Localization and quantitative co-localization of enamelin with amelogenin.

Enamelin and amelogenin are vital proteins in enamel formation. The cooperative function of these two proteins controls crystal nucleation and morphology in vitro. We quantitatively analyzed the co-localization between enamelin and amelogenin by confocal microscopy and using two antibodies, one raised against a sequence in the porcine 32 kDa enamelin region and the other raised against full-length recombinant mouse amelogenin. We further investigated the interaction of the porcine 32 kDa enamelin and recombinant amelogenin using immuno-gold labeling. This study reports the quantitative co-localization results for postnatal days 1-8 mandibular mouse molars. We show that amelogenin and enamelin are secreted into the extracellular matrix on the cuspal slopes of the molars at day 1 and that secretion continues to at least day 8. Quantitative co-localization analysis (QCA) was performed in several different configurations using large (45 µm height, 33 µm width) and small (7 µm diameter) regions of interest to elucidate any patterns. Co-localization patterns in day 8 samples revealed that enamelin and amelogenin co-localize near the secretory face of the ameloblasts and appear to be secreted approximately in a 1:1 ratio. The degree of co-localization decreases as the enamel matures, both along the secretory face of ameloblasts and throughout the entire thickness of the enamel. Immuno-reactivity against enamelin is concentrated along the secretory face of ameloblasts, supporting the theory that this protein together with amelogenin is intimately involved in mineral induction at the beginning of enamel formation.

Immunohistochemical and biochemical evidence of ameloblastic origin of amyloid-producing odontogenic tumors in cats.

Amyloid-producing odontogenic tumors (APOT) are rare, and in cats, the histogenesis of the amyloid remains undetermined. In the present study, APOTs in 3 cats were characterized by immunohistochemistry, and the amyloid components analyzed using tandem mass spectrometry. Antiameloblastin antibodies labeled both neoplastic epithelial cells and amyloid in all cases. Neoplastic epithelial cells had strong, diffuse immunoreactivity to antibodies against cytokeratin AE1/AE3, cytokeratin 14, and cytokeratin 19 in all cases and focal immunoreactivity to nerve growth factor receptor antibodies in 2 of 3 cases. Amyloid and some tumor stromal cells were weakly positive for laminin. Calretinin, amelogenin, S100, and glial fibrillary acidic protein antibodies did not label neoplastic epithelial cells or amyloid. Extracted amyloid peptide sequences were compared to the porcine database because the cat genome is not yet complete. Based on this comparison, 1 identical ameloblastin peptide was detected in each tumor. These results suggest that feline APOTs and the amyloid they produce are of ameloblastic lineage.

Enamel defects associated with coeliac disease: putative role of antibodies against gliadin in pathogenesis.

Enamel defects in the permanent teeth of patients with coeliac disease (CD) are often reported as an atypical manifestation, sometimes being suggestive of an undiagnosed atypical disease. We proposed to explore the pathogenesis of these oral defects, which are poorly studied. Sequence analyses of proteins from gluten (gliadins) and of proline-rich enamel proteins (amelogenin and ameloblastin) suggested the presence of common antigenic motifs. Therefore, we analyzed, by ELISA and western blotting, the reactivity of sera from patients with CD against gliadin and enamel-derived peptides. Correlation analyses between the levels of specific antibodies against gliadin and enamel derived peptides and inhibition experiments confirmed the presence of cross-reactive antibodies. Immunoblot analysis revealed that the most prominent component in enamel matrix derivative (of approximately 18.6 kDa), identified by an amelogenin-specific antibody, is recognized by sera from patients with CD; in addition, several fractions of pure gliadin were recognized by amelogenin-specific antibody. In agreement, sera from mice immunized with enamel matrix-derived proteins generated antibodies that recognized a peptide (of approximately 21.2 kDa) derived from gliadin. In conclusion, antibodies against gliadin generated in patients with CD can react in vitro with a major enamel protein. The involvement of anti-gliadin serum in the pathogenesis of enamel defects in children with untreated CD can be hypothesized on the basis of these novel results.

Immunohistochemical localization of amelogenins in enameloid of lower vertebrate teeth.

The indirect method of immunofluorescence was used to demonstrate the presence of amelogenins in the enameloid of teeth and dermal denticles of Chondrichthyes; in the enameloid of Teleostei and Amphibia; and in the enamel of Reptilia. Nonmammalian amelogenins are formed in the ectodermal cells of tooth organs and chemically are so similar to mammalian amelogenins that they interact with antiserum prepared from bovine enamel matrix.

The induction and possible subsequent effect of human antibodies against porcine enamel matrix derivative.

BACKGROUND: The amino acid sequence of porcine amelogenin, the major component of enamel matrix derivative (EMD), is approximately 91% identical to that of its human counterpart. Whether porcine EMD (pEMD) can elicit neutralizing antibodies after the first surgery, thereby reducing the clinical effect of secondary surgery, has not been established. METHODS: The sera of patients receiving periodontal surgery with or without pEMD were collected before and after surgery. The pEMD product was subjected to electrophoresis and transferred for a Western blot using the purified antibodies from patients as the primary antibodies. To clarify whether the antibodies in patients could inhibit the production of transforming growth factor-beta1 (TGF-beta1), we added different amounts of purified antibodies from pEMD patients (before versus after surgery) into the cell cultures of periodontal ligament fibroblasts. The conditioned media were then collected for an immunoassay of TGF-beta1. RESULTS: The result of the Western blot demonstrated that human antibodies against pEMD were elicited 10 days after surgery. Using mass spectrometry, a non-specific band on the Western blot appeared to be porcine immunoglobulin G (IgG). The results of the immunoassay showed the antibodies from pEMD-treated patients did not hinder the subsequent production of TGF-beta1. CONCLUSIONS: The pEMD product was contaminated with porcine IgG. The application of the product could induce antibodies against different isoforms of porcine amelogenin in humans. However, the increased antibodies did not hinder the production of TGF-beta1, one of the established in vitro functions of pEMD on periodontal ligament fibroblasts.

Human and mouse cementum proteins immunologically related to enamel proteins.

SDS-polyacrylamide gel electrophoresis, immunoblot and amino acid composition analyses were applied to human and mouse acellular cementum proteins immunologically related to enamelins and amelogenins. In this analysis, anti-mouse amelogenin, anti-human enamelin and synthetic peptide (e.g., -LPPHPGHPGYIC-) antibodies were shown to cross-react with tooth crown-derived enamelin with a molecular mass of 72,000 Da (72 kDa), amelogenins (26 kDa), and also to four human cementum proteins (72, 58, 50 and 26 kDa) and two mouse cementum proteins (72 and 26 kDa). Each of the antibodies recognized tooth root-derived cementum polypeptides which share one or more epitopes with tooth crown-derived enamel proteins. The molecular mass and isoelectric points for crown-derived and root-derived enamel-related proteins were similar. Analysis of human and mouse cementum proteins revealed a characteristic amino acid composition enriched in glutamyl, serine, glycine, alanine, proline, valine and leucine residues; compared to the major enamel protein amelogenin, cementum proteins were low in proline, histidine and methionine. The human and mouse putative intermediate cementum proteins appear to represent a distinct class of enamel-related proteins. Moreover, these results support the hypothesis that epithelial root sheath epithelia express several cementum proteins immunologically related to canonical enamel proteins.

[Preparation and application of polyclonal antibody against amelotin polypeptide].

OBJECTIVE: To prepare and identify the rabbit anti-mouse polyclonal antibody against amelotin polypeptide. METHODS: The polypeptide was synthesized based on the bioinformatics analysis of amelotin, and coupled with keyhole limpet hemocyanin (KLH) for immunization. The amelotin polypeptide-KLH was injected into New Zealand rabbits to prepare the polyclonal antibody. ELISA technology was used to detect the titer of the antibody. The specificity of the polyclonal antibody was identified by Western blotting. The expression of amelotin in the submandibular tissue of mice was observed by immunohistochemistry. RESULTS: ELISA showed that the titer of amelotin antibody was 1:1,000,000. Western blotting verified that the antibody had a high specificity. Immunohistochemistry indicated that amelotin was highly expressed in odontoprisis full-thick enamel of 3-day-old and 7-day-old mice, and also expressed in duct epithelial cytoplasm of submandibular glands of 7-day-old mice. CONCLUSION: The polyclonal antibody against amelotin polypeptide has been prepared successfully with high titer and high specificity.

Selective but nonspecific immunolabeling of enamel protein-associated compartments by a monoclonal antibody against vimentin.

Vimentin, an intermediate filament component, has been identified in many mesenchymal cells by a variety of LM and EM immunolabeling techniques. In our study, several tissue-processing conditions and monoclonal and polyclonal antibodies against vimentin were screened for immunostaining of rat incisor odontoblasts. Using postembedding colloidal gold immunocytochemistry, we were unable to detect any convincing vimentin antigenicity in these cells, but one of the monoclonal antibodies (V9-S) unexpectedly resulted in intense labeling over intra- and extracellular compartments that normally are strongly immunoreactive with anti-amelogenin antibodies. Blocking experiments showed that V9-S binding was competed by anti-amelogenin antibody. Immunoblots indicated that enamel proteins reacted with this anti-vimentin antibody after fixation with glutaraldehyde. These data suggest that the observed immunoreaction is directed against an epitope apparently created by crosslinking of enamel proteins during fixation. Although the labeling cannot be considered specific, it is nevertheless selective because it is very precisely localized over compartments containing enamel proteins and shows no binding to other calcified dental tissues, including dentin and bone. The V9-S antibody can therefore be used as a reliable probe to identify the presence and distribution of amelogenins in fixed tissues. (J Histochem Cytochem 47:1237-1245, 1999)

Enamel protein biosynthesis and secretion in mouse incisor secretory ameloblasts as revealed by high-resolution immunocytochemistry.

Mouse secretory ameloblasts express a number of enamel proteins, which have been divided into amelogenin and enamelin subfamilies. We have used polyclonal antibodies to murine amelogenins to reveal enamel proteins in mouse ameloblasts using the protein A-gold immunocytochemical technique. Specific immunolabeling was detected over the extracellular enamel matrix and over the rough endoplasmic reticulum, the saccules of the Golgi apparatus, and the secretory granules of the ameloblasts. In addition, some lysosome-like granules were also labeled. Only background labeling was obtained over mitochondria, nuclei, cytosol, adjacent odontoblasts, and dentin. Quantitation of the intensity of labeling showed the presence of an increasing gradient along the secretory pathway, which may correspond to the concentration or the maturation of these proteins as they are processed by the cell. These findings indicate that the ameloblast displays an intracellular distribution of its secretory products similar to that of other merocrine secreting cells. The presence of enamel proteins in lysosomes suggests that crinophagy and/or resorption occurs in these cells.

Immunochemical characterization of a chicken egg yolk antibody to secretory forms of rat incisor amelogenin.

Amelogenins represent the major component of the organic matrix of enamel, and consist of several intact and degraded forms. A precise knowledge of their respective distributions throughout the enamel layer could provide some insight into their functions. To date, no antibody exists that can selectively detect the secretory forms of amelogenin. In this study we used the chicken egg yolk system to generate an antibody to recombinant mouse amelogenin. Immunoblots of whole homogenates from rat incisor enamel organs and enamel showed that the resulting antibody (M179y) recognized proteins corresponding to the five known secretory forms of rat amelogenin. Immunogold cytochemistry demonstrated that reactivity was restricted to ameloblasts and enamel. Secretory forms of amelogenin persisted in significant amounts throughout the enamel layer. The density of labeling was highest over the surface portion of the enamel layer, but enamel growth sites in this region showed a localized paucity of gold particles. Immunoreactivity was lowest over the mid-portion of the layer and increased moderately near the dentino-enamel junction. These results indicate that intact forms of amelogenin probably have a more complex distribution in the enamel layer than was heretofore suspected.

Synthesis, secretion, degradation, and fate of ameloblastin during the matrix formation stage of the rat incisor as shown by immunocytochemistry and immunochemistry using region-specific antibodies.

Rat ameloblastin is a recently cloned tooth-specific enamel matrix protein containing 422 amino acid residues. We investigated the expression of this protein during the matrix formation stage of the rat incisor immunohistochemically and immunochemically, using anti-synthetic peptide antibodies that recognize residues 27-47 (Nt), 98-107 (M-1), 224-232 (M-2), 386-399 (M-3), and 406-419 (Ct) of ameloblastin. Immunohistochemical preparations using antibodies Nt and M-1 stained the Golgi apparatus and secretory granules of the secretory ameloblast and the entire thickness of the enamel matrix. Only M-1 intensely stained the peripheral region of the enamel rods. Immunostained protein bands were observed near 65, 55, and below 22 kD. Immunohistochemical preparations using antibodies M-2 and Ct stained the Golgi apparatus and secretory granules of the ameloblast and the immature enamel adjacent to the secretion sites, but not deeper enamel layers. Immunostaining using M-2 and Ct revealed protein bands near 65 and 40-56 kD, and 65, 55, 48, 36, and 25 kD, respectively. M-3 stained the cis side of the Golgi apparatus but not the enamel matrix. This antibody recognized a protein band near 55 kD, but none larger. After brefeldin A treatment, immunoreaction of the 55-kD protein band intensified, and dilated cisternae of rER of the secretory ameloblast contained immunoreactive material irrespective of the antibodies used. These data indicate that ameloblastin is synthesized as a 55-kD core protein and then is post-translationally modified with O-linked oligosaccharides to become the 65-kD secretory form. Initial cleavages of the 65-kD protein generate N-terminal polypeptides, some of which concentrate in the prism sheath, and C-terminal polypeptides, which are rapidly degraded and lost from the enamel matrix soon after secretion.

An antisera for the fluorescent labeling of mouse amelogenesis.

Embryonic mammalian enamel extracellular matrix is immunogenic. Antisera has been produced in New Zealand white rabbits using 5-day-old (post-natal) C57B1/6J mandibular and maxillary incisor and molar tooth organs as immunogens. The expression of secretory amelogenesis in mouse molar tooth organs was studied from the "cap stage" (circa 17-day fetus) to the fifth day of postnatal odontogenesis using indirect immunofluorescent microscopy. The specificity of the antisera for enamel matrix secretion was unequivocal. Secretory amelogenesis was observed in molar tooth organs as early as day-2 postnatal age. These reagents and methods provide a significant strategy in studies of epithelial-mesenchymal interactions during tooth development.

Immunofluorescent evidence for the similarity of amelogenins in calf, mouse and pig teeth.

Antiserum was prepared to fetal bovine enamel matrix and was used to localize the amelogenins in developing bovine molars by immunofluorescent microscopy. Amelogenins could be identified to preameloblasts, secretory ameloblasts, stratum intermedium cells, and the newly deposited enamel matrix. Mature enamel matrix did not fluoresce except in a thin line along the DEJ and adjacent to the ameloblasts. Immature enamel matrix of murine and porcine teeth fluoresced when treated with antiserum to bovine enamel matrix. No other portions of tooth buds or other tissues reacted with the specific antiserum.

Immunogenicity of enamel protein.

Antisera to enamel proteins have been used primarily as a probe for biochemical and developmental studies of enamel proteins and tooth formation. More recently, the inherent immunogenicity of enamel protein has become a subject for investigation. Papers are reviewed which show that soluble enamel proteins are immunogenic across species barriers, and that solid-phase enamel proteins are immunogenic within a species. New data are presented which demonstrate that enamel proteins are autoantigenic both in vivo and in vitro. Future areas of research are suggested.

Demonstration of an alloimmune response to embryonic enamel matrix proteins.

Young adult rabbits were immunized with enamel matrix proteins from embryonic tooth organs of the same strain of rabbit. These proteins elicited an alloimmune response as demonstrated by the specific binding of antiserum to enamel matrix which was visualized by indirect immunofluorescent microscopy. The labial and lingual surfaces of embryonic incisor tooth organs were found to share common antigenic determinants. The observations suggest that enamel proteins could possibly be autoantigens.

Sheathlin: cloning, cDNA/polypeptide sequences, and immunolocalization of porcine enamel sheath proteins.

Sheath proteins designate low-molecular-weight non-amelogenin enamel polypeptides and their parent protein, which concentrate in the sheath space separating rod and inter-rod enamel (Uchida et al., 1995). Two porcine sheath proteins, with apparent molecular weights of 13 and 15 kDa, are characterized by protein sequencing. The primary structures of these polypeptides match a portion of the derived amino acid sequences of clones isolated from a porcine enamel organ epithelia-specific cDNA library. Sheath protein RNA messages differ by the inclusion or deletion of a 45-nucleotide segment and by the use of three alternative polyadenylation/cleavage sites. The secreted proteins are 395 and 380 residues in length, with molecular masses of 42,358 and 40,279 Daltons and calculated isoelectric points of 6.3 and 6.7, respectively. Polyclonal antibodies were raised against a synthetic peptide having the sheathlin-specific sequence EHETQQYEYSGGC. Immunohistochemistry with this antibody demonstrates that the protein encoded by the sheathlin cDNA is preferentially localized in the sheath space. We propose that the porcine sheath proteins and their proteolytic cleavage products be designated "sheathlin".

A multicenter study evaluating the sensitization potential of enamel matrix derivative after treatment of two infrabony defects.

BACKGROUND: Several studies reported some success toward regeneration in infrabony defects using enamel matrix derivative (EMD). Clinically and statistically significant improvements in probing depth reduction, clinical attachment levels, and bone fill have been demonstrated. This multi-center study evaluated the potential for sensitization to EMD in a subgroup of periodontal patients treated at least twice with at least 2 months between treatments. METHODS: Three hundred seventy-six (376) patients in 11 university-based postgraduate periodontics programs and five private practices were selected. Surgeries were performed on infrabony defects. Following reflection of mucoperiosteal flaps and debridement of the root surface and defect, root conditioning (either citric acid pH = 1 or 24% EDTA) was performed and the site was irrigated with sterile saline. Enamel matrix derivative was reconstituted and applied to the exposed root surface and the bony defect. Flaps were sutured and pressure applied for 5 minutes. The second test defect was treated in a similar manner at least 8 weeks after the first surgery. The patient was given a diary card where any subjective adverse events (erythema, swelling, itching, headache, root hypersensitivity, or pain) were recorded at weeks 1 and 2 post-surgery. In addition, objective adverse events (gingival inflammation, ulcers, abscess, cratering, and lesions) were recorded by the investigator on an adverse event form. RESULTS: No clinical adverse reactions to multiple applications of EMD were noted. Of 376 patients, two were referred to a dermatologist for evaluation, but neither had signs indicating any adverse events due to EMD treatment. Instead their reactions were classified as a small local abscess and tinea cruris. The single immunoassay performed (on the patient with a small local abscess) did not demonstrate any EMD-reactive antibodies, neither IgE nor IgG. Other subjective/objective reactions that occurred during this study were of the type that are commonly experienced by patients immediately following periodontal surgery, but were not related to EMD. They included headache, swelling, itching, pain, and root hypersensitivity. CONCLUSIONS: This study demonstrated a lack of clinical adverse reactions following two separate applications of EMD. Any subjective/objective adverse reactions experienced by the patient were typical complications following routine periodontal surgery and were not directly related to the use of enamel matrix derivative.

Monoclonal antibodies to different epitopes in amelogenins from fetal bovine teeth recognize high-molecular-weight components.

Enamel proteins were extracted and partitioned into amelogenin and enamelin fractions. Although several attempts were made to raise monoclonal antibodies to each protein fraction, monoclonal antibodies were only obtained against the amelogenin fraction. Six monoclonal antibodies were generated, and these could be classified into three groups recognizing different epitopes by a competitive enzyme-linked immuno-absorbent assay. A model for the arrangement of the epitopes is proposed. In Western-blotting experiments, all six monoclonal antibodies recognized amelogenin components of approx. 45,000 and 60,000 daltons as well as lower molecular-weight components of 10,000 to 30,000. It is proposed that the 45,000 and 60,000 dalton components are precursors of the lower molecular-weight components.

Production of a monoclonal antibody to bovine tooth enamel proteins.

Several monoclonal antibodies to bovine enamel proteins were produced using the mouse myeloma system. Each antibody recognized the same two protein bands on gel electrophoresis. The clones were tested in situ and clone 185 localized specifically in the enamel layer. Clones 185 and 121 were shown to recognize different antigenic determinants.

Determination of bovine enamel protein by enzyme-linked immuno-adsorbent assay.

A specific enzyme-linked immuno-adsorbent assay for enamel protein of the developing tooth was devised, using antibody against antigen prepared from immature bovine enamel. Samples containing 5-500 ng of amelogenin protein in 50 microliters gave consistent results. Bovine enamelin cross-reacted with the antibody but was less reactive than amelogenin. Proteins in other tissues or fluids did not react to the antibody.