these transcripts have yet to be observed because of the limitations of currently used detection methodologies. For this study our objective was to create an amelogenin minigene to study amelogenin RNA splicing events in cell lines of diverse character. Mouse genomic DNA was used as a PCR template to amplify the amelogenin DNA sequence spanning exons 2-7. The resulting PCR-generated DNA was subcloned in an expression vector. This resulting amelogenin minigene was shown to be functionally active by transfection into multiple cell lines. We have successfully cloned an amelogenin minigene, and as a result we describe and discuss novel amelogenin alternatively spliced transcripts.