Proteomic Mapping of Dental Enamel Matrix from Inbred Mouse Strains: Unraveling Potential New Players in Enamel.

Enamel formation is a complex 2-step process by which proteins are secreted to form an extracellular matrix, followed by massive protein degradation and subsequent mineralization. Excessive systemic exposure to fluoride can disrupt this process and lead to a condition known as dental fluorosis. The genetic background influences the responses of mineralized tissues to fluoride, such as dental fluorosis, observed in A/J and 129P3/J mice. The aim of the present study was to map the protein profile of enamel matrix from A/J and 129P3/J strains. Enamel matrix samples were obtained from A/J and 129P3/J mice and analyzed by 2-dimensional electrophoresis and liquid chromatography coupled with mass spectrometry. A total of 120 proteins were identified, and 7 of them were classified as putative uncharacterized proteins and analyzed in silico for structural and functional characterization. An interesting finding was the possibility of the uncharacterized sequence Q8BIS2 being an enzyme involved in the degradation of matrix proteins. Thus, the results provide a comprehensive view of the structure and function for putative uncharacterized proteins found in the enamel matrix that could help to elucidate the mechanisms involved in enamel biomineralization and genetic susceptibility to dental fluorosis.

Changes in the Proteomic Profile of Acquired Enamel Pellicles as a Function of Their Time of Formation and Hydrochloric Acid Exposure.

OBJECTIVE: Changes in the protein profile of acquired enamel pellicles (AEP) formed in vivo over different time periods were evaluated after the application of hydrochloric acid (HCl). METHODS: Nine subjects were submitted to dental prophylaxis with pumice. After 3 or 120 min, the teeth were isolated with cotton rolls and 50 µL of 0.1 M HCl (pH 1.0), 0.01 M HCl (pH 2.0), or deionized water were applied on the buccal surface of the teeth for 10 s. The AEP was then collected using an electrode filter paper presoaked in 3% citric acid. After protein extraction, the samples were submitted to reverse-phase liquid chromatography coupled to mass spectrometry (nano LC-ESI-MS/MS). Label-free quantification was performed (Protein Lynx Global Service software). RESULTS: A total of 180 proteins were successfully identified in the AEP samples. The number of identified proteins increased with the time of pellicle formation. Only 4 proteins were present in all the groups (isoforms of IgA, serum albumin, and statherin). The greatest number of proteins identified uniquely in one of the groups was obtained for the groups treated with HCl after 2 h of pellicle formation (approx. 50 proteins). CONCLUSION: Proteins resistant to removal by HCl, such as serum albumin and statherin, were identified even in the short-term AEP. In addition, 120-min pellicles present many proteins that are resistant to removal by HCl. This suggests an increase in protection against intrinsic acids with the time of pellicle formation, which should be evaluated in future studies.

Enamel proteins and proteases in Mmp20 and Klk4 null and double-null mice.

Matrix metalloproteinase 20 (MMP20) and kallikrein-related peptidase 4 (KLK4) are thought to be necessary to clear proteins from the enamel matrix of developing teeth. We characterized Mmp20 and Klk4 null mice to better understand their roles in matrix degradation and removal. Histological examination showed retained organic matrix in Mmp20, Klk4, and Mmp20/Klk4 double-null mouse enamel matrix, but not in the wild-type. X-gal histostaining of Mmp20 null mice heterozygous for the Klk4 knockout/lacZ knockin showed that Klk4 is expressed normally in the Mmp20 null background. This finding was corroborated by zymogram and western blotting, which discovered a 40-kDa protease induced in the maturation stage of Mmp20 null mice. Proteins were extracted from secretory-stage or maturation-stage maxillary first molars from wild-type, Mmp20 null, Klk4 null, and Mmp20/Klk4 double-null mice and were analyzed by SDS-PAGE and western blotting. Only intact amelogenins and ameloblastin were observed in secretory-stage enamel of Mmp20 null mice, whereas the secretory-stage matrix from Klk4 null mice was identical to the matrix from wild-type mice. More residual matrix was observed in the double-null mice compared with either of the single-null mice. These results support the importance of MMP20 during the secretory stage and of KLK4 during the maturation stage and show there is only limited functional redundancy for these enzymes.

In vitro study on the interaction between the 32 kDa enamelin and amelogenin.

Enamel extracelluar matrix components play vital roles in controlling crystal nucleation and growth during enamel formation. We investigated the interaction between the 32 kDa enamelin fragment and amelogenin using immunochemical and biophysical methods. Immunoprecipitation studies revealed that the 32 kDa enamelin and amelogenin eluted together from a Protein A column. Dynamic light scattering results showed that the 32 kDa enamelin had a profound effect on amelogenin assembly at pH 8.0, causing partial dissociation of the nanospheres, in a dose-dependent manner. The appearance of an isodichroic point and the shifting and intensity decrease of the ellipticity minima in the circular dichroism spectra of amelogenin following the addition of the 32 kDa enamelin were indicative of conformational changes in amelogenin and of a direct interaction between the two macromolecules. Our results collectively demonstrate that the 32 kDa enamelin has a direct interaction with amelogenin in vitro. Our current studies provide novel insights into understanding possible cooperation between enamelin and amelogenin in macromolecular self-assembly and in controlling enamel mineral formation.

Purification of Developing Enamel Matrix Proteins Using Preparative SDS-PAGE.

In this chapter we discuss the potential of preparative SDS-PAGE for use in purifying native developing enamel matrix proteins. We believe that the methodology has the potential to provide the relatively large-scale single-step purification of any enamel protein that can be resolved as a single band during analytical SDS-PAGE. Of course, a single band on analytical SDS-PAGE does not guarantee absolute purity as the band may be comprised of two or more proteins migrating at the same apparent molecular weight on the gel. Where absolute purity is required, the methodology can be used in conjunction with other techniques such as ion-exchange chromatography or reverse-phase chromatography. We do not see preparative SDS-PAGE replacing chromatographic methodologies but believe that it can provide another powerful tool to add to the battery of purification techniques already available to researchers in the field.

The Expression and Purification of Recombinant Mouse Ameloblastin in E. coli.

Ameloblastin is the second most abundant enamel matrix protein, and is thought to be essential for ameloblast cell polarization, cell adhesion, and enamel mineralization. However, studies of ameloblastin's function and its molecular mechanism have been limited due to difficulty in obtaining recombinant ameloblastin in vitro. Here, we present a protocol for successful ameloblastin expression and purification in E. coli.

A study of enamel matrix proteins on differentiation of porcine bone marrow stromal cells into cementoblasts.

OBJECTIVE: To further explore the role of enamel matrix proteins (EMPs) in periodontal regeneration, we have used porcine bone marrow-derived stromal cells (BMSCs) to observe whether the EMPs could have an effect on their differentiation into cementoblasts. MATERIALS AND METHODS: In this study, EMPs were extracted from porcine tooth germs by the use of acetic acid. BMSCs obtained from porcine iliac marrow aspiration were inoculated onto the surface of autologous root slices treated with or without EMPs. Following 7-day co-culture, all the BMSC-seeded root slices, with their respective non-cell-inoculated control specimens, were pocketed with expanded polytetrafluoroethylene membrane and were transplanted subcutaneously into 11 nude mice. The animals were sacrificed after 3 and 8 weeks, and the new specimens were processed for haematoxylin and eosin staining. RESULTS: Histological analysis demonstrated new cellular cementum-like tissue formed along EMP-treated root slices. CONCLUSION: Our work has indicated for the first time, differentiation of BMSCs into cementoblasts using an EMP-based protocol.

Purification and analysis of a 5kDa component of enamel matrix derivative.

High performance liquid chromatography (HPLC) methods were used to analyse a 5kDa component purified from enamel matrix derivative (EMD), the active ingredient in Emdogain, a commercial product for periodontal tissue regeneration. After initial purification by size-exclusion chromatography (SEC) on a 100 cm x 5 cm column (Bio-Gel P-30 Fine, 280 nm), collected fractions were analysed by size-exclusion HPLC (SE HPLC; TSK-Gel Super SW2000, 220 nm). The fractions containing only the 5kDa component were analysed by reversed-phase high-pressure chromatography (RP HPLC; YMC-Pack ODS-A, 200 nm), revealing four peaks of the 5kDa component. From 1200 mg of EMD (of which 9% is the 5kDa component), approximately 65 mg of lyophilised 5kDa component were obtained, corresponding to a recovery of 60%. The SE HPLC method was mainly suitable for qualitative analysis, whereas the RP HPLC method was appropriate for both qualitative and quantitative analysis.

Porcine enamel protein fractions contain transforming growth factor-beta1.

BACKGROUND: Enamel extracts are biologically active and capable of inducing osteogenesis and cementogenesis, but the specific molecules carrying these activities have not been ascertained. The purpose of this study was to identify osteogenic factors in porcine enamel extracts. METHODS: Enamel proteins were separated by size-exclusion chromatography into four fractions, which were tested for their osteogenic activity on osteoblast-like cells (ST2) and human periodontal ligament (HPDL) cells. RESULTS: Fraction 3 (Fr.3) and a transforming growth factor-beta 1 (TGF-beta1) control reduced alkaline phosphatase (ALP) activity in ST2 but enhanced ALP activity in HPDL cells. The enhanced ALP activity was blocked by anti-TGF-beta antibodies. Furthermore, using a dual-luciferase reporter assay, we demonstrated that Fr.3 can induce the promoter activity of the plasminogen activator inhibitor type 1 (PAI-1) gene. CONCLUSION: These results show that the osteoinductive activity of enamel extracts on HPDL cells is mediated by TGF-beta1.

Enamel matrix derivative gel stimulates signal transduction of BMP and TGF-{beta}.

It has been shown that Emdogain Gel (Emd-Gel) containing enamel matrix proteins promotes biomineralization, such as osteogenesis and cementogenesis, during the regeneration of periodontal tissues. However, the growth factors involved in these activities of Emd-Gel remain unclear. In this study, Emd-Gel was fractionated into 22 sub-fractions by size exclusion chromatography. The osteoinductive factors, TGF-beta and BMP, were examined by a specific luciferase reporter gene assay. In the unfractionated Emd-Gel, TGF-beta-like activity was detected, while BMP activity was not. In contrast, in the fractionated Emd-Gel samples, TGF-beta-like activity was detected from fractions 8 to 13, and BMP-like activity was detected from fractions 4 to 6. Also, it was confirmed that the BMP-like activity in Emd-Gel was inhibited by authentic TGF-beta1 and TGF-beta-like activity. These results indicate that Emd-Gel contains both TGF-beta- and BMP-like growth factors that contribute to the induction of biomineralization during periodontal regeneration.

Relative molecular mass determination of a major, highest relative molecular mass extracellular amelogenin of developing bovine enamel.

Proteins of developing bovine enamel were fractionated by molecular sieving and ion-exchange chromatography. The major fraction corresponding to the highest Mr amelogenin of Mr approximately 26 000-30 000 was isolated and its Mr determined by SDS-PAGE, molecular sieving on G-100 resin and high performance liquid chromatography and by sedimentation-equilibrium ultracentrifugation, the latter three procedures in guanidine hydrochloride. SDS-PAGE and HPLC molecular sieving, employing commonly used Mr standards, gave Mr values of approximately 22 000-26 000. SDS-PAGE and HPLC molecular sieving, using proline-rich CNBr peptides of collagen as standards, and sedimentation-equilibrium ultracentrifugation, gave Mr values of approximately 15 000-18 000 and approximately 17 385, respectively. These latter values correspond well with those reported earlier and with the Mr of the major amelogenin computed from recent amino acid sequence data (approximately 19 000). It is concluded that the recently described, highest Mr amelogenin of Mr = 26 000-30 000 is not a new component but is identical to the proline-rich components having relative molecular masses ranging from 15 000 to 18 000 described much earlier by several groups of workers.

Dental enamel matrix--isolation and partial sequence of a peptide component.

Chromatography of demineralized bovine fetal enamel matrix proteins on 'Biogel P6' was shown to yield a trailing peak which contained a single electrophoretic component. This polypeptide, further purified by chromatography on 'Biogel P4', was found to be homogeneous by disc electrophoresis and gel isoelectric focussing. Amino acid analyses indicated this component (designated: "E5') to be similar to some of the phosphopeptides previously described. Cyanogen bromide cleavage of E5 yielded three principal products whose amino acid compositions and N-terminal residues were investigated. A partial sequence for component E5 was proposed and comparison with previous bovine matrix isolates was made.

Mass spectrometry of native rat amelogenins: primary transcripts, secretory isoforms, and C-terminal degradation.

Cloning technologies have established unambiguously that amelogenins always seem larger in molecular weight (Mr) by gel electrophoresis (SDS-PAGE) than by mass spectrometry (MS). This has caused many problems relating cloned versions of amelogenin to proteins actually secreted by ameloblasts in vivo. In this study, discrete protein fractions at 31-20 kDa (Mr(SDS)) were prepared from freeze-dried rat incisor enamel by techniques optimized for preserving protein integrity. N-terminal sequence and amino acid compositional analyses indicated that the major protein forming these fractions was amelogenin. As expected, the molecular weights estimated by matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) MS were significantly less than their apparent molecular weights estimated by SDS-PAGE. Plots of Mr(SDS) vs. Mr(MS) for all fractions showed high linear correlation (r = 0.992). Analysis of MS data further indicated that the major protein in the 27-kDa fraction corresponded to the R180 secretory isoform of rat amelogenin, whereas some minor proteins in the 23-kDa fraction likely corresponded to a R156 secretory isoform. This was in contrast to major proteins forming the 25-, 24-, and 23-kDa fractions (Mr(SDS)), which seemed to represent proteolytic fragments of R180 progressively altered at the P169-A170, P164-L165, and F151-S152 C-terminal cleavage sites, respectively. Proteins in the 20-kDa fraction (Mr(SDS)) most closely matched by ESI-MS fragments of the R156 secretory isoform that were C-terminally-modified at the equivalent P164-L165 site.

Immunochemical and biochemical studies of human enamel proteins during neonatal development.

The present communication provides descriptions of the developmental, biochemical, and immunological properties of the human enamel extracellular matrix proteins. We report the isolation and partial characterization of the major human enamel proteins, the production of polyclonal antibodies directed against the human enamelins, and a comparison between the immunogenicity of enamelins and amelogenins from human and mouse enamel extracellular matrices. Our results indicate that although enamelins and amelogenins share some epitopes, each one of these proteins appears to invoke a different degree of immunogenicity.

Enamel proteins: biosynthesis and chemistry.

Bovine ameloblast cell layer was incubated in 3H-proline containing medium and the incorporation of radioactivity into protein fractions of the cell and the medium was investigated by SDS- polyacrylamide gel electrophoresis. The results suggest that the ameloblasts synthesize and secrete a protein having a molecular weight of 25,000 daltons which is supposed to be the prototype of enamel proteins. This protein was degraded into dialyzable sizes by the cell extract. After a longer period of incubation another low molecular weight protein fraction appeared in the medium. Purification procedure with SDS gel filtration of the enamel protein is reported.

Purification, characterization, and cloning of enamel matrix serine proteinase 1.

The maturation of dental enamel succeeds the degradation of organic matrix. Inhibition studies have shown that this degradation is accomplished by a serine-type proteinase. To isolate and characterize cDNA clones encoding this proteinase, we used two degenerate primer approaches to amplify part of the coding region using polymerase chain-reaction (PCR). First, we purified the proteinase from porcine transition-stage enamel matrix and characterized it by partial protein sequencing. The enzyme was isolated from the neutral soluble enamel extract by successive ammonium sulfate precipitations, hydroxyapatite HPLC, reverse-phase HPLC, DEAE ion exchange, and affinity chromatography with a Benzamidine Sepharose 6B column. The intact protein and lysylendopeptidase-generated cleavage products were characterized by amino acid sequence analyses. Degenerate oligonucleotide primers encoding two of the polypeptide sequences were synthesized. In a complementary strategy, degenerate oligonucleotide primers were designed against highly conserved active-site regions of chymotrypsin-like proteinases. Both approaches yielded PCR amplification products that served as probes for screening a porcine enamel organ epithelia-specific cDNA library. The longest full-length clone is 1133 nucleotides and encodes a preproprotein of 254 amino acids. We designate this protein enamel matrix serine proteinase 1 or EMSP1. The active protein has 224 amino acids, an isotope-averaged molecular mass of 24.1 kDa, and an isoelectric point of 6.0. Multiple-tissue Northern analysis indicates that EMSP1 is a tooth-specific protein. Gelatin enzymography shows a dramatic increase in EMSP1 activity in the transition-stage enamel matrix. EMSP1 is most homologous to kallikriens and trypsins.

Amino acid sequence of a major human amelogenin protein employing Edman degradation and cDNA sequencing.

The abundant hydrophobic, proline-glutamine, and histidine-rich (over 90%) amelogenins constitute the major class of proteins in forming extracellular enamel matrix. These are thought to play a major role in the structural organization and mineralization of developing enamel. The present report describes the successful sequencing of the major human amelogenin protein, by use of both Edman degradation and cDNA sequencing. When Edman degradation was used, over 75% of the primary structure of the protein was determined. This sequence was supplemented with cDNA sequencing studies, which revealed the predicted sequence of this protein. Together, they provide the complete sequence of an important human enamel protein. The information complements recent studies on bovine and human amelogenin genes. A comparison between the present results and the protein sequences predicted from the corresponding human amelogenin genomic coding regions and that of cDNA sequences of other species is described.

Attachment of human periodontal ligament cells to enamel matrix-derived protein is mediated via interaction between BSP-like molecules and integrin alpha(v)beta3.

BACKGROUND: Although enamel matrix-derived protein (EMD) can stimulate attachment of human periodontal ligament (HPDL) cells to the root surface, the biological mechanism of this phenomenon is unclear. The purpose of this study was to determine which molecules in EMD are involved in the attachment of HPDL cells, and which types of integrins on the cell surface mediate the interaction between the cells and EMD. METHODS: HPDL explants were obtained from tooth surfaces extracted from 4 individuals, and cells taken from the individual explants were separately harvested and subcultured through as many as 5 passages. Cells were incubated on EMD-coated culture plates with and without neutral antibodies for integrins or RGD-sequence blocking peptides and stained with toluidine blue. Proteins in EMD that were able to induce cell attachment were identified by incubating sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) replicas with HPDL cells; the cell-binding regions were detected by staining the cells with toluidine blue. Characteristics of the cell-binding proteins in the EMD were identified by Western blot analysis. RESULTS: It was shown that anti-alpha(v)beta3 antibody and GRGDSP peptide markedly reduced attachment of HPDL cells to EMD. When the cells were incubated with SDS-PAGE replicas, distinct cell attachment was observed at a molecular mass of approximately 55 kDa. The cell-binding ability of this protein was completely blocked by treatment with anti-alpha(v)beta3 antibody or GRGDSP peptide. In the Western blot analysis, the 55 kDa protein was recognized by anti-bone sialoprotein (BSP) antibody as a single band. CONCLUSIONS: Our study provides the first evidence that the attachment of HPDL cells to EMD can be mediated by interaction between a BSP-like molecule and integrin alpha(v)beta3 on the cell surface.

D-Aspartic acid in bovine dentine non-collagenous phosphoprotein.

In tooth dentine, owing to its slow metabolism after its formation, racemized and transformed D-aspartic acid remains in the tissue and accumulates with age. However, no dentinal proteins which contain D-aspartic acid have been identified. In this study, a non-collagenous phosphoprotein was purified from bovine dentine. Its molecular mass was about 130 kDa and its amino acid composition was very similar to that of bovine dentine phosphophoryn. The purified protein contained a large proportion of aspartic acid residues and some of them were stereoinverted from the L-isomer to the D-isomer. The D-/L-aspartic acid ratio of dentine non-collagenous phosphoproteins purified from 8-month-old fetal, postnatal and 1-year-old bovine first incisors showed that the stereoinversion tended to increase with age. These results suggest that the purified non-collagenous phosphoprotein is a candidate for the protein in dentine containing D-aspartic acid.

Anti-peptide antibodies reactive with epitopic domains of porcine amelogenins at the C-terminus.

This was an immunological investigation of the processing of porcine amelogenins in situ. Rabbit and rat anti-peptide sera reacted specifically with the hydrophilic segment of the intact amelogenins at the C-terminus. The immunogens used were the synthetic peptides: (a) C13 composed of PATDKTKREEVDC and (b) C25 composed of MQSLLPDLPLEAWPATDKTKREEVD. These peptides correspond to the C-terminal 12- and 25-residue segments of porcine amelogenin, respectively. Cystine was introduced at the C-terminus of C12 for KLH-binding (C13). Western blot analysis disclosed that: (i) both rabbit and rat anti-C13 sera reacted selectively with the 25-kDa porcine amelogenin and three other minor components (27, 22 and 18 kDa); (ii) anti-C25 peptide sera, additionally, reacted with the 23-kDa amelogenins (a degradation derivative of the 25-kDa protein, lacking the 12-residue segment at the C-terminus) and as trace components, 20-, 16- and 14-kDa moieties. Importantly, all the proteins reactive with the anti-C13 serum were concentrated in the outer secretory enamel adjacent to the ameloblasts, decreasing significantly in the underlying inner secretory enamel. Immunohistochemical studies applying the anti-peptide sera to the developing tooth germs of a minipig also confirmed the localization of reactivity in the outer secretory region. Neither anti-peptide serum reacted with porcine non-amelogenins, serum proteins nor dentine matrix proteins at the dilutions tested. however, it was found that both the anti-C13 and C25 sera reacted with human keratin.(ABSTRACT TRUNCATED AT 250 WORDS)