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.
- MeSH
- cirkulární dichroismus MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- exony * MeSH
- gelová chromatografie MeSH
- konformace proteinů MeSH
- mikroskopie atomárních sil MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- proteiny zubní skloviny chemie genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- transmisní elektronová mikroskopie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- biofyzikální jevy MeSH
- chlorid sodný farmakologie MeSH
- chlorid vápenatý farmakologie MeSH
- cirkulární dichroismus MeSH
- interakční proteinové domény a motivy MeSH
- konformace proteinů účinky léků MeSH
- lidé MeSH
- osmolární koncentrace MeSH
- proteiny zubní skloviny chemie MeSH
- radiační rozptyl MeSH
- reagencia zkříženě vázaná MeSH
- rekombinantní proteiny chemie MeSH
- sekundární struktura proteinů MeSH
- sekvenční analýza proteinů MeSH
- spektrální analýza MeSH
- teplota MeSH
- terciární struktura proteinů MeSH
- trifluorethanol farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
Novel mRNA isoforms encoding the enamel matrix proteins amelin-1, amelin-2 and ameloblastin have been recently described. We have applied detailed immunohistochemical as well as non-radioactive in situ hybridization analyses to follow amelin-1 expression in developing rat incisors and molars. We constructed an expression vector, overproduced recombinant amelin in Escherichia coli and prepared an antibody. In addition to the previously reported amelin mRNA expression patterns in ameloblasts, the amelin message was also detected in pulpal mesenchymal cells including preodontoblasts and young odontoblasts. The signal in these cells persisted until deposition of mantle dentin became evident. The immunolocalization of amelin-1 in preodontoblasts and ameloblasts essentially followed the pattern of mRNA expression. The most intense staining was found in the enamel matrix adjacent to secretory ameloblasts. Focal accumulations of immunoreactive material were found at the dentinoenamel junction during the maturation stage. Also, using 5'-RACE (Rapid Amplification of cDNA Ends) we could confirm only amelin-1 and ameloblastin messages in the total RNA pool from rat molars and conclude that amelin-2 is a truncated form of ameloblastin. The sequential expression of amelin in mesenchymal and epithelial cells suggests it plays a role in cell differentiation during early tooth development.
- MeSH
- ameloblasty metabolismus MeSH
- DNA primery genetika MeSH
- epitelové buňky metabolismus MeSH
- hybridizace in situ MeSH
- imunohistochemie MeSH
- komplementární DNA genetika MeSH
- krysa rodu rattus MeSH
- messenger RNA genetika MeSH
- mezoderm metabolismus MeSH
- odontoblasty metabolismus MeSH
- odontogeneze * genetika MeSH
- potkani Sprague-Dawley MeSH
- proteiny zubní skloviny * genetika metabolismus MeSH
- rekombinantní fúzní proteiny genetika MeSH
- sekvence nukleotidů MeSH
- vývojová regulace genové exprese MeSH
- zubní sklovina cytologie metabolismus růst a vývoj MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Two variants of an mRNA sequence are identified that are expressed at high levels in rat ameloblasts during the formation of the enamel matrix. The sequences contain open reading frames for 407 and 324 amino acid residues, respectively. The encoded proteins, which we call amelins, are rich in proline, glycine, leucine, and alanine residues and contain the peptide domain DGEA, an integrin recognition sequence. The sequences coding for the C-terminal 305 amino acid residues, the 3' nontranslated part, and a microsatellite repeat at the nontranslated 5' region are identical in both mRNA variants. The remaining 5' regions contain 338 nucleotides unique to the long variant, 54 common nucleotides, and 46 nucleotides present only in the short variant. Eleven nucleotides have the potential to code for 5 amino acids of both proteins in different reading frames. The reading frame of the longer variant includes codons for a typical N-terminal signal peptide. The amelins are likely to be constituents of the enamel matrix and the only proteins that have so far been implicated in binding interactions between the ameloblast surface and its extracellular matrix.
- MeSH
- ameloblasty * fyziologie MeSH
- cytoskeletální proteiny * genetika metabolismus MeSH
- genetický kód MeSH
- genomová knihovna * MeSH
- hybridizace in situ MeSH
- krysa rodu rattus MeSH
- molekulární sekvence - údaje MeSH
- oligonukleotidové sondy MeSH
- potkani Sprague-Dawley MeSH
- proteiny nervové tkáně * genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- terciární struktura proteinů * MeSH
- vazba proteinů MeSH
- vývojová regulace genové exprese * fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
2. vyd. 70 s. : il.
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- chemie, klinická chemie
- zdravotní výchova
- biochemie
- NLK Publikační typ
- učebnice vysokých škol
