Ameloblastin (Ambn) as an intrinsically disordered protein (IDP) stands for an important role in the formation of enamel-the hardest biomineralized tissue commonly formed in vertebrates. The human ameloblastin (AMBN) is expressed in two isoforms: full-length isoform I (AMBN ISO I) and isoform II (AMBN ISO II), which is about 15 amino acid residues shorter than AMBN ISO I. The significant feature of AMBN-its oligomerization ability-is enabled due to a specific sequence encoded by exon 5 present at the N-terminal part in both known isoforms. In this study, we characterized AMBN ISO I and AMBN ISO II by biochemical and biophysical methods to determine their common features and differences. We confirmed that both AMBN ISO I and AMBN ISO II form oligomers in in vitro conditions. Due to an important role of AMBN in biomineralization, we further addressed the calcium (Ca2+)-binding properties of AMBN ISO I and ISO II. The binding properties of AMBN to Ca2+ may explain the role of AMBN in biomineralization and more generally in Ca2+ homeostasis processes.

• Keywords
• ameloblastin, biomineralization, calcium binding, intrinsically disordered protein (IDPs), oligomerization,
• MeSH
• Models, Biological MeSH
• Hydrodynamics MeSH
• Humans MeSH
• Protein Multimerization MeSH
• Protein Isoforms MeSH
• Calcium-Binding Proteins chemistry   metabolism   MeSH
• Dental Enamel Proteins chemistry   metabolism   MeSH
• Spectrum Analysis MeSH
• Temperature MeSH
• Calcium metabolism   MeSH
• Protein Binding MeSH
• Intrinsically Disordered Proteins metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• AMBN protein, human MeSH Browser
• Protein Isoforms MeSH
• Calcium-Binding Proteins MeSH
• Dental Enamel Proteins MeSH
• Calcium MeSH
• Intrinsically Disordered Proteins MeSH

OBJECTIVE: Transcriptional regulatory elements in the ameloblastin (AMBN) promoter indicate that adipogenesis may influence its expression. The objective here was to investigate if AMBN is expressed in adipose tissue, and have a role during differentiation of adipocytes. DESIGN: AMBN expression was examined in adipose tissue and adipocytes by real-time PCR and ELISA. Distribution of ameloblastin was investigated by immunofluorescence in sections of human subcutaneous adipose tissue. The effect of recombinant proteins resembling AMBN and its processed products on proliferation of primary human pre-adipocytes and murine 3T3-L1 cell lines was measured by [3H]-thymidine incorporation. The effect on adipocyte differentiation was evaluated by the expression profile of the adipogenic markers PPARγ and leptin, and the content of lipids droplets (Oil-Red-O staining). RESULTS: AMBN was found to be expressed in human adipose tissue, human primary adipocytes, and in 3T3-L1 cells. The C-terminus of the AMBN protein and a 45 bp shorter splice variant was identified in human subcutaneous adipose tissue. The expression of AMBN was found to increase four-fold during differentiation of 3T3-L1 cells. Administration of recombinant AMBN reduced the proliferation, and enhanced the expression of PPARγ and leptin in 3T3-L1 and human pre-adipocytes, respectively. CONCLUSIONS: The AMBN C-terminus variant was identified in adipocytes. This variant may be encoded from a short splice variant. Increased expression of AMBN during adipogenesis and its effect on adipogenic factors suggests that AMBN also has a role in adipocyte development.

• Keywords
• Biochemistry, Cell biology, Molecular biology, Physiology,
• Publication type
• Journal Article MeSH

Ameloblastin (AMBN), an important component of the self-assembled enamel extra cellular matrix, contains several in silico predicted phosphorylation sites. However, to what extent these sites actually are phosphorylated and the possible effects of such post-translational modifications are still largely unknown. Here we report on in vitro experiments aimed at investigating what sites in AMBN are phosphorylated by casein kinase 2 (CK2) and protein kinase A (PKA) and the impact such phosphorylation has on self-assembly and calcium binding. All predicted sites in AMBN can be phosphorylated by CK2 and/or PKA. The experiments show that phosphorylation, especially in the exon 5 derived part of the molecule, is inversely correlated with AMBN self-assembly. These results support earlier findings suggesting that AMBN self-assembly is mostly dependent on the exon 5 encoded region of the AMBN gene. Phosphorylation was significantly more efficient when the AMBN molecules were in solution and not present as supramolecular assemblies, suggesting that post-translational modification of AMBN must take place before the enamel matrix molecules self-assemble inside the ameloblast cell. Moreover, phosphorylation of exon 5, and the consequent reduction in self-assembly, seem to reduce the calcium binding capacity of AMBN suggesting that post-translational modification of AMBN also can be involved in control of free Ca2+ during enamel extra cellular matrix biomineralization. Finally, it is speculated that phosphorylation can provide a functional crossroad for AMBN either to be phosphorylated and act as monomeric signal molecule during early odontogenesis and bone formation, or escape phosphorylation to be subsequently secreted as supramolecular assemblies that partake in enamel matrix structure and mineralization.

• Keywords
• Ca2+- binding, ameloblastin, casein kinase 2, enamel, intrinsically disordered proteins, phosphorylation, protein kinase A, self-assembly,
• Publication type
• Journal Article MeSH

Using the distal molar of a minipig as a model, we studied changes in the microstructural characteristics of apatite crystallites during enamel maturation (16-23 months of postnatal age), and their effects upon the mechanical properties of the enamel coat. The slow rate of tooth development in a pig model enabled us to reveal essential heterochronies in particular components of the maturation process. The maturation changes began along the enamel-dentine junction (EDJ) of the trigonid, spreading subsequently to the outer layers of the enamel coat to appear at the surface zone with a 2-month delay. Correspondingly, at the distal part of the tooth the timing of maturation processes is delayed by 3-5 month compared to the mesial part of the tooth. The early stage of enamel maturation (16-20 months), when the enamel coat is composed almost exclusively of radial prismatic enamel, is characterized by a gradual increase in crystallite thickness (by a mean monthly increment of 3.8 nm); and an increase in the prism width and thickness of crystals composed of elementary crystallites. The late stage of maturation (the last two months prior to tooth eruption), marked with the rapid appearance of the interprismatic matrix (IPM) during which the crystals densely infill spaces between prisms, is characterized by an abrupt decrease in microstrain and abrupt changes in the micromechanical properties of the enamel: a rapid increase in its ability to resist long-term load and its considerable hardening. The results suggest that in terms of crystallization dynamics the processes characterizing the early and late stage of mammalian enamel maturation represent distinct entities. In regards to common features with enamel formation in the tribosphenic molar we argue that the separation of these processes could be a common apomorphy of mammalian amelogenetic dynamics in general.

• MeSH
• Dentin diagnostic imaging   metabolism   MeSH
• Crystallography MeSH
• Swine, Miniature MeSH
• Molar diagnostic imaging   growth & development   MeSH
• Swine MeSH
• Tooth Eruption physiology   MeSH
• Dental Enamel diagnostic imaging   metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

During amelogenesis the extracellular enamel matrix protein AMBN is quickly processed into 17 kDa (N-terminus) and 23 kDa (C-terminus) fragments. In particular, alternatively spliced regions derived by exon 5/6 within the N-terminus region are known to be critical in biomineralization. Human mesenchymal stem cells (hMSC) also express and secrete AMBN, but it is unclear if this expression has effects on the hMSC themselves. If, as suggested from previous findings, AMBN act as a signaling molecule, such effects could influence hMSC growth and differentiation, as well as promoting the secretion of other signaling proteins like cytokines and chemokines. If AMBN is found to modulate stem cell behavior and fate, it will impact our understanding on how extracellular matrix molecules can have multiple roles during development ontogenesis, mineralization and healing of mesenchymal tissues. Here we show that synthetic peptides representing exon 5 promote hMSC proliferation. Interestingly, this effect is inhibited by the application of a 15 aa peptide representing the alternatively spliced start of exon 6. Both peptides also influence gene expression of RUNX2 and osteocalcin, and promote calcium deposition in cultures, indicating a positive influence on the osteogenic capacity of hMSC. We also show that the full-length AMBN-WT and N-terminus region enhance the secretion of RANTES, IP-10, and IL-8. In contrast, the AMBN C-terminus fragment and the exon 5 deleted AMBN (DelEx5) have no detectable effects on any of the parameters investigated. These findings suggest the signaling effect of AMBN is conveyed by processed products, whereas the effect on proliferation is differentially modulated through alternative splicing during gene expression.

• Keywords
• ameloblastin, biomineralization, bone growth, exon 5, human mesenchymal stem cells, osteogenesis, proliferation,
• Publication type
• Journal Article MeSH

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.

• Keywords
• Ameloblastin, Amelogenin, Extracellular Matrix Proteins, Intrinsically Disordered Proteins, Protein Purification, Protein Self-assembly, Tooth,
• MeSH
• Circular Dichroism MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Exons * MeSH
• Chromatography, Gel MeSH
• Protein Conformation MeSH
• Microscopy, Atomic Force MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Dental Enamel Proteins chemistry   genetics   metabolism   MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Microscopy, Electron, Transmission MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• AMBN protein, human MeSH Browser
• Dental Enamel Proteins MeSH
• Recombinant Proteins MeSH