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Janet Moradianoldak - One of the best experts on this subject based on the ideXlab platform.

  • control of calcium phosphate nucleation and transformation through interactions of enamelin and Amelogenin exhibits the goldilocks effect
    Crystal Growth & Design, 2018
    Co-Authors: Jinhui Tao, Janet Moradianoldak, Andreas Fijneman, Jiaqi Wan, Saumya Prajapati, Kaushik Mukherjee, Alejandro Fernandezmartinez, James J De Yoreo
    Abstract:

    Although Amelogenin comprises the vast majority of the matrix that templates calcium phosphate nucleation during enamel formation, other proteins, particularly enamelin, are also known to play an important role in the formation of enamel's intricate architecture. However, there is little understanding of the interplay between Amelogenin and enamelin in controlling processes of mineral nucleation and growth. Here, we used an in vitro model to investigate the impact of enamelin interaction with Amelogenin on calcium phosphate nucleation for a range of enamelin-to-Amelogenin ratios. We found that Amelogenin alone is a weak promoter of nucleation, but addition of enamelin enhanced nucleation rates in a highly nonlinear, nonmonotonic manner reaching a sharp maximum at a ratio of 1:50 enamelin/Amelogenin. We provide a phenomenological model to explain this effect that assumes only isolated enamelin proteins can act as sites of enhanced nucleation, while enamelin oligomers cannot. Even when interaction is random, the model reproduces the observed behavior, suggesting a simple means to tightly control the timing and extent of nucleation and phase transformation by Amelogenin and enamelin.

  • dissecting Amelogenin protein nanospheres characterization of metastable oligomers
    Journal of Biological Chemistry, 2011
    Co-Authors: Keith M Bromley, Andrew Kiss, Sowmya Bekshe Lokappa, Rajamani Lakshminarayanan, Daming Fan, Moise Ndao, John Spencer Evans, Janet Moradianoldak
    Abstract:

    Amelogenin self-assembles to form an extracellular protein matrix, which serves as a template for the continuously growing enamel apatite crystals. To gain further insight into the molecular mechanism of Amelogenin nanosphere formation, we manipulated the interactions between Amelogenin monomers by altering pH, temperature, and protein concentration to create isolated metastable Amelogenin oligomers. Recombinant porcine Amelogenins (rP172 and rP148) and three different mutants containing only a single tryptophan (Trp161, Trp45, and Trp25) were used. Dynamic light scattering and fluorescence studies demonstrated that oligomers were metastable and in constant equilibrium with monomers. Stable oligomers with an average hydrodynamic radius (RH) of 7.5 nm were observed at pH 5.5 between 4 and 10 mg·ml−1. We did not find any evidence of a significant increase in folding upon self-association of the monomers into oligomers, indicating that they are disordered. Fluorescence experiments with single tryptophan Amelogenins revealed that upon oligomerization the C terminus of Amelogenin (around residue Trp161) is exposed at the surface of the oligomers, whereas the N-terminal region around Trp25 and Trp45 is involved in protein-protein interaction. The truncated rP148 formed similar but smaller oligomers, suggesting that the C terminus is not critical for Amelogenin oligomerization. We propose a model for nanosphere formation via oligomers, and we predict that nanospheres will break up to form oligomers in mildly acidic environments via histidine protonation. We further suggest that oligomeric structures might be functional components during maturation of enamel apatite.

  • the tooth enamel protein porcine Amelogenin is an intrinsically disordered protein with an extended molecular configuration in the monomeric form
    Biochemistry, 2009
    Co-Authors: Katya Delak, Janet Moradianoldak, Rajamani Lakshminarayanan, Zhi Sun, Craig Harcup, Yuwwei Fan, John Spencer Evans
    Abstract:

    Amelogenins make up a class of proteins associated with the formation of mineralized enamel in vertebrates, possess highly conserved N- and C-terminal sequence regions, and represent an interesting model protein system for understanding biomineralization and protein assembly. Using bioinformatics, we report here the identification of molecular traits that classify 12 Amelogenin proteins as members of the intrinsically disordered or unstructured protein family (IDPs), a group of proteins that normally exist as unfolded species but are capable of transformation to a folded state as part of their overall function. Using biophysical techniques (CD and NMR), we follow up on our bioinformatics studies and confirm that one of the Amelogenins, recombinant porcine rP172, exists in an extended, unfolded state in the monomeric form. This protein exhibits evidence of conformational exchange between two states, and this exchange may be mediated by Pro residues in the sequence. Although the protein is globally unfolded...

  • immunogold labeling of Amelogenin in developing porcine enamel revealed by field emission scanning electron microscopy
    Cells Tissues Organs, 2009
    Co-Authors: Daming Fan, Yuwei Fan, Rajamani Lakshminarayanan, Zhi Sun, Janet Moradianoldak
    Abstract:

    The present study describes a method using immunohistochemical labeling in combination with high-resolution imaging (field emission scanning electron microscopy) to investigate the spatial localization of Amelogenins on apatite crystallites in developing porcine enamel. Cross-sections of developing enamel tissue from freeze-fractured pig third molar were treated with antiserum against recombinant mouse Amelogenin and immunoreactivity confirmed by Western blot analysis. The samples were then treated with the goat anti-rabbit IgG conjugated with 10-nm gold particles. The control samples were treated with the secondary antibody only. The in-lens secondary electrons detector and quadrant back-scattering detector were employed to reveal the high-resolution morphology of enamel structures and gold particle distribution. The immunolabeling showed a preference of the gold particle localization along the side faces of the ribbon-like apatite crystals. The preferential localization of Amelogenin in vivo on enamel crystals strongly supports its direct function in controlling crystal morphology.

  • the role of secondary structure in the entropically driven Amelogenin self assembly
    Biophysical Journal, 2007
    Co-Authors: Rajamani Lakshminarayanan, Daming Fan, Janet Moradianoldak
    Abstract:

    Amelogenin, the major extracellular enamel matrix protein, plays critical roles in controlling enamel mineralization. This generally hydrophobic protein self-assembles to form nanosphere structures under certain solution conditions. To gain clearer insight into the mechanisms of Amelogenin self-assembly, we first investigated the occurrences of secondary structures within its sequence. By applying isothermal titration calorimetry (ITC), we determined the thermodynamic parameters associated with protein-protein interactions and with conformational changes during self-assembly. The recombinant porcine full length (rP172) and a truncated Amelogenin lacking the hydrophilic C-terminal (rP148) were used. Circular dichroism (CD) measurements performed at low concentrations (,5 mM) revealed the presence of the polyproline-type II (PPII) conformation in both Amelogenins in addition to a-helix and unordered conformations. Structural transition from PPII/unordered to b-sheet was observed for both proteins at higher concentrations (.62.5 mM) and upon self-assembly. ITC measurements indicated that the self-assembly of rP172 and rP148 is entropically driven (1DSA) and energetically favorable (� DGA). The magnitude of enthalpy (DHA) and entropy changes of assembly (DSA) were smaller for rP148 than rP172, whereas the Gibbs free energy change of assembly (DGA) was not significantly different. It was found that rP172 had higher PPII content than rP148, and the monomer-multimer equilibrium for rP172 was observed in a narrower protein concentration range when compared to rP148. The large positive enthalpy and entropy changes in both cases are attributed to the release of ordered water molecules and the associated entropy gain (due to the hydrophobic effect). These findings suggest that PPII conformation plays an important role in Amelogenin self-assembly and that rP172 assembly is more favorable than rP148. The data are direct evidence for the notion that hydrophobic interactions are the main driving force for Amelogenin self-assembly.

James P. Simmer - One of the best experts on this subject based on the ideXlab platform.

  • protein phosphorylation and mineral binding affect the secondary structure of the leucine rich Amelogenin peptide
    Frontiers in Physiology, 2017
    Co-Authors: Yasuo Yamakoshi, James P. Simmer, Henry C Margolis, Elia Beniash, Hajime Yamazaki
    Abstract:

    Previously, we have shown that serine-16 phosphorylation in native full-length porcine Amelogenin (P173) and the Leucine-Rich Amelogenin Peptide (LRAP(+P)), an alternative Amelogenin splice product, affects protein assembly and mineralization in vitro. Notably, P173 and LRAP(+P) stabilize amorphous calcium phosphate (ACP) and inhibit hydroxyapatite (HA) formation, while non-phosphorylated counterparts (rP172, LRAP(-P)) guide the growth of ordered bundles of HA crystals. Based on these findings, we hypothesize that the phosphorylation of full-length Amelogenin and LRAP induces conformational changes that critically affect its capacity to interact with forming calcium phosphate mineral phases. To test this hypothesis, we have utilized Fourier transform infrared spectroscopy (FTIR) to determine the secondary structure of LRAP(-P) and LRAP(+P) in the absence/presence of calcium and selected mineral phases relevant to amelogenesis; i.e., hydroxyapatite (HA: an enamel crystal prototype) and amorphous calcium phosphate (ACP: an enamel crystal precursor phase). Aqueous solutions of LRAP(-P) or LRAP(+P) were prepared with or without 7.5 mM of CaCl2 at pH 7.4. FTIR spectra of each solution were obtained using attenuated total reflectance, and amide-I peaks were analyzed to provide secondary structure information. Secondary structures of LRAP(+P) and LRAP(-P) were similarly assessed following incubation with suspensions of HA and pyrophosphate-stabilized ACP. Amide I spectra of LRAP(-P) and LRAP(+P) were found to be distinct from each other in all cases. Spectra analyses showed that LRAP(-P) is comprised mostly of random coil and β-sheet, while LRAP(+P) exhibits more β-sheet and α-helix with little random coil. With added Ca, the random coil content increased in LRAP(-P), while LRAP(+P) exhibited a decrease in α-helix components. Incubation of LRAP(-P) with HA or ACP resulted in comparable increases in β-sheet structure. Notably, however, LRAP(+P) secondary structure was more affected by ACP, primarily showing an increase in β-sheet structure, compared to that observed with added HA. These collective findings indicate that phosphorylation induces unique secondary structural changes that may enhance the functional capacity of native phosphorylated Amelogenins like LRAP to stabilize an ACP precursor phase during early stages of enamel mineral formation.

  • cryotem study of effects of phosphorylation on the hierarchical assembly of porcine Amelogenin and its regulation of mineralization in vitro
    Journal of Structural Biology, 2013
    Co-Authors: Ping An Fang, James P. Simmer, Henry C Margolis, James F Conway, Elia Beniash
    Abstract:

    Amelogenin, the major extracellular enamel matrix protein, plays a critical role in regulating the growth and organization of enamel. Assembly and mineralization of full-length native (P173) and recombinant (rP172) porcine Amelogenins were studied by cryogenic Transmission Electron Microscopy (cryoTEM). The cryoTEM revealed that both native and recombinant porcine Amelogenins undergo step-wise self-assembly. Although the overall structural organization of P173 and rP172 oligomers was similar and resembled oligomers of murine recombinant Amelogenin rM179, there were subtle differences suggesting that a single phosphorylated serine present in P173 might affect Amelogenin self-assembly. Our mineralization studies demonstrated that both P173 and rP172 oligomers stabilize initial mineral clusters. Importantly, however, rP172 regulated the organization of initial mineral clusters into linear chains and guided the formation of parallel arrays of elongated mineral particles, which are the hallmark of enamel structural organization. These results are similar to those obtained previously using full-length recombinant murine Amelogenin (Fang et al., 2011a). In contrast to that seen with rP172, phosphorylated P173 strongly inhibits mineralization for extended periods of time. We propose that these differences might be due to the differences in the structural organization and charge distribution between P173 and rP172. Overall our studies indicate that self-assembly of Amelogenin and the mechanisms of its control over mineralization might be universal across different mammalian species. Our data also provide new insight into the effect of phosphorylation on Amelogenin self-assembly and its regulation of mineralization.

  • potential role of the Amelogenin n terminus in the regulation of calcium phosphate formation in vitro
    Cells Tissues Organs, 2011
    Co-Authors: Le E Norcy, Yasuo Yamakoshi, James P. Simmer, Felicitas B Wiedemannbidlack, Elia Beniash, S Y Kwak, Henry C Margolis
    Abstract:

    N-terminal and C-terminal (CT) domains of Amelogenin have been shown to be essential for proper enamel formation. Recent studies have also suggested that although the C-terminus plays an apparent role in protein-mineral interactions, other Amelogenin structural domains are involved. The objective was to explore the role of the Amelogenin N-terminus in the regulation of calcium phosphate formation in vitro. Spontaneous mineralization studies were carried out using the phosphorylated (+P) and nonphosphorylated (–P) N-terminus of the leucine-rich Amelogenin peptide (LRAP) that lacks the hydrophilic CT domain. Mineralization progress was monitored via changes in solution pH. Mineral phases formed were characterized using TEM, selected area electron diffraction, and FT-IR. In controls, amorphous calcium phosphate was initially formed and subsequently transformed to randomly oriented hydroxyapatite (HA) plate-like crystals. In contrast to the control, LRAP(+P)-CT stabilized ACP formation for >1 day, while LRAP(–P)-CT accelerated the transformation of ACP to HA but had little effect on crystal shape or orientation. In conclusion, the N-terminal domain found in LRAP, as in Amelogenins, appears to have the capacity to interact with forming calcium phosphate mineral phases. Results suggest that the N-terminal domain of Amelogenin may play a direct role in early stages of enamel formation.

  • Amelogenin nanoparticles in suspension deviations from spherical shape and ph dependent aggregation
    Biomacromolecules, 2010
    Co-Authors: Barbara Aichmayer, Yasuo Yamakoshi, James P. Simmer, Felicitas B Wiedemannbidlack, Christoph Gilow, Franziska Emmerling, Henry C Margolis, Peter Fratzl
    Abstract:

    It is well-known that Amelogenin self-assembles to form nanoparticles, usually referred to as Amelogenin nanospheres, despite the fact that not much is known about their actual shape in solution. In the current paper, we combine SAXS and DLS to study the three-dimensional shape of the recombinant Amelogenins rP172 and rM179. Our results show for the first time that Amelogenins build oblate nanoparticles in suspension using experimental approaches that do not require the proteins to be in contact with a support material surface. The SAXS studies give evidence for the existence of isolated Amelogenin nano-oblates with aspect ratios in the range of 0.45−0.5 at pH values higher than pH 7.2 and show an aggregation of these nano-oblates at lower pH values. The role of the observed oblate shape in the formation of chain-like structures at physiological conditions is discussed as a key factor in the biomineralization of dental enamel.

  • mmp 20 and klk4 cleavage site preferences for Amelogenin sequences
    Journal of Dental Research, 2009
    Co-Authors: Yasuo Yamakoshi, John D. Bartlett, T Nagano, Ayako Kakegawa, Shuhei Tsuchiya, Kazuhiro Gomi, Takashi Arai, James P. Simmer
    Abstract:

    Mmp-20 and Klk4 are the two key enamel proteases. Can both enzymes process Amelogenin to generate the major cleavage products that accumulate during the secretory stage of amelogenesis? We isolated Mmp-20 and Klk4 from developing pig teeth and used them to digest the tyrosine-rich Amelogenin polypeptide (TRAP), the leucine-rich Amelogenin protein (LRAP), and 5 fluorescence peptides. We characterized the digestion products by LC-MSMS, SDS-PAGE, and C18 RP-HPLC monitored with fluorescence and UV detectors. Mmp-20 cleaves Amelogenin sequences after Pro162, Ser148, His62, Ala63, and Trp45. These cleavages generate all of the major cleavage products that accumulate in porcine secretory-stage enamel: the 23-kDa, 20-kDa, 13-kDa, 11-kDa, and 6-kDa (TRAP) Amelogenins. Mmp-20 cleaves LRAP after Pro45 and Pro40, producing the two LRAP products previously identified in tooth extracts. Among these key cleavage sites, Klk4 was able to cleave only after His62. We propose that Mmp-20 alone processes Amelogenin during the secretory stage.

A. G. Fincham - One of the best experts on this subject based on the ideXlab platform.

  • elongated growth of octacalcium phosphate crystals in recombinant Amelogenin gels under controlled ionic flow
    Journal of Dental Research, 2002
    Co-Authors: Mayumi Iijima, A. G. Fincham, H.b. Wen, Yutaka Moriwaki, Janet Moradianoldak
    Abstract:

    Amelogenin proteins constitute the primary structural entity of the extracellular protein framework of the developing enamel matrix. Recent data on the interactions of Amelogenin with calcium phosphate crystals support the hypothesis that Amelogenins control the oriented and elongated growth of enamel carbonate apatite crystals. To exploit further the molecular mechanisms involved in Amelogenin-calcium phosphate mineral interactions, we conducted in vitro experiments to examine the effect of Amelogenin on synthetic octacalcium phosphate (OCP) crystals. A 10% (wt/vol) recombinant murine Amelogenin (rM179, rM166) gel was constructed with nanospheres of about 10- to 20-nm diameter, as observed by atomic force microscopy. The growth of OCP was modulated uniquely in 10% rM179 and rM166 Amelogenin gels, regardless of the presence of the hydrophilic C-terminal residues. Fibrous crystals grew with large length-to-width ratio and small width-to-thickness ratio. Both rM179 and rM166 enhanced the growth of elongated OCP crystals, suggesting a relationship to the initial elongated growth of enamel crystals.

  • self assembly properties of recombinant engineered Amelogenin proteins analyzed by dynamic light scattering and atomic force microscopy
    Journal of Structural Biology, 2000
    Co-Authors: Janet Moradianoldak, A. G. Fincham, Michael L Paine, Yaping Lei, Malcolm L Snead
    Abstract:

    Abstract Dynamic light scattering (DLS) analysis together with atomic force microscopy (AFM) imaging was applied to investigate the supramolecular self-assembly properties of a series of recombinant Amelogenins. The overall objective was to ascertain the contribution of certain structural motifs in Amelogenin to protein–protein interactions during the self-assembly process. Mouse Amelogenins lacking either amino- or carboxy-terminal domains believed to be involved in self-assembly and Amelogenins having single or double amino acid mutations identical to those found in cases of amelogenesis imperfecta were analyzed. The polyhistidine-containingfull-length recombinant Amelogenin protein [rp(H)M180] generated nanospheres with monodisperse size distribution (hydrodynamic radius of 20.7 ± 2.9 nm estimated from DLS and 16.1 ± 3.4 nm estimated from AFM images), comparable to nanospheres formed by full-length Amelogenin rM179 without the polyhistidine domain, indicating that this histidine modification did not interfere with the self-assembly process. Deletion of the N-terminal self-assembly domain from Amelogenin and their substitution by a FLAG epitope (“A”-domain deletion) resulted in the formation of assemblies with a heterogeneous size distribution with the hydrodynamic radii of particles ranging from 3 to 38 nm. A time-dependent dynamic light scattering analysis of Amelogenin molecules lacking amino acids 157 through 173 and containing a hemagglutinin epitope (“B”-domain deletion) resulted in the formation of particles (21.5 ± 6.8 nm) that fused to form larger particles of 49.3 ± 4.3 nm within an hour. Single and double point mutations in the N-terminal region resulted in the formation of larger and more heterogeneous nanospheres. The above data suggest that while the N-terminal A-domain is involved in the molecular interactions for the formation of nanospheres, the carboxy-terminal B-domain contributes to the stability and homogeneity of the nanospheres, preventing their fusion to larger assemblies. These in vitro findings support the notion that the proteolytic cleavage of Amelogenin at amino- and carboxy-terminii occurring during enamel formation influences Amelogenin to Amelogenin interactions during self-assembly and hence alters the structural organization of the developing enamel extracellular matrix, thus affecting enamel biomineralization.

  • the structural biology of the developing dental enamel matrix
    Journal of Structural Biology, 1999
    Co-Authors: A. G. Fincham, Janet Moradianoldak, James P. Simmer
    Abstract:

    The biomineralization of the dental enamel matrix with a carbonated hydroxyapatite mineral generates one of the most remarkable examples of a vertebrate mineralized tissue. Recent advances in the molecular biology of ameloblast gene products have now revealed the primary structures of the principal proteins involved in this extracellular mineralizing system, Amelogenins, tuftelins, ameloblastins, enamelins, and proteinases, but details of their secondary, tertiary, and quaternary structures, their interactions with other matrix and or cell surface proteins, and their functional role in dental enamel matrix mineralization are still largely unknown. This paper reviews our current knowledge of these molecules, the probable molecular structure of the enamel matrix, and the functional role of these extracellular matrix proteins. Recent studies on the major structural role played by the Amelogenin proteins are discussed, and some new data on synthetic Amelogenin matrices are reviewed.

  • interaction of Amelogenin with hydroxyapatite crystals an adherence effect through Amelogenin molecular self association
    Biopolymers, 1998
    Co-Authors: Janet Moradianoldak, J Tan, A. G. Fincham
    Abstract:

    At the secretory stage of tooth enamel formation the majority of the organic matrix is composed of Amelogenin proteins that are believed to provide the scaffolding for the initial carbonated hydroxyapatite crystals to grow. The primary objective of this study was to investigate the interaction between Amelogenins and growing apatite crystals. Two in vitro strategies were used: first, we examined the influence of Amelogenins as compared to two other macromolecules, on the kinetics of seeded growth of apatite crystals; second, using transmission electron micrographs of the crystal powders, based on a particle size distribution study, we evaluated the effect of the macromolecules on the aggregation of growing apatite crystals. Two recombinant Amelogenins (rM179, rM166), the synthetic leucine-rich Amelogenin polypeptide (LRAP), poly(L-proline), and phosvitin were used. It was shown that the rM179 Amelogenin had some inhibitory effect on the kinetics of calcium hydroxyapatite seeded growth. The inhibitory effect, however, was not as destructive as that of other macromolecules tested. The degree of inhibition of the macromolecules was in the order of phosvitin > LRAP > poly(L-proline) > rM179 > rM166. Analysis of particle size distribution of apatite crystal aggregates indicated that the full-length Amelogenin protein (rM179) caused aggregation of the growing apatite crystals more effectively than other macromolecules. We propose that during the formation of hydroxyapatite crystal clusters, the growing apatite crystals adhere to each other through the molecular self-association of interacting Amelogenin molecules. The biological implications of this adherence effect with respect to enamel biomineralization are discussed.

  • comparative immunochemical analyses of the developmental expression and distribution of ameloblastin and Amelogenin in rat incisors
    Journal of Histochemistry and Cytochemistry, 1998
    Co-Authors: Antonio Nanci, James P. Simmer, S Zalzal, P Lavoie, M Kunikata, W Y Chen, P H Krebsbach, Yoshihiko Yamada, L Hammarstrom, A. G. Fincham
    Abstract:

    SUMMARY Mineralized tissues are unique in using proteins to attract and organize calcium and phosphate ions into a structured mineral phase. A precise knowledge of the expression and extracellular distribution of matrix proteins is therefore very important in understanding their function. The purpose of this investigation was to obtain comparative information on the expression, intracellular and extracellular distribution, and dynamics of proteins representative of the two main classes of enamel matrix proteins. Amelogenins were visualized using an antibody and an mRNA probe prepared against the major alternatively spliced isoform in rodents, and nonAmelogenins by antibodies and mRNA probes specific to one enamel protein referred to by three names: ameloblastin, amelin, and sheathlin. Qualitative and quantitative immunocytochemistry, in combination with immunoblotting and in situ hybridization, indicated a correlation between mRNA signal and sites of protein secretion for Amelogenin, but not for ameloblas...

Harold C. Slavkin - One of the best experts on this subject based on the ideXlab platform.

  • evidence for Amelogenin nanospheres as functional components of secretory stage enamel matrix
    Journal of Structural Biology, 1995
    Co-Authors: A. G. Fincham, Thomas G.h. Diekwisch, J. T. Wright, Pablo Bringas, Janet Moradianoldak, D M Lyaruu, Harold C. Slavkin
    Abstract:

    Abstract Amelogenins are the principal proteins of the extracellular matrix of developing dental enamel and are postulated to function in the processes of biomineralization of the developing tooth although the molecular mechanisms concerned are poorly understood. Recent imaging studies, employing dynamic light scattering, atomic force, and transmission electron microscopy (TEM) have shown that a recombinant Amelogenin ( M r -20 000 Da) spontaneously forms supramolecular quasi-spherical aggregates ("nanospheres") of 15-20 nm in diameter. By comparison with in vitro experiments employing the recombinant Amelogenin we show that the nanospheres appear as electron-lucent structures when treated with conventional electron microscopy contrast reagents (phosphotungstate or uranyl acetate) and we speculate that this property derives from the hydrophobic nature of the Amelogenin protein. Employing TEM preparations of developing enamel from mouse, bovine, and hamster we demonstrate that the Amelogenin nanospheres occur as beaded rows of electron-lucent structures aligned with, and separating, the enamel mineral crystallites. We postulate that the Amelogenin monmers self-assemble to form nanospheres which function to space the intial crystallites, control crystal habit, inhibit intercrystalline fusion, and through the apposition of their surfaces, create anionic channels which facilitate ion transport within the mineralizing matrix.

  • evidence for Amelogenin nanospheres as functional components of secretory stage enamel matrix
    Journal of Structural Biology, 1995
    Co-Authors: A. G. Fincham, Thomas G.h. Diekwisch, J. T. Wright, Pablo Bringas, Janet Moradianoldak, D M Lyaruu, Harold C. Slavkin
    Abstract:

    Amelogenins are the principal proteins of the extracellular matrix of developing dental enamel and are postulated to function in the processes of biomineralization of the developing tooth although the molecular mechanisms concerned are poorly understood. Recent imaging studies, employing dynamic light scattering, atomic force, and transmission electron microscopy (TEM) have shown that a recombinant Amelogenin (M(r) approximately 20,000 Da) spontaneously forms supramolecular quasi-spherical aggregates ("nanospheres") of 15-20 nm in diameter. By comparison with in vitro experiments employing the recombinant Amelogenin we show that the nanospheres appear as electron-lucent structures when treated with conventional electron microscopy contrast reagents (phosphotungstate or uranyl acetate) and we speculate that this property derives from the hydrophobic nature of the Amelogenin protein. Employing TEM preparations of developing enamel from mouse, bovine, and hamster we demonstrate that the Amelogenin nanospheres occur as beaded rows of electron-lucent structures aligned with, and separating, the enamel mineral crystallites. We postulate that the Amelogenin monomers self-assemble to form nanospheres which function to space the initial crystallites, control crystal habit, inhibit intercrystalline fusions, and, through the apposition of their surfaces, create anionic channels which facilitate ion transport within the mineralizing matrix.

  • detection of monodisperse aggregates of a recombinant Amelogenin by dynamic light scattering
    Biopolymers, 1994
    Co-Authors: Janet Moradianoldak, Eduardo C. Lau, James P. Simmer, Harold C. Slavkin, P E Sarte, A. G. Fincham
    Abstract:

    Recombinant murine Amelogenins M179 and M166 were expressed in Escherichia coli and purified. The aggregation properties of these Amelogenins have been investigated in aqueous solutions as well as acetonitrile-containing solutions using dynamic light scattering. Dynamic light scattering provides direct measurement of the translational diffusion coefficient and hydrodynamic radius, and of an estimate of the molecular weight. Polydispersity and statistical parameters of how to interpret the analysis are also provided. Amelogenin aggregation was examined in solutions of a range of pH, ionic strengths, and protein concentrations. It was shown that at pH 7.8–8 and ionic strength of 0.02–0.05M the M179 molecules form monodispersed aggregates with hydrodynamic radii ranging from 15 to 19 nm. Analysis of hydrodynamic radii and size distribution of M179 aggregates in acetonitrile-containing solvents compared to that in aqueous solutions indicated a primary role for hydrophobic interactions in the association process of Amelogenin molecules to form aggregates. Comparison between the aggregates formed by M179 and M166, which lacks the hydrophilic carboxy-terminal 13 residue sequence of M179, suggested that the self-assembly of Amelogenin molecules to form stable and monodisperse aggregates requires the presence of the hydrophilic carboxy-terminal sequence of M179. © 1994 John Wiley & Sons, Inc.

  • isolation and characterization of a mouse Amelogenin expressed in escherichia coli
    Calcified Tissue International, 1994
    Co-Authors: James P. Simmer, Malcolm L Snead, Eduardo C. Lau, Harold C. Slavkin, T Aoba, M Lacey, D Nelson, Margarita Zeichnerdavid, A. G. Fincham
    Abstract:

    A mouse cDNA encoding a 180 amino acid Amelogenin was subcloned into the pET expression plasmid (Novagen, Madison, WI) for production in Escherichia coli. A simple growth and purification protocol yields 20–50 mg of 95–99% pure recombinant Amelogenin from a 4.5-liter culture. This is the first heterologous expression of an enamel protein. The expressed protein was characterized by partial Edman sequencing, amino acid composition analysis, SDS-PAGE, Western blotting, laser desorption mass spectrometry, and hydroxyapatite binding. The recombinant Amelogenin is 179 amino acids in length, has a molecular weight of 20,162 daltons, and hydroxyapatite binding properties similar to the porcine 173 residue Amelogenin. Solubility analyses showed that the bacterially expressed protein is only sparingly soluble in the pH range of 6.4–8.0 or in solutions 20% saturated with ammonium sulfate. The purified protein was used to generate rabbit polyclonal anti-Amelogenin antibodies which show specific reaction to Amelogenins in both Western blot analyses of enamel extracts and in immunostaining of developing mouse molars.

  • self assembly of a recombinant Amelogenin protein generates supramolecular structures
    Journal of Structural Biology, 1994
    Co-Authors: A. G. Fincham, Thomas G.h. Diekwisch, Eduardo C. Lau, James P. Simmer, Janet Moradianoldak, P E Sarte, Harold C. Slavkin
    Abstract:

    Amelogenin proteins are the principal constituents of the extracellular organic matrix associated with the nucleation and growth of the carbonated calcium hydroxyapatite (HAP)-containing mineral phase of dental enamel. Amelogenins are believed to function in controlling the sizes and organization of the developing enamel crystals. Previous studies have shown that enamel proteins exhibit unusual reversible aggregation properties. The present studies were designed to test the hypothesis that self-assembly of recombinant Amelogenin generates supramolecular structures that are indistinguishable from the electron-dense particles associated with HAP crystal growth in vivo. A recombinant Amelogenin analog of the murine 180-residue protein was analyzed by high-resolution size exclusion chromatography, atomic force (AFM), and transmission electron (TEM) microscopy. It was found that the Amelogenin formed supramolecular aggregates which were in a concentration-dependent equilibrium with protein monomers. Imaging of the Amelogenin by both AFM and TEM techniques revealed spherical aggregate structures of about 18 nm diameter which were seen to be similar to electron-dense enamel structures observed in vivo. We interpret these results to suggest that, in vivo, the Amelogenin protein self-assembles through functional motifs of the protein primary structure, generating specific supramolecular aggregates which we hypothesize function to control the ultrastructural organization of the developing enamel crystallites.

Malcolm L Snead - One of the best experts on this subject based on the ideXlab platform.

  • full length Amelogenin binds to cell surface lamp 1 on tooth root periodontium associated cells
    Archives of Oral Biology, 2010
    Co-Authors: Hai Zhang, Carolyn W Gibson, Malcolm L Snead, Kevin A Tompkins, Jacques Garrigues, Martha J Somerman
    Abstract:

    Objectives Lysosome-associated membrane protein-1 (LAMP-1) has been suggested to be a cell surface receptor for a specific Amelogenin isoform, leucine-rich Amelogenin peptide or LRAP. However, it is unclear if LAMP-1 is an Amelogenin receptor for dental mesenchymal cells. The goal of this study was to determine if LAMP-1 serves as a cell surface binding site for full length Amelogenin on tooth root/periodontium associated mesenchymal cells.

  • Cellular uptake of Amelogenin, and its localization to CD63, and Lamp1-positive vesicles
    Cellular and molecular life sciences : CMLS, 2006
    Co-Authors: Jason L. Shapiro, Malcolm L Snead, Michel Goldberg, Xin Wen, Curtis T. Okamoto, Hongjun Wang, Staale Petter Lyngstadaas, Michael L Paine
    Abstract:

    Proteins of the developing enamel matrix include Amelogenin, ameloblastin and enamelin. Of these three proteins Amelogenin predominates. Protein-protein interactions are likely to occur at the ameloblast Tomes’ processes between membrane-bound proteins and secreted enamel matrix proteins. Such protein-protein interactions could be associated with cell signaling or endocytosis. CD63 and Lamp1 are ubiquitously expressed, are lysosomal integral membrane proteins, and localize to the plasma membrane. CD63 and Lamp1 interact with Amelogenin in vitro. In this study our objective was to study the molecular events of intercellular trafficking of an exogenous source of Amelogenin, and related this movement to the spatiotemporal expression of CD63 and Lamp1 using various cell lineages. Exogenously added Amelogenin moves rapidly into the cell into established Lamp1-positive vesicles that subsequently localize to the perinuclear region. These data indicate a possible mechanism by which Amelogenin, or degraded Amelogenin peptides, are removed from the extracellular matrix during enamel formation and maturation.

  • Amelogenins regulate expression of genes associated with cementoblasts in vitro.
    European journal of oral sciences, 2006
    Co-Authors: Erica C Swanson, Carolyn W Gibson, Malcolm L Snead, Michael L Paine, Brian L Foster, Hanson K Fong, Martha J Somerman
    Abstract:

    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.

  • Amelogenin a potential regulator of cementum associated genes
    Journal of Periodontology, 2003
    Co-Authors: Hema Viswanathan, Malcolm L Snead, Janice E Berry, Brian L Foster, Martha J Somerman
    Abstract:

    Background: Studies suggest that enamel matrix proteins induce differentiation and mineralization of a variety of mesenchymal cells, including odontoblasts, osteoblasts, and cementoblasts. It has been postulated that this activity could be due to Amelogenin-like proteins, known to be present in some mixtures of enamel matrix derivatives. Amelogenins have been reported to induce expression of a mineralized tissue-specific marker, bone sialoprotein (BSP), indicating that epithelial products can regulate the activity of mesenchyme-derived cells. Methods: To explore the molecular mechanisms involved in BSP regulation, a clonal population of immortalized murine cementoblasts (OCCM-30) was exposed to full-length murine Amelogenin protein (rp(H)M180), 0.1 µg/ml to 10.0 µg/ml, for 8 days in vitro. To further investigate the potential epithelial-mesenchymal interaction, an Amelogenin knockout mouse model was used to examine expression of BSP and other markers, including Type I collagen, in tissue samples. Results: The lowest dose of Amelogenin slightly enhanced BSP expression, whereas at the highest dose, a dramatic decrease (three-fold) in BSP expression was observed. Parallel experiments showed a corresponding decrease in mineral nodule formation in vitro for cells treated with the higher dose of rp(H)M180. In situ hybridization and immunohistochemical analysis of sections from Amelogenin null mice revealed a dramatic reduction in expression of BSP mRNA and protein in cementoblasts and surrounding osteoblasts in comparison to age-matched controls. In contrast, the expression of Type I collagen was not significantly different from controls. Conclusion: These data suggest that Amelogenin may be a critical signaling molecule required for appropriate development of the periodontium. J Periodontol 2003;74:1423-1431.

  • self assembly properties of recombinant engineered Amelogenin proteins analyzed by dynamic light scattering and atomic force microscopy
    Journal of Structural Biology, 2000
    Co-Authors: Janet Moradianoldak, A. G. Fincham, Michael L Paine, Yaping Lei, Malcolm L Snead
    Abstract:

    Abstract Dynamic light scattering (DLS) analysis together with atomic force microscopy (AFM) imaging was applied to investigate the supramolecular self-assembly properties of a series of recombinant Amelogenins. The overall objective was to ascertain the contribution of certain structural motifs in Amelogenin to protein–protein interactions during the self-assembly process. Mouse Amelogenins lacking either amino- or carboxy-terminal domains believed to be involved in self-assembly and Amelogenins having single or double amino acid mutations identical to those found in cases of amelogenesis imperfecta were analyzed. The polyhistidine-containingfull-length recombinant Amelogenin protein [rp(H)M180] generated nanospheres with monodisperse size distribution (hydrodynamic radius of 20.7 ± 2.9 nm estimated from DLS and 16.1 ± 3.4 nm estimated from AFM images), comparable to nanospheres formed by full-length Amelogenin rM179 without the polyhistidine domain, indicating that this histidine modification did not interfere with the self-assembly process. Deletion of the N-terminal self-assembly domain from Amelogenin and their substitution by a FLAG epitope (“A”-domain deletion) resulted in the formation of assemblies with a heterogeneous size distribution with the hydrodynamic radii of particles ranging from 3 to 38 nm. A time-dependent dynamic light scattering analysis of Amelogenin molecules lacking amino acids 157 through 173 and containing a hemagglutinin epitope (“B”-domain deletion) resulted in the formation of particles (21.5 ± 6.8 nm) that fused to form larger particles of 49.3 ± 4.3 nm within an hour. Single and double point mutations in the N-terminal region resulted in the formation of larger and more heterogeneous nanospheres. The above data suggest that while the N-terminal A-domain is involved in the molecular interactions for the formation of nanospheres, the carboxy-terminal B-domain contributes to the stability and homogeneity of the nanospheres, preventing their fusion to larger assemblies. These in vitro findings support the notion that the proteolytic cleavage of Amelogenin at amino- and carboxy-terminii occurring during enamel formation influences Amelogenin to Amelogenin interactions during self-assembly and hence alters the structural organization of the developing enamel extracellular matrix, thus affecting enamel biomineralization.