A solution NMR investigation into the murine amelogenin splice-variant LRAP (Leucine-Rich Amelogenin Protein)

Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel biomineralization, making up > 90% of the matrix protein. Along with the full-length protein there are several splice-variant isoforms of amelogenin present including LRAP (Leucine-Rich Amelogenin Protein), a protein that consists of the first 33 and the last 26 residues of full-length amelogenin. Using solution-state NMR spectroscopy we have assigned the ¹H-¹⁵N HSQC spectrum of murine LRAP (rp(H)LRAP) in 2% acetic acid at pH 3.0 by making extensive use of previous chemical shift assignments for full-length murine amelogenin (rp(H)M180). This correlation was possible because LRAP, like the full-length protein, is intrinsically disordered under these solution conditions. The major difference between the ¹H-¹⁵N HSQC spectra of rp(H)M180 and rp(H)LRAP was an additional set of amide resonances for each of the seven non-proline residues between S12* and Y12 near the N-terminus of rp(H)LRAP indicating that the N-terminal region of LRAP exists in two different conformations. Analysis of the proline carbon chemical shifts suggests that the molecular basis for the two states is not a cis-trans isomerization of one or more of the proline residues in the N-terminal region. Starting from 2% acetic acid, where rp(H)LRAP was monomeric in solution, NaCl addition effected residue specific changes in molecular dynamics manifested by the reduction in intensity and disappearance of ¹H-¹⁵N HSQC cross peaks. As observed for the full-length protein, these perturbations may signal early events governing supramolecular self-assembly of rp(H)LRAP into nanospheres. However, the different patterns of ¹H-¹⁵N HSQC cross peak perturbation between rp(H)LRAP and rp(H)M180 in high salt suggest that the termini may behave differently in their respective nanospheres, and perhaps, these differences contribute to the cell signaling properties attributable to LRAP but not to the full-length protein.     

Truncated amelogenin and LRAP transgenes improve Amelx null mouse enamel

Amelogenin is the most abundant enamel protein involved in enamel mineralization. Our goal was to determine whether all three regions of amelogenin (N-terminus, C-terminus, central core) are required for enamel formation. Amelogenin RNA is alternatively spliced, resulting in at least 16 different amelogenin isoforms in mice, with M180 and LRAP expressed most abundantly. Soon after secretion by ameloblasts, M180 is cleaved by MMP20 resulting in C-terminal truncated (CTRNC) amelogenin. We aimed to determine whether the 2 transgenes (Tg), LRAP and CTRNC together, can improve LRAPTg/Amelx −/− and CTRNCTg/Amelx −/− enamel thickness and prism organization, which were not rescued in Amelx −/− enamel. We generated CTRNCTg/LRAPTg/Amelx −/− mice and analyzed developing and mature incisor and molar enamel histologically, by microCT, SEM and microhardness testing. CTRNCTg and LRAPTg overexpression together significantly improved the enamel phenotype of LRAPTg/Amelx −/− and CTRNCTg/Amelx −/− mouse enamel, however enamel microhardness was recovered only when M180Tg was expressed, alone or with LRAPTg. We determined that both LRAP and CTRNC, which together express all three regions of the amelogenin protein (N-terminus, C-terminus and hydrophobic core) contribute to the final enamel thickness and prism organization in mice.     

The receptor activator of nuclear factor-kappa B ligand-mediated osteoclastogenic pathway is elevated in amelogenin-null mice

Amelogenins, major components of developing enamel, are predominantly involved in the formation of tooth enamel. Although amelogenins are also implicated in cementogenesis, their precise spatial expression pattern and molecular role are not clearly understood. Here, we report for the first time the expression of two alternate splice forms of amelogenins, M180 and the leucine-rich amelogenin peptide (LRAP), in the periodontal region of mouse tooth roots. Lack of M180 and LRAP mRNA expression correlated with cementum defects observed in the amelogenin-null mice. The cementum defects were characterized by an increased presence of multinucleated cells, osteoclasts, and cementicles. These defects were associated with an increased expression of the receptor activator of the nuclear factor-kappa B ligand (RANKL), a critical regulator of osteoclastogenesis. These findings indicate that the amelogenin splice variants, M180 and LRAP, are critical in preventing abnormal resorption of cementum.     

Leucine-rich amelogenin peptide induces osteogenesis by activation of the Wnt pathway

We previously showed that one of the amelogenin splicing isoforms, Leucine-rich amelogenin peptide (LRAP), induced osteogenic differentiation of mouse embryonic stem cells; however, the signaling pathway(s) activated by LRAP remained unknown. Here, we demonstrated that the canonical Wnt/β-catenin signaling is activated upon LRAP treatment, as evidenced by elevated β-catenin level and increased Wnt reporter gene activity. Furthermore, a specific Wnt inhibitor sFRP-1 completely blocks the LRAP-mediated Wnt signaling. However, exogenous recombinant Wnt3a alone was less effective at osteogenic induction of mouse ES cells in comparison to LRAP. Using a quantitative real-time PCR array, we discovered that LRAP treatment up-regulated the expression of Wnt agonists and down-regulated the expression of Wnt antagonists. We conclude that LRAP activates the canonical Wnt signaling pathway to induce osteogenic differentiation of mouse ES cells through the concerted regulation of Wnt agonists and antagonists.     

Leucine-rich amelogenin peptide induces osteogenesis in mouse embryonic stem cells

Leucine-rich amelogenin peptide (LRAP), an alternatively spliced amelogenin protein, possesses a signaling property shown to induce osteogenic differentiation. In the current study, we detected LRAP expression during osteogenesis of wild-type (WT) embryonic stem (ES) cells and observed the absence of LRAP expression in amelogenin-null (KO) ES cells. We explored the signaling effect of LRAP on wild-type ES cells, and the ability of LRAP to rescue the impaired osteogenesis phenotype observed in KO ES cells. Our data indicate that LRAP treatment of WT and KO ES cells induces a significant increase in mineral matrix formation, and significant increases in bone sialoprotein and osterix gene expression. In addition, the amelogenin KO phenotype is partially rescued by the addition of exogenous LRAP. These data suggest a unique function of LRAP during ES cell differentiation along osteogenic lineage.     

The leucine rich amelogenin protein (LRAP) adsorbs as monomers or dimers onto surfaces

Amelogenin is believed to be involved in controlling the formation of the highly anisotropic and ordered hydroxyapatite crystallites that form enamel. The adsorption behavior of amelogenin proteins onto substrates is very important because protein–surface interactions are critical to its function. We have previously used LRAP, a splice variant of amelogenin, as a model protein for the full-length amelogenin in solid-state NMR and neutron reflectivity studies at interfaces. In this work, we examined the adsorption behavior of LRAP in greater detail using model self-assembled monolayers containing COOH, CH₃, and NH₂ end groups as substrates. Dynamic light scattering (DLS) experiments indicated that LRAP in phosphate buffered saline and solutions containing low concentrations of calcium and phosphate consisted of aggregates of nanospheres. Null ellipsometry and atomic force microscopy (AFM) were used to study protein adsorption amounts and quaternary structures on the surfaces. Relatively high amounts of adsorption occurred onto the CH₃ and NH₂ surfaces from both buffer solutions. Adsorption was also promoted onto COOH surfaces only when calcium was present in the solutions suggesting an interaction that involves calcium bridging with the negatively charged C-terminus. The ellipsometry and AFM studies revealed that LRAP adsorbed onto the surfaces as small subnanosphere-sized structures such as monomers or dimers. We propose that the monomers/dimers were present in solution even though they were not detected by DLS or that they adsorbed onto the surfaces by disassembling or “shedding” from the nanospheres that are present in solution. This work reveals the importance of small subnanosphere-sized structures of LRAP at interfaces.     

Dental enamel matrix: Sequences of two amelogenin polypeptides

The amino acid sequences of a leucine-rich amelogenin polypeptide (LRAP) and a tyrosine-rich amelogenin polypeptide (TRAP), isolated from foetal bovine enamel matrix, were determined. Both LRAP and TRAP occurred in two forms; in each case, one of the molecular species appeared to be shortened at the COOH terminus by 2 and 4 residues, respectively. A striking finding was that LRAP and TRAP had identical sequences for the first 33 residues but were almost completely different for the remaining 12 amino acids.     

The exon 6ABC region of amelogenin mRNA contribute to increased levels of amelogenin mRNA through amelogenin protein-enhanced mRNA stabilization

We recently demonstrated that the reuptake of full-length amelogenin protein results in increased levels of amelogenin mRNA through enhanced mRNA stabilization (Xu, L., Harada, H., Tamaki, T. Y., Matsumoto, S., Tanaka, J., and Taniguchi, A. (2006) J. Biol. Chem. 281, 2257-2262). Here, we examined the molecular mechanism of enhanced amelogenin mRNA stabilization. To identify the cis-regulatory region within amelogenin mRNA, we tested various reporter systems using a deletion series of reporter plasmids. A deletion at exon 6ABC of amelogenin mRNA resulted in a 2.5-fold increase in the amelogenin mRNA expression level when compared with that of full-length mRNA, indicating that a cis-element exists in exon 6ABC of amelogenin mRNA. Furthermore, Northwestern analysis demonstrated that amelogenin protein binds directly to its mRNA in vitro, suggesting that amelogenin protein acts as a trans-acting protein that specifically binds to this cis-element. Moreover, recombinant mouse amelogenin protein extended the half-life of full-length amelogenin mRNA but did not significantly alter the half-life of exon 6ABC-deletion mutant mRNA. The splice products produced by deletion of exon 6ABC are known as leucine-rich amelogenin peptides and have signaling effects on cells. Our findings also suggest that the regulation of full-length amelogenin protein expression differs from the regulation of leucine-rich amelogenin peptide expression.     

Interaction of amelogenin with hydroxyapatite crystals: An adherence effect through amelogenin molecular self-association

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. © 1998 John Wiley & Sons, Inc. Biopoly 46: 225–238, 1998     

Histidine tag fusion increases expression levels of active recombinant amelogenin in Escherichia coli

Amelogenin is a dental enamel matrix protein involved in formation of dental enamel. In this study, we have expressed two different recombinant murine amelogenins in Escherichia coli: the untagged rM179, and the histidine tagged rp(H)M180, identical to rM179 except that it carries the additional N-terminal sequence MRGSHHHHHHGS. The effects of the histidine tag on expression levels, and on growth properties of the amelogenin expressing cells were studied. Purification of a crude protein extract containing rp(H)M180 was also carried out using IMAC and reverse-phase HPLC. The results of this study showed clearly that both growth properties and amelogenin expression levels were improved for E. coli cells expressing the histidine tagged amelogenin rp(H)M180, compared to cells expressing the untagged amelogenin rM179. The positive effect of the histidine tag on amelogenin expression is proposed to be due to the hydrophilic nature of the histidine tag, generating a more hydrophilic amelogenin, which is more compatible with the host cell. Human osteoblasts treated with the purified rp(H)M180 showed increased levels of secreted osteocalcin, compared to untreated cells. This response was similar to cells treated with enamel matrix derivate, mainly composed by amelogenin, suggesting that the recombinant protein is biologically active. Thus, the histidine tag favors expression and purification of biologically active recombinant amelogenin.     

Partial rescue of the amelogenin null dental enamel phenotype

The amelogenins are the most abundant secreted proteins in developing dental enamel. Enamel from amelogenin (Amelx) null mice is hypoplastic and disorganized, similar to that observed in X-linked forms of the human enamel defect amelogenesis imperfecta resulting from amelogenin gene mutations. Both transgenic strains that express the most abundant amelogenin (TgM180) have relatively normal enamel, but strains of mice that express a mutated amelogenin (TgP70T), which leads to amelogenesis imperfecta in humans, have heterogeneous enamel structures. When Amelx null (KO) mice were mated with transgenic mice that produce M180 (TgM180), the resultant TgM180KO offspring showed evidence of rescue in enamel thickness, mineral density, and volume in molar teeth. Rescue was not observed in the molars from the TgP70TKO mice. It was concluded that a single amelogenin protein was able to significantly rescue the KO phenotype and that one amino acid change abrogated this function during development.     

The COOH terminus of the amelogenin, LRAP, is oriented next to the hydroxyapatite surface

The organic matrix in forming enamel consists largely of the amelogenin protein self-assembled into nanospheres that are necessary to guide the formation of the unusually long and highly ordered hydroxyapatite (HAP) crystallites that constitute enamel. Despite its ability to direct crystal growth, the interaction of the amelogenin protein with HAP is unknown. However, the demonstration of growth restricted to the c-axis suggests a specific protein-crystal interaction, and the charged COOH terminus is often implicated in this function. To elucidate whether the COOH terminus is important in the binding and orientation of amelogenin onto HAP, we have used solid state NMR to determine the orientation of the COOH terminus of an amelogenin splice variant, LRAP (leucine-rich amelogenin protein), which contains the charged COOH terminus of the full protein, on the HAP surface. These experiments demonstrate that the methyl 13C-labeled side chain of Ala46 is 8.0 A from the HAP surface under hydrated conditions, for the protein with and without phosphorylation. The experimental results provide direct evidence orienting the charged COOH-terminal region of the amelogenin protein on the HAP surface, optimized to exert control on developing enamel crystals.     

Effects of human full-length amelogenin on the proliferation of human mesenchymal stem cells derived from bone marrow

Amelogenins are enamel matrix proteins that play a crucial role in enamel formation. Recent studies have revealed that amelogenins also have cell signaling properties. Although amelogenins had been described as specific products of ameloblasts, recent research has demonstrated their expression in bone marrow stromal cells. In this study, we examined the effect of recombinant human full-length amelogenin (rh174) on the proliferation of human mesenchymal stem cells (MSCs) derived from bone marrow and characterized the associated changes in intracellular signaling pathways. MSCs were treated with rh174 ranging in dose from 0 to 1,000 ng/ml. Cell proliferative activity was analyzed by bromodeoxyuridine (BrdU) immunoassay. The expression of lysosomal-associated membrane protein 1 (LAMP1), a possible amelogenin receptor, in MSCs was analyzed. Anti-LAMP1 antibody was used to block the binding of rh174 to LAMP1. The MAPK-ERK pathway was examined by Cellular Activation of Signaling ELISA (CASE) kit and western blot analysis. A specific MAPK inhibitor, U0126, was used to block ERK activity. It was shown that rh174 increased the proliferation of MSCs and MAPK-ERK activity. The MSC proliferation and MAPK-ERK activity enhanced by rh174 were reduced by the addition of anti-LAMP1 antibody. Additionally, the increased proliferation of MSCs induced by rh174 was inhibited in the presence of U0126. In conclusion, it is demonstrated that rh174 increases the proliferation of MSCs by interaction with LAMP1 through the MAPK-ERK signaling pathway, indicating the possibility of MSC application to tissue regeneration in the orofacial region.     

Enamel biomineralization defects result from alterations to amelogenin self-assembly

Enamel formation is a powerful model for the study of biomineralization. A key feature common to all biomineralizing systems is their dependency upon the biosynthesis of an extracellular organic matrix that is competent to direct the formation of the subsequent mineral phase. The major organic component of forming mouse enamel is the 180-amino-acid amelogenin protein (M180), whose ability to undergo self-assembly is believed to contribute to biomineralization of vertebrate enamel. Two recently defined domains (A and B) within amelogenin appear essential for this self-assembly. The significance of these two domains has been demonstrated previously by the yeast two-hybrid system, atomic force microscopy, and dynamic light scattering. Transgenic animals were used to test the hypothesis that the self-assembly domains identified with in vitro model systems also operate in vivo. Transgenic animals bearing either a domain-A-deleted or domain-B-deleted amelogenin transgene expressed the altered amelogenin exclusively in ameloblasts. This altered amelogenin participates in the formation an organic enamel extracellular matrix and, in turn, this matrix is defective in its ability to direct enamel mineralization. At the nanoscale level, the forming matrix adjacent to the secretory face of the ameloblast shows alteration in the size of the amelogenin nanospheres for either transgenic animal line. At the mesoscale level of enamel structural hierarchy, 6-week-old enamel exhibits defects in enamel rod organization due to perturbed organization of the precursor organic matrix. These studies reflect the critical dependency of amelogenin self-assembly in forming a competent enamel organic matrix and that alterations to the matrix are reflected as defects in the structural organization of enamel.     

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.     

Endo-lysosomal vesicles positive for Rab7 and LAMP1 are terminal vesicles for the transport of dextran

The endo-lysosomal pathway is essential for intracellular transport and the degradation of extracellular cargo. The relationship between three populations of endo-lysosomal vesicles--Rab7-positive, LAMP1-positive, and both Rab7- and LAMP1-postive--was probed with fluorescence microscopy and single particle tracking. Of specific interest was determining if these vesicles were intermediate or terminal vesicles in the transport of extracellular cargo. We find that the major organelle in the endo-lysosomal pathway, both in terms of population and cargo transport, is positive for Rab7 and LAMP1. Dextran, a fluid phase cargo, shifts from localization within all three populations of vesicles at 30 minutes and 1 hour to primarily LAMP1- and Rab7/LAMP1-vesicles at longer times. This demonstrates that LAMP1- and Rab7/LAMP1-vesicles are terminal vesicles in the endo-lysosomal pathway. We tested two possible mechanisms for this distribution of cargo, delivery to mannose 6-phosphate receptor (M6PR)-negative vesicles and the fusion dynamics of individual vesicles. We find no correlation with M6PR but do find that Rab7-vesicles undergo significantly fewer fusion events than LAMP1- or Rab7/LAMP1-vesicles suggesting that the distribution of fluid phase cargo is driven by vesicle dynamics.