Polyelectrolyte-mediated adsorption of amelogenin monomers and nanospheres forming mono- or multilayers

We have applied optical waveguide lightmode spectroscopy combined with streaming potential measurements and Fourier-transformed infrared spectroscopy to investigate adsorption of amelogenin nanospheres onto polyelectrolytes. The long-term objective was to better understand the chemical nature of these assemblies and to gain further insight into the molecular mechanisms involved during self-assembly. It was found that monolayers of monomers and negatively charged nanospheres of a recombinant amelogenin (rM179) irreversibly adsorbed onto a positively charged polyelectrolyte multilayer films. On the basis of measurements performed at different temperatures, it was demonstrated that intermolecular interactions for the formation of nanospheres were not affected by their adsorption onto polyelectrolytes. Consecutive adsorption of nanospheres resulting in the formation of multilayer structures was possible by using cationic poly(l-lysine) as mediators. N-Acetyl-d-glucosamine (GlcNac) did not disturb the nanosphere-assembled protein's structure, and it only affected the adsorption of monomeric amelogenin. Infrared spectroscopy of adsorbed amelogenin revealed conformational differences between the monomeric and assembled forms of rM179. While there was a considerable amount of alpha-helices in the monomers, beta-turn and beta-sheet structures dominated the assembled proteins. Our work constitutes the first report on a structurally controlled in vitro buildup of an rM179 nanosphere monolayer-based matrix. Our data support the notion that amelogenin self-assembly is mostly driven by hydrophobic interactions and that amelogenin/PEM interactions are dominated by electrostatic forces. We suggest that similar forces can govern amelogenin interactions with non-amelogenins or the mineral phase during enamel biomineralization.     

Structure characterization of the central repetitive domain of high molecular weight gluten proteins. I. Model studies using cyclic and linear peptides.

The high molecular weight (HMW) proteins from wheat contain a repetitive domain that forms 60-80% of their sequence. The consensus peptides PGQGQQ and GYYPTSPQQ form more than 90% of the domain; both are predicted to adopt beta-turn structure. This paper describes the structural characterization of these consensus peptides and forms the basis for the structural characterization of the repetitive HMW domain, described in the companion paper. The cyclic peptides cyclo-[PGQGQQPGQGQQ] (peptide 1), cyclo-[GYYPTSPQQGA] (peptide 2), and cyclo-[PGQGQQGYYPTSPQQ] (peptide 3) were prepared using a novel synthesis route. In addition, the linear peptides (PGQGQQ)n (n = 1, 3, 5) were prepared. CD, FTIR, and NMR data demonstrated a type II beta-turn structure at QPGQ in the cyclic peptide 1 that was also observed in the linear peptides 9PGQGQQ)n. A type I beta-turn was observed at YPTS and SPQQ in peptides 2 and 3, with additional beta-turns of either type I or II at GAGY (peptide 2) and QQGY (peptide 3). The proline in YPTS showed considerable cis/trans isomerization, with up to 50% of the population in the cis-conformation; the other prolines were more than 90% in the trans conformation. The conversion from trans to cis destroys the type I beta-turn at YPTS, but leads to an increase in turn character at SPQQ and GAGY (peptide 2) or QQGY (peptide 3).     

Identification and characterization of a squamate reptilian amelogenin gene: Iguana iguana

As the principal components of the developing tooth enamel matrix, amelogenins play a significant role in tooth enamel formation and organization. In order to elucidate the structure and function of amelogenins in the evolution of enamel, we have selected the Iguana iguana as a squamate model organism. Here we report the first complete squamate amelogenin sequence available as of yet and document unique features of Iguana amelogenins and enamel. Transmission electron microscopy documented randomly oriented Iguana enamel crystals during the elongation phase compared with organized enamel crystal patterns at comparable stages in mammals. Sequencing of PCR amplified products revealed a full-length I. iguana amelogenin cDNA containing 877 nucleotides with a 564 nucleotide coding sequence encoding 187 amino acids. The homologies of the newly discovered I. iguana amelogenin amino acid sequence with the published mouse, caiman (Palaeosuchus), and snake (Elaphe) amelogenin were 41.3%, 53.5%, and 55.5%, respectively. On Western blots one major protein with a molecular weight of 24 kDa, and two minor proteins with molecular weights of 28 and 13.5 kDa, respectively, were detected based on the cross-reactivity of antisera against recombinant Rana pipiens amelogenin proteins. Sequence analysis revealed a moderate sequence homology between mammalian and reptilian amelogenin genes. A significant alteration was the deletion of the hydrophilic GSP sequence from exon 3 in the mouse sequence resulting in a conversion to a hydrophobic region in Iguana. Together, these findings identified a novel amelogenin cDNA sequence in the squamate reptilian I. iguana and functional implications for the evolution of amelogenins and enamel in squamates.     

Reuptake of extracellular amelogenin by dental epithelial cells results in increased levels of amelogenin mRNA through enhanced mRNA stabilization

Amelogenin is an extracellular matrix protein secreted by ameloblasts and is a major component of enamel matrix. Recently, in addition to their role in enamel formation, the biological activity of enamel proteins in the process of cell differentiation has recently become widely appreciated. In this study, we examined the biological activity of amelogenin on ameloblast differentiation. Recombinant mouse amelogenin (rm-amelogenin) enhanced the expression of endogenous amelogenin mRNA in a cultured dental epithelial cell line (HAT-7), despite a lack of increased amelogenin promoter activity. To solve this discrepancy, we analyzed the effects of rm-amelogenin on the stability of amelogenin mRNA. The half-life of amelogenin mRNA is extremely short, but in the presence of rm-amelogenin its half-life was extended three times longer than the control. Furthermore, we showed the entry of exogenous fluorescein isothiocyanate-conjugated rm-amelogenin into the cytoplasm of HAT-7 cells. It follows from our results that exogenous amelogenin increases amelogenin mRNA levels through stabilization of mRNA in the cytoplasm of HAT-7 cells. Here we speculated that during differentiation, dental epithelial cells utilize a unique mechanism for increasing the production of amelogenin, the reuptake of secreted amelogenin.     

A 27-mer tandem repeat polypeptide in bovine amelogenin: synthesis and CD spectra.

CD spectra of a tandem 27-mer repeat polypeptide, Gln-Pro-His-Gln-Pro-Leu-Gln-Pro-His-Gln-Pro-Leu-Gln-Pro-Met-(Gln-Pro-Leu)4, from bovine amelogenin synthesized by standard solid-phase synthesis manifests an archtypical CD pattern of a beta-spiral structure in phosphate buffer at pH 5.2 and trifluoroethanol (TFE), CF3OH. beta-spiral structure is unique to a class of diverse proteins including amelogenins conferring unusual physicochemical properties.     

Structure characterization of the central repetitive domain of high molecular weight gluten proteins. II. Characterization in solution and in the dry state.

The structure of the central repetitive domain of high molecular weight HMW) wheat gluten proteins was characterized in solution and in the dry state using HMW proteins Bx6 and Bx7 and a subcloned, bacterially expressed part of the repetitive domain of HMW Dx5. Model studies of the HMW consensus peptides PGQGQQ and GYYPTSPQQ formed the basis for the data analysis (van Dijk AA et al., 1997, Protein Sci 6:637-648). In solution, the repetitive domain contained a continuous nonoverlapping series of both type I and type II II beta-turns at positions predicted from the model studies; type II beta-turns occurred at QPGQ and QQGY sequences and type I beta-turns at YPTS and SPQQ. The subcloned part of the HMW Dx5 repetitive domain sometimes migrated as two bands on SDS-PAGE; we present evidence that this may be caused by a single amino acid insertion that disturbs the regular structure of beta-turns. The type I beta-turns are lost when the protein is dried on a solid surface, probably by conversion to type II beta-turns. The homogeneous type II beta-turn distribution is compatible with the formation of a beta-spiral structure, which provides the protein with elastic properties. The beta-turns and thus the beta-spiral are stabilized by hydrogen bonds within and between turns. Reformation of this hydrogen bonding network after, e.g., mechanical disruption may be important for the elastic properties of gluten proteins.     

Towards assignment of secondary structures by anti-peptide antibodies. Specificity of the immune response to a beta-turn.

In an attempt to assign secondary structure elements to protein primary structures with antibodies, we synthesized a model peptide (beta-peptide: TVTVTDPGQTVTY) with a putative beta-turn structure and analysed the anti-peptide antibodies for their specificity towards the turn sequence. At least 50% of the peptide fraction adopts the intended conformation of a beta-turn (DPGQ) inserted between the two segments of an antiparallel beta-sheet structure. The specific anti-beta-peptide antibodies of the hyperimmune response bind the beta-turn containing epitope of the immunogenic beta-peptide with a three orders of magnitude higher affinity than the synthetic control peptide (Gly-peptide: GGGGGDPGQGGGG). The affinity of the antibodies with specificity for the beta-turn region increases from the primary to the hyperimmune response. Therefore, probing of secondary structure elements, i.e., of individual beta-turn regions, by anti-peptide antibodies now seems feasible for proteins of known sequence and may result in sequence assignments of secondary structures.     

Evidence for regulation of amelogenin gene expression by 1,25-dihydroxyvitamin D(3) in vivo

The unique hereditary enamel defect clearly related to the disturbance of one enamel matrix protein is X-linked amelogenesis imperfecta (AI), in which several mutations of amelogenin gene have been identified. The clinical phenotype of many of these subjects shows similarities with enamel defects related to rickets. Therefore, we hypothesized that rachitic dental dysplasia is related to disturbances in the amelogenin pathway. In order to test this hypothesis, combined qualitative and quantitative studies in experimental vitamin D-deficient (-D) rat model systems were performed. First, Western blot analysis of microdissected enamel matrix (secretion and maturation stages) showed no clear evidence of dysregulation of amelogenin protein processing in -D rats as compared with the controls. Second, the ultrastructural investigation permitted identification of the internal tissular defect of rachitic enamel, the irregular absence of intraprismatic enamel observed in -D animals, suggesting a possible link between prism morphogenesis and vitamin D. In addition, the steady-state levels of amelogenin mRNAs measured in microdissected dental cells was decreased in -D rats and up-regulated by an unique injection of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). The present study shows evidences that amelogenin expression is regulated by vitamin D. This is the first study of an hormonal regulation of tooth-specific genes.     

The epitopes of apolipoprotein A-I define distinct structural domains including a mobile middle region.

The definition of epitopes on apoA-I provides evidence for a very dense packing of the peptide chain and supports the proposed supersecondary structure, made of repetitive antiparallel helices. Screening with overlapping synthetic hexapeptides shows that only a few epitopes are continuous and that all continuous contact sequences identified within the epitopes coincide or are contiguous to a putative beta-turn, such as residues 1-8, 48-55, 98-104, 118-123, and 135-140. On the N-terminal half of apoA-I we identified 6 overlapping tertiary discontinuous epitopes, uniquely constituted by amino acids and discontinuous sequences on helical segments that are far apart and which define a particular region with a complex tertiary structure. Among these are the epitopes for antibody 5G6, which reacts with residues 45-51, 83-92, 119-126, and 136-143; for A16, which reacts with 14-19, 23-28, and 60-82; and for r-FC1, which reacts with 1-8, 29-35, 78-83, and 98-121. The very far apart discontinuous sequences included in these epitopes can be explained by the predicted turns and coiled domains, and thus provide evidence for such a tertiary structure. Alternatively, these results could also be explained by intermolecular epitopes involving the N-terminal region. In contrast, in the middle of apoA-I, all identified epitopes are shorter and discontinuous within the secondary structure and are constituted by residues forming a beta-turn with all or part of an adjacent alpha-helix. We hypothesize that these multiple epitopes, that are mostly limited to a single helix, reflect the existence of a very mobile domain, possibly with hinged pairs of adjacent alpha-helices. On the C-terminal half of apoA-I, several monoclonal antibodies react with overlapping epitopes located between residues 149 and 186, which probably reflects 2 antiparallel alpha-helices interrupted by a beta-turn.     

In vitro preparation of amelogenin nanoparticles carrying nucleic acids

Amelogenin, a matrix protein involved in biomineralization of enamel, can self-assemble to form nanospheres in a pH-dependent manner. Nucleic acids (single-stranded, double-stranded, and plasmid DNA, as well as RNA) could be co-precipitated with amelogenin, demonstrating a strong binding of nucleic acids to amelogenin. The amounts of co-precipitated nucleic acids were analyzed and binding levels upto 90 μg DNA/mg amelogenin was achieved. The co-precipitation could also be carried out in a bacterial cell homogenate, and no bacterial proteins were found in the amelogenin aggregates, suggesting specificity for nucleic acid binding. Dynamic light scattering showed that amelogenin nanosphere structure is maintained upon DNA binding with an upto 2.6 nm increase in diameter. The reported binding of nucleic acids to amelogenin can be explored practically for nucleic acid separation.     

Evidence for regulation of amelogenin gene expression by 1,25‐dihydroxyvitamin D3 in vivo

The unique hereditary enamel defect clearly related to the disturbance of one enamel matrix protein is X‐linked amelogenesis imperfecta (AI), in which several mutations of amelogenin gene have been identified. The clinical phenotype of many of these subjects shows similarities with enamel defects related to rickets. Therefore, we hypothesized that rachitic dental dysplasia is related to disturbances in the amelogenin pathway. In order to test this hypothesis, combined qualitative and quantitative studies in experimental vitamin D‐deficient (−D) rat model systems were performed. First, Western blot analysis of microdissected enamel matrix (secretion and maturation stages) showed no clear evidence of dysregulation of amelogenin protein processing in −D rats as compared with the controls. Second, the ultrastructural investigation permitted identification of the internal tissular defect of rachitic enamel, the irregular absence of intraprismatic enamel observed in −D animals, suggesting a possible link between prism morphogenesis and vitamin D. In addition, the steady‐state levels of amelogenin mRNAs measured in microdissected dental cells was decreased in −D rats and up‐regulated by an unique injection of 1,25‐dihydroxyvitamin D₃ (1,25(OH)₂D₃). The present study shows evidences that amelogenin expression is regulated by vitamin D. This is the first study of an hormonal regulation of tooth‐specific genes. J. Cell. Biochem. 76:194–205, 1999. © 1999 Wiley‐Liss, Inc.     

Amelogenin sequence and enamel biomineralization in Rana pipiens

The amelogenin gene contributes the majority of tooth enamel proteins and plays a significant role in enamel biomineralization. While several mammalian and reptilian amelogenins have been cloned and sequenced, basal vertebrate amelogenin evolution remains to be understood. In order to start elucidating the structure and function of amelogenins in the evolution of enamel, the leopard frog (Rana pipiens) was used as a model. Tissues from Rana pipiens teeth were analyzed for enamel structure and RNA extracts were processed for sequence analysis. Electron microscopy revealed that Rana pipiens enamel contains long and parallel crystals similar to mammalian enamel, while immunoreactions confirmed the site-specific localization of cross-reactive amelogenins in Rana pipiens enamel. Sequencing of amelogenin PCR products revealed a 782bp cDNA with a 546-nucleotide coding sequence encoding 181 amino acids. The homology of the newly discovered Rana pipiens amelogenin nucleotide and amino acid sequence with the published mouse amelogenin was 38.6% and 45%, respectively. These findings report the first complete amelogenin cDNA sequence in amphibians and indicate a close homology between mammalian enamel formation and Rana pipiens enamel biomineralization.     

Expression and characterisation of a highly repetitive peptide derived from a wheat seed storage protein

The high molecular weight (HMW) subunit group of wheat seed storage proteins impart elasticity to wheat doughs and glutens. They consist of three domains: non-repetitive N- and C-terminal domains, which contain cysteine residues for covalent cross-linking, and a central domain consisting of repeated sequences. The circular dichroism and infrared (IR) spectra of an intact HMW subunit were compared with those of a peptide corresponding to the central repetitive domain expressed in Escherichia coli. This allowed the structure of the central domain to be studied in the absence of the N- and C-terminal domains and the contributions of these domains to the structure of the whole protein to be determined. In solution the peptide showed the presence of beta-turns and polyproline II-like structure. Variable temperature studies indicated an equilibrium between these two structures, the polyproline II conformation predominating at low temperatures and the beta-turn conformation at higher temperatures. IR in the hydrated solid state also indicated the presence of beta-turns and intermolecular beta-sheet structures. In contrast, spectroscopy of the whole subunit showed the presence of alpha-helix in the N- and C-terminal domains. The content of beta-sheet was also higher in the whole subunit, indicating that the N- and C-terminal domains may promote the formation of intermolecular beta-sheet structures between the repetitive sequences, perhaps by aligning the molecules to promote interaction.     

Conservation and variation in enamel protein distribution during vertebrate tooth development

Vertebrate enamel formation is a unique synthesis of the function of highly specialized enamel proteins and their effect on the growth and organization of apatite crystals. Among tetrapods, the physical structure of enamel is highly conserved, while there is a greater variety of enameloid tooth coverings in fish. In the present study, we postulated that in enamel microstructures of similar organization, the principle components of the enamel protein matrix would have to be highly conserved. In order to identify the enamel proteins that might be most highly conserved and thus potentially most essential to the process of mammalian enamel formation, we used immunoscreening with enamel protein antibodies as a means to assay for degrees of homology to mammalian enamel proteins. Enamel preparations from mouse, gecko, frog, lungfish, and shark were screened with mammalian enamel protein antibodies, including amelogenin, enamelin, tuftelin, MMP20, and EMSP1. Our results demonstrated that amelogenin was the most highly conserved enamel protein associated with the enamel organ, enamelin featured a distinct presence in shark enameloid but was also present in the enamel organ of other species, while the other enamel proteins, tuftelin, MMP20, and EMSP1, were detected in both in the enamel organ and in other tissues of all species investigated. We thus conclude that the investigated enamel proteins, amelogenin, enamelin, tuftelin, MMP20, and EMSP1, were highly conserved in a variety of vertebrate species. We speculate that there might be a unique correlation between amelogenin-rich tetrapod and lungfish enamel with long and parallel crystals and enamelin-rich basal vertebrate enameloid with diverse patterns of crystal organization.     

Increasing protein conformational stability by optimizing beta-turn sequence

Protein conformational stability is an important concern in many fields. Here, we describe a strategy for significantly increasing conformational stability by optimizing beta-turn sequence. Proline and glycine residues are statistically preferred at several beta-turn positions, presumably because their unique side-chains contribute favorably to conformational stability in certain beta-turn positions. However, beta-turn sequences often deviate from preferred proline or preferred glycine. Therefore, our strategy involves replacing non-proline and non-glycine beta-turn residues with preferred proline or preferred glycine residues. Here, we develop guidelines for selecting appropriate mutations, and present results for five mutations (S31P, S42G, S48P, T76P, and Q77G) that significantly increase the conformational stability of RNase Sa. The increases in stability ranged from 0.7 kcal/mol to 1.3 kcal/mol. The strategy was successful in overlapping or isolated beta-turns, at buried (up to 50%) or completely exposed sites, and at relatively flexible or inflexible sites. Considering the significant number of beta-turn residues in every globular protein and the frequent deviation of beta-turn sequences from preferred proline and preferred glycine residues, this simple, efficient strategy will be useful for increasing the conformational stability of proteins.     

Different period gene repeats take 'turns' at fine-tuning the circadian clock

The repetitive region of the circadian clock gene period in Drosophila pseudoobscura consists predominantly of a pentapeptide sequence whose consensus is NSGAD. In D. melanogaster, this region is replaced by a dipeptide Thr-Gly repeat, which plays a role in the thermal stability of the circadian phenotype. The Thr-Gly repeat has been shown to form a type II or III beta-turn, whose conformational monomer is (Thr-Gly)3. Here we report, using conformational analyses, that both an NSGAD pentapeptide, and a polymer of the same sequence, form type II beta-turns. Thus two peptide sequences, whose amino-acid composition is very different, nevertheless form the same secondary structure. The implications of these structures for clock function are discussed.     

Structural engineering of the HIV-1 protease molecule with a beta-turn mimic of fixed geometry.

An important goal in the de novo design of enzymes is the control of molecular geometry. To this end, an analog of the protease from human immunodeficiency virus 1 (HIV-1 protease) was prepared by total chemical synthesis, containing a constrained, nonpeptidic type II' beta-turn mimic of predetermined three-dimensional structure. The mimic beta-turn replaced residues Gly16,17 in each subunit of the homodimeric molecule. These residues constitute the central amino acids of two symmetry-related type I' beta-turns in the native, unliganded enzyme. The beta-turn mimic-containing enzyme analog was fully active, possessed the same substrate specificity as the Gly16,17-containing enzyme, and showed enhanced resistance to thermal inactivation. These results indicate that the precise geometry of the beta-turn at residues 15-18 in each subunit is not critical for activity, and that replacement of the native sequence with a rigid beta-turn mimic can lead to enhanced protein stability. Finally, the successful incorporation of a fixed element of secondary structure illustrates the potential of a "molecular kit set" approach to protein design and synthesis.     

Improved method for predicting beta-turn using support vector machine

MOTIVATION: Numerous methods for predicting beta-turns in proteins have been developed based on various computational schemes. Here, we introduce a new method of beta-turn prediction that uses the support vector machine (SVM) algorithm together with predicted secondary structure information. Various parameters from the SVM have been adjusted to achieve optimal prediction performance. RESULTS: The SVM method achieved excellent performance as measured by the Matthews correlation coefficient (MCC = 0.45) using a 7-fold cross validation on a database of 426 non-homologous protein chains. To our best knowledge, this MCC value is the highest achieved so far for predicting beta-turn. The overall prediction accuracy Qtotal was 77.3%, which is the best among the existing prediction methods. Among its unique attractive features, the present SVM method avoids overtraining and compresses information and provides a predicted reliability index.