ARTICLE |
Correspondence to: Takashi Uchida, Dept. of Oral Anatomy, Hiroshima U. School of Dentistry, Kasumi 1-2-3, Minami-ku, Hiroshima 734, Japan.
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Summary |
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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. (J Histochem Cytochem 45:1329-1340, 1997)
Key Words: ameloblastin, sheath protein, immunocytochemistry, amelogenesis, post-translational modification, postsecretory modification
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Introduction |
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Dental enamel is initially formed as a partially mineralized matrix which contains about 30% enamel proteins by weight (
Recent investigations of rat cDNA libraries revealed full-length sequences of tooth-specific proteins homologous to porcine sheath protein, named ameloblastin (
We also characterized two novel clones from a porcine enamel organ-specific cDNA library encoding sheath proteins, and proposed that the porcine sheath proteins and their proteolytic cleavage products be designated sheathlin (
Ameloblastin and amelin contain two consensus sequences for peptide domains, DGEA and VTKG, interacting with cell surface proteins (Cerny et al. 1996), whereas sheathlin contains no such sequence (
We have already demonstrated immunolocalization of sheathlin in the porcine tooth germ using region-specific antibodies. N-terminal-specific anti-sheathlin antibodies stained the prism sheath throughout the full thickness of immature enamel at the stages of matrix formation, transition, and early maturation (
In this study we investigated the synthesis, secretion, degradation, and fate of ameloblastin at the matrix formation stage of the rat incisor by immunochemical and immunohistochemical methods, using affinity-purified antibodies raised against synthetic peptides.
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Materials and Methods |
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Antibodies
Five antibodies were used in this study. Because the amino acid sequence of ameloblastin is similar to that of sheathlin (
Because antibodies Nt, M-1 and M-2 were raised against BSA-peptide conjugates, they sometimes reacted faintly with rat serum albumin, which appeared as a protein band near 57 kD in immunochemical analyses under nonreducing condition. Therefore, these antibodies were preabsorbed with an excess amount of BSA before use. Antibodies M-3 and Ct did not react with rat serum albumin because they were generated against ovalbumin-peptide conjugates.
Animals
Male Wistar rats weighting 100-150 g, purchased from Charles River Japan (Osaka, Japan), were used throughout this study. Experimental animals were anesthetized with sodium pentobarbital (Abbott; North Chicago, Illinois; 5 mg/100 g bw) and injected with brefeldin A (Sigma; 0.2 mg/100 g bw) dissolved in 0.1-0.15 ml of distilled water containing 10% methanol and 40% propylene glycol via the external jugular vein 1 hr before sacrifice. Control animals were injected with the same amount of this solution without brefeldin A. Brefeldin A is a potent inhibitor of protein transport from the rough endoplasmic reticulum (rER) to the Golgi apparatus (
Immunochemical Analysis
For immunochemical analyses, four untreated, four experimental (brefeldin A-injected), and two control rats were used. The animals were anesthetized with sodium pentobarbital and decapitated. The upper and lower incisors were removed from the alveolar bone and the enamel organ of the matrix formation stage was dissected with a fine forceps. The immature enamel surface was then gently wiped with a small cotton ball to remove remnants of secretory ameloblasts, and the immature enamel was dissected with scalpels. The samples obtained from each rat were further processed individually. Total protein was extracted with 0.5 M acetic acid and lyophilized. Samples were electrophoresed on 15 % polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS-PAGE) under nonreducing conditions and then transblotted onto nitrocellulose membranes as described previously (
Immunohistochemical Analysis
For immunohistochemical analyses, five untreated, four experimental (brefeldin A-injected), and two control rats were used. The animals were anesthetized with sodium pentobarbital and perfusion-fixed with 4% paraformaldehyde/1% glutaraldehyde in 0.067 M phosphate buffer, pH 7.4. The lower incisors were removed from the alveolar bone and decalcified with 10 % EDTA for about 1 week at 4C. Without postfixation in osmium tetroxide, they were dehydrated with graded N,N-dimethylformamide at progressively lower temperatures from 4 to -20C and embedded in a glycol methacrylate mixture at -20C (
The region of the inner and outer enamel secretion (
Immunohistochemical specificity was checked by the absorption test as described previously (
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Results |
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Immunochemical Analysis
Figure 1 shows the results of SDS-PAGE and Western blot analysis. There was almost no variation among the four animals used in this study. In the enamel organ sample, antibody Nt reacted with protein bands having molecular weights of 65 kD and 15 to 18 kD, and a faint band near 55 kD. In the enamel matrix sample, the 65-kD protein band stained weakly, and proteins ranging from 10 to 22 kD stained intensely. No reaction was observed near 55 kD (Figure 1B). Antibody M-1 stained many protein bands ranging from 10 kD to 23 kD in the enamel matrix and a single protein band near 18 kD in the enamel organ. The 65-kD and 55-kD protein bands were scarcely discernible in the enamel organ sample and not at all in the enamel matrix sample (Figure 1C). Several faintly stained protein bands ranging from 23 to 50 kD appeared after staining with antibodies Nt and M-1, especially in the enamel matrix sample (Figure 1B and Figure 1C). Electrophoresis using a freshly prepared sample showed that almost no band was found between 23-50 kD. Therefore, the proteins ranging from 23 to 50 kD stained by the antibodies Nt and M-1 were probably aggregates of lower molecular weight proteins. Antibody M-2 faintly reacted with a protein band near 65 kD and a diffuse band around 40-56 kD of the enamel organ. In the enamel matrix sample the 65-kD protein band was not detected, but a diffuse band around 40-56 kD was observed (Figure 1D). Antibody M-3 stained a protein band near 55 kD distinctly and 33 kD faintly from the enamel organ. No staining band was found in the enamel matrix (Figure 1E). Antibody Ct stained protein bands near 65 kD, 55 kD, 48 kD, 41 kD, 36 kD, 35 kD, 33 kD, and 25 kD of the enamel organ. In the enamel matrix, a similar staining pattern was obtained. However, no band was detected near 55 kD and 33 kD and the immunoreaction of the 65-kD protein band was reduced, whereas that of the 25-kD protein was intensified (Figure 1E). The 57-kD protein band, corresponding to rat serum albumin under nonreducing conditions, was distinct in the enamel organ and the enamel sample after staining with CBB (Figure 1A), but was not reactive with all the antibodies used (Figure 1B-F). Only antibody M-2 faintly stained a protein band near 56 kD. However, comparing the staining patterns after CBB and M-2, it was clear that the 56-kD protein band stained by the antibody M-2 did not correspond to rat serum albumin (Figure 1A and Figure 1D).
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Light Microscopic Immunocytochemistry
The five antibodies displayed characteristic staining patterns. There was no variation in the staining pattern among animals. Antibody Nt intensely stained the Golgi apparatus of the ameloblast and the enamel matrix. Over the enamel matrix, this antibody produced a decreasing gradient from the surface towards the dentino-enamel junction (Figure 2B). Antibody M-1 weakly stained the Golgi apparatus. Staining over the enamel matrix was intense and was similar to that produced by antibody Nt. However, in the middle- to inner-layer enamel, immunoreaction concentrated over the peripheral region of the enamel rod (Figure 2C). This staining pattern was clearer at the stage of outer enamel secretion (Figure 3B). Antibody M-2 stained the Golgi apparatus and the superficial layer of enamel matrix. The middle and inner layers were almost devoid of immunoreactivity (Figure 2D). Antibody M-3 stained the Golgi apparatus, but no reaction was found over the enamel matrix (Figure 2E). Antibody Ct intensely stained the Golgi apparatus of the ameloblast and immature enamel near the Tomes' processes. At the middle layer of immature enamel, the periphery of the enamel rod was also stained (Figure 2F). In addition to the Golgi apparatus and enamel matrix, antibodies Nt, M-1, M-2, and Ct occasionally stained small spherical bodies in the ameloblast. All the antibodies used stained neither dentin nor odontoblasts (Figure 2B-F).
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Sections cut parallel to the long axis of the interdigitating portion of the Tomes' process clearly demonstrated the relationship between the immunoreaction over the immature enamel and Tomes' processes, which are divided into proximal and interdigitating portions in the rat incisor (
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Electron Microscopic Immunocytochemistry
At ultrastructural resolution, silver grains representing immunoreactivity in the secretory ameloblast were found over the cell organelles involved in the synthesis and secretion as well as the degradation and absorption of proteins. A few silver grains were occasionally found over nuclei, mitochondria, and cytoplasmic matrix after staining with all the antibodies (Figure 5 Figure 6 Figure 7).
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The Golgi apparatus of a secretory ameloblast is a long, cylindrical structure (
The immunolocalization over immature enamel around the Tomes' process was mostly identical to that found at the light microscopic level, with the following additional observations obtained by immunoelectron microscopy. After staining with antibody M-2 and Ct, silver grains were located along the lateral plasma membrane of the interdigitating portion of the Tomes' process (Figure 6B). Many secretory granules in the core region of the interdigitating portion were intensely stained by antibodies Nt, M-2, and Ct (Figure 6), whereas most secretory granules were unlabeled or only weakly labeled after staining with antibody M-1. Tubulo-vesicular structures in the interdigitating portion of the Tomes' process were not labeled (Figure 6). In the proximal cytoplasm of the Tomes' process, coated and uncoated pits and vesicles, both of which apparently differed from the secretory granules, sometimes showed immunoreactivity after staining with antibodies Nt and Ct (Figure 7). No immunoreaction was found over the enamel matrix or secretory granules after staining with antibody M-3.
Effects of Brefeldin A
After the treatment with brefeldin A, immunochemical analysis of enamel organ samples revealed that the immunoreactivity of the 55-kD protein band was intensified, whereas the 65-kD protein bands were reduced or unchanged (Figure 8). The 15-20-kD protein bands stained with antibody Nt were dramatically reduced by the brefeldin A injection. In light microscopic immunocytochemical preparations, all of the antibodies stained granular structures diffusely distributed in the cytoplasm of secretory ameloblasts affected by brefeldin A. These structures were very clear when antibodies M-2, M-3, and Ct were used. The Golgi apparatus could not be recognized at the light microscopic level (Figure 9). At electron microscopic resolution, rER located in supra- and infranuclear compartments was sometimes dilated and contained flocculent material showing immunoreactivity (Figure 10). The Golgi apparatus was small and undulated but appeared to retain its normal organization. Secretory granules in the Golgi area were significantly reduced in number but were numerous in the Tomes' process. Mitochondria and nuclei remained unstained (Figure 10). There was almost no variation among the experimental animals concerning the results of immunochemical and immunohistochemical analyses. The immunochemical and immunohistochemical data obtained from the control rats injected with solutions used for dissolving brefeldin A were the same as those of nontreated rats.
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Specificity Control
Immunostaining over the ameloblast and the immature enamel almost disappeared after preabsorption of the antibodies with purified peptide.
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Discussion |
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Immunochemical analyses showed that the highest molecular weight protein band that reacted to all the antibodies used (excepting M-3) was 65 kD in each case. In addition, all antibodies stained cell organelles involved in the production and secretion of proteins in secretory ameloblasts, although intensities varied. Therefore, we conclude that the 65-kD protein is likely to be the largest form of ameloblastin.
Immunoelectron microscopy demonstrated that antibody M-3, which specifically recognizes residues 386-399 of ameloblastin, stained only the rER and the cis side of the Golgi apparatus. Downstream Golgi elements, secretory granules, and enamel matrix showed no immunoreactivity. In immunochemical preparations of the enamel organ, the M-3 antibody reacted to a protein band near 55 kD but not the 65-kD band. After treatment with brefeldin A, which inhibits protein transport from the rER to the Golgi apparatus (
The calculated molecular mass of ameloblastin in the absence of post-translational modifications is 45 kD (
In the present Western blot analyses, no immunoreactive protein band larger than 65 kD was found in either the enamel organ sample or the enamel matrix sample. On the other hand,
Immunochemical analyses showed that the antibodies that specifically recognize ameloblastin residues 27-48 (Nt) and 98-107 (M-1) reacted with 10-kD to 23-kD proteins but not with proteins whose molecular weight was higher than 25 kD, except for the 55-kD and 65-kD proteins. In immunohistochemical preparations, these antibodies stained the entire thickness of immature enamel. On the other hand, the antibody that recognizes residues 224-232 (M-2) stained protein bands near 65 kD and diffuse bands from 56 to 40 kD. The antibody that recognizes residues 406-419 (Ct) stained several protein bands ranging from 25 kD to 65 kD. These two antibodies (M-2 and Ct) stained enamel matrix located near the putative secretion sites in immunocytochemical preparations. These results indicate that the initial cleavages of the 65-kD ameloblastin generate relatively small polypeptides containing the N-terminal and relatively large polypeptides containing the C-terminal portions of the secreted protein. The N-terminal polypeptides appear to be rather stable and are gradually degraded, but are not lost, from the enamel matrix during the matrix formation stage. The C-terminal large polypeptides appear to be degraded rapidly and are lost from the immature enamel soon after secretion.
Electron microscopic immunocytochemistry identified coated pits or vesicles containing ameloblastin and/or its cleaved polypeptides. These data support the hypothesis that secretory ameloblasts absorb enamel matrix proteins (
Our previous immunohistochemical study using antiserum against porcine low molecular weight sheath proteins demonstrated no specific staining pattern over the immature enamel of the rat incisor (
Silver deposits representing immunoreactivity of the C-terminal region of ameloblastin were found along the plasma membrane of the interdigitating portion of the Tomes' process. These results, together with the fact that ameloblastin contains consensus sequences for peptide domains interacting with cell surface proteins (Cerny et al. 1996), suggest that the plasma membrane of the interdigitating portion of the Tomes' process is connected to the enamel matrix via ameloblastin and/or its cleavage products. The functional significance of ameloblastin as a cell binding protein has not been elucidated. It is well known that the Tomes' process in rat is extremely long relative to other animal species such as pig, cat, and human, and is deeply embedded in immature enamel. Hence, the distal portion of the Tomes' process of the rat has been called the interdigitating portion (
Using autoradiography of sections of rat incisor and fluorographs of proteins extracted from rat enamel organ and immature enamel after injection of various radioactive molecules, Smith and co-workers have revealed basic biochemical properties of short-lived sulfated glycoproteins (
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Acknowledgments |
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Supported in part by Grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (Nos. 08672071, 09671855 and 09771502).
We thank Profs J.P. Simmer and J.C-C. Hu, Department of Pediatric Dentistry, University of Texas Health Science Center at San Antonio, School of Dentistry, who read the manuscript and made helpful comments. We also thank the Research Center for Molecular Medicine, Hiroshima University, for use of the peptide synthesizer PSSM-8.
Received for publication January 17, 1997; accepted May 23, 1997.
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